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Sample records for adult skin fibroblasts

  1. Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

    SciTech Connect

    Wharton, W.

    1984-01-01

    Cultures of human fibroblasts were prepared from chest skin obtained either from newborns (less than 3 months old) or adults (more than 35 years old) and maintained in vitro until they senesced. Adult cells grew logarithmically in medium supplemented with whole blood serum but not with platelet-poor plasma. Early passage cells obtained from newborns grew equally well in either plasma- or serum-supplemented medium. The difference in growth factor requirements between adult and newborn cells persisted through the lifespan of the cells; i.e., newborn cells did not develop adult hormonal requirements when maintained in culture. Thus, in vitro cellular aging can be distinguished from some types of differentiation.

  2. Fibroblast heterogeneity: more than skin deep.

    PubMed

    Sorrell, J Michael; Caplan, Arnold I

    2004-02-15

    Dermal fibroblasts are a dynamic and diverse population of cells whose functions in skin in many respects remain unknown. Normal adult human skin contains at least three distinct subpopulations of fibroblasts, which occupy unique niches in the dermis. Fibroblasts from each of these niches exhibit distinctive differences when cultured separately. Specific differences in fibroblast physiology are evident in papillary dermal fibroblasts, which reside in the superficial dermis, and reticular fibroblasts, which reside in the deep dermis. Both of these subpopulations of fibroblasts differ from the fibroblasts that are associated with hair follicles. Fibroblasts engage in fibroblast-epidermal interactions during hair development and in interfollicular regions of skin. They also play an important role in cutaneous wound repair and an ever-increasing role in bioengineering of skin. Bioengineered skin currently performs important roles in providing (1) a basic understanding of skin biology, (2) a vehicle for testing topically applied products and (3) a resource for skin replacement.

  3. Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models.

    PubMed

    Samluk, Anna; Zakrzewska, Karolina Ewa; Pluta, Krzysztof Dariusz

    2013-07-01

    Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.

  4. Alteration of Skin Properties with Autologous Dermal Fibroblasts

    PubMed Central

    Thangapazham, Rajesh L.; Darling, Thomas N.; Meyerle, Jon

    2014-01-01

    Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices. PMID:24828202

  5. Alteration of skin properties with autologous dermal fibroblasts.

    PubMed

    Thangapazham, Rajesh L; Darling, Thomas N; Meyerle, Jon

    2014-05-13

    Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA) while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices.

  6. Key Regulatory Role of Dermal Fibroblasts in Pigmentation as Demonstrated Using a Reconstructed Skin Model: Impact of Photo-Aging

    PubMed Central

    Duval, Christine; Cohen, Catherine; Chagnoleau, Corinne; Flouret, Virginie; Bourreau, Emilie; Bernerd, Françoise

    2014-01-01

    To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is

  7. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    SciTech Connect

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-02-01

    The metabolism of (1,2,6,7-3H)testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC.

  8. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    PubMed

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes.

  9. Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

    PubMed

    Berning, Manuel; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-09-01

    Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

  10. Histamine inhibits differentiation of skin fibroblasts into myofibroblasts.

    PubMed

    Lin, Lin; Yamagata, Kaoru; Nakayamada, Shingo; Sawamukai, Norifumi; Yamaoka, Kunihiro; Sakata, Kei; Nakano, Kazuhisa; Tanaka, Yoshiya

    2015-07-31

    Histamine and TGF-β, major mediators secreted by mast cells, are involved in skin inflammation and play critical roles in the pathogenesis of systemic sclerosis. However, the roles of signaling mechanisms in the development of skin fibrosis remain largely unclear. Here we show that histamine suppressed the expression of α smooth muscle actin (αSMA), a marker of myofibroblasts, induced by TGF-β1 in skin fibroblasts. Histamine H1-receptor (H1R), but not H2-receptor (H2R) or H4-receptor (H4R), was expressed on skin fibroblasts at both mRNA and protein levels. Interestingly, an H1R antagonist, but not H2R or H4R antagonists, antagonized the histamine-mediated suppression of αSMA expression by TGF-β1. Correspondingly, phosphorylated Smad2 was detected after treatment with TGF-β1, whereas the addition of histamine inhibited this phosphorylation. Taken together, histamine-H1R decreased TGF-β1-mediated Smad2 phosphorylation and inhibited differentiation of skin fibroblasts into myofibroblasts.

  11. Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

    SciTech Connect

    Nakamura, T.; Takagaki, K.; Kubo, K.; Morikawa, A.; Tamura, S.; Endo, M. )

    1990-10-15

    The chain length of ({sup 3}H)hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of ({sup 3}H)glucosamine was investigated. ({sup 3}H)Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.

  12. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

    PubMed Central

    Saini, Natalie; Chan, Kin; Grimm, Sara A.; Dai, Shuangshuang; Fargo, David C.; Kaufmann, William K.; Taylor, Jack A.; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J.; Schurman, Shepherd H.; Malc, Ewa P.; Mieczkowski, Piotr A.

    2016-01-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307 PMID:27788131

  13. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

    PubMed

    Saini, Natalie; Roberts, Steven A; Klimczak, Leszek J; Chan, Kin; Grimm, Sara A; Dai, Shuangshuang; Fargo, David C; Boyer, Jayne C; Kaufmann, William K; Taylor, Jack A; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J; Schurman, Shepherd H; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

    2016-10-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

  14. Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts

    SciTech Connect

    Mattes, P.M.; Maloney, P.C.; Littlefield, J.W.

    1987-05-01

    An abnormal regulation of chloride permeability has been described for epithelial cells from patients with cystic fibrosis (CF). To learn more about the biochemical basis of this inherited disease, the authors have studied chloride metabolism in cultured CF fibroblasts by comparing the efflux of /sup 36/Cl/sup -/ from matched pairs of CF and normal fibroblasts. The rate constants describing /sup 36/Cl/sup -/ efflux did not differ between the two cell types, but in each of the four pairs tested the amount of /sup 36/Cl/sup -/ contained within CF cells was consistently reduced, by 25-30%, relative to normal cells. Comparisons of cell water content and /sup 22/Na/sup +/ efflux showed no differences between the two cell types, suggesting that overall intracellular chloride concentration is lower than normal in CF fibroblasts. Such data suggest that the CF gene defect is expressed in skin fibroblasts and that this defect may alter the regulation of intracellular Cl/sup -/ concentration, perhaps through changes in Cl/sup -/ permeability.

  15. Leopard syndrome associated with hyperelastic skin: analysis of collagen metabolism in cultured skin fibroblasts.

    PubMed

    Ohkura, T; Ohnishi, Y; Kawada, A; Tajima, S; Ishibashi, A; Ono, K

    1999-01-01

    We present a patient with Leopard syndrome and hyperelastic skin. Biochemical analysis using cultured skin fibroblasts showed normal type III and V collagen synthesis, lysyl hydroxylation level of type I procollagen and processing of pro-alpha(1) and alpha(2)(I). Our results suggest that molecular defects of hyperelasticity in Leopard syndrome are not related to abnormal collagen metabolism, although not all steps of collagen synthesis have been investigated.

  16. Tattoo ink nanoparticles in skin tissue and fibroblasts

    PubMed Central

    Twigg, Peter C; Baker, Richard; Tobin, Desmond J

    2015-01-01

    Summary Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells. PMID:26171294

  17. Tattoo ink nanoparticles in skin tissue and fibroblasts.

    PubMed

    Grant, Colin A; Twigg, Peter C; Baker, Richard; Tobin, Desmond J

    2015-01-01

    Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells.

  18. Skin telocytes versus fibroblasts: two distinct dermal cell populations

    PubMed Central

    Kang, Yuli; Zhu, Zaihua; Zheng, Yonghua; Wan, Weiguo; Manole, Catalin G; Zhang, Qiangqiang

    2015-01-01

    It is already accepted that telocytes (TCs) represent a new type of interstitial cells in human dermis. In normal skin, TCs have particular spatial relations with different dermal structures such as blood vessels, hair follicles, arrector pili muscles or segments of sebaceous and/or eccrine sweat glands. The distribution and the density of TCs is affected in various skin pathological conditions. Previous studies mentioned the particular (ultra)structure of TCs and also their immunophenotype, miR imprint or proteome, genome or secretome features. As fibroblast is the most common intersitital cell (also in human dermis), a dedicated comparison between human skin TCs and fibroblasts (Fbs) was required to be performed. In this study, using different techniques, we document several points of difference between human dermis TCs and Fbs. By transmission electron microscopy (TEM) and scanning electron microscopy (SEM), we demonstrated TCs with their hallmark cellular prolongations – telopodes. Thus, we showed their ultrastructural distinctiveness from Fbs. By RayBio Human Cytokine Antibody Array V analyses performed on the supernatant from separately cultured TCs and Fbs, we detected the cytokine profile of both cell types, individually. Two of 79 detected cytokines – epithelial-derived neutrophil-activating peptide 78 and granulocyte chemotactic protein-2 – were 1.5 times higher in the supernatant of TCs (comparing with Fbs). On the other hand, 37 cytokines were at least 1.5 higher in Fbs supernatant (comparing with TCs), and among them six cytokines – interleukin 5, monocyte chemotactic protein-3 (MCP-3), MCP-4, macrophage inflammatory protein-3, angiogenin, thrombopoietin – being 9.5 times higher (results also confirmed by ELISA testing). In summary, using different techniques, we showed that human dermal TCs and Fbs are different in terms of ultrastructure and cytokine profile. PMID:26414534

  19. Deletion of the homeobox gene PRX-2 affects fetal but not adult fibroblast wound healing responses.

    PubMed

    White, Philip; Thomas, David W; Fong, Steven; Stelnicki, Eric; Meijlink, Fritz; Largman, Corey; Stephens, Phil

    2003-01-01

    The phenotype of fibroblasts repopulating experimental wounds in vivo has been shown to influence both wound healing responses and clinical outcome. Recent studies have demonstrated that the human homeobox gene PRX-2 is strongly upregulated in fibroblasts within fetal, but not adult, mesenchymal tissues during healing. Differential homeobox gene expression by fibroblasts may therefore be important in mediating the scarless healing exhibited in early fetal wounds. RNase protection analysis demonstrated that murine Prx-2 expression was involved in fetal but not adult wound healing responses in vitro. Using fibroblasts established from homozygous mutant (Prx-2-/-) and wild-type (Prx-2+/+) murine skin tissues it was demonstrated that Prx-2 affected a number of fetal fibroblastic responses believed to be important in mediating scarless healing in vivo; namely cellular proliferation, extracellular matrix reorganization, and matrix metalloproteinase 2 and hyaluronic acid production. These data demonstrate how Prx-2 may contribute to the regulation of fetal, but not adult, fibroblasts and ultimately the wound healing phenotype. This study provides further evidence for the importance of homeobox transcription factors in the regulation of scarless wound healing. A further understanding of these processes will, it is hoped, enable the targeting of specific therapies in wound healing, both to effect scarless healing and to stimulate healing in chronic, nonhealing wounds such as venous leg ulcers.

  20. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    SciTech Connect

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J. )

    1990-08-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation.

  1. Exposure to Varying Strain Magnitudes Influences the Conversion of Normal Skin Fibroblasts Into Hypertrophic Scar Cells.

    PubMed

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Cai, Xia; Liu, Tao

    2016-04-01

    Mechanical strain is a key contributor in the pathogenesis of hypertrophic scarring, whose optimal stretch magnitudes to initiate the differentiation of normal skin fibroblasts into aberrant fibroblasts phenotype remains largely unresolved. Influence of varying cyclic strain magnitudes on cultured human normal skin fibroblasts and its transformation into hypertrophic scar fibroblast-like phenotype is investigated in this study. Cultured fibroblasts isolated from hypertrophic scar and normal skin tissue were subjected to cyclic mechanical stretching under individual 10%, 15%, and 20% strain magnitudes at a frequency of 0.1 Hz for 24 hours. Stretched normal skin fibroblasts demonstrated significantly increased rates of cell proliferation, and also apparently oriented away nearly perpendicular to the applied stretching direction. Interestingly, the applied 10% strains magnitude resulted in a markedly enhanced cell proliferative ability compared with that of 20% strain magnitude. Parameters involving the mechanotransduction signaling, such as integrin β1 and P130Cas, were significantly improved at both mRNA and protein levels in the stretched normal skin fibroblasts, which was demonstrated in a negative magnitude-dependent manner. In addition, 10% strains magnitude triggered the highest expression levels of growth factor TGF-β1 and collagen matrix in stretched normal skin fibroblasts. Collectively, these results indicate that the 10% stretching magnitude, of the 3 strain magnitudes studied, is most effective for triggering the optimal mechanotransduction effects and biological responses inside cultured skin fibroblasts. The demonstrable conversion of normal skin fibroblasts into hypertrophic scar fibroblasts was also observed when 10% stretching magnitude was applied to cultured fibroblasts in vitro.

  2. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

    PubMed Central

    Yao, Zizhen; Jaeger, Jochen C; Ruzzo, Walter L; Morale, Cecile Z; Emond, Mary; Francke, Uta; Milewicz, Dianna M; Schwartz, Stephen M; Mulvihill, Eileen R

    2007-01-01

    Background Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 × 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater. PMID:17850668

  3. Expression of catalytically active Matrix Metalloproteinase-1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

    PubMed Central

    Xia, Wei; Hammerberg, Craig; Li, Yong; He, Tianyuan; Quan, Taihao; Voorhees, John J; Fisher, Gary J

    2013-01-01

    Summary Increased expression of matrix metalloproteinase-1 (MMP-1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP-1-mediated collagen fibril fragmentation is a key driver of age-related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP-1 mutant (MMP-1 V94G) in adult human skin in organ culture and fibroblasts in three dimensional collagen lattice cultures. Expression of MMP-1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP-1 V94G in dermal fibroblasts cultured in three-dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP-1 siRNA-mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP-1 V94G-fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF-β pathway, and reduced collagen production. These results support the concept that MMP-1-mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts, and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age-related decline of fibroblast function. PMID:23601157

  4. Abnormal Collagen Metabolism in Cultured Skin Fibroblasts from Patients with Duchenne Muscular Dystrophy

    NASA Astrophysics Data System (ADS)

    Rodemann, H. Peter; Bayreuther, Klaus

    1984-08-01

    Total collagen synthesis is decreased by about 29% (P < 0.01) in skin fibroblasts established in vitro from male patients with Duchenne muscular dystrophy (DMD) as compared with that in normal male skin fibroblasts in vitro. The reduction in collagen synthesis is associated with an approximately 2-fold increase in collagen degradation in DMD fibroblasts. Correlated to these alterations in the metabolism of collagen, DMD fibroblasts express a significantly higher hydroxyproline/proline ratio (DMD: 1.36-1.45; P < 0.01) than do normal fibroblasts (controls: 0.86-0.89). The increased hydroxylation of proline residues of collagen (composed of type I and type III) could be the cause for the enhanced degradation of collagen in DMD fibroblasts.

  5. Mercury-induced micronuclei in skin fibroblasts of beluga whales

    SciTech Connect

    Gauthier, J.M.; Dubeau, H.; Rassart, E.

    1998-12-01

    Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high incidence of cancer observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei assay was used to test the genotoxic potential of mercury compounds in skin fibroblasts of an Arctic beluga whale. Both mercuric chloride (Hg) and methylmercury (MeHg) induced a highly significant dose-response increase of micronucleated cells. Statistically significant increases in micronucleated cells were observed for 0.5, 5, and 20 {micro}g/ml Hg and 0.05, 0.5, and 2 {micro}g/ml MeHg when compared to control cultures. Concentrations of 0.5, 5, and 20 {micro}g/ml Hg induced a two-, three- and fourfold increase of micronucleated cells, respectively. Treatment with MeHg was one order of magnitude more potent in inducing micronuclei and in inhibiting cell proliferation than Hg. Although results of this in vitro study do not imply that mercury compounds are involved in the etiology of cancer in St. Lawrence beluga whales, significant increases in micronuclei frequency were found at low concentrations of MeHg that are believed to be comparable to concentrations present in certain whales of this population.

  6. AGE-RELATED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS INDUCED BY MMS

    EPA Science Inventory

    Age-Related Gene Expression Changes In Human Skin Fibroblasts Induced By methyl methanesulfonate. Geremy W. Knapp, Alan H. Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Prote...

  7. CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Robichaud, Patrick; Voorhees, John J; Fisher, Gary J

    2011-08-01

    Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482-90, 2006, J Invest Dermatol 130:1697-706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an "Age-Associated Secretory Phenotype", in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.

  8. Constituents from the roots of Taraxacum platycarpum and their effect on proliferation of human skin fibroblasts.

    PubMed

    Warashina, Tsutomu; Umehara, Kaoru; Miyase, Toshio

    2012-01-01

    A MeOH extract from the roots of Taraxacum platycarpum has shown significant effects on the proliferation of normal human skin fibroblasts. Chemical analysis of the extract resulted in the isolation of 26 compounds, including eight new triterpenes, one new sesquiterpene glycoside, and seventeen known compounds. The structure of each new compound was established using NMR spectroscopy. Some triterpenes had a significant effect on the proliferation of normal human skin fibroblasts.

  9. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  10. Properties of sulfatases in cultured skin fibroblasts of multiple sulfatase deficient patients.

    PubMed

    Yutaka, T; Okada, S; Kato, T; Inui, K; Yabuuchi, H

    1981-10-01

    Various sulfatase activities were assayed in cultured skin fibroblasts from patients with multiple sulfatase deficiency (MSD). MSD cell lines displayed deficiencies of arylsulfatase A and iduronate sulfatase, but activities of arylsulfatase B, N-acetylgalactosamine 6-sulfate sulfatase and N-acetylglucosamine 6-sulfate sulfatase were within normal ranges, but not consistently. Arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD cell lines had similar Km, pH optima, inhibitory or activator sensitivity to that of normal skin fibroblasts. Arylsulfatase B in MSD cell lines also had properties similar to that of normal skin fibroblasts, except an abnormal heat stability. From our results, we conclude that properties of arylsulfatase A, minor anionic arylsulfatase and N-acetylgalactosamine 6-sulfate sulfatase in MSD fibroblasts were intact. On the other hand, arylsulfatase B in MSD might be a functionally abnormal enzyme.

  11. Skin Temperature Biofeedback in Children and Adults.

    ERIC Educational Resources Information Center

    Suter, Steve; Loughry-Machado, Glenna

    1981-01-01

    Skin temperature biofeedback performance was studied in 38 6- to 10-year-old children and 38 of their parents across two sessions of audio biofeedback segments in which participants alternately attempted hand-warming and hand-cooling. Children were superior to adults in controlling skin temperature in the presence of biofeedback. (Author/DB)

  12. Human gingival fibroblasts display a non-fibrotic phenotype distinct from skin fibroblasts in three-dimensional cultures.

    PubMed

    Mah, Wesley; Jiang, Guoqiao; Olver, Dylan; Cheung, Godwin; Kim, Ben; Larjava, Hannu; Häkkinen, Lari

    2014-01-01

    Scar formation following skin injury can be a major psychosocial and physiological problem. However, the mechanisms of scar formation are still not completely understood. Previous studies have shown that wound healing in oral mucosa is faster, associates with a reduced inflammatory response and results to significantly reduced scar formation compared with skin wounds. In the present study, we hypothesized that oral mucosal fibroblasts from human gingiva are inherently distinct from fibroblasts from breast and abdominal skin, two areas prone to excessive scar formation, which may contribute to the preferential wound healing outcome in gingiva. To this end, we compared the phenotype of human gingival and skin fibroblasts cultured in in vivo-like three-dimensional (3D) cultures that mimic the cells' natural extracellular matrix (ECM) niche. To establish 3D cultures, five parallel fibroblast lines from human gingiva (GFBLs) and breast skin (SFBLs) were seeded in high density, and cultured for up to 21 days in serum and ascorbic acid containing medium to induce expression of wound-healing transcriptome and ECM deposition. Cell proliferation, morphology, phenotype and expression of wound healing and scar related genes were analyzed by real-time RT-PCR, Western blotting and immunocytochemical methods. The expression of a set of genes was also studied in three parallel lines of human abdominal SFBLs. Findings showed that GFBLs displayed morphologically distinct organization of the 3D cultures and proliferated faster than SFBLs. GFBLs expressed elevated levels of molecules involved in regulation of inflammation and ECM remodeling (MMPs) while SFBLs showed significantly higher expression of TGF-β signaling, ECM and myofibroblast and cell contractility-related genes. Thus, GFBLs display an inherent phenotype conducive for fast resolution of inflammation and ECM remodeling, characteristic for scar-free wound healing, while SFBLs have a profibrotic, scar-prone phenotype.

  13. Collagen Fragmentation Promotes Oxidative Stress and Elevates Matrix Metalloproteinase-1 in Fibroblasts in Aged Human Skin

    PubMed Central

    Fisher, Gary J.; Quan, Taihao; Purohit, Trupta; Shao, Yuan; Cho, Moon Kyun; He, Tianyuan; Varani, James; Kang, Sewon; Voorhees, John J.

    2009-01-01

    Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and α2β1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and α2β1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ10 significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging. PMID:19116368

  14. Full-thickness skin wound healing using autologous keratinocytes and dermal fibroblasts with fibrin: bilayered versus single-layered substitute.

    PubMed

    Idrus, Ruszymah Bt Hj; Rameli, Mohd Adha bin P; Low, Kiat Cheong; Law, Jia Xian; Chua, Kien Hui; Latiff, Mazlyzam Bin Abdul; Saim, Aminuddin Bin

    2014-04-01

    Split-skin grafting (SSG) is the gold standard treatment for full-thickness skin defects. For certain patients, however, an extensive skin lesion resulted in inadequacies of the donor site. Tissue engineering offers an alternative approach by using a very small portion of an individual's skin to harvest cells for propagation and biomaterials to support the cells for implantation. The objective of this study was to determine the effectiveness of autologous bilayered tissue-engineered skin (BTES) and single-layer tissue-engineered skin composed of only keratinocytes (SLTES-K) or fibroblasts (SLTES-F) as alternatives for full-thickness wound healing in a sheep model. Full-thickness skin biopsies were harvested from adult sheep. Isolated fibroblasts were cultured using medium Ham's F12: Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, whereas the keratinocytes were cultured using Define Keratinocytes Serum Free Medium. The BTES, SLTES-K, and SLTES-F were constructed using autologous fibrin as a biomaterial. Eight full-thickness wounds were created on the dorsum of the body of the sheep. On 4 wounds, polyvinyl chloride rings were used as chambers to prevent cell migration at the edge. The wounds were observed at days 7, 14, and 21. After 3 weeks of implantation, the sheep were euthanized and the skins were harvested. The excised tissues were fixed in formalin for histological examination via hematoxylin-eosin, Masson trichrome, and elastin van Gieson staining. The results showed that BTES, SLTES-K, and SLTES-F promote wound healing in nonchambered and chambered wounds, and BTES demonstrated the best healing potential. In conclusion, BTES proved to be an effective tissue-engineered construct that can promote the healing of full-thickness skin lesions. With the support of further clinical trials, this procedure could be an alternative to SSG for patients with partial- and full-thickness burns.

  15. Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts

    PubMed Central

    Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

    2017-01-01

    Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts. PMID:28217097

  16. Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts.

    PubMed

    Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

    2017-01-01

    Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine(9) (pGSK3β Ser(9)) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

  17. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Głuszuk, Katarzyna; Surażyński, Arkadiusz

    2014-01-01

    Aim The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Results Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts. PMID:25342885

  18. Senescent phenotypes of skin fibroblasts from patients with Tangier disease

    SciTech Connect

    Matsuura, Fumihiko . E-mail: fumihiko@imed2.med.osaka-u.ac.jp; Hirano, Ken-ichi; Ikegami, Chiaki; Sandoval, Jose C.; Oku, Hiroyuki; Yuasa-Kawase, Miyako; Tsubakio-Yamamoto, Kazumi; Koseki, Masahiro; Masuda, Daisaku; Tsujii, Ken-ichi; Shimomura, Iichiro; Hori, Masatsugu; Yamashita, Shizuya; Ishigami, Masato; Nishida, Makoto

    2007-06-01

    Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) {approx}10 compared with control cells. TD cells practically ceased proliferation at PDL {approx}30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated {beta}-galactosidase (SA-{beta}-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients.

  19. Multiple sulfatase deficiency (mucosulfatidosis): impaired degradation of labeled sulfated compounds in cultured skin fibroblasts in vivo.

    PubMed

    Eto, Y; Numaguchi, S; Tahara, T; Rennert, O M

    1980-10-01

    Skin fibroblasts from a Japanese patient with multiple sulfatase deficiency (MSD) (Mucosulfatidosis) were studied with regard to metabolism of various sulfated compounds in vivo. Several sulfatase activities (arylsulfatases A, B and C, cholesterol sulfatase, heparin N-sulfatase) were deficient in skin fibroblasts grown in F-10 CO2 medium. The accumulation and degradation of 35S-sulfatide, 35S-mucopolysaccharides, 14C-cholesterol sulfate by MSD cells were also studied, comparing them to control, Hunter and metachromatic leukodystrophy cells. MSD fibroblasts accumulated and failed to degrade these compounds in vivo. Cholesterol sulfate was also incorporated into the control and pathological cells, and MSD cells were unable to hydrolyze cholesterol sulfate, though cholesterol sulfate is known to be hydrolyzed in the non-lysosomal subfraction. From these data it is clear that multiple enzyme deficiencies in MSD fibroblasts can be demonstrated in vivo.

  20. The effect of keratinocytes on the biomechanical characteristics and pore microstructure of tissue engineered skin using deep dermal fibroblasts.

    PubMed

    Varkey, Mathew; Ding, Jie; Tredget, Edward E

    2014-12-01

    Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing.

  1. Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

    DTIC Science & Technology

    2008-06-01

    diversity in its structure and function across anatomic sites. Scalp skin is easily recognizable by the numerous terminal hair follicles ; in contrast...forming the placodes that are progeni- tors of hair follicles (Millar, 2002). Classic heterotopic recombination PERSPECTIVE 776 Journal of...to the development of scales rather than feathers (Dhouailly, 1973, 1984). Specialized fibroblasts from the dermal papilla of hair follicles (Jahoda

  2. Effects of cigarette smoke residues from textiles on fibroblasts, neurocytes and zebrafish embryos and nicotine permeation through human skin.

    PubMed

    Hammer, Timo R; Fischer, Kirsten; Mueller, Marina; Hoefer, Dirk

    2011-09-01

    Toxic substances from cigarette smoke can attach to carpets, curtains, clothes or other surfaces and thus may pose risks to affected persons. The phenomenon itself and the potential hazards are discussed controversially, but scientific data are rare. The objective of this study was to examine the potential of textile-bound nicotine for permeation through human skin and to assess the effects of cigarette smoke extracts from clothes on fibroblasts, neurocytes and zebrafish embryos. Tritiated nicotine from contaminated cotton textiles penetrated through adult human full-thickness skin as well as through a 3D in vitro skin model in diffusion chambers. We also observed a significant concentration-dependent cytotoxicity of textile smoke extracts on fibroblast viability and structure as well as on neurocytes. Early larval tests with zebrafish embryos were used as a valid assay for testing acute vertebrate toxicity. Zebrafish development was delayed and most of the embryos died when exposed to smoke extracts from textiles. Our data show that textiles contaminated with cigarette smoke represent a potential source of nicotine uptake and can provoke adverse health effects.

  3. Caffeine protects human skin fibroblasts from acute reactive oxygen species-induced necrosis.

    PubMed

    Silverberg, Jonathan I; Patel, Mital; Brody, Neil; Jagdeo, Jared

    2012-11-01

    Oxidative damage by reactive oxygen species (ROS) plays a major role in aging and carcinogenesis. Little is known about either the effects of acute ROS in necrosis and inflammation of skin or the therapeutic agents for prevention and treatment. Previously, our laboratory identified caffeine as an inhibitor of hydrogen peroxide (H2O2)-generated lipid peroxidation products in human skin fibroblasts. Here, we study effects of caffeine on acute ROS-mediated necrosis. Human skin fibroblasts were incubated with caffeine, followed by H2O2 challenge. Flow cytometry was used to analyze cell morphology, counts, apoptosis and necrosis, and ROS. We found that caffeine protects from H2O2 cell damage at lower (0.01 mM) and intermediate (0.1 mM) doses. The beneficial effects of caffeine appear to be mediated by a mechanism other than antioxidant function.

  4. Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts.

    PubMed

    Opländer, Christian; Wetzel, Wiebke; Cortese, Miriam M; Pallua, Norbert; Suschek, Christoph V

    2008-05-01

    Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

  5. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts

    PubMed Central

    Schenkel, Laila C.; Singh, Ratnesh K.; Michel, Vera; Zeisel, Steven H.; da Costa, Kerry-Ann; Johnson, Amy R.; Mudd, Harvey S.; Bakovic, Marica

    2015-01-01

    Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2–3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.—Schenkel, L. C., Singh, R. K., Michel, V., Zeisel, S. H., da Costa, K.-A., Johnson, A. R., Mudd, H. S., Bakovic, M. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts. PMID:25466896

  6. Genotoxicity of alpha particles in human embryonic skin fibroblasts

    SciTech Connect

    Chen, D.J.; Strniste, G.F.; Tokita, N.

    1984-11-01

    Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to ..cap alpha.. particles from /sup 238/ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, D/sub 0/, is approximately 1.3 Gy for X rays and 0.25 Gy for ..cap alpha.. particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for ..cap alpha.. particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to ..cap alpha.. particles than to X rays.

  7. DNA damage in human skin fibroblasts exposed to UVA light used in clinical PUVA treatment

    SciTech Connect

    Bredberg, A.

    1981-06-01

    Human skin fibroblasts were irradiated with a clinically used UVA light source. The doses (1.1 and 3 J/cm2) were similar to those reaching the dermis during clinical PUVA treatment of psoriasis. DNA strand breaks, as determined by alkaline elution, were formed in a dose-dependent way and disappeared within 1 hr of postincubation at 37 degrees C. These findings have clinical implications since UVA-induced DNA damage may be accompanied by mutagenic and tumor promoting effects.

  8. Impacts of antibiotics on in vitro UVA-susceptibility of human skin fibroblasts.

    PubMed

    Le Gall, Rozenn; Marchand, Cécile; Rees, Jean-François

    2005-01-01

    Many studies of UVA-induced cell damage use skin cells obtained during plastic surgery. As the skin is contaminated by micro-organisms, antibiotics need to be added to primary skin cell culture media. This study analysed the impact of the most widely used agents, penicillin, streptomycin, and amphotericin B deoxycholate (amB), on UVA-irradiated human skin fibroblasts. The results show that the presence of amB in cell culture media increases the susceptibility of fibroblasts to UVA and the intracellular level of reactive oxygen species, even when cells are irradiated in amB-free saline. This photosensitising effect of amB can be prevented if the antifungal agent is removed from the culture medium at least 24 hours before irradiation. Moreover, the use of streptomycin during cell culture partly protects cells against the UVA-induced mortality linked to amB. Acellular tests on lipid micelles suggest that this protective effect could result from an inhibition of lipid peroxidation by the antibacterial agent. In conclusion, antibiotics should be used with care in cell culture media if the cells are to be used in physiological studies of fine mechanisms in UVA-susceptibility of skin cells. In other cases, cells should be maintained in antibiotic-free media for 24 hours before irradiation.

  9. Fibroblast-dependent induction of a murine skin lesion with similarity to human common blue nevus.

    PubMed Central

    Prouty, S. M.; Lawrence, L.; Stenn, K. S.

    1996-01-01

    In an attempt to define epithelial-mesenchymal interactions in skin appendage formation, we have been studying a nude mouse grafting model that permits the combination of heterotypic and heterochronic epithelial and mesenchymal cells. In this study using neonatal hair bud cells combined with various mesenchymal cell preparations, we show that one can regenerate near-complete skin with intact epidermal and dermal layers plus mature hair follicles. It was determined that the character of the resulting regenerated skin could be manipulated as a function of the specific mesenchymal component. Lack of dermal cells resulted in a scar, whereas inclusion of a suspension of dissociated total dermal cells resulted in near-complete skin regeneration, and in the presence of follicular papilla fibroblasts (both hair-inductive and non-hair-inductive) or NIH3T3 fibroblasts, the reconstitution had similarity to the common blue nevus. The results indicate that 1) a stimulant of human common blue nevus can be produced in an animal model, 2) the underlying disorder of the lesion in mice appears to be entirely dermal in origin, arising independent of the epidermal component, and 3) complex dermal cell interactions involving lesion-initiative and lesion-suppressive activities underlie the pathogenesis. This experimental system will serve as a valuable tool in elucidating cutaneous dermal-epidermal signals in normal skin as well as the alteration of these signals in malformations such as the hamartoma described here. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8669473

  10. Far-infrared suppresses skin photoaging in ultraviolet B-exposed fibroblasts and hairless mice

    PubMed Central

    Chiu, Hui-Wen; Chen, Cheng-Hsien; Chen, Yi-Jie; Hsu, Yung-Ho

    2017-01-01

    Ultraviolet (UV) induces skin photoaging, which is characterized by thickening, wrinkling, pigmentation, and dryness. Collagen, which is one of the main building blocks of human skin, is regulated by collagen synthesis and collagen breakdown. Autophagy was found to block the epidermal hyperproliferative response to UVB and may play a crucial role in preventing skin photoaging. In the present study, we investigated whether far-infrared (FIR) therapy can inhibit skin photoaging via UVB irradiation in NIH 3T3 mouse embryonic fibroblasts and SKH-1 hairless mice. We found that FIR treatment significantly increased procollagen type I through the induction of the TGF-β/Smad axis. Furthermore, UVB significantly enhanced the expression of matrix metalloproteinase-1 (MMP-1) and MMP-9. FIR inhibited UVB-induced MMP-1 and MMP-9. Treatment with FIR reversed UVB-decreased type I collagen. In addition, FIR induced autophagy by inhibiting the Akt/mTOR signaling pathway. In UVB-induced skin photoaging in a hairless mouse model, FIR treatment resulted in decreased skin thickness in UVB irradiated mice and inhibited the degradation of collagen fibers. Moreover, FIR can increase procollagen type I via the inhibition of MMP-9 and induction of TGF-β in skin tissues. Therefore, our study provides evidence for the beneficial effects of FIR exposure in a model of skin photoaging. PMID:28301572

  11. Inhibition of hydrogen peroxide induced injuring on human skin fibroblast by Ulva prolifera polysaccharide.

    PubMed

    Cai, Chuner; Guo, Ziye; Yang, Yayun; Geng, Zhonglei; Tang, Langlang; Zhao, Minglin; Qiu, Yuyan; Chen, Yifan; He, Peimin

    2016-10-01

    Ulva prolifera can protect human skin fibroblast from being injured by hydrogen peroxide. This work studied the composition of Ulva prolifera polysaccharide and identified its physicochemical properties. The results showed that the cell proliferation of 0.5mg/mL crude polysaccharide was 154.4% of that in negative control group. Moreover, ROS detection indices, including DCFH-DA, GSH-PX, MDA and CAT, indicated that crude polysaccharide could improve cellular ability to scavenge free radical and decrease the injury on human skin fibroblast by hydrogen peroxide. In purified polysaccharide, the activity of fraction P1-1 was the highest, with 174.6% of that in negative control group. The average molecular weight of P1-1 was 137kD with 18.0% of sulfate content. This work showed the inhibition of hydrogen peroxide induced injuries on human skin fibroblast by Ulva prolifera polysaccharide, which may further evaluate the application of U. prolifera on cosmetics.

  12. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    PubMed Central

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  13. Age-associated increase of skin fibroblast-derived prostaglandin E2 contributes to reduced collagen levels in elderly human skin

    PubMed Central

    Li, Yong; Lei, Dan; Swindell, William R; Xia, Wei; Weng, Shinuo; Fu, Jianping; Worthen, Christal A; Okubo, Toru; Johnston, Andrew; Gudjonsson, Johann E; Voorhees, John J; Fisher, Gary J

    2015-01-01

    Production of type I collagen declines during aging, leading to skin thinning and impaired function. Prostaglandin E2 (PGE2) is a pleiotropic lipid mediator that is synthesized from arachidonic acid by the sequential actions of cyclooxygenases (COX) and PGE synthases (PTGES). PGE2 inhibits collagen production by fibroblasts in vitro. We report that PTGES1 and COX2 progressively increase with aging in sun-protected human skin. PTGES1 and COX2 mRNA was increased 3.4-fold and 2.7-fold, respectively, in the dermis of elderly (>80 years) versus young (21-30 years) individuals. Fibroblasts were the major cell source of both enzymes. PGE2 levels were increased 70% in elderly skin. Fibroblasts in aged skin display reduced spreading due to collagen fibril fragmentation. To investigate the relationship between spreading and PGE2 synthesis, fibroblasts were cultured on micropost arrays or hydrogels of varying mechanical compliance. Reduced spreading/mechanical force resulted in increased expression of both PTGES1 and COX2 and elevated levels of PGE2. Inhibition of PGE2 synthesis by diclofenac enhanced collagen production in skin organ cultures. These data suggest that reduced spreading/mechanical force of fibroblasts in aged skin elevates PGE2 production, contributing to reduced collagen production. Inhibition of PGE2 production may be therapeutically beneficial for combating age-associated collagen deficit in human skin. PMID:25905589

  14. Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

    PubMed

    Yamaba, Hiroyuki; Haba, Manami; Kunita, Mayumi; Sakaida, Tsutomu; Tanaka, Hiroshi; Yashiro, Youichi; Nakata, Satoru

    2016-08-01

    Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.

  15. Role of rat autologous skin fibroblasts and mechanism underlying the repair of depressed scars

    PubMed Central

    Zhao, Juan; Liu, Yan-Chun; Shi, Yan-Hua; Xie, Ya-Qin; Cui, Hai-Peng; Li, Ying; Li, Xiang-Jun; Ren, Li-Qun

    2016-01-01

    The aim of the present study was to provide reliable experimental evidence for the application of autologous skin fibroblasts (asFbs) in the repair of depressed scars. In the experiments, depressed trauma was induced in male Wistar rats, and fibroblasts were separated from the removed skin tissues to culture in medium. In vitro cultured asFbs were injected into the depressed scar sites of rats, and the repair function of asFbs in the depressed scars was then examined at the cellular and whole-animal levels. The expression levels of type I and type III collagen in the dermal layer of the skin injected with asFb cells were significantly higher, as compared with those of the control, and type I collagen expression was significantly higher compared with Type III. Re-injection of asFbs into the dermal layer of depressed scars can markedly improve their repair. These results may prove useful for skin repair in clinical settings. PMID:27446300

  16. Transcriptome-Wide Expression Profiling in Skin Fibroblasts of Patients with Joint Hypermobility Syndrome/Ehlers-Danlos Syndrome Hypermobility Type

    PubMed Central

    Chiarelli, Nicola; Carini, Giulia; Zoppi, Nicoletta; Dordoni, Chiara; Ritelli, Marco; Venturini, Marina; Castori, Marco; Colombi, Marina

    2016-01-01

    Joint hypermobility syndrome/Ehlers–Danlos syndrome hypermobility type (JHS/EDS-HT), is likely the most common systemic heritable connective tissue disorder, and is mostly recognized by generalized joint hypermobility, joint instability complications, minor skin changes and a wide range of satellite features. JHS/EDS-HT is considered an autosomal dominant trait but is still without a defined molecular basis. The absence of (a) causative gene(s) for JHS/EDS-HT is likely attributable to marked genetic heterogeneity and/or interaction of multiple loci. In order to help in deciphering such a complex molecular background, we carried out a comprehensive immunofluorescence analysis and gene expression profiling in cultured skin fibroblasts from five women affected with JHS/EDS-HT. Protein study revealed disarray of several matrix structural components such as fibrillins, tenascins, elastin, collagens, fibronectin, and their integrin receptors. Transcriptome analysis indicated perturbation of different signaling cascades that are required for homeostatic regulation either during development or in adult tissues as well as altered expression of several genes involved in maintenance of extracellular matrix architecture and homeostasis (e.g., SPON2, TGM2, MMP16, GPC4, SULF1), cell-cell adhesion (e.g., CDH2, CHD10, PCDH9, CLDN11, FLG, DSP), immune/inflammatory/pain responses (e.g., CFD, AQP9, COLEC12, KCNQ5, PRLR), and essential for redox balance (e.g., ADH1C, AKR1C2, AKR1C3, MAOB, GSTM5). Our findings provide a picture of the gene expression profile and dysregulated pathways in JHS/EDS-HT skin fibroblasts that correlate well with the systemic phenotype of the patients. PMID:27518164

  17. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization

    PubMed Central

    Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

    2016-01-01

    Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts. PMID:26966384

  18. Aluminum is More Cytotoxic than Lunar Dust in Human Skin and Lung Fibroblasts

    NASA Technical Reports Server (NTRS)

    Hammond, D.; Shehata, T.; Hammond, D.; Shehata, T.; Wise, J.P.; Martino, J; Wise, J.P.; Wise, J.P.

    2009-01-01

    NASA plans to build a permanent space station on the moon to explore its surface. The surface of the moon is covered in lunar dust, which consists of fine particles that contain silicon, aluminum and titanium, among others. Because this will be a manned base, the potential toxicity of this dust has to be studied. Also, toxicity standards for potential exposure have to be set. To properly address the potential toxicity of lunar dust we need to understand the toxicity of its individual components, as well as their combined effects. In order to study this we compared NASA simulant JSC-1AVF (volcanic ash particles), that simulates the dust found on the moon, to aluminum, the 3rd most abundant component in lunar dust. We tested the cytotoxicity of both compounds on human lung and skin fibroblasts (WTHBF-6 and BJhTERT cell lines, respectively). Aluminum oxide was more cytotoxic than lunar dust to both cell lines. In human lung fibroblasts 5, 10 and 50 g/sq cm of aluminum oxide induced 85%, 61% and 30% relative survival, respectively. For human skin fibroblasts the same concentrations induced 58%, 41% and 58% relative survival. Lunar dust was also cytotoxic to both cell lines, but its effects were seen at higher concentrations: 50, 100, 200 and 400 g/sq cm of lunar dust induced a 69%, 46%, 35% and 30% relative survival in the skin cells and 53%, 16%, 8% and 2% on the lung cells. Overall, for both compounds, lung cells were more sensitive than skin cells. This work was supported by a NASA EPSCoR grant through the Maine Space Grant Consortium (JPW), the Maine Center for Toxicology and Environmental Health., a Fulbright Grant (JM) and a Delta Kappa Gamma Society International World Fellowship (JM).

  19. Redox imbalance and morphological changes in skin fibroblasts in typical Rett syndrome.

    PubMed

    Signorini, Cinzia; Leoncini, Silvia; De Felice, Claudio; Pecorelli, Alessandra; Meloni, Ilaria; Ariani, Francesca; Mari, Francesca; Amabile, Sonia; Paccagnini, Eugenio; Gentile, Mariangela; Belmonte, Giuseppe; Zollo, Gloria; Valacchi, Giuseppe; Durand, Thierry; Galano, Jean-Marie; Ciccoli, Lucia; Renieri, Alessandra; Hayek, Joussef

    2014-01-01

    Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (-43.6%) and GSH/GSSG ratio (-3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

  20. Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome

    PubMed Central

    Amabile, Sonia; Belmonte, Giuseppe; Valacchi, Giuseppe; Galano, Jean-Marie; Ciccoli, Lucia; Renieri, Alessandra; Hayek, Joussef

    2014-01-01

    Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients. PMID:24987493

  1. Proteomic identification of cathepsin B and nucleophosmin as novel UVA-targets in human skin fibroblasts.

    PubMed

    Lamore, Sarah D; Qiao, Shuxi; Horn, David; Wondrak, Georg T

    2010-01-01

    Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA-targets in human dermal fibroblasts following a two-dimensional-difference-gel-electrophoresis (2D-DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye-labeling followed by 2D-DIGE/mass-spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA-induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA-induced downregulation was identified as cathepsin B, a lysosomal cysteine-protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin-like autofluorescence and osmiophilic material occurred in UVA-exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA-induced upregulation of oxidative stress response gene expression, and UVA-induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA-induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA-induced photo-oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin.

  2. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts.

    PubMed

    Schenkel, Laila C; Singh, Ratnesh K; Michel, Vera; Zeisel, Steven H; da Costa, Kerry-Ann; Johnson, Amy R; Mudd, Harvey S; Bakovic, Marica

    2015-05-01

    Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.

  3. Smad3-dependent CCN2 mediates fibronectin expression in human skin dermal fibroblasts

    PubMed Central

    Purohit, Trupta; Qin, Zhaoping; Quan, Chunji; Lin, Zhenhua

    2017-01-01

    The potential involvement of connective tissue growth factor (CCN2/CTGF) in extracellular matrix (ECM) production is recognized. However, the role CCN2 in fibronectin (FN) gene expression has remained incompletely understood and even controversial. Here we report that CCN2 is absolutely necessary for FN expression in primary human skin dermal fibroblasts, the major cells responsible for ECM production in skin. Gain- and loss-of-function approaches demonstrate that CCN2 is an essential component of FN expression in both basal and stimulation by TGF-β signaling, the major regulator of FN expression. CCN2 is significantly induced by Smad3, a critical mediator of TGF-β signaling. CCN2 acts as a downstream mediator of TGF-β/Smad signaling and acting synergistically with TGF-β to regulate FN gene expression. Finally, we observed that CCN2 and FN predominantly expressed in the dermis of normal human skin, stromal tissues of skin squamous cell carcinoma (SCC), and simultaneously induced in wounded human skin in vivo. These findings provide evidence that CCN2 is responsible for mediating the stimulatory effects of TGF-β/Smad on FN gene expression, and attenuation of CCN2 expression may benefit to reduce fibrotic ECM microenvironment in disease skin. PMID:28267785

  4. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast.

    PubMed

    Seol, Ja Young; Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami; Choi, Soon Young

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin's elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity.

  5. Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Voorhees, John J; Fisher, Gary J

    2012-09-01

    Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

  6. Microporous Dermal-Mimetic Electrospun Scaffolds Pre-Seeded with Fibroblasts Promote Tissue Regeneration in Full-Thickness Skin Wounds

    PubMed Central

    Bonvallet, Paul P.; Schultz, Matthew J.; Mitchell, Elizabeth H.; Bain, Jennifer L.; Culpepper, Bonnie K.; Thomas, Steven J.; Bellis, Susan L.

    2015-01-01

    Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone) electrospun scaffold (70:30 col/PCL) containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM), and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344) rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14%) over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold). Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration. Collectively these

  7. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast

    PubMed Central

    Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin’s elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity. PMID:27194933

  8. Silver nanoparticles mediate differential responses in keratinocytes and fibroblasts during skin wound healing.

    PubMed

    Liu, Xuelai; Lee, Pui-Yan; Ho, Chi-Ming; Lui, Vincent C H; Chen, Yan; Che, Chi-Ming; Tam, Paul K H; Wong, Kenneth K Y

    2010-03-01

    With advances in nanotechnology, pure silver has been recently engineered into nanometer-sized particles (diameter <100 nm) for use in the treatment of wounds. In conjunction with other studies, we previously demonstrated that the topical application of silver nanoparticles (AgNPs) can promote wound healing through the modulation of cytokines. Nonetheless, the question as to whether AgNPs can affect various skin cell types--keratinocytes and fibroblasts--during the wound-healing process still remains. Therefore, the aim of this study was to focus on the cellular response and events of dermal contraction and epidermal re-epithelialization during wound healing under the influence of AgNPs; for this we used a full-thickness excisional wound model in mice. The wounds were treated with either AgNPs or control with silver sulfadiazine, and the proliferation and biological events of keratinocytes and fibroblasts during healing were studied. Our results confirm that AgNPs can increase the rate of wound closure. On one hand, this was achieved through the promotion of proliferation and migration of keratinocytes. On the other hand, AgNPs can drive the differentiation of fibroblasts into myofibroblasts, thereby promoting wound contraction. These findings further extend our current knowledge of AgNPs in biological and cellular events and also have significant implications for the treatment of wounds in the clinical setting.

  9. Cell surface abnormality in clones of skin fibroblasts from a carrier of Duchenne muscular dystrophy.

    PubMed Central

    Hillier, J; Jones, G E; Statham, H E; Witkowski, J A; Dubowitz, V

    1985-01-01

    We have previously reported that skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) have a lower intercellular adhesiveness than control cells, and that cells from carriers of DMD have normal adhesiveness instead of the expected intermediate value. We have now cloned skin fibroblasts from a carrier of DMD (subject AS) who is also heterozygous for G6PD B/G6PD Mediterranean and determined the intercellular adhesiveness and G6PD phenotypes of the clones. G6PD activity was determined using the 2d-G6P/G6P ratio method. Normal cells had a percentage utilisation of 7.31% and uncloned cells from AS a value of 25.16%. Of 16 clones, 15 had normal values (mean 8.72%) while one clone was G6PD Med with a value of 57.5%. Mean intercellular adhesiveness of normal and uncloned cells from AS were 2.95 and 2.90 respectively. Of 11 clones tested, nine had normal values of adhesiveness (mean 3.1) and all these clones were G6PD B. The single G6PD Med clone had a value of 0.88, compared with 1.39 for DMD cells. We have no explanation at present for the single clone that was G6PD B but DMD-like on aggregation. PMID:3989823

  10. Keratinocytes and fibroblasts in a human skin equivalent model enhance melanocyte survival and melanin synthesis after ultraviolet irradiation.

    PubMed

    Archambault, M; Yaar, M; Gilchrest, B A

    1995-05-01

    To investigate paracrine effects of fibroblasts and keratinocytes on melanocyte behavior after ultraviolet (UV) irradiation, we compared an in vitro skin equivalent model with melanocyte cultures. Human melanocytes were maintained alone in monolayer cultures or on dermal equivalents with or without keratinocytes and were irradiated daily with solar-simulated light. After seven daily UV irradiations, monolayer melanocytes displayed dose-dependent increases in cellular damage. In contrast, melanocytes on dermal equivalents survived strikingly better. Moreover, UV-irradiated skin equivalent melanocytes became highly dendritic as compared with sham-irradiated cells, closely mimicking their morphology in UV-irradiated skin. In addition, in skin equivalents melanocytes migrated from the center to the periphery of the keratinocyte layer after UV irradiation. Melanin production per culture, as measured by 14C-dihydroxyphenylalanine incorporation, was consistently higher in skin equivalent melanocytes than in monolayer melanocytes from the same donor, and it was highest in melanocytes from skin equivalents containing both keratinocytes and fibroblasts. Our data strongly suggest that fibroblasts and keratinocytes modulate melanocyte function in skin. The skin equivalent is a valuable model for investigating paracrine effects on melanocytes after UV irradiation.

  11. Sensitivity of cultured skin fibroblasts from patients with neurofibromatosis to DNA-damaging agents

    SciTech Connect

    Woods, W.G.; McKenzie, B.; Letourneau, M.A.; Byrne, T.D.

    1986-01-01

    Neurofibromatosis (NF) is an autosomal dominant disorder associated with various constitutional abnormalities as well as a striking predisposition for malignant and nonmalignant neoplasms, both in cells originating in and not originating in the neural crest. We have examined the sensitivity of cultured skin fibroblasts from patients with neurofibromatosis to several types of DNA damage. Fibroblasts in Dulbecco's modified Eagle's medium were plated at 10(2) to 2 X 10(4) cells per 75 cm2 tissue culture plates, and exposed to various doses of gamma radiation (leads to DNA scission), actinomycin D, or mitomycin C. Cells were reincubated for 15 to 40 days until surviving colonies exhibited greater than 30-50 cells. Plates were then stained with 1% methylene blue and the colonies counted, with surviving fraction determined relative to plating efficiency. Nine skin fibroblast cell strains from normal individuals were studied as controls. One neurofibromatosis (NF) cell strain, SB23, exhibited normal sensitivity to all three DNA-damaging agents studied in early (7-8) and middle (12-13) in vitro passage. Strain GM0622, on the other hand, exhibited normal sensitivity to the three DNA-damaging agents studied at early passage, but showed a significant decrease in survival after exposure to both gamma radiation (D0 = 106 rad) and actinomycin D (D0 = 0.024 mcg/ml) with increasing passage. Strain GM1639 exhibited decreased survival after actinomycin D exposure at early passage (D0 = 0.017 mcg/ml), with normal survival after exposure to gamma radiation and mitomycin C at the same passage.

  12. Succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells. A methodological study.

    PubMed

    Hansen, T L; Andersen, H

    1983-01-01

    Through a methodological evaluation, reliable histochemical and biochemical methods for succinate dehydrogenase activity in cultured human skin fibroblasts and amniotic fluid cells were developed. The histochemical method includes a cleaning of the cultured cells in 1 mM malonate in 0.9% NaCl, air-drying and fixation in acetone (5 min at -20 degrees C), coating of cells with CoQ10 (0.2 mg/ml in ether/acetone) and incubation for 1 h at 37 degrees C in 50 mM succinate and 0.5 mg/ml Nitro BT in 200 mM phosphate buffer, pH 7.6 PMS as an intermediate electron carrier was found inferior to exogenous CoQ10. Both types of cells exhibit equal activity. In the biochemical method homogenizing was performed in 50 mM Tris-HCl buffer, pH 7.5, and 200 mM sucrose. The standard incubation was 2.0 mM INT and 10 mM succinate in 10 mM Tris-HCl buffer, pH 7.5 for 1 h at 37 degrees C. The apparent Km values for INT and succinate were estimated to 0.39 mM and 0.13 mM, respectively, while I0.5 for malonate was 0.46 mM. Activity in amniotic fluid cells was 18.1 pkat/mg protein and in human skin fibroblasts 20.3 pkat/mg protein. Specificity of the methods was tested by use of a Chinese hamster fibroblast strain B9 known to be succinate dehydrogenase deficient in addition to various control experiments. Congruent results were obtained with the two methods.

  13. Protective effect of porphyra-334 on UVA-induced photoaging in human skin fibroblasts.

    PubMed

    Ryu, Jina; Park, Su-Jin; Kim, In-Hye; Choi, Youn Hee; Nam, Taek-Jeong

    2014-09-01

    The significant increase in life expectancy is closely related to the growing interest in the impact of aging on the function and appearance of the skin. Skin aging is influenced by several factors, and solar ultraviolet (UV) irradiation is considered one of the most important causes of skin photoaging. The aim of this study was to examine the anti-photoaging role of porphyra-334 from Porphyra (P.) yezoensis, a mycosporine-like amino acid (MAA), using high-performance liquid chromatography (HPLC), and electrospray ionization‑mass spectrometry (ESI-MS). In the present study, extracted UV‑absorbing compounds from P. yezoensis included palythine, asterina-330 and porphyra-334. Porphyra-334 was the most abundant MAA in P. yezoensis, and it was therefore used for conducting antiphotoaging experiments. The effect of porphyra-334 on the prevention of photoaging was investigated by measuring reactive oxygen species (ROS) production and matrix metalloproteinase (MMP) levels, as well as extracellular matrix (ECM) components and protein expression in UVA‑irradiated human skin fibroblasts. Porphyra-334 suppressed ROS production and the expression of MMPs following UVA irradiation, while increasing levels of ECM components, such as procollagen, type I collagen, elastin. These results suggest that porphyra-334 has various applications in cosmetics and toiletries because of its anti‑photoaging activities and may serve as a novel anti-aging agent.

  14. Lovastatin and sodium phenylacetate normalize the levels of very long chain fatty acids in skin fibroblasts of X- adrenoleukodystrophy.

    PubMed

    Singh, I; Pahan, K; Khan, M

    1998-04-24

    The present study underlines the importance of lovastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, and the sodium salt of phenylacetic acid (NaPA), an inhibitor of mevalonate pyrophosphate decarboxylase, in normalizing the pathognomonic accumulation of saturated very long chain fatty acids (VLCFA) in cultured skin fibroblasts of X-adrenoleukodystrophy (X-ALD) in which the ALD gene is either mutated or deleted. Lovastatin or NaPA alone or in combination stimulated the beta-oxidation of lignoceric acid (C24:0) and normalized the elevated levels of VLCFA in skin fibroblasts of X-ALD. Ability of lovastatin and NaPA to normalize the pathognomonic accumulation of VLCFA in skin fibroblasts of X-ALD may identify these drugs as possible therapeutics for X-ALD.

  15. Zinc and propolis reduces cytotoxicity and proliferation in skin fibroblast cell culture: total polyphenol content and antioxidant capacity of propolis.

    PubMed

    Tyszka-Czochara, Małgorzata; Paśko, Paweł; Reczyński, Witold; Szlósarczyk, Marek; Bystrowska, Beata; Opoka, Włodzimierz

    2014-07-01

    It has been demonstrated that zinc exerts its beneficial influence on skin fibroblasts. Propolis, a complex mixture of plant-derived and bees' products, was reported to stimulate cicatrization processes in skin and prevent infections. The aim of this study was to find out how zinc and propolis influence human skin fibroblasts in cell culture and to compare the effect of individual compounds to the effect of a mixture of zinc and propolis. In this study, zinc, as zinc aspartate, at a concentration of 16 μM, increased human fibroblasts proliferation in cell culture, whereas propolis at a concentration of 0.01% (w/v) revealed antiproliferative and cytotoxic action followed by mild cell necrosis. In culture, zinc was effectively transported into fibroblasts, and propolis inhibited the amount of zinc incorporated into the cells. An addition of propolis to the medium caused a decrease in the Zn(II) amount incorporated into fibroblasts. The obtained results also indicate an appreciable antioxidant property of propolis and revealed its potential as a supplement when applied at doses lower than 0.01% (w/v). In conclusion, the present study showed that zinc had a protective effect on human cultured fibroblasts' viability, although propolis revealed its antiproliferative action and caused mild necrosis.

  16. Resveratrol Prevents High Fluence Red Light-Emitting Diode Reactive Oxygen Species-Mediated Photoinhibition of Human Skin Fibroblast Migration

    PubMed Central

    Mamalis, Andrew; Koo, Eugene; Isseroff, R. Rivkah; Murphy, William; Jagdeo, Jared

    2015-01-01

    Background Skin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL) is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease. Objective The goal of our study was to investigate the how reactive oxygen species (ROS) free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed. Methods High fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR). For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2) on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2. Results High fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158

  17. Protein, RNA, and DNA synthesis in cultures of skin fibroblasts from healthy subjects and patients with rheumatic diseases

    SciTech Connect

    Abakumova, O.Y.; Kutsenko, N.G.; Panasyuk, A.F.

    1985-07-01

    To study the mechanism of the lasting disturbance of fibroblast function, protein, RNA and DNA synthesis was investigated in skin fibroblasts from patients with rheumatoid arthritis (RA) and systemic scleroderma (SS). The labeled precursors used to analyze synthesis of protein, RNA, and DNA were /sup 14/C-protein hydrolysate, (/sup 14/C)uridine, and (/sup 14/C) thymidine. Stimulation was determined by measuring incorporation of (/sup 14/C)proline into fibroblast proteins. During analysis of stability of fast-labeled RNA tests were carried out to discover whether all measurable radioactivity belonged to RNA molecules.

  18. Various sulfatase activities in leukocytes and cultured skin fibroblasts from heterozygotes for the multiple sulfatase deficiency (mukosulfatidosis).

    PubMed

    Eto, Y; Tahara, T; Tokoro, T; Maekawa, K

    1983-02-01

    In heterozygotes for multiple sulfatase deficiency (MSD), several sulfatase activities including arylsulfatases A, B1, B2, and C, and cholesterol sulfatase were 40-50% of normals in cultured skin fibroblasts and 70-80% of normals in leukocytes. In MSD patients, these enzyme activities were deficient or reduced. DEAE-Sepharose column chromatographic patterns of 4-methylumbelliferyl sulfatases A, B1, and B2 in leukocytes and cultured skin fibroblasts from MSD patients and heterozygotes were also consistent with the above data. These data indicate that several sulfatase activities in heterozygotes of MSD exhibited intermediate activities as observed in the heterozygote state of other autosomal recessive inherited diseases.

  19. LOXL as a target to increase the elastin content in adult skin: a dill extract induces the LOXL gene expression.

    PubMed

    Cenizo, Valérie; André, Valérie; Reymermier, Corinne; Sommer, Pascal; Damour, Odile; Perrier, Eric

    2006-08-01

    The lysyl oxidases lysyl oxidase (LOX) and lysyl oxidase-like (LOXL) are responsible for elastin cross-linking. It was shown recently that LOXL is essential for the elastic fibres homeostasis and for their maintenance at adult age. We first determined whether or not elastin, LOX and LOXL are less expressed during adulthood. The LOX and LOXL mRNA level, quantified by real-time reverse transcriptase-polymerase chain reaction decreased in adult skin fibroblasts compared with fibroblasts from children. In contrast, the elastin mRNA level remains stable at all ages. The goal of this study was to induce elastogenesis at the adult age. Therefore, both enzymes, and in particular LOXL, of which expression is the most affected by age, could be targeted to induce elastogenesis in adult skin. We screened a library of about 1000 active ingredients to find activators capable to stimulate specifically the LOXL gene expression in adult dermal fibroblasts. The positive effect of selected active ingredients was confirmed on fibroblasts grown on monolayers and on dermal and skin equivalent cultures. One extract, obtained from dill (LYS'LASTINE V, Engelhard, Lyon, France), stimulates the LOXL gene expression in dermal equivalents (+64% increase in the LOXL mRNA level when compared with control). At the same time, the elastin detection is increased in dermal equivalents and under the dermal-epidermal junction of skin equivalents, without increase of the elastin mRNA. In conclusion, LOXL can be considered as a new target to reinduce elastogenesis. Its stimulation by a dill extract is correlated with increased elastin detection, suggesting an increase in elastogenesis efficiency.

  20. Passiflora tarminiana fruits reduce UVB-induced photoaging in human skin fibroblasts.

    PubMed

    Bravo, Karent; Duque, Luisa; Ferreres, Federico; Moreno, Diego A; Osorio, Edison

    2017-03-01

    Skin aging is a complex process that is strongly affected by UV radiation, which stimulates the production of reactive oxygen species (ROS) in the epidermis and dermis and subsequently causes skin damage. Among the major consequences are increased collagen degradation and reduced collagen synthesis. Previous reports have demonstrated the beneficial effects of polyphenols for healthy skin. Passiflora tarminiana Coppens & V.E. Barney, a species of the Passifloraceae family, is widely distributed in South America and is rich in flavonoids. We show that UVB radiation increases metalloproteinase 1 (MMP-1) and reduces procollagen production in human dermal fibroblast (HDF) cells in a dose- and time-dependent manner. We examined the antioxidant and antiaging effects of the extract and fractions of P. tarminiana fruits. The fractions showed high polyphenol content (620mg EAG/g) and antioxidant activity, as measured by ORAC (4097μmol ET/g) and ABTS (2992μmol ET/g) assays. The aqueous fraction drastically inhibited the collagenase enzyme (IC50 0.43μg/mL). The extract and fractions presented photoprotective effects by reducing UVB-induced MMP-1 production, increasing UVB-inhibited procollagen production, and decreasing ROS production after UVB irradiation in HDF. Finally, the polyphenol contents of the extracts and fractions from P. tarminiana were analyzed by HPLC-DAD-ESI-MS(n), and procyanidins and glycosylated flavonoids were identified.

  1. Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

    SciTech Connect

    Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

    2010-04-15

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-{beta}1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

  2. Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

    PubMed

    Singh, Mahipal; Sharma, Anil K

    2011-02-01

    Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

  3. FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

    PubMed Central

    2013-01-01

    Background Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. Results We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. Conclusions Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential. PMID:24066673

  4. 5-azacytidine affects TET2 and histone transcription and reshapes morphology of human skin fibroblasts

    PubMed Central

    Manzoni, Elena F. M.; Pennarossa, Georgia; deEguileor, Magda; Tettamanti, Gianluca; Gandolfi, Fulvio; Brevini, Tiziana A. L.

    2016-01-01

    Phenotype definition is controlled by epigenetic regulations that allow cells to acquire their differentiated state. The process is reversible and attractive for therapeutic intervention and for the reactivation of hypermethylated pluripotency genes that facilitate transition to a higher plasticity state. We report the results obtained in human fibroblasts exposed to the epigenetic modifier 5-azacytidine (5-aza-CR), which increases adult cell plasticity and facilitates phenotype change. Although many aspects controlling its demethylating action have been widely investigated, the mechanisms underlying 5-aza-CR effects on cell plasticity are still poorly understood. Our experiments confirm decreased global methylation, but also demonstrate an increase of both Formylcytosine (5fC) and 5-Carboxylcytosine (5caC), indicating 5-aza-CR ability to activate a direct and active demethylating effect, possibly mediated via TET2 protein increased transcription. This was accompanied by transient upregulation of pluripotency markers and incremented histone expression, paralleled by changes in histone acetylating enzymes. Furthermore, adult fibroblasts reshaped into undifferentiated progenitor-like phenotype, with a sparse and open chromatin structure. Our findings indicate that 5-aza-CR induced somatic cell transition to a higher plasticity state is activated by multiple regulations that accompany the demethylating effect exerted by the modifier. PMID:27841324

  5. Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

    PubMed

    Speer, Allison L; Al Alam, Denise; Sala, Frederic G; Ford, Henri R; Bellusci, Saverio; Grikscheit, Tracy C

    2012-01-01

    The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10) and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b), in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22) except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.

  6. The genotoxicity of particulate and soluble chromate in sperm whale (physeter macrocephalus) skin fibroblasts.

    PubMed

    Wise, John Pierce; Wise, Sandra S; LaCerte, Carolyne; Wise, John Pierce; Aboueissa, AbouEl-Makarim

    2011-01-01

    Hexavalent chromium is a marine pollutant of concern, both for the health of ocean ecosystems and for public health. Hexavalent chromium is known to induce genotoxicity in human and other terrestrial mammals. It is also known to be present in both water and air in the marine environment. However, currently there are limited data concerning both chromium levels and its toxicological effects in marine mammals. This study investigated the cytotoxic and genotoxic effects of soluble and particulate hexavalent chromium in sperm whale skin fibroblasts. Both forms of hexavalent chromium induced concentration-dependent increases in cytotoxicity and genotoxicity indicating that these compounds can be a health risk if the whales are exposed to them. These data support a hypothesis that chromium is a concern in the marine environment in general and for the health of sperm whales in particular.

  7. Establishment, characterization, and toxicological application of loggerhead sea turtle (Caretta caretta) primary skin fibroblast cell cultures.

    PubMed

    Webb, Sarah J; Zychowski, Gregory V; Bauman, Sandy W; Higgins, Benjamin M; Raudsepp, Terje; Gollahon, Lauren S; Wooten, Kimberly J; Cole, Jennifer M; Godard-Codding, Céline

    2014-12-16

    Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Characterization included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions by use of growth curve assays with a fractional factorial experimental design. Two cell viability assays, MTT and lactate dehydrogenase (LDH), and an assay measuring cytochrome P4501A (CYP1A) expression by quantitative PCR were optimized in the characterized cells. MTT and LDH assays confirmed cytotoxicity of perfluorooctanoic acid at 500 μM following 72 and 96 h exposures while CYP1A5 induction was detected after 72 h exposure to 0.1-10 μM benzo[a]pyrene. This research demonstrates the validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells.

  8. Dramatic Increase in Oxidative Stress in Carbon-Irradiated Normal Human Skin Fibroblasts

    PubMed Central

    Laurent, Carine; Leduc, Alexandre; Pottier, Ivannah; Prévost, Virginie; Sichel, François; Lefaix, Jean-Louis

    2013-01-01

    Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D0 (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D10% (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D0% (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this

  9. Phenotypic markers in human skin fibroblasts as possible diagnostic indices of hereditary adenomatosis of the colon and rectum.

    PubMed

    Kopelovich, L

    1977-11-01

    Hereditary adenomatosis of the colon and rectum (ACR) and its Gardner's syndrome variant, an autosomal dominant trait, indicate a propensity for neoplasia. The present study describes the growth abnormalities of cultured human skin fibroblasts derived from normal-appearing cutaneous biopsies of ACR genotypes and a portion of the clinically asymptomatic ACR progeny, first filial generation, and their differential susceptibility to transformation by Kirsten murine sarcoma virus. These skin fibroblasts, but not cells derived from unaffected individuals, showed lack of contact inhibition, decreased serum requirement for growth, elevated levels of plasminogen activator, and alterations in the intracellular distribution of actin cables; they did not, however, grow in the absence of anchorage, nor did they form palpable tumors in congenitally athymic BALB/c nu/nu mice, and they were normal with regard to cholesterol feedback regulation. Skin fibroblasts from ACR subjects were 100- to 1000-fold more susceptible to transformation by the Kirsten murine sarcoma virus than were normal cells. The virally transformed skin fibroblasts were anchorage-independent and formed tumors in athymic mice. These growth abnormalities represent steps in the changing phenotypic expression of cells undergoing neoplastic transformation. Identification of abnormal expressions associated with oncogenesis may facilitate their use as diagnostic indices for the detection of latent forms of colon cancer in man.

  10. Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors.

    PubMed

    Huang, H M; Gibson, G E

    1993-07-15

    An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

  11. Attachment, skin deep? Relationships between adult attachment and skin barrier recovery.

    PubMed

    Robles, Theodore F; Brooks, Kathryn P; Kane, Heidi S; Schetter, Christine Dunkel

    2013-06-01

    This study examined the relationship between individual differences in adult attachment and skin barrier recovery. Dating couples (N = 34) completed a self-report measure of attachment anxiety and avoidance, and during two separate laboratory visits, normal skin barrier function was disrupted using a tape-stripping procedure, followed by a 20 min discussion of personal concerns in one visit and relationship problems in the other, counterbalanced randomly across visits. Skin barrier recovery was assessed by measuring transepidermal water loss up to 2 h after skin disruption. Multilevel modeling showed that skin barrier recovery did not differ between the personal concern or relationship problem discussions. Among women, greater attachment anxiety predicted faster skin barrier recovery across the two visits, while greater attachment avoidance predicted slower skin barrier recovery. Among men, greater attachment anxiety predicted slower skin barrier recovery during the personal concern discussion only. The observed effects remained significant after controlling for transepidermal water loss in undisturbed skin, suggesting that the relationship between attachment security and skin barrier recovery was not due to other skin-related factors like sweating. Cortisol changes, self-reported emotions, stress appraisals, and supportiveness ratings were tested as potential mediators, and none explained the relationships between attachment and skin barrier recovery. These findings are the first to demonstrate associations between individual differences in attachment style and restorative biological processes in the skin, even in a sample of young dating couples in satisfied relationships.

  12. Curcumin induces stress response and hormetically modulates wound healing ability of human skin fibroblasts undergoing ageing in vitro.

    PubMed

    Demirovic, Dino; Rattan, Suresh I S

    2011-10-01

    Wound healing becomes impaired in several diseases and during ageing. A commonly used model for the study of wound healing is a scratched monolayer of cells in vitro, which is convenient for the analysis of the cellular and molecular changes occurring during the two phases of wound healing, namely cell migration and cell proliferation. Cell migration, which is the primary event to occur during initial wound healing, is inversely dependent on the number of focal adhesions (FA) that attach cells to the extracellular matrix. Here we report that the number of FA, measured by determining the levels of FA-proteins paxillin and talin, increase with increasing population doubling level of the serially passaged normal adult skin fibroblasts, and that this increase may account for the age-related slowing down of wound healing in vitro. We also report that curcumin, a component of the widely used spice turmeric, modulates wound healing in vitro in a biphasic dose response manner, being stimulatory at low doses (between 1 and 5 μM), and inhibitory at higher doses. Furthermore, our results show that the hormetic effects of low levels of curcumin are achieved by virtue of it being a hormetin in terms of the induction of stress response pathways, including Nrf2 and HO-1 in human cells.

  13. 3,3'-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts.

    PubMed

    Wagener, Sarah; Völker, Tanja; De Spirt, Silke; Ernst, Hansgeorg; Stahl, Wilhelm

    2012-08-01

    Skin cancer is among the most frequent neoplastic malignancies and exposure to UV irradiation is a major risk factor. In addition to topical sunscreens, photoprotection by dietary antioxidants such as carotenoids or polyphenols has been suggested as a means of prevention. Isorenieratene (IR) and dihydroxyisorenieratene (DHIR) are aromatic carotenoids with particular antioxidant properties produced by Brevibacterium linens. The aim of this study was to investigate the photoprotective and antioxidant activities of DHIR and IR in comparison to the nonaromatic carotenoid lutein in human dermal fibroblasts. Incubation of the cells with DHIR and IR significantly decreased the UV-induced formation of cyclobutane pyrimidine dimers and formation of DNA strand breaks. Lipid oxidation was lowered as determined by the formation of malondialdehyde as a biomarker. Both aromatic carotenoids also prevented oxidatively generated damage to DNA as demonstrated by a decrease in DNA strand breaks associated with the formation of oxidized DNA bases. These data highlight the multifunctional photoprotective properties of aromatic carotenoids, which may be suitable natural compounds for the prevention of skin cancer.

  14. Cytotoxicity in human skin fibroblasts induced by photoactivated polycyclic aromatic hydrocarbons

    SciTech Connect

    Strniste, G.F.; Brake, R.J.

    1980-01-01

    Polycyclic aromatic hydrocarbons (PAH) require chemical modification in order to exert their mutagenic/carcinogenic activity on biological systems. The mode of activation which has been most extensively studied involves enzyme-catalyzed oxidation reactions but PAH can also be modified into oxidized and radical intermediates upon exposure to various radiations. The resulting products have been shown to be cytotoxic, mutagenic and carcinogenic in a variety of test systems. A number of recent reports have examined the process of sunlight-activation of airborne particles contaminated with PAH. The concern here is the photoactivation of PAH into reactive intermediates which could act directly on target organs such as the skin and lungs. We have been studying the molecular and cellular effects of photoactivated PAH and complex organic mixtures (shale oil byproducts). In this report we present data concerning the cytotoxicity of near ultraviolet light (NUV)-activated PAH in cultured human skin fibroblasts, comparing normal cells with cells obtained from patients with the rare, autosomal recessive disease, xeroderma pigmentosum. In addition, we have quantitated the products of reactions of light-activated benzo(a)pyrene (B(a)P) with DNA in vitro, and have attempted to identify the photoproduct(s) of B(a)P that are important in these reactions.

  15. Luteolin decreases the UVA-induced autophagy of human skin fibroblasts by scavenging ROS

    PubMed Central

    Yan, Miaomiao; Liu, Zhongrong; Yang, Huilan; Li, Cuihua; Chen, Hulin; Liu, Yan; Zhao, Minling; Zhu, Yingjie

    2016-01-01

    Luteolin (LUT) is a flavone, which is universally present as a constituent of traditional Chinese herbs, and certain vegetables and spices, and has been demonstrated to exhibit potent radical scavenging and cytoprotective properties. Although LUT has various beneficial effects on health, the effects of LUT on the protection of skin remain to be fully elucidated. The present study investigated whether LUT can protect human skin fibroblasts (HSFs) from ultraviolet (UV) A irradiation. It was found that, following exposure to different doses of UVA irradiation, the HSFs exhibited autophagy, as observed by fluorescence and transmission electron microscopy, and reactive oxygen species (ROS) bursts, analyzed by flow cytometry, to differing degrees. Following incubation with micromolar concentrations of LUT, ROS production decreased and autophagy gradually declined. In addition, the expression of hypoxia-inducible factor-1α and the classical autophagy-associated proteins, LC3 and Beclin 1 were observed by western blotting. Western blot analysis showed that the expression levels of HIF-1α, LC3-II and Beclin 1 gradually decreased in the UVA-irradiated HSFs following treatment with LUT. These data indicated that UVA-induced autophagy was mediated by ROS, suggesting the possibility of resistance against UV by certain natural antioxidants, including LUT. PMID:27430964

  16. Iloprost suppresses connective tissue growth factor production in fibroblasts and in the skin of scleroderma patients

    PubMed Central

    Stratton, Richard; Shiwen, Xu; Martini, Giorgia; Holmes, Alan; Leask, Andrew; Haberberger, Thomas; Martin, George R.; Black, Carol M.; Abraham, David

    2001-01-01

    Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud’s phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-β to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-β. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-β. PMID:11457877

  17. Antibiotic consumption and Enterobacteriaceae skin colonization in hospitalized adults.

    PubMed

    Kirby, A; Berry, C; West, R

    2017-01-01

    Enterobacteriaceae are increasingly antibiotic resistant, and skin colonization may contribute to their spread in hospitals. This study screened 100 hospitalized adults for Enterobacteriaceae skin colonization, and assessed potential risk factors, including antibiotic consumption. Multi-variable analysis found that antibiotic consumption whilst an inpatient [odds ratio (OR) 3.16, 95% confidence interval (CI) 1.19-8.4] and male sex (OR 2.92, 95% CI 1.06-8.4) were risk factors for Enterobacteriaceae skin colonization. If these risk factors are confirmed, work to understand the biological mechanism involved may lead to the development of interventions to prevent Enterobacteriaceae skin colonization.

  18. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products.

    PubMed

    El Ghalbzouri, Abdoelwaheb; Commandeur, Suzan; Rietveld, Marion H; Mulder, Aat A; Willemze, Rein

    2009-01-01

    Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.

  19. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

    PubMed Central

    Reijnders, Christianne M.A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin–eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely

  20. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts.

    PubMed

    Reijnders, Christianne M A; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J; Gibbs, Susan

    2015-09-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin-eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely

  1. Particulate hexavalent chromium is cytotoxic and genotoxic to the North Atlantic right whale (Eubalaena glacialis) lung and skin fibroblasts.

    PubMed

    Li Chen, Tânia; Wise, Sandra S; Kraus, Scott; Shaffiey, Fariba; Levine, Kaitlynn M; Thompson, W Douglas; Romano, Tracy; O'Hara, Todd; Wise, John Pierce

    2009-06-01

    Hexavalent chromium compounds are present in the atmosphere and oceans and are established mutagens and carcinogens in human and terrestrial mammals. However, the adverse effects of these toxicants in marine mammals are uncertain. Previously, we reported that North Atlantic right whales, one of the most endangered great whales, have tissue chromium levels that are high, levels that may pose a risk to the whale's health. Furthermore, the study suggested that inhalation may be an important exposure route. Exposure to chromium through inhalation is mainly because of particulate compounds. However, the toxicity of particulate chromium compounds in marine mammal cells is unknown. Accordingly, in this study, we tested the cytotoxic and genotoxic effects of particulate hexavalent chromium in primary cultured lung and skin fibroblasts from the endangered North Atlantic right whale. Cytotoxicity was measured by clonogenic survival assay, and genotoxicity was measured as production of chromosome aberrations. Particulate hexavalent chromium induced cytotoxicity and genotoxicity in a concentration-dependent manner in both right whale lung and skin fibroblasts. Lung fibroblasts were more resistant to chromium cytotoxicity, but presented with more chromosome damage than skin fibroblasts. These data further support the hypothesis that chromium may be a health concern for the endangered North Atlantic right whale.

  2. Transforming growth factor-beta reverses a posttranscriptional defect in elastin synthesis in a cutis laxa skin fibroblast strain.

    PubMed Central

    Zhang, M C; Giro, M; Quaglino, D; Davidson, J M

    1995-01-01

    Skin fibroblasts from two cases of autosomal recessive cutis laxa (CL), having insignificant elastin production and mRNA levels, were challenged with transforming growth factor beta-1 (TGF-beta 1). Elastin production was brought from undetectable values to amounts typical of normal human skin fibroblasts in a dose-dependent fashion. Basic fibroblast growth factor (100 ng/ml) alone or in combination with TGF-beta 1 reduced elastin production and mRNA expression in CL skin fibroblasts more extensively than in normal cells. In situ hybridization showed that these effects were at the transcript level. One of the CL strains was examined in detail. Transcription rates for elastin were similar in normal and CL and unchanged by TGF-beta 1 or TGF-beta 2 (10 ng/ml), while in CL elastin mRNA half-life was increased > 10-fold by TGF-beta 2 and reduced 6-fold after TGF-beta 2 withdrawal, as compared with a control strain. Cycloheximide partially reversed elastin mRNA instability. These data are consistent with a defect in elastin mRNA stability that requires synthesis of labile factors or intact translational machinery, resulting in an extremely low steady state level of mRNA present in this strain of CL. Furthermore, TGF-beta can relieve elastin mRNA instability in at least one CL strain and elastin production defects in both CL strains. Images PMID:7884000

  3. Myofibroblasts are distinguished from activated skin fibroblasts by the expression of AOC3 and other associated markers

    PubMed Central

    Hsia, Lin-ting; Ashley, Neil; Ouaret, Djamila; Wang, Lai Mun; Wilding, Jennifer; Bodmer, Walter F.

    2016-01-01

    Pericryptal myofibroblasts in the colon and rectum play an important role in regulating the normal colorectal stem cell niche and facilitating tumor progression. Myofibroblasts previously have been distinguished from normal fibroblasts mostly by the expression of α smooth muscle actin (αSMA). We now have identified AOC3 (amine oxidase, copper containing 3), a surface monoamine oxidase, as a new marker of myofibroblasts by showing that it is the target protein of the myofibroblast-reacting mAb PR2D3. The normal and tumor tissue distribution and the cell line reactivity of AOC3 match that expected for myofibroblasts. We have shown that the surface expression of AOC3 is sensitive to digestion by trypsin and collagenase and that anti-AOC3 antibodies can be used for FACS sorting of myofibroblasts obtained by nonenzymatic procedures. Whole-genome microarray mRNA-expression profiles of myofibroblasts and skin fibroblasts revealed four additional genes that are significantly differentially expressed in these two cell types: NKX2-3 and LRRC17 in myofibroblasts and SHOX2 and TBX5 in skin fibroblasts. TGFβ substantially down-regulated AOC3 expression in myofibroblasts but in skin fibroblasts it dramatically increased the expression of αSMA. A knockdown of NKX2-3 in myofibroblasts caused a decrease of myofibroblast-related gene expression and increased expression of the fibroblast-associated gene SHOX2, suggesting that NKX2-3 is a key mediator for maintaining myofibroblast characteristics. Our results show that colorectal myofibroblasts, as defined by the expression of AOC3, NKX2-3, and other markers, are a distinctly different cell type from TGFβ-activated fibroblasts. PMID:27036009

  4. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  5. Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

    PubMed

    Lippolis, Rosa; Siciliano, Rosa Anna; Pacelli, Consiglia; Ferretta, Anna; Mazzeo, Maria Fiorella; Scacco, Salvatore; Papa, Francesco; Gaballo, Antonio; Dell'Aquila, Claudia; De Mari, Michele; Papa, Sergio; Cocco, Tiziana

    2015-09-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

  6. Inhibition of androgen receptor binding by natural and synthetic steroids in cultured human genital skin fibroblasts.

    PubMed

    Breiner, M; Romalo, G; Schweikert, H U

    1986-08-15

    The ability of various natural and synthetic steroids (some of which are widely used in clinical practice) to compete with dihydrotestosterone receptor binding in human genital skin fibroblasts was studied. Binding was assessed in fibroblast monolayers after incubation for 1 h at 37 degrees C with 2 nM 3H-dihydrotestosterone in the presence or absence of increasing concentrations of the steroid to be tested. Inhibition constants (Ki) were determined as the concentration of competitor-required for 50% inhibition of 3H-dihydrotestosterone binding. In addition, relative binding activity (RBA) of each test compound was calculated. Each competitor was tested in at least two different cell strains. The concentrations of unlabeled methyltrienolone (a synthetic nonmetabolizable androgen) and dihydrotestosterone for 50% inhibition of 3H-dihydrotestosterone binding were in the same order of magnitude, namely, 2 nM (2.2 respectively, 2.4 nM), whereas the affinity of testosterone was approximately one-fifth that of dihydrotestosterone. Other potent competitors for dihydrotestosterone binding were three progestins (norgestrel, gestoden, and medroxyprogesterone acetate) which have Ki values similar to testosterone. An order of magnitude lower Ki values (around 10(-7) M) were found for the androgen 17 alpha-propylmesterolone, the antiandrogen cyproterone acetate, and the progestin norethisterone acetate. Binding affinities of all other steroids to the androgen receptor were markedly lower and showed the following order of potency: estrogens (estradiol, ethinyl estradiol, diethylstilbestrol) greater than glucocorticoids as well as aromatase inhibitors and potassium canrenoate.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Cell cycle synchronization of skin fibroblast cells in four species of family Felidae.

    PubMed

    Wittayarat, M; Thongphakdee, A; Saikhun, K; Chatdarong, K; Otoi, T; Techakumphu, M

    2013-04-01

    This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 μm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 μm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.

  8. Previous chronic exogenous glucocorticoid administration in vivo does not affect functional characteristics and cellular lifespan of human skin fibroblasts in vitro.

    PubMed

    Pratsinis, Harris; Dimozi, Anastasia; Pilichos, Konstantinos; Tsagarakis, Stylianos; Yiacoumettis, Andreas M; Kletsas, Dimitris

    2011-06-01

    Excess of glucocorticoids (GCs) has been reported to lead to skin atrophy and impaired wound healing. The present study investigates whether human skin fibroblasts suffer permanent damages due to a long-term exposure to GC excess. Fibroblasts obtained from patients being under GC treatment for periods over one year were cultured under standard conditions in vitro, and studied regarding pivotal parameters involved in skin homeostasis and aging, i.e. collagen production, cell proliferation, and cellular replicative lifespan. No statistical differences were observed regarding these functions compared to those of normal human skin fibroblasts. Furthermore, no differences between normal and patient-derived cells were observed regarding their sensitivity to a supra-physiological cortisol concentration. In conclusion, the prolonged exposure of human skin fibroblasts in vivo to high concentrations of exogenously-administered GC does not lead to persistent adverse effects on their physiology.

  9. Double trisomy mosaic (47,XXX/48,XXX,+13) confirmed by FISH and skin fibroblast culture

    SciTech Connect

    Lieber, E.; Grady, V.; Dosik, H.

    1994-09-01

    A 4 lb 8 oz female was born to a 49-year-old woman (P1200G12) at 40 weeks. The baby had tetralogy of Fallot, polydactyly, microcephaly, low set simple ears, posterior cleft of the soft palate and overlapping flexion deformities of both hands. The eyes were deep set. The clinical impression was trisomy 13. The baby is not doing well and needs a gastrotomy tube for feeding. Sucking is allright but swallowing is impeded. An MRI showed an anomaly of the corpus callosum. The ophthalmological examination showed no abnormalities. A chromosome study on a 2-day peripheral blood sample resulted in poor growth and poor morphology; however, 20 Giemsa-banded cells revealed a 47,XXX karyotype. A second specimen was obtained to search for mosaicism and a blood smear revealed nuclear projections on the neutrophils. FISH analysis using whole chromosome painting probe (Life Technologies) first identified the extra chromosome number 13, the final results showing five of sixty metaphase cells (8.3%) with trisomy 13. Cytogenetic analysis using Giemsa-banding technique revealed four cells in fifty examined (8.0%) with a 48,XXX,+13 karyotype. In order to further evaluate the mosaicism, cytogenetic analysis of a skin fibroblast culture was performed. Twenty one of twenty three cells examined (91.3%) showed the 48,XXX,+13 karyotype. FISH analysis of the skin biopsy revealed eighteen of twenty cells (90.9%) with the trisomy 13. The FISH technique is an important enhancement to routine cytogenetic studies when they do not immediately correlate with clinical impressions.

  10. Inhibition of oxidative stress by low-molecular-weight polysaccharides with various functional groups in skin fibroblasts.

    PubMed

    Chen, Szu-Kai; Hsu, Chu-Hsi; Tsai, Min-Lang; Chen, Rong-Huei; Drummen, Gregor P C

    2013-09-25

    The aim of this study was to evaluate the in cellulo inhibition of hydrogen-peroxide-induced oxidative stress in skin fibroblasts using different low-molecular-weight polysaccharides (LMPS) prepared from agar (LMAG), chitosan (LMCH) and starch (LMST), which contain various different functional groups (i.e., sulfate, amine, and hydroxyl groups). The following parameters were evaluated: cell viability, intracellular oxidant production, lipid peroxidation, and DNA damage. Trolox was used as a positive control in order to allow comparison of the antioxidant efficacies of the various LMPS. The experimentally determined attenuation of oxidative stress by LMPS in skin fibroblasts was: LMCH > LMAG > LMST. The different protection levels of these LMPS may be due to the physic-chemical properties of the LMPS' functional groups, including electron transfer ability, metal ion chelating capacities, radical stabilizing capacity, and the hydrophobicity of the constituent sugars. The results suggest that LMCH might constitute a novel and potential dermal therapeutic and sun-protective agent.

  11. Defective mitochondrial fusion, altered respiratory function, and distorted cristae structure in skin fibroblasts with heterozygous OPA1 mutations.

    PubMed

    Agier, Virginie; Oliviero, Patricia; Lainé, Jeanne; L'Hermitte-Stead, Caroline; Girard, Samantha; Fillaut, Sandrine; Jardel, Claude; Bouillaud, Frédéric; Bulteau, Anne Laure; Lombès, Anne

    2012-10-01

    Deleterious consequences of heterozygous OPA1 mutations responsible for autosomal dominant optic atrophy remain a matter of debate. Primary skin fibroblasts derived from patients have shown diverse mitochondrial alterations that were however difficult to resolve in a unifying scheme. To address the potential use of these cells as disease model, we undertook parallel and quantitative analyses of the diverse reported alterations in four fibroblast lines harboring different OPA1 mutations, nonsense or missense, in the guanosine triphosphatase or the C-terminal coiled-coil domains. We tackled several factors potentially underlying discordant reports and showed that fibroblasts with heterozygous OPA1 mutations present with several mitochondrial alterations. These included defective mitochondrial fusion during pharmacological challenge with the protonophore carbonyl cyanide m-chlorophenyl hydrazone, significant mitochondrial elongation with decreased OPA1 and DRP1 proteins, and abnormal mitochondrial fragmentation during glycolysis shortage or exogenous oxidative stress. Respiratory complex IV activity and subunits steady-state were decreased without alteration of the mitochondrial deoxyribonucleic acid size, amount or transcription. Physical link between OPA1 protein and oxidative phosphorylation was shown by reciprocal immunoprecipitation. Altered cristae structure coexisted with normal response to pro-apoptotic stimuli and expression of Bax or Bcl2 proteins. Skin fibroblasts with heterozygous OPA1 mutations thus share significant mitochondrial remodeling, and may therefore be useful for analyzing disease pathophysiology. Identifying whether the observed alterations are also present in ganglion retinal cells, and which of them underlies their degeneration process remains however an essential goal for therapeutic strategy.

  12. Photobiomodulation on the proliferation and collagen synthesis of normal human skin fibroblast cells

    NASA Astrophysics Data System (ADS)

    Cheng, Lei; Liu, Timon Cheng-Yi; Chi, Jin-Quan; Li, Yan; Jin, Hua

    2006-01-01

    Background and Objective: Cultured normal human skin fibroblast cells (HSFs) were once used to study the mechanism of the effects of low intensity He-Ne laser irradiation (LHNL) on wound healing. The proliferation and collagen synthesis of HFSs were modulated by LHNL in different papers, respectively, and both of them are studied in this paper. Study Design/Materials and Methods: The dosage was studied for the same radiation time 300s. The proliferation and collagen synthesis were measured by 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and the spectrophotometric method for the determination of hydroxyproline, respectively. Results: The dose zones were called dose 1, dose 2 and dose 3 from low dose on so that HSF proliferation was inhibited in dose 1 (16, 24 mJ/cm2), and promoted in dose 2 (298, 503, 597mJ/cm2), and the collagen synthesis was inhibited in dose 2 (401, 526 mJ/cm2), and promoted in dose 3 (714, 926, 1539, 1727mJ/cm2), which supports our biological model of photobiomodulation. It was found there is the linear relationship of the effect with dose with dose in each dose zone. Conclusions: The photobiomodulation on the proliferation and collagen synthesis of HSFs might be linearly dose-dependent in limited dosage with radiation time kept constant, which provides a foundation to discuss photobiomodulation on wound healing.

  13. Wound healing effect of acellular artificial dermis containing extracellular matrix secreted by human skin fibroblasts.

    PubMed

    Seo, Young-Kwon; Song, Kye-Yong; Kim, Young-Jin; Park, Jung-Keug

    2007-07-01

    In this study, an acellular artificial dermis, composed of human collagen and glycosaminoglycan (GAG) secreted by cultured human fibroblasts on a bovine collagen sponge, was developed. Much of the newly secreted extracellular matrix (ECM) remained after the cell removal process. The main theme of this study focused on the matrix, rather than the viable cell components of the skin, as the major dermal deficit in the wound. Both the acellular artificial and bioartificial dermises, containing viable cells with ECM, were significantly less soluble than the collagen sponge, and the relative GAG content in the bioartificial and acellular artificial dermises was approximately 115-120% of the chondroitin-6-sulfate (CS) content found in the collagen sponge. In the group receiving the collagen sponge, the wound area gradually decreased to approximately 10% of its original area, while in the groups receiving the bioartificial and acellular artificial dermises, the wound area also gradually decreased to approximately 60 and 50%, respectively, of the original size over the 5 weeks after grafting. Both the bioartificial and acellular artificial dermises formed thicker, denser collagen fibers; more new blood vessel formation was observed in both cases. The basement membrane of the regenerated epidermal-dermal junction was thicker and more linear in the acellular artificial dermis graft than in the collagen sponge graft. In conclusion, the wound healing effects of acellular artificial dermis are no less than those of the bioartificial dermis, and much better than the collagen sponge graft with respect to wound contraction, angiogenesis, collagen formation, and basement membrane repair.

  14. Different oxidative stress response in keratinocytes and fibroblasts of reconstructed skin exposed to non extreme daily-ultraviolet radiation.

    PubMed

    Marionnet, Claire; Pierrard, Cécile; Lejeune, François; Sok, Juliette; Thomas, Marie; Bernerd, Françoise

    2010-08-10

    Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as "daily UV radiation" (DUVR) with a higher UVA (320-400 nm) to UVB (280-320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure.

  15. Different Oxidative Stress Response in Keratinocytes and Fibroblasts of Reconstructed Skin Exposed to Non Extreme Daily-Ultraviolet Radiation

    PubMed Central

    Marionnet, Claire; Pierrard, Cécile; Lejeune, François; Sok, Juliette; Thomas, Marie; Bernerd, Françoise

    2010-01-01

    Experiments characterizing the biological effects of sun exposure have usually involved solar simulators. However, they addressed the worst case scenario i.e. zenithal sun, rarely found in common outdoor activities. A non-extreme ultraviolet radiation (UV) spectrum referred as “daily UV radiation” (DUVR) with a higher UVA (320–400 nm) to UVB (280–320 nm) irradiance ratio has therefore been defined. In this study, the biological impact of an acute exposure to low physiological doses of DUVR (corresponding to 10 and 20% of the dose received per day in Paris mid-April) on a 3 dimensional reconstructed skin model, was analysed. In such conditions, epidermal and dermal morphological alterations could only be detected after the highest dose of DUVR. We then focused on oxidative stress response induced by DUVR, by analyzing the modulation of mRNA level of 24 markers in parallel in fibroblasts and keratinocytes. DUVR significantly modulated mRNA levels of these markers in both cell types. A cell type differential response was noticed: it was faster in fibroblasts, with a majority of inductions and high levels of modulation in contrast to keratinocyte response. Our results thus revealed a higher sensitivity in response to oxidative stress of dermal fibroblasts although located deeper in the skin, giving new insights into the skin biological events occurring in everyday UV exposure. PMID:20706594

  16. Screening for disorders of pyruvate metabolism by measuring the ratio of the rates of lactate production and pyruvate decarboxylation in cultured skin fibroblasts.

    PubMed

    Kuroda, Y; Kawakami, I; Kobashi, H; Naito, E; Ito, M; Saijo, T; Yokota, I; Takeda, E

    1991-05-31

    We assayed the rates of lactate production from [1-14C]pyruvate and decarboxylation of [1-14C]pyruvate in cultured skin fibroblasts from 8 patients with disorders of pyruvate metabolism and 16 control subjects. The disorders of pyruvate metabolism could be more readily detected by measuring the ratio between the rates of lactate production and pyruvate decarboxylation by cultured skin fibroblasts than by measuring either the rate in isolation.

  17. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    PubMed

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  18. Enhanced reactivation of ultraviolet-damaged herpes virus in ultraviolet pretreated skin fibroblasts of cancer prone donors

    SciTech Connect

    Coppey, J.; Menezes, S.

    1981-01-01

    An enhanced reactivation of ultraviolet-damaged (u.v. at 254 nm) unclear replicating double-stranded DNA viruses occurs when corresponding host cells are treated with radiation or carcinogens prior to infection. This phenomenon seems to be due to an induced DNA repair activity the nature of which is yet unknown. The u.v.-induced enhanced reactivation (ER) of u.v.-damaged herpes simplex virus (u.v. - HSV) was compared in dividing skin fibroblasts of 30 donors either normal or afflicted by genetic disorders, some of which confer a high risk for sunlight induced skin cancers. Cultures were exposed to a single dose of 1.0-25 J.m-2 from 0-60 h before infection with u.v.-HSV (at about 10-3 survival) and the rate of viral production was determined. ER was maximal for a 36 h time interval in all lines. The u.v. dose eliciting maximal ER was 15 J.m-2 in fibroblasts from normal donors, xeroderma pigmentosum (XP) heterozygotes, Mibelli's porokeratosis, diffused naevomatosis, Down's syndrome, xerodermoids, XP variants and epidermodysplasia verruciformis. However, in the latter 3 cases, ER was almost 10 times more pronounced than in the normal cases. The u.v. dose eliciting maximal ER was 0.1, 0.3 and 2 J.m-2 in excision deficient XP fibroblasts from groups A, D and C, respectively, 2.5 J.m-2 in 11961 fibroblasts and 5 J.m-2 in fibroblast lines from cockayne s syndrome.

  19. Comparison of the Transcriptomes of Mouse Skin Derived Precursors (SKPs) and SKP-Derived Fibroblasts (SFBs) by RNA-Seq

    PubMed Central

    Mao, Yujie; Xiong, Lidan; Wang, Siyu; Zhong, Jianqiao; Zhou, Rongying; Li, Li

    2015-01-01

    Skin-derived precursors (SKPs) from dermis possess the capacities of self-renewal and multipotency. In vitro and in vivo studies demonstrated that they can differentiate into fibroblasts. However, little is known about the molecular mechanism of the differentiation of SKPs into fibroblasts. Here we compare the transcriptomes of mouse SKPs and SKP-derived fibroblasts (SFBs) by RNA-Seq analysis, trying to find differences in gene expression between the two kinds of cells and then elucidate the candidate genes that may play important roles in the differentiation of SKPs into fibroblasts. A total of 1971 differentially expressed genes (DEGs) were identified by RNA-Seq, which provided abundant data for further analysis. Gene Ontology enrichment analysis revealed that genes related to cell differentiation, cell proliferation, protein binding, transporter activity and membrane were significantly enriched. The most significantly up-regulated genes Wnt4, Wisp2 and Tsp-1 and down-regulated genes Slitrk1, Klk6, Agtr2, Ivl, Msx1, IL15, Atp6v0d2, Kcne1l and Thbs4 may play important roles in the differentiation of SKPs into fibroblasts. KEGG analysis showed that DEGs were significantly enriched in the TGF-β signaling pathway, Wnt signaling pathway and Notch signaling pathway, which have been previously proven to regulate the differentiation and self-renewal of various stem cells. These identified DEGs and pathways could facilitate further investigations of the detailed molecular mechanisms, making it possible to take advantage of the potential therapeutic applications of SKPs in skin regeneration in the future. PMID:25719759

  20. Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing.

    PubMed

    Ghosh, Kaustabh; Ren, Xiang-Dong; Shu, Xiao Zheng; Prestwich, Glenn D; Clark, Richard A F

    2006-03-01

    Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

  1. Influence of beam shape on in-vitro cellular transformations in human skin fibroblasts

    NASA Astrophysics Data System (ADS)

    Mthunzi, Patience; Forbes, Andrew; Hawkins, Denise; Abrahamse, Heidi; Karsten, Aletta E.

    2005-08-01

    A variety of strategies have been utilised for prevention and treatment of chronic wounds such as leg ulcers, diabetic foot ulcers and pressure sores1. Low Level Laser Therapy (LLLT) has been reported to be an invaluable tool in the enhancement of wound healing through stimulating cell proliferation, accelerating collagen synthesis and increasing ATP synthesis in mitochondria to name but a few2. This study focused on an in-vitro analysis of the cellular responses induced by treatment with three different laser beam profiles namely, the Gaussian (G), Super Gaussian (SG) and Truncated Gaussian (TG), on normal wounded irradiated (WI) and wounded non-irradiated (WNI) human skin fibroblast cells (WS1), to test their influence in wound healing at 632.8 nm using a helium neon (HeNe) laser. For each beam profile, measurements were made using average energy densities over the sample ranging from 0.2 to 1 J, with single exposures on normal wounded cells. The cells were subjected to different post irradiation incubation periods, ranging from 0 to 24 hours to evaluate the duration (time) dependent effects resulting from laser irradiation. The promoted cellular alterations were measured by increase in cell viability, cell proliferation and cytotoxicity. The results obtained showed that treatment with the G compared to the SG and TG beams resulted in a marked increase in cell viability and proliferation. The data also showed that when cells undergo laser irradiation some cellular processes are driven by the peak energy density rather than the energy of the laser beam. We show that there exist threshold values for damage, and suggest optimal operating regimes for laser based wound healing.

  2. LET and ion species dependence for cell killing in normal human skin fibroblasts.

    PubMed

    Tsuruoka, Chizuru; Suzuki, Masao; Kanai, Tatsuaki; Fujitaka, Kazunobu

    2005-05-01

    We studied the LET and ion species dependence of the RBE for cell killing to clarify the differences in the biological effects caused by the differences in the track structure that result from the different energy depositions for different ions. Normal human skin fibroblasts were irradiated with heavy-ion beams such as carbon, neon, silicon and iron ions that were generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Science (NIRS) in Japan. Cell killing was measured as reproductive cell death using a colony formation assay. The RBE-LET curves were different for carbon ions and for the other ions. The curve for carbon ions increased steeply up to around 98 keV/microm. The RBE of carbon ions at 98 keV/microm was 4.07. In contrast, the curves for neon, silicon and iron ions had maximum peaks around 180 keV/microm, and the RBEs at the peak position ranged from 3.03 to 3.39. When the RBEs were plotted as a function of Z*2/beta2 (where Z* is the effective charge and beta is the relative velocity of the ion) instead of LET, the discrepancies between the RBE-LET curves for the different ion beams were reduced, but branching of the RBE-Z*2/beta2 curves still remained. When the inactivation cross section was plotted as a function of either LET or Z*2/beta2, it increased with increasing LET. However, the inactivation cross section was always smaller than the geometrical cross section. These results suggest that the differences in the energy deposition track structures of the different ion sources have an effect on cell killing.

  3. Cell therapy for skin wound using fibroblast encapsulated poly(ethylene glycol)-poly(L-alanine) thermogel.

    PubMed

    Yun, Eun Jung; Yon, Bora; Joo, Min Kyung; Jeong, Byeongmoon

    2012-04-09

    As a new application of a thermogel, a poly(ethylene glycol)-b-poly(L-alanine) (PEG-L-PA) gel encapsulating fibroblasts was investigated for wound healing. The fibroblasts were encapsulated by the temperature sensitive sol-to-gel transition of the polymer aqueous solution. Under the in vitro three-dimensional (3D) cell culture condition, the PEG-L-PA thermogel was comparable with Matrigel for cell proliferation and was significantly better than Matrigel for collagen types I and III formation. After confirming the excellent 3D microenvironment of the PEG-L-PA thermogel for fibroblasts, in vivo wound healing was investigated by injecting the cell-suspended polymer aqueous solution on incisions of rat skin, where the cell-encapsulated gel was formed in situ. Compared with the phosphate buffered saline treated system and the cell-free PEG-L-PA thermogel, the cell-encapsulated PEG-L-PA thermogel not only accelerated the wound closure but also improved epithelialization and the formation of skin appendages such as keratinocyte layer (epidermis), hair follicles, and sebaceous glands. The results demonstrate the potential of thermogels for cell therapy as an injectable tissue-engineering scaffold.

  4. Stimulation of Skin and Wound Fibroblast Migration by Mesenchymal Stem Cells Derived from Normal Donors and Chronic Wound Patients

    PubMed Central

    Rodriguez-Menocal, Luis; Salgado, Marcela; Ford, Dwayne

    2012-01-01

    Chronic wounds continue to be a major cause of morbidity for patients and an economic burden on the health care system. Novel therapeutic approaches to improved wound healing will need, however, to address cellular changes induced by a number of systemic comorbidities seen in chronic wound patients, such as diabetes, chronic renal failure, and arterial or venous insufficiency. These effects likely include impaired inflammatory cell migration, reduced growth factor production, and poor tissue remodeling. The multifunctional properties of bone marrow-derived mesenchymal stem cells (MSCs), including their ability to differentiate into various cell types and capacity to secrete factors important in accelerating healing of cutaneous wounds, have made MSCs a promising agent for tissue repair and regeneration. In this study we have used an in vitro scratch assay procedure incorporating labeled MSCs and fibroblasts derived from normal donors and chronic wound patients in order to characterize the induction of mobilization when these cells are mixed. A modified Boyden chamber assay was also used to examine the effect of soluble factors on fibroblast migration. These studies suggest that MSCs play a role in skin wound closure by affecting dermal fibroblast migration in a dose-dependent manner. Deficiencies were noted, however, in chronic wound patient fibroblasts and MSCs as compared with those derived from normal donors. These findings provide a foundation to develop therapies targeted specifically to the use of bone marrow-derived MSCs in wound healing and may provide insight into why some wounds do not heal. PMID:23197781

  5. Acute leukemia after radiotherapy in a patient with Turcot's syndrome. Impaired colony formation in skin fibroblast cultures after irradiation

    SciTech Connect

    Li, F.P.; Little, J.B.; Bech-Hansen, N.T.; Paterson, M.C.; Arlett, C.; Garnick, M.B.; Mayer, R.J.

    1983-02-01

    Colonic polyposis and carcinoma developed in a woman with Turcot's syndrome at the age of 31 years; astrocytoma developed when she was 37. Her brother and sister had died of astrocytoma at the ages of 18 and 33 years, respectively. Progressive neutropenia developed in the patient three months after radiotherapy for her brain tumor and acute myelomonocytic leukemia 19 months after treatment. Three laboratories independently evaluated cultures of her skin fibroblasts for in vitro sensitivity to cell killing (loss of colony-forming ability) by x-rays. Survival assays consistently revealed slight but significant radiosensitivity in an early-passage (six to 10 doublings) fibroblast subculture. A later subculture (21 to 29 doublings) showed no abnormality, a possible effect of selective in vitro loss of radiosensitive cells.

  6. Effect of wavelength and fluence on morphology, cellular and genetic integrity of diabetic wounded human skin fibroblasts

    NASA Astrophysics Data System (ADS)

    Abrahamse, H.; Hawkins, D.; Houreld, N.

    2006-02-01

    An alternative treatment modality for diabetic wound healing includes low level laser therapy (LLLT). Biostimulation of such wounds may be of benefit to patients by reducing healing time. Structural, cellular and genetic events in diabetic wounded human skin fibroblasts (WS1) were evaluated after exposing cells in culture to a Helium-Neon (632.8nm), a Diode laser (830nm) and a Nd:YAG (Neodynium:Yttrium-Allumina-Gallium) laser (1064nm) at either 5J/cm2 or 16J/cm2. Cells were exposed twice a week and left 24 hours post-irradiation prior to measuring effects. Structural changes were evaluated by assessing colony formation, haptotaxis and chemotaxis. Cellular changes were evaluated using cell viability, (adenosine-triphosphate, ATP production), and proliferation, (alkaline phosphatase, ALP and basic fibroblast growth factor, bFGF expression), while the Comet assay evaluated DNA damage and cytotoxicity was determined assessing membrane permeability for lactate dehydrogenase (LDH). Caspase 3/7 activity was used as an estimate of apoptosis as a result of irradiation. The irradiated diabetic wounded cells showed structural, cellular as well as molecular resilience comparable to that of unwounded normal skin fibroblast cells. With regards to fluence, 5J/cm2 elicit positive cellular and structural responses while 16J/cm2 increases cellular and genetic damage and cellular morphology is altered. Different wavelengths of LLLT influences the beneficial outcomes of diabetic wounded cells and although all three wavelengths elicit cellular effects, the penetration depth of 830nm plays a significant role in the healing of diabetic wounded human fibroblast cells. Results from this study validate the contribution of LLLT to wound healing and elucidate the biochemical effects at a cellular level while highlighting the role of different dosages and wavelengths in LLLT.

  7. Characterization of the maitotoxin-activated cationic current from human skin fibroblasts

    PubMed Central

    Martínez-François, Juan Ramón; Morales-Tlalpan, Verónica; Vaca, Luis

    2002-01-01

    The maitotoxin (MTX)-induced cationic current (Imtx) from human skin fibroblasts was characterized using the patch-clamp technique in whole-cell configuration. Under resting conditions (absence of MTX), the main current observed is produced by an outwardly rectifying K+ channel which is inhibited by 1 mm TEA. The current reversal potential was −86 mV (n = 12). MTX (500 pm) activated a current with a linear current–voltage relationship and a reversal potential of −10 mV (n = 10). Replacing the extracellular Na+ and K+ with N-methyl-d-glucamine (NMDG) caused a shift of the reversal potential to a value below −100 mV, indicating that Na+ and K+, but not NMDG, carry Imtx. Further ion selectivity experiments showed that Ca2+ carries Imtx also. The resulting permeability sequence obtained with the Goldman–Hodgkin–Katz equation yielded Na+ (1) ≈ K+ (1) > Ca2+ (0.87). The Imtx activation time course reflected the changes in intracellular Ca2+ and Na+ measured with the fluorescent indicators fura-2 and SBFI, respectively, suggesting that the activation of Imtx brings about an increment in intracellular Ca2+ and Na+. Reducing the extracellular Ca2+ concentration below 1.8 mm prevented the activation of Imtx and the increment in intracellular Na+ induced by MTX. Mn2+ and Mg2+ could not replace Ca2+, but Ba2+ could replace Ca2+. MTX activation of current in 10 mm Ba2+ was approximately 50 % of that induced in the presence of 1.8 mm Ca2+. When 5 mm of the Ca2+ chelator BAPTA was included in the patch pipette, MTX either failed to activate the current or induced a small current (less than 15 % of the control), indicating that intracellular Ca2+ is also required for the activation of Imtx. Intracellular Ba2+ can replace Ca2+ as an activator of Imtx. However, in the presence of 10 mm Ba2+ the activation by MTX of the current was 50 % less than the activation with nm concentrations of free intracellular Ca2+. PMID:11773318

  8. DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low laser irradiation

    NASA Astrophysics Data System (ADS)

    Hawkins Evans, D.; Mbene, A.; Zungu, I.; Houreld, N.; Abrahamse, H.

    2009-02-01

    Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2

  9. Site-Specific Differentiation of Fibroblasts in Normal and Scleroderma Skin

    DTIC Science & Technology

    2010-06-01

    expression of one HOX lncRNA, termed nc-HOXC10, is correlated with fibrotic gene expression in fibroblasts. In our initial study, we found nc-HOXC10...anatomic expression pattern of Hox genes from embryonic development through adulthood. The ongoing Hox expression endowed fibroblasts with site...specific inductive activities that control the homeostasis and regeneration of epithelia throughout the body. The epigenetic memory of Hox genes depend on

  10. All-trans retinoic acid (RA) stimulates events in organ-cultured human skin that underlie repair. Adult skin from sun-protected and sun-exposed sites responds in an identical manner to RA while neonatal foreskin responds differently.

    PubMed Central

    Varani, J; Perone, P; Griffiths, C E; Inman, D R; Fligiel, S E; Voorhees, J J

    1994-01-01

    Adult human skin from a sun-protected site (hip) and from a sun-exposed site (forearm) was maintained in organ culture for 12 d in the presence of a serum-free, growth factor-free basal medium. Cultures were incubated under conditions optimized for keratinocyte growth (i.e., in 0.15 mM extracellular Ca2+) or for fibroblast growth (i.e., in 1.4 mM extracellular Ca2+). Treatment with all-trans retinoic acid (RA) induced histological changes in the organ-cultured skin under both conditions which were similar to the changes seen in intact skin after topical application. These included expansion of the viable portion of the epidermis and activation of cells in the dermis. In sun-damaged skin samples, which were characterized by destruction of normal connective tissue elements and presence of thick, dark-staining elastotic fibers, a zone of healthy connective tissue could be seen immediately below the dermo-epidermal junction. This zone was more prominent in RA-treated organ cultures than in matched controls. Associated with these histological changes was an increase in overall protein and extracellular matrix synthesis. In concomitant studies, it was found that RA treatment enhanced survival and proliferation of adult keratinocytes and adult dermal fibroblasts under both low- and high-Ca2+ conditions. In all of these assays, responses of sun-protected and sun-exposed skin were identical. In contrast, responses of neonatal foreskin to RA were similar to those of adult skin in the presence of low-Ca2+ culture medium, but under conditions of high extracellular Ca2+ RA provided little or no additional stimulus. Together these studies suggest that the ability of RA to enhance repair of sun-damaged skin (documented in previous studies) may reflect its ability to influence the behavior of skin in a manner that is age dependent but independent of sun-exposure status. Images PMID:7962521

  11. Molecular Profiling Reveals Diversity of Stress Signal Transduction Cascades in Highly Penetrant Alzheimer's Disease Human Skin Fibroblasts

    PubMed Central

    Mendonsa, Graziella; Dobrowolska, Justyna; Lin, Angela; Vijairania, Pooja; Jong, Y.-J. I.; Baenziger, Nancy L.

    2009-01-01

    The serious and growing impact of the neurodegenerative disorder Alzheimer's disease (AD) as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer's disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R) inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival pathway in normal

  12. Molecular profiling reveals diversity of stress signal transduction cascades in highly penetrant Alzheimer's disease human skin fibroblasts.

    PubMed

    Mendonsa, Graziella; Dobrowolska, Justyna; Lin, Angela; Vijairania, Pooja; Jong, Y-J I; Baenziger, Nancy L

    2009-01-01

    The serious and growing impact of the neurodegenerative disorder Alzheimer's disease (AD) as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer's disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R) inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival pathway in normal

  13. Chromosomal radiosensitivity during the G2 cell-cycle period of skin fibroblasts from individuals with familial cancer

    SciTech Connect

    Parshad, R.; Sanford, K.K.; Jones, G.M.

    1985-08-01

    The authors reported previously that human cells after neoplastic transformation in culture had acquired an increased susceptibility to chromatid damage induced by x-irradiation during the G2 phase of the cell cycle. Evidence suggested that this results from deficient DNA repair during G2 phase. Cells derived from human tumors also showed enhanced G2-phase chromosomal radiosensitivity. Furthermore, skin fibroblasts from individuals with genetic diseases predisposing to a high risk of cancer, including ataxia-telangiectasia, Bloom syndrome, Fanconi anemia, and xeroderma pigmentosum exhibited enhanced G2-phase chromosomal radiosensitivity. The present study shows that apparently normal skin fibroblasts from individuals with familial cancer--i.e., from families with a history of neoplastic disease--also exhibit enhanced G2-phase chromosomal radiosensitivity. This radiosensitivity appears, therefore, to be associated with both a genetic predisposition to cancer and a malignant neoplastic state. Furthermore, enhanced G2-phase chromosomal radiosensitivity may provide the basis for an assay to detect genetic susceptibility to cancer.

  14. X ray sensitivity of diploid skin fibroblasts from patients with Fanconi's anemia

    NASA Technical Reports Server (NTRS)

    Kale, Ranjini

    1989-01-01

    Experiments were performed on Fanconi's anemia and normal human fibroblast cell lines growing in culture in an attempt to correlate cell cycle kinetics with genomic damage and determine their bearing on the mechanism of chromosome aberration induction. FA fibroblasts showed a significantly increased susceptibility to chromosomal breakage by x rays in the G2 phase of the cell cycle. No such response was observed in fibroblasts irradiated in the G0 phase. The observed increases in achromatic lesions and in chromatid deletions in FA cells as compared with normal cells appear to indicate that FA cells are deficient in strand break repair and also possibly in base damage excision repair. Experiments are now in progress to further elucidate the mechanisms involved.

  15. TGFbeta and TGFalpha, antagonistic effect in vitro on extracellular matrix accumulation by chick skin fibroblasts at two distinct embryonic stages.

    PubMed

    Locci, P; Baroni, T; Lilli, C; Martinese, D; Marinucci, L; Bellocchio, S; Calvitti, M; Becchetti, E

    1999-03-01

    ECM macromolecules create a specific environment that participates in the control of cell proliferation and differentiation during embryogenesis. Quantitative and qualitative alterations in the ECM may depend on several growth factors that modify cell metabolism. Since transforming growth factor beta (TGFbeta) and alpha (TGFalpha) are abundantly expressed during embryonic development in organs in which epithelial-mesenchymal interactions occur, the aim of this study was to determine: a) the effect of TGFbeta on the phenotype of 7 and 14 day chick embryo back skin (CEBS) fibroblasts by evaluating the neosynthesis of GAG, collagen and fibronectin; b) whether TGFalpha and TGFbeta production, in particular TGFbeta3 and TGFbeta4, and the number of TGFbeta receptors change during these two stages of embryonic development. The results show that the neosynthesis of ECM macromolecules, tested using radiolabelled precursors, is increased by TGFbeta. The growth factor generally favours cellular accumulation more than secretion. As far as GAG is concerned, TGFbeta has a greater stimulatory effect on sulphated GAG than on HA. Specific bioassay shows that TGFbeta3 and TGFbeta4 activity is higher in 7 day than 14 day CEBS fibroblasts. Moreover, TGFbeta3 and TGFbeta4 mRNA expression is increased in the first stages of development. Instead, the level of TGFalpha increases in successive developmental stages. Since TGFalpha stimulates the synthesis and secretion of HA, and HA binds and inactivates TGFbeta, the greater quantity of HA in 14 day fibroblasts may contribute to reducing the TGFbeta effect. Overall our data suggest that the production of TGFbeta and TGFalpha are age-dependent and that the balance between the two growth factors may be a mechanism for controlling skin differentiation.

  16. Glucocorticoid coordinate regulation of type I procollagen gene expression and procollagen DNA-binding proteins in chick skin fibroblasts

    SciTech Connect

    Cockayne, D.; Cutroneo, K.R.

    1988-04-19

    Nuclei were isolated from control and dexamethasone-treated (2 h) embryonic chick skin fibroblasts and transcribed in vitro. Nuclei isolated from dexamethasone-treated fibroblasts transcribed less pro..cap alpha..1(I) and pro..cap alpha..2(I) mRNAs but not ..beta..-actin mRNA. Fibroblasts receiving dexamethasone and (5,6-/sup 3/H)uridine also demonstrated decreased synthesis of nuclear type I procollagen mRNAs but not ..beta..-actin mRNA. In fibroblasts treated with cycloheximide the newly synthesized nuclear type I procollagen mRNA species were markedly decreased. An enhanced inhibitory effect was observed when fibroblasts were treated with cycloheximide plus dexamethasone. Since the studies above demonstrate that active protein synthesis is required to maintain the constitutive expression of the type I procollagen genes, the authors determined if glucocorticoids regulate DNA-binding proteins with sequence specificity for the ..cap alpha..2(I) procollagen gene. Nuclear protein blots were probed with the /sup 32/P-end-labeled pBR322 vector DNA and /sup 32/P-end-labeled ..cap alpha..2(I) procollagen promoter containing DNA. Nonhistone proteins remained bound to labeled DNA at stringency washes of 0.05 and 0.1 M NaCl. As the ionic strength was increased to 0.2 and 0.3 M NaCl, the nonhistone-protein DNA binding was preferentially lost. Only the low molecular weight proteins remained bound to labeled DNA at the highest ionic strength, indicating nonspecific binding of these nuclear proteins. Dexamethasone treatment resulted in an increase of binding of nonhistone proteins to vector- and promoter-labeled DNAs over that observed in control fibroblasts at stringency washes of 0.05 and 0.1 M NaCl and to a lesser extent at 0.2 M NaCl. The binding specificities of nonhistone proteins for the ..cap alpha..2(I) procollagen promoter containing DNA were calculated.

  17. High concentrations of mast cell chymase facilitate the transduction of the transforming growth factor-β1/Smads signaling pathway in skin fibroblasts.

    PubMed

    Dong, Xianglin; Zhang, Chuanshan; Ma, Shaolin; Wen, Hao

    2015-03-01

    The aim of the present study was to investigate the effect of different concentrations of mast cell chymase on the transforming growth factor (TGF)-β1/Smad signaling pathway in skin fibroblasts. Cultured skin fibroblasts were treated with various concentrations of chymase for different time periods. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the rate of cell proliferation. In addition, protein expression in the fibroblasts was measured using western blot analysis. Chymase was shown to enhance the proliferation of skin fibroblasts following incubation for 48, 72 and 96 h (P<0.01). Furthermore, high concentrations of mast cell chymase were shown to enhance the mRNA and protein expression levels of TGF-β1 after long-term (≥6 h) incubation. In addition, high concentrations of mast cell chymase increased P-Smad2/3 and Smad2/3 protein expression. By contrast, low concentrations of mast cell chymase increased Smad7 protein expression. Therefore, the results demonstrated that high concentrations of mast cell chymase facilitated the transduction of the TGF-β1/Smad signaling pathway in skin fibroblasts.

  18. Effects of cathepsin D inhibitor from Vicia sativa L. seed hulls on human skin fibroblasts and breast cancer cells (in vitro studies).

    PubMed

    Roszkowska-Jakimiec, W; Wołczyński, S; Chlabicz, M

    2004-01-01

    Cathepsin D is a lysosomal protease which plays an important role in cancer invasion and metastasis. There are known inhibitors of that enzyme, such as pepstatin and potato inhibitor. In this study, we examined effects of the cathepsin D inhibitor from Vicia sativa L. seed hulls on cell cultures of human skin fibroblasts and breast cancer cells. There is no effect of the D-cathepsin inhibitor from Vicia sativa L. seed hulls on the proliferative activity of either human skin fibroblasts or breast cancer cells, measured by the [3H] thymidine incorporation assay.

  19. Dermal fibroblast expression of stromal cell-derived factor-1 (SDF-1) promotes epidermal keratinocyte proliferation in normal and diseased skin.

    PubMed

    Quan, Chunji; Cho, Moon Kyun; Shao, Yuan; Mianecki, Laurel E; Liao, Eric; Perry, Daniel; Quan, Taihao

    2015-12-01

    Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.

  20. Enzyme-Treated Asparagus Extract Attenuates Hydrogen Peroxide-Induced Matrix Metalloproteinase-9 Expression in Murine Skin Fibroblast L929 Cells.

    PubMed

    Shirato, Ken; Takanari, Jun; Ogasawara, Junetsu; Sakurai, Takuya; Imaizumi, Kazuhiko; Ohno, Hideki; Kizaki, Takako

    2016-05-01

    Enzyme-treated asparagus extract (ETAS) exerts a wide variety of beneficial biological actions including facilitating anti-cortisol stress and neurological anti-aging responses. However, the anti-skin aging effects of ETAS remain to be elucidated. Reactive oxygen species (ROS) play pivotal roles in skin aging. Increased ROS levels in fibroblasts in response to ultraviolet irradiation activate c-Jun N-terminal kinase (JNK) and its downstream transcription factor activator protein-1 (AP-1), and the resultant gene expression of matrix metalloproteinase (MMP) isoforms accelerates collagen breakdown in the dermis. Therefore, we explored whether ETAS has anti-skin aging effects by attenuating the oxidative stress responses in fibroblasts. Simultaneous treatment of murine skin L929 fibroblasts with hydrogen peroxide (H2O2) and either ETAS or dextrin showed that ETAS significantly suppressed H2O2-induced expression of MMP-9 mRNA as measured by real-time polymerase chain reaction. ETAS also clearly suppressed H2O2-stimulated phosphorylation of c-Jun (AP-1 subunit) and JNK as determined by Western blot. However, ETAS did not affect the increased amounts of carbonyl proteins in response to H2O2, also as determined by Western blotting. These results suggest that ETAS diminishes cellular responsiveness to ROS but does not scavenge ROS. Thus, ETAS has the potential to prevent skin aging through attenuating the oxidative stress responses in dermal fibroblasts.

  1. Reduction of fibroblast size/mechanical force down-regulates TGF-β type II receptor: implications for human skin aging.

    PubMed

    Fisher, Gary J; Shao, Yuan; He, Tianyuan; Qin, Zhaoping; Perry, Daniel; Voorhees, John J; Quan, Taihao

    2016-02-01

    The structural integrity of human skin is largely dependent on the quality of the dermal extracellular matrix (ECM), which is produced, organized, and maintained by dermal fibroblasts. Normally, fibroblasts attach to the ECM and thereby achieve stretched, elongated morphology. A prominent characteristic of dermal fibroblasts in aged skin is reduced size, with decreased elongation and a more rounded, collapsed morphology. Here, we show that reduced size of fibroblasts in mechanically unrestrained three-dimensional collagen lattices coincides with reduced mechanical force, measured by atomic force microscopy. Reduced size/mechanical force specifically down-regulates TGF-β type II receptor (TβRII) and thus impairs TGF-β/Smad signaling pathway. Both TβRII mRNA and protein were decreased, resulting in 90% loss of TGF-β binding to fibroblasts. Down-regulation of TβRII was associated with significantly decreased phosphorylation, DNA-binding, and transcriptional activity of its key downstream effector Smad3 and reduced expression of Smad3-regulated essential ECM components type I collagen, fibronectin, and connective tissue growth factor (CTGF/CCN2). Restoration of TβRII significantly increased TGF-β induction of Smad3 phosphorylation and stimulated expression of ECM components. Reduced expression of TβRII and ECM components in response to reduced fibroblast size/mechanical force was fully reversed by restoring size/mechanical force. Reduced fibroblast size was associated with reduced expression of TβRII and diminished ECM production, in aged human skin. Taken together, these data reveal a novel mechanism that provides a molecular basis for loss of dermal ECM, with concomitant increased fragility, which is a prominent feature of human skin aging.

  2. Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells.

    PubMed

    Song, Eunju; Chung, Haeyon; Shim, Eugene; Jeong, Jung-Ky; Han, Bok-Kyung; Choi, Hyuk-Joon; Hwang, Jinah

    2016-11-01

    Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

  3. Increase in the radioresistance of normal skin fibroblasts but not tumor cells by mechanical injury.

    PubMed

    Chen, Zelin; Wang, Xin; Jin, Taotao; Wang, Yu; Hong, Christopher S; Tan, Li; Dai, Tingyu; Wu, Liao; Zhuang, Zhengping; Shi, Chunmeng

    2017-02-02

    The timing of radiation after mechanical injury such as in the case of surgery is considered a clinical challenge because radiation is assumed to impair wound healing. However, the physiological responses and underlying mechanisms of this healing impairment are still unclear. Here, we show that mechanical injury occurring before ionizing radiation decreases radiation-induced cell damage and increases cell repair in normal fibroblasts but not tumor cells in vitro and in vivo. At the molecular level, mechanical injury interrupts focal adhesion complexes and cell-cell cadherin interactions, transducing mechanical signals into intracellular chemical signals via activation of the phosphatidylinositol 3-kinase (PI3K), Akt, and glycogen synthase kinase 3 beta (GSK-3β) pathways. We show that subsequent nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and β-catenin strengthen the stemness, antioxidant capabilities, and DNA double-strand break repair abilities of fibroblasts, ultimately contributing to increased radioresistance. Our findings demonstrate that mechanical injury to normal fibroblasts enhances radioresistance and may therefore question conventional wisdom surrounding the timing of radiation after surgery.

  4. The Effect of Phototherapy on Cancer Predisposition Genes of Diabetic and Normal Human Skin Fibroblasts

    PubMed Central

    Tangtrakulwanich, Boonsin; Sangkhathat, Surasak

    2017-01-01

    The purpose of this study was to investigate whether LED light at different wavelengths affects the expression profile of 143 cancer predisposition genes in both diabetic and normal human fibroblasts. In this study, both diabetic and normal fibroblast cell lines were cultured and irradiated with red (635 nm), green (520 nm), and blue (465 nm) LED light for 10 minutes at 0.67 J/cm2 each. After that, mRNA from all cell lines was extracted for microarray analysis. We found that green light activates EPHB2, KIT, ANTXR2, ESCO2, MSR1, EXT1, TSC1, KIT, NF1, BUB1B, FANCD2, EPCAM, FANCD2, NF, DIS3L2, and RET in normal fibroblast cells, while blue and red light can upregulate RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, KIT, FANCD2, BMPR1A, BUB1B, PDE11A, and RET. Therefore, genetic screening before phototherapy treatment may be required. PMID:28386563

  5. Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts

    PubMed Central

    Horiba, Minori; Kamiya, Tetsuro; Hara, Hirokazu; Adachi, Tetsuo

    2017-01-01

    Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues and has emerged as a novel technology for medical applications. NTAPP affects cells not only directly, but also indirectly with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the preconditioning effects of “mild PAM” which was prepared under relatively mild conditions, on fibroblasts against cellular injury generated by a high dose of hydrogen peroxide (H2O2). We observed the preconditioning effects of mild PAM containing approximately 50 μM H2O2. Hydrogen peroxide needs to be the main active species in mild PAM for it to exert preconditioning effects because the addition of catalase to mild PAM eliminated these effects. The nuclear translocation and recruitment of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response elements (ARE) in heme oxygenase 1 (HO-1) promoters and the up-regulation of HO-1 were detected in fibroblasts treated with mild PAM. The addition of ZnPP, a HO-1-specific inhibitor, or the knockdown of Nrf2 completely abrogated the preconditioning effects. Our results demonstrate that mild PAM protects fibroblasts from oxidative stress by up-regulating HO-1, and the H2O2-induced activation of the Nrf2-ARE pathway needs to be involved in this reaction. PMID:28169359

  6. A comparative study of baby immature and adult shoots of Aloe vera on UVB-induced skin photoaging in vitro.

    PubMed

    Hwang, Eunson; Kim, Su Hyeon; Lee, Sarah; Lee, Choong Hwan; Do, Seon-Gil; Kim, Jinwan; Kim, Sun Yeou

    2013-12-01

    Ultraviolet (UV) irradiation induces photo-damage of the skin, which in turn causes depletion of the dermal extracellular matrix and chronic alterations in skin structure. Skin wrinkle formations are associated with collagen synthesis and matrix metalloproteinase (MMP) expression. The production of type I procollagen is regulated by transforming growth factor-β1 (TGF-β1) expression; the activation of MMP is also correlated with an increase of interleukin-6 (IL-6). Aloe barbadensis M. (Aloe vera) is widely used in cosmetic and pharmaceutical products. In this study, we examined whether baby aloe shoot extract (BAE, immature aloe extract), which is from the one-month-old shoots of Aloe vera, and adult aloe shoot extract (AE), which is from the four-month-old shoots of Aloe vera, have a protective effect on UVB-induced skin photoaging in normal human dermal fibroblasts (NHDFs). The effects of BAE and AE on UVB-induced photoaging were tested by measuring the levels of reactive oxygen species, MMP-1, MMP-3, IL-6, type I procollagen, and TGF-β1 after UVB irradiation. We found that NHDF cells treated with BAE after UVB-irradiation suppressed MMP-1, MMP-3, and IL-6 levels compared to the AE-treated cells. Furthermore, BAE treatment elevated type I procollagen and TGF-β1 levels. Our results suggest that BAE may potentially protect the skin from UVB-induced damage more than AE.

  7. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    SciTech Connect

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  8. Abnormal sensitivity of skin fibroblasts from familial polyposis patients to DNA alkylating agents

    SciTech Connect

    Barfknecht, T.R.; Little, J.B.

    1982-04-01

    Fibroblast cell strains derived from different patients all afflicted with genetic predisposing to the development of intestinal polyposis and cancer were tested for their sensitivity to the lethal effects of the DNA alkylating agents methylmethanesulfonate (MMS), ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, and 4-nitroquinoline 1-oxide. The genetic syndromes studied were: (a) adenomatosis of the colon and rectum only, an autosomal dominant trait; (b) Turcot's syndrome, a rare autosomal recessive polyposis syndrome also characterized by central nervous system tumors; and (c) Gardner's syndrome, an autosomal dominant syndrome which, in addition to intestinal polyposis, is also clinically characterized by osteomas and soft tissue tumors. Fibroblasts from a patient with Turcot's syndrome were hypersensitive to MMS, having a D0 value of 0.24 mM (p less than 0.01) versus the normal average D0 of 0.36 mM and a D10 value of 0.95 mM (p less than 0.01) compared with the normal average value of 1.3 mM. Fibroblasts from the Gardner's syndrome proband were moderately sensitive to MMS, ethyl methanesulfonate, and N-methyl-N'-nitro-N-nitrosoguanidine due to significant differences of D10 values of 0.60 mM (p less than 0.01), 15 mM (p less than 0.01), and 4.8 microM (p less than 0.025), respectively, versus the normal average values of 1.3 mM, 28 mM, and 9.4 microM. Fibroblasts from the clinically affected Gardner's syndrome daughter of the proband were significantly more sensitive to MMS treatment, D0 of 0.22 mM (p less than 0.01) versus the normal average D0 of 0.36 mM and a D10 of 0.97 mM (p less than 0.01) versus the normal average. This differential sensitivity to the several DNA alkylating agents suggests that different mechanisms of hypersensitivity to these chemicals may be associated with fibroblasts from the various forms of familial polyposis.

  9. Inhibition of prolyl hydroxylation during collagen biosynthesis in human skin fibroblast cultures by ethyl 3,4-dihydroxybenzoate.

    PubMed

    Majamaa, K; Sasaki, T; Uitto, J

    1987-10-01

    The enzymatically catalyzed formation of 4-hydroxyproline plays a key role in the intracellular biosynthesis of collagen, since a critical number of 4-hydroxyprolyl residues is required for synthesis and secretion of triple-helical procollagen molecules under physiologic conditions. The enzyme catalyzing the conversion of prolyl residues to 4-hydroxyproline, prolyl 4-hydroxylase, requires ferrous ion, alpha-ketoglutarate, and ascorbate for its activity. 3,4-Dihydroxybenzoic acid has been known to act as potent competitive inhibitor of purified prolyl 4-hydroxylase with respect to one or several of the cofactors or cosubstrates of the enzyme. 3,4-Dihydroxybenzoic acid, however, is a poor inhibitor of prolyl hydroxylation in intact cells, probably due to its polarity not allowing it to enter the cells. In this study, several hydrophobic modifications of 3,4-dihydroxybenzoic acid were tested in human skin fibroblast cultures for their efficacy to inhibit the synthesis of 4-hydroxyproline. The results indicated that the ethyl ester of 3,4-dihydroxybenzoic acid was an efficient inhibitor of prolyl hydroxylation in fibroblast cultures, with Ki of approximately 0.4 mM. Ethyl 3,4-dihydroxybenzoate had little, if any, effect on the hydroxylation of lysyl residues, and it did not affect total protein synthesis or DNA replication in these cells. To test the hypothesis that ethyl 3,4-dihydroxybenzoate might serve as a potential antifibrotic agent, its efficacy in inhibiting prolyl hydroxylation in scleroderma fibroblasts was also tested. The results indicated that the synthesis of 4-hydroxyproline in scleroderma cell cultures was similarly reduced by ethyl 3,4-dihydroxybenzoate. Thus, structural analogs of the cofactors or cosubstrates of prolyl 4-hydroxylase, such as ethyl 3,4-dihydroxybenzoate tested here or its further modifications, may serve as inhibitors of posttranslational hydroxylation of prolyl residues also in vivo. These compounds could potentially provide a novel

  10. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    PubMed

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

  11. Characterization of lymphangiogenesis in a model of adult skin regeneration.

    PubMed

    Rutkowski, Joseph M; Boardman, Kendrick C; Swartz, Melody A

    2006-09-01

    To date, adult lymphangiogenesis is not well understood. In this study we describe the evolution of lymphatic capillaries in regenerating skin and correlate lymphatic migration and organization with the expression of matrix metalloproteinases (MMPs), immune cells, the growth factors VEGF-A and VEGF-C, and the heparan sulfate proteogylcan perlecan, a key component of basement membrane. We show that while lymphatic endothelial cells (LECs) migrate and organize unidirectionally, in the direction of interstitial fluid flow, they do not sprout into the region but rather migrate as single cells that later join together into vessels. Furthermore, in a modified "shunted flow" version of the model, infiltrated LECs fail to organize into functional vessels, indicating that interstitial fluid flow is necessary for lymphatic organization. Perlecan expression on new lymphatic vessels was only observed after vessel organization was complete and also appeared first in the distal region, consistent with the directionality of lymphatic migration and organization. VEGF-C expression peaked at the initiation of lymphangiogenesis but was reduced to lower levels throughout organization and maturation. In mice lacking MMP-9, lymphatics regenerated normally, suggesting that MMP-9 is not required for lymphangiogenesis, at least in mouse skin. This study thus characterizes the process of adult lymphangiogenesis and differentiates it from sprouting blood angiogenesis, verifies its dependence on interstitial fluid flow for vessel organization, and correlates its temporal evolution with those of relevant environmental factors.

  12. The spleen and skin wound healing in Xenopus adults.

    PubMed

    Franchini, Antonella; Della Rocca, Annalaura; Bertolotti, Evelina

    2016-07-01

    In most vertebrates, the regenerative capacity to restore lost/damage tissues to original structure and functionality decreases at some time during ontogenesis. To evaluate the role of the acquired immunity in the decline of regenerative potential, we examined the cellular responses elicited in the spleen during skin repair in Xenopus adults. Modifications in the architecture were found to be induced and were remarkable 14 days postinjury when the spleen increased significantly in size. In white pulp, the periarteriolar lymphoid sheaths were associated with follicles having central light zones, morphologically similar to germinal centers. With the progress of healing, pigment-containing cells were seen to accumulate in both white and red pulp regions. Moreover, compared to controls, the cells immunoreactive to anti-cytokines (TNF-α, TGF-β1) and -iNOS increased from the first days after wounding. The 14th day, the positive cells formed a dense network of reticular cells in central regions of lymphoid follicles and more frequent reactive leukocytes were detected within the red pulp. A higher number of lymphoid cells immunostained with anti-CD3ε were also observed in the perifollicular zone. The results suggest that the spleen of adult frogs is involved in skin wound healing with the expansion of lymphoid compartments. J. Morphol. 277:888-895, 2016. © 2016 Wiley Periodicals, Inc.

  13. Shell Extracts from the Marine Bivalve Pecten maximus Regulate the Synthesis of Extracellular Matrix in Primary Cultured Human Skin Fibroblasts

    PubMed Central

    Latire, Thomas; Legendre, Florence; Bigot, Nicolas; Carduner, Ludovic; Kellouche, Sabrina; Bouyoucef, Mouloud; Carreiras, Franck; Marin, Frédéric; Lebel, Jean-Marc; Galéra, Philippe; Serpentini, Antoine

    2014-01-01

    Mollusc shells are composed of more than 95% calcium carbonate and less than 5% of an organic matrix consisting mostly of proteins, glycoproteins and polysaccharides. Previous studies have elucidated the biological activities of the shell matrices from bivalve molluscs on skin, especially on the expression of the extracellular matrix components of fibroblasts. In this work, we have investigated the potential biological activities of shell matrix components extracted from the shell of the scallop Pecten maximus on human fibroblasts in primary culture. Firstly, we demonstrated that shell matrix components had different effects on general cellular activities. Secondly, we have shown that the shell matrix components stimulate the synthesis of type I and III collagens, as well as that of sulphated GAGs. The increased expression of type I collagen is likely mediated by the recruitment of transactivating factors (Sp1, Sp3 and human c-Krox) in the −112/−61 bp COL1A1 promoter region. Finally, contrarily to what was obtained in previous works, we demonstrated that the scallop shell extracts have only a small effect on cell migration during in vitro wound tests and have no effect on cell proliferation. Thus, our research emphasizes the potential use of shell matrix of Pecten maximus for dermo-cosmetic applications. PMID:24949635

  14. Functional differences between neonatal and adult fibroblasts and keratinocytes: Donor age affects epithelial-mesenchymal crosstalk in vitro

    PubMed Central

    Mateu, Rosana; Živicová, Veronika; Krejčí, Eliška Drobná; Grim, Miloš; Strnad, Hynek; Vlček, Čestmír; Kolář, Michal; Lacina, Lukáš; Gál, Peter; Borský, Jiří; Smetana, Karel; Dvořánková, Barbora

    2016-01-01

    Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial-mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period. PMID:27513730

  15. Functional differences between neonatal and adult fibroblasts and keratinocytes: Donor age affects epithelial-mesenchymal crosstalk in vitro.

    PubMed

    Mateu, Rosana; Živicová, Veronika; Krejčí, Eliška Drobná; Grim, Miloš; Strnad, Hynek; Vlček, Čestmír; Kolář, Michal; Lacina, Lukáš; Gál, Peter; Borský, Jiří; Smetana, Karel; Dvořánková, Barbora

    2016-10-01

    Clinical evidence suggests that healing is faster and almost scarless at an early neonatal age in comparison with that in adults. In this study, the phenotypes of neonatal and adult dermal fibroblasts and keratinocytes (nestin, smooth muscle actin, keratin types 8, 14 and 19, and fibronectin) were compared. Furthermore, functional assays (proliferation, migration, scratch wound closure) including mutual epithelial‑mesenchymal interactions were also performed to complete the series of experiments. Positivity for nestin and α smooth muscle actin was higher in neonatal fibroblasts (NFs) when compared with their adult counterparts (adult fibroblasts; AFs). Although the proliferation of NFs and AFs was similar, they significantly differed in their migration potential. The keratinocyte experiments revealed small, poorly differentiated cells (positive for keratins 8, 14 and 19) in primary cultures isolated from neonatal tissues. Moreover, the neonatal keratinocytes exhibited significantly faster rates of healing the experimentally induced in vitro defects in comparison with adult cells. Notably, the epithelial/mesenchymal interaction studies showed that NFs in co-culture with adult keratinocytes significantly stimulated the adult epithelial cells to acquire the phenotype of small, non-confluent cells expressing markers of poor differentiation. These results indicate the important differences between neonatal and adult cells that may be associated with improved wound healing during the early neonatal period.

  16. Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through αVβ3 integrin in human skin fibroblasts.

    PubMed

    Qin, Zhaoping; Fisher, Gary J; Quan, Taihao

    2013-04-26

    Human skin largely comprises collagenous extracellular matrix. The hallmark of skin aging is fragmentation of collagen fibrils. Matrix metalloproteinases (MMPs) are largely responsible for collagen degradation. MMP-1, principally derived from dermal fibroblasts, is the major protease capable of initiating degradation of native fibrillar collagens. Presently, we report that CCN1, a secreted and extracellular matrix-associated protein, is elevated in aged human skin dermal fibroblasts in vivo and stimulates MMP-1 expression through functional interaction with αVβ3 integrin in human dermal fibroblasts. CCN1 contains four conserved structural domains. Our results indicate that the three N-terminal domains (IGFBP, VWC, and TSP1), but not the C-terminal CT domain, are required for CCN1 to stimulate MMP-1 expression. This stimulation is dependent on interaction between the active structural domains and αVβ3 integrin. The interaction of VWC domain with integrin αVβ3 is necessary and requires functional cooperation with adjacent IGFBP and TSP1 domains to stimulate MMP-1 expression. Finally, induction of MMP-1 expression in dermal fibroblasts by CCN1 N-terminal domains resulted in fragmentation of type I collagen fibrils in a three-dimensional collagen lattice model. These data suggest that domain-specific interactions of CCN1 with αVβ3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

  17. ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY

    EPA Science Inventory

    ANALYSIS OF DNA DAMAGE AND REPAIR IN SKIN FIBROBLASTS OF INFANT AND OLDER CHILDREN USING THE IN VITRO ALKALINE COMET ASSAY, Alan H. Tennant1, Geremy W. Knapp1 and Andrew D. Kligerman1, 1Environmental Carcinogenesis Division, National Health and Environmental Effects Research Lab...

  18. Effect of culture conditions on microRNA expression in primary adult control and COPD lung fibroblasts in vitro.

    PubMed

    Ikari, Jun; Smith, Lynette M; Nelson, Amy J; Iwasawa, Shunichiro; Gunji, Yoko; Farid, Maha; Wang, Xingqi; Basma, Hesham; Feghali-Bostwick, Carol; Liu, Xiangde; DeMeo, Dawn L; Rennard, Stephen I

    2015-04-01

    In vitro cell cultures, including lung fibroblasts, have been used to identify microRNAs (miRNAs) associated with chronic obstructive pulmonary disease (COPD) pathogenesis. However, culture conditions may affect miRNA expression. We examined whether miRNA expression in primary adult lung fibroblasts varies with cell density or passage in vitro and whether culture conditions confound the identification of altered miRNA expression in COPD lung fibroblasts. Primary adult control and COPD lung fibroblasts were cultured until passage 3 or 8, after which cells were further cultured for 3 or 7 d (low vs. high density). Then, cells at low density were cultured with serum-free media, and those at high density were cultured with serum-free media in the absence or presence of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) for 24 h. RNA was extracted to perform miRNA microarray from which 1.25-fold differential expression and 10% false discovery rate were applied to identify "invariant" and "variant" miRNA for the various culture conditions. Of the 2226 miRNAs evaluated, 39.0% for cell density, 40.7% for cell passage, and 29.4% for both conditions were identified as "invariant" miRNAs. Furthermore, 38.1% of the evaluated miRNAs were "invariant" for cell passage with IL-1β and TNF-α. Differentially expressed miRNAs between control and COPD lung fibroblasts were identified with and without IL-1β and TNF-α, and of these, 32 out of the 34 top-ranked miRNAs exceeded the differences due to culture conditions. Thus, culture conditions may affect miRNA expression of adult human lung fibroblasts. Nevertheless, in vitro cultures can be used to assess differential miRNA expression in COPD lung fibroblasts.

  19. Electrophysiological Profiles of Induced Neurons Converted Directly from Adult Human Fibroblasts Indicate Incomplete Neuronal Conversion

    PubMed Central

    Koppensteiner, Peter; Boehm, Stefan

    2014-01-01

    Abstract The direct conversion of human fibroblasts to neuronal cells, termed human induced neuronal (hiN) cells, has great potential for future clinical advances. However, previous studies have not provided an in-depth analysis of electrophysiological properties of adult fibroblast-derived hiN cultures. We have examined the electrophysiological profile of hiN cells by measuring passive and active membrane properties, as well as spontaneous and evoked neurotransmission. We found that hiN cells exhibited passive membrane properties equivalent to perinatal rodent neurons. In addition, 30% of hiN cells were incapable of action potential (AP) generation and did not exhibit rectifying membrane currents, and none of the cells displayed firing patterns of typical glutamatergic pyramidal neurons. Finally, hiN cells exhibited neither spontaneous nor evoked neurotransmission. Our results suggest that current methods used to produce hiN cells provide preparations in which cells do not achieve the cellular properties of fully mature neurons, rendering these cells inadequate to investigate pathophysiological mechanisms. PMID:25437871

  20. Efficient Uptake and Dissemination of Scrapie Prion Protein by Astrocytes and Fibroblasts from Adult Hamster Brain

    PubMed Central

    Hollister, Jason R.; Lee, Kil Sun; Dorward, David W.; Baron, Gerald S.

    2015-01-01

    Prion infections target neurons and lead to neuronal loss. However, the role of non-neuronal cells in the initiation and spread of infection throughout the brain remains unclear despite the fact these cells can also propagate prion infectivity. To evaluate how different brain cells process scrapie prion protein (PrPres) during acute infection, we exposed neuron-enriched and non-neuronal cell cultures from adult hamster brain to fluorescently-labeled purified PrPres and followed the cultures by live cell confocal imaging over time. Non-neuronal cells present in both types of cultures, specifically astrocytes and fibroblasts, internalized PrPres more efficiently than neurons. PrPres was trafficked to late endosomal/lysosomal compartments and rapidly transported throughout the cell bodies and processes of all cell types, including contacts between astrocytes and neurons. These observations suggest that astrocytes and meningeal fibroblasts play an as yet unappreciated role in prion infections via efficient uptake and dissemination of PrPres. PMID:25635871

  1. Efficient uptake and dissemination of scrapie prion protein by astrocytes and fibroblasts from adult hamster brain.

    PubMed

    Hollister, Jason R; Lee, Kil Sun; Dorward, David W; Baron, Gerald S

    2015-01-01

    Prion infections target neurons and lead to neuronal loss. However, the role of non-neuronal cells in the initiation and spread of infection throughout the brain remains unclear despite the fact these cells can also propagate prion infectivity. To evaluate how different brain cells process scrapie prion protein (PrPres) during acute infection, we exposed neuron-enriched and non-neuronal cell cultures from adult hamster brain to fluorescently-labeled purified PrPres and followed the cultures by live cell confocal imaging over time. Non-neuronal cells present in both types of cultures, specifically astrocytes and fibroblasts, internalized PrPres more efficiently than neurons. PrPres was trafficked to late endosomal/lysosomal compartments and rapidly transported throughout the cell bodies and processes of all cell types, including contacts between astrocytes and neurons. These observations suggest that astrocytes and meningeal fibroblasts play an as yet unappreciated role in prion infections via efficient uptake and dissemination of PrPres.

  2. Specific toxicity of aphidicolin to ultraviolet-irradiated excision proficient human skin fibroblasts

    SciTech Connect

    Tyrrell, R.M.

    1983-01-01

    Aphidicolin, a specific inhibitor of the eucaryotic alpha polymerase, has been employed to study the role of this enzyme in repair of potentially lethal damage (PLD) induced by far u.v. (254 nm) radiation in normal and repair defective primary human fibroblasts. There is strong concentration dependent specific toxicity to cells treated with a fluence of 6 Jm-2 of far-u.v. radiation and incubated with aphidicolin for 2 days over the concentration range 0.0025-2.5 micrograms/ml. A similar effect is seen with a xeroderma pigmentosum (XP) variant (excision proficient) strain but there is no specific toxicity to u.v. irradiated excision deficient XP cells of complementation group A. Inactivation of irradiated excision proficient fibroblasts is rapid over the first 6 h of aphidicolin (1 microgram/ml) treatment but the reaction takes 2 days or longer to complete depending on the u.v. dose. These results demonstrate that the apparent uncoupling of excision repair seen previously by other investigators prevents repair of PLD and is lethal to the cells.

  3. Second generation codon optimized minicircle (CoMiC) for nonviral reprogramming of human adult fibroblasts.

    PubMed

    Diecke, Sebastian; Lisowski, Leszek; Kooreman, Nigel G; Wu, Joseph C

    2014-01-01

    The ability to induce pluripotency in somatic cells is one of the most important scientific achievements in the fields of stem cell research and regenerative medicine. This technique allows researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful tool for disease modeling and drug screening approaches. However, using viruses for the delivery of reprogramming genes and transcription factors may result in integration into the host genome and cause random mutations within the target cell, thus limiting the use of these cells for downstream applications. To overcome this limitation, various non-integrating techniques, including Sendai virus, mRNA, minicircle, and plasmid-based methods, have recently been developed. Utilizing a newly developed codon optimized 4-in-1 minicircle (CoMiC), we were able to reprogram human adult fibroblasts using chemically defined media and without the need for feeder cells.

  4. The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

    PubMed

    Opländer, Christian; Cortese, Miriam M; Korth, Hans-Gert; Kirsch, Michael; Mahotka, Csaba; Wetzel, Wiebke; Pallua, Norbert; Suschek, Christoph V

    2007-09-01

    Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.

  5. Recovery of fibroblast-like cells from refrigerated goat skin up to 41 d of animal death.

    PubMed

    Okonkwo, Charles; Singh, Mahipal

    2015-05-01

    Successful cloning of animals using somatic cell nuclear transfer requires undamaged nuclear DNA from desired donor cell types. In vitro culture of cells is one way of ensuring nuclear integrity. The goal of this study was to evaluate the limits of postmortem cell survival/culture in refrigerated goat ear skin tissues which could be used for long-term storage and cloning of animals in future. To achieve this, 60 explants from 6 different goats were cultured after 0, 3, 6, 9, 13, 16, 20, 23, 27, 30, 33, 37, and 41 d postmortem and observed under inverted microscope for outgrowth of fibroblast-like cells, after 10-12 d of culture. Explants from all time points including 19% from 41-dpm tissues exhibited outgrowth. However, the percentage of outgrowth positive explants, as well as culture confluence, reduced with increasing postmortem time interval. Cell cultures established from primary outgrowth of 41-dpm tissues when compared for their growth profile with similarly obtained 0-dpm cultures revealed similar growth curve and cell morphology. Cytogenetic analysis of 41-dpm tissue-derived cell populations revealed a normal female karyotype with 60 XX homologous chromosomes indicating genetic stability of the cell population. In conclusion, these results show that refrigerated skin tissue remains alive for more than a month and that the cells derived from such tissues are normal and can be cryopreserved for long-term storage and future cloning of animals with desired genetics.

  6. Downregulation of let-7b promotes COL1A1 and COL1A2 expression in dermis and skin fibroblasts during heat wound repair.

    PubMed

    Liu, Jinyan; Luo, Chengqun; Yin, Zhaoqi; Li, Ping; Wang, Shaohua; Chen, Jia; He, Quanyong; Zhou, Jianda

    2016-03-01

    MicroRNAs (miRs), a class of non‑coding RNAs 18‑25 nucleotides in length, generally serve suppressive role in the regulation of gene expression via directly binding to the 3'‑untranslated region (UTR) of their target mRNA. Previous studies have identified several miRs to be involved in thermal injury repair. However, the role of miR let‑7b during the recovery of thermal injury, in addition to the underlying mechanisms, has not previously been studied. In the present study, the expression of let‑7b was observed to be significantly increased in skin tissue shortly following thermal injury, however, gradually reduced during the recovery of thermal injury. Notably, similar findings were observed in heat‑denatured skin fibroblasts. Furthermore, collagen, type I, alpha 1 (COL1A1) and collagen, type I, alpha 2 (COL1A2), which are associated with the synthesis of type I collagen, were identified as two targets of let‑7b in skin fibroblasts. The overexpression of let‑7b was observed to upregulate the protein expression levels of COL1A1 and COL1A2, while knockdown of let‑7b reduced the levels of COL1A1 and COL1A2 in skin fibroblasts. Furthermore, COL1A1 and COL1A2 were significantly downregulated shortly following thermal injury, while gradually upregulated during healing, in heat‑damaged skin tissue and skin fibroblasts, with the expression profiles opposite to that of let‑7b. Taken together, this suggests that the downregulation of let‑7b in heat‑damaged dermis promotes the synthesis of type I collagen and thus aids in burn wound repair.

  7. Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women

    PubMed Central

    Tanaka, Miyuki; Misawa, Eriko; Yamauchi, Koji; Abe, Fumiaki; Ishizaki, Chiaki

    2015-01-01

    Background Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. Methods First, we investigated the capability of Aloe sterols (cycloartenol and lophenol) to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP) containing 40 μg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. Results After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake period. Conclusion The present study confirms that daily oral Aloe sterol-containing AVGP significantly reduced facial wrinkles in women aged ≥40 years, and Aloe sterols stimulate collagen and hyaluronic acid production by human dermal fibroblasts. PMID:25759593

  8. Thrombin-activated platelets induce proliferation of human skin fibroblasts by stimulating autocrine production of insulin-like growth factor-1.

    PubMed

    Giacco, Ferdinando; Perruolo, Giuseppe; D'Agostino, Elio; Fratellanza, Giorgio; Perna, Enzo; Misso, Saverio; Saldalamacchia, Gennaro; Oriente, Francesco; Fiory, Francesca; Miele, Claudia; Formisano, Salvatore; Beguinot, Francesco; Formisano, Pietro

    2006-11-01

    Platelet components have found successful clinical utilization to initiate or to accelerate tissue-repair mechanisms. However, the molecular pathways by which platelet factors contribute to tissue regeneration have not been fully elucidated. We have studied the effect of thrombin-activated platelets (TAPs) on cell growth in vivo and in cultured cell systems. Application of TAPs to ulcerative skin lesions of diabetic patients induced local activation of ERK1/2 and Akt/PKB. Moreover, when applied to cultured human skin fibroblasts, TAPs promoted cell growth and DNA synthesis and activated platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1 receptor tyrosine kinases. PDGF was released by TAPs and rapidly achieved a plateau. At variance, the release of IGF-1 was mainly provided by the TAPs-stimulated fibroblasts and progressively increased up to 48 h. The PDGF-R blocker Ag1296 reduced the activation of Akt/PKB and, at a lesser extent, of ERK1/2. Conversely, inhibition of IGF-1 signaling by Ag1024 and expression of a dominant-negative IGF-1R mutant selectively reduced the stimulation of ERK1/2 by TAPs and fibroblast-released factors, with minor changes of Akt/PKB activity. Thus, platelet factors promote fibroblast growth by acutely activating Akt/PKB and ERK1/2. Sustained activation of ERK1/2, however, requires autocrine production of IGF-1 by TAPs-stimulated fibroblasts.

  9. Interleukin-13 modulates collagen homeostasis in human skin and keloid fibroblasts.

    PubMed

    Oriente, A; Fedarko, N S; Pacocha, S E; Huang, S K; Lichtenstein, L M; Essayan, D M

    2000-03-01

    Interleukin (IL)-13 has been implicated in the pathogenesis of various diseases characterized by fibrosis. We describe the effects of IL-13 on collagen homeostasis from normal (NF) and keloid (KF) fibroblasts and compare these effects with those of IL-4 and transforming growth factor (TGF)-beta(1). Total collagen generation was up-regulated in NF after 48 h of stimulation by IL-13; in KF, IL-13 stimulated a more rapid collagen response. The kinetics and magnitude of collagen generation induced by IL-13 were equivalent to those induced by similar concentrations of IL-4 and TGF-beta(1). Collagen type I production paralleled total collagen generation from both NF and KF; however, IL-4-induced collagen type I and total collagen production from KF was more transient than that induced by either IL-13 or TGF-beta(1). Procollagen 1alpha1 gene expression was induced in KF by stimulation with IL-13 for 24 h. Moreover, IL-13 was unique among these three cytokines in its ability to induce gene expression for procollagen 3alpha1. Finally, IL-13 inhibited IL-1beta-induced matrix metalloproteinase (MMP)-1 and MMP-3 production and enhanced tissue inhibitor of metalloproteinase (TIMP)-1 generation from NF; although similar effects were observed with IL-4, TGF-beta(1) transiently enhanced MMP-1 and MMP-3 generation without effecting TIMP-1. In KF, IL-13 and IL-4 inhibited MMP-3, whereas TGF-beta(1) enhanced MMP-3; TIMP-1 was unaffected by any of the three cytokines. These data demonstrate both the profibrotic effects of IL-13 on collagen homeostasis and the potential differential regulation of collagen homeostasis in fibroblast subtypes by IL-13.

  10. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence

    PubMed Central

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption. PMID:26576225

  11. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence.

    PubMed

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption.

  12. Research Resource: Comprehensive Expression Atlas of the Fibroblast Growth Factor System in Adult Mouse

    PubMed Central

    Fon Tacer, Klementina; Bookout, Angie L.; Ding, Xunshan; Kurosu, Hiroshi; John, George B.; Wang, Lei; Goetz, Regina; Mohammadi, Moosa; Kuro-o, Makoto; Mangelsdorf, David J.; Kliewer, Steven A.

    2010-01-01

    Although members of the fibroblast growth factor (FGF) family and their receptors have well-established roles in embryogenesis, their contributions to adult physiology remain relatively unexplored. Here, we use real-time quantitative PCR to determine the mRNA expression patterns of all 22 FGFs, the seven principal FGF receptors (FGFRs), and the three members of the Klotho family of coreceptors in 39 different mouse tissues. Unsupervised hierarchical cluster analysis of the mRNA expression data reveals that most FGFs and FGFRs fall into two groups the expression of which is enriched in either the central nervous system or reproductive and gastrointestinal tissues. Interestingly, the FGFs that can act as endocrine hormones, including FGF15/19, FGF21, and FGF23, cluster in a third group that does not include any FGFRs, underscoring their roles in signaling between tissues. We further show that the most recently identified Klotho family member, Lactase-like, is highly and selectively expressed in brown adipose tissue and eye and can function as an additional coreceptor for FGF19. This FGF atlas provides an important resource for guiding future studies to elucidate the physiological functions of FGFs in adult animals. PMID:20667984

  13. Cardiolipin content in mitochondria from cultured skin fibroblasts harboring mutations in the mitochondrial ATP6 gene.

    PubMed

    El-Hafidi, Mohammed; Meschini, Maria Chiara; Rizza, Teresa; Santorelli, Filippo M; Bertini, Enrico; Carrozzo, Rosalba; Vázquez-Memije, Martha Elisa

    2011-12-01

    The role of phospholipids in normal assembly and organization of the membrane proteins has been well documented. Cardiolipin, a unique tetra-acyl phospholipid localized in the inner mitochondrial membrane, is implicated in the stability of many inner-membrane protein complexes. Loss of cardiolipin content, alterations in its acyl chain composition and/or cardiolipin peroxidation have been associated with dysfunction in multiple tissues in a variety of pathological conditions. The aim of this study was to analyze the phospholipid composition of the mitochondrial membrane in the four most frequent mutations in the ATP6 gene: L156R, L217R, L156P and L217P but, more importantly, to investigate the possible changes in the cardiolipin profile. Mitochondrial membranes from fibroblasts with mutations at codon 217 of the ATP6 gene, showed a different cardiolipin content compared to controls. Conversely, results similar to controls were obtained for mutations at codon 156. These findings may be attributed to differences in the biosynthesis and remodeling of cardiolipin at the level of the inner mitochondrial transmembrane related to some mutations of the ATP6 gene.

  14. Effect of Fe3O4 Nanoparticles on Skin Tumor Cells and Dermal Fibroblasts

    PubMed Central

    Alili, Lirija; Chapiro, Swetlana; Marten, Gernot U.; Schmidt, Annette M.; Zanger, Klaus; Brenneisen, Peter

    2015-01-01

    Iron oxide (Fe3O4) nanoparticles have been used in many biomedical approaches. The toxicity of Fe3O4 nanoparticles on mammalian cells was published recently. Though, little is known about the viability of human cells after treatment with Fe3O4 nanoparticles. Herein, we examined the toxicity, production of reactive oxygen species, and invasive capacity after treatment of human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) with Fe3O4 nanoparticles. These nanoparticles had an average size of 65 nm. Fe3O4 nanoparticles induced oxidative stress via generation of reactive oxygen species (ROS) and subsequent initiation of lipid peroxidation. Furthermore, the question was addressed of whether Fe3O4 nanoparticles affect myofibroblast formation, known to be involved in tumor invasion. Herein, Fe3O4 nanoparticles prevent the expression alpha-smooth muscle actin and therefore decrease the number of myofibroblastic cells. Moreover, our data show in vitro that concentrations of Fe3O4 nanoparticles, which are nontoxic for normal cells, partially reveal a ROS-triggered cytotoxic but also a pro-invasive effect on the fraction of squamous cancer cells surviving the treatment with Fe3O4 nanoparticles. The data herein show that the Fe3O4 nanoparticles appear not to be adequate for use in therapeutic approaches against cancer cells, in contrast to recently published data with cerium oxide nanoparticles. PMID:26090418

  15. Plasmid-Based Generation of Induced Neural Stem Cells from Adult Human Fibroblasts

    PubMed Central

    Capetian, Philipp; Azmitia, Luis; Pauly, Martje G.; Krajka, Victor; Stengel, Felix; Bernhardi, Eva-Maria; Klett, Mariana; Meier, Britta; Seibler, Philip; Stanslowsky, Nancy; Moser, Andreas; Knopp, Andreas; Gillessen-Kaesbach, Gabriele; Nikkhah, Guido; Wegner, Florian; Döbrössy, Máté; Klein, Christine

    2016-01-01

    Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV)-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC) 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72%) and glial cells (9% astrocytes, 6% oligodendrocytes). Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts). Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial) pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside the cells at

  16. Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity

    PubMed Central

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Ahn, Jin Seop; Ryu, Junghyun; Heo, Soon Young; Park, Sang-Min; Kang, Jee Hyun; Choi, You Jung; Shim, Hosup

    2017-01-01

    Objective Production of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods Miniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets. Results Monoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous αGT knockout piglets. Conclusion The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a

  17. Efficacy of a single high dose versus multiple low doses of LLLT on wounded skin fibroblasts

    NASA Astrophysics Data System (ADS)

    Hawkins, Denise H.; Abrahamse, Heidi

    2007-07-01

    Background/purpose: In vivo studies have demonstrated that phototherapy accelerates wound healing in the clinical environment; however the exact mechanism is still not completely understood. The main focus of this study was to use in vitro laboratory results to establish an effective treatment regimen that may be practical and applicable to the clinical environment. This in vitro study aimed to compare the cellular responses of wounded fibroblasts following a single exposure of 5 J/cm2 or multiple exposures of low doses (2.5 J/cm2 or 5 J/cm2) on one day of the week to a single application of a higher dose (16 J/cm2) on day 1 and day 4. Methodology: Cellular responses to Helium-Neon (632.8 nm) laser irradiation were evaluated by measuring changes in cell morphology, cell viability, cell proliferation, membrane integrity and DNA damage. Results: Wounded cells exposed to 5 J/cm2 on day 1 and day 4 showed an increase in cell viability, increase in the release of bFGF, increase in cell density, decrease in ALP enzyme activity and decrease in caspase 3/7 activity indicating a stimulatory effect. Wounded cells exposed to three doses of 5 J/cm2 on day 1 showed a decrease in cell viability and cell proliferation and an increase in LDH cytotoxicity and DNA damage indicating an inhibitory effect. Conclusion: Results indicate that cellular responses are influenced by the combination of dose administered, number of exposures and time between exposures. Single doses administered with sufficient time between exposures is more beneficial to restoring cell function than multiple doses within a short period. Although this work confirms previous reports on the cumulative effect of laser irradiation it provides essential information for the initiation of in vivo clinical studies.

  18. Photobiomodulation of distinct lineages of human dermal fibroblasts: a rational approach towards the selection of effective light parameters for skin rejuvenation and wound healing

    NASA Astrophysics Data System (ADS)

    Mignon, Charles; Uzunbajakava, Natallia E.; Raafs, Bianca; Moolenaar, Mitchel; Botchkareva, Natalia V.; Tobin, Desmond J.

    2016-03-01

    Distinct lineages of human dermal fibroblasts play complementary roles in skin rejuvenation and wound healing, which makes them a target of phototherapy. However, knowledge about differential responses of specific cell lineages to different light parameters and moreover the actual molecular targets remain to be unravelled. The goal of this study was to investigate the impact of a range of parameters of light on the metabolic activity, collagen production, and cell migration of distinct lineages of human dermal fibroblasts. A rational approach was used to identify parameters with high therapeutic potential. Fibroblasts exhibited both inhibitory and cytotoxic change when exposed to a high dose of blue and cyan light in tissue culture medium containing photo-reactive species, but were stimulated by high dose red and near infrared light. Cytotoxic effects were eliminated by refreshing the medium after light exposure by removing potential ROS formed by extracellular photo-reactive species. Importantly, distinct lineages of fibroblasts demonstrated opposite responses to low dose blue light treatment when refreshing the medium after exposure. Low dose blue light treatment also significantly increased collagen production by papillary fibroblasts; high dose significantly retarded closure of the scratch wound without signs of cytotoxicity, and this is likely to have involved effects on both cell migration and proliferation. We recommend careful selection of fibroblast subpopulations and their culture conditions, a systematic approach in choosing and translating treatment parameters, and pursuit of fundamental research on identification of photoreceptors and triggered molecular pathways, while seeking effective parameters to address different stages of skin rejuvenation and wound healing.

  19. Skin Fibroblasts from Patients with Type 1 Diabetes (T1D) Can Be Chemically Transdifferentiated into Insulin-Expressing Clusters: A Transgene-Free Approach

    PubMed Central

    Pereyra-Bonnet, Federico; Gimeno, María L.; Argumedo, Nelson R.; Ielpi, Marcelo; Cardozo, Johana A.; Giménez, Carla A.; Hyon, Sung-Ho; Balzaretti, Marta; Loresi, Mónica; Fainstein-Day, Patricia; Litwak, León E.; Argibay, Pablo F.

    2014-01-01

    The conversion of differentiated cells into insulin-producing cells is a promising approach for the autologous replacement of pancreatic cells in patients with type 1 diabetes (T1D). At present, cellular reprogramming strategies encompass ethical problems, epigenetic failure or teratoma formation, which has prompted the development of new approaches. Here, we report a novel technique for the conversion of skin fibroblasts from T1D patients into insulin-expressing clusters using only drug-based induction. Our results demonstrate that skin fibroblasts from diabetic patients have pancreatic differentiation capacities and avoid the necessity of using transgenic strategies, stem cell sources or global demethylation steps. These findings open new possibilities for studying diabetes mechanisms, drug screenings and ultimately autologous transgenic-free regenerative medicine therapies in patients with T1D. PMID:24963634

  20. Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence.

    PubMed

    Carvalho, Ana C; Gomes, Andreia C; Pereira-Wilson, Cristina; Lima, Cristovao F

    2015-06-01

    Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

  1. Autophagy in human skin fibroblasts: Comparison between young and aged cells and evaluation of its cellular rhythm and response to Ultraviolet A radiation.

    PubMed

    Pernodet, Nadine; Dong, Kelly; Pelle, Edward

    2016-01-01

    Autophagic mechanisms play critical roles in cell maintenance. Damaged organelles that are not removed by autophagosomes, which act by engulfing and degrading these cellular components, have been linked to various pathologies. Recently, the progression of aging has also been correlated to a compromised autophagic response. Here, we report for the first time a significant reduction in autophagic levels in synchronized aged normal human skin fibroblasts as compared to young fibroblasts. We measured a 77.9% reduction in autophagy as determined by reverse transcription-polymerase chain reaction for LC3B expression, a microtubule-associated protein correlated to late stage autophagosome formation. In addition, we visualized these same changes by immunocytofluorescence with antibodies directed against LC3B. By harvesting synchronized, as well as unsynchronized cells over time, we were also able to measure for the first time a nighttime peak in autophagy that was present in young but absent in aged fibroblasts. Finally, since human skin is constantly subjected to environmentally induced oxidative stress from sunlight, we exposed fibroblasts to 10 J/cm2 ultraviolet A and found, in good agreement with current literature, not only that irradiation could partially reactivate autophagy in the aged cells, but also that this increase was phase shifted earlier from its endogenous temporal pattern because of its loss of synchronization with circadian rhythm.

  2. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    PubMed

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  3. The histamine degradative uptake pathway in human vascular endothelial cells and skin fibroblasts is dependent on extracellular Na+ and Cl-

    SciTech Connect

    Haddock, R.C.; Mack, P.; Leal, S.; Baenziger, N.L. )

    1990-08-25

    We have previously reported that human vascular endothelial cells and skin fibroblasts carry out degradation of (3H)histamine by a mechanism involving two successive enzymatic steps: imidazole ring tele-methylation by the cells' endogenous methyltransferase and subsequent amine oxidation by an exogenous diamine oxidase. Both histamine and the exogenous second enzyme in the pathway associate with the cells via separate binding sites or receptors. The enzymatic degradation process results in cellular accumulation of the proximal and distal metabolites tele-methylhistamine and 1-methyl-4-imidazoleacetic acid (MIAA). We have now demonstrated that this two-stage histamine degradative pathway is dependent on Na+ and Cl- in the extracellular environment. Accumulation of (3H) histamine-derived products is partially inhibited under conditions of Na+ deprivation and more substantially when Cl- is also withdrawn. The individual tele-methylation and amine oxidation enzymatic reactions themselves are unaffected or actually facilitated under these conditions. This indicates that it is the cellular mechanism for uptake coupled to the degradative pathway which reflects the cation and anion dependency. Restoration of degradative uptake displays a biphasic Na+ concentration curve, suggesting that the uptake process may be driven by multiple components. These findings indicate a role for both inward Na+ and Cl- ion movement in this cellular degradative uptake mechanism.

  4. Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome

    PubMed Central

    Germinario, Ralph J.; Kahlenberg, Arthur; Pinsky, Leonard

    1973-01-01

    1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na235SO4 and d-[2-3H]glucose, were used and their intracellular fates during uptake and `chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [35S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [3H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased 35S/3H ratio (nmol of [35S]sulphate incorporated/nmol of [3H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of `chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [3H]glucose. 3. After 34h exposure to a `corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [35S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of `corrected' Hurler-syndrome cells. PMID:4269307

  5. Activities of sulfatases for the degradation of acidic glycosaminoglycans in cultured skin fibroblasts from two siblings with multiple sulfatase deficiency.

    PubMed

    Minami, R; Fujibayashi, S; Tachi, N; Wagatsuma, K; Nakao, T; Ikeno, T; Tsugawa, S; Sukegawa, K; Orii, T

    1983-04-01

    Cultured skin fibroblasts from two siblings with multiple sulfatase deficiency (MSD) were assayed for the activities of sulfatases known to degrade acidic glycosaminoglycans (AGAG). There were iduronate sulfatase, arylsulfatase B, heparan sulfate (HS) sulfatase, N-acetylgalactosamine-6-sulfate sulfatase, HS-derived N-acetylglucosamine-6-sulfate sulfatase, and two keratan sulfate (KS)-derived N-acetylglucosamine-6-sulfate sulfatases. The activities of sulfatases required for the degradation of HS were reduced to a greater extent than those for the degradation of dermatan sulfate (DS), and those of sulfatases associated with basic defect of Morquio disease type A were moderately decreased or normal. On the other hand, urinary excretion of AGAG in both patients was increased about 10-fold compared to controls, and especially, the excretion of HS and DS was increased about 150-fold and 50-fold, respectively. Keratan sulfate was not detected. The results suggest that in patients with MSD the degradation of HS might be affected to a greater extent than that of DS.

  6. Altered binding of /sup 125/I-labeled calmodulin to a 46. 5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

    SciTech Connect

    Tallant, E.A.; Wallace, R.W.

    1987-02-01

    The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca/sup 2 +//calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblasts or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of /sup 125/I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.

  7. Radiosensitivity of fibroblasts obtained from a cafe-au-lait spot and normal-appearing skin of a patient with neurofibromatosis (NF-6)

    SciTech Connect

    Hannan, M.A.; Smith, B.P.; Sigut, D.; Sackey, K. )

    1990-07-15

    Fibroblast cells derived from a cafe-au-lait spot and normal-appearing skin of a neurofibromatosis (NF-6) patient were studied for radiosensitivity in comparison with two normal cell lines used as controls. No difference in radiosensitivity was observed between the patient's cell lines and the controls using acute gamma-irradiation. However, a markedly increased radiosensitivity of the fibroblasts obtained from the patient's skin of normal appearance was demonstrated after chronic gamma-irradiation. The cells from the cafe-au-lait spot showed intermediate sensitivity to chronic irradiation as compared with the control cell lines and the fibroblasts derived from the normal skin of the patient. These results showed the usefulness of chronic irradiation in detecting increased cellular radiosensitivity which may result from a unique DNA repair defect in an NF patient. We suggest that enhanced genetic changes in radiosensitive NF patients may lead to formation of cafe-au-lait lesions and certain tumors. Such a transformation may be associated with production of radiotolerant cells.

  8. Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

    PubMed

    Wu, Shuai Shuai; Li, Dong Jiang; Lv, Chun Rong; Yang, Hong Yuan; Zhu, Lan; Li, Wei Juan; Quan, Guo Bo; Hong, Qiong Hua

    2013-01-01

    In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P < 0.05). In conclusion, the skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in

  9. Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts.

    PubMed

    French, Andrew J; Adams, Catharine A; Anderson, Linda S; Kitchen, John R; Hughes, Marcus R; Wood, Samuel H

    2008-02-01

    Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20-24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.

  10. "Mirror, mirror...." a preliminary investigation of skin tone dissatisfaction and its impact among British adults.

    PubMed

    Swami, Viren; Henry, Amy; Peacock, Nicola; Roberts-Dunn, Ahkin; Porter, Alan

    2013-10-01

    This study examined skin tone dissatisfaction, measured using a skin tone chart, among a multiethnic sample of British adults. A total of 648 British White individuals, 292 British South Asians, and 260 British African Caribbean participants completed a visual task in which they were asked to indicate their actual and ideal skin tones. They also completed measures of body appreciation, self-esteem, and ethnic identity attachment. Results showed that Asians had a lighter skin tone ideal than White and African Caribbean participants. Conversely, White participants had higher skin tone dissatisfaction (preferring a darker skin tone) than Asian and African Caribbean participants, who preferred a lighter skin tone. Results also showed that skin tone dissatisfaction predicted body appreciation once the effects of participant ethnicity, age, ethnic identity attachment, and self-esteem had been accounted for. Implications of our findings and suggestions for future research are discussed.

  11. Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts

    PubMed Central

    Park, Jinny; Song, Hwa-Ryung; Lee, Minok; Hong, On-Yu; Whang, Pyoung H.; Han, Myung-Kwan; Kwon, Kang-Beom

    2016-01-01

    Reactive oxygen species (ROS) play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3) metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC) or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts. PMID:28003865

  12. Photosensitization of skin fibroblasts and HeLa cells by three chlorin derivatives: Role of chemical structure and delivery vehicle.

    PubMed

    Postigo, Fernando; Sagristá, M Luisa; De Madariaga, M Africa; Nonell, Santi; Mora, Margarita

    2006-05-01

    The chemical nature of the sensitizer and its selective uptake by malignant cells are decisive to choose an appropriate biocompatible carrier, able to preserve the photosensitizing characteristics of the dye. In this paper we demonstrate the photodynamic properties of three chlorins, derived from chlorophyll a, and the usefulness of liposomal carriers to design pharmaceutical formulations. The chlorins have been quantitatively incorporated into stable liposomes obtained from a mixture of L-alpha-palmitoyloleoylphosphatidylcholine and L-alpha-dioleoylphosphatidylserine in a 13.5:1.5 molar ratio (POPC/OOPS-liposomes). The chlorin uptake by skin fibroblasts increases steadily, reaching in all cases a plateau level dependent on both the chlorin structure and the vehicle employed. The photophysical properties of the three chlorins in THF are nearly identical and fulfill the requirements for a PDT photosensitizer. Incorporation of chlorins into liposomes induces important changes in their photophysics, but does not impair their cellular uptake or their cell photosensitization ability. In fact we observe in the cells the same photophysical behavior as in THF solution. Specifically, we demonstrate, by recording the near-IR phosphorescence of 1O2, that the chlorins are able to photosensitize the production of 1O2 in the cell membrane. The cell-photosensitization efficiency depended on the chlorin and cell line nature, the carrier, and the length of pre-incubation and post-irradiation periods. The high photodynamic activity of chlorin-loaded liposomes and the possibility to design liposomal carriers to achieve a specific target site favors this approach to obtain an eventual pharmaceutical formulation.

  13. Beliefs and Intentions for Skin Protection and UV Exposure in Young Adults

    ERIC Educational Resources Information Center

    Heckman, Carolyn J.; Manne, Sharon L.; Kloss, Jacqueline D.; Bass, Sarah Bauerle; Collins, Bradley; Lessin, Stuart R.

    2011-01-01

    Objective: To evaluate Fishbein's integrative model in predicting young adults' skin protection, sun exposure, and indoor tanning intentions. Methods: Two hundred twelve participants completed an online survey. Results: Damage distress, self-efficacy, and perceived control accounted for 34% of the variance in skin protection intentions. Outcome…

  14. The effects of macrophage-stimulating protein on the migration, proliferation, and collagen synthesis of skin fibroblasts in vitro and in vivo.

    PubMed

    Zhao, Jiajia; Hu, Li; Gong, Niya; Tang, Qingming; Du, Luyang; Chen, Lili

    2015-03-01

    Macrophage-stimulating protein (MSP), an important cytokine with multiple functions, is highly expressed in adipose-derived stem cells-conditioned medium (ASC-CM). ASCs can effectively promote wound healing through paracrine mechanism, suggesting that MSP may play a critical role in wound healing. Through binding to its receptor, RON (Receptuerd'OrigineNantaise, also called macrophage stimulation 1 receptor; MST1R), it can activate epithelial cells and work as an inflammatory mediator. In this study, we found RON was also expressed on dermal fibroblasts and investigated the effects of MSP on proliferation, migration, and collagen synthesis of fibroblasts. With the treatment of different concentrations of MSP (0, 1, 10, 20, 50, and 100 ng/mL) on fibroblasts, proliferation, migration, and collagen synthesis were analyzed by Cell Counting Kit-8 (CCK-8), transwell and real-time polymerase chain reaction. Under the treatment of MSP, the migration, Collagen I, III synthesis, and matrix metalloproteinase-1 (MMP-1) mRNA expression of fibroblasts were upregulated significantly, although there was no effect on fibroblasts proliferation, and the optimal concentration of MSP for migration and collagen synthesis was 10 ng/mL. In the in vivo study, 10 ng/mL MSP was applied to full-thickness skin wound with bacterial cellulose membranes, and this treatment could accelerate the wound healing rate and increased the collagen synthesis of wound sites. This study suggested that MSP appears to promote the migration of fibroblasts, enhances collagen synthesis and remodeling, and effectively improves wound healing.

  15. Exercise training and the control of skin blood flow in older adults.

    PubMed

    Tew, G A; Saxton, J M; Hodges, G J

    2012-03-01

    The ability to control skin blood flow decreases with primary aging, making older adults less able to adequately thermoregulate and repair cutaneous wounds. Lifestyle factors such as physical activity, diet, and smoking might interact with the aging process to modulate "normal" age-associated changes in the cutaneous microcirculation. The main focus of this brief review is the effects of exercise training on the control of skin blood flow in older adults.

  16. Murine Dermal Fibroblast Isolation by FACS.

    PubMed

    Walmsley, Graham G; Maan, Zeshaan N; Hu, Michael S; Atashroo, David A; Whittam, Alexander J; Duscher, Dominik; Tevlin, Ruth; Marecic, Owen; Lorenz, H Peter; Gurtner, Geoffrey C; Longaker, Michael T

    2016-01-07

    Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin(-)) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

  17. CopA3 peptide prevents ultraviolet-induced inhibition of type-I procollagen and induction of matrix metalloproteinase-1 in human skin fibroblasts.

    PubMed

    Kim, Dong-Hee; Kim, Han-Hyuk; Kim, Hyeon-Jeong; Jung, Hyun-Gug; Yu, Jae-Myo; Lee, Eun-Su; Cho, Yong-Hun; Kim, Dong-In; An, Bong-Jeun

    2014-05-20

    Ultraviolet (UV) exposure is well-known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1) activity. A 9-mer peptide, CopA3 (CopA3) was synthesized from a natural peptide, coprisin, which is isolated from the dung beetle Copris tripartitus. As part of our continuing search for novel bioactive natural products, CopA3 was investigated for its in vitro anti-skin photoaging activity. UV-induced inhibition of type-I procollagen and induction of MMP-1 were partially prevented in human skin fibroblasts by CopA3 peptide in a dose-dependent manner. At a concentration of 25 μM, CopA3 nearly completely inhibited MMP-1 expression. These results suggest that CopA3, an insect peptide, is a potential candidate for the prevention and treatment of skin aging.

  18. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    PubMed

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  19. Mécano-Stimulation™ of the skin improves sagging score and induces beneficial functional modification of the fibroblasts: clinical, biological, and histological evaluations

    PubMed Central

    Humbert, Philippe; Fanian, Ferial; Lihoreau, Thomas; Jeudy, Adeline; Elkhyat, Ahmed; Robin, Sophie; Courderot-Masuyer, Carol; Tauzin, Hélène; Lafforgue, Christine; Haftek, Marek

    2015-01-01

    Background Loss of mechanical tension appears to be the major factor underlying decreased collagen synthesis in aged skin. Numerous in vitro studies have shown the impact of mechanical forces on fibroblasts through mechanotransduction, which consists of the conversion of mechanical signals to biochemical responses. Such responses are characterized by the modulation of gene expression coding not only for extracellular matrix components (collagens, elastin, etc.) but also for degradation enzymes (matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). A new device providing a mechanical stimulation of the cutaneous and subcutaneous tissue has been used in a simple, blinded, controlled, and randomized study. Materials and methods Thirty subjects (aged between 35 years and 50 years), with clinical signs of skin sagging, were randomly assigned to have a treatment on hemiface. After a total of 24 sessions with Mécano-Stimulation™, biopsies were performed on the treated side and control area for in vitro analysis (dosage of hyaluronic acid, elastin, type I collagen, MMP9; equivalent dermis retraction; GlaSbox®; n=10) and electron microscopy (n=10). Furthermore, before and after the treatment, clinical evaluations and self-assessment questionnaire were done. Results In vitro analysis showed increases in hyaluronic acid, elastin, type I collagen, and MMP9 content along with an improvement of the migratory capacity of the fibroblasts on the treated side. Electron microscopy evaluations showed a clear dermal remodeling in relation with the activation of fibroblast activity. A significant improvement of different clinical signs associated with skin aging and the satisfaction of the subjects were observed, correlated with an improvement of the sagging cheek. Conclusion Mécano-Stimulation is a noninvasive and safe technique delivered by flaps microbeats at various frequencies, which can significantly improve the skin

  20. The involvement of integrin β1 signaling in the migration and myofibroblastic differentiation of skin fibroblasts on anisotropic collagen-containing nanofibers.

    PubMed

    Huang, Chengyang; Fu, Xiaoling; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2012-02-01

    Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic (i.e., fibers collected randomly with no alignment) or anisotropic (i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.

  1. UVA exposure of human skin reconstructed in vitro induces apoptosis of dermal fibroblasts: subsequent connective tissue repair and implications in photoaging.

    PubMed

    Bernerd, F; Asselineau, D

    1998-09-01

    The skin reconstructed in vitro has been previously shown to be a useful model to investigate the effects of UVB exposure (Bernerd and Asselineau, 1997). The present study describes the response to UVA irradiation. Major alterations were observed within the dermal compartment. Apoptosis of fibroblasts located in the superficial area of the dermal equivalent was observed as soon as 6 h after irradiation, leading to their disappearance after 48 h. This effect was obtained without major alterations of epidermal keratinocytes suggesting a differential cell type sensitivity to UVA radiations. In addition, collagenase I was secreted by dermal fibroblasts. The UVA dermal effects could be observed even after removal of the epidermis during the post irradiation period, demonstrating that they were independent of the keratinocyte response. The analysis of the tissue regeneration during the following 2 weeks revealed a connective tissue repair via fibroblasts proliferation, migration and active synthesis of extracellular matrix proteins such as fibronectin and procollagen I. This cellular recolonization of the superficial part of the dermal equivalent was due to activation of surviving fibroblasts located deeply in the dermal equivalent. The direct damage in the dermis and the subsequent connective tissue repair may contribute to the formation of UVA-induced dermal alterations.

  2. Rhamm−/− fibroblasts are defective in CD44-mediated ERK1,2 motogenic signaling, leading to defective skin wound repair

    PubMed Central

    Tolg, Cornelia; Hamilton, Sara R.; Nakrieko, Kerry-Ann; Kooshesh, Fatemeh; Walton, Paul; McCarthy, James B.; Bissell, Mina J.; Turley, Eva A.

    2006-01-01

    Rhamm (receptor for hyaluronan-mediated motility) is an hyaluronan binding protein with limited expression in normal tissues and high expression in advanced cancers. To understand its physiological functions and identify the molecular mechanisms underlying these functions, we created mice with a genetic deletion of Rhamm. We show that Rhamm−/− fibroblasts fail to resurface scratch wounds >3 mm or invade hyaluronan-supplemented collagen gels in culture. We identify a requirement for Rhamm in the localization of CD44 to the cell surface, formation of CD44–ERK1,2 (extracellular-regulated kinase 1,2) complexes, and activation/subcellular targeting of ERK1,2 to the cell nucleus. We also show that cell surface Rhamm, restricted to the extracellular compartment by linking recombinant protein to beads, and expression of mutant active mitogen-activated kinase kinase 1 (Mek1) are sufficient to rescue aberrant signaling through CD44–ERK1,2 complexes in Rh−/− fibroblasts. ERK1,2 activation and fibroblast migration/differentiation is also defective during repair of Rh−/− excisional skin wounds and results in aberrant granulation tissue in vivo. These results identify Rhamm as an essential regulator of CD44–ERK1,2 fibroblast motogenic signaling required for wound repair. PMID:17158951

  3. Pharmacological enhancement of beta-hexosaminidase activity in fibroblasts from adult Tay-Sachs and Sandhoff Patients.

    PubMed

    Tropak, Michael B; Reid, Stephen P; Guiral, Marianne; Withers, Stephen G; Mahuran, Don

    2004-04-02

    Tay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of beta-hexosaminidase A (alphabeta). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of approximately 5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases.

  4. Pharmacological Enhancement of β-Hexosaminidase Activity in Fibroblasts from Adult Tay-Sachs and Sandhoff Patients*

    PubMed Central

    Tropak, Michael B.; Reid, Stephen P.; Guiral, Marianne; Withers, Stephen G.; Mahuran, Don

    2010-01-01

    Tay-Sachs and Sandhoff diseases are lysosomal storage disorders that result from an inherited deficiency of β-hexosaminidase A (αβ). Whereas the acute forms are associated with a total absence of hexosaminidase A and early death, the chronic adult forms exist with activity and protein levels of ~5%, and unaffected individuals have been found with only 10% of normal levels. Surprisingly, almost all disease-associated missense mutations do not affect the active site of the enzyme but, rather, inhibit its ability to obtain and/or retain its native fold in the endoplasmic reticulum, resulting in its retention and accelerated degradation. By growing adult Tay-Sachs fibroblasts in culture medium containing known inhibitors of hexosaminidase we have raised the residual protein and activity levels of intralysosomal hexosaminidase A well above the critical 10% of normal levels. A similar effect was observed in fibroblasts from an adult Sandhoff patient. We propose that these hexosaminidase inhibitors function as pharmacological chaperones, enhancing the stability of the native conformation of the enzyme, increasing the amount of hexosaminidase A capable of exiting the endoplasmic reticulum for transport to the lysosome. Therefore, pharmacological chaperones could provide a novel approach to the treatment of adult Tay-Sachs and possibly Sandhoff diseases. PMID:14724290

  5. Nrf2 deficiency causes lipid oxidation, inflammation, and matrix-protease expression in DHA-supplemented and UVA-irradiated skin fibroblasts.

    PubMed

    Gruber, Florian; Ornelas, Cayo Mecking; Karner, Susanne; Narzt, Marie-Sophie; Nagelreiter, Ionela Mariana; Gschwandtner, Maria; Bochkov, Valery; Tschachler, Erwin

    2015-11-01

    Fish oil rich in docosahexaenoic acid (DHA) has beneficial effects on human health. Omega-3 polyunsaturated fatty acids are precursors of eicosanoids and docosanoids, signaling molecules that control inflammation and immunity, and their dietary uptake improves a range of disorders including cardiovascular diseases, ulcerative colitis, rheumatoid arthritis, and psoriasis. The unsaturated nature of these fatty acids, however, makes them prone to oxidation, especially when they are incorporated into (membrane) phospholipids. The skin is an organ strongly exposed to oxidative stress, mainly due to solar ultraviolet radiation. Thus, increased levels of PUFA in combination with oxidative stress could cause increased local generation of oxidized lipids, whose action spectrum reaches from signaling molecules to reactive carbonyl compounds that can crosslink biomolecules. Here, we investigated whether PUFA supplements to fibroblasts are incorporated into membrane phospholipids and whether an increase of PUFA within phospholipids affects the responses of the cells to UV exposure. The redox-sensitive transcription factor Nrf2 is the major regulator of the fibroblast stress response to ultraviolet radiation or exposure to oxidized lipids. Here we addressed how Nrf2 signaling would be affected in PUFA-supplemented human dermal fibroblasts and mouse dermal fibroblasts from Nrf2-deficient and wild type mice. We found, using HPLC-tandem MS, that DHA supplements to culture media of human and murine fibroblasts were readily incorporated into phospholipids and that subsequent irradiation of the supplemented cells with UVA resulted in an increase in 1-palmitoyl-2-(epoxyisoprostane-E2)-sn-glycero-3-phosphorylcholine and Oxo-DHA esterified to phospholipid, both of which are Nrf2 agonists. Also, induction of Nrf2 target genes was enhanced in the DHA-supplemented fibroblasts after UVA irradiation. In Nrf2-deficient murine fibroblasts, the expression of the target genes was, as expected

  6. Nacre-driven water-soluble factors promote wound healing of the deep burn porcine skin by recovering angiogenesis and fibroblast function.

    PubMed

    Lee, Kyunghee; Kim, Hyunsoo; Kim, Jin Man; Chung, Yeoun Ho; Lee, Tae Yoon; Lim, Hyun-Sook; Lim, Ji-Hye; Kim, Taewoon; Bae, Jin Seung; Woo, Chang-Hoon; Kim, Keuk-Jun; Jeong, Daewon

    2012-03-01

    To assess the recovery effect of water-soluble components of nacre on wound healing of burns, water-soluble nacre (WSN) was obtained from powdered nacre. Alterations to WSN-mediated wound healing characteristics were examined in porcine skin with deep second-degree burns; porcine skin was used as a proxy for human. When WSN was applied to a burned area, the burn-induced granulation sites were rapidly filled with collagen, and the damaged dermis and epidermis were restored to the appearance of normal skin. WSN enhanced wound healing recovery properties for burn-induced apoptotic and necrotic cellular damage and spurred angiogenesis. Additionally, WSN-treated murine fibroblast NIH3T3 cells showed increased proliferation and collagen synthesis. Collectively, the findings indicate that WSN improves the process of wound healing in burns by expeditiously restoring angiogenesis and fibroblast activity. WSN may be useful as a therapeutic agent, with superior biocompatibility to powdered nacre, and evoking less discomfort when applied to a wounded area.

  7. Effects of licarin E on expression of matrix metalloproteinase-1 and type-1 procollagen in UVB-irradiated human skin fibroblasts.

    PubMed

    Kwon, Yi-Young; Kim, Daeyoung; Kim, Jaekyung; Hwang, Jae-Kwan

    2011-12-01

    Ultraviolet B (UVB) radiation induces photoaging by upregulating the expression of matrix metalloproteinase (MMP) and decreasing collagen synthesis in human skin cells. This study evaluated the effects of licarin E isolated from mace, the aril of Myristica fragrans Houtt., on MMP-1 and type-1 procollagen levels in UVB-irradiated human skin fibroblasts. Powdered mace extracted with 95% ethanol was used and licarin E isolated by preparative high-performance liquid chromatography. In addition, western blot analysis, reverse transcription PCR and electrophoretic mobility shift assay were used to evaluate the effects of licarin E and its molecular mechanism. It was found that licarin E attenuated UVB-induced MMP-1 expression by inactivating mitogen-activated protein kinases (MAPKs), thereby inhibiting activator protein 1. Licarin E also increased type-1 procollagen expression by stimulating transforming growth factor β (TGFβ)/Smad signaling. The findings show that licarin E positively regulates the expression of MMP-1 and type-1 procollagen in UVB-irradiated human skin fibroblasts through MAPK and TGFβ signaling, suggesting its potential as a potent antiphotoaging agent.

  8. Effects of macelignan isolated from Myristica fragrans (Nutmeg) on expression of matrix metalloproteinase-1 and type I procollagen in UVB-irradiated human skin fibroblasts.

    PubMed

    Lee, Kyung-Eun; Mun, Sukyeong; Pyun, Hee-Bong; Kim, Myung-Suk; Hwang, Jae-Kwan

    2012-01-01

    Exposure to ultraviolet (UV) light causes premature skin aging that is associated with upregulated matrix metalloproteinases (MMPs) and decreased collagen synthesis. Macelignan, a natural lignan compound isolated from Myristica fragrans HOUTT. (nutmeg), has been reported to possess antioxidant and antiinflammatory activities. This study assessed the effects of macelignan on photoaging and investigated its mechanisms of action in UV-irradiated human skin fibroblasts (Hs68) by reverse transcription-polymerase chain reaction, Western blot analysis, 2',7'-dichlorofluorescein diacetate assay, and enzyme-linked immunosorbent assay. Our results show that macelignan attenuated UV-induced MMP-1 expression by suppressing phosphorylation of mitogen-activated protein kinases (MAPKs) induced by reactive oxygen species. Macelignan also increased type I procollagen expression and secretion through transforming growth factor β (TGF-β)/Smad signaling. These findings indicate that macelignan regulates the expression of MMP-1 and type I procollagen in UV-irradiated human skin fibroblasts by modulating MAPK and TGF-β/Smad signaling, suggesting its potential as an efficacious antiphotoaging agent.

  9. Ultraviolet irradiation induces CYR61/CCN1, a mediator of collagen homeostasis, through activation of transcription factor AP-1 in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Xu, Yiru; He, Tianyuan; Kang, Sewon; Voorhees, John J; Fisher, Gary J

    2010-06-01

    UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

  10. Role of CD14 and TLR4 in type I, type III collagen expression, synthesis and secretion in LPS-induced normal human skin fibroblasts

    PubMed Central

    Yang, Hongming; Li, Juncong; Wang, Yihe; Hu, Quan

    2015-01-01

    Objectives: The primary aim of this study was to investigate the role of CD14 and TLR4 in type I, type III collagen expression, synthesis and secretion in LPS-induced normal human skin fibroblasts. The secondary aim was to provide theoretical basis for the molecular mechanisms of scar formation induced by LPS. Methods: The normal skin fibroblasts cultured in vitro were randomly divided into four groups: 0.1 μg/mL LPS reference group, CD14 pretreatment + LPS, TLR4 pretreatment + LPS, CD14 and TLR4 pretreatment + LPS. The collagen DNA synthesis was assessed by 3H-proline incorporation method. Real-time Quantitative PCR was used to detect type I, type III collagen mRNA expression. Results: Similar results were revealed for mRNA expression levels. The immunofluorescence staining suggested that type I and type III collagen were expressed in all investigated groups and that the expression was differentially downregulated in groups B, C, D. ELISA demonstrated markedly decreased levels in secreting type I, type III collagens and hydroxyproline in groups B, C, D (P<0.05), and the lowest level was detected in group D (P<0.01). Conclusion: Pretreatment with CD14 or TLR4 alone or their combination can significantly reduce the levels of type I and type III collagen expression, synthesis and secretion, with the most notable reduction detected in case of CD14 and TLR4 combined. We could thus conclude that both CD14 and TLR4 are involved in type I and type III collagen expression, synthesis and secretion in LPS-induced skin fibroblasts. PMID:25932184

  11. A role for Nrf2 in UVA-mediated heme oxygenase induction and protection from membrane damage in human skin fibroblasts

    NASA Astrophysics Data System (ADS)

    Li, Haibin; Li, Linhao; Deng, Linhong; Singh, Gurinder; Tyrrell, Rex M.; Zhong, J. Li

    2010-11-01

    Our previous study has shown that Ultraviolet-A (UVA) irradiation induces heme oxygenase 1 (HO-1) expression in cultured human primary skin fibroblasts FEK4. In the present study, we demonstrate a coordinated induction of HO-1 and NF-E2-related factor 2 (Nrf2) following UVA irradiation or hemin treatment. The induction of HO-1 by either UVA irradiation or hemin treatment was largely abolished by down-regulation of Nrf2 with its targeted short interfering RNA (siNrf2). The study further reveals that knockdown of Nrf2 protein increased UVA-induced cell death measured by MTS assay. These findings together indicate that Nrf2-mediated induction of HO-1 expression may provide a cytoprotection for human skin cells from oxidative damage.

  12. Specific high-affinity binding of high density lipoproteins to cultured human skin fibroblasts and arterial smooth muscle cells.

    PubMed

    Biesbroeck, R; Oram, J F; Albers, J J; Bierman, E L

    1983-03-01

    Binding of human high density lipoproteins (HDL, d = 1.063-1.21) to cultured human fibroblasts and human arterial smooth muscle cells was studied using HDL subjected to heparin-agarose affinity chromatography to remove apoprotein (apo) E and B. Saturation curves for binding of apo E-free 125I-HDL showed at least two components: low-affinity nonsaturable binding and high-affinity binding that saturated at approximately 20 micrograms HDL protein/ml. Scatchard analysis of high-affinity binding of apo E-free 125I-HDL to normal fibroblasts yielded plots that were significantly linear, indicative of a single class of binding sites. Saturation curves for binding of both 125I-HDL3 (d = 1.125-1.21) and apo E-free 125I-HDL to low density lipoprotein (LDL) receptor-negative fibroblasts also showed high-affinity binding that yielded linear Scatchard plots. On a total protein basis, HDL2 (d = 1.063-1.10), HDL3 and very high density lipoproteins (VHDL, d = 1.21-1.25) competed as effectively as apo E-free HDL for binding of apo E-free 125I-HDL to normal fibroblasts. Also, HDL2, HDL3, and VHDL competed similarly for binding of 125I-HDL3 to LDL receptor-negative fibroblasts. In contrast, LDL was a weak competitor for HDL binding. These results indicate that both human fibroblasts and arterial smooth muscle cells possess specific high affinity HDL binding sites. As indicated by enhanced LDL binding and degradation and increased sterol synthesis, apo E-free HDL3 promoted cholesterol efflux from fibroblasts. These effects also saturated at HDL3 concentrations of 20 micrograms/ml, suggesting that promotion of cholesterol efflux by HDL is mediated by binding to the high-affinity cell surface sites.

  13. Two-stage implantation of the skin and bone integrated pylon (SBIP) seeded with autologous fibroblasts induced into osteoblast differentiation for direct skeletal attachment of limb prostheses

    PubMed Central

    Shevtsov, Maxim A.; Galibin, Oleg V.; Yudintceva, Nataliya M.; Blinova, Miralda I.; Pinaev, Grigoriy P.; Ivanova, Anna A.; Savchenko, Olga N.; Suslov, Dmitriy N.; Potokin, Igor L.; Pitkin, Emil; Raykhtsaum, Grigory; Pitkin, Mark R.

    2013-01-01

    Angio- and osteogenesis following the two-stage implantation of the Skin and Bone Integrated Pylon (SBIP) seeded with autologous fibroblasts was evaluated. Two consecutive animal substudies were undertaken: intramedullary subcutaneous implantation (fifteen rabbits) and a two-stage transcutaneous implantation (twelve rabbits). We observed enhanced osseointegrative properties of the intramedullary porous component seeded with fibroblasts induced into osteoblast differentiation, as compared to the untreated porous titanium pylon. The three-phase scintigraphy and subsequent histological analysis showed that the level of osteogenesis was 1.5-fold higher than in the control group, and significantly so (P<0.05). The biocompatibility was further proved by the absence of inflammatory response or encapsulation and sequestration on the histology assay. Treatment of the transcutaneous component with autologous fibroblasts was associated with nearly a 2-fold decrease in the period required for the ingrowth of dermal and subdermal soft tissues into the implant surface, as compared to the untreated porous titanium component. Direct dermal attachment to the transcutaneous implant prevented superficial and deep periprosthetic infections in rabbits in vivo. PMID:24115308

  14. Induction of 6-thioguanine-resistant mutations by rat-liver homogenate (S9)-activated promutagens in human embryonic skin fibroblasts.

    PubMed

    Chen, D J; Okinaka, R T; Strniste, G F; Barnhart, B J

    1982-03-01

    Most normal human fibroblasts grown in culture do not metabolize promutagens/procarcinogens. Thus screening assays employing normal human fibroblasts have only been successful for direct-acting chemical mutagens and various radiations. In this report we describe a mutation assay (HGPRT locus) employing a normal human embryonic skin fibroblast and a rat-liver homogenate (S9) mixture. 3 model promutagens, benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3MC), and dimethylnitrosamine (DMN) have been utilized in these studies. In addition to discussing conditions for optimizing the response of this assay, our results indicate that at constant amount of S9 protein concentration, there exists a linear correlation between mutagenicity and dose. At 50% survival, the mutant frequencies induced by B[a]P and 3MC (5 micrograms/ml) are 60 and 30 times the background mutant frequency, respectively. Similarly, at 50% survival, DMN (5 mg/ml) induced 6-TGr mutant frequencies are 25-fold over the background frequency. The increase in cytotoxicity resulting from exposure of cells to these 'activated' chemicals is also a linear dose response. At high S9 concentrations a deactivation or detoxification phenomenon occurs. However, the mutagenic efficiency of S9-activated chemicals when plotted as the number of induced mutations versus log survival is unaffected by the deactivating capacity of S9 proteins. This study demonstrates a quantitative mutation assay using an early passage human culture with an exogenous rat-liver microsomal preparation providing activating enzymes.

  15. Lymphoblastoid lines and skin fibroblasts from patients with tuberous sclerosis are abnormally sensitive to ionizing radiation and to a radiomimetic chemical

    SciTech Connect

    Scudiero, D.A.; Moshell, A.N.; Scarpinato, R.G.; Meyer, S.A.; Clatterbuck, B.E.; Tarone, R.E.; Robbins, J.H.

    1982-03-01

    Lymphoblastoid lines, derived by transforming peripheral blood lymphocytes with Epstein-Barr virus, and skin fibroblast lines were established from two patients with tuberous sclerosis. The number of viable lymphoblastoid cells was determined by their ability to exclude the vital dye trypan blue after their irradiation with x-rays or 254 nm ultraviolet light. The growth of fibroblasts was determined by their ability to form colonies after treatment with the radiomimetic, DNA-damaging chemical N-methyl-N'-nitro-N-nitrosoguanidine. The tuberous sclerosis lymphoblastoid lines were hypersensitive to x-rays but had normal sensitivity to the ultraviolet radiation. The tuberous sclerosis fibroblast lines were hypersensitive to the N-methyl-N'-nitro-N-nitrosoguanidine. The hypersensitivity of tuberous sclerosis cells to x-rays and to N-methyl-N'-nitro-N-nitrosoguanidine is believed to reflect defective repair of DNA damaged by these agents and may provide the basis for in vitro, including prenatal, diagnostic tests for tuberous sclerosis.

  16. Recombinant growth factor mixtures induce cell cycle progression and the upregulation of type I collagen in human skin fibroblasts, resulting in the acceleration of wound healing processes.

    PubMed

    Lee, Do Hyun; Choi, Kyung-Ha; Cho, Jae-We; Kim, So Young; Kwon, Tae Rin; Choi, Sun Young; Choi, Yoo Mi; Lee, Jay; Yoon, Ho Sang; Kim, Beom Joon

    2014-05-01

    Application of growth factor mixtures has been used for wound healing and anti-wrinkles agents. The aim of this study was to evaluate the effect of recombinant growth factor mixtures (RGFM) on the expression of cell cycle regulatory proteins, type I collagen, and wound healing processes of acute animal wound models. The results showed that RGFM induced increased rates of cell proliferation and cell migration of human skin fibroblasts (HSF). In addition, expression of cyclin D1, cyclin E, cyclin-dependent kinase (Cdk)4, and Cdk2 proteins was markedly increased with a growth factor mixtures treatment in fibroblasts. Expression of type I collagen was also increased in growth factor mixtures-treated HSF. Moreover, growth factor mixtures-induced the upregulation of type I collagen was associated with the activation of Smad2/3. In the animal model, RGFM-treated mice showed accelerated wound closure, with the closure rate increasing as early as on day 7, as well as re-epithelization and reduced inflammatory cell infiltration than phosphate-buffered saline (PBS)-treated mice. In conclusion, the results indicated that RGFM has the potential to accelerate wound healing through the upregulation of type I collagen, which is partly mediated by activation of Smad2/3-dependent signaling pathway as well as cell cycle progression in HSF. The topical application of growth factor mixtures to acute and chronic skin wound may accelerate the epithelization process through these molecular mechanisms.

  17. Studies of DNA and chromosome damage in skin fibroblasts and blood lymphocytes from psoriasis patients treated with 8-methoxypsoralen and UVA irradiation

    SciTech Connect

    Bredberg, A.; Lambert, B.; Lindblad, A.; Swanbeck, G.; Wennersten, G.

    1983-08-01

    Exposure of human lymphocytes and skin fibroblasts in vitro to a single, clinically used dose of PUVA, i.e., 0.1 micrograms/ml of 8-methoxypsoralen (8-MOP) plus 0.9-4 J/cm2 of longwave ultraviolet radiation (UVA), lead to the formation of DNA damage as determined by alkaline elution, and to chromosome aberrations and sister chromatid exchanges (SCE). When lymphocyte-enriched plasma was obtained from psoriasis patients 2 h after oral intake of 8-MOP and then UVA irradiated (1.8-3.6 J/cm2) in vitro, an increased frequency of chromosome aberrations and SCE was observed. Normal levels of chromosome aberrations and SCE were found in lymphocytes of psoriasis patients after 3-30 weeks of PUVA treatment in vivo. A small but statistically significant increase in the SCE frequency was observed in the lymphocytes of psoriasis patients treated for 1-6 years with PUVA (mean 18.0 SCE/cell) as compared with before PUVA (mean 15.8, p less than 0.05). Skin fibroblasts of psoriasis patients analyzed 5 years after the start of PUVA treatment showed a normal number of SCE but a high fraction of filter-retained DNA in the alkaline elution assay, suggesting the presence of cross-linked DNA.

  18. Nrf2 and NF-κB Signaling Pathways Contribute to Porphyra-334-Mediated Inhibition of UVA-Induced Inflammation in Skin Fibroblasts.

    PubMed

    Ryu, Jina; Kwon, Mi-Jin; Nam, Taek-Jeong

    2015-07-31

    In this study, we examined the protective effects of porphyra-334 against UVA-irradiated cellular damage and elucidated the underlying mechanisms. Porphyra-334 prevented UVA-induced cell death and exhibited scavenging activities against intracellular oxidative stress induced by UVA irradiation in skin fibroblasts. We found that porphyra-334 significantly reduced the secretion and expression of IL-6 and TNF-α, reduced nuclear expression of Nuclear factor-κB (NF-κB), and sustained NF-E2-related factor 2 (Nrf2) activation. Further mechanism research revealed that porphyra-334 promoted the Nrf2 signaling pathway in UVA-irradiated skin fibroblasts. Our results show that the antioxidant effect of porphyra-334 is due to the direct scavenging of oxidative stress and its inhibitory effects on NF-κB-dependent inflammatory genes, such as IL-6 and TNF-κ. Therefore, we hypothesize that boosting the Nrf2- NF-κB-dependent response to counteract environmental stress is a promising strategy for the prevention of UVA-related damage.

  19. Effect of olive and sunflower seed oil on the adult skin barrier: implications for neonatal skin care.

    PubMed

    Danby, Simon G; AlEnezi, Tareq; Sultan, Amani; Lavender, Tina; Chittock, John; Brown, Kirsty; Cork, Michael J

    2013-01-01

    Natural oils are advocated and used throughout the world as part of neonatal skin care, but there is an absence of evidence to support this practice. The goal of the current study was to ascertain the effect of olive oil and sunflower seed oil on the biophysical properties of the skin. Nineteen adult volunteers with and without a history of atopic dermatitis were recruited into two randomized forearm-controlled mechanistic studies. The first cohort applied six drops of olive oil to one forearm twice daily for 5 weeks. The second cohort applied six drops of olive oil to one forearm and six drops of sunflower seed oil to the other twice daily for 4 weeks. The effect of the treatments was evaluated by determining stratum corneum integrity and cohesion, intercorneocyte cohesion, moisturization, skin-surface pH, and erythema. Topical application of olive oil for 4 weeks caused a significant reduction in stratum corneum integrity and induced mild erythema in volunteers with and without a history of atopic dermatitis. Sunflower seed oil preserved stratum corneum integrity, did not cause erythema, and improved hydration in the same volunteers. In contrast to sunflower seed oil, topical treatment with olive oil significantly damages the skin barrier, and therefore has the potential to promote the development of, and exacerbate existing, atopic dermatitis. The use of olive oil for the treatment of dry skin and infant massage should therefore be discouraged. These findings challenge the unfounded belief that all natural oils are beneficial for the skin and highlight the need for further research.

  20. Sun exposure, skin color and vitamin D status in Arab children and adults.

    PubMed

    Al-Daghri, Nasser M; Al-Saleh, Yousef; Khan, Nasiruddin; Sabico, Shaun; Aljohani, Naji; Alfawaz, Hanan; Alsulaimani, Maha; Al-Othman, Abdulaziz M; Alokail, Majed S

    2016-11-01

    Accumulating evidence suggests an increased prevalence of vitamin D deficiency in the Middle East and North African countries. Sunlight has long been recognized as a major provider of vitamin D and lighter skin color has been associated with better vitamin D status. In this context, we aimed to determine whether 25-hydroxyvitamin D[25-(OH)D] concentrations are related to skin color, sun exposure and gender among healthy Saudi children and adults. A total of 808 Saudi children (age=14.6±0.04years) and 561 (age=31.4±0.3years) adults of both genders were included in this study. Levels of sun exposure and skin color were determined using a standard questionnaire. Anthropometry and plasma 25-(OH)D concentrations were analyzed. On the basis of duration of sun exposure (<20min vs. >20min), a significantly lower concentration of 25-(OH)D (40.9±1.2 vs. 35.5±1.8nmol/l; p<0.019) was demonstrated in dark-skinned boys with exposure time less than 20min than those exposed longer than 20min. We were unable to show an effect of sunlight exposure or skin color on vitamin D status of children or adults, except in dark-skinned boys who had lower 25(OH)D concentrations associated with limited sun exposure.

  1. Zinc l-pyrrolidone carboxylate inhibits the UVA-induced production of matrix metalloproteinase-1 by in vitro cultured skin fibroblasts, whereas it enhances their collagen synthesis.

    PubMed

    Takino, Y; Okura, F; Kitazawa, M; Iwasaki, K; Tagami, H

    2012-02-01

    Reduced collagen matrix in the dermis constitutes one of the characteristic features of chronologically aged skin, which is further enhanced on the sun-exposed portions of the body by chronic ultraviolet light (UV) irradiation, inducing the unique changes associated with skin photoageing. The zinc salt of l-pyrrolidone carboxylate (Zinc PCA) has long been used as a cosmetic ingredient, because of its astringent and anti-microbial properties. In the present study, by employing cultured normal human dermal fibroblasts, we found that Zinc PCA suppressed UVA-induced activation of activator protein-1 (AP-1) and reduced matrix metalloproteinase-1 production in these cells, which is thought to be involved in collagen degradation in photoaged skin. Moreover, Zinc PCA treatment of the cells increased the expression of an ascorbic acid transporter mRNA, SVCT2, but not SVCT1, resulting in the enhanced production of type I collagen. Based on these in vitro findings, we consider Zinc PCA to be a promising candidate for an anti-skin ageing agent.

  2. Skin fibroblasts from pantothenate kinase-associated neurodegeneration patients show altered cellular oxidative status and have defective iron-handling properties.

    PubMed

    Campanella, Alessandro; Privitera, Daniela; Guaraldo, Michela; Rovelli, Elisabetta; Barzaghi, Chiara; Garavaglia, Barbara; Santambrogio, Paolo; Cozzi, Anna; Levi, Sonia

    2012-09-15

    Pantothenate kinase-associated neurodegeneration (PKAN) is a neurodegenerative disease belonging to the group of neurodegeneration with brain iron accumulation disorders. It is characterized by progressive impairments in movement, speech and cognition. The disease is inherited in a recessive manner due to mutations in the Pantothenate Kinase-2 (PANK2) gene that encodes a mitochondrial protein involved in Coenzyme A synthesis. To investigate the link between a PANK2 gene defect and iron accumulation, we analyzed primary skin fibroblasts from three PKAN patients and three unaffected subjects. The oxidative status of the cells and their ability to respond to iron were analyzed in both basal and iron supplementation conditions. In basal conditions, PKAN fibroblasts show an increase in carbonylated proteins and altered expression of antioxidant enzymes with respect to the controls. After iron supplementation, the PKAN fibroblasts had a defective response to the additional iron. Under these conditions, ferritins were up-regulated and Transferrin Receptor 1 (TfR1) was down-regulated to a minor extent in patients compared with the controls. Analysis of iron regulatory proteins (IRPs) reveals that, with respect to the controls, PKAN fibroblasts have a reduced amount of membrane-associated mRNA-bound IRP1, which responds imperfectly to iron. This accounts for the defective expression of ferritin and TfR1 in patients' cells. The inaccurate quantity of these proteins produced a higher bioactive labile iron pool and consequently increased iron-dependent reactive oxygen species formation. Our results suggest that Pank2 deficiency promotes an increased oxidative status that is further enhanced by the addition of iron, potentially causing damage in cells.

  3. Substance P induces inflammatory responses involving NF-κB in genetically diabetic mice skin fibroblasts co-cultured with macrophages

    PubMed Central

    Ni, Tao; Liu, Yushu; Peng, Yinbo; Li, Ming; Fang, Yong; Yao, Min

    2016-01-01

    Purpose: Delayed wound healing is an intractable complex of diabetes and substance P (SP) is proved to benefit wound healing, whose functioning mechanism remains elusive. This study aims at revealing whether the influence of SP on diabetic wound healing is dependent on inflammatory responses, particularly NF-κB. Methods: Skin fibroblasts of genetically diabetic mice were co-cultured with bone marrow-derived macrophages, and treated with SP, SP + L703,606 (a neurokinin-1 receptor antagonist), or SP + MG132 (an inhibitor of NF-κB). For macrophages, their migration ability was assessed by Transwell experiments, and their M2 polarization was analyzed by flow cytometry and markers for M2 phenotype. Pro-inflammatory factors in the supernatant were detected by enzyme-linked immunosorbent assay. In fibroblasts, the transcription levels of the four pro-inflammatory factors and the protein levels of NF-κB regulators like inhibitor of NF-κB alpha (IκBα) and IκB kinases (IKKs) were monitored by real-time quantitative PCR and western blot, respectively. Results: SP could significantly induce migration to fibroblasts (P<0.01), M2 polarization (P<0.001) and pro-inflammatory factor concentration (P<0.01) in the co-culture system. It also promotes the transcription process of pro-inflammatory factors in fibroblasts (P<0.01), and induce activation of IKKα/β and phosphorylation of IκBα, which caused NF-κB activation. All these effects were reversed if NF-κB was inhibited. Conclusion: The promoting effects of SP on diabetic wound healing was dependent on enhanced inflammatory responses, especially the activation of NF-κB. This study provided evidence for the potential usage of SP in accelerating diabetic wound healing. PMID:27347325

  4. Association of skin color, race/ethnicity, and hearing loss among adults in the USA.

    PubMed

    Lin, Frank R; Maas, Paige; Chien, Wade; Carey, John P; Ferrucci, Luigi; Thorpe, Roland

    2012-02-01

    Epidemiologic studies of hearing loss in adults have demonstrated that the odds of hearing loss are substantially lower in black than in white individuals. The basis of this association is unknown. We hypothesized that skin pigmentation as a marker of melanocytic functioning mediates this observed association and that skin pigmentation is associated with hearing loss independent of race/ethnicity. We analyzed cross-sectional data from 1,258 adults (20-59 years) in the 2003-2004 cycle of the National Health and Nutritional Examination Survey who had assessment of Fitzpatrick skin type and pure-tone audiometric testing. Audiometric thresholds in the worse hearing ear were used to calculate speech- (0.5-4 kHz) and high-frequency (3-8 kHz) pure-tone averages (PTA). Regression models were stratified by Fitzpatrick skin type or race/ethnicity to examine the association of each factor with hearing loss independent of the other. Models were adjusted for potential confounders (demographic, medical, and noise exposure covariates). Among all participants, race/ethnicity was associated with hearing thresholds (black participants with the best hearing followed by Hispanics and then white individuals), but these associations were not significant in analyses stratified by skin color. In contrast, in race-stratified analyses, darker-skinned Hispanics had better hearing than lighter-skinned Hispanics by an average of -2.5 dB hearing level (HL; 95% CI, -4.8 to -0.2) and -3.1 dB HL (95% CI, -5.3 to -0.8) for speech and high-frequency PTA, respectively. Associations between skin color and hearing loss were not significant in white and black participants. Our results demonstrate that skin pigmentation is independently associated with hearing loss in Hispanics and suggest that skin pigmentation as a marker of melanocytic functioning may mediate the strong association observed between race/ethnicity and hearing loss.

  5. Expression of gelatinase A and TIMP-2 mRNAs in desmoplastic fibroblasts in both mammary carcinomas and basal cell carcinomas of the skin.

    PubMed Central

    Poulsom, R; Hanby, A M; Pignatelli, M; Jeffery, R E; Longcroft, J M; Rogers, L; Stamp, G W

    1993-01-01

    AIMS--To compare the localisation of mRNAs for the basement membrane degrading enzyme gelatinase A (72 kilodalton type IV collagenase) and its inhibitor TIMP-2 in carcinomas of the breast and basal cell carcinomas of the skin which have little or no ability to metastasize. METHODS--In situ hybridisation was performed on formalin fixed, paraffin wax embedded blocks using 35S-labelled riboprobes on 16 mammary carcinomas, three fibroadenomas, and a benign phyllodes tumour, and on 15 basal cell carcinomas of the skin (BCC). RESULTS--Labelling for both mRNAs was detectable in 14 of 16 mammary carcinomas and in 13 of 15 BCC, most often over organising desmoplastic fibroblasts in the stroma around invasive epithelial aggregates. Some sparse labelling was seen over malignant epithelial cells in six of the mammary carcinomas but not in the BCC. Some expression of gelatinase A mRNA was also seen in fibroblasts of breast lobules adjacent to the mammary carcinomas and around engulfed adnexal elements in the BCC, but not in unaffected breast tissues, fibroadenomas, the phyllodes tumour or unaffected skin. CONCLUSIONS--Maximal expression of gelatinase A and TIMP-2 mRNAs occurs in malignant neoplasms as part of the host response to the presence of established neoplastic cells rather than as an initial response to invasion. The degree to which this is present suggests this may be a highly relevant mechanism modulating tumour differentiation, growth and progression, possibly entailing uptake via specific receptors on the tumour cell surface. Images PMID:8391548

  6. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    PubMed

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  7. Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats

    PubMed Central

    Li, Na; Luo, Heng-Cong; Yang, Chuan; Deng, Jun-Jie; Ren, Meng; Xie, Xiao-Ying; Lin, Diao-Zhu; Yan, Li; Zhang, Li-Ming

    2014-01-01

    Background Excessive expression of matrix metalloproteinase-9 (MMP-9) is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of β-cyclodextrin (β-CD) core and poly(amidoamine) dendron arms (β-CD-[D3]7) could be used as the gene carrier of small interfering RNA (siRNA) to reduce MMP-9 expression for enhanced diabetic wound healing. Methods The cytotoxicity of β-CD-(D3)7 was investigated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MMT) method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of β-CD-(D3)7/MMP-9-small interfering RNA (siRNA) complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT) polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by β-CD-(D3)7/MMP-9-siRNA complexes. The β-CD-(D3)7/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. Results β-CD-(D3)7 exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The β-CD-(D3)7/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01). Animal experiments revealed that the treatment by β-CD-(D3)7/MMP-9-siRNA complexes enhanced wound closure in diabetic rats on day 7 post-wounding (P<0.05). Conclusion β-CD-(D3)7 may be used as an efficient carrier for the delivery of MMP-9-siRNA to reduce MMP-9 expression in skin fibroblast cells and promote wound healing in diabetic rats. PMID:25075185

  8. Protective and restorative effects of a Commiphora mukul gum resin and triheptanoin preparation on the CCL-110 skin fibroblast cell line.

    PubMed

    Ramachandran, C; Quirin, K-W; Resek, A; Melnick, S J

    2012-04-01

    Coenzyme Q10 (CoQ10) is a major ingredient in skin care products because of its anti-wrinkle effects, although it has some side effects especially at higher amounts. In this study, we compare the anti-wrinkle related properties of CoQ10 and a proprietary Commiphora mukul gum resin (guggul) and triheptanoin preparation (GU-TC7). GU-TC7 is prepared with a supercritical CO₂-co-solvent extraction with ethanol, standardized to 2% guggulsterones and triheptanoin, a triglyceride composed of three 7-carbon fatty acids. Treatment of CCL-110 skin fibroblasts with GU-TC7 demonstrates a mild proliferative effect compared to CoQ10 and increased type I collagen synthesis. Additionally, GU-TC7 inhibited matrix metalloproteinase-1 (MMP-1) expression in a dose-dependent manner at 20-100 μg mL⁻¹ and inhibited human elastase expression by more than 50% as compared to no elastase inhibition with CoQ10 treatment. These results suggest that GU-TC7 possesses properties that are applicable to the treatment of wrinkles and may be considered for its further evaluation in skin care products.

  9. Antiageing Mechanisms of a Standardized Supercritical CO2 Preparation of Black Jack (Bidens pilosa L.) in Human Fibroblasts and Skin Fragments

    PubMed Central

    Dieamant, Gustavo; Pereda, Maria Del Carmen V.; Nogueira, Cecília; Eberlin, Samara; Facchini, Gustavo; Checon, Juliana Tibério; Cesar, Camila Kappke; Mussi, Lilian; Polezel, Márcio Antonio; Martins-Oliveira, Divino; Di Stasi, Luiz Claudio

    2015-01-01

    The use of topical retinoids to treat skin disorders and ageing can induce local reactions, while oral retinoids are potent teratogens and produce several unwanted effects. This way, efforts to explore complementary care resources should be supported. Based on this, we evaluate the antiageing effects of a supercritical CO2 extract from Bidens pilosa L. (BPE-CO2A) containing a standardized multicomponent mixture of phytol, linolenic, palmitic, linoleic, and oleic acids. BPE-CO2A was assessed for its effects on human dermal fibroblasts (TGF-β1 and FGF levels using ELISA; collagen, elastin, and glycosaminoglycan by colorimetric assays, and mRNA expression of RXR, RAR, and EGFr by qRT-PCR) and human skin fragments (RAR, RXR, collagen, elastin, and glycosaminoglycan by immunohistochemical analysis). Levels of extracellular matrix elements, TGF-β1 and FGF, and EGFr gene expression were significantly increased by BPE-CO2A. The modulation of RXR and RAR was positively demonstrated after the treatment with BPE-CO2A or phytol, a component of BPE-CO2A. The effects produced by BPE-CO2A were similar to or better than those produced by retinol and retinoic acid. The ability to stimulate extracellular matrix elements, increase growth factors, and modulate retinoid and rexinoid receptors provides a basis for the development of preparation containing BPE-CO2A as an antiageing/skin-repair agent. PMID:25883669

  10. Factitious disorders in adults: two cases of unusual skin ulcers.

    PubMed

    Giuliodori, Katia; Campanati, Anna; Rosa, Laura; Marconi, Barbara; Cellini, Andreina; Brandozzi, Giuliano; Ganzetti, Giulia; Simoncini, Annalisa; Offidani, Annamaria

    2014-03-01

    The factitious disorders (FDs) include a large group of dermatological conditions characterized by skin lesions that patients themselves self-inflict but deny their active participation. FDs usually represent a diagnostic challenge, and their therapeutic management is usually based on a multidisciplinary approach because most FRD patients also suffer from undiagnosed behavior and personality disorders. We report two cases of FD: one patient that was suffering from severe depression and induced third-degree chemical burns on his own lower limbs, and another patient affected by obsessive-compulsive disorder and anorexia nervosa that presented with deep self-inflicted ulcers on her face. Our multispecialist approach, based on close cooperation between dermatologists and psychiatrists, led to a significant improvement of clinical conditions in both cases. Dermatologists need to be aware that FDs are complex disorders and a multidisciplinary approach is usually recommended to control their clinical course.

  11. Sox2-mediated regulation of adult neural crest precursors and skin repair.

    PubMed

    Johnston, Adam P W; Naska, Sibel; Jones, Karen; Jinno, Hiroyuki; Kaplan, David R; Miller, Freda D

    2013-01-01

    Nerve-derived neural crest cells are essential for regeneration in certain animals, such as newts. Here, we asked whether they play a similar role during mammalian tissue repair, focusing on Sox2-positive neural crest precursors in skin. In adult skin, Sox2 was expressed in nerve-terminal-associated neural crest precursor cells (NCPCs) around the hair follicle bulge, and following injury was induced in nerve-derived cells, likely dedifferentiated Schwann cell precursors. At later times postinjury, Sox2-positive cells were scattered throughout the regenerating dermis, and lineage tracing showed that these were all neural-crest-derived NCPCs. These Sox2-positive NCPCs were functionally important, since acute deletion of Sox2 prior to injury caused a decrease of NCPCs in the wound and aberrant skin repair. These data demonstrate that Sox2 regulates skin repair, likely by controlling NCPCs, and raise the possibility that nerve-derived NCPCs may play a general role in mammalian tissue repair.

  12. Generation and Characterization of Leukemia Inhibitory Factor-Dependent Equine Induced Pluripotent Stem Cells from Adult Dermal Fibroblasts

    PubMed Central

    Ovchinnikov, Dmitry A.; Sun, Jane; Fortuna, Patrick R.J.; Wolvetang, Ernst J.

    2014-01-01

    In this study we have reprogrammed dermal fibroblasts from an adult female horse into equine induced pluripotent stem cells (equiPSCs). These equiPSCs are dependent only on leukemia inhibitory factor (LIF), placing them in striking contrast to previously derived equiPSCs that have been shown to be co-dependent on both LIF and basic fibroblast growth factor (bFGF). These equiPSCs have a normal karyotype and have been maintained beyond 60 passages. They possess alkaline phosphatase activity and express eqNANOG, eqOCT4, and eqTERT mRNA. Immunocytochemistry confirmed that they produce NANOG, REX1, SSEA4, TRA1-60, and TRA1-81. While our equiPSCs are LIF dependent, bFGF co-stimulates their proliferation via the PI3K/AKT pathway. EquiPSCs lack expression of eqXIST and immunostaining for H3K27me3, suggesting that during reprogramming the inactive X chromosome has likely been reactivated to generate cells that have two active X chromosomes. EquiPSCs form embryoid bodies and in vitro teratomas that contain derivatives of all three germ layers. These LIF-dependent equiPSCs likely reflect a more naive state of pluripotency than equiPSCs that are co-dependent on both LIF and bFGF and so provide a novel resource for understanding pluripotency in the horse. PMID:24555755

  13. Prebiotic effects of almonds and almond skins on intestinal microbiota in healthy adult humans.

    PubMed

    Liu, Zhibin; Lin, Xiuchun; Huang, Guangwei; Zhang, Wen; Rao, Pingfan; Ni, Li

    2014-04-01

    Almonds and almond skins are rich in fiber and other components that have potential prebiotic properties. In this study we investigated the prebiotic effects of almond and almond skin intake in healthy humans. A total of 48 healthy adult volunteers consumed a daily dose of roasted almonds (56 g), almond skins (10 g), or commercial fructooligosaccharides (8 g) (as positive control) for 6 weeks. Fecal samples were collected at defined time points and analyzed for microbiota composition and selected indicators of microbial activity. Different strains of intestinal bacteria had varying degrees of growth sensitivity to almonds or almond skins. Significant increases in the populations of Bifidobacterium spp. and Lactobacillus spp. were observed in fecal samples as a consequence of almond or almond skin supplementation. However, the populations of Escherichia coli did not change significantly, while the growth of the pathogen Clostridum perfringens was significantly repressed. Modification of the intestinal microbiota composition induced changes in bacterial enzyme activities, specifically a significant increase in fecal β-galactosidase activity and decreases in fecal β-glucuronidase, nitroreductase and azoreductase activities. Our observations suggest that almond and almond skin ingestion may lead to an improvement in the intestinal microbiota profile and a modification of the intestinal bacterial activities, which would induce the promotion of health beneficial factors and the inhibition of harmful factors. Thus we believe that almonds and almond skins possess potential prebiotic properties.

  14. UVA light stimulates the production of cathepsin G and elastase-like enzymes by dermal fibroblasts: a possible contribution to the remodeling of elastotic areas in sun-damaged skin.

    PubMed

    Cavarra, Eleonora; Fimiani, Michele; Lungarella, Giuseppe; Andreassi, Lucio; de Santi, Margherita; Mazzatenta, Carlo; Ciccoli, Lucia

    2002-01-01

    Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.

  15. Synthesis and protective effects of bis{4-[N,N-di-(carboxymethyl)amino]phenoxy}alkane derivatives on UVA-induced production of MMP-1 in human skin fibroblasts.

    PubMed

    Hsu, Ling-Yih; Nien, Chih-Ying; Huang, Wei-Ming; Hsu, Shou-Che; Chang, Tsu-Chung

    2014-01-01

    UV-induced matrix metalloproteinase (MMP) production is considered a cause of skin aging. In this study, a number of novel bis{4-[N,N-di-(carboxymethyl)amino]phenoxy}alkane derivatives were synthesized and evaluated as UVA-protective agents. These compounds significantly protected human dermal fibroblast (HDF) cells from UVA-induced cytotoxicity and inhibited MMP-1 activation and expression with potency comparable to desferoxamine (DFO). Promoter activity assay indicated that they inhibited MMP-1 expression at the transcriptional level. Further studies revealed that the mechanism of these compounds may include blockage of the UVA-induced activation of the p38/mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. Together, these results suggest that further development of these compounds may be of interest.

  16. The surface glycoproteins of human skin fibroblasts detected after electrophoresis by the binding of peanut (Arachis hypogaea) agglutinin and Ricinus communis (castor-bean) agglutinin I.

    PubMed

    Gordon, B B; Pena, S D

    1982-11-15

    A new methodology was developed to study the cell-surface glycoproteins of cultured human skin fibroblasts. This was based on the binding of a variety of biotinyl-lectins to nitrocellulose electrophoretic transfers of total fibroblast lysates after separation in sodium dodecyl sulphate/polyacrylamide gels, followed by reaction with avidin-biotinyl-peroxidase complexes and detection with 3,3'-diaminobenzidine. The technique proved to be very sensitive and a large number of glycoproteins were detected by binding of concanavalin A and wheat-germ agglutinin. Binding of peanut agglutinin and to a lesser extent of Ricinus communis agglutinin I were found to be dependent on prior removal of sialic acid residues from the glycoproteins. Since by treatment of intact viable cells with neuraminidase only external sialic acid residues were removed, peanut agglutinin and Ricinus communis agglutinin I could thus be utilized for selective detection of cell-surface glycoproteins. Also, because peanut agglutinin was known to bind preferentially to oligosaccharides of the O-glycosidic type, and Ricinus communis agglutinin I to those of the N-glycosidic type, the two lectins were complementary in displaying the surface glycoproteins and in providing information about their oligosaccharide composition.

  17. Comparison of protective effect of ascorbic acid on redox and endocannabinoid systems interactions in in vitro cultured human skin fibroblasts exposed to UV radiation and hydrogen peroxide.

    PubMed

    Gęgotek, Agnieszka; Bielawska, Katarzyna; Biernacki, Michał; Zaręba, Ilona; Surażyński, Arkadiusz; Skrzydlewska, Elżbieta

    2017-03-11

    The mechanisms of biological activity of commonly used natural compounds are constantly examined. Therefore, the aim of this study was to compare ascorbic acid efficacy in counteracting the consequences of UV and hydrogen peroxide treatment on lipid mediators and their regulative action on antioxidant abilities. Skin fibroblasts exposed to UVA and UVB irradiation, treated with hydrogen peroxide and ascorbic acid. The redox system was estimated through reactive oxygen species (ROS) generation (electron spin resonance spectrometer) and antioxidants level/activity (HPLC/spectrometry) which activity was evaluated by the level of phospholipid metabolites: 4-hydroxynonenal, malondialdehyde, 8-isoprostanes and endocannabinoids (GC/LC-MS) in the human skin fibroblasts. Protein and DNA oxidative modifications were also determined (LC). The expression of nuclear factor erythroid 2-related factor 2 (Nrf2), its activators and inhibitors as well as pro/anti-apoptotic proteins and endocannabinoid receptors was examined (Western blot) and collagen metabolism was evaluated by collagen biosynthesis and prolidase activity (spectrometry). UVA and UVB irradiation and hydrogen peroxide treatment enhanced activity of xanthine and NADPH oxidases resulting in ROS generation as well as diminution of antioxidant phospholipid protection (glutathione peroxidase-glutathione-vitamin E), what led to increased lipid peroxidation and decreased endocannabinoids level. Dysregulation of cannabinoid receptors expression and environment of transcription factor Nrf2 caused apoptosis induction. Ascorbic acid partially prevented ROS generation, antioxidant capacity diminution and endocannabinoid systems disturbances but only slightly protected macromolecules such as phospholipid, protein and DNA against oxidative modifications. However, ascorbic acid significantly prevented decrease in collagen type I biosynthesis. Ascorbic acid in similar degree prevents UV (UVA and UVB) and hydrogen peroxide

  18. Nanoscale gelatinase A (MMP-2) inhibition on human skin fibroblasts of Longkong (Lansium domesticum Correa) leaf extracts for anti-aging.

    PubMed

    Manosroi, Aranya; Kumguan, Kulthida; Chankhampan, Charinya; Manosroi, Worapaka; Manosroi, Jiradej

    2012-09-01

    Leaves of Longkong which collected from Chantaburi in Thailand were extracted by the hot and cold processes using three different solvents including water, chloroform and methanol. The crude extracts were tested for antioxidative activities, tyrosinase inhibition and in vitro cytotoxicity as well as the MMP-2 inhibition activity on human skin fibroblasts for anti-aging evaluation. The hot water crude extract showed the highest antioxidative activities (DPPH radical scavenging, metal ion chelating and lipid peroxidation inhibition) with the SC50, CC50 and IPC50 values of 5.40 +/- 1.23, 32.31 +/- 0.84 and 3.29 +/- 0.30 mg/ml, respectively, and the highest tyrosinase inhibition activity with the IC50 value of 0.49 +/- 0.23 mg/ml. The extract also showed no cytotoxicity on human skin fibroblasts with the cell viability of 80.52 +/- 15.16%. It demonstrated the anti-aging potential by having the pro and active MMP-2 inhibition activity, but lower than ascorbic acid of 1.28 and 1.12 times, respectively. The semi-purified extracts were prepared from this crude extract by solvent-solvent partition. The ethyl acetate soluble fraction showed higher activities (DPPH radical scavenging, metal ion chelating and tyrosinase inhibition) than the crude extract of 23.48, 71.80 and 2.58 times, respectively. This fraction exhibited similar pro and active MMP-2 inhibitory effect to the crude extract. The results from this study have indicated the possible application of the ethyl acetate fraction of the hot water crude extract from leaves of Longkong to be developed as an anti-aging product.

  19. H2O2 treatment or serum deprivation induces autophagy and apoptosis in naked mole-rat skin fibroblasts by inhibiting the PI3K/Akt signaling pathway.

    PubMed

    Zhao, Shanmin; Li, Li; Wang, Shiyong; Yu, Chenlin; Xiao, Bang; Lin, Lifang; Cong, Wei; Cheng, Jishuai; Yang, Wenjing; Sun, Wei; Cui, Shufang

    2016-12-20

    Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.

  20. Ultrastructural analysis between fetal and adult wound healing process of marsupial opossum skin.

    PubMed

    Matsuno, Kei; Ihara, Setsunosuke

    2015-01-01

    The opossum delivers a newborn baby equivalent to tremature fetus state by postpregnancy. The peculiarity is advantageous for studies of fetus, because operations to take out fetus from the uterus of a mother are not necessary. When mammalian skin is wounded by full-thickness excision, fetal and adult wound healing processes differ. Fetal-type wound healing does not leave a scar. However, studies of how the fetal wound healing process differs in detail from the adult type are not advanced. We first observed the normal skin development of the gray short-tailed opossum (Monodelphis domestica) using an electron microscope. As for normal skin, an epidermis became multi-layered, and thickened from birth through to 7 days after birth. The quantity of extracellular matrix of the dermis increased thereafter, and several types of cells were found in the dermis. To examine the wound healing, we used material from a 1 day-old newborn baby, and from another 15 days after birth, and compared the wound healing style morphologically. Differences in the constitution of cells and fine structures of the skin were observed, it was obviously suggested that change in the wound healing style from fetal-type to adult-type occurred between 1 to 15 days after birth.

  1. Development of an Internet Intervention to Address Behaviors Associated with Skin Cancer Risk among Young Adults

    PubMed Central

    Heckman, Carolyn; Darlow, Susan; Munshi, Teja; Caruso, Carolyn; Ritterband, Lee; Raivitch, Stephanie; Fleisher, Linda; Manne, Sharon

    2015-01-01

    Purpose Skin cancer is the most common cancer in the US, and its incidence is increasing. The major risk factor for skin cancer is exposure to ultraviolet radiation (UV). Young adults tend to expose themselves to large amounts of UV and engage in minimal skin protection, which increases their skin cancer risk. Interventions are needed to address risk behaviors among young adults that may lead to skin cancer. The nternet offers a cost-effective way to widely disseminate efficacious interventions. The current paper describes the development of an online skin cancer risk reduction intervention (UV4.me) for young adults. Procedures The iterative development process for UV4.me followed best-practice guidelines and included the following activities: individual interviews, focus groups, content development by the expert team, acceptability testing, cognitive interviewing for questionnaires, quality control testing, usability testing, and a pilot randomized controlled trial. Participant acceptability and usability feedback was assessed. Principal Results The development process produced an evidence-informed intervention that is individually-tailored, interactive, and multimedia in nature based on the Integrative Model of Behavior Prediction, a model for internet interventions, and other best-practice recommendations, expert input, as well as user acceptability and usability feedback gathered before, during, and after development. Major Conclusions Development of an acceptable intervention intended to have a significant public health impact requires a relatively large investment in time, money, expertise, and ongoing user input. Lessons learned and recommendations are discussed. The comprehensive process used may help prepare others interested in creating similar behavioral health interventions. PMID:26640776

  2. Studies on the conservation of epidermal specificies of skin and certain mucosas in adult mammals.

    PubMed

    Billingham, R E; Silvers, W K

    1967-03-01

    To determine whether the factor(s) responsible for the conservation of epidermal specificities in adult guinea pigs and hamsters resides in the germinal layer of the epidermis or in the dermis, thin grafts of skin, possessing qualitatively distinct regional characteristics, were separated into their superficial epidermal and dermal components with the aid of trypsin. Dermis of one type was combined with epidermis of another to produce "recombinant" grafts which were then transplanted to small, full thickness cutaneous sites on the thorax of geneticaily compatible hosts. A variant of this procedure involved transplanting sheets of superficial epidermis of various types to shallow split thickness recipient areas in the skin of the thorax. All grafts were maintained for 100 days before they were excised and examined histologically. The results indicate that, whereas the dermis determines the kind of epidermis produced in recombinant grafts involving the ear, the sole of the foot, and the trunk, this is not the case in recombinants which include tongue, esophageal, or cheek pouch epithelia. The one exception to this occurred when tongue or esophagus epithelia were transplanted to split thickness beds in trunk skin. Here they appeared to produce an epidermis characteristic of their new location. It is believed that this exception is probably due to the fact that the native follicular epidermis present in trunk dermis made such a substantial contribution to the new superficial epidermis that it behaved overtly as body skin epidermis. Taken together, these results suggest that basal layer cells of the superficial epidermis of sole of foot skin, ear skin, and the hair-bearing skin of the general integument behave as if they are equipotential, and that in adult life maintenance of these particular epidermal specificities is the outcome of persistent specific inductive stimuli from the underlying dermis. The results of subsidiary experiments are reported which indicate that

  3. Comparative skin permeability of neonatal and adult timber rattlesnakes (Crotalus horridus).

    PubMed

    Agugliaro, Joseph; Reinert, Howard K

    2005-05-01

    Skin permeability and lipid content were determined using shed epidermis of neonatal and adult timber rattlesnakes (Crotalus horridus) from the Coastal Plain Pine Barrens of New Jersey and from the Appalachian Mountains of northern Pennsylvania. Differences between populations due to habitat and within populations due to age were tested. Skin permeability was not found to differ according to locality (P>0.05), but rates were significantly different for age. Permeability of adult epidermis was greater than that of neonates (P<0.01). Lipid content did not differ by locality (P>0.05), but differed between ages, paralleling the results found for permeation rates. Neonate sheds had a greater amount of extractable lipids than adult sheds (P<0.01). Despite the lower skin permeability of neonates, our estimates indicate that the percentage of their total body water content lost per hour may still be 2.2 times that of adults. Resistance to cutaneous water loss may be advantageous to neonates given their relatively large surface area-to-volume ratio.

  4. Absorption of sunscreens across human skin: an evaluation of commercial products for children and adults

    PubMed Central

    Jiang, R; Roberts, M S; Collins, D M; Benson, H A E

    1999-01-01

    Aims Topical sunscreens are routinely applied to the skin by a large percentage of the population. This study assessed the extent of absorption of a number of common chemical sunscreen agents into and through human skin following application of commercially available products. Methods Sunscreen products were applied to excised human epidermis in Franz diffusion cells with the amount penetrating into and across the epidermis assessed by h.p.l.c. for 8 h following application. Results All sunscreen agents investigated penetrated into the skin (0.25 g m−2 or 14% of applied dose), but only benzophenone-3 passed through the skin in significant amounts (0.08 g m−2 or 10% of the applied dose). With one exception, suncreen agents in corresponding products marketed for adults and children had similar skin penetration profiles. Conclusions Whilst limited absorption across the skin was observed for the majority of the sunscreens tested, benzophenone-3 demonstrated sufficiently high penetration to warrant further investigation of its continued application. PMID:10583038

  5. Systemic evening primrose oil improves the biophysical skin parameters of healthy adults.

    PubMed

    Muggli, R

    2005-08-01

    Biophysical skin parameters are indicators of age-related structural and functional changes in skin tissues. This randomized, double-blind, placebo-controlled study in healthy adults tested the effect of Efamol evening primrose oil [EPO, a gamma-linolenic acid (GLA) containing vegetable oil] on skin moisture, transepidermal water loss (TEWL), redness, firmness, elasticity, fatigue resistance and roughness. Efamol EPO was administered orally in soft gel capsules, 3 x 500 mg b.i.d. for 12 weeks. Measurements were taken at baseline and at weeks 4 and 12. The two treatment groups did not differ at baseline and at week 4. At week 12, however, all measured variables, with the exception of skin redness, were significantly different in the EPO group compared with placebo. Skin moisture, TEWL, elasticity, firmness, fatigue resistance and roughness had significantly improved by 12.9, 7.7, 4.7, 16.7, 14.2 and 21.7%, respectively. The two-sided levels of significance in favor of the EPO treatment ranged between 0.034 and 0.001. These findings lend further support to the notion that GLA is a conditionally essential fatty acid for the skin, i.e. it is unable to synthesize GLA, and therefore depends on preformed GLA for optimal structure and function.

  6. SOS1 over-expression in genital skin fibroblasts from hirsute women: a putative role of the SOS1/RAS pathway in the pathogenesis of hirsutism.

    PubMed

    Minella, D; Wannenes, F; Biancolella, M; Amati, F; Testa, B; Nardone, A; Bueno, S; Fabbri, A; Lauro, D; Novelli, G; Moretti, C

    2011-01-01

    Hirsutism is the development of androgen-dependent terminal body hair in women in places in which terminal hair are normally not found. It is often associated with hyperandrogenemia and/or polycystic ovary syndrome (PCOS), but the existence of uncommom hirsutism forms that are not related to altered androgen plasma levels lead also to the definition of - idiopathic hirsutism. Although the pathophysiology of hirsutism has been linked to increasing 5-alpha reductase (SRD5A) activity and to an alteration of the androgen receptor (AR) transcriptional machinery, many aspects remain unclear. In particular, the relationships between androgens and local factors are poorly understood. In the present paper, we selected for a genital skin biopsy, 8 women affected with severe hirsutism (Ferriman-Gallway score greater than 25) but with normal plasma androgen levels, with the exception of slightly higher serum 3alpha-diol-glucuronide levels, and 6 healthy controls and analyzed their androgen- and insulin-specific transcriptional profile using a specific custom low density microarray (AndroChip 2, GPL9164). We identified the over-expression of the Son of Sevenless-1 (SOS1) gene in all of the hirsute skin fibroblast primary cell cultures compared to control healthy women. Since SOS1 is a guanine nucleotide exchange factor that couples receptor tyrosine kinases to the RAS signaling pathway that controls cell proliferation and differentiation, we further analyzed SOS1 expression, protein level and RAS signaling activation pathway in an in vitro model (NHDF, normal human dermal fibroblast cell line). NHDF treated for 24 h with different concentrations of DHT and T showed an increase in SOS1 levels (both mRNA and protein) and also an activation of the RAS pathway. Our in vivo and in vitro data represent a novel preliminary observation that factors activating SOS1 could act as local proliferative modulators linked to the androgen pathway in the pilosebaceous unit. SOS1 over

  7. Profile of skin barrier proteins and cytokines in adults with atopic dermatitis.

    PubMed

    Orfali, Raquel L; Zaniboni, Mariana C; Aoki, Valeria

    2017-04-01

    Atopic dermatitis (AD), an inflammatory skin disorder with chronic course and characterized by intense pruritus, is a dermatosis of high prevalence of childhood. However, persistence of the disease in adolescents and adults may occur, and more studies regarding the interactions of the complex triggering factors, especially between the adaptive and innate immune alterations and skin barrier defects are needed. In this review the authors summarize the major novel findings of a dysfunctional skin barrier in AD, with emphasis on tight junction components, such as claudins and on proteins of the keratinocyte differentiation, such as filaggrin. This review also provides an update on the characterization of immune response in adults with atopic dermatitis. The adaptive immune dysfunction in AD, classically known as a Th2/Th1 model, has changed its profile, with recent reported cytokines such as interleukins 17, 22, and 31; as for the innate immune system scenario in AD, the characterization of skin microbiome opens new frontiers for the understanding of such a complex inflammatory disease.

  8. Synthesis and Characterization of a Model Extracellular Matrix that Induces Partial Regeneration of Adult Mammalian Skin

    NASA Astrophysics Data System (ADS)

    Yannas, I. V.; Lee, E.; Orgill, D. P.; Skrabut, E. M.; Murphy, G. F.

    1989-02-01

    Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen--glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 μ m, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was >5 × 104 cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physicochemical structure, functioning as a transient basal lamina during morphogenesis of skin.

  9. Synthesis and characterization of a model extracellular matrix that induces partial regeneration of adult mammalian skin.

    PubMed Central

    Yannas, I V; Lee, E; Orgill, D P; Skrabut, E M; Murphy, G F

    1989-01-01

    Regeneration of the dermis does not occur spontaneously in the adult mammal. The epidermis is regenerated spontaneously provided there is a dermal substrate over which it can migrate. Certain highly porous, crosslinked collagen-glycosaminoglycan copolymers have induced partial morphogenesis of skin when seeded with dermal and epidermal cells and then grafted on standard, full-thickness skin wounds in the adult guinea pig. A mature epidermis and a nearly physiological dermis, which lacked hair follicles but was demonstrably different from scar, were regenerated over areas as large as 16 cm2. These chemical analogs of extracellular matrices were morphogenetically active provided that the average pore diameter ranged between 20 and 125 microns, the resistance to degradation by collagenase exceeded a critical limit, and the density of autologous dermal and epidermal cells inoculated therein was greater than 5 x 10(4) cells per cm2 of wound area. Unseeded copolymers with physical structures that were within these limits delayed the onset of wound contraction by about 10 days but did not eventually prevent it. Seeded copolymers not only delayed contraction but eventually arrested and reversed it while new skin was being regenerated. The data identify a model extracellular matrix that acts as if it were an insoluble growth factor with narrowly specified physiochemical structure, functioning as a transient basal lamina during morphogenesis of skin. Images PMID:2915988

  10. Comparison of impedance measurements near the skin of newborns and adults.

    PubMed

    Amm, Bruce; Kao, Tzu-Jen; Newell, Jonathan; Isaacson, David; Saulnier, Gary; Shoudy, David; Boverman, Greg; Sahni, Rakesh; Weindler, Marilyn; Chong, David; DiBardino, David; Davenport, David; Ashe, Jeffrey

    2016-06-01

    Electrical impedance tomography (EIT) is a non-invasive imaging technology that has been extensively studied for monitoring lung function of neonatal and adult subjects, especially in neonatal intensive care unit (NICU) and intensive care unit (ICU) environments. The sources of the total impedance in these applications include internal organs, near-boundary tissues, electrode-skin impedance, electrodes and conducting wires. This total impedance must be considered for system design and setting voltage gain since it will contribute to the measured voltage. To adapt a single instrument for use on infants and adults, we studied the difference between the impedance near the skin in both classes of patients. We used a simultaneous multi-source EIT (SMS-EIT) system to make impedance measurements. Characteristic resistance was calculated for two different current patterns: one that is more sensitive to boundary region impedance and another that is more sensitive to interior changes. We present ratios of these resistances to assess the relative contribution of near-skin effects to the overall impedance. Twenty adult ICU subjects (10 male, 10 female, age: 49.05  ±  16.32 years (mean  ±  standard deviation)) and 45 neonates (23 male, 22 female, gestational age: 37.67  ±  2.11 weeks, postnatal age, 2.56  ±  2.67 d) were studied at Columbia University Medical Center. Impedance measurements at 10 kHz were collected for approximately one hour from each subject. The characteristic resistance ratio for each subject was computed and analyzed. The result shows the impedance at or near the skin of newborns is significantly higher than in adult subjects.

  11. Profiling of Sox4-dependent transcriptome in skin links tumour suppression and adult stem cell activation.

    PubMed

    Foronda, Miguel; Morgado-Palacin, Lucia; Gómez-López, Gonzalo; Domínguez, Orlando; Pisano, David G; Blasco, Maria A

    2015-12-01

    Adult stem cells (ASCs) reside in specific niches in a quiescent state in adult mammals. Upon specific cues they become activated and respond by self-renewing and differentiating into newly generated specialised cells that ensure appropriate tissue fitness. ASC quiescence also serves as a tumour suppression mechanism by hampering cellular transformation and expansion (White AC et al., 2014). Some genes restricted to early embryonic development and adult stem cell niches are often potent modulators of stem cell quiescence, and derailed expression of these is commonly associated to cancer (Vervoort SJ et al., 2013). Among them, it has been shown that recommissioned Sox4 expression facilitates proliferation, survival and migration of malignant cells. By generating a conditional Knockout mouse model in stratified epithelia (Sox4 (cKO) mice), we demonstrated a delayed plucking-induced Anagen in the absence of Sox4. Skin global transcriptome analysis revealed a prominent defect in the induction of transcriptional networks that control hair follicle stem cell (HFSC) activation such as those regulated by Wnt/Ctnnb1, Shh, Myc or Sox9, cell cycle and DNA damage response-associated pathways. Besides, Sox4 (cKO) mice are resistant to skin carcinogenesis, thus linking Sox4 to both normal and pathological HFSC activation (Foronda M et al., 2014). Here we provide additional details on the analysis of Sox4-regulated transcriptome in Telogen and Anagen skin. The raw and processed microarray data is deposited in GEO under GSE58155.

  12. Tolerance of fragranced and fragrance-free facial cleansers in adults with clinically sensitive skin.

    PubMed

    Draelos, Zoe D; Fowler, Joseph; Larsen, Walter G; Hornby, Sidney; Walters, Russel M; Appa, Yohini

    2015-10-01

    Although mild, fragrance-free, nonfoaming cleansers generally are recommended for individuals with sensitive skin, many consumers choose fragranced foaming cleansers. The addition of hydrophobically modified polymers (HMPs) to mild facial cleansers has been shown to improve product tolerability in individuals with sensitive skin while facilitating foaming. The objective of the 2 studies reported here was to assess the tolerability of a mild, HMP-containing, foaming facial cleanser with a fragrance that was free of common allergens and irritating essential oils in patients with sensitive skin. In the first study, 8 participants with clinically diagnosed fragrance sensitivity used a gentle foaming HMP-containing facial cleanser with or without fragrance for 3 weeks. Both cleansers improved global disease severity, irritation, and erythema with similar cleansing effectiveness. The second study was a 3-week, prospective, double-blind, randomized, 2-center study of 153 participants with clinically diagnosed sensitive skin. In this study, the fragranced gentle foaming cleanser with HMP was as well tolerated as a benchmark gentle, fragrance-free, nonfoaming cleanser. Itching, irritation, and desquamation were most improved from baseline in both groups. The participant-rated effectiveness of the cleanser with HMP was similar or better than the benchmark cleanser after 3 weeks of use. In conclusion, the gentle facial cleanser with HMPs and a fragrance offers a new option for adults with sensitive skin who may prefer, and commonly use, a fragranced and foaming product.

  13. Acculturation, Skin Tone Preferences, and Tanning Behaviours Among Young Adult Asian Australians.

    PubMed

    Day, Ashley K; Wilson, Carlene J; Hutchinson, Amanda D; Roberts, Rachel M

    2016-10-01

    Australia has a significant proportion of residents of Asian heritage. Although the incidence of skin cancer is lower in those of Asian heritage than Caucasians, their prognosis is often worse. Sociocultural variables are central to the tanning behaviours of individuals from Western cultures. We examined the role of sociocultural variables in the tanning behaviours (outdoor tanning, indoor/solarium and fake tan use) among Asian Australians. A sample of 399 young adults identifying either as a person of Asian heritage or as Asian Australian participated in an online survey. Our results suggest that Asian Australians are at risk of skin cancer; over 35 % of the sample reported engaging in outdoor tanning and over 10 % in solarium tanning. After controlling for demographic factors and skin cancer knowledge, preferring a darker skin tone and being acculturated to Australia were significantly associated with tanning behaviour. Participants' low levels of skin cancer knowledge are of concern, and possibilities for improving knowledge levels in this group are considered. Further, we recommended that future research studies investigate sociocultural and appearance-related beliefs associated with tanning behaviours in this population, in order to determine best avenues for intervention.

  14. Analysis of carnitine esters by radio-high performance liquid chromatography in cultured skin fibroblasts from patients with mitochondrial fatty acid oxidation disorders.

    PubMed

    Schmidt-Sommerfeld, E; Bobrowski, P J; Penn, D; Rhead, W J; Wanders, R J; Bennett, M J

    1998-08-01

    Acylcarnitines are important diagnostic markers for inborn errors of fatty acid oxidation, but their analysis in body fluids may not always be reliable. Recently, disease-specific acylcarnitine profiles generated by cultured skin fibroblasts were reported to facilitate the diagnosis by localizing a specific enzymatic defect in the mitochondrial beta-oxidation pathway. Using a novel methodologic approach, fibroblasts from 16 patients with inborn errors of fatty acid oxidation and 13 control subjects were preincubated with L-[3H]carnitine to label the intracellular carnitine pool. Cells were subsequently incubated with unlabeled palmitic acid and, after methanol extraction of cells and media, labeled free carnitine and acylcarnitines were analyzed by radio-HPLC. Quantitation was based on the integrated radioactivity of individual peaks relative to the total radioactivity recovered. In control cell lines, all saturated acylcarnitines were detected, and reference values were established. With the exception of one cell line deficient in electron transfer flavoprotein, all mutant cell lines showed abnormal and disease-specific relative concentrations of acylcarnitines. Advantages of the method include use of a small number of cells, no need for trypsinization and permeabilization of cells before incubation, simple extraction without purification of the specimen before HPLC, and relatively inexpensive equipment. The method allows a focused approach to the subsequent, more laborious confirmation of a particular disease by direct enzymatic and/or molecular analysis. It remains to be established whether the method can replace widely used global measurements of fatty acid oxidation rates in vitro that do not provide specific information about the enzyme deficiency involved.

  15. Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

    SciTech Connect

    Silbert, C.K.; Humphries, D.E.; Palmer, M.E.; Silbert, J.E. )

    1991-02-15

    Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with (3H)glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of (3H)chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

  16. Cholesterol-deprivation increases mono-unsaturated very long-chain fatty acids in skin fibroblasts from patients with X-linked adrenoleukodystrophy.

    PubMed

    Engelen, M; Ofman, R; Mooijer, P A W; Poll-The, B T; Wanders, R J A; Kemp, S

    2008-03-01

    X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder and is characterized by a striking and unpredictable variation in phenotypic expression. It ranges from a rapidly progressive and fatal cerebral demyelinating disease in childhood (CCALD), to the milder slowly progressive form in adulthood (AMN). X-ALD is caused by mutations in the ABCD1 gene that encodes a peroxisomal membrane located ABC half-transporter named ALDP. Mutations in ALDP result in reduced beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbon atoms) in peroxisomes and elevated levels of VLCFA in plasma and tissues. Previously, it has been shown that culturing skin fibroblasts from X-ALD patients in lipoprotein-deficient medium results in reduced VLCFA levels and increased expression of the functionally redundant ALD-related protein (ALDRP). The aim of this study was to further resolve the interaction between cholesterol and VLCFA metabolism in X-ALD. Our data show that the reduction in 26:0 in X-ALD fibroblasts grown in lipoprotein-deficient culture medium (free of cholesterol) is offset by a significant increase in both the level and synthesis of 26:1. We also demonstrate that cholesterol-deprivation results in increased expression of stearoyl-CoA-desaturase (SCD) and increased desaturation of 18:0 to 18:1. Finally, there was no increase in [1-(14)C]-26:0 beta-oxidation. Taken together, we conclude that cholesterol-deprivation reduces saturated VLCFA, but increases mono-unsaturated VLCFA. These data may have implications for treatment of X-ALD patients with lovastatin.

  17. Activation of homologous recombination DNA repair in human skin fibroblasts continuously exposed to X-ray radiation

    PubMed Central

    Osipov, Andreyan N.; Grekhova, Anna; Pustovalova, Margarita; Ozerov, Ivan V.; Eremin, Petr; Vorobyeva, Natalia; Lazareva, Natalia; Pulin, Andrey; Zhavoronkov, Alex; Roumiantsev, Sergey; Klokov, Dmitry; Eremin, Ilya

    2015-01-01

    Molecular and cellular responses to protracted ionizing radiation exposures are poorly understood. Using immunofluorescence microscopy, we studied the kinetics of DNA repair foci formation in normal human fibroblasts exposed to X-rays at a dose rate of 4.5 mGy/min for up to 6 h. We showed that both the number of γH2AX foci and their integral fluorescence intensity grew linearly with time of irradiation up to 2 h. A plateau was observed between 2 and 6 h of exposure, indicating a state of balance between formation and repair of DNA double-strand breaks. In contrast, the number and intensity of foci formed by homologous recombination protein RAD51 demonstrated a continuous increase during 6 h of irradiation. We further showed that the enhancement of the homologous recombination repair was not due to redistribution of cell cycle phases. Our results indicate that continuous irradiation of normal human cells triggers DNA repair responses that are different from those elicited after acute irradiation. The observed activation of the error-free homologous recombination DNA double-strand break repair pathway suggests compensatory adaptive mechanisms that may help alleviate long-term biological consequences and could potentially be utilized both in radiation protection and medical practices. PMID:26337087

  18. Impaired colony-forming ability following. gamma. irradiation of skin fibroblasts from tuberous scierosis patients. [/sup 60/Co

    SciTech Connect

    Paterson, M.C.; Sell, B.M.; Smith, B.P.; Bech-Hansen, N.T.

    1982-05-01

    The radiosensitivity of cultured dermal fibroblasts from human subjects afflicted with tuberous sclerosis (TS), a hereditary neurocutaneous syndrome, was assessed by assaying loss of colony-forming ability in response to acute ..gamma..-ray exposure. Related to control strains from clinically normal donors, three cell lines (GM1635, GM1643, GM2333) from two affected patients displayed enhanced sensitivity to inactivation by /sup 60/Co ..gamma..-ray treatment, whether administered oxically (air-saturated) or hypoxically (N/sub 2/-gassed); a fourth strain (GM1644) from a third patient exhibited normal radiosensitivity under both treatment conditions. The post-..gamma..-irradiaton colony-forming ability of the three hypersensitive TS strains was intermediate between that of normal controls and that of strains from patients inheriting the radiotherapy-sensitive neurovascular disorder ataxia telangiectasia. The variability in the radioresponse of the TS stains (three sensitive and one normal) is not surprising, considering the widely recognized clinical heterogeneity in the disease. Our findings, aside from providing a laboratory marker for early (possible presymptomatic) detection of persons at high risk for TS, may lead to a better understanding of the origin and progressive development of this multifaceted syndrome.

  19. Upregulation of PIM2 by Underexpression of MicroRNA-135-5p Improves Survival Rates of Skin Allografts by Suppressing Apoptosis of Fibroblast Cells

    PubMed Central

    Zhang, Hongtu

    2017-01-01

    Background It has been reported that miR-135-5p is involved with many diseases. In this study, we aimed at define the relationship between miR-135-5p level and burn patient survival after skin transplantation. Material/Methods Expression of miR-135-5p and PIM2 was measured using real-time PCR and Western blot analysis in the skin samples collected from burn patients who received skin graft or in the fibroblast cells transfected with miR-135-5p mimics or inhibitors. The regulatory association between miR-135-5p and PIM2 was verified using bioinformatics analysis and luciferase assay. Results The expression level of miR-135-5p was determined in 60 tissue samples divided into 2 groups based on the presence of rejection (long survival n=30, and short survival n=30). We found that miR-135-5p was substantially downregulated in the long survival group. We then searched the miRNA database online with the “seed sequence” located within the 3′-UTR of the target gene, and then validated PIM2 to be the direct gene via luciferase reporter assay system. We also established the negative regulatory relationship between miR-135-5p and PIM2 via studying the relative luciferase activity. We also conducted real-time PCR and Western blot analysis to study the mRNA and protein expression level of PIM2 among different groups (long survival n=30, short survival n=30) or cells treated with scramble control, miR-135-5p mimics, PIM2 siRNA, and miR-135-5p inhibitors, indicating the negative regulatory relationship between MiR-135-5p and PIM2. We also conducted experiments to investigate the influence of miR-135-5p and PIM2 on viability and apoptosis of cells. The results showed miR-135-5p reduced the viability of cells, while PIM2 negatively interfered with the viability of cells, and miR-135-5p inhibited apoptosis and PIM2 suppressed apoptosis. Conclusions MiR-135-5p is involved with the prognosis of burn patients after skin transplantation. PIM2 is a virtual target of miR-135-5p, and

  20. Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

    PubMed Central

    Collins, Charlotte A.; Kretzschmar, Kai; Watt, Fiona M.

    2011-01-01

    Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis. PMID:22031549

  1. Presynaptic size of associational/commissural CA3 synapses is controlled by fibroblast growth factor 22 in adult mice.

    PubMed

    Pasaoglu, Taliha; Schikorski, Thomas

    2016-02-01

    Associational/commissural CA3-CA3 synapses define the recurrent CA3 network that generates the input to CA1 pyramidal neurons. We quantified the fine structure of excitatory synapses in the stratum radiatum of the CA3d area in adult wild type (WT) and fibroblast growth factor 22 knock-out (FGF22KO) mice by using serial 3D electron microscopy. WT excitatory CA3 synapses are rather small yet range 10 fold in size. Spine size, however, was small and uniform and did not correlate with the size of the synaptic junction. To reveal mechanisms that regulate presynaptic structure, we investigated the role of FGF22, a target-derived signal specific for the distal part of area CA3 (CA3d). In adult FGF22KO mice, postsynaptic properties of associational CA3 synapses were unaltered. Presynaptically, the number of synaptic vesicles (SVs), the bouton volume, and the number of vesicles in axonal regions (the super pool) were reduced. This concurrent decrease suggests concerted control by FGF22 of presynaptic size. This hypothesis is supported by the finding that WT presynapses in the proximal part of area CA3 (CA3p) that do not receive FGF22 signaling in WT mice were smaller than presynapses in CA3d in WT but of comparable size in CA3d of FGF22KO mice. Docked SV density was decreased in CA1, CA3d, and CA3p in FGF22KO mice. Because CA1 and CA3p are not directly affected by the loss of FGF22, the smaller docked SV density may be an adaptation to activity changes in the CA3 network. Thus, docked SV density potentially is a long-term regulator for the synaptic release probability and/or the strength of short-term depression in vivo.

  2. Fibroblast growth factor 10 alters the balance between goblet and Paneth cells in the adult mouse small intestine.

    PubMed

    Al Alam, Denise; Danopoulos, Soula; Schall, Kathy; Sala, Frederic G; Almohazey, Dana; Fernandez, G Esteban; Georgia, Senta; Frey, Mark R; Ford, Henri R; Grikscheit, Tracy; Bellusci, Saverio

    2015-04-15

    Intestinal epithelial cell renewal relies on the right balance of epithelial cell migration, proliferation, differentiation, and apoptosis. Intestinal epithelial cells consist of absorptive and secretory lineage. The latter is comprised of goblet, Paneth, and enteroendocrine cells. Fibroblast growth factor 10 (FGF10) plays a central role in epithelial cell proliferation, survival, and differentiation in several organs. The expression pattern of FGF10 and its receptors in both human and mouse intestine and their role in small intestine have yet to be investigated. First, we analyzed the expression of FGF10, FGFR1, and FGFR2, in the human ileum and throughout the adult mouse small intestine. We found that FGF10, FGFR1b, and FGFR2b are expressed in the human ileum as well as in the mouse small intestine. We then used transgenic mouse models to overexpress Fgf10 and a soluble form of Fgfr2b, to study the impact of gain or loss of Fgf signaling in the adult small intestine. We demonstrated that overexpression of Fgf10 in vivo and in vitro induces goblet cell differentiation while decreasing Paneth cells. Moreover, FGF10 decreases stem cell markers such as Lgr5, Lrig1, Hopx, Ascl2, and Sox9. FGF10 inhibited Hes1 expression in vitro, suggesting that FGF10 induces goblet cell differentiation likely through the inhibition of Notch signaling. Interestingly, Fgf10 overexpression for 3 days in vivo and in vitro increased the number of Mmp7/Muc2 double-positive cells, suggesting that goblet cells replace Paneth cells. Further studies are needed to determine the mechanism by which Fgf10 alters cell differentiation in the small intestine.

  3. IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes.

    PubMed

    Wang, Xiaoyong; Bi, Zhigang; Chu, Wenming; Wan, Yinsheng

    2005-12-01

    Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.

  4. Phospholipase D from Loxosceles laeta Spider Venom Induces IL-6, IL-8, CXCL1/GRO-α, and CCL2/MCP-1 Production in Human Skin Fibroblasts and Stimulates Monocytes Migration.

    PubMed

    Rojas, José M; Arán-Sekul, Tomás; Cortés, Emmanuel; Jaldín, Romina; Ordenes, Kely; Orrego, Patricio R; González, Jorge; Araya, Jorge E; Catalán, Alejandro

    2017-04-05

    Cutaneous loxoscelism envenomation by Loxosceles spiders is characterized by the development of a dermonecrotic lesion, strong inflammatory response, the production of pro-inflammatory mediators, and leukocyte migration to the bite site. The role of phospholipase D (PLD) from Loxosceles in the recruitment and migration of monocytes to the envenomation site has not yet been described. This study reports on the expression and production profiles of cytokines and chemokines in human skin fibroblasts treated with catalytically active and inactive recombinant PLDs from Loxosceles laeta (rLlPLD) and lipid inflammatory mediators ceramide 1-phosphate (C1P) and lysophosphatidic acid (LPA), and the evaluation of their roles in monocyte migration. Recombinant rLlPLD1 (active) and rLlPLD2 (inactive) isoforms induce interleukin (IL)-6, IL-8, CXCL1/GRO-α, and CCL2/monocyte chemoattractant protein-1 (MCP-1) expression and secretion in fibroblasts. Meanwhile, C1P and LPA only exhibited a minor effect on the expression and secretion of these cytokines and chemokines. Moreover, neutralization of both enzymes with anti-rLlPLD1 antibodies completely inhibited the secretion of these cytokines and chemokines. Importantly, conditioned media from fibroblasts, treated with rLlPLDs, stimulated the transmigration of THP-1 monocytes. Our data demonstrate the direct role of PLDs in chemotactic mediator synthesis for monocytes in human skin fibroblasts and indicate that inflammatory processes play an important role during loxoscelism.

  5. Histology and Ultrastructure of Transitional Changes in Skin Morphology in the Juvenile and Adult Four-Striped Mouse (Rhabdomys pumilio)

    PubMed Central

    Stewart, Eranée; Ajao, Moyosore Salihu

    2013-01-01

    The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin. PMID:24288469

  6. Histology and ultrastructure of transitional changes in skin morphology in the juvenile and adult four-striped mouse (Rhabdomys pumilio).

    PubMed

    Stewart, Eranée; Ajao, Moyosore Salihu; Ihunwo, Amadi Ogonda

    2013-01-01

    The four-striped mouse has a grey to brown coloured coat with four characteristic dark stripes interspersed with three lighter stripes running along its back. The histological differences in the skin of the juvenile and adult mouse were investigated by Haematoxylin and Eosin and Masson Trichrome staining, while melanocytes in the skin were studied through melanin-specific Ferro-ferricyanide staining. The ultrastructure of the juvenile skin, hair follicles, and melanocytes was also explored. In both the juvenile and adult four-striped mouse, pigment-containing cells were observed in the dermis and were homogeneously dispersed throughout this layer. Apart from these cells, the histology of the skin of the adult four-striped mouse was similar to normal mammalian skin. In the juvenile four-striped mouse, abundant hair follicles of varying sizes were observed in the dermis and hypodermis, while hair follicles of similar size were only present in the dermis of adult four-striped mouse. Ultrastructural analysis of juvenile hair follicles revealed that the arrangement and differentiation of cellular layers were typical of a mammal. This study therefore provides unique transition pattern in the four-striped mouse skin morphology different from the textbook description of the normal mammalian skin.

  7. The role of Rho kinase (Rock) in re-epithelialization of adult zebrafish skin wounds.

    PubMed

    Richardson, Rebecca; Hammerschmidt, Matthias

    2016-08-03

    Complete re-epithelialization of full-thickness skin wounds in adult mammals takes days to complete and relies on numerous signaling cues and multiple overlapping cellular processes that take place both within the epidermis itself and in other participating tissues. We have previously shown that re-epithelialization of full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing allowing for the dissection of specific processes that occur in, or have a direct effect on, re-epithelializing keratinocytes. Recently, we have shown that, in addition to lamellipodial crawling at the leading edge, re-epithelialization of zebrafish partial- and full-thickness wounds requires long-range epithelial rearrangements including radial intercalations, flattening and directed elongation and that each of these processes involves Rho kinase (Rock) signaling. Our studies demonstrate how these coordinated signaling events allow for the rapid collective cell migration observed in adult zebrafish wound healing. Here we discuss the particular contribution of Rock to each of these processes.

  8. Patterns of Sunscreen Use on the Face and Other Exposed Skin among US Adults

    PubMed Central

    Holman, Dawn M.; Berkowitz, Zahava; Guy, Gery P.; Hawkins, Nikki A.; Saraiya, Mona; Watson, Meg

    2015-01-01

    Background Sunscreen is a common form of sun protection, but little is known about patterns of use. Objective To assess patterns of sunscreen use on the face and other exposed skin among US adults. Methods Using cross-sectional data from the 2013 Summer ConsumerStyles survey (N= 4,033), we calculated descriptive statistics and adjusted risk ratios to identify characteristics associated with regular sunscreen use (always/most of the time when outside on a warm sunny day for 1+ hour). Results Few adults regularly used sunscreen on the face (men: 18.1%, 95% Confidence Interval [CI]: 15.8–20.6; women: 42.6%, 95% CI 39.5–46.7), other exposed skin (men: 19.9%, 95% CI 17.5–22.6; women: 34.4%, 95% CI 31.5–37.5), or both the face and other exposed skin (men: 14.3%, 95% CI: 12.3–16.6; women: 29.9%, 95% CI: 27.2–32.8). Regular use was associated with sun-sensitive skin, a household income ≥$60,000, and meeting aerobic activity guidelines (Ps < 0.05). Nearly 40% of users were unsure if their sunscreen provided broad spectrum protection. Limitations Reliance on self-report and lack of information on sunscreen reapplication or other sun-safety practices. Conclusion Sunscreen use is low, especially among certain demographic groups. These findings can inform sun-safety interventions and the interpretation of surveillance data on sunscreen use. PMID:26002066

  9. Photoprotective Potential of Penta-O-Galloyl-β-DGlucose by Targeting NF-κB and MAPK Signaling in UVB Radiation-Induced Human Dermal Fibroblasts and Mouse Skin.

    PubMed

    Kim, Byung-Hak; Choi, Mi Sun; Lee, Hyun Gyu; Lee, Song-Hee; Noh, Kum Hee; Kwon, Sunho; Jeong, Ae Jin; Lee, Haeri; Yi, Eun Hee; Park, Jung Youl; Lee, Jintae; Joo, Eun Young; Ye, Sang-Kyu

    2015-11-01

    Exposure of the skin to ultraviolet radiation can cause skin damage with various pathological changes including inflammation. In the present study, we identified the skin-protective activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (pentagalloyl glucose, PGG) in ultraviolet B (UVB) radiation-induced human dermal fibroblasts and mouse skin. PGG exhibited antioxidant activity with regard to intracellular reactive oxygen species (ROS) generation as well as ROS and reactive nitrogen species (RNS) scavenging. Furthermore, PGG exhibited anti-inflammatory activity, inhibiting the activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, resulting in inhibition of the expression of pro-inflammatory mediators. Topical application of PGG followed by chronic exposure to UVB radiation in the dorsal skin of hairless mice resulted in a significant decrease in the progression of inflammatory skin damages, leading to inhibited activation of NF-κB signaling and expression of pro-inflammatory mediators. The present study demonstrated that PGG protected from skin damage induced by UVB radiation, and thus, may be a potential candidate for the prevention of environmental stimuli-induced inflammatory skin damage.

  10. Photoprotective Potential of Penta-O-Galloyl-β-DGlucose by Targeting NF-κB and MAPK Signaling in UVB Radiation-Induced Human Dermal Fibroblasts and Mouse Skin

    PubMed Central

    Kim, Byung-Hak; Choi, Mi Sun; Lee, Hyun Gyu; Lee, Song-Hee; Noh, Kum Hee; Kwon, Sunho; Jeong, Ae Jin; Lee, Haeri; Yi, Eun Hee; Park, Jung Youl; Lee, Jintae; Joo, Eun Young; Ye, Sang-Kyu

    2015-01-01

    Exposure of the skin to ultraviolet radiation can cause skin damage with various pathological changes including inflammation. In the present study, we identified the skin-protective activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (pentagalloyl glucose, PGG) in ultraviolet B (UVB) radiation-induced human dermal fibroblasts and mouse skin. PGG exhibited antioxidant activity with regard to intracellular reactive oxygen species (ROS) generation as well as ROS and reactive nitrogen species (RNS) scavenging. Furthermore, PGG exhibited anti-inflammatory activity, inhibiting the activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, resulting in inhibition of the expression of pro-inflammatory mediators. Topical application of PGG followed by chronic exposure to UVB radiation in the dorsal skin of hairless mice resulted in a significant decrease in the progression of inflammatory skin damages, leading to inhibited activation of NF-κB signaling and expression of pro-inflammatory mediators. The present study demonstrated that PGG protected from skin damage induced by UVB radiation, and thus, may be a potential candidate for the prevention of environmental stimuli-induced inflammatory skin damage. PMID:26537189

  11. Patient fibroblasts-derived induced neurons demonstrate autonomous neuronal defects in adult-onset Krabbe disease

    PubMed Central

    Choi, Won Jun; Oh, Ki-Wook; Nahm, Minyeop; Xue, Yuanchao; Choi, Jae Hyeok; Choi, Ji Young; Kim, Young-Eun; Chung, Ki Wha; Fu, Xiang-Dong; Ki, Chang-Seok; Kim, Seung Hyun

    2016-01-01

    Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by defective β-galactosylceramidase (GALC), a lysosomal enzyme responsible for cleavage of several key substrates including psychosine. Accumulation of psychosine to the cytotoxic levels in KD patients is thought to cause dysfunctions in myelinating glial cells based on a comprehensive study of demyelination in KD. However, recent evidence suggests myelin-independent neuronal death in the murine model of KD, thus indicating defective GALC in neurons as an autonomous mechanism for neuronal cell death in KD. These observations prompted us to generate induced neurons (iNeurons) from two adult-onset KD patients carrying compound heterozygous mutations (p.[K563*];[L634S]) and (p.[N228_S232delinsTP];[G286D]) to determine the direct contribution of autonomous neuronal toxicity to KD. Here we report that directly converted KD iNeurons showed not only diminished GALC activity and increased psychosine levels, as expected, but also neurite fragmentation and abnormal neuritic branching. The lysosomal-associated membrane proteins 1 (LAMP1) was expressed at higher levels than controls, LAMP1-positive vesicles were significantly enlarged and fragmented, and mitochondrial morphology and its function were altered in KD iNeurons. Strikingly, we demonstrated that psychosine was sufficient to induce neurite defects, mitochondrial fragmentation, and lysosomal alterations in iNeurons derived in healthy individuals, thus establishing the causal effect of the cytotoxic GALC substrate in KD and the autonomous neuronal toxicity in KD pathology. PMID:27780934

  12. Association of 25-hydroxyvitamin D3)levels in adult New Zealanders with ethnicity, skin color and self-reported skin sensitivity to sun exposure.

    PubMed

    Nessvi, Sofia; Johansson, Lisa; Jopson, Jan; Stewart, Alistair; Reeder, Anthony; McKenzie, Richard; Scragg, Robert K

    2011-01-01

    The study aim was to determine the contribution of ethnicity, objectively measured skin color and skin reaction-to-sun exposure to variations in 25-hydroxyvitamin D(3) [25(OH)D(3) ]. A multiethnic sample (European, Maori, Pacific and Asian) of 503 adult volunteers aged 18-85 years, recruited from Auckland and Dunedin in New Zealand, answered a questionnaire on sun exposure and self-defined ethnicity. Skin color was measured using a spectrophotometer and the Individual Typology Angle (ITA) calculated. A blood sample was collected 4 weeks later to measure 25(OH)D(3). 25(OH)D(3) was associated with ethnicity, but not self-reported skin reaction-to-sun exposure. Amongst the ethnic groups, Asians had the lowest mean 25(OH)D level (37.0 nmol L(-1)) and Europeans with lighter colored skin had the highest (57.9 nmol L(-1)). An association also was seen between 25(OH)D(3) and skin color, with an increase of 2-3 nmol L(-1) per 10° increase in ITA value, indicating higher 25(OH)D(3) with lighter skin color; but much of this association disappeared after adjusting for ethnicity. In contrast, ethnicity remained associated with 25(OH)D(3) after adjusting for ITA skin color and skin reaction-to-sun exposure. These results indicate that self-defined ethnicity was a major determinant of variations in serum 25(OH)D(3), while objective measures of skin color explained relatively little additional variation.

  13. Use of Amniotic Microparticles Coated With Fibroblasts Overexpressing SDF-1α to Create an Environment Conducive to Neovascularization for Repair of Full-Thickness Skin Defects.

    PubMed

    Zhang, Yun-qing; Ji, Shi-zhao; Fang, He; Zheng, Yong-jun; Luo, Peng-fei; Wu, Hai-bin; Wu, Min-juan; Wang, Zhi-hong; Xiao, Shi-chu; Xia, Zhao-fan

    2016-01-01

    As angiogenesis and vasculogenesis involve the complex network structures of various types of cells, extracellular matrix components, and cytokines, it is still difficult to exactly mimic the microenvironment of vascularization in vivo. In our study, we constructed a complex containing highly proliferative fibroblasts that can secrete extracellular matrix components and growth factors to chemotaxize endothelial progenitor cells (EPCs) in an attempt to create an ideal microenvironment for quick vascularization. Amniotic membrane microparticles (mAM) rich in type IV collagen (COL IV) and laminin (LN) were prepared, and human dermal fibroblasts (HDF) were infected with lentivirus (LV) of overexpression of SDF-1α to construct SDF-1α(ov)HDF. Using the rotary cell culture system (RCCS), mAM was loaded with HDF or SDF-1α(ov)HDF to construct HDF-mAM and SDF-1α(ov)HDF-mAM complexes. The complexes were able to secrete various types of active peptides (IL-6, IL-8, TGF-β, and bFGF) during in vitro culture. In addition, SDF-1α(ov)HDF-mAM complex highly expressed SDF-1α. Transwell assay showed SDF-1α(ov)HDF-mAM complex had an apparent chemotactic effect on EPCs. Transplantation of complexes onto full-thickness skin defects of C57BL mice further demonstrated that SDF-1α expression and the number of peripheral EPCs at days 3, 5, and 7 in the SDF-1α(ov)HDF-mAM group were significantly higher than that in other groups (p < 0.01). The local microvascular density at day 10 of transplantation showed that the microvascular density in the SDF-1α(ov)HDF-mAM group was significantly higher than that in HDF-mAM group (p < 0.01). In conclusion, HDF-mAM had a strong proliferative activity and could be used to create a sound microenvironment for quick vascularization by secreting multiple cytokines and extracellular matrix components. Overexpression of SDF-1α could chemotaxize EPCs to reach local wounds, thus further accelerating angiogenesis in the transplant site. The

  14. Chemokines and skin diseases.

    PubMed

    Sugaya, Makoto

    2015-04-01

    Chemokines are small molecules that induce chemotaxis and activation of certain subsets of leukocytes. The expression patterns of chemokines and chemokine receptors are specific to certain organs and cells. Therefore, chemokines are important to elucidate the mechanism of organ-specific human diseases. CCL17 expressed by Langerhans cells, blood endothelial cells, and fibroblasts plays a key role in attracting Th2 cells and tumor cells of adult T-cell leukemia/lymphoma and mycosis fungoides/Sézary syndrome into the skin, developing various Th2-type inflammatory skin diseases as well as cutaneous lymphoma. CCL11 and CCL26 expressed by skin-resident cells, such as fibroblasts, blood endothelial cells, and keratinocytes, induce infiltration of CCR3-expressing cells such as Th2 cells and eosinophils. CCL11 may also serve as an autocrine as well as a paracrine in anaplastic large cell lymphoma. CX3CL1 expressed on blood endothelial cells leads to infiltration of CX3CR1(+) immune cells, such as mast cells, neutrophils, and macrophages, playing important roles in wound healing, tumor immunity, and vasculitis. Biologics targeting chemokines and their receptors are promising strategies for various skin diseases that are resistant to the current therapy.

  15. A source of healthcare disparity: race, skin color, and injuries after rape among adolescents and young adults.

    PubMed

    Baker, Rachel B; Fargo, Jamison D; Shambley-Ebron, Donna; Sommers, Marilyn S

    2010-01-01

    Differences in anogenital injury resulting from rape may occur because of racial or skin color differences in adult women. It is critical to determine if these differences also are associated with differences in injury prevalence and frequency in adolescents and young adults. In a retrospective review of medical records, we examined whether Black adolescent/young adult females had different anogenital injuries as compared to White females following rape. Next, we examined whether skin color differences explained a significant amount of the racial difference in injuries. We reviewed charts of 234 female victims of rape ages 14 to 29. Overall injury prevalence was 62.8%. Race was significantly associated with frequency of injuries in several anatomical locations, with White victims having a higher frequency of injuries than Black victims. Skin color was significantly associated with injury frequency in many anatomical locations, with victims with light skin sustaining more injuries than victims with dark skin. Even when skin color was included in the relationship, race remained a statistically significant factor, suggesting that the relationship between race and injuries may be more complicated than merely a skin color difference that has been mislabeled a racial difference.

  16. Arsenic methylation and skin lesions in migrant and native adult women with chronic exposure to arsenic from drinking groundwater.

    PubMed

    Wei, Binggan; Yu, Jiangping; Yang, Linsheng; Li, Hairong; Chai, Yuanqing; Xia, Yajuan; Wu, Kegong; Gao, Jianwei; Guo, Zhiwei; Cui, Na

    2017-02-01

    In order to figure out the prevalence of skin lesions and methylation capacity for migrant and native adult women in an endemic area for arsenic poisoning in Inner Mongolia, China, 207 adult women were selected for study subjects. The results showed that the prevalence of skin lesions for the external group, provincial group and native group was 36.54, 26.15 and 35.56 %, respectively. The nail content of arsenic and urinary concentrations of dimethylarsenic (DMA), monomethylarsenic (MMA) and inorganic arsenic (iAs) were significantly higher in women with skin lesions than in those without skin lesions. The highest urinary concentrations of DMA, MMA and iAs were 213.93, 45.72 and 45.01 μg/L in the native group. The arsenic methylation capacity index revealed that the external group had the greatest capacity, while the native group had the lowest. The odds ratios of skin lesions in relation to arsenic metabolites and arsenic methylation capacity varied widely among the three groups. Urinary MMA and iAs concentrations were positively associated with risk of skin lesions in the three groups of adult women, while primary and secondary methylation capacities were negatively related to risk of skin lesions in native and provincial groups. The external group might be more susceptible to MMA and iAs, while the provincial and native groups were more tolerance to MMA and iAs. Lower primary and secondary arsenic methylation capacities increased the risk of skin lesions in native and provincial groups. Moreover, higher nail arsenic concentration increased the risk of skin lesions of adult women.

  17. Effects of DNA methyltransferase inhibitor RG108 on methylation in buffalo adult fibroblasts and subsequent embryonic development following somatic cell nuclear transfer.

    PubMed

    Sun, H L; Meng, L N; Zhao, X; Jiang, J R; Liu, Q Y; Shi, D S; Lu, F H

    2016-09-02

    Buffalo are characteristic livestock of the Guangxi Zhuang Autonomous Region of China, but their low reproductive capacity necessitates the use of somatic cell nuclear transfer (SCNT). We investigated the effects of RG108 on DNA methylation in buffalo adult fibroblasts, and on subsequent SCNT embryo development. RG108 treatment (0, 5, 10, 20, and 100 mM) had no effect on cell morphology, viability, or karyotype (2n = 48), and cell growth followed a typical "S" curve. Immunohistochemistry showed that relative DNA methylation gradually decreased as RG108 concentration increased, and was significantly lower in the 20 and 100 mM groups compared to the 0, 5, and 10 mM treatments (0.94 ± 0.03 and 0.92 ± 0.05 vs 1.0 ± 0.02, 0.98 ± 0.05, and 0.98 ± 0.09, respectively; P < 0.05). Quantitative polymerase chain reaction revealed that DNMT1 gene expression of fibroblasts administered 10, 20, and 100 mM RG108 was significantly lower than those in the 0 and 5 mM groups (0.2 ± 0.05, 0.18 ± 0.07, and 0.3 ± 0.09 vs 1.0 ± 0.12 and 1.4 ± 0.12, respectively; P < 0.05). Treatment with 20 mM RG108 resulted in the lowest expression levels. Fibroblasts incubated with 20 mM RG108 for 72 h were used as donor cells to generate SCNT embryos. A greater number of such embryos developed into blastocysts compared to the non-treated group (28.9 ± 3.9 vs 15.3 ± 3.4%; P < 0.05). RG108 treatment can modify DNA methylation in buffalo adult fibroblasts and promote development of subsequent SCNT embryos.

  18. Molybdenum nanoparticles-induced cytotoxicity, oxidative stress, G2/M arrest, and DNA damage in mouse skin fibroblast cells (L929).

    PubMed

    Siddiqui, Maqsood A; Saquib, Quaiser; Ahamed, Maqusood; Farshori, Nida N; Ahmad, Javed; Wahab, Rizwan; Khan, Shams T; Alhadlaq, Hisham A; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; Pant, Aditya B

    2015-01-01

    The present investigation was aimed to study the cytotoxicity, oxidative stress, and genotoxicity induced by molybdenum nanoparticles (Mo-NPs) in mouse skin fibroblast cells (L929). Cells were exposed to different concentrations (1-100 μg/ml) of Mo-NPs (size 40 nm) for 24 and 48 h. After the exposure, different cytotoxicity assays (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide, MTT; neutral red uptake, NRU; and cellular morphology) and oxidative stress markers (lipid peroxidation, LPO; glutathione, GSH; and catalase) were studied. Further, Mo-NPs-induced intracellular reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), cell cycle arrest, and DNA damage were also studied. L929 cells treated with Mo-NPs showed a concentration- and time-dependent decrease in cell viability and a loss of the normal cell morphology. The percentage cell viability was recorded as 25%, 42%, and 58% by MTT assay and 24%, 46%, and 56% by NRU assay at 25, 50, and 100 μg/ml of Mo-NPs, respectively after 48 h exposure. Furthermore, the cells showed a significant induction of oxidative stress. This was confirmed by the increase in LPO and ROS generation, as well as the decrease in the GSH and catalase levels. The decrease in MMP also confirms the impaired mitochondrial membrane. The cell cycle analysis and comet assay data revealed that Mo-NPs induced G2/M arrest and DNA damage in a concentration-dependent manner. Our results demonstrated, for the first time, Mo-NPs induced cytotoxicity, oxidative stress and genotoxicity in L929 cells. Thus, data suggest the potential hazardous nature of Mo-NPs.

  19. Skin incision induces expression of axonal regeneration-related genes in adult rat spinal sensory neurons

    PubMed Central

    Hill, Caitlin E.; Harrison, Benjamin J.; Rau, Kris K.; Hougland, M. Tyler; Bunge, Mary Bartlett; Mendell, Lorne M.; Petruska, Jeffrey C.

    2010-01-01

    Skin incision and nerve injury both induce painful conditions. Incisional and post-surgical pain is believed to arise primarily from inflammation of tissue and the subsequent sensitization of peripheral and central neurons. The role of axonal regeneration-related processes in development of pain has only been considered when there has been injury to the peripheral nerve itself, even though tissue damage likely induces injury of resident axons. We sought to determine if skin incision would affect expression of regeneration-related genes such as activating transcription factor 3 (ATF3) in dorsal root ganglion (DRG) neurons. ATF3 is absent from DRG neurons of the normal adult rodent, but is induced by injury of peripheral nerves and modulates the regenerative capacity of axons. Image analysis of immunolabeled DRG sections revealed that skin incision led to an increase in the number of DRG neurons expressing ATF3. RT-PCR indicated that other regeneration-associated genes (galanin, GAP-43, Gadd45a) were also increased, further suggesting an injury-like response in DRG neurons. Our finding that injury of skin can induce expression of neuronal injury/regeneration-associated genes may impact how clinical post-surgical pain is investigated and treated. Perspective Tissue injury, even without direct nerve injury, may induce a state of enhanced growth capacity in sensory neurons. Axonal regeneration-associated processes should be considered alongside nerve signal conduction and inflammatory/sensitization processes as possible mechanisms contributing to pain, particularly the transition from acute to chronic pain. PMID:20627820

  20. IN VIVO EVALUATION OF SKIN IRRITATION POTENTIAL, MELASMA AND SEBUM CONTENT FOLLOWING LONG TERM APPLICATION OF SKIN CARE CREAM IN HEALTHY ADULTS, USING NON-INVASIVE BIOMETROLOGICAL TECHNIQUES.

    PubMed

    Arshad, Atif I; Khan, Shoaib H M; Akhtar, Naveed; Mahmood, Asif; Sarfraz, Rai Muhammad

    2016-01-01

    The present investigation was conducted to evaluate non-invasively, various functional skin parameters i.e., irritation potential, melasma and sebum contents following long term application of topical cream (w/o) loaded with 2% methanolic extract of Ananas comosus L. versus placebo control (base) in healthy adults. Healthy human volunteers (n = 11, aged 20-30 years) were recruited for investigation and written informed consent was taken from each volunteer. In this single blinded study every volunteer applied formulation on one side of face and placebo on the other side of face twice daily for a period of 12 weeks (three months). Different skin parameters i.e., skin irritancy, melasma, and sebum contents were measured on both sides of face at baseline and after two weeks interval, using photometric device Mexameter and Sebumeter in a draught free room with modulated conditions of temperature (22-25°C) and humidity (55-60%). It was evident from the results that no primary skin irritancy was observed with patch test. Besides, statistical interpretation indicates that treatment with formulation is superior to placebo because it significantly (p ≤ 0.05) reduced the skin irritancy, melasma and sebum secretions throughout the study and reaching maximum -20.76 ± 0.89, -54.2 ± 0.37 and -40.71 ± 0.75%, respectively, at the end of study period. Antioxidant activity of extract was 92% compared to standard antioxidant. Conclusively, active cream loaded with fruit extract was well tolerated by all the volunteers and suitable to treat contact dermatitis, greasy skin, acne and seborrheic dermatitis and augmenting beauty and attraction by depigmentation of human skin. So, in the future, there is need to clinically evaluate these formulations in patients with compromised skin functions i.e., contact dermatitis, melasma, and acne vulgaris in order to explore the actual potential of this fruit.

  1. Effects of low-level laser therapy, electroacupuncture, and radiofrequency on the pigmentation and skin tone of adult women

    PubMed Central

    Kim, Hee-Kyoung; Min, Kyoung-Ok; Choi, Jung-Hyun; Kim, Soon-Hee

    2016-01-01

    [Purpose] In this study, the effects of low-level laser therapy (LLLT), electroacupuncture (EA), and radiofrequency (RF), which are used in physical therapy, on the pigmentation and skin tone of adult women’s faces were investigated to provide basic data for skin interventions. [Subjects and Methods] Thirty adult females were assigned to either an LLLT group (n=10), an EA group (n=10), or an RF group (n=10). The intervention was performed in two 15-minute sessions per week for six weeks. Subjects’ skin tone and pigmentation were observed before and after the intervention. [Results] The EA group showed significant reductions in pigmentation in the left and right eye rims, as well as in the left cheek. The RF group showed significant post-intervention reductions in pigmentation under the left eye, as well as in the left and right eye rims and the left cheek. The LLLT group showed significant increases in skin tone in the forehead and both eye rims. The RF group showed significant increases in skin tone under both eyes. [Conclusion] The application of LLLT, EA, and RF had positive effects on pigmentation and skin tone of adult women’s faces. PMID:27313340

  2. Basic fibroblast growth factor has a differential effect on MyoD conversion of cultured aortic smooth muscle cells from newborn and adult rats.

    PubMed Central

    van Neck, J. W.; Medina, J. J.; Onnekink, C.; van der Ven, P. F.; Bloemers, H. P.; Schwartz, S. M.

    1993-01-01

    MyoD is a master regulatory gene for myogenesis that also converts many mesoderm-derived cells into the skeletal muscle phenotype. Rat aortic smooth muscle cells do not contain MyoD homologous mRNA. However, expression of an exogenously supplied MyoD gene in aortic smooth muscle cells cultured from newborn and adult animals converts these cells to elongated myoblasts and myotubes expressing the skeletal muscle genes for titin, nebulin, myosin, and skeletal alpha-actin. The presence of basic fibroblast growth factor during growth and serum starvation completely inhibits MyoD-mediated conversion in cultures of newborn smooth muscle cells. However, in smooth muscle cell cultures derived from adult rats the presence of fibroblast growth factor increases the conversion frequency. The differential response of exogenous MyoD suggests that the two morphological types of aortic smooth muscle cells, one typical for the newborn rat, the other for the adult rat, represent two distinctive states of differentiation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 PMID:8391216

  3. Chemical Characterization of Lipophilic Constituents in the Skin of Migratory Adult Sea Lamprey from the Great Lakes Region

    PubMed Central

    Dissanayake, Amila A.; Wagner, C. Michael; Nair, Muraleedharan G.

    2016-01-01

    The sea lamprey (Petromzons marinus) is an invasive ectoparasite of large-bodied fishes that adversely affects the fishing industry and ecology of the Laurentian Great Lakes. Lipid content in the whole sea lamprey and muscles, liver and kidney of metamorphosing larval stages has been reported. Similarly, the fatty acid profile of the rope tissues of sexually-mature male sea lampreys has also been reported. The average body weight of a sub-adult migratory sea lamprey is 250 g, which includes 14.4% skin (36 g). Our preliminary extraction data of an adult sea lamprey skin revealed that it contained approximately 8.5% of lipophilic compounds. Lamprey skin is home to a naturally aversive compound (an alarm cue) that is being developed into a repellent for use in pest management. As part of an ongoing investigation to identify the chemical structure of the sea lamprey alarm cue, we extracted the skin with water and methanol, respectively. The methanolic extract (1.55%) contained exclusively lipophilic compounds and did not include the alarm cue. We chemically characterized all compounds present in the methanolic extract as cholesterol esters (CE), tri- and di-glycerides (TG and DG), cholesterol, free fatty acids (FFA) and minor amounts of plasticizers. The free fatty acids fraction was composed of saturated (41.8%), monounsaturated (40.7%) and polyunsaturated (17.4%) fatty acids, respectively. The plasticizers characterized were phthalate and benzoate and found to be 0.95 mg and 2.54 mg, respectively, per adult sea lamprey skin. This is the first report of the chemical characterization of all the lipophilic constituents in the skin of sub-adult migratory sea lamprey. The CEs isolated and characterized from sea lamprey skin are also for the first time. PMID:27992570

  4. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin

    PubMed Central

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-01-01

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts. PMID:27428951

  5. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    PubMed

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-07-14

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

  6. Metabolic conversion of 12-O-tetradecanoylphorbol-13-acetate in adult and newborn mouse skin and mouse liver microsomes.

    PubMed

    Berry, D L; Bracken, W M; Fischer, S M; Viaje, A; Slaga, T J

    1978-08-01

    Tritiated 12-O-tetradecanoylphorbol-13-acetate (TPA) was applied to adult mouse skin; at specified time intervals the mice were killed, and the labeled phorbol was extracted and subjected to separation and quantitation by high-pressure liquid chromatography. After 24 hr, TPA comprised greater than 96% of the recovered label from the skin, and its apparent half-life was 17.8 hr. Pretreatment of adult skin with TPA for 4 weeks before treatment with labeled TPA resulted in an increase in the clearance rate of TPA from the skin. Skin from newborn mice was capable of converting TPA into monoesters and phorbol, but the clearance rate in the adult was about 12 times more rapid than it was in the newborn. Epidermal homogenates converted TPA into 12-O-tetradecanoylphorbol, phorbol-13-acetate, and phorbol. Hepatic homogenates were able to convert TPA to monoesters and phorbol at rates 14 to 15 times faster than were epidermal homogenates. Attempts to isolate any previously undescribed metabolites of TPA by use of liver homogenates were unsuccessful, and mixed-function oxidation did not contribute to the metabolism of TPA. From inhibitor studies it was judged that esterases were implicated in the conversion of TPA to monoesters and phorbol. The results support the hypothesis that the tumor-promoting activity of TPA is directly related to its concentration in a specific tissue and that conversion of TPA to an active metabolite probably does not occur.

  7. CAF-like state in primary skin fibroblasts with constitutional BRCA1 epimutation sheds new light on tumor suppressor deficiency-related changes in healthy tissue

    PubMed Central

    Etzold, Anna; Galetzka, Danuta; Weis, Eva; Bartsch, Oliver; Haaf, Thomas; Spix, Claudia; Itzel, Timo; Schweiger, Susann; Strand, Dennis; Strand, Susanne; Zechner, Ulrich

    2016-01-01

    ABSTRACT Constitutive epimutations of tumor suppressor genes are increasingly considered as cancer predisposing factors equally to sequence mutations. In light of the emerging role of the microenvironment for cancer predisposition, initiation, and progression, we aimed to characterize the consequences of a BRCA1 epimutation in cells of mesenchymal origin. We performed a comprehensive molecular and cellular comparison of primary dermal fibroblasts taken from a monozygous twin pair discordant for recurrent cancers and BRCA1 epimutation, whose exceptional clinical case we previously reported in this journal. Comparative transcriptome analysis identified differential expression of extracellular matrix-related genes and pro-tumorigenic growth factors, such as collagens and CXC chemokines. Moreover, genes known to be key markers of so called cancer-associated fibroblasts (CAFs), such as ACTA2, FAP, PDPN, and TNC, were upregulated in fibroblasts of the affected twin (BRCA1mosMe) in comparison to those of the healthy twin (BRCA1wt). Further analyses detected CAF-typical cellular features, including an elevated growth rate, enhanced migration, altered actin architecture and increased production of ketone bodies in BRCA1mosMe fibroblasts compared to BRCA1wt fibroblasts. In addition, conditioned medium of BRCA1mosMe fibroblasts was more potent than conditioned medium of BRCA1wt fibroblasts to promote cell proliferation in an epithelial and a cancer cell line. Our data demonstrate, that a CAF-like state is not an exclusive feature of tumor-associated tissue but also exists in healthy tissue with tumor suppressor deficiency. The naturally occurring phenomenon of twin fibroblasts differing in their BRCA1 methylation status revealed to be a unique powerful tool for exploring tumor suppressor deficiency-related changes in healthy tissue, reinforcing their significance for cancer predisposition. PMID:26949839

  8. Cardiac fibroblast-derived extracellular matrix (biomatrix) as a model for the studies of cardiac primitive cell biological properties in normal and pathological adult human heart.

    PubMed

    Castaldo, Clotilde; Di Meglio, Franca; Miraglia, Rita; Sacco, Anna Maria; Romano, Veronica; Bancone, Ciro; Della Corte, Alessandro; Montagnani, Stefania; Nurzynska, Daria

    2013-01-01

    Cardiac tissue regeneration is guided by stem cells and their microenvironment. It has been recently described that both cardiac stem/primitive cells and extracellular matrix (ECM) change in pathological conditions. This study describes the method for the production of ECM typical of adult human heart in the normal and pathological conditions (ischemic heart disease) and highlights the potential use of cardiac fibroblast-derived ECM for in vitro studies of the interactions between ECM components and cardiac primitive cells responsible for tissue regeneration. Fibroblasts isolated from adult human normal and pathological heart with ischemic cardiomyopathy were cultured to obtain extracellular matrix (biomatrix), composed of typical extracellular matrix proteins, such as collagen and fibronectin, and matricellular proteins, laminin, and tenascin. After decellularization, this substrate was used to assess biological properties of cardiac primitive cells: proliferation and migration were stimulated by biomatrix from normal heart, while both types of biomatrix protected cardiac primitive cells from apoptosis. Our model can be used for studies of cell-matrix interactions and help to determine the biochemical cues that regulate cardiac primitive cell biological properties and guide cardiac tissue regeneration.

  9. Spinal dorsal horn cell receptive field size is increased in adult rats following neonatal hindpaw skin injury.

    PubMed

    Torsney, Carole; Fitzgerald, Maria

    2003-07-01

    Local tissue damage in newborn rats can lead to changes in skin sensitivity that last into adulthood and this is likely to be due to plasticity of developing peripheral and central sensory connections. This study examines the functional connections of dorsal horn neurons in young and adult rats that have undergone local skin damage at birth. Newborn rat pups were halothane anaesthetised and received either a unilateral subcutaneous plantar injection of 1 % lambda-carrageenan or a unilateral plantar foot injury made by removal of 2 mm x 2 mm of skin. At 3 weeks, (postnatal day (P) 19-23) and 6 weeks (P40-44) in vivo extracellular recordings of single dorsal horn cells with plantar cutaneous receptive fields were made under urethane anaesthesia (2 g kg-1) and responses to mechanical and electrical stimulation of the skin were assessed. Following neonatal carrageenan inflammation, dorsal horn neuron properties and receptive field sizes at 3 weeks were the same as those of controls. In contrast, following neonatal skin injury, dorsal horn cell receptive field sizes were significantly greater than those of controls at 3 weeks (2.5-fold) and at 6 weeks (2.2-fold). Mechanical thresholds, mechanical response magnitudes and evoked responses to single and repeated A and C fibre stimulation remained unaffected. These results show that early skin injury can cause prolonged changes in central sensory connections that persist into adult life, long after the skin has healed. Enlarged dorsal horn neuron receptive field sizes provide a physiological mechanism for the persistent behavioural hypersensitivity that follows neonatal skin injury in rats and for the prolonged sensory changes reported in human infants after early pain and injury.

  10. Practical application of cellular bioenergetics to the care of aged skin.

    PubMed

    Osborne, R; Carver, R S; Mullins, L A; Finlay, D R

    2013-07-01

    In human skin fibroblasts in vitro, procollagen-1 and NAD(+)/NADH were reduced in three strains of adult fibroblasts compared with neonatal fibroblasts. The levels of both procollagen-1 and NAD(+)/NADH were increased in the adult fibroblasts by treatment for 24 (NAD energy) or 48 h (procollagen-1) with a complex containing niacinamide, Pal-KTTKS peptide and an olive oil fatty acid derivative (Olivem(®)), especially in combination with a natural extract from dill (Lys'lastine V(®)). In one of the adult fibroblast strains evaluated, these changes in procollagen-1 and NAD(+)/NADH in response to the complex of bioactives were in parallel with increased expression of mRNA biomarkers related primarily to dermal matrix and basement membrane structure, including COL1A1, COL3A1, COL5A1, COL14A1, ELN and LOXL2, in addition to SOD2, NAMPT and TGFBR3; MMP1 was decreased in expression. In general, these mRNA biomarker effects were maintained or boosted by the addition of Lys'lastine V, particularly at 1%, and were similar to the fold changes in mRNA expression in neonatal compared with adult fibroblasts. These results indicate that the complex of niacinamide, Pal-KTTKS and Olivem, especially with addition of Lys'lastine V, increases the NAD(+)/NADH bioenergy level of adult skin fibroblasts in parallel with increased expression of skin structure biomarkers in vitro to levels similar to those in younger fibroblasts. Thus, niacinamide, Pal-KTTKS, Olivem and Lys'lastine V are promising bioactive candidates for inclusion in cosmetic formulations.

  11. Effects of transforming growth factor-beta1 and tumour necrosis factor-alpha on cultured fibroblasts from skin fibroma as modulated by toremifene.

    PubMed

    Lilli, Cinzia; Marinucci, Lorella; Bellocchio, Silvia; Ribatti, Domenico; Balducci, Chiara; Baroni, Tiziano; Cagini, Lucio; Giustozzi, Giammario; Locci, Paola

    2002-04-20

    To determine how toremifene, an anti-oestrogen triphenylethylene derivate, reduces tumour mass, we investigated its modulation of TGF-beta1 and TNF-alpha in fibroma fibroblasts. Normal and fibroma fibroblasts, isolated from patients affected by Gardner's syndrome without or with fibroma manifestation, were cultured in vitro. Secretion of GAG, collagen and TGF-beta1 was increased in fibroma fibroblasts compared to healthy cells. The increase in TGF-beta1 secretion into the medium was associated with a parallel increase in TGF-beta1 gene expression and receptor number. Receptor cross-linking studies using radiolabelled TGF-beta1 revealed more receptors, particularly types I and II, in fibroma fibroblasts than in normal cells. Normal and fibroma fibroblasts did not synthesise TNF-alpha, but they had TNF-alpha membrane receptors, as shown by TNF-alpha assay. TNF-alpha secreted by human monocytes, which may be present in the peritumoral area, increased cell proliferation and GAG accumulation and was, in turn, enhanced by TGF-beta1 treatment. Both growth factors increased angiogenesis, as shown by the CAM assay. Toremifene reduced TGF-beta1 secretion by fibroma fibroblasts and TNF-alpha secretion by monocytes, thus downregulating cell proliferation, ECM macromolecule accumulation and angiogenic progression. We hypothesise that increased TGF-beta1 gene expression and TGF-beta1 secretion in fibroma fibroblasts as well as the subsequent rise in TNF-alpha production by monocytes may facilitate fibroma growth and that toremifene inhibits autocrine and paracrine growth factor production.

  12. Inhibition of β-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing

    PubMed Central

    Gomez, Celine; Pisco, Angela Oliveira; Rawlins, Emma L.; Simons, Ben D.

    2016-01-01

    New hair follicles (HFs) do not form in adult mammalian skin unless epidermal Wnt signalling is activated genetically or within large wounds. To understand the postnatal loss of hair forming ability we monitored HF formation at small circular (2 mm) wound sites. At P2, new HFs formed in back skin, but HF formation was markedly decreased by P21. Neonatal tail also formed wound-associated HFs, albeit in smaller numbers. Postnatal loss of HF neogenesis did not correlate with wound closure rate but with a reduction in Lrig1-positive papillary fibroblasts in wounds. Comparative gene expression profiling of back and tail dermis at P1 and dorsal fibroblasts at P2 and P50 showed a correlation between loss of HF formation and decreased expression of genes associated with proliferation and Wnt/β-catenin activity. Between P2 and P50, fibroblast density declined throughout the dermis and clones of fibroblasts became more dispersed. This correlated with a decline in fibroblasts expressing a TOPGFP reporter of Wnt activation. Surprisingly, between P2 and P50 there was no difference in fibroblast proliferation at the wound site but Wnt signalling was highly upregulated in healing dermis of P21 compared with P2 mice. Postnatal β-catenin ablation in fibroblasts promoted HF regeneration in neonatal and adult mouse wounds, whereas β-catenin activation reduced HF regeneration in neonatal wounds. Our data support a model whereby postnatal loss of hair forming ability in wounds reflects elevated dermal Wnt/β-catenin activation in the wound bed, increasing the abundance of fibroblasts that are unable to induce HF formation. PMID:27287810

  13. Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts

    SciTech Connect

    Antoniades, H.N. Center for Blood Research, Boston, MA Inst. of Molecular Biology, Boston, MA ); Galanopoulos, T.; Neville-Golden, J.; Kiritsy, C.P. Inst. of Molecular Biology, Boston, MA ); Lynch, S.E. Inst. of Molecular Biology, Boston, MA Harvard School of Dental Medicine, Boston, MA )

    1991-01-15

    Platelet-derived growth factor (PDGF) stimulates many of the processes important in tissue repair, including proliferation of fibroblasts and synthesis of extracellular matrices. In this study, the authors have demonstrated with in situ hydridization and immunocytochemistry the reversible expression of 3-sis/PDGF-2 and PDGF receptor (PDGF-R) b mRNAs and their respective protein products in epithelial cells and fibroblasts following cutaneous injury in pigs. Epithelial cells in control, unwounded skin did not express c-sis and PDGF-R mRNAs, and fibroblasts expressed only PDGF-R mRNA. The expression levels in the injured site were correlated with the stage of tissue repair, being highest during the initial stages of the repair process and declining at the time of complete re-epithelialization and tissue remodeling. These studies provide a mulecular basis for understanding the mechanisms contributing to normal tissue repair. They suggest the possibility that a defect in these mechanisms may be associated with defective wound healing. It is also conceivable that chronic injury may induce irreversible gene expression leading to pathologic, unregulated cell growth.

  14. Chitosan/TiO2 composite membrane improves proliferation and survival of L929 fibroblast cells: Application in wound dressing and skin regeneration.

    PubMed

    Behera, Sudhanshu Shekhar; Das, Urmimala; Kumar, Awanish; Bissoyi, Akalabya; Singh, Abhishek Kumar

    2017-05-01

    The microbial infection and growth of fibroblasts are the critical factors for the effective wound healing. The natural polymer-based dressing membranes may mimic extracellular matrix to support the survival, proliferation and differentiation of fibroblasts. The present study deals with the preparation of chitosan/titanium dioxide (CS/TiO2) composite membranes with different degree of TiO2 incorporation, and their characterization in terms of morphology, ultrastructure, thermal behavior and mechanical properties with SEM, FTIR, XRD and tensile strength analyses. The data demonstrated the formation of strong O-Ti-O bonding between TiO2 and CS resulting in superior porosity, mechanical strength, crystallinity and flexibility of the composite membranes. Further, the cyto-compatibility, proliferation, oxidative stress, cell cycle and apoptosis analyses of fibroblast L929 cells demonstrated the enhanced proliferation and survival, and decreased oxidative stress and apoptosis in L929 cells grown on CS/TiO2 membrane incorporated with 025% TiO2. Next, we measured the significant up-regulation in the expression of fibroblast-markers in L929 cells cultured on CS/TiO2 (0.25%) membrane. Furthermore, the CS/TiO2 composite membranes exhibited a superior antibacterial activity against Staphylococcus aureus. Taken together, the data confirmed that CS/TiO2 (0.25%) membrane improved the growth, survival and functional integrity of fibroblasts, and exerted antibacterial activity which may be utilized as potential dressing materials.

  15. CD99 Is Strongly Expressed in Basal Cells of the Normal Adult Epidermis and Some Subpopulations of Appendages: Comparison with Developing Fetal Skin

    PubMed Central

    Choi, Gawon; Roh, Jin; Park, Chan-Sik

    2016-01-01

    Background CD99 is a cell surface transmembrane glycoprotein expressed in various tissues. CD99 is differentially expressed between subpopulations of each tissue and is highly expressed in certain hematopoietic and precursor cells. However, there has been no comprehensive study of CD99 expression in normal skin. We evaluated CD99 expression in normal human skin and developing fetal skin. Methods Seventy-five adult skin samples containing normal skin and eight fetal skin samples of different gestational ages were collected. CD99 immunohistochemical staining was performed to evaluate expression pattern in adult and fetal skin samples. CD99 and CD34 expression were compared by double immunofluorescence. Results In normal adult skin, CD99 was strongly expressed in the membrane of epidermal basal keratinocytes, hair follicle bulges and outer root sheaths, and inner secretory cells of eccrine sweat glands. In fetal skin, CD99 was not expressed on the periderm at 16 weeks of gestation but was expressed in basal cells of fetal skin at around 19 weeks of gestation. CD99 expression became comparable to that of the adult skin after 20 weeks of gestation. CD99 and CD34 were co-expressed in hair follicle outer root sheaths, as seen by double immunofluorescence study. Conclusions This is the first study examining CD99 expression pattern in normal adult and fetal skin. CD99 tends to be expressed in the basal/precursor cells of epidermis and in hair follicles. These results provide a basis for future investigation on functions of CD99 in the skin and provide a novel potential target for the treatment of dermatologic lesions. PMID:27498544

  16. Functional analysis of keratinocyte and fibroblast gene expression in skin and keloid scar tissue based on deviation analysis of dynamic capabilities

    PubMed Central

    Li, Mingming; Wu, Lei

    2016-01-01

    The aim of the present study was to select key genes that are associated with fibroblasts and keratinocytes during keloid scar progression and development. The gene expression profile of GSE44270, which includes 32 samples, was downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) in case samples compared with control samples were screened using the Limma R package followed by hierarchical clustering analysis. Protein-protein interaction (PPI) networks of the total selected DEGs were constructed using Cytoscape. Moreover, the Gene Ontology biological processes and significant Kyoto Encyclopedia of Genes and Genomes pathways of the total selected DEGs were enriched using the Database for Annotation, Visualization and Integrated Discovery. Significant pathways that may be associated with keloid scar were analyzed using deviation analysis of dynamic capabilities. There were 658 DEGs in fibroblast keloid vs. normal, 112 DEGs in fibroblast non-lesion vs. normal, 439 DEGs in fibroblast keloid vs. non-lesion, 523 DEGs in keratocyte keloid vs. normal, 186 DEGs in keratocyte non-lesion vs. normal, and 963 DEGs in keratocyte keloid vs. non-lesion groups. HOXA9, BMP4, CDKN1A and SMAD2 in fibroblasts, and HOXA7, MCM8, PSMA4 and PSMB2 in keratinocytes were key genes in the PPI networks. Moreover, the amino sugar and nucleotide sugar metabolism pathway, cell cycle, and extracellular matrix (ECM)-receptor interaction pathway were significant pathways. This study suggests that several key genes (BMP4, HOXA9, SMAD2, CDKN1A, HOXA7, PSMA4 and PSMB2) that participate in some significant pathways (cell cycle and ECM-receptor interaction pathways) may be potential therapeutic targets for keloid scars. PMID:28101157

  17. Visualization of the initiation and sequential expansion of the metamorphic conversion of anuran larval skin into the precursor of adult type.

    PubMed

    Tamakoshi, T; Oofusa, K; Yoshizato, K

    1998-02-01

    A tadpole of bullfrog, Rana catesbeiana, is originally covered with the larval skin over its entire body. Drastic changes arise in both the epidermis and the subcutaneous connective tissue at an early developmental stage, producing the precursor of adult type skin (pre-adult skin). It was found that calcium is a useful probe to detect the region where the precursor formation has occurred because its deposition in the upper part of subcutaneous collagen bundles coincides with the appearance of the pre-adult skin. Whole-mount in situ staining of tadpoles with alizarin red S revealed the initiation site of the premetamorphic transformation of the larval skin into the adult precursor and its ensuing region-dependent expansion. The pre-adult skin first emerged at TK II to III (TK, Taylor and Kollros staging) t lateral sides of the body, which led us to postulate that 'the center for premetamorphic skin transformation' is formed at the specific site in this region. This center moved dorsally and then ventrally, then reached to the most proximal region of the tail, yielding a unique sequential conversion pattern by around TK V when the conversion was completed in the trunk. The present study also visualized the process of the hindlimb skin transformation.

  18. Inactivation of fibroblast growth factor receptor signaling in myelinating glial cells results in significant loss of adult spiral ganglion neurons accompanied by age-related hearing impairment.

    PubMed

    Wang, S J; Furusho, M; D'Sa, C; Kuwada, S; Conti, L; Morest, D K; Bansal, R

    2009-11-15

    Hearing loss has been attributed to many factors, including degeneration of sensory neurons in the auditory pathway and demyelination along the cochlear nerve. Fibroblast growth factors (FGFs), which signal through four receptors (Fgfrs), are produced by auditory neurons and play a key role in embryonic development of the cochlea and in neuroprotection against sound-induced injury. However, the role of FGF signaling in the maintenance of normal auditory function in adult and aging mice remains to be elucidated. Furthermore, the contribution of glial cells, which myelinate the cochlear nerves, is poorly understood. To address these questions, we generated transgenic mice in which Fgfr1 and Fgfr2 were specifically inactivated in Schwann cells and oligodendrocytes but not in neurons. Adult mutant mice exhibited late onset of hearing impairment, which progressed markedly with age. The hearing impairment was accompanied by significant loss of myelinated spiral ganglion neurons. The pathology extended into the cochlear nucleus, without apparent loss of myelin or of the deletion-bearing glial cells themselves. This suggests that perturbation of FGF receptor-mediated glial function leads to the attenuation of glial support of neurons, leading to their loss and impairment of auditory functions. Thus, FGF/FGF receptor signaling provides a potentially novel mechanism of maintaining reciprocal interactions between neurons and glia in adult and aging animals. Dysfunction of glial cells and FGF receptor signaling may therefore be implicated in neurodegenerative hearing loss associated with normal aging.

  19. The use of teledermoscopy in the accurate identification of cancerous skin lesions in the adult population: a systematic review.

    PubMed

    Bruce, Amy F; Mallow, Jennifer A; Theeke, Laurie A

    2017-01-01

    Background The use of teledermoscopy in the diagnostic management of pre-cancerous and cancerous skin lesions involves digital dermoscopic images transmitted over telecommunication networks via email or web applications. Teledermoscopy may improve the accuracy in clinical diagnoses of melanoma skin cancer if integrated into electronic medical records and made available to rural communities, potentially leading to decreased morbidity and mortality. Objective and method The purpose of this paper is to present a systematic review of evidence on the use of teledermoscopy to improve the accuracy of skin lesion identification in adult populations. The PRISMA method guided the development of this systematic review. A total of seven scholarly databases were searched for articles published between the years of 2000 and 2015. All studies were critically appraised using the Rosswurm and Larrabee critique worksheet, placed in a matrix for comparison evaluating internal and external validity and inspected for homogeneity of findings. Results Sixteen articles met inclusion criteria for this review. A majority of the studies were cross-sectional and non-experimental. Ten of the 16 focused on interobserver concordance and diagnostic agreement between teledermoscopy and another comparator. Instrumentation in conducting the studies showed inconsistency with reported results. Discussion Higher level evidence is needed to support clinical application of teledermoscopy for accuracy of diagnostic measurement in the treatment of pre-cancerous and cancerous skin lesions in adults. Future research is needed to develop a standardized, reliable and valid measurement tool for implementation in clinical practice.

  20. Light Emitting Diode-Generated Blue Light Modulates Fibrosis Characteristics: Fibroblast Proliferation, Migration Speed, and Reactive Oxygen Species Generation

    PubMed Central

    Mamalis, Andrew; Garcha, Manveer; Jagdeo, Jared

    2016-01-01

    Background and Objective Blue light is part of the visible light spectrum that does not generate harmful DNA adducts associated with skin cancer and photoaging, and may represent a safer therapeutic modality for treatment of keloid scars and other fibrotic skin diseases. Our laboratory previously demonstrated that light-emitting diode (LED) red and infrared light inhibits proliferation of skin fibroblasts. Moreover, different wavelengths of light can produce different biological effects. Furthermore, the effects of LED blue light (LED-BL) on human skin fibroblasts are not well characterized. This study investigated the effects of LED-BL on human skin fibroblast proliferation, viability, migration speed, and reactive oxygen-species (ROS) generation. Methods and Materials Irradiation of adult human skin fibroblasts using commercially-available LED-BL panels was performed in vitro, and modulation of proliferation and viability was quantified using the trypan blue dye exclusion assay, migratory speed was assessed using time-lapse video microscopy, and intracellular ROS generation was measured using the dihydrorhodamine flow cytometry assay. Statistical differences between groups were determined by ANOVA and Student s t-test. Results Human skin fibroblasts treated with LED-BL fluences of 5, 30, 45, and 80 J/cm2 demonstrated statistically significant dose-dependent decreases in relative proliferation of 8.4%, 29.1%, 33.8%, 51.7%, and 55.1%, respectively, compared to temperature and environment matched bench control plates, respectively. LED-BL fluences of 5, 30, 45 and 80 J/cm2 decreased fibroblast migration speed to 95 ± 7.0% (p = 0.64), 81.3 ± 5.5% (p = 0.021), 48.5 ± 2.7% (p < 0.0001), and 32.3 ± 1.9% (p < 0.0001), respectively, relative to matched controls. LED fluences of 5, 10, 30, and 80 J/cm2 resulted in statistically significant increases in reactive oxygen species of 110.4%, 116.6%, 127.5%, and 130%, respectively, relative to bench controls. Conclusion At

  1. Environment and activity affect skin temperature in breeding adult male elephant seals (Mirounga angustirostris).

    PubMed

    Norris, A L; Houser, D S; Crocker, D E

    2010-12-15

    The large body size and high rates of metabolic heat production associated with male mating success in polygynous systems creates potential thermoregulatory challenges for species breeding in warm climates. This is especially true for marine predators carrying large blubber reserves intended for thermoregulation in cold water and fuel provision during extended fasts. Thermographic images were used to measure changes in skin temperature (T(S)) in adult male northern elephant seals (Mirounga angustirostris) over the breeding season. Environmental variables, primarily ambient temperature and solar radiation, were the principal determinants of mean and maximum T(S). When controlled for environmental variables, dominance rank significantly impacted mean T(S), being highest in alpha males. Behavioral activity significantly influenced T(S) but in a counter-intuitive way, with inactive males exhibiting the highest T(S). This was likely due to strong impacts of environmental variables on the kinds of behavior exhibited, with males being less active on warm, humid days at peak solar radiation. We classified thermal windows as areas in which T(S) was one standard deviation greater than mean T(S) for the individual seal within a thermograph. Thermal features suggest active physiological thermoregulation during and after combat and significant circulatory adaptations for heat dumping, as evidenced by recurring locations of thermal windows representing widely varying T(S) values. Frequent observations of localized T(S) above 37°C, particularly after combat, suggest the production of thermoregulatory stress during breeding behavior. Our findings demonstrate the importance of environmental drivers in shaping activity patterns during breeding and provide evidence for thermoregulatory costs of successful breeding in large polygynous males.

  2. Preliminary Study of Altered Skin Temperature at Body Sites Associated with Self-Injurious Behavior in Adults Who Have Developmental Disabilities.

    ERIC Educational Resources Information Center

    Symons, Frank J.; Sutton, Kelly A.; Bodfish, James W.

    2001-01-01

    The sensory status of four nonverbal adults with mental retardation and severe self-injury was examined using skin temperature measures prior to opiate antagonist treatment. For each participant, the body site targeted most frequently for self-injury was associated with altered skin temperature and reduced by naltrexone treatment. In all cases,…

  3. Adult human brain neural progenitor cells (NPCs) and fibroblast-like cells have similar properties in vitro but only NPCs differentiate into neurons.

    PubMed

    Park, Thomas In-Hyeup; Monzo, Hector; Mee, Edward W; Bergin, Peter S; Teoh, Hoon H; Montgomery, Johanna M; Faull, Richard L M; Curtis, Maurice A; Dragunow, Mike

    2012-01-01

    The ability to culture neural progenitor cells from the adult human brain has provided an exciting opportunity to develop and test potential therapies on adult human brain cells. To achieve a reliable and reproducible adult human neural progenitor cell (AhNPC) culture system for this purpose, this study fully characterized the cellular composition of the AhNPC cultures, as well as the possible changes to this in vitro system over prolonged culture periods. We isolated cells from the neurogenic subventricular zone/hippocampus (SVZ/HP) of the adult human brain and found a heterogeneous culture population comprised of several types of post-mitotic brain cells (neurons, astrocytes, and microglia), and more importantly, two distinct mitotic cell populations; the AhNPCs, and the fibroblast-like cells (FbCs). These two populations can easily be mistaken for a single population of AhNPCs, as they both proliferate under AhNPC culture conditions, form spheres and express neural progenitor cell and early neuronal markers, all of which are characteristics of AhNPCs in vitro. However, despite these similarities under proliferating conditions, under neuronal differentiation conditions, only the AhNPCs differentiated into functional neurons and glia. Furthermore, AhNPCs showed limited proliferative capacity that resulted in their depletion from culture by 5-6 passages, while the FbCs, which appear to be from a neurovascular origin, displayed a greater proliferative capacity and dominated the long-term cultures. This gradual change in cellular composition resulted in a progressive decline in neurogenic potential without the apparent loss of self-renewal in our cultures. These results demonstrate that while AhNPCs and FbCs behave similarly under proliferative conditions, they are two different cell populations. This information is vital for the interpretation and reproducibility of AhNPC experiments and suggests an ideal time frame for conducting AhNPC-based experiments.

  4. The expression of β-galactosidase during long-term cultured goat skin fibroblasts and the effect of donor cell passage on in vitro development of nuclear transfer embryos.

    PubMed

    Liu, Haijun; Peng, Hui; Liu, Fang; Ma, Qun; Zhang, Wenchang

    2016-05-01

    The present study aimed to detect the expression of β-galactosidase during long-term cultured goat skin fibroblasts and investigate the effects of donor goat age, sex, and cell passage on senescence and the effects of donor cell passage on in vitro development of nuclear transfer embryos. The results showed that, in the same cell passage, more β-galactosidase-positive cells were detected in cells from older donors than younger donors. Irrespective of the donor age, the number of positive cells was higher in later passages from passages 20 to 50. In the same passage from 20 to 50, the β-galactosidase-positive rate was higher in cells from 5-yr female goat than 5-yr male goat. Using fibroblasts from male goats at various passages as donor cells, reconstructed embryos had similar fusion and cleavage rates, but the blastocyst rate was higher for cells at passages 10 and 20 than passage 30. In conclusion, donor goat age and cell passage had significant effects on the β-galactosidase-positive rate; also, cells from 5-yr female goat had a higher β-galactosidase-positive rate than those from 5-yr male goat, and the donor cell passage affected the developmental potential of nuclear transfer embryos.

  5. The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity

    PubMed Central

    Plikus, Maksim V.; Van Spyk, Elyse Noelani; Pham, Kim; Geyfman, Mikhail; Kumar, Vivek; Takahashi, Joseph S.; Andersen, Bogi

    2015-01-01

    Historically work on peripheral circadian clocks has been focused on organs and tissues that have prominent metabolic functions, such as liver, fat and muscle. In recent years, skin is emerging as a model for studying circadian clock regulation of cell proliferation, stem cell functions, tissue regeneration, aging and carcinogenesis. Morphologically skin is complex, containing multiple cell types and structures, and there is evidence for a functional circadian clock in most, if not all, of its cell types. Despite the complexity, skin stem cell populations are well defined, experimentally tractable and exhibit prominent daily cell proliferation cycles. Hair follicle stem cells also participate in recurrent, long-lasting cycles of regeneration -- the hair growth cycles. Among other advantages of skin is a broad repertoire of available genetic tools enabling the creation of cell-type specific circadian mutants. Also, due to the accessibility of the skin, in vivo imaging techniques can be readily applied to study the circadian clock and its outputs in real time, even at the single-cell level. Skin provides the first line of defense against many environmental and stress factors that exhibit dramatic diurnal variations such as solar UV radiation and temperature. Studies have already linked the circadian clock to the control of UVB-induced DNA damage and skin cancers. Due to the important role that skin plays in the defense against microorganisms, it represents a promising model system to further explore the role of the clock in the regulation of the body's immune functions. To that end, recent studies have already linked the circadian clock to psoriasis, one of the most common immune-mediated skin disorders. The skin also provides opportunities to interrogate clock regulation of tissue metabolism in the context of stem cells and regeneration. Furthermore, many animal species feature prominent seasonal hair molt cycles, offering an attractive model for investigating the

  6. The circadian clock in skin: implications for adult stem cells, tissue regeneration, cancer, aging, and immunity.

    PubMed

    Plikus, Maksim V; Van Spyk, Elyse N; Pham, Kim; Geyfman, Mikhail; Kumar, Vivek; Takahashi, Joseph S; Andersen, Bogi

    2015-06-01

    Historically, work on peripheral circadian clocks has been focused on organs and tissues that have prominent metabolic functions, such as the liver, fat, and muscle. In recent years, skin has emerged as a model for studying circadian clock regulation of cell proliferation, stem cell functions, tissue regeneration, aging, and carcinogenesis. Morphologically, skin is complex, containing multiple cell types and structures, and there is evidence for a functional circadian clock in most, if not all, of its cell types. Despite the complexity, skin stem cell populations are well defined, experimentally tractable, and exhibit prominent daily cell proliferation cycles. Hair follicle stem cells also participate in recurrent, long-lasting cycles of regeneration: the hair growth cycles. Among other advantages of skin is a broad repertoire of available genetic tools enabling the creation of cell type-specific circadian mutants. Also, due to the accessibility of skin, in vivo imaging techniques can be readily applied to study the circadian clock and its outputs in real time, even at the single-cell level. Skin provides the first line of defense against many environmental and stress factors that exhibit dramatic diurnal variations such as solar ultraviolet (UV) radiation and temperature. Studies have already linked the circadian clock to the control of UVB-induced DNA damage and skin cancers. Due to the important role that skin plays in the defense against microorganisms, it also represents a promising model system to further explore the role of the clock in the regulation of the body's immune functions. To that end, recent studies have already linked the circadian clock to psoriasis, one of the most common immune-mediated skin disorders. Skin also provides opportunities to interrogate the clock regulation of tissue metabolism in the context of stem cells and regeneration. Furthermore, many animal species feature prominent seasonal hair molt cycles, offering an attractive model

  7. Development of a chemically defined in vitro culture system to effectively stimulate the proliferation of adult human dermal fibroblasts.

    PubMed

    Kim, Min Seong; Yun, Jung Im; Gong, Seung Pyo; Ahn, Ji Yeon; Lim, Jeong Mook; Song, Young Han; Park, Kyu Hyun; Lee, Seung Tae

    2015-07-01

    Despite the fact that dermal fibroblasts are a practical model for research related to cell physiology and cell therapy, an in vitro culture system excluding serum, which complicates standardization and specificity and induces variability and unwanted effects, does not exist. We tried to establish a CDCS that supports effective proliferation of aHDFs. KDMEM supplemented with 5% (v/v) KSR, 12 ng/ml bFGF, 5 ng/ml EGF and 1 μg/ml hydrocortisone supported sufficient proliferation of aHDFs for 1 week. However, aHDF proliferation was decreased greatly after subculture. This problem could be overcome by culturing aHDFs in CDCM in culture plates coated with 10 μg/ml FN. Long-term culture of aHDFs was achieved using CDCM and FN-coated culture plates for 7 weeks. The optimized CDCS increased the proliferation of aHDFs significantly, without any increase in the senescence rate or alteration in morphology of aHDFs, despite long-term culture. In conclusion, we established a CDCS that improved proliferation of aHDFs while inhibiting cellular senescence. The CDCS will contribute to advances in various future research related to clinical skin regeneration.

  8. MicroRNA-29a in Adult Muscle Stem Cells Controls Skeletal Muscle Regeneration During Injury and Exercise Downstream of Fibroblast Growth Factor-2.

    PubMed

    Galimov, Artur; Merry, Troy L; Luca, Edlira; Rushing, Elisabeth J; Mizbani, Amir; Turcekova, Katarina; Hartung, Angelika; Croce, Carlo M; Ristow, Michael; Krützfeldt, Jan

    2016-03-01

    The expansion of myogenic progenitors (MPs) in the adult muscle stem cell niche is critical for the regeneration of skeletal muscle. Activation of quiescent MPs depends on the dismantling of the basement membrane and increased access to growth factors such as fibroblast growth factor-2 (FGF2). Here, we demonstrate using microRNA (miRNA) profiling in mouse and human myoblasts that the capacity of FGF2 to stimulate myoblast proliferation is mediated by miR-29a. FGF2 induces miR-29a expression and inhibition of miR-29a using pharmacological or genetic deletion decreases myoblast proliferation. Next generation RNA sequencing from miR-29a knockout myoblasts (Pax7(CE/+) ; miR-29a(flox/flox) ) identified members of the basement membrane as the most abundant miR-29a targets. Using gain- and loss-of-function experiments, we confirm that miR-29a coordinately regulates Fbn1, Lamc1, Nid2, Col4a1, Hspg2 and Sparc in myoblasts in vitro and in MPs in vivo. Induction of FGF2 and miR-29a and downregulation of its target genes precedes muscle regeneration during cardiotoxin (CTX)-induced muscle injury. Importantly, MP-specific tamoxifen-induced deletion of miR-29a in adult skeletal muscle decreased the proliferation and formation of newly formed myofibers during both CTX-induced muscle injury and after a single bout of eccentric exercise. Our results identify a novel miRNA-based checkpoint of the basement membrane in the adult muscle stem cell niche. Strategies targeting miR-29a might provide useful clinical approaches to maintain muscle mass in disease states such as ageing that involve aberrant FGF2 signaling.

  9. Culturing adult human bone marrow stem cells on gelatin scaffold with pNIPAAm as transplanted grafts for skin regeneration.

    PubMed

    Perng, Cherng-Kang; Kao, Chung-Lan; Yang, Yi-Ping; Lin, Han-Tso; Lin, Wen-Bin; Chu, Yue-Ru; Wang, Hsiao-Jung; Ma, Hsu; Ku, Hung-Hai; Chiou, Shih-Hwa

    2008-03-01

    Skin tissue engineering is a possible solution for the treatment of extensive skin defect. The ultimate goal of skin tissue engineering is to restore the complete functions of native skin, but until now the structures and functions of skins are only partially restored. By negative immunoselection (CD45 and glycophorin A), we isolated and cultivated adult human bone marrow stem cells (hBMSCs) that are of multilineage differentiation potential. In this study, we first demonstrated that by using gelatin/thermo-sensitive poly N-isopropylacrylamide (pNIPAAm) and the immunocompromised mice model, the hBMSCs possess the differentiation potential of epidermis and the capability of healing skin wounds. The in vitro observations and the results of the scanning electron microscope showed that the hBMSCs can attach and proliferate in the gelatin/thermo-sensitive pNIPAAm. To further monitor the in vivo growth effect of the hBMSCs in the skin-defected nude mice, the green fluorescence protein (GFP) gene was transduced into the hBMSCs by the murine stem cell viral vector. The results showed that the rates of cell growth and wound recovery in the hBMSC-treated group were significantly higher than those in the control group, which was only treated with the gelatin/pNIPAAm (p < 0.01). More importantly, the re-epithelialization markers of human pan-cytokeratin and E-cadherin were significantly increased on day 7, day 14, and day 21 after the hBMSC-scaffold with the pNIPAAM in the mice with skin defects (p < 0.05). Moreover, the stem cell markers of human CD13 and CD105 were gradually decreased during the period of wound healing. In sum, this novel method provides a transferring system for cell therapies and maintains its temperature-sensitive property of easy-peeling by lower-temperature treatment. In addition, the in vitro and in vivo GFP imaging systems provide a new imaging modality for understanding the differentiation process and the effective expression of stem cells in wound

  10. Odorous and Non-Fatal Skin Secretion of Adult Wrinkled Frog (Rana rugosa) Is Effective in Avoiding Predation by Snakes

    PubMed Central

    Yoshimura, Yuri; Kasuya, Eiiti

    2013-01-01

    The roles played by nonfatal secretions of adult anurans in the avoidance of predation remain unknown. The adult Wrinkled frog (Rana rugosa) has warty skin with the odorous mucus secretion that is not fatal to the snake Elaphe quadrivirgata. We fed R. rugosa or Fejervarya limnocharis, which resembles R. rugosa in appearance and has mucus secretion, to snakes and compared the snakes’ responses to the frogs. Compared to F. limnocharis, R. rugosa was less frequently bitten or swallowed by snakes. The snakes that bit R. rugosa spat out the frogs and showed mouth opening (gaping) behavior, while the snakes that bit F. limnocharis did not show gaping behavior. We also compared the responses of the snakes to R. rugosa and F. limnocharis secretions. We coated palatable R. japonica with secretions from R. rugosa or F. limnocharis. The frogs coated by R. rugosa secretion were less frequently bitten or swallowed than those coated by F. limnocharis secretion. We concluded that compared to different frog species of similar sizes, the adult R. rugosa was less frequently preyed upon by, and that its skin secretion was effective in avoiding predation by snakes. PMID:24278410

  11. Odorous and non-fatal skin secretion of adult wrinkled frog (Rana rugosa) is effective in avoiding predation by snakes.

    PubMed

    Yoshimura, Yuri; Kasuya, Eiiti

    2013-01-01

    The roles played by nonfatal secretions of adult anurans in the avoidance of predation remain unknown. The adult Wrinkled frog (Rana rugosa) has warty skin with the odorous mucus secretion that is not fatal to the snake Elaphe quadrivirgata. We fed R. rugosa or Fejervarya limnocharis, which resembles R. rugosa in appearance and has mucus secretion, to snakes and compared the snakes' responses to the frogs. Compared to F. limnocharis, R. rugosa was less frequently bitten or swallowed by snakes. The snakes that bit R. rugosa spat out the frogs and showed mouth opening (gaping) behavior, while the snakes that bit F. limnocharis did not show gaping behavior. We also compared the responses of the snakes to R. rugosa and F. limnocharis secretions. We coated palatable R. japonica with secretions from R. rugosa or F. limnocharis. The frogs coated by R. rugosa secretion were less frequently bitten or swallowed than those coated by F. limnocharis secretion. We concluded that compared to different frog species of similar sizes, the adult R. rugosa was less frequently preyed upon by, and that its skin secretion was effective in avoiding predation by snakes.

  12. Comparison between Siriraj mite allergen vaccine and standardized commercial mite vaccine by skin prick testing in normal Thai adults.

    PubMed

    Visitsunthorn, Nualanong; Pacharn, Punchama; Jirapongsananuruk, Orathai; Weeravejsukit, Sirirat; Sripramong, Chaweewan; Sookrung, Nitat; Bunnag, Chaweewan

    2010-03-01

    House dust mite is a major cause of allergic asthma and rhinitis in Thai population. Skin prick test (SPT) is a useful tool for the diagnosis of the IgE-mediated reactions. The imported commercial mite vaccine for SPT is available but it is relatively expensive. Aim of this study is to compare Siriraj Mite Allergen Vaccine (SMAV) with standardized commercial mite allergen vaccine by skin prick testing in normal Thai adults. A double blind, self-controlled study between the SMAV and standardized commercial mite allergen vaccine was performed by SPT in 17 normal Thai adult males and non-pregnant or non-lactating females aged 18-60 years. The study showed that 35.29 % of non atopic adults had positive SPT reaction to Dp and Df of both SMAV and standardized commercial mite allergen vaccine. Mean wheal and flare diameters from SPT of Dp and Df of SMAV showed strong correlation with standardized commercial mite allergen vaccine (r= 0.768 and 0.897 in Dp and Df respectively, p <0.001). The intraclass correlation was also excellent (0.893 and 0.775 in Dp and Df respectively). There was no significant difference in wheal and flare diameter between SMAV and standardized commercial mite allergen vaccine. No systemic or large local reaction was found in any of the study cases.

  13. Evaluation of the effect of laser radiation on fibroblast proliferation in repair of skin wounds of rats with iron deficiency anemia

    NASA Astrophysics Data System (ADS)

    DeCastro, Isabele C. V.; Oliveira-Sampaio, Susana C. P.; Monteiro, Juliana S. de C.; Ferreira, Maria de Fátima L.; Cangussu, Maria T.; N. dos Santos, Jean; Pinheiro, Antonio Luiz B.

    2011-03-01

    The aim of this study was to assess the effect of low- level laser therapy (LLLT) on fibroblast proliferation on wound repair of rats with Iron deficiency anemia since there is no reports on literature about this subject. Iron deficiency anemia was induced on 36 newborn rats then an excisional wound was created on the dorsum of the animals which were divided into four groups: (I) - non-anemic, (II) - Anemic, (III) - non-anemic + LLLT, (IV) Anemic+ LLLT. The animals in each group were sacrificed at 7, 14 and 21 days. Laser irradiation was performed on each group (λ660nm,40Mw,CW) by contact mode with a dose of 2,5J/ cm2 in four points on the area of the wound and total of 10J/cm2 per session. Data were evaluated by analysis of variance (ANOVA) followed by Paired t-test. The results showed LLLT was able to stimulate fibroblastic proliferation in rats with iron deficiency anemia at the 21st day while at control group (III) no statistically significant differences was found.

  14. New Wine in Old Skins: Changing Patterns in the Governing of the Adult Learner in Sweden

    ERIC Educational Resources Information Center

    Fejes, Andreas

    2005-01-01

    This paper explores the ways in which the adult learner has been governed in recent years and whether the techniques for doing this have changed over the last 50 years. The focus is first on which adult subject (adult learner) is constructed in the material analysed. What kinds of subjects are governed? This is followed by an analysis of what…

  15. The Skin-Ego: Dyadic Sensuality, Trauma in Infancy, and Adult Narcissistic Issues.

    PubMed

    Anzieu-Premmereur, Christine

    2015-10-01

    The skin-ego is a metaphor created by the French psychoanalyst Didier Anzieu to describe the process by which the infant's emerging ego develops a container for psychic contents and achieves a secure feeling of well-being. The ego encloses the psychic apparatus as the skin encloses the body. The ego becomes able to fix barriers protecting the internal world and to screen exchanges with the id, the superego, and the outside world. The skin-ego is an envelope that contains thoughts and gives to thinking activity some limits, continuity, and a protection against the instincts. The functions of the skin-ego are to maintain thoughts, to contain ideas and affects, to provide a protective shield, to register traces of primary communication with the outside world, to manage inter-sensorial correspondences, to individuate, to support sexual excitation, and to recharge the libido. The skin-ego is the foundation of the container-contained relationship. An important part of psychoanalytic work with borderline patients is the reconstruction of the earliest phases of the skin-ego and their consequences for mental organization.

  16. A study of IgE sensitization and skin response to histamine in Asian-Pacific American adults.

    PubMed

    Lee-Wong, Mary; Chou, Vivian; Silverberg, Jonathan I

    2012-01-01

    Allergic disorders and skin response to histamine have been noted to vary in different ethnicities. We investigated IgE-mediated allergic sensitization and skin response to histamine in Asian Pacific Americans (APAs), black and Hispanic Americans, and white adults. A retrospective questionnaire-based study was performed of 2222 adults presenting at a New York City allergy referral center from 1994 to 2003. Questionnaire data included sex, age, and ethnicity and personal and family history of atopic disorders. Skin-prick test (SPT) data included saline and histamine controls and response to a standardized panel of 10 aeroallergens. APA patients had a lower odds of asthma (adjusted odds ratio [aOR], 0.68; 95% confidence interval [CI], 0.52-0.89; p = 0.005) and/or animal allergies (aOR, 0.64; 95% CI, 0.50-0.82; p = 0.0003). Histamine response was not significantly different in APA (aOR, 0.90; 95% CI, 0.73-1.12; p = 0.36) or Hispanic Americans (aOR, 1.03; 95% CI, 0.85-1.24; p = 0.76), but was higher in black Americans (aOR, 2.32; 95% CI, 1.67-3.21; p < 0.0001). APA had higher odds of a positive SPT to trees (aOR, 1.49; 95% CI, 1.16-1.91; p = 0.002), grasses (aOR, 1.32; 95% CI, 1.05-1.43; p = 0.02), feathers (aOR, 1.65; 95% CI, 1.31-2.09; p < 0.0001), and cockroaches (aOR, 1.37; 95% CI, 1.10-1.62; p = 0.005). Moreover, APA had a higher total number of positive SPTs when compared with white patients (5.5 ± 3.2 versus 4.9 ± 3.3; aOR, 1.34; 95% CI, 1.10-1.62 p = 0.004). APA adults in our patient population had more IgE sensitizations but not an increased skin response to histamine. In contrast, black Americans had increased skin response to histamine.

  17. Seminoma in a postmenopausal woman with a Y;15 translocation in peripheral blood lymphocytes and a t(Y;15)/45,X Turner mosaic pattern in skin fibroblasts.

    PubMed Central

    Hoshi, N; Fujita, M; Mikuni, M; Fujino, T; Okuyama, K; Handa, Y; Yamada, H; Sagawa, T; Hareyama, H; Nakahori, Y; Fujieda, K; Kant, J A; Nagashima, K; Fujimoto, S

    1998-01-01

    We report an unusual case of a 55 year old Japanese woman with a seminoma but relatively normal menses. The patient was a phenotypic female with late onset menarche (18 years of age), who was amenorrhoeic for the first year, followed by menses of one to three days' slight flow with dysmenorrhoea, but an otherwise normal menstrual history. A typical seminoma was removed from the left adnexal region and an immature testis was identified separately as an associated right adnexal mass. Repeated karyotypic studies on peripheral blood lymphocyte cultures showed only 46,X,-Y,t(Y;15)(q12;p13). Cytogenetic examination of the patient's younger brother, who had fathered three healthy children, showed an identical karyotype. Mosaicism of 46,X,-Y,t(Y;15)(q12;p13)/45,X cell lines was found in skin samples from the patient's elbow and genital regions, although there were no clinical stigmata of Turner syndrome. An androgen receptor binding assay of cultured genital skin fibroblasts was negative. Molecular analysis using Southern blot hybridisation, PCR, and direct DNA sequencing showed that neither the patient nor her brother had a detectable deletion or other abnormalities of Y chromosome sequences, including the SRY (sex determining region of the Y chromosome) gene sequence. These findings suggest that Turner mosaicism of the 45,X cell line may have contributed to this atypical presentation in an XY female, although we cannot exclude abnormalities of other genes related to sex differentiation. Images PMID:9783712

  18. Seminoma in a postmenopausal woman with a Y;15 translocation in peripheral blood lymphocytes and a t(Y;15)/45,X Turner mosaic pattern in skin fibroblasts.

    PubMed

    Hoshi, N; Fujita, M; Mikuni, M; Fujino, T; Okuyama, K; Handa, Y; Yamada, H; Sagawa, T; Hareyama, H; Nakahori, Y; Fujieda, K; Kant, J A; Nagashima, K; Fujimoto, S

    1998-10-01

    We report an unusual case of a 55 year old Japanese woman with a seminoma but relatively normal menses. The patient was a phenotypic female with late onset menarche (18 years of age), who was amenorrhoeic for the first year, followed by menses of one to three days' slight flow with dysmenorrhoea, but an otherwise normal menstrual history. A typical seminoma was removed from the left adnexal region and an immature testis was identified separately as an associated right adnexal mass. Repeated karyotypic studies on peripheral blood lymphocyte cultures showed only 46,X,-Y,t(Y;15)(q12;p13). Cytogenetic examination of the patient's younger brother, who had fathered three healthy children, showed an identical karyotype. Mosaicism of 46,X,-Y,t(Y;15)(q12;p13)/45,X cell lines was found in skin samples from the patient's elbow and genital regions, although there were no clinical stigmata of Turner syndrome. An androgen receptor binding assay of cultured genital skin fibroblasts was negative. Molecular analysis using Southern blot hybridisation, PCR, and direct DNA sequencing showed that neither the patient nor her brother had a detectable deletion or other abnormalities of Y chromosome sequences, including the SRY (sex determining region of the Y chromosome) gene sequence. These findings suggest that Turner mosaicism of the 45,X cell line may have contributed to this atypical presentation in an XY female, although we cannot exclude abnormalities of other genes related to sex differentiation.

  19. Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

    PubMed Central

    Jiménez Pérez, Zuly Elizabeth; Mathiyalagan, Ramya; Markus, Josua; Kim, Yeon-Ju; Kang, Hyun Mi; Abbai, Ragavendran; Seo, Kwang Hoon; Wang, Dandan; Soshnikova, Veronika; Yang, Deok Chun

    2017-01-01

    There has been a growing interest in the design of environmentally affable and biocompatible nanoparticles among scientists to find novel and safe biomaterials. Panax ginseng Meyer berries have unique phytochemical profile and exhibit beneficial pharmacological activities such as antihyperglycemic, antiobesity, antiaging, and antioxidant properties. A comprehensive study of the biologically active compounds in ginseng berry extract (GBE) and the ability of ginseng berry (GB) as novel material for the biosynthesis of gold nanoparticles (GBAuNPs) and silver nanoparticles (GBAgNPs) was conducted. In addition, the effects of GBAuNPs and GBAgNPs on skin cell lines for further potential biological applications are highlighted. GBAuNPs and GBAgNPs were synthesized using aqueous GBE as a reducing and capping agent. The synthesized nanoparticles were characterized for their size, morphology, and crystallinity. The nanoparticles were evaluated for antioxidant, anti-tyrosinase, antibacterial, and cytotoxicity activities and for morphological changes in human dermal fibroblast and murine melanoma skin cell lines. The phytochemicals contained in GBE effectively reduced and capped gold and silver ions to form GBAuNPs and GBAgNPs. The optimal synthesis conditions (ie, temperature and v/v % of GBE) and kinetics were investigated. Polysaccharides and phenolic compounds present in GBE were suggested to be responsible for stabilization and functionalization of nanoparticles. GBAuNPs and GBAgNPs showed increased scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radicals compared to GBE. GBAuNPs and GBAgNPs effectively inhibited mushroom tyrosinase, while GBAgNPs showed antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, GBAuNPs were nontoxic to human dermal fibroblast and murine melanoma cell lines, and GBAgNPs showed cytotoxic effect on murine melanoma cell lines. The current results evidently suggest that GBAgNPs can act as potential

  20. Tooth, skin, hair and eye colour interrelationships in Greek young adults.

    PubMed

    Lagouvardos, Panagiotis E; Tsamali, Ioana; Papadopoulou, Christine; Polyzois, Gregory

    2013-01-01

    The purpose of this study was to investigate the possible interrelationships of teeth, skin, eye and hair colour. A portable colorimeter (Shade Eye NCC/Shofu) was used to record the colour in the CIELAB system of the upper right incisors in 150 dental school students, along with their skin colour at three different areas. Natural hair and eye colour was classified into several categories by a trained examiner (ICC 0.93-0.99). One-way ANOVA and correlation tests were used to statistically analyse the data. Skin was found to have significantly higher L*, b* but lower a* values than teeth (p < 0.05). A significant correlation (p < 0.05) of teeth to skin L* and a*colour coordinate was found, but not to b* coordinate (p > 0.05). Hair tones were not correlated to teeth L* or b*, but only to a*coordinate. Teeth and eye colour coordinates were not correlated (p > 0.05). Eye and hair tones were found to have the highest significant correlation (ρ = 0.369). In conclusion, teeth of this cohort were found to be lighter, less red and yellow than the skin. Teeth colour was not related to eye colour, but lighter teeth were found to be associated with lighter skins, and redder lateral incisors to lighter hair. Darker facial skins or yellower forehead areas were also associated with darker hair and vice versa. The clinical relevance of the study is that the investigated facial characteristics are inter-correlated weakly to moderately, and for this reason predicting the colour parameters of one facial characteristic by another would not be accurate, but helpful for a rough colour selection as associations show.

  1. 3,3'-Dihydroxyisorenieratene prevents UV-induced formation of reactive oxygen species and the release of protein-bound zinc ions in human skin fibroblasts.

    PubMed

    Lutter, Kaya; De Spirt, Silke; Kock, Sebastian; Kröncke, Klaus-Dietrich; Martin, Hans-Dieter; Wagener, Tanja; Stahl, Wilhelm

    2010-02-01

    3,3'-Dihydroxyisorenieratene (DHIR) is a structurally unusual carotenoid exhibiting bifunctional antioxidant properties. It is synthesized by Brevibacterium linens, used in dairy industry for the production of red smear cheeses. The compound protects cellular structures against photo-oxidative damage and inhibits the UV-dependent formation of thymidine dimers. Here we show that DHIR prevents a UV-induced intracellular release of zinc ions from proteins in human dermal fibroblasts. The effect is correlated with a decreased formation of intracellular reactive oxygen species. In contrast, zinc release from cellular proteins induced by hyperthermia is not affected by pretreatment of cells with the antioxidant DHIR. It is suggested that the intracellular zinc release upon UV irradiation is due to oxidative modifications of the zinc ligands in proteins (e.g. cysteine) and that protection by DHIR is due to intracellular scavenging of reactive oxygen species generated in photo-oxidation.

  2. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

  3. The potentiation by caffeine of X-ray damage to cultured human skin fibroblasts from normal subjects and ataxia-telangiectasia patients

    SciTech Connect

    Furcinitti, P.S.

    1983-07-01

    Caffeine was found to potentiate X-ray-induced killing of human diploid fibroblasts from a normal subject and an ataxia-telangiectasia (AT) patient when it was present at 2 mM concentration for 30 to 66 hr postirradiation. The dose-modifying factor for caffeine-treated normal cells had an average value of 1.26 +/- 0.13 which did not vary significantly with treatment time or X-ray dose. The dose-modifying factor for caffeine-treated AT cells was 1.12 +/- 0.12 at 30 hr, rose to 1.66 +/- 0.17 at 41 hr, and decreased to 1.31 +/- 0.13 at 66 hr. Thus no clear difference was observed between these two cell strains' susceptibility to postirradiation caffeine treatment.

  4. Potentiation by caffeine of x-ray damage to cultured human skin fibroblasts from normal subjects and ataxia-telangiectasia patients

    SciTech Connect

    Furcinitti, P.S.

    1983-07-01

    Caffeine was found to potentiate x-ray-induced killing of human diploid fibroblasts from a normal subject and an ataxia-telangiectasia (AT) patient when it was present at 2 mM concentration for 30 to 66 h postirradiation. The dose-modifying factor for caffeine-treated normal cells had an average value of 1.26 +- 0.13 which did not vary significantly with treatment time or x-ray dose. The dose-modifying factor for caffeine-treated AT cells was 1.12 +- 0.12 at 30 h, rose to 1.66 +- 0.17 at 41 h, and decreased to 1.31 +- 0.13 at 66 h. Thus no clear difference was observed between these two cell strains' susceptibility to postirradiation caffeine treatment.

  5. Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsy

    SciTech Connect

    Tyrrell, R.M.; Pidoux, M.

    1986-06-01

    Repair efficiency of cultured cells may be estimated by measuring the ability of a particular cell type to support virus damaged by an appropriate agent. In this study we have compared the inactivation of ultraviolet (254 nm)-damaged herpes simplex virus in human fibroblast and epidermal keratinocyte cell lines derived from the same foreskin biopsy and found the epithelial cells to be a factor of 3 times less efficient in supporting the damaged virus. The two different cell types show comparable ultraviolet inactivation of clone-forming ability, indicating that the difference is specific to viral host cell reactivation. This study required the development of a quantitative infectious centers assay for the measurement of viral titer in human epithelial cells, a system which may be of more general application in studies of potential human carcinogens.

  6. Ammonia transport across the skin of adult rainbow trout (Oncorhynchus mykiss) exposed to high environmental ammonia (HEA).

    PubMed

    Zimmer, Alex M; Brauner, Colin J; Wood, Chris M

    2014-01-01

    Recent molecular evidence points towards a capacity for ammonia transport across the skin of adult rainbow trout. A series of in vivo and in vitro experiments were conducted to understand the role of cutaneous ammonia excretion (J amm) under control conditions and after 12-h pre-exposure to high environmental ammonia (HEA; 2 mmol/l NH4HCO3). Divided chamber experiments with bladder-catheterized, rectally ligated fish under light anesthesia were performed to separate cutaneous J amm from branchial, renal, and intestinal J amm. Under control conditions, cutaneous J amm accounted for 4.5 % of total J amm in vivo. In fish pre-exposed to HEA, plasma total ammonia concentration increased 20-fold to approximately 1,000 μmol/l, branchial J amm increased 1.5- to 2.7-fold, and urinary J amm increased about 7-fold. Urinary J amm still accounted for less than 2 % of total J amm. Cutaneous J amm increased 4-fold yet amounted to only 5.7 % of total J amm in these fish. Genes (Rhcg1, Rhcg2, Rhbg, NHE-2, v-type H(+)-ATPase) known to be involved in ammonia excretion at the gills of trout were all expressed at the mRNA level in the skin, but their expression did not increase with HEA pre-exposure. In vitro analyses using [(14)C] methylamine (MA), an ammonia analog which is transported by Rh proteins, demonstrated that MA permeability in isolated skin sections was higher in HEA pre-exposed fish than in control fish. The addition of basolateral ammonia (1,000 μmol/l) to this system abolished this increase in permeability, suggesting ammonia competition with MA for Rh-mediated transport across the skin of HEA pre-exposed trout; this did not occur in skin sections from control trout. Moreover, in vitro J amm by the skin of fish which had been pre-exposed to HEA was also higher than in control fish in the absence of basolateral ammonia, pointing towards a possible cutaneous ammonia loading in response to HEA. In vitro MA permeability was reduced upon the addition of amiloride (10

  7. Initiation of antiretroviral therapy in HIV-infected adults with skin complaints in northern Tanzania

    PubMed Central

    Mavura, Daudi R.; Masenga, E. John; Minja, Eli; Grossmann, Henning; Crump, John A.; Bartlett, John A.

    2015-01-01

    Abnormal skin findings are identified in over 90% of human immunodeficiency virus (HIV)-infected persons globally. A prospective cohort study of HIV-infected patients with skin complaints commencing antiretroviral therapy (ART) in northern Tanzania was undertaken. Consecutive HIV-infected subjects presenting with skin complaints, who met criteria for ART initiation, were recruited at a Tanzanian Regional Dermatology Training Center. A single dermatologist evaluated all subjects; baseline skin biopsies were performed, and CD4+ cell counts and plasma HIV RNA levels were measured. All subjects received a fixed-dose combination of stavudine, lamivudine, and nevirapine. A total of 100 subjects were enrolled; 86 subjects completed six months of follow-up. Median baseline CD4+ cell counts and plasma HIV RNA levels were 120 cells/μl and 5.2 log10 copies/ml. The most common dermatologic condition was papular pruritic eruption (47%). The median baseline score on the Burn Scale was 38%. After six months, 10 subjects had achieved the complete resolution of skin abnormalities. In those without complete resolution, the median Burn Scale score improved to 7%. Five patients developed new eruptions by month 3, which in two cases were attributed to drug reactions. In the 86 subjects remaining on ART after six months, the median CD4+ cell count had increased to 474 cells/μl, and plasma HIV RNA levels were <400 copies/ml in 85 (99%) subjects. Patients with HIV infection with skin complaints experienced marked clinical improvements following ART initiation. PMID:25256912

  8. Periplanosides A-C: new insect-derived dihydroisocoumarin glucosides from Periplaneta americana stimulating collagen production in human dermal fibroblasts.

    PubMed

    Yang, Yong-Xun; Luo, Qi; Hou, Bo; Yan, Yong-Ming; Wang, Yue-Hu; Tang, Jian-Jun; Dong, Xiao-Ping; Ma, Xiu-Ying; Yang, Tong-Hua; Zuo, Zhi-Li; Cheng, Yong-Xian

    2015-01-01

    Three new dihydroisocoumarin glucosides, termed periplanosides A-C (1-3), a known analog, pericanaside (4), and the other twenty known compounds were isolated from the insect Periplaneta americana. Their structures including absolute configurations were determined by comprehensive spectroscopic analyses and computational methods. Biological evaluation showed that compound 2 could stimulate collagen production by 31.2% in human dermal fibroblasts-adult (HDFa) at the concentration of 30 μM, indicating its significance in skin repair and ulcer.

  9. Targeted Disruption of the Lama3 Gene in Adult Mice Is Sufficient to Induce Skin Inflammation and Fibrosis.

    PubMed

    Pesch, Monika; König, Sabrina; Aumailley, Monique

    2017-02-01

    Genetic, clinical, and biochemical studies have shown that integrity of the dermal-epidermal junction requires a particular subset of laminins, that is, those containing the α3 chain encoded by the Lama3 gene. Inherited mutations in the human gene or introduction of constitutive mutations in the mouse gene prevent expression of these laminins, causing junctional epidermolysis bullosa, a very severe, often lethal disorder characterized by detachment of the epidermis from the dermis. This has precluded in vivo functional analysis of α3 chain-containing laminins, and it is still unknown whether and how they contribute to adult skin homeostasis. To address this question, we have disrupted the Lama3 gene in basal keratinocytes of adult mice. This led to the gradual disappearance of α3 chain-containing laminins along the dermal-epidermal junction and formation of subepidermal blisters like in congenital junctional epidermis bullosa. The mice lose their nails and have bullae and erosions on the footpads. Because the blistering is restricted to the interfollicular epidermis, the animals do not lose the epidermis and are viable. There is abundant and scattered deposition of collagen VII on the dermal side of the blisters, inflammation, and development of skin fibrosis with extensive accumulation of interstitial and microfibrillar collagens.

  10. Self-Antigen Presentation by Keratinocytes in the Inflamed Adult Skin Modulates T-Cell Auto-Reactivity.

    PubMed

    Meister, Michael; Tounsi, Amel; Gaffal, Evelyn; Bald, Tobias; Papatriantafyllou, Maria; Ludwig, Julia; Pougialis, Georg; Bestvater, Felix; Klotz, Luisa; Moldenhauer, Gerhard; Tüting, Thomas; Hämmerling, Günter J; Arnold, Bernd; Oelert, Thilo

    2015-08-01

    Keratinocytes have a pivotal role in the regulation of immune responses, but the impact of antigen presentation by these cells is still poorly understood, particularly in a situation where the antigen will be presented only in adult life. Here, we generated a transgenic mouse model in which keratinocytes exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II β-chain, solely under inflammatory conditions. In these mice, inflammation caused by epicutaneous contact sensitizer treatment resulted in keratinocyte-mediated expansion of MBP-specific CD4(+) T cells in the skin. Moreover, repeated contact sensitizer application preceding a systemic MBP immunization reduced the reactivity of the respective CD4(+) T cells and lowered the symptoms of the resulting experimental autoimmune encephalomyelitis. This downregulation was CD4(+) T-cell-mediated and dependent on the presence of the immune modulator Dickkopf-3. Thus, presentation of a neo self-antigen by keratinocytes in the inflamed, adult skin can modulate CD4(+) T-cell auto-aggression at a distal organ.

  11. Different cryopreservation requirements in foetal versus adult skin cells from an endangered mammal, the Iberian lynx (Lynx pardinus).

    PubMed

    León-Quinto, Trinidad; Simón, Miguel A; Cadenas, Rafael; Martínez, Africa; Serna, Arturo

    2014-04-01

    Cryobanking somatic foetal cells acquire much relevance in endangered species for biodiversity conservation purposes. Such cells could be later used to reintroduce the lost genes into the breeding pool, by inducing pluripotency and/or nuclear transfer if necessary. Since requirements for preserving foetal cells are not always the same as for adult ones, we evaluated the cryosensitivity of foetal skin cells in comparison with adult ones from the critically endangered Iberian lynx. Responses to cryoinjury were analyzed in both thawed cell types by means of cell viability and functionality (by analyzing their membrane integrity, metabolic activity, glycosaminoglycan content and proliferative activity). Freezing media included the permeating cryoprotectant Me2SO, either alone or along with the non-permeating cryoprotectant sucrose at 0.1 or 0.2M. When Me2SO was the only cryoprotectant, survival rate fell in thawed foetal cells to 54±4% (against 89±6% for thawed adult ones) and both proliferative and metabolic activities remained significantly lower than values for thawed adult cells. However, the combination of sucrose (both 0.1 as 0.2) and Me2SO in foetal cells significantly increased their survival rates (to 71±4% and 73±5%, respectively), proliferative activities (partially at day 7 and completely at day 14 after thawing) and metabolic activities. Our findings clearly show a difference between foetal and adult cells concerning their cryopreservation sensitivity and requirements, as well as their recovery time after thawing. These results are of relevance for the cryopreservation of foetal and adult cells from the Iberian lynx and could be also useful for other mammals.

  12. In Vitro and In Vivo Development of Horse Cloned Embryos Generated with iPSCs, Mesenchymal Stromal Cells and Fetal or Adult Fibroblasts as Nuclear Donors

    PubMed Central

    Olivera, Ramiro; Moro, Lucia Natalia; Jordan, Roberto; Luzzani, Carlos; Miriuka, Santiago; Radrizzani, Martin; Donadeu, F. Xavier; Vichera, Gabriel

    2016-01-01

    The demand for equine cloning as a tool to preserve high genetic value is growing worldwide; however, nuclear transfer efficiency is still very low. To address this issue, we first evaluated the effects of time from cell fusion to activation (<1h, n = 1261; 1-2h, n = 1773; 2-3h, n = 1647) on in vitro and in vivo development of equine embryos generated by cloning. Then, we evaluated the effects of using different nuclear donor cell types in two successive experiments: I) induced pluripotent stem cells (iPSCs) vs. adult fibroblasts (AF) fused to ooplasts injected with the pluripotency-inducing genes OCT4, SOX2, MYC and KLF4, vs. AF alone as controls; II) umbilical cord-derived mesenchymal stromal cells (UC-MSCs) vs. fetal fibroblasts derived from an unborn cloned foetus (FF) vs. AF from the original individual. In the first experiment, both blastocyst production and pregnancy rates were higher in the 2-3h group (11.5% and 9.5%, respectively), respect to <1h (5.2% and 2%, respectively) and 1-2h (5.6% and 4.7%, respectively) groups (P<0.05). However, percentages of born foals/pregnancies were similar when intervals of 2-3h (35.2%) or 1-2h (35.7%) were used. In contrast to AF, the iPSCs did not generate any blastocyst-stage embryos. Moreover, injection of oocytes with the pluripotency-inducing genes did not improve blastocyst production nor pregnancy rates respect to AF controls. Finally, higher blastocyst production was obtained using UC-MSC (15.6%) than using FF (8.9%) or AF (9.3%), (P<0.05). Despite pregnancy rates were similar for these 3 groups (17.6%, 18.2% and 22%, respectively), viable foals (two) were obtained only by using FF. In summary, optimum blastocyst production rates can be obtained using a 2-3h interval between cell fusion and activation as well as using UC-MSCs as nuclear donors. Moreover, FF line can improve the efficiency of an inefficient AF line. Overall, 24 healthy foals were obtained from a total of 29 born foals. PMID:27732616

  13. In vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and produce a fibrotic-like environment upon stimulation with TGF-β1: Is there a thin line between fetal scarless healing and fibrosis?

    PubMed

    Walraven, M; Akershoek, J J; Beelen, R H J; Ulrich, M M W

    2017-03-01

    Transforming growth factor-β (TGF-β) is a cytokine occurring in three isoforms with an important function in development and wound healing. In wound healing, prolonged TGF-β signaling results in myofibroblast differentiation and fibrosis. In contrast, the developing second-trimester fetal skin contains high levels of all three TGF-β isoforms but still has the intrinsic capacity to heal without scarring. Insight into TGF-β signal transduction during fetal wound healing might lead to methods to control the signaling pathway during adult wound healing. In this study, we imitated wound healing in vitro by stimulating fibroblasts with TGF-β1 and examining myofibroblast differentiation. The aim was to gain insight into TGF-β signaling in human fibroblasts from fetal and adult dermis. First, TGF-β1 stimulation resulted in similar or even more severe upregulation of myofibroblast-associated genes in fetal fibroblasts compared to adult fibroblasts. Second, fetal fibroblasts also had higher protein levels of myofibroblast-marker α-smooth muscle actin (α-SMA). Third, stimulated fetal fibroblasts in collagen matrices had higher protein levels of α-SMA, produced more of the fibrotic protein fibronectin splice-variant extra domain A (FnEDA), and showed enhanced contraction. Finally, fetal fibroblasts also produced significant higher levels of TGF-β1. Altogether, these data show that in vitro cultured fetal fibroblasts have myofibroblast-associated characteristics and do produce a fibrotic environment. As healthy fetal skin has high levels of TGF-β1, FnEDA, and collagen-III as well, these findings correlate with the in vivo situation. Therefore, our study demonstrates that there are similarities between fetal skin development and fibrosis and shows the necessity to discriminate between these processes.

  14. Prolidase-dependent mechanism of (Z)-8,9-epoxyheptadeca-1,11,14-triene-induced inhibition of collagen biosynthesis in cultured human skin fibroblasts.

    PubMed

    Szoka, Lukasz; Karna, Ewa; Nazaruk, Jolanta; Palka, Jerzy A

    2016-01-01

    The effects of polyolefinic compound from roots of Cirsium palustre, (Z)-8,9-epoxyheptadeca-1,11,14-triene (EHT) on collagen biosynthesis, prolidase activity, expression of insulin-like growth factor receptor (IGF-IR), β1 integrin, MAP kinases (pERK1/2), the transcription factors such as nuclear factor kappa B (NF-κB) and hypoxia-inducible factor-1α (HIF-1α) were evaluated in human dermal fibroblasts treated with micromolar concentrations (40-200 μM) for 24 h. It was found that EHT-dependent inhibition of collagen biosynthesis was accompanied by parallel inhibition in prolidase activity. Since IGF-I is the most potent regulator of both processes and prolidase is regulated by β1 integrin signalling, the effect of EHT on IGF-IR and β1 integrin receptor expressions were evaluated. Exposure of the cells to EHT contributed to distinct increase in IGF-IR and slight increase in β1 integrin receptor expressions. It was accompanied by decrease in expression of pERK1/2, HIF-1α and NF-κB. EHT-dependent inhibition of collagen biosynthesis results from inhibition of prolidase activity, the enzyme involved in collagen biosynthesis.

  15. Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

    SciTech Connect

    Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

    1987-12-01

    A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.

  16. Estrogens and aging skin

    PubMed Central

    Thornton, M. Julie

    2013-01-01

    Estrogen deficiency following menopause results in atrophic skin changes and acceleration of skin aging. Estrogens significantly modulate skin physiology, targeting keratinocytes, fibroblasts, melanocytes, hair follicles and sebaceous glands, and improve angiogenesis, wound healing and immune responses. Estrogen insufficiency decreases defense against oxidative stress; skin becomes thinner with less collagen, decreased elasticity, increased wrinkling, increased dryness and reduced vascularity. Its protective function becomes compromised and aging is associated with impaired wound healing, hair loss, pigmentary changes and skin cancer.   Skin aging can be significantly delayed by the administration of estrogen. This paper reviews estrogen effects on human skin and the mechanisms by which estrogens can alleviate the changes due to aging. The relevance of estrogen replacement, selective estrogen receptor modulators (SERMs) and phytoestrogens as therapies for diminishing skin aging is highlighted. Understanding estrogen signaling in skin will provide a basis for interventions in aging pathologies. PMID:24194966

  17. Antibiotic allergies in children and adults: from clinical symptoms to skin testing diagnosis.

    PubMed

    Romano, Antonino; Caubet, Jean-Christoph

    2014-01-01

    Hypersensitivity reactions to β-lactam and non-β-lactam antibiotics are commonly reported. They can be classified as immediate or nonimmediate according to the time interval between the last drug administration and their onset. Immediate reactions occur within 1 hour after the last drug administration and are manifested clinically by urticaria and/or angioedema, rhinitis, bronchospasm, and anaphylactic shock; they may be mediated by specific IgE-antibodies. Nonimmediate reactions occur more than 1 hour after the last drug administration. The most common manifestations are maculopapular exanthems; specific T lymphocytes may be involved in this type of manifestation. The diagnostic evaluation of hypersensitivity reactions to antibiotics is usually complex. The patient's history is fundamental; the allergic examination is based mainly on in vivo tests selected on the basis of the clinical features and the type of reaction, immediate or nonimmediate. Immediate reactions can be assessed by immediate-reading skin tests and, in selected cases, drug provocation tests. Nonimmediate reactions can be assessed by delayed-reading skin tests, patch tests, and drug provocation tests. However, skin tests have been well validated mainly for β-lactams but less for other classes of antibiotics.

  18. Bioglass Activated Skin Tissue Engineering Constructs for Wound Healing.

    PubMed

    Yu, Hongfei; Peng, Jinliang; Xu, Yuhong; Chang, Jiang; Li, Haiyan

    2016-01-13

    Wound healing is a complicated process, and fibroblast is a major cell type that participates in the process. Recent studies have shown that bioglass (BG) can stimulate fibroblasts to secrete a multitude of growth factors that are critical for wound healing. Therefore, we hypothesize that BG can stimulate fibroblasts to have a higher bioactivity by secreting more bioactive growth factors and proteins as compared to untreated fibroblasts, and we aim to construct a bioactive skin tissue engineering graft for wound healing by using BG activated fibroblast sheet. Thus, the effects of BG on fibroblast behaviors were studied, and the bioactive skin tissue engineering grafts containing BG activated fibroblasts were applied to repair the full skin lesions on nude mouse. Results showed that BG stimulated fibroblasts to express some critical growth factors and important proteins including vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, collagen I, and fibronectin. In vivo results revealed that fibroblasts in the bioactive skin tissue engineering grafts migrated into wound bed, and the migration ability of fibroblasts was stimulated by BG. In addition, the bioactive BG activated fibroblast skin tissue engineering grafts could largely increase the blood vessel formation, enhance the production of collagen I, and stimulate the differentiation of fibroblasts into myofibroblasts in the wound site, which would finally accelerate wound healing. This study demonstrates that the BG activated skin tissue engineering grafts contain more critical growth factors and extracellular matrix proteins that are beneficial for wound healing as compared to untreated fibroblast cell sheets.

  19. Caffeine does not cause override of the G2/M block induced by UVc or gamma radiation in normal human skin fibroblasts

    PubMed Central

    Deplanque, G; Vincent, F; Mah-Becherel, M C M; Cazenave, J-P; Bergerat, J-P; Klein-Soyer, C

    2000-01-01

    Caffeine has for many years been known to be involved in the sensitization of DNA to damage. One potential mechanism recently put forward is an override of the G2/M block induced by irradiation, which would leave the cells less time for DNA repair prior to mitosis. However, different cell types display a variety of responses and no clear pathway has yet emerged, especially as little is known about the capacity of this agent to enhance DNA damage in normal, untransformed cells. Continuous exposure to commonly used caffeine concentrations (1–5 mM) inhibited the proliferation of normal human fibroblasts (NHFs) in a dose-dependent manner to up to 80% at 5 mM. Exposure of exponentially growing NHFs to UVc radiation (20 J m–2) or γ radiation (2.5–8 Gy) led to a 45–60% inhibition of proliferation and protracted accumulation of cells in the G2/M phase. Addition of 2 mM caffeine after irradiation induced slowing of the S phase passage, with a resultant delay in G2/M accumulation mimicking a G2/M block override. These results were confirmed by stathmokinetic studies, which showed delayed entry of the cells into mitosis in the presence of caffeine. Our data demonstrate that caffeine primarily inhibits replicative DNA synthesis and suggest that, at least in normal cells, caffeine potentiates the cytotoxicity of radiation by intervening in DNA repair rather than by overriding the G2/M block. © 2000 Cancer Research Campaign PMID:10917550

  20. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    PubMed

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  1. Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

    PubMed

    Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

    2010-02-01

    Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

  2. Distinct phenotype and therapeutic potential of gingival fibroblasts.

    PubMed

    Häkkinen, Lari; Larjava, Hannu; Fournier, Benjamin P J

    2014-09-01

    Gingiva of the oral mucosa provides a practical source to isolate fibroblasts for therapeutic purposes because the tissue is easily accessible, tissue discards are common during routine clinical procedures and wound healing after biopsy is fast and results in complete wound regeneration with very little morbidity or scarring. In addition, gingival fibroblasts have unique traits, including neural crest origin, distinct gene expression and synthetic properties and potent immunomodulatory functions. These characteristics may provide advantages for certain therapeutic approaches over other more commonly used cells, including skin fibroblasts, both in intraoral and extra-oral sites. However, identity and phenotype of gingival fibroblasts, like other fibroblasts, are still not completely understood. Gingival fibroblasts are phenotypically heterogeneous, and these…fibroblast subpopulations may play different roles in tissue maintenance, regeneration and pathologies. The purpose of this review is to summarize what is currently known about gingival fibroblasts, their distinct potential for tissue regeneration and their potential therapeutic uses in the future.

  3. The Skin Cancer and Sun Knowledge (SCSK) Scale: Validity, Reliability, and Relationship to Sun-Related Behaviors Among Young Western Adults.

    PubMed

    Day, Ashley K; Wilson, Carlene; Roberts, Rachel M; Hutchinson, Amanda D

    2014-08-01

    Increasing public knowledge remains one of the key aims of skin cancer awareness campaigns, yet diagnosis rates continue to rise. It is essential we measure skin cancer knowledge adequately so as to determine the nature of its relationship to sun-related behaviors. This study investigated the psychometric properties of a new measure of skin cancer knowledge, the Skin Cancer and Sun Knowledge (SCSK) scale. A total of 514 Western young adults (females n = 320, males n = 194) aged 18 to 26 years completed measures of skin type, skin cancer knowledge, tanning behavior, sun exposure, and sun protection. Two-week test-retest of the SCSK was conducted with 52 participants. Internal reliability of the SCSK scale was acceptable (KR-20 = .69), test-retest reliability was high (r = .83, n = 52), and acceptable levels of face, content, and incremental validity were demonstrated. Skin cancer knowledge (as measured by SCSK) correlated with sun protection, sun exposure, and tanning behaviors in the female sample, but not in the males. Skin cancer knowledge appears to be more relevant to the behavior of young women than that of young males. We recommend that future research establish the validity of the SCSK across a range of participant groups.

  4. Ganglioside GM2 N-acetyl-beta-D-galactosaminidase activity in cultured fibroblasts of late-infantile and adult GM2 gangliosidosis patients and of healthy probands with low hexosaminidase level.

    PubMed Central

    Conzelmann, E; Kytzia, H J; Navon, R; Sandhoff, K

    1983-01-01

    A sensitive assay was developed to assess the ability of extracts from cultured fibroblasts to catabolize ganglioside GM2, in the presence of the natural activator protein but without detergents. This method, which permitted the reliable determination of residual activities as low as 0.1% of normal controls, was then used to measure ganglioside GM2 hydrolase activities in fibroblasts from several hexosaminidase variants. The residual activities thus determined correlated well with the clinical status of the respective proband: infantile Tay-Sachs (0.1% of normal controls), late-infantile (0.5%), and adult GM2 gangliosidoses (2%-4%) and healthy probands with "low hexosaminidase" (11% and 20%). In contrast, beta-hexosaminidase A levels as measured with the synthetic substrate 4-MU-GlcNAc could not be relied on for diagnostic purposes (the late-infantile patient studied retained 80% of the activity of controls). PMID:6614006

  5. Differences of cell surface marker expression between bone marrow- and kidney-derived murine mesenchymal stromal cells and fibroblasts.

    PubMed

    Cakiroglu, F; Osbahr, J W; Kramer, J; Rohwedel, J

    2016-10-31

    Mesenchymal stromal cells (MSC) are undifferentiated, multipotent adult cells with regenerative properties. They are particularly relevant for therapeutic approaches due to the simplicity of their isolation and cultivation. Since MSC show an expression pattern of cell surface marker, which is almost identical to fibroblasts, many attempts have been made to address the similarities and differences between MSC and fibroblasts. In this study we aimed to isolate murine MSC from bone marrow (BM) and kidney to characterize them in comparison to fibroblasts. Cells were isolated from murine kidney, BM and abdominal skin by plastic adherence and subsequently characterized by analysing their capability to build colony-forming unit-fibroblasts (CFU-F), their morphology, their proliferation, expression of telomerase activity and cell surface antigens as well as their differentiation capacity. Plastic adherent cells from the 3 mouse tissues showed similar morphology, proliferation profiles and CFU-F building capacities. However, while MSC from BM and kidney differentiated into the adipogenic, chondrogenic and osteogenic direction, fibroblasts were not able to do so efficiently. In addition, a tendency for lower expression of telomerase was found in the fibroblast population. Proliferating cells from kidney and BM expressed the MSC-specific cell surface markers CD105 and Sca-1 on a significantly higher and CD117 on a significantly lower level compared to fibroblasts and were thereby distinguishable from fibroblasts. Furthermore, we found that certain CD markers were specifically expressed on a higher level, either in BM-derived cells or fibroblasts. This study demonstrates that murine MSC isolated from different organs express certain specific markers, which enable their discrimination.

  6. Prolongation of GFP-expressed skin graft after intrathymic injection of GFP positive splenocytes in adult rat

    NASA Astrophysics Data System (ADS)

    Hakamata, Yoji; Igarashi, Yuka; Murakami, Takashi; Kobayashi, Eiji

    2006-02-01

    GFP is a fluorescent product of the jellyfish Aequorea victoria and has been used for a variety of biological experiments as a reporter molecule. While GFP possesses advantages for the non-invasive imaging of viable cells, GFP-positive cells are still considered potential xeno-antigens. It is difficult to observe the precise fate of transplanted cells/organs in recipients without immunological control. The aim of this study was to determine whether intrathymic injection of GFP to recipients and the depletion of peripheral lymphocytes could lead to donor-specific unresponsiveness to GFP-expressed cell. LEW rats were administered intraperitoneally with 0.2 ml of anti-rat lymphocyte serum (ALS) 1 day prior to intrathymic injection of donor splenocytes or adeno-GFP vector. Donor cells and vector were non-invasively inoculated into the thymus under high frequency ultrasound imaging using an echo-guide. All animals subsequently received a 7 days GFP-expressed skin graft from the same genetic background GFP LEW transgenic rat. Skin graft survival was greater in rats injected with donor splenocytes (23.6+/-9.1) compared with adeno-GFP (13.0+/-3.7) or untreated control rats (9.5+/-1.0). Intrathymic injection of donor antigen into adult rats can induce donor-specific unresponsiveness. Donor cells can be observed for a long-term in recipients with normal immunity using this strategy.

  7. Smooth muscle differentiation in scleroderma fibroblastic cells.

    PubMed Central

    Sappino, A. P.; Masouyé, I.; Saurat, J. H.; Gabbiani, G.

    1990-01-01

    Using antibodies to alpha-smooth muscle actin and desmin on paraffin-embedded formalin-fixed tissue sections, the authors demonstrate that fibroblastic cells of localized and systemic scleroderma lesions express features of smooth muscle differentiation. Eleven of eleven skin specimens of systemic sclerosis patients and two of four skin specimens of localized scleroderma displayed the presence of fibroblasts expressing alpha-smooth muscle actin, a cell population that predominated in areas of prominent collagen deposition. A similar fibroblastic phenotype was found in the esophagus, the liver, and the lung specimens obtained from four patients who died of progressive systemic sclerosis. Immunostaining for desmin, performed on adjacent tissue sections, demonstrated that a minority of these fibroblastic cells present in skin and visceral lesions contained this protein. The authors' observations indicate that scleroderma fibroblasts are phenotypically related to the stromal cells previously identified in hypertrophic scars, fibromatoses, and desmoplasia; they might provide novel criteria for the characterization of scleroderma lesions and help to identify the factors responsible for phenotypic modulations in fibroblastic cells. Images Figure 1 Figure 2 Figure 3 PMID:1698026

  8. Contractile properties of skinned muscle fibres from young and adult normal and dystrophic (mdx) mice.

    PubMed Central

    Williams, D A; Head, S I; Lynch, G S; Stephenson, D G

    1993-01-01

    1. Single muscle fibres were enzymatically isolated from the soleus and extensor digitorum longus (EDL) muscles of genetically dystrophic mdx and normal (C57BL/10) mice aged 3-6 or 17-23 weeks. 2. Fibres of both muscles were chemically skinned with the non-ionic detergent Triton X-100 (2% v/v). Ca(2+)- and Sr(2+)-activated contractile responses were recorded and comparisons were made between several contractile parameters of various fibre types of normal and dystrophic mice of similar age. 3. There were no significant differences in the following contractile parameters of skinned fibres of normal and mdx mice of the same age: sensitivity to activating Ca2+ (pCa50) or Sr2+ (pSr50) and differential sensitivity to the activating ions (pCa50-pSr50). However the maximum isometric tension (Po) and the frequency of myofibrillar force oscillations in EDL fast-twitch fibres of young mdx mice were significantly lower than those of soleus fast-twitch fibres of the same animals, or fast-twitch fibres (EDL or soleus) of normal mice. 4. Age-related differences were apparent in some contractile parameters of both normal and mdx mice. In particular the steepness of force-pCa and force-pSr curves increased with age in normal mice, yet decreased with age in fibres of mdx mice. 5. A fluorescent probe, ethidium bromide, which interchelates with DNA, was used with laser-scanning confocal microscopy to determine the distribution of myonuclei in fibres. Fibres isolated from either muscle type of normal animals displayed a characteristic peripheral spiral of myonuclei. Fibres from muscles of mdx mice displayed three major patterns of nuclear distribution; the normal peripheral spiral, long central strands of nuclei, and a mixture of these two patterns. 6. The contractile characteristics of mdx fibres were not markedly influenced by the nuclear distribution pattern in that there were no discernible differences in the major contractile parameters (the Hill coefficients nCa and nSr, which

  9. Management of adult concealed penis using a meshed, split-thickness skin graft

    PubMed Central

    Boonjindasup, Aaron; Pinsky, Michael; Stewart, Carrie; Trost, Landon; Chaffin, Abigail; Jansen, David; Hellstrom, Wayne

    2016-01-01

    Introduction Concealed penis (CP) is a rare problem faced by urologists and plastic surgeons. CP occurs secondary to trauma, obesity, or infection. Surgical treatment is individualized and based on patient and provider variables. We aim to review our recent experience using meshed split-thickness skin grafting (STSG) for CP management. Methods A retrospective review was performed on patients who underwent STSG for CP at our institution. Records were reviewed for demographic, operative, and postoperative variables. Preoperative and postoperative photos were obtained to monitor cosmetic results. Results Eleven patients underwent CP release with meshed STSG placement. All cases showed improved functional phallic length and good cosmetic results, regardless of etiology. Conclusions STSG is a viable option for penile coverage for management of this difficult-to-treat CP population. This primary or salvage modality offers excellent cosmetic results and may be used following prior reconstructive attempts. PMID:28096930

  10. IgE Sensitization Patterns to Commonly Consumed Foods Determined by Skin Prick Test in Korean Adults

    PubMed Central

    2016-01-01

    Offending food allergens can vary with regional preferences in food consumption. In this study, we analysed sensitization rates to commonly consumed foods in Korean adults suspected of having food allergy. One hundred and thirty four subjects underwent a skin prick test (SPT) with 55 food allergens, of which 13 were made by our laboratory and the rest were commercially purchased. Of the 134 patients, 73 (54.5%) were sensitized to one or more food allergens. Sensitization to chrysalis was detected most frequently, at a rate of 25.4%. Sensitization rates to other food allergens were as follows: maize grain (13.4%), shrimp (11.9%), almond (11.1%), wheat flour (8.2%), lobster (8.2%), buckwheat (8.2%), mackerel (5.2%), pollack (5.2%), halibut (4.5%), peanut (4.5%), anchovy (4.4%), squid (3.7%), saury (3.0%), common eel (3.0%), yellow corvina (3.0%), hairtail (2.2%), octopus (2.2%), and others. In addition to well-known food allergens, sensitivity to mackerel, chrysalis, pollack, and halibut, which are popular foods in Korea, was observed at high rates in Korean adults. We suggest that the SPT panel for food allergy in Korea should include these allergens. PMID:27478328

  11. IgE Sensitization Patterns to Commonly Consumed Foods Determined by Skin Prick Test in Korean Adults.

    PubMed

    Kim, Sung Ryeol; Park, Hye Jung; Park, Kyung Hee; Lee, Jae-Hyun; Park, Jung-Won

    2016-08-01

    Offending food allergens can vary with regional preferences in food consumption. In this study, we analysed sensitization rates to commonly consumed foods in Korean adults suspected of having food allergy. One hundred and thirty four subjects underwent a skin prick test (SPT) with 55 food allergens, of which 13 were made by our laboratory and the rest were commercially purchased. Of the 134 patients, 73 (54.5%) were sensitized to one or more food allergens. Sensitization to chrysalis was detected most frequently, at a rate of 25.4%. Sensitization rates to other food allergens were as follows: maize grain (13.4%), shrimp (11.9%), almond (11.1%), wheat flour (8.2%), lobster (8.2%), buckwheat (8.2%), mackerel (5.2%), pollack (5.2%), halibut (4.5%), peanut (4.5%), anchovy (4.4%), squid (3.7%), saury (3.0%), common eel (3.0%), yellow corvina (3.0%), hairtail (2.2%), octopus (2.2%), and others. In addition to well-known food allergens, sensitivity to mackerel, chrysalis, pollack, and halibut, which are popular foods in Korea, was observed at high rates in Korean adults. We suggest that the SPT panel for food allergy in Korea should include these allergens.

  12. A highly enriched niche of precursor cells with neuronal and glial potential within the hair follicle dermal papilla of adult skin.

    PubMed

    Hunt, David P J; Morris, Paul N; Sterling, Jane; Anderson, Jane A; Joannides, Alexis; Jahoda, Colin; Compston, Alastair; Chandran, Siddharthan

    2008-01-01

    Skin-derived precursor cells (SKPs) are multipotent neural crest-related stem cells that grow as self-renewing spheres and are capable of generating neurons and myelinating glial cells. SKPs are of clinical interest because they are accessible and potentially autologous. However, although spheres can be readily isolated from embryonic and neonatal skin, SKP frequency falls away sharply in adulthood, and primary sphere generation from adult human skin is more problematic. In addition, the culture-initiating cell population is undefined and heterogeneous, limiting experimental studies addressing important aspects of these cells such as the behavior of endogenous precursors in vivo and the molecular mechanisms of neural generation. Using a combined fate-mapping and microdissection approach, we identified and characterized a highly enriched niche of neural crest-derived sphere-forming cells within the dermal papilla of the hair follicle of adult skin. We demonstrated that the dermal papilla of the rodent vibrissal follicle is 1,000-fold enriched for sphere-forming neural crest-derived cells compared with whole facial skin. These "papillaspheres" share a phenotypic and developmental profile similar to that of SKPs, can be readily expanded in vitro, and are able to generate both neuronal and glial cells in response to appropriate cues. We demonstrate that papillaspheres can be efficiently generated and expanded from adult human facial skin by microdissection of a single hair follicle. This strategy of targeting a highly enriched niche of sphere-forming cells provides a novel and efficient method for generating neuronal and glial cells from an accessible adult somatic source that is both defined and minimally invasive.

  13. Looking older: Fibroblast Collapse and Therapeutic Implications

    PubMed Central

    Fisher, Gary J.; Varani, James; Voorhees, John J.

    2010-01-01

    Background Skin appearance is a primary indicator of age. During the last decade, substantial progress has been made towards understanding underlying mechanisms of human skin aging. This understanding provides the basis for current use and new development of anti-aging treatments. Objective To present state of the art knowledge pertaining to mechanisms involved in skin aging, with specific focus on the dermal collagen matrix. Results A major feature of aged skin is fragmentation of the dermal collagen matrix. Fragmentation results from actions of specific enzymes (matrix metalloproteinases), and impairs the structural integrity of the dermis. Fibroblasts that produce and organize the collagen matrix cannot attach to fragmented collagen. Loss of attachment prevents fibroblasts from receiving mechanical information from their support and they collapse. Stretch is critical for normal balanced production of collagen and collagen-degrading enzymes. In aged skin, collapsed fibroblasts produce low levels of collagen and high levels of collagen–degrading enzymes. This imbalance advances the aging process, in a self-perpetuating, never-ending deleterious cycle. Clinically-proven anti-aging treatments such as topical retinoic acid, CO2 laser resurfacing, and intradermal injection of cross-linked hyaluronic acid stimulate production of new undamaged collagen. Attachment of fibroblasts to this new collagen allows stretch, which in turn balances collagen production/degradation and thereby slows the aging process. Conclusion Collagen fragmentation is responsible for loss of structural integrity and impairment of fibroblast function in aged human skin. Treatments that stimulate production of new, non-fragmented collagen should provide substantial improvement to the appearance and health of aged skin. PMID:18490597

  14. Role of transiently altered sarcolemmal membrane permeability and basic fibroblast growth factor release in the hypertrophic response of adult rat ventricular myocytes to increased mechanical activity in vitro.

    PubMed Central

    Kaye, D; Pimental, D; Prasad, S; Mäki, T; Berger, H J; McNeil, P L; Smith, T W; Kelly, R A

    1996-01-01

    One of the trophic factors that has been implicated in initiating or facilitating growth in response to increased mechanical stress in several tissues and cell types is basic fibroblast growth factor (bFGF; FGF-2). Although mammalian cardiac muscle cells express bFGF, it is not known whether it plays a role in mediating cardiac adaptation to increased load, nor how release of the cytosolic 18-kD isoform of bFGF would be regulated in response to increased mechanical stress. To test the hypothesis that increased mechanical activity induces transient alterations in sarcolemmal permeability that allow cytosolic bFGF to be released and subsequently to act as an autocrine and paracrine growth stimulus, we examined primary isolates of adult rat ventricular myocytes maintained in serum-free, defined medium that were continually paced at 3 Hz for up to 5 d. Paced myocytes, but not nonpaced control cells, exhibited a "hypertrophic" response, which was characterized by increases in the rate of phenylalanine incorporation, total cellular protein content, and cell size. These changes could be mimicked in control cells by exogenous recombinant bFGF and could be blocked in continually paced cells by a specific neutralizing anti-bFGF antibody. In addition, medium conditioned by continually paced myocytes contained significantly more bFGF measured by ELISA and more mitogenic activity for 3T3 cells, activity that could be reduced by a neutralizing anti-bFGF antibody. The hypothesis that transient membrane disruptions sufficient to allow release of cytosolic bFGF occur in paced myocytes was examined by monitoring the rate of uptake into myocytes from the medium of 10-kD dextran linked to fluorescein. Paced myocytes exhibited a significantly higher rate of fluoresceinlabeled dextran uptake. These data are consistent with the hypothesis that nonlethal, transient alterations in sarcolemmal membrane permeability with release of cytosolic bFGF is one mechanism by which increased

  15. The Skin-Brain Connection Hypothesis, Bringing Together CCL27-Mediated T-Cell Activation in the Skin and Neural Cell Damage in the Adult Brain.

    PubMed

    Blatt, Nataliya L; Khaiboullin, Timur I; Lombardi, Vincent C; Rizvanov, Albert A; Khaiboullina, Svetlana F

    2016-01-01

    Recent discovery of an association of low serum melatonin levels with relapse in multiple sclerosis (MS) opens a new horizon in understanding the pathogenesis of this disease. Skin is the main organ for sensing seasonal changes in duration of sunlight exposure. Level of melatonin production is dependent on light exposure. The molecular mechanisms connecting peripheral (skin) sensing of the light exposure and developing brain inflammation (MS) have not been investigated. We hypothesize that there is a connection between the reaction of skin to seasonal changes in sunlight exposure and the risk of MS and that seasonal changes in light exposure cause peripheral (skin) inflammation, the production of cytokines, and the subsequent inflammation of the brain. In skin of genetically predisposed individuals, cytokines attract memory cutaneous lymphocyte-associated antigen (CLA+) T lymphocytes, which then maintain local inflammation. Once inflammation is resolved, CLA+ lymphocytes return to the circulation, some of which eventually migrate to the brain. Once in the brain these lymphocytes may initiate an inflammatory response. Our observation of increased CC chemokine ligand 27 (CCL27) in MS sera supports the involvement of skin in the pathogenesis of MS. Further, the importance of our data is that CCL27 is a chemokine released by activated keratinocytes, which is upregulated in inflamed skin. We propose that high serum levels of CCL27 in MS are the result of skin inflammation due to exposure to seasonal changes in the sunlight. Future studies will determine whether CCL27 serum level correlates with seasonal changes in sunlight exposure, MS exacerbation, and skin inflammation.

  16. Wnt5a Exhibits Layer-Specific Expression in Adult Skin, Is Upregulated in Psoriasis, and Synergizes with Type 1 Interferon

    PubMed Central

    Romanowska, Malgorzata; Evans, Alan; Kellock, David; Bray, Susan E.; McLean, Kathleen; Donandt, Susanne; Foerster, John

    2009-01-01

    Background Wnt5a is a member of the wingless-type patterning regulators important in pre-natal development. The expression and distribution of Wnt5a and its receptors frizzled (fzd) 3 and fzd 5 in adult human skin have not been comprehensively studied to date. Methodology/Principal Findings We here show that Wnt5a, fzd3, fzd5, as well as fzd6 are restricted to specific layers in normal epidermis, analogous to their zonal distribution in hair follicles, suggesting a role in adult skin differentiation. In line, Wnt5a and fzd5 are both overexpressed and re-distributed in the epidermis of psoriasis which involves disturbed keratinocyte differentiation. Functionally, Wnt5a lowers the concentration of IFN required to induce target genes, and increases the magnitude of IFN target gene induction, suggesting a molecular mechanism underlying IFN hypersensitivity in psoriasis. Finally, we identify nedd8 and the amyloid precursor APP, previously shown to be upregulated in psoriasis, as targets of synergistic IFNα/Wnt5a induction. Conclusions/Significance The present data (i) suggest that Wnt5a regulates epidermal differentiation even in adult skin and (ii) identify synergistic induction of type 1 IFN target genes as a novel mode of Wnt5a action. Targeting Wnt5a in the skin may reduce IFN hypersensitivity and be of therapeutical value. PMID:19399181

  17. Immortalized skin fibroblasts expressing conditional MyoD as a renewable and reliable source of converted human muscle cells to assess therapeutic strategies for muscular dystrophies: validation of an exon-skipping approach to restore dystrophin in Duchenne muscular dystrophy cells.

    PubMed

    Chaouch, Soraya; Mouly, Vincent; Goyenvalle, Aurélie; Vulin, Adeline; Mamchaoui, Kamel; Negroni, Elisa; Di Santo, James; Butler-Browne, Gillian; Torrente, Yvan; Garcia, Luis; Furling, Denis

    2009-07-01

    Abstract Numerous strategies are under development for the correction of deleterious effects of mutations in muscular dystrophies, and these strategies must be validated in compelling models. Cellular models seem straightforward to set up; however, the proliferative capacity of muscle cells isolated from dystrophic patients is limited, and in addition it is difficult to envisage the use of large muscle biopsies from patients to obtain enough cells for ex vivo assessments. To overcome these problems, we have devised a strategy to obtain, from a patient with Duchenne muscular dystrophy (DMD), an inexhaustible source of myogenic progenitor cells with a deletion of exons 49 and 50 in the dystrophin gene. Starting material consisted of dermal fibroblasts isolated from a skin biopsy taken in a noninvasive way. These fibroblasts were first immortalized by telomerase gene transfer. Subsequent cell lines were converted into myogenic cells by means of a lentiviral vector encoding an inducible MyoD construct. Before myogenic induction, engineered DMD fibroblasts were able to proliferate infinitely. Under induction conditions, they were converted into myogenic cells, which differentiated into large multinucleated myotubes. We used these DMD fibroblast cell lines to assess dystrophin rescue by using engineered U7 small nuclear RNAs harboring antisense sequences required to restore an in-frame dystrophin mRNA by skipping exon 51. Further molecular analyses showed dystrophin rescue ex vivo as well as in vivo after engrafting of treated cells into regenerating muscles in immunodeficient mice.

  18. Serum-free bioprocessing of adult human and rodent skin-derived Schwann cells: implications for cell therapy in nervous system injury.

    PubMed

    Mirfeizi, Leila; Stratton, Jo Anne; Kumar, Ranjan; Shah, Prajay; Agabalyan, Natacha; Stykel, Morgan G; Midha, Rajiv; Biernaskie, Jeff; Kallos, Michael S

    2017-02-08

    Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long-lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily-accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier-based bioreactor system is proposed as a means to scale-up large numbers of adult skin-derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static-grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.

  19. Optimization of an ex vivo wound healing model in the adult human skin: Functional evaluation using photodynamic therapy.

    PubMed

    Mendoza-Garcia, Jenifer; Sebastian, Anil; Alonso-Rasgado, Teresa; Bayat, Ardeshir

    2015-09-01

    Limited utility of in vitro tests and animal models of human repair, create a demand for alternative models of cutaneous healing capable of functional testing. The adult human skin Wound Healing Organ Culture (WHOC) provides a useful model, to study repair and enable evaluation of therapies such as the photodynamic therapy (PDT). Thus, the aim here was to identify the optimal WHOC model and to evaluate the role of PDT in repair. Wound geometry, system of support, and growth media, cellular and matrix biomarkers were investigated in WHOC models. Subsequently, cellular activity, extracellular matrix remodeling, and oxidative stress plus gene and protein levels of makers of wound repair measured the effect of PDT on the optimized WHOC. WHOCs embedded in collagen and supplemented DMEM were better organized showing stratified epidermis and compact dermis with developing neo-epidermis. Post-PDT, the advancing reepithelialization tongue was 3.5 folds longer, and was highly proliferative with CK-14 plus p16 increased (p < 0.05) compared to controls. The neo-epidermis was fully differentiated forming neo-collagen. Proliferating nuclear antigen, p16, COLI, COLIII, MMP3, MMP19, and α-SMA were significantly more expressed (p < 0.05) in dermis surrounding the healing wound. In conclusion, an optimal model of WHOC treated with PDT shows increased reepithelialization and extracellular matrix reconstruction and remodeling, supporting evidence toward development of an optimal ex vivo wound healing model.

  20. Rho kinase inhibitor Y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    PubMed

    van den Bogaard, Ellen H; Rodijk-Olthuis, Diana; Jansen, Patrick A M; van Vlijmen-Willems, Ivonne M J J; van Erp, Piet E; Joosten, Irma; Zeeuwen, Patrick L J M; Schalkwijk, Joost

    2012-09-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.

  1. Wound healing in a fetal, adult, and scar tissue model: a comparative study.

    PubMed

    Coolen, Neeltje A; Schouten, Kelly C W M; Boekema, Bouke K H L; Middelkoop, Esther; Ulrich, Magda M W

    2010-01-01

    Early gestation fetal wounds heal without scar formation. Understanding the mechanism of this scarless healing may lead to new therapeutic strategies for improving adult wound healing. The aims of this study were to develop a human fetal wound model in which fetal healing can be studied and to compare this model with a human adult and scar tissue model. A burn wound (10 x 2 mm) was made in human ex vivo fetal, adult, and scar tissue under controlled and standardized conditions. Subsequently, the skin samples were cultured for 7, 14, and 21 days. Cells in the skin samples maintained their viability during the 21-day culture period. Already after 7 days, a significantly higher median percentage of wound closure was achieved in the fetal skin model vs. the adult and scar tissue model (74% vs. 28 and 29%, respectively, p<0.05). After 21 days of culture, only fetal wounds were completely reepithelialized. Fibroblasts migrated into the wounded dermis of all three wound models during culture, but more fibroblasts were present earlier in the wound area of the fetal skin model. The fast reepithelialization and prompt presence of many fibroblasts in the fetal model suggest that rapid healing might play a role in scarless healing.

  2. Galvanic microparticles increase migration of human dermal fibroblasts in a wound-healing model via reactive oxygen species pathway

    PubMed Central

    Tandon, Nina; Cimetta, Elisa; Villasante, Aranzazu; Kupferstein, Nicolette; Southall, Michael D.; Fassih, Ali; Xie, Junxia; Sun, Ying; Vunjak-Novakovic, Gordana

    2015-01-01

    Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing. PMID:24113575

  3. Improvement of green tea polyphenol with milk on skin with respect to antioxidation in healthy adults: a double-blind placebo-controlled randomized crossover clinical trial.

    PubMed

    Chiu, Hui-Fang; Lin, Tung-Yi; Shen, You-Cheng; Venkatakrishnan, Kamesh; Wang, Chin-Kun

    2016-02-01

    Green tea polyphenols (GTP) have been widely tested for their effects on several metabolic syndromes and degenerative diseases such as cancer, cardiovascular diseases, and diabetes. The present study was formulated to assess the physiological efficacy of green tea polyphenol infused with milk (GTPM) on skin integrity in correlation with antioxidative status in healthy adults. Forty-four healthy voluntary subjects were recruited and assigned to two groups, who drank 240 ml of mineral water mixed with either an experimental (GTPM) or placebo package (2 packs per day) for the following 6 months. The experimental group then switched to the placebo package, and vice versa, for a further 6 months, with one month of washout period in between. During the initial, 3(rd), 6(th), 10(th), and 13(th) month anthropometric measurements were performed and fasting blood samples were withdrawn for various biochemical assays. Skin examination was performed at the initial, 6(th) and 13(th) month. No significant alterations were observed in any of the anthropometric measurements. Administration of GTPM significantly increased (p < 0.05) the antioxidant index and antioxidant enzyme activities when compared with the placebo group, whereas a concomitant decrease in the levels of lipid peroxidation were noted. Moreover, GTPM intake notably improved skin integrity and texture by markedly lowering (p < 0.05) skin wrinkles and roughness in elderly subjects. GTPM proved to be an effective antioxidant by lowering oxidative stress and thereby ameliorating skin texture and integrity.

  4. Distally based venous flap: a new technique for the correction of syndactyly without skin graft in adult patients.

    PubMed

    Cil, Yakup; Kocman, Atacan Emre; Yapici, Abdul Kerim

    2009-12-01

    Skin grafts and local flaps are conventional methods for repairing simple syndactyly. Skin grafts usually leave unsightly appearance and contracture formation. In this study, unipedicled distally based venous flap were raised from third or fourth metacarpal area of the hand for syndactyly treatment. The distally based venous flap was to provide skin coverage to one side of the finger, in order to avoid complications arising from using skin graft. Nine patients' syndactylies (5 simple incomplete and 4 simple complete syndactyly) were treated using this method. The mean follow-up period of the flaps was 14 months, ranging from 12 to 16 months. Mild edema and venous congestion occurred in all flaps. Superficial necrosis involving two flaps did not affect flap survival. All flaps survived completely. In this article, we have described a new surgical technique for the correction of syndactyly in a single surgical procedure that utilizes a distally based venous flap to provide skin coverage without skin graft.

  5. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration.

    PubMed

    Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

    2017-03-28

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy.

  6. Transcriptional control of cardiac fibroblast plasticity.

    PubMed

    Lighthouse, Janet K; Small, Eric M

    2016-02-01

    Cardiac fibroblasts help maintain the normal architecture of the healthy heart and are responsible for scar formation and the healing response to pathological insults. Various genetic, biomechanical, or humoral factors stimulate fibroblasts to become contractile smooth muscle-like cells called myofibroblasts that secrete large amounts of extracellular matrix. Unfortunately, unchecked myofibroblast activation in heart disease leads to pathological fibrosis, which is a major risk factor for the development of cardiac arrhythmias and heart failure. A better understanding of the molecular mechanisms that control fibroblast plasticity and myofibroblast activation is essential to develop novel strategies to specifically target pathological cardiac fibrosis without disrupting the adaptive healing response. This review highlights the major transcriptional mediators of fibroblast origin and function in development and disease. The contribution of the fetal epicardial gene program will be discussed in the context of fibroblast origin in development and following injury, primarily focusing on Tcf21 and C/EBP. We will also highlight the major transcriptional regulatory axes that control fibroblast plasticity in the adult heart, including transforming growth factor β (TGFβ)/Smad signaling, the Rho/myocardin-related transcription factor (MRTF)/serum response factor (SRF) axis, and Calcineurin/transient receptor potential channel (TRP)/nuclear factor of activated T-Cell (NFAT) signaling. Finally, we will discuss recent strategies to divert the fibroblast transcriptional program in an effort to promote cardiomyocyte regeneration. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling".

  7. Skin Dictionary

    MedlinePlus

    ... your skin, hair, and nails Skin dictionary Camp Discovery Good Skin Knowledge lesson plans and activities Video library Find a ... your skin, hair, and nails Skin dictionary Camp Discovery Good Skin Knowledge lesson plans and activities Video library Find a ...

  8. Skin conductance biofeedback training in adults with drug-resistant temporal lobe epilepsy and stress-triggered seizures: a proof-of-concept study.

    PubMed

    Micoulaud-Franchi, Jean-Arthur; Kotwas, Iliana; Lanteaume, Laura; Berthet, Christelle; Bastien, Mireille; Vion-Dury, Jean; McGonigal, Aileen; Bartolomei, Fabrice

    2014-12-01

    The present proof-of-concept study investigated the feasibility of skin conductance biofeedback training in reducing seizures in adults with drug-resistant temporal lobe epilepsy (TLE), whose seizures are triggered by stress. Skin conductance biofeedback aims to increase levels of peripheral sympathetic arousal in order to reduce cortical excitability. This might seem somewhat counterintuitive, since such autonomic arousal may also be associated with increased stress and anxiety. Thus, this sought to verify that patients with TLE and stress-triggered seizures are not worsened in terms of stress, anxiety, and negative emotional response to this nonpharmacological treatment. Eleven patients with drug-resistant TLE with seizures triggered by stress were treated with 12 sessions of biofeedback. Patients did not worsen on cognitive evaluation of attentional biases towards negative emotional stimuli (P>.05) or on psychometric evaluation with state anxiety inventory (P = .059); in addition, a significant improvement was found in the Negative Affect Schedule (P = .014) and in the Beck Depression Inventory (P = .009). Biofeedback training significantly reduced seizure frequency with a mean reduction of -48.61% (SD = 27.79) (P = .005). There was a correlation between the mean change in skin conductance activity over the biofeedback treatment and the reduction of seizure frequency (r(11) = .62, P = .042). Thus, the skin conductance biofeedback used in the present study, which teaches patients to achieve an increased level of peripheral sympathetic arousal, was a well-tolerated nonpharmacological treatment. Further, well-controlled studies are needed to confirm the therapeutic value of this nonpharmacological treatment in reducing seizures in adults with drug-resistant TLE with seizures triggered by stress.

  9. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    SciTech Connect

    Tashiro, Kanae; Shishido, Mayumi; Fujimoto, Keiko; Hirota, Yuko; Yo, Kazuyuki; Gomi, Takamasa; Tanaka, Yoshitaka

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  10. Skin Diseases: Skin Health and Skin Diseases

    MedlinePlus

    Skip Navigation Bar Home Current Issue Past Issues Skin Diseases Skin Health and Skin Diseases Past Issues / Fall 2008 Table of Contents ... acne to wrinkles Did you know that your skin is the largest organ of your body? It ...

  11. The effects of feeding with synbiotic (Pediococcus acidilactici and fructooligosaccharide) enriched adult Artemia on skin mucus immune responses, stress resistance, intestinal microbiota and performance of angelfish (Pterophyllum scalare).

    PubMed

    Azimirad, Mahmood; Meshkini, Saeed; Ahmadifard, Nasrollah; Hoseinifar, Seyed Hossein

    2016-07-01

    The aim of this study was to evaluate the effects of feeding on synbiotic (Pediococcus acidilactici and fructooligosaccharide) enriched adult Artemia franciscana on skin mucus immune responses, stress resistance, intestinal microbiota and growth performance of angelfish (Pterophyllum scalare). Three hundred and sixty fish with initial weight 3.2 ± 0.13 g were randomly divided into twelve aquaria (50 L) assigned to four groups in triplicates. Fish were fed for 7 weeks with dietary treatments, including treatment 1: feeding adult Artemia without enrichment (control group), treatment 2: feeding adult Artemia enriched with lyophilised probiotic P. acidilactici (700 mg L(-1)), 3: feeding adult Artemia enriched with prebiotic fructooligosaccharide (FOS) (100 mg L(-1)), group 4: feeding adult Artemia enriched with synbiotic (P. acidilactici (700 mg L(-1)) + FOS (100 mg L(-1))). Skin mucus immune responses (lysozyme activity, total Immunoglobulin and protease), stress resistance against environmental stress (acute decrease of temperature and increase salinity), intestinal microbiota as well as growth indices were measured at the end of feeding trial. Artemia enriched with synbiotic significantly improved growth performance compared to other treatments (P < 0.05). The highest weight gain and specific growth rate (SGR) was observed in synbiotic fed fish (P < 0.05). Compared to the other treatments, the population of lactic acid bacteria was significantly higher in the intestinal microbiota of fish fed synbiotic supplemented diet (P < 0.05). In the environmental stress challenge test, the maximum resistance to abrupt decrease of temperature (17 °C) or elevation of salinity (12 g per liter) was observed in the synbiotic treatment. Also, the total immunoglobulin and lysozyme activity level of skin mucus was significantly elevated in fish fed Artemia enriched with synbiotic (P < 0.05). These results revealed that feeding angelfish with synbiotic

  12. Beryllium nitrate inhibits fibroblast migration to disrupt epimorphic regeneration.

    PubMed

    Cook, Adam B; Seifert, Ashley W

    2016-10-01

    Epimorphic regeneration proceeds with or without formation of a blastema, as observed for the limb and skin, respectively. Inhibition of epimorphic regeneration provides a means to interrogate the cellular and molecular mechanisms that regulate it. In this study, we show that exposing amputated limbs to beryllium nitrate disrupts blastema formation and causes severe patterning defects in limb regeneration. In contrast, exposing full-thickness skin wounds to beryllium only causes a delay in skin regeneration. By transplanting full-thickness skin from ubiquitous GFP-expressing axolotls to wild-type hosts, we demonstrate that beryllium inhibits fibroblast migration during limb and skin regeneration in vivo Moreover, we show that beryllium also inhibits cell migration in vitro using axolotl and human fibroblasts. Interestingly, beryllium did not act as an immunostimulatory agent as it does in Anurans and mammals, nor did it affect keratinocyte migration, proliferation or re-epithelialization, suggesting that the effect of beryllium is cell type-specific. While we did not detect an increase in cell death during regeneration in response to beryllium, it did disrupt cell proliferation in mesenchymal cells. Taken together, our data show that normal blastema organogenesis cannot occur without timely infiltration of local fibroblasts and highlights the importance of positional information to instruct pattern formation during regeneration. In contrast, non-blastemal-based skin regeneration can occur despite early inhibition of fibroblast migration and cell proliferation.

  13. Influence of three laser wavelengths on human fibroblasts cell culture.

    PubMed

    Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Campian, Radu; Crisan, Liana; Baciut, Grigore

    2013-02-01

    Although experimental studies in vitro and vivo have been numerous, the effect of laser wavelength irradiation on human fibroblast cell culture is poorly understood. This emphasizes the need of additional cellular and molecular research into laser influence with low energy and power. The aim of this study was to assess the influence of three different laser wavelengths on the human skin fibroblasts cell culture. We wanted to evaluate if near infrared lasers had any influence in healing of wounds by stimulating mitochondrial activity of fibroblasts. The cells were irradiated using 830-, 980- and 2,940-nm laser wavelengths. The irradiated cells were incubated and their mitochondrial activity was assessed by the MTT assay at 24, 48 and 72 h. Simultaneously, an apoptosis assay was assessed on the irradiated fibroblasts. It can be concluded that laser light of the near-infrared region (830 and 980 nm) influences fibroblasts mitochondrial activity compared to the 2,940-nm wavelength which produces apoptosis.

  14. Collagen Expression in Fibroblasts with a Novel LMNA Mutation

    PubMed Central

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko

    2007-01-01

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy. PMID:17150192

  15. Collagen expression in fibroblasts with a novel LMNA mutation

    SciTech Connect

    Nguyen, Desiree; Leistritz, Dru F.; Turner, Lesley; MacGregor, David; Ohson, Kamal; Dancey, Paul; Martin, George M.; Oshima, Junko . E-mail: picard@u.washington.edu

    2007-01-19

    Laminopathies are a group of genetic disorders caused by LMNA mutations; they include muscular dystrophies, lipodystrophies, and progeroid syndromes. We identified a novel heterozygous LMNA mutation, L59R, in a patient with the general appearance of mandibuloacral dysplasia and progeroid features. Examination of the nuclei of dermal fibroblasts revealed the irregular morphology characteristic of LMNA mutant cells. The nuclear morphological abnormalities of LMNA mutant lymphoblastoid cell lines were less prominent compared to those of primary fibroblasts. Since it has been reported that progeroid features are associated with increased extracellular matrix in dermal tissues, we compared a subset of these components in fibroblast cultures from LMNA mutants with those of control fibroblasts. There was no evidence of intracellular accumulation or altered mobility of collagen chains, or altered conversion of procollagen to collagen, suggesting that skin fibroblast-mediated matrix production may not play a significant role in the pathogenesis of this particular laminopathy.

  16. Short-Term Exposure to Coal Combustion Waste Has Little Impact on the Skin Microbiome of Adult Spring Peepers (Pseudacris crucifer)

    PubMed Central

    Walke, Jenifer B.; Becker, Matthew H.; Umile, Thomas P.; Burzynski, Elizabeth A.; Minbiole, Kevin P. C.; Iannetta, Anthony A.; Santiago, Celina N.; Hopkins, William A.; Belden, Lisa K.

    2016-01-01

    ABSTRACT Disruptions to the microbiome can impact host health as can exposure to environmental contaminants. However, few studies have addressed how environmental contaminants impact the microbiome. We explored this question for frogs that breed in wetlands contaminated with fly ash, a by-product of coal combustion that is enriched in trace elements. We found differences in the bacterial communities among a fly ash-contaminated site and several reference wetlands. We then experimentally assessed the impacts of fly ash on the skin microbiome of adult spring peepers (Pseudacris crucifer). Frogs were exposed to fly ash in the laboratory for 12 h, the duration of a typical breeding event, and the skin microbiome was assessed after 5 days (experiment 1) or after 5 and 15 days (experiment 2). We examined bacterial community structure using 16S rRNA gene amplicon sequencing and metabolite profiles using high-pressure liquid chromatography-mass spectrometry (HPLC-MS). We found little impact as the result of acute exposure to fly ash on the bacterial communities or metabolite profiles in either experiment, suggesting that the bacterial symbiont communities of adults may be relatively resistant to brief contaminant exposure. However, housing frogs in the laboratory altered bacterial community structure in the two experiments, which supports prior research suggesting that environmental source pools are important for maintaining the amphibian skin microbiome. Therefore, for contaminants like fly ash that may alter the potential source pool of symbionts, we think it may be important to explore how contaminants affect the initial assembly of the amphibian skin microbiome in larval amphibians that develop within contaminated sites. IMPORTANCE Animals are hosts to many symbiotic microorganisms, collectively called the microbiome, that play critical roles in host health. Therefore, environmental contaminants that alter the microbiome may impact hosts. Some of the most widespread

  17. Basic fibroblast growth factor induces matrix metalloproteinase-13 via ERK MAP kinase-altered phosphorylation and sumoylation of Elk-1 in human adult articular chondrocytes.

    PubMed

    Im, Hee-Jeong; Sharrocks, Andrew D; Lin, Xia; Yan, Dongyao; Kim, Jaesung; van Wijnen, Andre J; Hipskind, Robert A

    2009-01-01

    Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) and release of basic fibroblast growth factor (bFGF) are principal aspects of the pathology of osteoarthritis (OA). ECM disruption leads to bFGF release, which activates the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway and its downstream target the Ets-like transcription factor Elk-1. Previously we demonstrated that the bFGF-ERK-Elk-1 signaling axis is responsible for the potent induction of MMP-13 in human primary articular chondrocytes. Here we report that, in addition to phosphorylation of Elk-1, dynamic posttranslational modification of Elk-1 by small ubiquitin-related modifier (SUMO) serves as an important mechanism through which MMP-13 gene expression is regulated. We show that bFGF activates Elk-1 mainly through the ERK pathway and that increased phosphorylation of Elk-1 is accompanied by decreased conjugation of SUMO to Elk-1. Reporter gene assays reveal that phosphorylation renders Elk-1 competent for induction of MMP-13 gene transcription, while sumoylation has the opposite effect. Furthermore, we demonstrate that the SUMO-conjugase Ubc9 acts as a key mediator for Elk-1 sumoylation. Taken together, our results suggest that sumoylation antagonizes the phosphorylation-dependent transactivation capacity of Elk-1. This attenuates transcription of its downstream target gene MMP-13 to maintain the integrity of cartilage ECM homeostasis.

  18. Induction of plasminogen activator by UV light in normal and xeroderma pigmentosum fibroblasts

    SciTech Connect

    Miskin, R.; Ben-Ishai, R.

    1981-10-01

    Normal and DNA repair-deficient human fibroblasts have been used to study induction of plasminogen activator (PA) by DNA damage. UV light induced the synthesis of PA in skin fibroblasts of all types of xeroderma pigmentosum (XP) in XP heterozygotes and in human amniotic cells. Enzyme induction was, however, not observed in fibroblasts of normal adults. In classical XP, which are deficient in excision repair, PA synthesis occurred in a narrow range of low-UV fluences. In such strains, the level of enzyme produced was correlated with the extent of repair deficiency. UV fluences required for PA induction in XP variants and XP heteozygotes were at least 10 times those inducing enzyme synthesis in excision-deficient XP. Maximum enzyme induction occurred 48 hr after irradiation, and the highest levels of enzyme produced were 15-20 times those of PA baseline levels. Electrophoretic analysis showed that UV irradiation enhances the synthesis of the M/sub r/ 60,000 human urokinase-type PA, which is present in low amounts in untreated cells. Our results suggest that PA induction in human cells is caused by unrepaired DNA damage and represents a eukaryotic SOS-like function. In addition, PA induction may provide a sensitive assay for detection of cellular DNA repair deficiencies and identification of XP heterozygotes.

  19. Small Molecules Modulate Chromatin Accessibility to Promote NEUROG2-Mediated Fibroblast-to-Neuron Reprogramming.

    PubMed

    Smith, Derek K; Yang, Jianjing; Liu, Meng-Lu; Zhang, Chun-Li

    2016-11-08

    Pro-neural transcription factors and small molecules can induce the reprogramming of fibroblasts into functional neurons; however, the immediate-early molecular events that catalyze this conversion have not been well defined. We previously demonstrated that neurogenin 2 (NEUROG2), forskolin (F), and dorsomorphin (D) can reprogram fibroblasts into functional neurons with high efficiency. Here, we used this model to define the genetic and epigenetic events that initiate an acquisition of neuronal identity. We demonstrate that NEUROG2 is a pioneer factor, FD enhances chromatin accessibility and H3K27 acetylation, and synergistic transcription activated by these factors is essential to successful reprogramming. CREB1 promotes neuron survival and acts with NEUROG2 to upregulate SOX4, which co-activates NEUROD1 and NEUROD4. In addition, SOX4 targets SWI/SNF subunits and SOX4 knockdown results in extensive loss of open chromatin and abolishes reprogramming. Applying these insights, adult human glioblastoma cell and skin fibroblast reprogramming can be improved using SOX4 or chromatin-modifying chemicals.

  20. Senescent fibroblasts in melanoma initiation and progression: an integrated theoretical, experimental, and clinical approach.

    PubMed

    Kim, Eunjung; Rebecca, Vito; Fedorenko, Inna V; Messina, Jane L; Mathew, Rahel; Maria-Engler, Silvya S; Basanta, David; Smalley, Keiran S M; Anderson, Alexander R A

    2013-12-01

    We present an integrated study to understand the key role of senescent fibroblasts in driving melanoma progression. Based on the hybrid cellular automata paradigm, we developed an in silico model of normal skin. The model focuses on key cellular and microenvironmental variables that regulate interactions among keratinocytes, melanocytes, and fibroblasts, key components of the skin. The model recapitulates normal skin structure and is robust enough to withstand physical as well as biochemical perturbations. Furthermore, the model predicted the important role of the skin microenvironment in melanoma initiation and progression. Our in vitro experiments showed that dermal fibroblasts, which are an important source of growth factors in the skin, adopt a secretory phenotype that facilitates cancer cell growth and invasion when they become senescent. Our coculture experiments showed that the senescent fibroblasts promoted the growth of nontumorigenic melanoma cells and enhanced the invasion of advanced melanoma cells. Motivated by these experimental results, we incorporated senescent fibroblasts into our model and showed that senescent fibroblasts transform the skin microenvironment and subsequently change the skin architecture by enhancing the growth and invasion of normal melanocytes. The interaction between senescent fibroblasts and the early-stage melanoma cells leads to melanoma initiation and progression. Of microenvironmental factors that senescent fibroblasts produce, proteases are shown to be one of the key contributing factors that promoted melanoma development from our simulations. Although not a direct validation, we also observed increased proteolytic activity in stromal fields adjacent to melanoma lesions in human histology. This leads us to the conclusion that senescent fibroblasts may create a prooncogenic skin microenvironment that cooperates with mutant melanocytes to drive melanoma initiation and progression and should therefore be considered as a

  1. Bioengineered Self-assembled Skin as an Alternative to Skin Grafts

    PubMed Central

    Climov, Mihail; Medeiros, Erika; Farkash, Evan A.; Qiao, Jizeng; Rousseau, Cecile F.; Dong, Shumin; Zawadzka, Agatha; Racki, Waldemar J.; Al-Musa, Ahmad; Sachs, David H.; Randolph, Mark A.

    2016-01-01

    For patients with extensive burns or donor site scarring, the limited availability of autologous and the inevitable rejection of allogeneic skin drive the need for new alternatives. Existing engineered biologic and synthetic skin analogs serve as temporary coverage until sufficient autologous skin is available. Here we report successful engraftment of a self-assembled bilayered skin construct derived from autologous skin punch biopsies in a porcine model. Dermal fibroblasts were stimulated to produce an extracellular matrix and were then seeded with epidermal progenitor cells to generate an epidermis. Autologous constructs were grafted onto partial- and full-thickness wounds. By gross examination and histology, skin construct vascularization and healing were comparable to autologous skin grafts and were superior to an autologous bilayered living cellular construct fabricated with fibroblasts cast in bovine collagen. This is the first demonstration of spontaneous vascularization and permanent engraftment of a self-assembled bilayered bioengineered skin that could supplement existing methods of reconstruction. PMID:27482479

  2. Pyruvate Dehydrogenase Complex Activity in Normal and Deficient Fibroblasts

    PubMed Central

    Sheu, Kwan-Fu Rex; Hu, Chii-Whei C.; Utter, Merton F.

    1981-01-01

    Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37°C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-14C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of 14CO2 production on the presence of thiamin-PP, coenzyme A (CoA), Mg++, and NAD+. Also, it has been shown that acetyl-CoA and 14CO2 are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg++ and Ca++, or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency

  3. LEOPARD syndrome with partly normal skin and sex chromosome mosaicism.

    PubMed

    Writzl, Karin; Hoovers, Jan; Sistermans, Erik A; Hennekam, Raoul C M

    2007-11-01

    We report on a family with LEOPARD syndrome which was molecularly proven (p.Thr468Met in PTPN11) in a father and his adult son. The father had multiple lentigines dispersed equally over his body; the son was similarly affected except for the left part of thorax, back and left arm, which were completely devoid of lentigines and only showed a few nevi. In addition, the son was found to have a mosaic karyotype, 47,XYY/46,XY, in lymphocytes. Skin biopsies from the pigmented and unpigmented forearm showed that mainly a 47,XYY karyotype was present in the pigmented skin and mainly a 46,XY karyotype in the unpigmented skin. In both fibroblast cultures the PTPN11 mutation was present, and no additional mutation could be detected. We discuss the various possible explanations for this phenotype, which include the possibility of coincidence; revertant mosaicism; silencing of a second PTPN11 mutation; gene(s) located on a sex chromosome influencing the phenotype; and epigenetic influences. We favor that the co-occurrence of a sex chromosome mosaicism and mosaicism for skin symptoms in a single patient with LEOPARD syndrome is coincidence, but that mosaicism for LEOPARD skin symptoms in itself may well be more frequent and needs additional studies. Each of the above-hypothesized mechanisms may then remain possible.

  4. Serotoninergic and melatoninergic systems are fully expressed in human skin.

    PubMed

    Slominski, Andrzej; Pisarchik, Alexander; Semak, Igor; Sweatman, Trevor; Wortsman, Jacobo; Szczesniewski, Andre; Slugocki, George; McNulty, John; Kauser, Söbia; Tobin, Desmond J; Jing, Chen; Johansson, Olle

    2002-06-01

    We investigated the cutaneous expression of genes and enzymes responsible for the multistep conversion of tryptophan to serotonin and further to melatonin. Samples tested were human skin, normal and pathologic (basal cell carcinoma and melanoma), cultured normal epidermal and follicular melanocytes, melanoma cell lines, normal neonatal and adult epidermal and follicular keratinocytes, squamous cell carcinoma cells, and fibroblasts from dermis and follicular papilla. The majority of the samples showed simultaneous expression of the genes for tryptophan hydroxylase, arylalkylamine N-acetyltransferase (AANAT), and hydroxyindole-O-methyltransferase (HIOMT). The products of AANAT activity were identified by RP-HPLC with fluorimetric detection in human skin and in cultured normal and malignant melanocytes and immortalized keratinocytes; HIOMT activity was detected in human skin, keratinocytes, and melanoma cells. N-acetylserotonin (NAS) was detected by RP-HPLC in human skin extracts. NAS identity was confirmed further by LC/MS in keratinocytes. In conclusion, we provide evidence that the human skin expresses intrinsic serotonin and melatonin biosynthetic pathways.

  5. Distribution of type XV collagen transcripts in human tissue and their production by muscle cells and fibroblasts.

    PubMed Central

    Kivirikko, S.; Saarela, J.; Myers, J. C.; Autio-Harmainen, H.; Pihlajaniemi, T.

    1995-01-01

    Type XV collagen is a recently identified member of the diverse family of collagens, its structure being characterized by extensive interruptions in the collagenous sequences. A combination of Northern blot hybridization of fetal and adult human tissues and in situ hybridization analyses of a fetus with Down's syndrome, several placentas, and adult skin were used to localize expression of its mRNAs. Northern blot analysis revealed marked expression in heart, skeletal muscle, and placenta tissues and moderate levels in the kidney and pancreas. Clear in situ hybridization signals were detected in fibroblasts and endothelial cells in all tissues studied. Examination of fetal heart, skeletal muscle, and smooth muscle tissues showed that the high type XV collagen mRNA level in the muscle RNA was localized not only to fibroblasts residing in the endomysium but also to myoblasts. Interestingly, type XV collagen mRNAs were also synthesized by certain epithelial cells in kidney, lung, pancreas, and placenta. It was the morphologically immature glomeruli in the kidney and the lower parts of the nephron, especially the collecting ducts, that contained these mRNAs but not the mature glomeruli or proximal tubules, suggesting differences in expression during development. These findings indicate a wide distribution of type XV collagen transcripts, the main producers being mesenchymally derived cells, particularly muscle cells and fibroblasts. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:7485412

  6. Mechanisms of referred visceral pain: uterine inflammation in the adult virgin rat results in neurogenic plasma extravasation in the skin.

    PubMed

    Wesselmann, U; Lai, J

    1997-12-01

    The purpose of this study was to investigate the mechanisms of referred pain observed in female patients with pain from the reproductive organs. We developed a model of inflammatory uterine pain in the rat. Inflammation of the uterus in rats pretreated with Evans Blue Dye resulted in dye extravasation in the skin over the abdomen, groin, lower back, thighs, perineal area and proximal tail, thus providing for the first time evidence for the trophic changes observed in the area of referred visceral pain in an animal model of uterine pain. The neuronal pathways mediating the observed dye extravasation in the skin after uterine inflammation may include dichotomizing afferent fibers, afferent-afferent interactions via a spinal cord pathway or a sympathetic reflex. This model will allow to gain further insight into the mechanisms of referred pain and the trophic changes observed in the area of referred pain in visceral disease.

  7. Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

    PubMed Central

    Mias, Céline; Coatrieux, Christelle; Denis, Colette; Genet, Gaël; Seguelas, Marie-Hélène; Laplace, Nathalie; Rouzaud-Laborde, Charlotte; Calise, Denis; Parini, Angelo; Cussac, Daniel; Pathak, Atul; Sénard, Jean-Michel; Galés, Céline

    2013-01-01

    Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic

  8. Skin Cancer

    MedlinePlus

    ... version of this page please turn Javascript on. Skin Cancer What is Skin Cancer? Skin cancer is the most common type ... of approximately 9,480 Americans in 2013. Can Skin Cancer Be Treated? Most basal cell and squamous ...

  9. Decreased lysosomal storage in the adult MPS VII mouse brain in the vicinity of grafts of retroviral vector-corrected fibroblasts secreting high levels of beta-glucuronidase.

    PubMed

    Taylor, R M; Wolfe, J H

    1997-07-01

    A deficiency of beta-glucuronidase (GUSB) causes the multisystem progressive degenerative syndrome, mucopolysaccharidosis (MPS) type VII (Sly disease), which includes mental retardation. Animal homologues of MPS VII (ref. 3, 4) are models for testing somatic gene transfer approaches to treat the central nervous system in this and other lysosomal storage disorders. Previous attempts to correct murine MPS VII by gene therapy have successfully treated lesions in some organs but not in the brain. Other experimental modalities have forestalled some disease progression in the brain, but only if done at birth, before the onset of severe lesions, when the animals are phenotypically normal. We tested whether therapeutic amounts of GUSB could be delivered to the diseased adult brain by transplanting cells engineered to super-secrete the normal enzyme for export to surrounding neural tissues. Lysosomal distention was cleared from neurons and glial cells in the vicinity of the grafts, showing that the secreted enzyme could reach the diseased cells and reverse lesions in the severely diseased brain. The ability to correct established lesions will be important for the treatment of many lysosomal storage diseases affecting the brain, because most patients are not diagnosed until lesions are advanced enough to affect phenotype or developmental milestones in early childhood, and some forms of the diseases do not become apparent until later in life.

  10. Myosin synthesis in embryonic chicken fibroblasts

    PubMed Central

    1979-01-01

    The rate of constitutive myosin synthesis was measured in cultures of replicating embryonic chicken skin fibroblasts by pulse labeling with [3H]leucine. These cells synthesized the 200,000-dalton heavy chain of myosin (MHC) at a rate of 3.2 x 10(3) molecules/cell/min. Additionally, an independent estimate of the MHC synthesis rate needed to maintain a constant level of constitutive MHC/cell was calculated from total protein content, percentage MHC, fibroblast doubling time, and MHC half- life. This calculated rate of approximately 2.9 x 10(3) molecules/cell/min was in close agreement with the measured rate. By comparison, the synthesis rate of myofibrillar MHC in fully activated muscle cell cultures was approximately 2.9 x 10(4) molecules/nucleus/min. PMID:479285

  11. Epidermal growth factor improves the migration and contractility of aged fibroblasts cultured on 3D collagen matrices.

    PubMed

    Kim, Daehwan; Kim, So Young; Mun, Seog Kyun; Rhee, Sangmyung; Kim, Beom Joon

    2015-04-01

    Epidermal growth factor (EGF) plays a critical role in fibroblasts by stimulating the production of collagen and supports cell renewal through the interaction between keratinocytes and fibroblasts. It is well known that the contractile activity of fibroblasts is required for the remodeling of the extracellular matrix (ECM), which contributes to skin elasticity. However, the role of EGF in the contraction of aged fibroblasts under 3-dimensional (3D) culture conditions is not yet fully understood. In the present study, we demonstrated that young fibroblasts spread and proliferated more rapidly than aged fibroblasts under 2-dimensional (2D) culture conditions. Cell migration assay using a nested collagen matrix revealed that the migration of young fibroblasts was also greater than that of aged fibroblasts under 3D culture conditions. However, the addition of recombinant human EGF (rhEGF) resulted in the enhanced migration of aged fibroblasts; the migration rate was similar to that of the young fibroblasts. The aged fibroblasts showed decreased cluster formation compared with the young fibroblasts on the collagen matrix, which was improved by the addition of rhEGF. Furthermore, cell contraction assay revealed that the basal contractility of the aged fibroblasts was lower than that of the young fibroblasts; however, following treatment with rhEGF, the contractility was restored to levels similar or even higher to those of the young fibroblasts. Taken together, our results suggest that rhEGF is a potential renewal agent that acts to improve the migration and contraction of aged fibroblasts more efficiently than young fibroblasts under 3D culture conditions; thus, EGF may have valuable regenerative effects on aged skin.

  12. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

  13. Dupuytren's Contracture: Fibroblast Contraction?

    PubMed Central

    Gabbiani, Giulio; Majno, Guido

    1972-01-01

    In 6 cases of Dupuytren's disease and 1 of Ledderhose's disease, the nodules of the palmar and plantar aponeurosis were examined by light and electron microscopy. The cells composing these nodules, presumably fibroblasts, showed three significant ultrastructural features: (1) a fibrillar system similar to that of smooth muscle cells; (2) nuclear deformations such as are found in contracted cells, the severest being recognizable by light microscopy (cross-banded nuclei); (3) cell-to-cell and cell-to-stroma attachments. Based on these data and on recent information about the biology of the fibroblasts, it is suggested that these cells are fibroblasts that have modulated into contractile cells (myofibroblasts), and that their contraction plays a role in the pathogenesis of the contracture observed clinically. ImagesFig 10Fig 5Fig 11Fig 6 and 7Fig 8Fig 1Fig 2Fig 9Fig 3Fig 4 PMID:5009249

  14. The elemental analysis of normal and Menkes' fibroblast cells with the SPMP

    NASA Astrophysics Data System (ADS)

    Allan, G. L.; Camakaris, J.; Legge, G. J. F.

    1991-03-01

    A scanning proton microprobe has been used to study the elemental distributions, including trace elements, in human skin fibroblast cells. Both normal fibroblasts and fibroblasts cultured from patients with Menkes' disease, an X-linked genetic disorder known to be associated with defective copper metabolism, were examined by the probe. Cells grown in a copper-rich medium exhibited higher levels of intracellular copper but were found to be susceptible to the toxic effect of the elevated copper level. In normal maintenance medium the Menkes' cells recorded an average intracellular copper level six times higher than normal fibroblasts.

  15. The onion skin-like organization of the septum arises from multiple embryonic origins to form multiple adult neuronal fates.

    PubMed

    Wei, B; Huang, Z; He, S; Sun, C; You, Y; Liu, F; Yang, Z

    2012-10-11

    In the past several decades, tremendous progress has been achieved through developmental studies of the central nervous system structures such as the cerebral cortex. The septum, which receives reciprocal connections from a variety of brain structures, contains diverse projection neurons but few interneurons. However, the mechanisms underlying its development remain poorly understood. Here we show that the septum is organized into an onion skin-like structure composed of five groups of neurons. These neurons are parvalbumin, choline acetyltransferase, neuronal nitric oxide synthase, calretinin and calbindin immunoreactive. Using the BrdU birth-dating method, we found that these five groups of neurons in the septum are grossly generated following an outside-in pattern. Interestingly, the distinct molecular identities of these neuronal subtypes correspond to their heterogeneous subpallial origins. Using three specific transgenic mouse lines and focal in utero electroporation of Cre-reporter plasmid, we showed that septal neurons originate from not only local progenitor regions but also neighboring progenitor regions including the medial ganglionic eminence and preoptic area. Thus, the neuronal diversity of the septum is achieved through both temporal and spatial control. Our results also suggest that multiple neuronal subtypes arrive to the septum through both radial and tangential migration. Based on these findings, we proposed a novel developmental model involving multiple spatial-temporal origins of septal neurons. This study presents new perspectives for comprehensively exploring septal functions in brain circuits.

  16. HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.

    PubMed

    Kömüves, László G; Michael, Elias; Arbeit, Jeffrey M; Ma, Xiao-Kui; Kwong, Angela; Stelnicki, Eric; Rozenfeld, Sophia; Morimune, M; Yu, Qian-Chun; Largman, Corey

    2002-05-01

    The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha-2-integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA-positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human

  17. Enhanced G2 chromatid radiosensitivity in dyskeratosis congenita fibroblasts.

    PubMed Central

    DeBauche, D M; Pai, G S; Stanley, W S

    1990-01-01

    Dyskeratosis congenita (DC) is an inherited disorder characterized by reticular pigmentation of the skin, dystrophic nails, mucosal leukoplakia, and a predisposition to cancer in early adult life. In the majority of cases, DC is an X-linked recessive trait. However, one or more autosomal form(s) of DC may exist. Although excessive spontaneous chromatid breakage has been reported in DC, it is not a consistent cytological marker for this disorder. We examined the frequency and specificity of X-irradiation-induced G2 chromatid breakage in fibroblasts from three unrelated DC patients (two males and one female). Metaphase cells from DC patients had significantly more chromatid breaks (16-18-fold and 17-26-fold at 50 and 100 rad X-irradiation, respectively) and chromatid gaps (10-12-fold and 6-7-fold at 50 and 100 rad, respectively) than those from two different controls. Analysis of banded chromosomes revealed a nonrandom distribution of chromatid aberrations in DC but not in controls, a distribution corresponding to some of the known breakpoints for cancer-specific rearrangements, constitutive fragile sites, and/or loci for cellular proto-oncogenes. The significance of this finding for cancer predisposition in DC patients is uncertain, but the increased susceptibility of X-irradiation-induced chromatid breakage may serve as a cellular marker of diagnostic value. PMID:2301400

  18. Skin Conditions

    MedlinePlus

    Your skin is your body's largest organ. It covers and protects your body. Your skin Holds body fluids in, preventing dehydration Keeps harmful ... it Anything that irritates, clogs, or inflames your skin can cause symptoms such as redness, swelling, burning, ...

  19. Aging Skin

    MedlinePlus

    ... email address Submit Home > Healthy Aging > Wellness Healthy Aging Aging skin More information on aging skin When it ... treated early. Return to top More information on Aging skin Read more from womenshealth.gov Varicose Veins ...

  20. [Dynamics of local expression of connexin-43 and basic fibroblast growth factor receptors in patients with skin and soft-tissue infections against the background of diabetes mellitus type II].

    PubMed

    Vinnik, Iu S; Salmina, A B; Tepliakova, O V; Drobushevskaia, A I; Malinovskaia, N A; Pozhilenkova, E A; Morgun, A V; Gitlina, A G

    2014-01-01

    Clinical results of wound healing dynamics were studied in 60 patients with soft-tissue infection against the background of diabetes mellitus type II. At the same time the study considered indices of intercellular contacts protein tissue expression such as connexin 43 (Cx43) and basic fibroblast growth factor receptors (bFGFR). The basic therapy of biopsy material of wound borders was applied. The reduction of bFGFR expression and the minor growth of Cx43 expression were observed. The pain syndrome proceeded for a long time and there were signs of perifocal inflammation, retard wound healing with granulation tissue. The application of combined method of ozone therapy which included autohemotherapy with ozone and an external management of wound by ozone-oxygen mixture facilitated to considerable shortening of inflammatory phase and regeneration. It was associated with increased Cx43 expression (in 1.9 times) in comparison with initial level and bFGFR was enlarged in 1.7 times to eighth day of postoperative period.

  1. Skin abscess

    MedlinePlus

    Abscess - skin; Cutaneous abscess; Subcutaneous abscess; MRSA - abscess; Staph infection - abscess ... Skin abscesses are common and affect people of all ages. They occur when an infection causes pus ...

  2. Rheumatologic Skin Disease.

    PubMed

    Kalus, Andrea

    2015-11-01

    In common rheumatologic diseases skin findings are an important diagnostic clue for astute clinicians. Skin manifestations can help identify systemic disease or may require therapy uniquely targeted at the cutaneous problem. This article discusses 3 common rheumatologic conditions seen in adults by dermatologists: cutaneous lupus, dermatomyositis, and morphea. The focus is on the cutaneous findings and clinical presentation. Some approaches to treatment are explored. Clues to help identify systemic disease are also highlighted.

  3. CRH stimulates POMC activity and corticosterone production in dermal fibroblasts.

    PubMed

    Slominski, Andrzej; Zbytek, Blazej; Semak, Igor; Sweatman, Trevor; Wortsman, Jacobo

    2005-05-01

    It has been previously documented that human skin cells including epidermal keratinocytes and dermal fibroblasts produce and process proopiomelanocortin (POMC), corticotropin releasing hormone (CRH), and express functional CRH receptors type-1 (CRH-R1). The skin also has corticosteroidogenic activity, suggesting a functional connection between these elements. In the current study, we found that human dermal fibroblasts (but not normal epidermal keratinocytes) respond to CRH with stimulation of cAMP, with POMC gene and protein expression, and ACTH production and release. Furthermore, CRH and ACTH stimulate production of corticosterone in fibroblasts, with ACTH being more potent. Although cortisol-immunoreactivity accumulation/production in fibroblasts has been detected by ELISA, it appears to be constitutive (not affected by CRH or ACTH). These effects are absent in keratinocytes. Therefore, we propose that fibroblasts but not keratinocytes display a functional CRH-POMC-corticosteroid axis organized similarly to the hypothalamus-pituitary-adrenal (HPA) axis. However, it diverges from the HPA organization in its distal step, where CRH and ACTH stimulate production of corticosterone, instead of cortisol.

  4. Adult skin-derived precursor Schwann cells exhibit superior myelination and regeneration supportive properties compared to chronically denervated nerve-derived Schwann cells.

    PubMed

    Kumar, Ranjan; Sinha, Sarthak; Hagner, Andrew; Stykel, Morgan; Raharjo, Eko; Singh, Karun K; Midha, Rajiv; Biernaskie, Jeff

    2016-04-01

    Functional outcomes following delayed peripheral nerve repair are poor. Schwann cells (SCs) play key roles in supporting axonal regeneration and remyelination following nerve injury, thus understanding the impact of chronic denervation on SC function is critical toward developing therapies to enhance regeneration. To improve our understanding of SC function following acute versus chronic-denervation, we performed functional assays of SCs from adult rodent sciatic nerve with acute- (Day 5 post) or chronic-denervation (Day 56 post), versus embryonic nerves. We also compared Schwann cells derived from adult skin-derived precursors (aSKP-SCs) as an accessible, autologous alternative to supplement the distal (denervated) nerve. We found that acutely-injured SCs and aSKP-SCs exhibited superior proliferative capacity, promotion of neurite outgrowth and myelination of axons, both in vitro and following transplant into a sciatic nerve crush injury model, while chronically-denervated SCs were severely impaired. Acute injury caused re-activation of transcription factors associated with an immature and pro-myelinating SC state (Oct-6, cJun, Sox2, AP2α, cadherin-19), but was diminished with prolonged denervation in vivo and could not be rescued following expansion in vitro suggesting that this is a permanent deficiency. Interestingly, aSKP-SCs closely resembled acutely injured and embryonic SCs, exhibiting elevated expression of these same transcription factors. In summary, prolonged denervation resulted in SC deficiency in several functional parameters that may contribute to impaired regeneration. In contrast, aSKP-SCs closely resemble the regenerative attributes ascribed to acutely-denervated or embryonic SCs emphasizing their potential as an accessible and autologous source of glia cells to enhance nerve regeneration, particularly following delays to surgical repair.

  5. Senescent fibroblast-derived Chemerin promotes squamous cell carcinoma migration

    PubMed Central

    Gatzka, Martina; Treiber, Nicolai; Schneider, Lars A.; Mulaw, Medhanie A.; Lucas, Tanja; Kochanek, Stefan; Dummer, Reinhard; Levesque, Mitchell P.; Wlaschek, Meinhard; Scharffetter-Kochanek, Karin

    2016-01-01

    Aging is associated with a rising incidence of cutaneous squamous cell carcinoma (cSCC), an aggressive skin cancer with the potential for local invasion and metastasis. Acquisition of a senescence-associated secretory phenotype (SASP) in dermal fibroblasts has been postulated to promote skin cancer progression in elderly individuals. The underlying molecular mechanisms are largely unexplored. We show that Chemerin, a previously unreported SASP factor released from senescent human dermal fibroblasts, promotes cSCC cell migration, a key feature driving tumor progression. Whereas the Chemerin abundance is downregulated in malignant cSCC cells, increased Chemerin transcripts and protein concentrations are detected in replicative senescent fibroblasts in vitro and in the fibroblast of skin sections from old donors, indicating that a Chemerin gradient is built up in the dermis of elderly. Using Transwell® migration assays, we show that Chemerin enhances the chemotaxis of different cSCC cell lines. Notably, the Chemerin receptor CCRL2 is remarkably upregulated in cSCC cell lines and human patient biopsies. Silencing Chemerin in senescent fibroblasts or the CCRL2 and GPR1 receptors in the SCL-1 cSCC cell line abrogates the Chemerin-mediated chemotaxis. Chemerin triggers the MAPK cascade via JNK and ERK1 activation, whereby the inhibition impairs the SASP- or Chemerin-mediated cSCC cell migration. Taken together, we uncover a key role for Chemerin, as a major factor in the secretome of senescent fibroblasts, promoting cSCC cell migration and possibly progression, relaying its signals through CCRL2 and GPR1 receptors with subsequent MAPK activation. These findings might have implications for targeted therapeutic interventions in elderly patients. PMID:27907906

  6. The Skin Cancer and Sun Knowledge (SCSK) Scale: Validity, Reliability, and Relationship to Sun-Related Behaviors among Young Western Adults

    ERIC Educational Resources Information Center

    Day, Ashley K.; Wilson, Carlene; Roberts, Rachel M.; Hutchinson, Amanda D.

    2014-01-01

    Increasing public knowledge remains one of the key aims of skin cancer awareness campaigns, yet diagnosis rates continue to rise. It is essential we measure skin cancer knowledge adequately so as to determine the nature of its relationship to sun-related behaviors. This study investigated the psychometric properties of a new measure of skin cancer…

  7. Dogs cloned from fetal fibroblasts by nuclear transfer.

    PubMed

    Hong, So Gun; Jang, Goo; Kim, Min Kyu; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Dae Yong; Lee, Byeong Chun

    2009-10-01

    Fetal fibroblasts have been considered as the prime candidate donor cells for the canine reproductive cloning by somatic cell nuclear transfer (SCNT) in regard to the future production of transgenic dogs, mainly due to their higher developmental competence and handling advantage in gene targeting. In this study, the cloning efficiency with canine fetal fibroblasts as donor cells was determined. A total of 50 presumptive cloned embryos were reconstructed, activated and transferred into the oviducts of naturally synchronous recipient bitches. While the fusion rate (76.9%) was similar to those of our earlier studies with adult fibroblasts as donor cells (73.9-77.1%), a high cloning efficiency (4.0%; 2 births/50 embryos transferred) was found compared to the previous success rate with adult fibroblasts (0.2-1.8%). The cloned beagles were healthy and genotypically identical to the donor fibroblast cells. This study shows that a fetal fibroblast cell would be an excellent donor for future production of transgenic dogs via gene targeting in this cell followed cloning using SCNT technology.

  8. Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes.

    PubMed Central

    Imokawa, G; Yada, Y; Morisaki, N; Kimura, M

    1998-01-01

    To clarify the paracrine linkage between human fibroblasts and melanocytes in cutaneous pigmentation, we studied the effects of human fibroblast-derived factors on the proliferation of human melanocytes. In medium conditioned for 4 days with human fibroblast culture, factors were produced that markedly stimulated DNA synthesis of human melanocytes. The stimulatory effect was higher in medium conditioned with fibroblasts from aged skin than in medium conditioned with fibroblasts from young skin, and was interrupted by inhibitors of tyrosine kinase, such as tyrphostin, genistein and herbimycin, but not by inhibitors of protein kinases C and A, such as H-7 and phloretin. The conditioned medium was also capable of activating mitogen-activated protein kinase of human melanocytes, with old fibroblasts being more effective than young ones. Analysis of factors released into the conditioned medium revealed that levels of hepatocyte growth factor (HGF) and stem cell factor (SCF) were increased in old-fibroblast-conditioned medium compared with young-fibroblast-conditioned medium. In contrast, levels of basic fibroblast growth factor (bFGF) were similar in both media. When the conditioned medium was treated with HGF antibody with or without SCF antibody, the increase in DNA synthesis by human melanocytes was decreased to 20% of the elevated level, whereas antibodies to bFGF had no effect. Analysis of the medium conditioned for 4 days after cytokine application demonstrated that, of the cytokines tested, interleukin 1alpha and tumour necrosis factor alpha are highly effective in stimulating HGF secretion by old fibroblasts. HGF and SCF, but not bFGF, were markedly increased in culture medium in the presence of IL-1alpha, and this stimulatory effect was confined to young human fibroblasts. These findings suggest that SCF and HGF derived from human fibroblasts may play a part in regulating cutaneous pigmentation during inflammation and aging. PMID:9494091

  9. Fibroblast activation protein is dispensable in the anti-influenza immune response in mice

    PubMed Central

    Chowdhury, Sumaiya; Polak, Natasa

    2017-01-01

    Fibroblast activation protein alpha (FAP) is a unique dual peptidase of the S9B serine protease family, being capable of both dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low level in healthy adult organs including the pancreas, cervix, uterus, submaxillary gland and the skin, and highly upregulated in embryogenesis, chronic inflammation and tissue remodelling. It is also expressed by cancer-associated stromal fibroblasts in more than 90% of epithelial tumours. FAP has enzymatic and non-enzymatic functions in the growth, immunosuppression, invasion and cell signalling of tumour cells. FAP deficient mice are fertile and viable with no gross abnormality, but little data exist on the role of FAP in the immune system. FAP is upregulated in association with microbial stimulation and chronic inflammation, but its function in infection remains unknown. We showed that major populations of immune cells including CD4+ and CD8+ T cells, B cells, dendritic cells and neutrophils are generated and maintained normally in FAP knockout mice. Upon intranasal challenge with influenza virus, FAP mRNA was increased in the lungs and lung-draining lymph nodes. Nonetheless, FAP deficient mice showed similar pathologic kinetics to wildtype controls, and were capable of supporting normal anti-influenza T and B cell responses. There was no evidence of compensatory upregulation of other DPP4 family members in influenza-infected FAP-deficient mice. FAP appears to be dispensable in anti-influenza adaptive immunity. PMID:28158223

  10. Proliferative response patterns of human fibroblasts after photoinjury with 4,5',8-trimethylpsoralen

    SciTech Connect

    Cohen, S.R.; Carter, D.M.; Gala, M.

    1981-01-01

    The extent of growth suppression and recovery following exposure to 4,5',8-trimethylpsoralen plus uv-A irradiation was studied in 3 diploid human fibroblast strains. Inhibition of cellular proliferation was dose-dependent within the concentration range of TMP that was tested, using a constant level of uv-A. The population generation times for all cell strains were progressively lengthened under these conditions while maximal cell densities were reduced. At 2 to 4 x 10(-7) M TMP in the presence of uv-A, there was a triphasic pattern of growth which consisted of proliferative activity during the first 24 to 36 h, followed by complete growth inhibition for variable periods of time and a recovery period of log phase proliferation that was not as vigorous as measured for untreated cells. There were also declines in the percentage of cells labeled with 3H-Tdr at various times after TMP-uv-A treatment. These measurements were essentially identical for the three fibroblast strains evaluated. In that the cells employed for these investigations were derived from embryonic pulmonary tissue, neonatal foreskin and the buttock skin of an adult male, it seems unlikely that donor age and tissue source were important variables in determining growth response patterns after TMP-uv-A exposure. Because proliferative recovery was attenuated after this photochemical injury, researchers conclude that the biologic effect(s) of TMP-uv-A extend beyond the known period of psoralen-DNA cross-link removal.

  11. Increased fibroblast functionality on CNN2-loaded titania nanotubes.

    PubMed

    Wei, Hongbo; Wu, Shuyi; Feng, Zhihong; Zhou, Wei; Dong, Yan; Wu, Guofeng; Bai, Shizhu; Zhao, Yimin

    2012-01-01

    Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant-skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor) fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.

  12. Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring.

    PubMed

    Crawford, Justin; Nygard, Karen; Gan, Bing Siang; O'Gorman, David Brian

    2015-02-01

    Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix-associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type-I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three-dimensional type-I collagen lattices. Inhibition of Rho-associated protein kinase activity significantly attenuated the effects of matrix-associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix-associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho-associated protein kinase-dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.

  13. Skin Biomes.

    PubMed

    Fyhrquist, N; Salava, A; Auvinen, P; Lauerma, A

    2016-05-01

    The cutaneous microbiome has been investigated broadly in recent years and some traditional perspectives are beginning to change. A diverse microbiome exists on human skin and has a potential to influence pathogenic microbes and modulate the course of skin disorders, e.g. atopic dermatitis. In addition to the known dysfunctions in barrier function of the skin and immunologic disturbances, evidence is rising that frequent skin disorders, e.g. atopic dermatitis, might be connected to a dysbiosis of the microbial community and changes in the skin microbiome. As a future perspective, examining the skin microbiome could be seen as a potential new diagnostic and therapeutic target in inflammatory skin disorders.

  14. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

    PubMed Central

    Janmaat, C. J.; de Rooij, K. E; Locher, H; de Groot, S. C.; de Groot, J. C. M. J.; Frijns, J. H. M.; Huisman, M. A.

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  15. Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

    PubMed Central

    Bustos-Arriaga, José; García-Machorro, Jazmín; León-Juárez, Moisés; García-Cordero, Julio; Santos-Argumedo, Leopoldo; Flores-Romo, Leopoldo; Méndez-Cruz, A. René; Juárez-Delgado, Francisco J.; Cedillo-Barrón, Leticia

    2011-01-01

    Background When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times (“probing”) before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. Methodology/Principal Findings Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts. Conclusions/Significance In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral

  16. Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

    PubMed

    Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

    2015-01-01

    Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings

  17. Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

    PubMed

    Canady, Johanna; Arndt, Stephanie; Karrer, Sigrid; Bosserhoff, Anja K

    2013-03-01

    Fibrotic disorders of the skin share the characteristic features of increased production and deposition of extracellular matrix components by activated fibroblasts. Their clinical course ranges from benign with localized cutaneous involvement to a systemic, life-threatening disease. The molecular cause for fibroblast activation remains unknown, yet epithelial-mesenchymal interactions draw mounting attention in the research field of fibrogenesis. We examined keratinocyte growth factor (KGF), a crucial molecule in fibroblast-keratinocyte cross talk, exemplarily in keloid and scleroderma, and found its expression to be increased in disease-derived fibroblasts and tissues compared with healthy controls. This overexpression induces fibroblast activation through a double paracrine mode of action. Upon KGF stimulation, the keratinocytes produced and secreted OSM (oncostatin M). Fibroblasts were in turn activated by OSM reacting with the increased expression of collagen type I-α1, fibroblast activation protein, and enhanced migration. The observed increase in collagen expression and fibroblast migration can be traced back to OSM-regulated STAT3 phosphorylation, leading to enhanced urokinase plasminogen activator expression. Hence, we propose a causative loop in the pathogenesis of fibrosing disorders of the skin mediated by the overexpression of KGF in mesenchymal cells.

  18. Ingested hyaluronan moisturizes dry skin

    PubMed Central

    2014-01-01

    Hyaluronan (HA) is present in many tissues of the body and is essential to maintain moistness in the skin tissues, which contain approximately half the body’s HA mass. Due to its viscosity and moisturizing effect, HA is widely distributed as a medicine, cosmetic, food, and, recently marketed in Japan as a popular dietary supplement to promote skin moisture. In a randomized, double-blind, placebo-controlled clinical study it was found that ingested HA increased skin moisture and improved treatment outcomes for patients with dry skin. HA is also reported to be absorbed by the body distributed, in part, to the skin. Ingested HA contributes to the increased synthesis of HA and promotes cell proliferation in fibroblasts. These effects show that ingestion of HA moisturizes the skin and is expected to improve the quality of life for people who suffer from dry skin. This review examines the moisturizing effects of dry skin by ingested HA and summarizes the series of mechanisms from absorption to pharmacological action. PMID:25014997

  19. Development of an innervated model of human skin.

    PubMed

    Khammo, Nancy; Ogilvie, Jane; Clothier, Richard H

    2007-10-01

    Neuronal cell responses and interactions with the epithelial and fibroblastic cells of the skin are a key factor in the production in vivo of the irritation/inflammatory response. Currently, few in vitro models are available that contain dermal, epidermal and the relevant neuronal components. The primary objective of this study was to produce and maintain a 3-D in vitro model of human skin containing these elements. The relevant neuronal component was supplied by adding sensory neurons derived from the dorsal root ganglion (DRG). Since adult neuronal cells do not grow significantly in vivo or in vitro, and since it is very difficult to obtain such cells from humans, it was necessary to employ embryonic rat DRG cells. The ultimate purpose of this model is to improve prediction of the in vivo skin irritancy potential of chemicals and formulations, without the need to use animal models. In addition, this approach has also been applied to the in vitro human eye and bronchial 3-D models being developed in the FRAME Alternatives Laboratory.

  20. Skin Infections

    MedlinePlus

    ... to the touch may have yellow drainage Of cellulitis: a red, inflamed area on the skin that is tender to the touch may occur in an area of a scratch or cut redness often spreads rapidly over the skin's surface ...

  1. Skin Pigment

    MedlinePlus

    ... Diseases Take Big Slice Out of America's Health, Economy (News) Health Tip: Use Caution When Applying Hair Dye Additional ... Skin Diseases Take Big Slice Out of America's Health, Economy THURSDAY, March 2, 2017 (HealthDay News) -- Skin diseases ...

  2. Sagging Skin

    MedlinePlus

    ... Stretch Marks Sun-damaged Skin Unwanted Hair Unwanted Tattoos Varicose Veins Vitiligo Wrinkles Treatments and Procedures Ambulatory ... Stretch Marks Sun-damaged Skin Unwanted Hair Unwanted Tattoos Varicose Veins Vitiligo Wrinkles Treatments and Procedures Ambulatory ...

  3. Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.

    PubMed

    Larouche, Danielle; Cuffley, Kristine; Paquet, Claudie; Germain, Lucie

    2011-03-01

    The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7-21 days of maturation at the air-liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10-26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.

  4. Differential Expression of Wound Fibrotic Factors between Facial and Trunk Dermal Fibroblasts

    PubMed Central

    Kurita, Masakazu; Okazaki, Mutsumi; Kaminishi-Tanikawa, Akiko; Niikura, Mamoru; Takushima, Akihiko; Harii, Kiyonori

    2012-01-01

    Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibro-blasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts. PMID:22260504

  5. Differential expression of wound fibrotic factors between facial and trunk dermal fibroblasts.

    PubMed

    Kurita, Masakazu; Okazaki, Mutsumi; Kaminishi-Tanikawa, Akiko; Niikura, Mamoru; Takushima, Akihiko; Harii, Kiyonori

    2012-01-01

    Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibroblasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts.

  6. Skin Aging

    MedlinePlus

    Your skin changes as you age. You might notice wrinkles, age spots and dryness. Your skin also becomes thinner and loses fat, making it ... heal, too. Sunlight is a major cause of skin aging. You can protect yourself by staying out ...

  7. Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload

    PubMed Central

    Fu, Lianwu; Wei, Chih-Chang; Powell, Pamela C.; Bradley, Wayne E.; Ahmad, Sarfaraz; Ferrario, Carlos M.; Collawn, James F.; Dell’Italia, Louis J.

    2016-01-01

    Background Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. Methods and results Isolated LV fibroblasts taken from 4 and 12 week ACF Sprague–Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24 h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase’s role in autophagy induction. Conclusion In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation. PMID:26807691

  8. Childhood Physical and Sexual Abuse in Caribbean Young Adults and Its Association with Depression, Post-Traumatic Stress, and Skin Bleaching

    PubMed Central

    James, Caryl; Seixas, Azizi A; Harrison, Abigail; Jean-Louis, Girardin; Butler, Mark; Zizi, Ferdinand; Samuels, Alafia

    2016-01-01

    Background The global prevalence of skin depigmentation/skin bleaching among blacks, estimated at 35%, is on the rise and is associated with a host of negative health and medical consequences. Current etiological approaches do not fully capture the emotional and psychological underpinnings of skin bleaching. The current study investigated the potential mediating role of depression, or post-traumatic stress symptoms (avoidance and hyperarousal) on the relationship between childhood physical and sexual abuse (CPSA) and skin bleaching. Methods A total of 1226 university participants (ages 18–30 years and 63.4% female) from three Caribbean countries (Jamaica, Barbados, and Grenada) provided data for the current analysis. They all completed self-reported measures of general demographic information along with the short screening scale for posttraumatic stress disorder (DSM-IV), childhood trauma, and skin bleaching questions. Results The prevalence of skin bleaching in our study was 25.4%. Our findings showed that individuals who bleached their skin were more likely to have been abused as children (21.6% versus 13.5%, p<0.001), were more likely to have significant symptoms of trauma (34.1% versus 24.0%, p=0.005), and were more likely to have significant depression (43.7% versus 35.1%, p=0.032). We found that trauma-related hyperarousal symptoms positively mediated the relationship between childhood physical and sexual abuse and skin bleaching (Indirect Effect=0.03, p<0.05), while avoidance (Indirect Effect=0.000, p>0.05) and depressive (Indirect Effect=0.005, p>0.05) symptoms did not. Conclusion The presence of trauma symptoms and childhood physical and sexual abuse (CPSA) may increase the likelihood of skin bleaching. Findings suggest that further exploration is needed to ascertain if the presence of skin bleaching warrants being also screened for trauma. PMID:27019771

  9. LIFE BEYOND LIFE - An Easy Way to Derive Lung Fibroblasts from Cadavers.

    PubMed

    Colomb, Sophie; Bareil, Corinne; Baccino, Eric; Berthet, Jean-Philippe; Knabe, Lucie; Vachier, Isabelle; Bourdin, Arnaud

    2017-02-10

    Several protocols have illustrated the possibility of deriving cells, such as fibroblasts, from different organs. These techniques generally concern organs sampled from living persons, but have already been described for cadavers, especially concerning the skin and tendons. We present, for the first time, an easy way to derive pulmonary fibroblasts from a lung tissue sampled from a cadaver and directly culture plated. The fibroblast output was checked daily. We obtained lung fibroblasts from 3 (60%) cadavers and 2 (100%) living persons. The fibroblast output took about 3 days for cells from living persons and took up to 39 days for those from cadavers. We did not clearly identify any parameters that could explain these differences. Nevertheless, these derived cells had the same features as the source cells, especially in terms of morphology and proliferation, and could potentially be used in different research domains such as forensic or regeneration medicine.

  10. CARFMAP: A Curated Pathway Map of Cardiac Fibroblasts

    PubMed Central

    Nim, Hieu T.; Furtado, Milena B.; Costa, Mauro W.; Kitano, Hiroaki; Rosenthal, Nadia A.; Boyd, Sarah E.

    2015-01-01

    The adult mammalian heart contains multiple cell types that work in unison under tightly regulated conditions to maintain homeostasis. Cardiac fibroblasts are a significant and unique population of non-muscle cells in the heart that have recently gained substantial interest in the cardiac biology community. To better understand this renaissance cell, it is essential to systematically survey what has been known in the literature about the cellular and molecular processes involved. We have built CARFMAP (http://visionet.erc.monash.edu.au/CARFMAP), an interactive cardiac fibroblast pathway map derived from the biomedical literature using a software-assisted manual data collection approach. CARFMAP is an information-rich interactive tool that enables cardiac biologists to explore the large body of literature in various creative ways. There is surprisingly little overlap between the cardiac fibroblast pathway map, a foreskin fibroblast pathway map, and a whole mouse organism signalling pathway map from the REACTOME database. Among the use cases of CARFMAP is a common task in our cardiac biology laboratory of identifying new genes that are (1) relevant to cardiac literature, and (2) differentially regulated in high-throughput assays. From the expression profiles of mouse cardiac and tail fibroblasts, we employed CARFMAP to characterise cardiac fibroblast pathways. Using CARFMAP in conjunction with transcriptomic data, we generated a stringent list of six genes that would not have been singled out using bioinformatics analyses alone. Experimental validation showed that five genes (Mmp3, Il6, Edn1, Pdgfc and Fgf10) are differentially regulated in the cardiac fibroblast. CARFMAP is a powerful tool for systems analyses of cardiac fibroblasts, facilitating systems-level cardiovascular research. PMID:26673252

  11. Protective effects of tea polysaccharides and polyphenols on skin.

    PubMed

    Wei, Xinlin; Liu, Ying; Xiao, Jianbo; Wang, Yuanfeng

    2009-09-09

    The protective effects of tea polysaccharides (TPS) and polyphenols (TPP) on skin were investigated. TPS1 (92% TPS), crude TPS2 (20% TPS), and TPP (98%) were tested. The abilities of TPS and TPP to protect the skin were assessed in four aspects: moisture absorption and retention, sunscreen, promoting the proliferation of fibroblast cells, and tyrosinase inhibitory effect. TPS and TPP absorbed and reserved moisture perfectly. TPS with higher purity had better moisture absorption and retention abilities. TPS1 hardly protected the skin from the sun's ultraviolet (UV) rays and had little promoting effect on fibroblasts proliferation. TPP, however, protected skin against UV rays and enhanced proliferation of fibroblast cells significantly. TPP had good tyrosinase inhibitory effects; TPS showed weaker tyrosinase inhibitory effects with the purity increased. The results indicated that TPP and TPS had complementary advantages and they should be appropriately combined to achieve higher performance when applied as active components of cosmetics.

  12. Conditional knockout of N-WASP in mouse fibroblast caused keratinocyte hyper proliferation and enhanced wound closure

    PubMed Central

    Jain, Neeraj; Kalailingam, Pazhanichamy; Tan, Kai Wei; Tan, Hui Bing; Sng, Ming Keat; Chan, Jeremy Soon Kiat; Tan, Nguan Soon; Thanabalu, Thirumaran

    2016-01-01

    Neural-Wiskott Aldrich Syndrome Protein (N-WASP) is expressed ubiquitously, regulates actin polymerization and is essential during mouse development. We have previously shown that N-WASP is critical for cell-ECM adhesion in fibroblasts. To characterize the role of N-WASP in fibroblast for skin development, we generated a conditional knockout mouse model in which fibroblast N-WASP was ablated using the Cre recombinase driven by Fibroblast Specific Protein promoter (Fsp-Cre). N-WASPFKO (N-WASPfl/fl; Fsp-cre) were born following Mendelian genetics, survived without any visible abnormalities for more than 1 year and were sexually reproductive, suggesting that expression of N-WASP in fibroblast is not critical for survival under laboratory conditions. Histological sections of N-WASPFKO mice skin (13 weeks old) showed thicker epidermis with higher percentage of cells staining for proliferation marker (PCNA), suggesting that N-WASP deficient fibroblasts promote keratinocyte proliferation. N-WASPFKO mice skin had elevated collagen content, elevated expression of FGF7 (keratinocyte growth factor) and TGFβ signaling proteins. Wound healing was faster in N-WASPFKO mice compared to control mice and N-WASP deficient fibroblasts were found to have enhanced collagen gel contraction properties. These results suggest that N-WASP deficiency in fibroblasts improves wound healing by growth factor-mediated enhancement of keratinocyte proliferation and increased wound contraction in mice. PMID:27909303

  13. Sensitive skin.

    PubMed

    Misery, L; Loser, K; Ständer, S

    2016-02-01

    Sensitive skin is a clinical condition defined by the self-reported facial presence of different sensory perceptions, including tightness, stinging, burning, tingling, pain and pruritus. Sensitive skin may occur in individuals with normal skin, with skin barrier disturbance, or as a part of the symptoms associated with facial dermatoses such as rosacea, atopic dermatitis and psoriasis. Although experimental studies are still pending, the symptoms of sensitive skin suggest the involvement of cutaneous nerve fibres and neuronal, as well as epidermal, thermochannels. Many individuals with sensitive skin report worsening symptoms due to environmental factors. It is thought that this might be attributed to the thermochannel TRPV1, as it typically responds to exogenous, endogenous, physical and chemical stimuli. Barrier disruptions and immune mechanisms may also be involved. This review summarizes current knowledge on the epidemiology, potential mechanisms, clinics and therapy of sensitive skin.

  14. Development of fibroblast culture in three-dimensional activated carbon fiber-based scaffold for wound healing.

    PubMed

    Huang, Wen-Ying; Yeh, Chia-Lin; Lin, Jui-Hsiang; Yang, Jai-Sing; Ko, Tse-Hao; Lin, Yu-Hsin

    2012-06-01

    This work developed a novel bi-layer wound dressing composed of 3D activated carbon fibers that allows facilitates fibroblast cell growth and migration to a wound site for tissue reconstruction, and the gentamicin is incorporated into a poly(γ-glutamic acid)/gelatin membrane to prevent bacterial infection. In an in vitro, field emission scanning electron microscopy shows that rat skin fibroblasts appeared and spread on the surface of activated carbon fibers, and penetrated the interior and exterior of the 3D activated carbon fiber construct to a depth of roughly 200 μm. An in vivo analysis shows that fibroblast cells containing the proposed 3D scaffold had the potential of a biologically functionalized dressing to accelerate wound closure. Additionally, fibroblasts migrated to the wound site in a bi-layer wound dressing containing fibroblasts, enhancing fibronectin and type I collagen expression, resulting in faster skin regeneration than that achieved with a Tegaderm™ hydrocolloid dressing or gauze.

  15. Primary fibroblasts of NDUFS4(-/-) mice display increased ROS levels and aberrant mitochondrial morphology.

    PubMed

    Valsecchi, Federica; Grefte, Sander; Roestenberg, Peggy; Joosten-Wagenaars, Jori; Smeitink, Jan A M; Willems, Peter H G M; Koopman, Werner J H

    2013-09-01

    The human NDUFS4 gene encodes an accessory subunit of the first mitochondrial oxidative phosphorylation complex (CI) and, when mutated, is associated with progressive neurological disorders. Here we analyzed primary muscle and skin fibroblasts from NDUFS4(-/-) mice with respect to reactive oxygen species (ROS) levels and mitochondrial morphology. NDUFS4(-/-) fibroblasts displayed an inactive CI subcomplex on native gels but proliferated normally and showed no obvious signs of apoptosis. Oxidation of the ROS sensor hydroethidium was increased and mitochondria were less branched and/or shorter in NDUFS4(-/-) fibroblasts. We discuss the relevance of these findings with respect to previous results and therapy development.

  16. Examination of HTLV-I integration in the skin lesions of various types of adult T-cell leukemia (ATL): independence of cutaneous-type ATL confirmed by Southern blot analysis.

    PubMed

    Dosaka, N; Tanaka, T; Miyachi, Y; Imamura, S; Kakizuka, A

    1991-02-01

    The various clinical features of adult T-cell leukemia/lymphoma (ATL) are frequently accompanied by skin eruptions. Recently, a cutaneous type of ATL has been proposed by clinical studies. We analyzed the viral integration of human T-cell leukemia virus-I (HTLV-I) and monoclonal rearrangement of T-cell receptor (TCR) gene in blood lymphocytes and the cutaneous infiltrated cells of nine ATL patients with various clinical features and skin eruptions. We classified them by the results of Southern blot analysis and propose a cutaneous-type ATL accordingly. In two of them, we could detect the monoclonal integration of HTLV-I and T-cell monoclonality only in the skin but not in the peripheral lymphocytes. We also demonstrated the time course study in one patient. Clinicians should be aware of the HTLV-I positive cutaneous T cell lymphoma that can be named cutaneous-type ATL. Examination of viral integration and T-cell monoclonality in skin lesions is required to make an exact diagnosis of cutaneous ATL.

  17. The Skin–Brain Connection Hypothesis, Bringing Together CCL27-Mediated T-Cell Activation in the Skin and Neural Cell Damage in the Adult Brain

    PubMed Central

    Blatt, Nataliya L.; Khaiboullin, Timur I.; Lombardi, Vincent C.; Rizvanov, Albert A.; Khaiboullina, Svetlana F.

    2017-01-01

    Recent d