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1

Radiation-induced radioresistance in a normal human skin fibroblast cell line  

International Nuclear Information System (INIS)

Damage to DNA resulting from ionizing radiation exposure may compromise cellular viability, contribute to malignancy and possibly to senescence. Experimental evidence indicates that several mechanisms exist to repair DNA damage but the molecular events underlying these types of repair in mammalian cells remain obscure. Using the human skin fibroblast line, AG1522, the authors have monitored the end-points of cellular survival, colony growth rate and micronucleus formation to asses adaptation to ionizing radiation. (author). 4 refs., 4 figs.

1992-01-01

2

Characterization of GSF289: a fibroblast cell line derived from goat ear skin explants  

Directory of Open Access Journals (Sweden)

Full Text Available Recently we established three fibroblast cell lines from ear skin explants of normal healthy dairy goats, of Kiko and Saanen breed (In Vitro Cell.Dev.Biol.-Animal; doi: 10.1007/s11626-010-9373-4). The cell lines revealed a viability rate of 96.2%, displayed a typical ‘S’ shaped growth curve with a population doubling time of 25 hours, and were free from microbial, fungal and mycoplasma contamination. We further characterized these cell lines and, in this communication we show the cytogenetic analysis and the genetic transfection of GSF289 cells. The GSF289 cell line which originated from Saanen breed of goats was successfully transfected with pcDNA3.1/NT-GFP plasmid vector containing green fluorescent protein (GFP) gene under human cytomegalovirus (CMV) promoter. The efficiency of transfection, as measured by flow cytometry, was 14.5% after 4 days of culture. The cytogenetic analysis performed on 29 G-banded metaphase cells revealed that the cell line has a normal male goat karyotype consisting of 58 autosomes and two XY sex chromosomes. These results suggest that GSF289 cell line with a normal karyotype, having a high rate of proliferation, and its ability to be easily transfected with plasmid DNA vectors is an additional tool to study molecular mechanisms that regulate fibroblast function as well as genetic manipulation of small ruminants.

Mahipal Singha; Anil K. Sharma; Pushpa Yadav

2011-01-01

3

Effect of enoxaparin and onion extract on human skin fibroblast cell line - Therapeutic implications for the treatment of keloids.  

UK PubMed Central (United Kingdom)

Abstract Context: Keloids and hypertrophic scars are hyperproliferative skin disorders resulting in abnormal wound healing. In the prevention and treatment of keloids and hypertrophic scars, ointments containing heparin and onion extract are very popular. Their therapeutic effects, however, are still controversial and the mechanism of action is not fully understood. Objective: The aim of this study was to assess the effect of enoxaparin and dry onion extract on proliferation, apoptosis and ?1 integrin expression in human fibroblasts. Materials and methods: Fibroblast human cell lines (46 BR.1?N) were treated for 48?h with various concentrations of enoxaparin sodium (20, 100, 500?µg/mL) and/or onion [Allium cepa L. (Alliaceae)] extract (50, 250, 1000?µg/mL). The cell proliferation was evaluated by [(3)H]-thymidine incorporation assay. Furthermore, the expression of ?1 integrin and apoptosis was determined by flow cytometry. Results and discussion: The results demonstrate that enoxaparin and onion extract inhibited the proliferation of human fibroblasts. Almost complete inhibition of cell proliferation was achieved by enoxaparin in 500?µg/mL concentration (91.5% reduction). The onion extract at a concentration of 250?µg/mL also strongly inhibited the proliferation of cells (50.8% reduction). Depending on concentration, enoxaparin and onion extract induced apoptosis (500 and 1000?µg/mL, respectively) and, depending on concentration, downregulated the expression of ?1 integrin on human fibroblasts. Conclusion: This work points at possible mechanism of action of enoxaparin and onion extract, when administered in the treatment of patients with keloids and hypertrophic scars.

Piku?a M; Zebrowska ME; Pob?ocka-Olech L; Krauze-Baranowska M; Sznitowska M; Trzonkowski P

2013-09-01

4

Critical role of GSH in Sulfur Mustard-induced Oxidative Stress and Cytotoxicity in Human Skin Fibroblast Cell Line  

Directory of Open Access Journals (Sweden)

Full Text Available In this study the role of glutathione (GSH) in sulfur mustard -induced oxidative stress and cytotoxicity, in human skin fibroblast cell line (HF2FF) was evaluated. Sulfur mustard-induced superoxide radical and hydrogen peroxide formation were evaluated by determination of superoxide dismutase and catalase activity in cell lysate. The cytotoxicity of sulfur mustard was estimated by lactate dehydrogenase leakage. The intracellular GSH content was modulated by N-acetylcysteine (NAC), a GSH precursor, and buthionine sulfoximine (BSO), a specific GSH synthesis inhibitor. It was found that sulfur mustard exposure led to a dose-and time-dependent decrease in GSH content in HF2FF cells. NAC increased intracellular GSH level and protected the cells against sulfur mustard-induced reactive oxygen species formation and lactate dehydrogenase leakage. In contrast, buthionine sulfoximine pretreatment depleted cellular GSH and enhanced the susceptibility of HF2FF to the cytotoxic effects of sulfur mustard. These results indicated that GSH plays a critical role in protecting HF2FF cell line against sulfur mustar-induced cell injury, most probably through its antioxidant activity.

Ali Beman Zaree Mahmoudabad; Mehdy Saberi; Jilla Pirzad

2008-01-01

5

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

UK PubMed Central (United Kingdom)

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.

Samluk A; Zakrzewska KE; Pluta KD

2013-07-01

6

Transcriptional profiling of human skin fibroblast cell line Hs27 induced by herbal formula Astragali Radix and Rehmanniae Radix.  

UK PubMed Central (United Kingdom)

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and serve as the principal herbs in treating diabetic foot ulcer. AIM OF THE STUDY: Chinese herbal formulus comprising Astragali Radix (AR) and Rehmanniae Radix (RR) have been shown to improve the healing of diabetic foot ulcer through enhancing the viability of primary fibroblasts in diabetic patients suffering insulin resistance. Our previous study demonstrated that the herbal formula NF3 comprising of AR and RR in the ratio of 2:1 was effective in promoting wound healing in diabetic rats, and in vitro data indicated that the wound healing effects of NF3 might be due to the regulation and coordination of inflammation, angiogenesis and tissue regeneration. However, the underlying molecular mechanism has not been well investigated. In this study, we investigated the cellular and molecular effects of the herbal formula NF3 on human skin fibroblast cells. MATERIALS AND METHODS: Human skin fibroblast cells Hs27 were treated with NF3 ranging from 0 to 8 mg/ml for 24h, and the cells without NF3 treatment were used as control. Cell proliferation assay and cell cycle analysis were performed. Transcriptional profiles of Hs27 cells upon NF3 treatment were acquired by using a human cDNA microarray containing 10,000 genes, and the signaling pathways differentially regulated by NF3 were identified and analyzed. RESULTS: NF3 promoted Hs27 cell proliferation and cell cycle progression. Microarray analysis revealed that 116 genes were differentially expressed upon NF3 treatment. Functional analysis of the genes indicated that NF3 mainly activated Wnt and angiogenesis related pathways, which are directly related to cell proliferation, angiogenesis, extracellular matrix (ECM) formation and inflammation during the process of wound healing. CONCLUSION: This study provides insight into the molecular mechanism of how the herbal formula Astragali Radix and Rehmanniae Radix may serve as potential therapeutics for wound healing.

Zhang Q; Wei F; Fong CC; Yu WK; Chen Y; Koon CM; Lau KM; Leung PC; Lau CB; Fung KP; Yang M

2011-12-01

7

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

1982-03-01

8

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

International Nuclear Information System (INIS)

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.

1982-01-01

9

Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis  

International Nuclear Information System (INIS)

[en] Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with 125I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture

1982-01-01

10

Cytoskeletal proteins from human skin fibroblasts, peripheral blood leukocytes, and a lymphoblastoid cell line compared by two-dimensional gel electrophoresis  

Energy Technology Data Exchange (ETDEWEB)

Differences in proteins between cells grown as suspension cultures and those grown as attached cultures were studied by comparing the proteins of detergent-resistant cytoskeletons prepared from peripheral blood leukocytes and a lymphoblastoid cell line (GM607) (both grown as suspension cultures) and those of human skin fibroblasts (grown as attached cultures) by two-dimensional gel electrophoresis. The major cytoskeletal proteins of the leukocytes were also present in the protein pattern of GM607 cytoskeletons. In contrast, the fibroblast cytoskeletal protein pattern contained four groups of proteins that differed from the patterns of the leukocytes and GM607. In addition, surface labeling of GM607 and human fibroblasts with /sup 125/I demonstrated that substantial amounts of vimentin and actin are exposed at the surface of the attached fibroblasts, but there is little evidence of similar exposure at the surface of the suspension-grown GM607. These results demonstrate some differences in cytoskeletal protein composition between different types of cells could be related to their ability or lack of ability to grow as attached cells in tissue culture.

Giometti, C.S.; Willard, K.E.; Anderson, N.L.

1982-04-01

11

Stiffening of Human Skin Fibroblasts with Age  

Science.gov (United States)

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ?60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Schulze, Christian; Wetzel, Franziska; Kueper, Thomas; Malsen, Anke; Muhr, Gesa; Jaspers, Soeren; Blatt, Thomas; Wittern, Klaus-Peter; Wenck, Horst; Kas, Josef A.

2010-01-01

12

Stiffening of human skin fibroblasts with age.  

UK PubMed Central (United Kingdom)

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ?60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Schulze C; Wetzel F; Kueper T; Malsen A; Muhr G; Jaspers S; Blatt T; Wittern KP; Wenck H; Käs JA

2012-01-01

13

Autologous fibroblast culture in the repair of aging skin.  

UK PubMed Central (United Kingdom)

BACKGROUND: Human cell cultures are being developed to replace various body tissues. OBJECTIVE: To assess the safety and efficacy of dermal regeneration with the injection of young autologous fibroblasts obtained from culture containing serum from patients themselves. MATERIALS AND METHODS: Dermal tissue from the groin of five patients was cultivated in M199 medium supplemented with 10% human serum. Four population doublings were obtained. The fibroblasts were injected intradermally into forehead wrinkles and periorbital and paranasal areas. RESULT: At the fourth population doubling, a mean of 3.85 × 10(6) cells/mL was obtained; viability was 98%. Sixty days after completing treatment, with four injections given at 15-day intervals, periorbital tonicity had improved significantly, although the quantity of fibroblasts used resulted in little improvement to surface lines and no improvement at all in deeper wrinkles. After 6 months, no further changes were found beyond the initial results obtained. CONCLUSION: Injection of skin fibroblasts cultivated in medium supplemented with human serum is a viable technique and provokes no side effects. Four injections given at 15-day intervals containing a total of 6.4 × 10(6) fibroblasts/mL resulted in significant improvement in periorbital skin flaccidity. Further studies should be conducted with larger sample sizes.

Eça LP; Pinto DG; de Pinho AM; Mazzetti MP; Odo ME

2012-02-01

14

Herbal formula Astragali Radix and Rehmanniae Radix exerted wound healing effect on human skin fibroblast cell line Hs27 via the activation of transformation growth factor (TGF-?) pathway and promoting extracellular matrix (ECM) deposition.  

Science.gov (United States)

Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and as the principal herbs in treating diabetic foot ulcer. In this study, we investigated the effect of NF3, which comprises of AR and RR in the ratio of 2:1(w/w), on skin fibroblast cell migration and the activation of selected genes and proteins related to wound healing. Human skin fibroblast cell line Hs27 was treated with NF3 at 4 mg/ml for 24h, and in vitro scratch wound healing and quantitative cell migration assays were performed, respectively. The expression of transformation growth factor (TGF-?1) and bone morphogenetic protein 6 (BMP6) in Hs27 cells with or without NF3 treatment was analyzed by western blot analysis. In addition, the expression of a panel of genes involved in human TGF-? signaling pathway was analyzed in Hs27 cells upon NF3 treatment (4 mg/ml, 24 h) by quantitative real-time PCR (qRT-PCR). Furthermore, the expression of several genes and proteins associated with ECM synthesis was investigated by qRT-PCR analysis or/and ELISA techniques. The results suggested that NF3 promoted the migration of human skin fibroblast cells. Western blot analysis demonstrated that NF3 up-regulated TGF-?1 and BMP-6 synthesis. qRT-PCR analysis revealed that the expression of 26 genes in Hs27 cells was changed upon NF3 induction, including TGF-? superfamily ligands and down stream effectors genes, and genes involved in TGF/Smad pathway, and Ras/MAPK (non-Smad) pathway. Among the extracellular matrix (ECM)-related molecules, it was found that NF3 up-regulated the expression of type I and III collagens, fibronectin as well as TIMP-1, and down-regulated the MMP-9 expression in skin fibroblast cells. This study demonstrated that herb formula NF3 could enhance skin fibroblast cell migration and activated genes involved in TGF-?1 pathway. NF3 could regulate gene transcription for extracellular matrix synthesis via the Smad pathway, and gene transcription for cell motility via the Ras/MAPK (non-Smad) pathway. PMID:23083814

Zhang, Qi; Fong, Chi Chun; Yu, Wai Kin; Chen, Yao; Wei, Fan; Koon, Chi Man; Lau, Kit Man; Leung, Ping Chung; Lau, Clara Bik San; Fung, Kwok Pui; Yang, Mengsu

2012-10-17

15

Herbal formula Astragali Radix and Rehmanniae Radix exerted wound healing effect on human skin fibroblast cell line Hs27 via the activation of transformation growth factor (TGF-?) pathway and promoting extracellular matrix (ECM) deposition.  

UK PubMed Central (United Kingdom)

Astragali Radix (AR) and Rehmanniae Radix (RR) have long been used in traditional Chinese Medicine and as the principal herbs in treating diabetic foot ulcer. In this study, we investigated the effect of NF3, which comprises of AR and RR in the ratio of 2:1(w/w), on skin fibroblast cell migration and the activation of selected genes and proteins related to wound healing. Human skin fibroblast cell line Hs27 was treated with NF3 at 4 mg/ml for 24h, and in vitro scratch wound healing and quantitative cell migration assays were performed, respectively. The expression of transformation growth factor (TGF-?1) and bone morphogenetic protein 6 (BMP6) in Hs27 cells with or without NF3 treatment was analyzed by western blot analysis. In addition, the expression of a panel of genes involved in human TGF-? signaling pathway was analyzed in Hs27 cells upon NF3 treatment (4 mg/ml, 24 h) by quantitative real-time PCR (qRT-PCR). Furthermore, the expression of several genes and proteins associated with ECM synthesis was investigated by qRT-PCR analysis or/and ELISA techniques. The results suggested that NF3 promoted the migration of human skin fibroblast cells. Western blot analysis demonstrated that NF3 up-regulated TGF-?1 and BMP-6 synthesis. qRT-PCR analysis revealed that the expression of 26 genes in Hs27 cells was changed upon NF3 induction, including TGF-? superfamily ligands and down stream effectors genes, and genes involved in TGF/Smad pathway, and Ras/MAPK (non-Smad) pathway. Among the extracellular matrix (ECM)-related molecules, it was found that NF3 up-regulated the expression of type I and III collagens, fibronectin as well as TIMP-1, and down-regulated the MMP-9 expression in skin fibroblast cells. This study demonstrated that herb formula NF3 could enhance skin fibroblast cell migration and activated genes involved in TGF-?1 pathway. NF3 could regulate gene transcription for extracellular matrix synthesis via the Smad pathway, and gene transcription for cell motility via the Ras/MAPK (non-Smad) pathway.

Zhang Q; Fong CC; Yu WK; Chen Y; Wei F; Koon CM; Lau KM; Leung PC; Lau CB; Fung KP; Yang M

2012-12-01

16

Hyaluronidase expression in human skin fibroblasts.  

Science.gov (United States)

Hyaluronidase activity has been detected for the first time in normal human dermal fibroblasts (HS27), as well as in fetal fibroblasts (FF24) and fibrosarcoma cells (HT1080). Enzymatic activity was secreted predominantly into the culture media, with minor amounts of activity associated with the cell layer. In both classes of fibroblasts, hyaluronidase expression was confluence-dependent, with highest levels of activity occurring in quiescent, post-confluent cells. However, in the fibrosarcoma cell cultures, expression was independent of cell density. The enzyme had a pH optimum of 3.7 and on hyaluronan substrate gel zymography, activity occurred as a single band corresponding to an approximate molecular size of 57 kDa. The enzyme could be immunoprecipitated in its entirety using monoclonal antibodies raised against Hyal-1, human plasma hyaluronidase. PCR confirmed that fibroblast hyaluronidase was identical to Hyal-1. The conclusion by previous investigators using earlier technologies that fibroblasts do not contain hyaluronidase activity should be reevaluated. PMID:10581201

Stair-Nawy, S; Csóka, A B; Stern, R

1999-12-01

17

Hyaluronidase expression in human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Hyaluronidase activity has been detected for the first time in normal human dermal fibroblasts (HS27), as well as in fetal fibroblasts (FF24) and fibrosarcoma cells (HT1080). Enzymatic activity was secreted predominantly into the culture media, with minor amounts of activity associated with the cell layer. In both classes of fibroblasts, hyaluronidase expression was confluence-dependent, with highest levels of activity occurring in quiescent, post-confluent cells. However, in the fibrosarcoma cell cultures, expression was independent of cell density. The enzyme had a pH optimum of 3.7 and on hyaluronan substrate gel zymography, activity occurred as a single band corresponding to an approximate molecular size of 57 kDa. The enzyme could be immunoprecipitated in its entirety using monoclonal antibodies raised against Hyal-1, human plasma hyaluronidase. PCR confirmed that fibroblast hyaluronidase was identical to Hyal-1. The conclusion by previous investigators using earlier technologies that fibroblasts do not contain hyaluronidase activity should be reevaluated.

Stair-Nawy S; Csóka AB; Stern R

1999-12-01

18

Alzheimer skin fibroblasts show increased susceptibility to free radicals.  

UK PubMed Central (United Kingdom)

We have studied the response to toxic oxygen metabolites of fibroblasts derived from skin biopsies of 5 patients with familial (FAD) and 4 with sporadic (AD) Alzheimer's disease compared with those derived from 4 normal controls. Fibroblasts were damaged by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by 50 munits of xanthine-oxidase (Xo). To quantify cell damage we measured lactate dehydrogenase (LDH) activity in the culture medium and cell viability in fibroblast cultures. We found a significant increase in LDH activity in the FAD vs. controls and also in the AD vs. controls.

Tesco G; Latorraca S; Piersanti P; Piacentini S; Amaducci L; Sorbi S

1992-11-01

19

Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts  

International Nuclear Information System (INIS)

The chain length of [3H]hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of [3H]glucosamine was investigated. [3H]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

1990-10-15

20

[Fibroblast subpopulations: a developmental approach of skin physiology and ageing].  

UK PubMed Central (United Kingdom)

Skin is an organ whose function is far beyond a physical barrier between the inside and the outside of the body. Skin as the whole organism is subjected to ageing which concerns skin mostly in its dermal and deepest component which is also its matricial component. The dermis is a tissue rich in matricial elements and poor in cellular content and it is generally admitted that modifications occurring in the matrix are those which mostly contribute to skin ageing, by altering its biomechanical properties. Therefore it is common to address questions related to skin ageing by considering alterations in matrix molecules like collagen. Actually the dermis is a complex tissue both matricial and cellular and is divided between a superficial dermis close to epidermis and a deep dermis much thicker and histologically different. Several years ago we have undertaken investigations related to fibroblasts which are the cells responsible for the formation and maintenance of the dermis, aiming at isolation, culture and characterization of the fibroblasts from the superficial dermis also called papillary dermis and fibroblasts from the deep dermis also called reticular dermis. We were able to show that these fibroblasts in classical culture on plastic exhibit very different morphologies associated with different secretion properties and we have confirmed and expanded such observations revealing different phenotypes by incorporating these cells in reconstructed skin which allows the reproduction of a three-dimensional architecture recalling skin in vivo especially after grafting onto the nude mouse. We also raise the question of how these two dermal regions appear during the formation of the dermis and the question of their fate during ageing. Progress in solving these questions would certainly appear to be very useful for a better understanding of skin physiology and ageing and would hopefully provide new strategies in anti-ageing research.

Asselineau D; Pageon H; Mine S

2008-01-01

 
 
 
 
21

Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts  

International Nuclear Information System (INIS)

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 36Cl- from matched pairs of CF and normal fibroblasts. The rate constants describing 36Cl- efflux did not differ between the two cell types, but in each of the four pairs tested the amount of 36Cl- contained within CF cells was consistently reduced, by 25-30%, relative to normal cells. Comparisons of cell water content and 22Na+ 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- concentration, perhaps through changes in Cl- permeability

1987-01-01

22

Effect of low-power red light laser irradiation on the viability of human skin fibroblast.  

UK PubMed Central (United Kingdom)

Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm2). The energy densities were 2 J/cm2 and 12 J/cm2, respectively, and the irradiation was carried out at a temperature of 22 degrees C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 degrees C, in the presence of 5% CO2, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation.

Bednarska K; Rózga B; Ko?odziejczyk K; Szosland D; Leyko W; Bryszewska M

1998-10-01

23

Studies of the in vivo radiosensitivity of human skin fibroblasts  

International Nuclear Information System (INIS)

Background and purpose: To examine the radiosensitivity of skin cells obtained directly from the irradiated skin of patients undergoing fractionated radiation treatment prior to surgery for treatment of soft tissue sarcoma (STS) and to determine if there was a relationship with the development of wound healing complications associated with the surgery post-radiotherapy. Methods: Micronucleus (MN) formation was measured in cells (primarily dermal fibroblasts) obtained from human skin at their first division after being removed from STS patients during post-radiotherapy surgery (2-9 weeks after the end of the radiotherapy). At the time of radiotherapy (planned tumor dose - 50 Gy in 25 daily fractions) measurements were made of surface skin dose at predetermined marked sites. Skin from these sites was obtained at surgery and cell suspensions were prepared directly for the cytokinesis-blocked MN assay. Cultured strains of the fibroblasts were also established from skin nominally outside the edge of the radiation beam and DNA damage (MN formation) was examined following irradiation in vitro for comparison with the results from the in situ irradiations. Results: Extensive DNA damage (MN) was detectable in fibroblasts from human skin at extended periods after irradiation (2-9 weeks after the end of the 5-week fractionated radiotherapy). Analysis of skin receiving a range of doses demonstrated that the level of damage observed was dose dependent. There was no clear correlation between the level of damage observed after irradiation in situ and irradiation of cell strains in culture. Similarly, there was no correlation between the extent of MN formation following in situ irradiation and the propensity for the patient to develop wound healing complications post-surgery. Conclusions: Despite the presence of DNA damage in dermal fibroblasts weeks after the end of the radiation treatment, there was no relationship between this damage and wound healing complications following surgery post-irradiation. These results suggest that factors other than the radiosensitivity of the skin fibroblasts likely also play a role in wound healing in deep wound sites associated with surgery for STS following radiation therapy.

2007-01-01

24

Acetyl-11-keto-beta-boswellic acid and arsenic trioxide regulate the productions and activities of matrix metalloproteinases in human skin fibroblasts and human leukemia cell line THP-1  

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Full Text Available Objective: In order to reveal the treatment mechanism of Chinese medicine with the effect of activating blood and resolving putridity, we selected acetyl-11-keto-beta-boswellic acid (AKBA) and arsenic trioxide (ATO), the main monomeric components of frankincense and arsenolite which are two most commonly used Chinese medicine with effect of activating blood and resolving putridity. We combined AKBA and ATO as a compound, and explored its regulatory role in productions and activities of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 in human skin fibroblasts (HSFbs) and human acute monocytic leukemia cell line THP-1 in inflammatory state.Methods: In order to simulate the inflammatory micro-environment of chronic wounds, we established 3 cell models: HSFb model activated by tumor necrosis factor-alpha (TNF-?), THP-1 cell model activated by phorbol-12-myristate-13-acetate (PMA) and HSFb-THP-1 cell coculture system. AKBA and ATO were cocultured with these cell models. Enzyme-linked immunosorbent assay (ELISA), gelatin zymography assay and reverse transcription-polymerase chain reaction (RT-PCR) were used to test the secretions, activities and mRNA expressions of MMP-1, MMP-2 and MMP-9. In the study of the regulatory mechanism of AKBA and ATO on MMPs, AKBA and ATO were cocultured with the cell models. ELISA was used to test the secretions of TNF-? and interleukin-1beta (IL-?) and Western blot was used to test the phosphorylation levels of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated proteinkinase (p38MAPK). Results: Compound of AKBA and ATO inhibited MMP-1, MMP-2 and MMP-9 mRNA expressions, secretions and activities respectively in HSFbs and THP-1 cells in inflammatory state (P?0.05, P?0.01). Also compound of AKBA and ATO inhibited secretions of TNF-? and IL-1? in THP-1 cells and cell coculture system (P?0.01). It also decreased the phosphorylation of ERK1/2 and p38 MAPK in HSFbs and THP-1 cells (P?0.05, P?0.01). Conclusion: The combined use of AKBA and ATO which in line with the rule of activating blood and resolving putridity inhibits fibroblasts and inflammatory cells in producing MMPs in inflammatory state through inhibiting the release of inflammatory factors and MAPK cascade pathway.

Ya-hui Liang; Ping Li

2010-01-01

25

Proliferation index of camel skin fibroblast cells as nuclear donor  

International Nuclear Information System (INIS)

Jaiselmeri is an excellent breed of riding camel, found in Jaiselmer and other adjoining districts of Western Rajasthan in India. Jaiselmeri camel like other pack animals are declining in India over the years due to increased mechanization and control of desert agriculture to some extent. The deep freezing technology on camel semen is poorly developed in India. The somatic cell technology has been developed at this Institute as an alternative tool of long-term conservation on endangered livestock breeds. For this study, samples of (0.25 cm2) skin tissue were collected from ear biopsy from elite male germplasm from National Research Centre on Camel, Bikaner. Skin tissues were cultured at 37 deg. C in Medium (DMEM+ Ham's F-12 nutritive mixture) supplemented with 10% fetal bovine serum, L-Glutamine and antibiotics in an incubator under 98% humidified and 5% Co2 atmosphere. The cell explants were visible from 12-16 days of culture. The cells were allowed to confluent in the TC flasks for additional 3-5 days till nearly 80% surface area is covered by the cells. The primary cells were harvested by usual trypsin-EDTA protocol. The cells were counted using Neubar's haemocytometer and cells were passaged subsequently. Since no reference values were available for camel skin fibroblasts, the present experiments were conducted to study the cell proliferation index, population doubling time, standard growth curve and cell viability using standard growth and MTT assays. It is shown that growth curves showed true sigmoid shape but a marked variation between the cell lines was observed. Moreover, cells, which grew faster attained plateau on day 6 while in slow growing cultures, the curve showed elevation even on day 8. This is probably due to non-availability of growing space for cells having faster growth rate. It was concluded that all animals do not produce karyoplast donors at equal rate or efficiency. Therefore, the growing cultures need to be compared with standard growth curve each time the cells are used as nuclear donor cells for cloning. Cell Proliferation Index: Cell multiplication rates vary considerably under different culture condition and slight change in environment or composition of medium may affect the proliferation of cells significantly. For camel skin fibroblast cells, the standard multiplication rate and the population doubling time was not known earlier. In order to study the proliferative indices of the growing cells using objective parameters, MTT assay was conducted. In this assay, the dividing and viable cells take up MTT [3- (4,5- dimethylthiozol- 2yl) 2,5 diphenyltetrazolium bromide] and a colour is developed. The intensity of colour is measured by ELISA reader at 540-570 nm. For this, 4000 cells per well were seeded in 96 well ELISA plate (flat bottom, Nunc) and cultured at 37 deg. C. First two rows of eight wells each were kept as negative and positive controls respectively. Rest of the 10 rows were kept as treatments. One row was harvested at an interval of 24 hours and adjoining row was treated with MTT solution for 4 hours. The MTT treated cells were fixed in 10% DMSO. Figures 2 and 3 show that the cell proliferation index both in terms of cell count and absorbance values in ELISA reader at appropriate wavelength was similar. From this study it is clear that MTT assay can give fairly accurate figures of cell proliferation rate of skin fibroblasts. Ploidy level: During long-term culture, the cells are likely to develop one or other type of chromosomal abnormalities. It must be ensured that the cells in different passages are checked for normal ploidy so that the viable clones can be developed from them. In order to see the utility of cells from Jaiselmeri camel as nuclear donor, the chromosomal profile was studied following the protocol described elsewhere. The 2N chromosomes up to passage No 4 (15th population doubling) was found to be normal (74XY) in 97% of the cells. From these preliminary studies it appears that camel skin fibroblast cells behave normally in culture and can serve as nuclear donors.

2003-01-01

26

Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts  

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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.

Nakamura, T.; Takagaki, K.; Kubo, K.; Morikawa, A.; Tamura, S.; Endo, M. (Hirosaki Univ. School of Medicine (Japan))

1990-10-15

27

Two different variants of the same tropomyosin polypeptide in clones from GM1386 human skin fibroblasts  

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A new protein observed in two-dimensional electrophoresis patterns of proteins from the human skin fibroblast line GM1386 has been identified as a charge and molecular-weight variant of the type of tropomyosin found in smooth muscle (Tm:3). This is the second variant of Tm:3 found in GM1386 and represents a second site mutation in one of the genes coding for Tm:3. 14 references, 4 figures.

Giometti, C.S.; Gemmell, M.A.; Anderson, N.L.

1985-05-01

28

Fibroblasts within concentrated collagen hydrogels favour chronic skin wound healing.  

Science.gov (United States)

Apligraf(®), a skin substitute currently used in skin chronic wound treatment, acts as a source of macromolecules and cytokines to promote wound healing. Normal collagen hydrogel (NCH), obtained from collagen at low concentration (0.66 mg/ml), is the base of the dermal layer. Apligraf has several drawbacks, such as poor persistence of fibroblasts within the normal collagen hydrogel. In the present study we have evaluated concentrated collagen hydrogels at 5 mg/ml (CCH5s) as dermal substitutes for the treatment of skin chronic wounds. The effect of raised collagen concentration on hydrogel stability, cell growth, apoptosis and fibroblast phenotype was evaluated over 21 days in culture. In contrast to NCHs, CCH5s were more stable because no contraction was observed during the first week. CCH5 favoured cell proliferation and protected fibroblasts against apoptosis. At day 21, cell number assessed in CCH5 was around one million, i.e about 10 times higher than in NCH. Matrix metalloproteinases detection appeared lower in CCH5 than in NCH. In CCH5, fibroblasts exhibited a sustained collagen I gene expression for 14 days, while it was inhibited from day 4 in NCH. Moreover, gene expression of KGF was constant in CCH5 and that of VEGFA increased from day 7. Taken together, our results demonstrate that concentrated collagen hydrogels at 5 mg/ml can be considered as new candidates for cell therapy in chronic skin wounds. They are stable, enhance cell viability and allow gene expression of matrix macromolecules and cytokines involved in re-epithelialization or neovascularization. PMID:22362469

Helary, Christophe; Zarka, Mylène; Giraud-Guille, Marie Madeleine

2011-03-28

29

Fibroblasts within concentrated collagen hydrogels favour chronic skin wound healing.  

UK PubMed Central (United Kingdom)

Apligraf(®), a skin substitute currently used in skin chronic wound treatment, acts as a source of macromolecules and cytokines to promote wound healing. Normal collagen hydrogel (NCH), obtained from collagen at low concentration (0.66 mg/ml), is the base of the dermal layer. Apligraf has several drawbacks, such as poor persistence of fibroblasts within the normal collagen hydrogel. In the present study we have evaluated concentrated collagen hydrogels at 5 mg/ml (CCH5s) as dermal substitutes for the treatment of skin chronic wounds. The effect of raised collagen concentration on hydrogel stability, cell growth, apoptosis and fibroblast phenotype was evaluated over 21 days in culture. In contrast to NCHs, CCH5s were more stable because no contraction was observed during the first week. CCH5 favoured cell proliferation and protected fibroblasts against apoptosis. At day 21, cell number assessed in CCH5 was around one million, i.e about 10 times higher than in NCH. Matrix metalloproteinases detection appeared lower in CCH5 than in NCH. In CCH5, fibroblasts exhibited a sustained collagen I gene expression for 14 days, while it was inhibited from day 4 in NCH. Moreover, gene expression of KGF was constant in CCH5 and that of VEGFA increased from day 7. Taken together, our results demonstrate that concentrated collagen hydrogels at 5 mg/ml can be considered as new candidates for cell therapy in chronic skin wounds. They are stable, enhance cell viability and allow gene expression of matrix macromolecules and cytokines involved in re-epithelialization or neovascularization.

Helary C; Zarka M; Giraud-Guille MM

2012-03-01

30

Free radical injury in skin cultured fibroblasts from Alzheimer's disease patients.  

UK PubMed Central (United Kingdom)

Oxygen radical production is postulated to be a major cause of cell damage in aging. We have studied the response to toxic oxygen metabolites of fibroblast cell lines derived from skin biopsies of patients with familial and sporadic Alzheimer's disease compared with those derived from normal controls. Fibroblasts were damaged by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by 50 mU of xanthine-oxidase. To quantify cell damage we measured lactate dehydrogenase activity in the culture medium and cell viability in fibroblast cultures from four normal subjects, five FAD, and four AD patients after 2 hours of Xo incubation. We found a significant increase of LDH activity in FAD vs. controls and also in AD vs. controls, suggesting that AD cells are more susceptible to oxygen radical damage than are normal controls.

Tesco G; Latorraca S; Piersanti P; Sorbi S; Piacentini S; Amaducci L

1992-12-01

31

Low dose rate ionizing radiation induces increased growth capacities of d-deletion retinoblastoma skin fibroblasts  

International Nuclear Information System (INIS)

[en] Skin fibroblasts from normal children and three children with a 13q deletion retinoblastoma (Rb) were exposed to cumulative low doses of gamma rays. The typical response of normal donors was a reduction in the lifespan of irradiated fibroblasts, the precocity of the decline being inversely related to the dose received. In contrast, the lifespan of one Rb cell line (Rb1) was prolonged; irradiated cells with an increased growth potential showed a higher number of cells at confluency and more cells were entering DNA synthesis phase than in non-irradiated cells. Another Rb cell line (Rb2) demonstrated a normal lifespan following irradiation but foci were observed in irradiated cultures. Cytogenetic analysis revealed no selection of abnormal clones in these cell populations. The third Rb line examined (Rb3) responded like a normal cell line. We suggest that irradiated skin fibroblasts derived from some patients with Rb are in certain cases able to express abnormal growth capacities which may be one of the manifestations of the high susceptibility of the individual's stromal cells to carcinogenic agents

1984-01-01

32

Human skin keloid fibroblasts display bioenergetics of cancer cells.  

UK PubMed Central (United Kingdom)

Cultured human skin keloid fibroblasts (KFs) showed bioenergetics similar to cancer cells in generating ATP mainly from glycolysis as demonstrated by increased lactate production. Activities of hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase were also significantly higher compared with normal fibroblasts (NFs). Inhibitors of glycolysis decreased the rate of ATP biosynthesis more significantly in KFs suggesting their reliance on glycolysis. In contrast, ATP generation in NFs was derived mainly from oxidative phosphorylation (OXPHOS), which was more compromised by mitochondrial/respiratory inhibitors. However, when fortified with excess exogenous respiratory substrates, ATP production was increased to a similar maximal level in both types of fibroblasts. In spite of this seemingly equal total capacity, ATP biosynthesis and intracellular ATP concentration were significantly higher in KFs, which further increased their ATP production when exposed to hypoxia and hypoxia-mimetics: desferrioxamine and cobalt chloride. This upregulation was again significantly compromised by glycolytic inhibitors. The rate of generation of reactive oxygen species was lower in KFs possibly due to their switch to aerobic glycolysis from mitochondrial OXPHOS. Thus, cultured skin KFs could provide a human cell model to study the de-regulation of bioenergetics of proliferative cells and their response to the HIF (hypoxia-inducible factor) signaling.

Vincent AS; Phan TT; Mukhopadhyay A; Lim HY; Halliwell B; Wong KP

2008-03-01

33

Modulation of clonogenicity, growth, and radiosensitivity of three human epidermoid tumor cell lines by a fibroblastic environment  

International Nuclear Information System (INIS)

Purpose: To develop a model vitro system to examine the influence of fibroblasts on the growth and survival of human tumor cells after exposure to ionizing radiation. Methods and Materials: The cell system consists of three epidermoid carcinoma cell lines derived from head and neck tumors having differing growth potentials and intrinsic radiosensitivities, as well as a low passage skin fibroblast strain from a normal human donor. The tumor cells were seeded for five days prior to exposure to radiation: (a) in the presence of different numbers of fibroblasts, (b) in conditioned medium from stationary fibroblast cultures, and (c) on an extracted fibroblastic matrix. Results: When grown with fibroblasts, all three tumor cell lines showed increased clonogenicity and increased radioresistance. The radioprotective effect was maximal at a density of approximately 105 fibroblasts/100 mm Petri dish, and was greatest in the intrinsically radiosensitive tumor cell line. On the other hand, the effects of incubation with conditioned medium or on a fibroblastic matrix varied among the tumor cell lines. Thus, the protective effect afforded by coculture with fibroblasts must involve several cellular factors related to the fibroblast itself. Conclusions: These observations emphasize the importance of cultural conditions on the apparent radiosensitivity of human tumor cell lines, and suggest that the fibroblastic connective tissue enveloping the malignant cells should be considered when the aim is to establish a radiopredictive assay from surgical tumors fragments

1996-03-15

34

Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts  

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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.

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

1987-05-01

35

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro  

International Nuclear Information System (INIS)

[en] The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern

1989-01-01

36

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro  

Energy Technology Data Exchange (ETDEWEB)

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific (35S)methionine-labeled polypeptide pattern.

Rodemann, H.P.; Bayreuther, K.; Francz, P.I.; Dittmann, K.; Albiez, M.

1989-01-01

37

Proteoglycan modifications in cultured osteogenesis imperfecta skin fibroblasts.  

UK PubMed Central (United Kingdom)

The PGs produced in the growth medium by skin fibroblast cultures from two O.I. affected patients were investigated. After density gradient centrifugation, in the most dense fraction two main families of molecules appeared. The patient with the more severe clinical picture showed a lower content of the PGs with the highest molecular weight. The GAG composition of PGs was different in the two patients. The more severely affected one showed an increase of HS and a decrease of ChS content, in agreement with the lower value of galactosamine to glucosamine ratio in urinary GAGs.

De Luca G; Tira ME; Rindi S; Salvini R; Cetta G; Castellani AA

1984-11-01

38

Fibrotic remodeling of tissue engineered skin with deep dermal fibroblasts is reduced by keratinocytes.  

UK PubMed Central (United Kingdom)

Two-thirds of burn patients with deep dermal injuries are affected by hypertrophic scars, and currently there are no clinically effective therapies. Tissue engineered skin is a very promising model for elucidation of the role of matrix microenvironment and biomechanical characteristics and could help in identification of new therapeutic targets for hypertrophic scars. Conventionally, tissue engineered skin is made of heterogeneous dermal fibroblasts and keratinocytes; however, recent work has shown that superficial and deep dermal fibroblasts are anti-fibrotic and pro-fibrotic, respectively. Further, keratinocytes are believed to regulate development and remodelling of fibrosis in skin. This study aimed to assess the influence of keratinocytes and layered fibroblasts on characteristics of tissue engineered skin. Layered fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of lower abdominal tissue were independently co-cultured on C-GAG scaffolds, and the resulting tissue engineered skin was assessed for differences in tissue remodelling based on the underlying specific dermal fibroblast subpopulation. Collagen production by deep fibroblasts but not superficial fibroblasts was significantly reduced upon co-culture with keratinocytes. Also, keratinocytes in the tissue engineered skin resulted in significantly reduced expression of pro-fibrotic CTGF and fibronectin, and increased expression of the anti-fibrotic MMP-1 by deep but not superficial fibroblasts. Tissue engineered skin made of deep fibroblasts and keratinocytes had lower levels of small proteoglycans, decorin and fibromodulin, and higher levels of large proteoglycan, versican, compared to tissue engineered skin made of superficial fibroblasts and keratinocytes. Tissue engineered skin made of deep fibroblasts and keratinocytes had lower expression of TGF-?, IL-1 and KGF but higher expression of PDGF and IL-6, compared to tissue engineered skin made of superficial fibroblasts and keratinocytes. Furthermore, co-culture with keratinocytes reduced TGF-?1 production of deep but not superficial fibroblasts. Additionally, keratinocytes reduced differentiation of deep fibroblasts to myofibroblasts in tissue engineered skin constructs, but not that of superficial fibroblasts. Taken together, keratinocytes reduce fibrotic remodeling of the scaffolds by deep dermal fibroblasts. Our results therefore demonstrate that tissue engineered skin made specifically with a homogeneous population of superficial fibroblasts and keratinocytes is less fibrotic than that with a heterogeneous population of fibroblasts and keratinocytes.

Varkey M; Ding J; Tredget E

2013-10-01

39

Distinct and complementary roles of papillary and reticular fibroblasts in skin morphogenesis and homeostasis.  

Science.gov (United States)

To study the biological properties of dermal fibroblast sub-populations, we used a reconstructed skin model with a dermal compartment populated with either papillary or reticular fibroblasts. The histological and immunohistological characterization of these reconstructed skins revealed distinct biological and structural differences, depending on the site-matched fibroblast population incorporated. Epidermal differentiation and maturation was favored and found optimum in the presence of papillary fibroblasts with little effect on ECM, as opposed to reticular fibroblasts, which had a significant positive effect on the production of the ECM molecules of the dermal epidermal junction and the dermis. In addition, the synthesis and release of MMPs and soluble factors like VEGF and KGF into the culture medium were influenced by the fibroblast population. MMP1 and VEGF were increased in the presence of papillary fibroblasts, whereas MMP3 and KGF levels were higher in the presence of reticular fibroblasts. Our results suggest that papillary and reticular fibroblasts exert distinct functions and activities in skin as revealed by the reconstructed skin model. These functional differences may have implications in wound healing and skin aging processes, considering the slow loss of papillary fibroblasts in human skin that occurs with age. PMID:22449755

Pageon, Hervé; Zucchi, Hélène; Asselineau, Daniel

40

Distinct and complementary roles of papillary and reticular fibroblasts in skin morphogenesis and homeostasis.  

UK PubMed Central (United Kingdom)

To study the biological properties of dermal fibroblast sub-populations, we used a reconstructed skin model with a dermal compartment populated with either papillary or reticular fibroblasts. The histological and immunohistological characterization of these reconstructed skins revealed distinct biological and structural differences, depending on the site-matched fibroblast population incorporated. Epidermal differentiation and maturation was favored and found optimum in the presence of papillary fibroblasts with little effect on ECM, as opposed to reticular fibroblasts, which had a significant positive effect on the production of the ECM molecules of the dermal epidermal junction and the dermis. In addition, the synthesis and release of MMPs and soluble factors like VEGF and KGF into the culture medium were influenced by the fibroblast population. MMP1 and VEGF were increased in the presence of papillary fibroblasts, whereas MMP3 and KGF levels were higher in the presence of reticular fibroblasts. Our results suggest that papillary and reticular fibroblasts exert distinct functions and activities in skin as revealed by the reconstructed skin model. These functional differences may have implications in wound healing and skin aging processes, considering the slow loss of papillary fibroblasts in human skin that occurs with age.

Pageon H; Zucchi H; Asselineau D

2012-05-01

 
 
 
 
41

The Level and Stability of Residual Catalase in Cultured Acatalasemic Skin Fibroblasts  

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Full Text Available In an attempt to determine the level and heat stability of residual catalase in somatic cells of acatalasemic Japanese, skin fibroblasts from an acatalasemic subject were cultured, and the catalase activity of the cultured fibroblasts was compared with that of cultured normal fibroblasts. Catalase activity was determined using an oxygen electrode. The residual catalase activity in cultured acatalasemic fibroblasts was 10% of the normal. The heat stability at 55 degrees C of residual catalase in the acatalasemic fibroblasts was similar to that of normal fibroblasts.

Ogata,Masana; Fujii,Yasuhito; Meguro,Tadamichi; Kira,Shohei; Matsuda,Akira; Izushi,Fumio; Kimoto,Tetsuo; Takahara,Shigeo

1987-01-01

42

Growth properties of familial Alzheimer skin fibroblasts during in vitro aging.  

UK PubMed Central (United Kingdom)

Human diploid fibroblasts undergo replicative senescence in vitro, which is strongly correlated with biological aging in vivo. In order to examine whether features compatible with a systemic premature aging are present in familial Alzheimer's disease (FAD) patients, we investigated the growth characteristics of three skin fibroblast lines from FAD patients and from three sex/age-matched controls at different passages until senescence was reached. A kinetic study of the replicative capacity was performed at different culture times by [3H]-thymidine incorporation and crystal violet staining. Data showed no significant difference between the two groups at any studied passage. The life span of the two types of cultures was also comparable. These results suggest that in familial Alzheimer patients there are not systemic signs of accelerated aging.

Tesco G; Vergelli M; Amaducci L; Sorbi S

1993-01-01

43

Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts.  

Science.gov (United States)

The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta cocoon has been assessed and compared with that of the mulberry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive control for the experiment. Our results showed that the sericin obtained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-incubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase; lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant. PMID:18377728

Dash, Rupesh; Acharya, Chitrangada; Bindu, P C; Kundu, S C

2008-03-31

44

Antioxidant potential of silk protein sericin against hydrogen peroxide-induced oxidative stress in skin fibroblasts.  

UK PubMed Central (United Kingdom)

The antioxidant potential of silk protein sericin from the non-mulberry tropical tasar silkworm Antheraea mylitta cocoon has been assessed and compared with that of the mulberry silkworm, Bombyx mori. Skin fibroblast cell line (AH927) challenged with hydrogen peroxide served as the positive control for the experiment. Our results showed that the sericin obtained from tasar cocoons offers protection against oxidative stress and cell viability is restored to that of control on pre-incubation with the sericin. Fibroblasts pre-incubated with non-mulberry sericin had significantly lower levels of catalase; lactate dehydrogenase and malondialdehyde activity when compared to untreated ones. This report indicates that the silk protein sericin from the non-mulberry tropical tasar silkworm, A. mylitta can serve as a valuable antioxidant.

Dash R; Acharya C; Bindu PC; Kundu SC

2008-03-01

45

A Marfan syndrome gene expression phenotype in cultured skin fibroblasts  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract 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 -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.

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

46

Characterization of deficient heme synthase activity in protoporphyria with cultured skin fibroblasts.  

UK PubMed Central (United Kingdom)

Heme synthase (ferrochelatase) activity, as determined by the chelation of ferrous iron to protoporphyrin or deuteroporphyrin, is reduced to 10-25% of normal in tissues of patients with protoporphyria. With cultured skin fibroblasts from seven patients with protoporphyria and six normal individuals, the present studies examined the enzymatic defect.Heme synthase activity in normal and protoporphyria fibroblasts had the same pH optimum, showed similar inhibition by divalent metals, and had the highest specific activity in the mitochondrial-enriched fraction. The ultrastructural features and other biochemical parameters of mitochondria were normal in protoporphyria cells, excluding a general mitochondrial defect. Measurement of the rate of deuteroheme formation at different concentrations of substrate demonstrated a significant reduction in the apparent K(m) for deuteroporphyrin in detergent-treated sonicates of protoporphyria fibroblasts compared to normal (7.5 +/- 0.9 muM, mean +/- SEM, vs. 17.4 +/- 1.8), as well as a decrease in the velocity of reaction (mean level was 21% of normal). Studies with intact cells, in which heme synthase activity was estimated indirectly, also indicated that the apparent K(m) for porphyrin substrate was significantly lower in protoporphyria lines. These data show that heme synthase in protoporphyria fibroblasts has markedly reduced catalytic activity despite an increased affinity for porphyrin substrate. This could be caused by either a change in the enzyme protein, or an alteration of its micro-environment.

Bloomer JR

1980-02-01

47

Mutagenic effects of alpha particles in normal human skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

Alpha-irradiation to the bronchial airways from inhaled radon progeny increases the risk of developing lung cancer. The molecular mechanism of radon-induced lung cancer is not clear, but one of the most important genetic effects of ionizing radiation is the induction of gene mutation. Mutations, especially those associated with visible chromosome abnormalities in humans, have been associated with cancer. Therefore, our objective is to use a well-defined model system to determine the mutagenic potential of alpha particles in normal human skin cells and to define this action at the molecular level. Normal human skin fibroblasts were irradiated with alpha particles (3.59 MeV, LET 115 keV {mu}m{sup {minus}1}) emitted from the decay of {sup 238}Pu. Mutagenicity was determined at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. Results from this study indicate that beta particles were more efficient in mutation induction than gamma rays. Based on the initial slopes of the dose-response curves, the RBE for mutation is about 8 for alpha particles. HPRT-deficient mutants which are resistant to 6-thioguanine have been isolated and analyzed by the Southern blot technique. To date, we have characterized 69 gamma-ray-induced and 195 alpha-particle-induced HPRT-deficient mutants. Our data indicate that more than 50% of all gamma-ray-induced mutants have band patterns identical to that observed for the normal structural HPRT gene, whereas the remaining mutants (45%) contain either a rearrangement, partial deletion, or total deletion of the HPRT gene. In contrast, only 30% of alpha-particle-induced human HPRT mutants contain a normal Southern blot pattern, and about 50% indicate total deletion of the HPRT gene. Our results support the notion that high-LET radiation produces more unrepaired or misrepaired DNA damage than do gamma rays.

Chen, D.J.; Carpenter, S.; Hanks, T. [Los Alamos National Lab., NM (United States)

1992-12-31

48

Mutagenic effects of alpha particles in normal human skin fibroblasts  

International Nuclear Information System (INIS)

Alpha-irradiation to the bronchial airways from inhaled radon progeny increases the risk of developing lung cancer. The molecular mechanism of radon-induced lung cancer is not clear, but one of the most important genetic effects of ionizing radiation is the induction of gene mutation. Mutations, especially those associated with visible chromosome abnormalities in humans, have been associated with cancer. Therefore, our objective is to use a well-defined model system to determine the mutagenic potential of alpha particles in normal human skin cells and to define this action at the molecular level. Normal human skin fibroblasts were irradiated with alpha particles (3.59 MeV, LET 115 keV ?m-1) emitted from the decay of 238Pu. Mutagenicity was determined at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. Results from this study indicate that beta particles were more efficient in mutation induction than gamma rays. Based on the initial slopes of the dose-response curves, the RBE for mutation is about 8 for alpha particles. HPRT-deficient mutants which are resistant to 6-thioguanine have been isolated and analyzed by the Southern blot technique. To date, we have characterized 69 gamma-ray-induced and 195 alpha-particle-induced HPRT-deficient mutants. Our data indicate that more than 50% of all gamma-ray-induced mutants have band patterns identical to that observed for the normal structural HPRT gene, whereas the remaining mutants (45%) contain either a rearrangement, partial deletion, or total deletion of the HPRT gene. In contrast, only 30% of alpha-particle-induced human HPRT mutants contain a normal Southern blot pattern, and about 50% indicate total deletion of the HPRT gene. Our results support the notion that high-LET radiation produces more unrepaired or misrepaired DNA damage than do gamma rays.

1992-01-01

49

HURLER'S SYNDROME : EFFECT OF RETINOL (VITAMIN A ALCOHOL) ON CELLULAR MUCOPOLYSACCHARIDES IN CULTURED HUMAN SKIN FIBROBLASTS  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Skin fibroblasts from eight families with Hurler's syndrome (X-linked recessive and autosomal recessive) and normal individuals have been studied in cell culture. A good correlation between cellular metachromasia and the quantitative estimation of intracellular mucopolysaccharides was observed prov...

Danes, B. Shannon; Bearn, Alexander G.

50

SKIN TOPICAL COMPOSITION CONTAINING EPIDERMAL GROWTH FACTOR AND FIBROBLAST GROWTH FACTOR  

UK PubMed Central (United Kingdom)

A skin topical composition comprising an epidermal growth factor and a fibroblast growth factor is provided to improve effects of skin whitening, melanin formation inhibition, skin moisturizing, skin wrinkle improvement and stretch mark and oxidation inhibition by optimizing the mixing ratio of effective ingredients. A skin topical composition for improving skin beauty comprises 0.01-1 ppm of epidermal growth factor, 0.01-1 ppm of fibroblast growth factor, 1.0-5.0 wt.% of amino acid peptide, 0.1-2.0 wt.% of glabridin, 0.1-3.0 wt.% of ubiquinone(coenzyme Q10) and 0.1-2.0 wt.% of vegetable collagen(extension N-200), and has a formulation selected from cosmetic lotion, cream, ointment, emulsion, foundation, oil, pack, soap, body soap, lipstick, nail cosmetic, eye cosmetic, perfume, washing cosmetic, toothpaste, mouth wash, deodorizer, bath agent, shampoo, rinse, hair tonic, hair spray or hair dye.

PARK HEE JUN

51

Elevated expression of hepatocyte and keratinocyte growth factor in cultured buccal-mucosa-derived fibroblasts compared with normal-skin-derived fibroblasts.  

UK PubMed Central (United Kingdom)

Oral mucosa heals faster with less scar formation than skin and a hypertrophic scar is very rare in the oral cavity, but its mechanism has not been elucidated enough. To elucidate whether or not there are differences in growth factor expression between fibroblasts derived from buccal mucosal and normal skin, we investigated the expression of hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and stem cell factor (SCF) by cultured fibroblasts. The semiquantitative RT-PCR revealed that the expression of HGF and KGF transcripts by buccal mucosal fibroblasts was significantly elevated compared with that by dermal fibroblasts. In parallel, ELISA revealed the significant increase of HGF production by buccal mucosal fibroblasts. The level of production of SCF protein did not differ significantly. Our study suggests that increased expression of HGF and KGF by buccal mucosal fibroblasts may partly be responsible for the faster wound healing with less scar formation in the oral cavity compared with normal skin.

Okazaki M; Yoshimura K; Uchida G; Harii K

2002-11-01

52

Senescent phenotypes of skin fibroblasts from patients with Tangier disease  

International Nuclear Information System (INIS)

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) ?10 compared with control cells. TD cells practically ceased proliferation at PDL ?30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated ?-galactosidase (SA-?-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.

2007-06-01

53

Hyaluronan uptake by adult human skin fibroblasts in vitro  

Directory of Open Access Journals (Sweden)

Full Text Available Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism.

MA Croce; F Boraldi; D Quaglino; R Tiozzo; I Pasquali-Ronchetti

2003-01-01

54

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

Energy Technology Data Exchange (ETDEWEB)

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.

Coppey, J.; Menezes, S.

1981-01-01

55

Trisomy 13 mosaicism demonstrated only in skin fibroblasts in a patient presenting psychomotor retardation, pigmentary dysplasia and some dysmorphic features  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english A Brazilian female infant presented delayed psychomotor development, skin pigmentary dysplasia and some dysmorphic features. Chromosome analysis from peripheral blood culture was normal, but the karyotype from skin fibroblasts revealed mosaicism for trisomy 13. This case demonstrates the relevance of performing chromosomal analysis of skin fibroblasts in patients with mental retardation, associated with pigmentary dysplasia of the skin and a normal karyotype in peripheral (more) blood lymphocytes. To our knowledge, it is the first report of trisomy 13 demonstrated only in skin fibroblasts.

Ferreira, A.P.S.; Mazzucatto, L.F.; Ramos, E.S.; Pina-Neto, J.M.

1996-01-01

56

Trisomy 13 mosaicism demonstrated only in skin fibroblasts in a patient presenting psychomotor retardation, pigmentary dysplasia and some dysmorphic features  

Directory of Open Access Journals (Sweden)

Full Text Available A Brazilian female infant presented delayed psychomotor development, skin pigmentary dysplasia and some dysmorphic features. Chromosome analysis from peripheral blood culture was normal, but the karyotype from skin fibroblasts revealed mosaicism for trisomy 13. This case demonstrates the relevance of performing chromosomal analysis of skin fibroblasts in patients with mental retardation, associated with pigmentary dysplasia of the skin and a normal karyotype in peripheral blood lymphocytes. To our knowledge, it is the first report of trisomy 13 demonstrated only in skin fibroblasts.

A.P.S. Ferreira; L.F. Mazzucatto; E.S. Ramos; J.M. Pina-Neto

1996-01-01

57

Altered chloride metabolism in cultured cystic fibrosis skin fibroblasts.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

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, we have studied chloride metabolism in cultured CF fibroblasts by comparing the efflux of 36Cl- from m...

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

58

Effects of cigarette smoke extracts on the growth and senescence of skin fibroblasts in vitro.  

Science.gov (United States)

Epidemiological studies have shown that cigarette smoke (CS), a very common environmental factor, plays an important role in skin aging. Although some in vivo studies have suggested that CS affects skin aging, the detailed effects of CS on skin cells in vitro remain largely unknown. In this study, we investigated the effects of cigarette smoke extract (CSE) on the growth, proliferation, and senescene of skin fibroblasts and the possible mechanism underlying these effects. Primary cultured human fibroblasts were exposed to a range of concentrations of CSE. Cell viability and cell proliferation after CSE exposure were analyzed with the methyl thiazolyl tetrazolium (MTT) assay and bromodeoxyuridine incorporation assay, respectively. Growth curves of fibroblasts exposed to different concentrations of CSE were developed and prolonged CSE-exposed cells were observed. Morphological and ultrastructural changes in fibroblasts were assessed by inverted light microscopy and transmission electron microscopy (TEM). Dying cells were stained with senescence-associated ?-galactosidase (SA ?-gal). Intracellular reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity were determined by a colorimetric method. We found that proliferative capacity and growth were inhibited by CSE exposure in a dose- and time-dependent manner. Fibroblasts exposed to even low concentrations of CSE for a long period of time (5 passages) showed significantly increased SA ?-gal activity and typical features of aging cells. Meanwhile, CSE inhibited superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and augmented ROS levels. Our observations suggest that CSE exposure impairs fibroblast growth and proliferation and leads to features similar to those seen in senescent cells. Oxidative stress injury and inhibition of antioxidant defense activity may be involved in CSE-induced fibroblast senescence. PMID:23847443

Yang, Gao-Yun; Zhang, Chun-Lei; Liu, Xiang-Chen; Qian, Ge; Deng, Dan-Qi

2013-06-28

59

Effects of cigarette smoke extracts on the growth and senescence of skin fibroblasts in vitro.  

UK PubMed Central (United Kingdom)

Epidemiological studies have shown that cigarette smoke (CS), a very common environmental factor, plays an important role in skin aging. Although some in vivo studies have suggested that CS affects skin aging, the detailed effects of CS on skin cells in vitro remain largely unknown. In this study, we investigated the effects of cigarette smoke extract (CSE) on the growth, proliferation, and senescene of skin fibroblasts and the possible mechanism underlying these effects. Primary cultured human fibroblasts were exposed to a range of concentrations of CSE. Cell viability and cell proliferation after CSE exposure were analyzed with the methyl thiazolyl tetrazolium (MTT) assay and bromodeoxyuridine incorporation assay, respectively. Growth curves of fibroblasts exposed to different concentrations of CSE were developed and prolonged CSE-exposed cells were observed. Morphological and ultrastructural changes in fibroblasts were assessed by inverted light microscopy and transmission electron microscopy (TEM). Dying cells were stained with senescence-associated ?-galactosidase (SA ?-gal). Intracellular reactive oxygen species (ROS) levels, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity were determined by a colorimetric method. We found that proliferative capacity and growth were inhibited by CSE exposure in a dose- and time-dependent manner. Fibroblasts exposed to even low concentrations of CSE for a long period of time (5 passages) showed significantly increased SA ?-gal activity and typical features of aging cells. Meanwhile, CSE inhibited superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities and augmented ROS levels. Our observations suggest that CSE exposure impairs fibroblast growth and proliferation and leads to features similar to those seen in senescent cells. Oxidative stress injury and inhibition of antioxidant defense activity may be involved in CSE-induced fibroblast senescence.

Yang GY; Zhang CL; Liu XC; Qian G; Deng DQ

2013-01-01

60

Possible role for metallothionein in the cellular defense mechanism against UVB irradiation in neonatal human skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

The role of metallothionein (MT) in protecting skin cells against UVB irradiation was investigated. Fibroblast strains from normal adult (HS-K) and neonatal (NB1RGB) human skins as well as keratinocyte strains from human skin (SV40-HSK) and newborn Balb/c mouse skin (Pam 212) were exposed to UVB irradiation. (Author).

Kobayashi, Shizuko; Hirota, Yumiko; Takehana, Makoto (Kyoritsu College of Pharmacy, Tokyo (Japan)); Sayato-Suzuki, Junko (Kyoritsu College of Pharmacy, Tokyo (Japan) National Inst. for Environmental Studies, Ibaraki (Japan). Environmental Health Sciences Div.); Nishimura, Hisao; Nishimura, Noriko (Aichi Medical Univ. (Japan). Dept. of Hygiene); Tohyama, Chiharu (National Inst. for Environmental Studies, Ibaraki (Japan). Environmental Health Sciences Div.)

1994-06-01

 
 
 
 
61

Possible role for metallothionein in the cellular defense mechanism against UVB irradiation in neonatal human skin fibroblasts  

International Nuclear Information System (INIS)

The role of metallothionein (MT) in protecting skin cells against UVB irradiation was investigated. Fibroblast strains from normal adult (HS-K) and neonatal (NB1RGB) human skins as well as keratinocyte strains from human skin (SV40-HSK) and newborn Balb/c mouse skin (Pam 212) were exposed to UVB irradiation. (Author).

1994-01-01

62

Effects of recombinant human epidermal growth factor on the proliferation and radiation survival of human fibroblast cell lines in vitro  

International Nuclear Information System (INIS)

To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. Number of fibroblast was significant more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing.

2006-01-01

63

Effects of recombinant human epidermal growth factor on the proliferation and radiation survival of human fibroblast cell lines in vitro  

Energy Technology Data Exchange (ETDEWEB)

To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. Number of fibroblast was significant more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing.

Kim, Hyun Sook; Kang, Ki Mun; Na, Jae Boem; Chai, Gyu Young [Gyeongsang National University College of Medicine, Jinju (Korea, Republic of); Lee, Sang Wook [University of Ulsan College of Medicine, Asan Medical Center, Seoul (Korea, Republic of)

2006-09-15

64

Trisomy 17 mosaicism in amniotic fluid cells not found at birth in blood but present in skin fibroblasts.  

UK PubMed Central (United Kingdom)

This article describes a case of fetal trisomy 17 mosaicism found in amniotic fluid cells in one of two bichorial biamniotic twins without any sonographic anomaly. The extra chromosome 17 was absent from cord blood cells at birth but present on karyotype and in situ hybridization in cultured fibroblasts from skin biopsy. Clinical examination showed a few mild dysmorphic features and a moderate neurological involvement which may rather be related to prematurity. It therefore seemed important to obtain the karyotype on fibroblasts when a trisomic cell line was found in amniocentesis and not confirmed on blood lymphocytes, even in the absence of dysmorphic features. This should help to differentiate a real mosaic from a mosaic restricted to extra-fetal tissues.

Lesca G; Boggio D; Bellec V; Magaud JP; Till M

1999-03-01

65

Curcumin-induced hormetic effects in human skin fibroblasts : implications toward anti-ageing interventions  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Ageing is associated with decreased cellular antioxidant defenses and with reduced ability to induce stress responses. We have tested the curcumin-induced hormetic stimulation of cellular stress responses in normal human skin fibroblasts, and associate those with potential anti-ageing effects. Curcu...

Lima, Cristóvão F.; Wilson, Cristina Pereira; Rattan, S. I. S.

66

Abnormal properties of collagen lysyl hydroxylase from skin fibroblasts of siblings with hydroxylysine-deficient collagen.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Skin fibroblasts from two siblings with hydroxylysine-deficient collagen collagen (Ehlers-Danlos syndrome, type VI) contained normal levels of collagen prolyl hydroxylase activity but were markedly deficient in collagen lysyl hydroxylase activity. The deficiency was evident in all fractions of cell ...

Quinn, R S; Krane, S M

67

IL-22 promotes fibroblast-mediated wound repair in the skin.  

Science.gov (United States)

Skin wound repair requires complex and highly coordinated interactions between keratinocytes, fibroblasts, and immune cells to restore the epidermal barrier and tissue architecture after acute injury. The cytokine IL-22 mediates unidirectional signaling from immune cells to epithelial cells during injury of peripheral tissues such as the liver and colon, where IL-22 causes epithelial cells to produce antibacterial proteins, express mucins, and enhance epithelial regeneration. In this study, we used IL-22(-/-) mice to investigate the in vivo role for IL-22 in acute skin wounding. We found that IL-22(-/-) mice displayed major defects in the skin's dermal compartment after full-thickness wounding. We also found that IL-22 signaling is active in fibroblasts, using in vitro assays with primary fibroblasts, and that IL-22 directs extracellular matrix (ECM) gene expression and myofibroblast differentiation both in vitro and in vivo. These data define roles of IL-22 beyond epithelial cross talk, and suggest that IL-22 has a previously unidentified role in skin repair by mediating interactions between immune cells and fibroblasts. PMID:23223145

McGee, Heather M; Schmidt, Barbara A; Booth, Carmen J; Yancopoulos, George D; Valenzuela, David M; Murphy, Andrew J; Stevens, Sean; Flavell, Richard A; Horsley, Valerie

2012-12-06

68

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.

Castellani AA; De Luca G; Rindi S; Salvini R; Tira ME

1986-09-01

69

Complementary antioxidant function of caffeine and green tea polyphenols in normal human skin fibroblasts.  

UK PubMed Central (United Kingdom)

The study of free radicals is particularly relevant in the context of human skin carcinogenesis and photoaging because of these oxidants' ability to induce DNA mutations and produce lipid peroxidation byproducts, including 4-hydroxy-2-nonenal (HNE). Therefore, it is important to identify and evaluate agents with the ability to modulate intracellular free radicals and HNE. The purpose of this research is to investigate the ability of antioxidants green tea polyphenols (GTPs) and caffeine, alone and in combination, to modulate the hydrogen peroxide (H2O2)-induced upregulation of reactive oxygen species (ROS) free radicals and HNE in normal human skin fibroblast WS-1 cells in vitro. GTPs and caffeine were selected for evaluation because these compounds have demonstrated antioxidative properties in various skin models. Furthermore, GTPs and caffeine share a close natural botanical association as caffeine is present in green tea, as well. Hydrogen peroxide is a well-known generator of free radicals that is produced during endogenous and UV-induced oxidation processes in human skin and was used to upregulate ROS and HNE in normal human fibroblast WS-1 cells. Using a flow cytometry-based assay, the results demonstrate that at 0.001% concentration, green tea polyphenols alone, and in combination with 0.1 mM caffeine, inhibited the upregulation of H2O2-generated free radicals and HNE in human skin fibroblasts in vitro. Caffeine alone demonstrated limited anti-oxidant properties.

Jagdeo J; Brody N

2011-07-01

70

Complementary antioxidant function of caffeine and green tea polyphenols in normal human skin fibroblasts.  

Science.gov (United States)

The study of free radicals is particularly relevant in the context of human skin carcinogenesis and photoaging because of these oxidants' ability to induce DNA mutations and produce lipid peroxidation byproducts, including 4-hydroxy-2-nonenal (HNE). Therefore, it is important to identify and evaluate agents with the ability to modulate intracellular free radicals and HNE. The purpose of this research is to investigate the ability of antioxidants green tea polyphenols (GTPs) and caffeine, alone and in combination, to modulate the hydrogen peroxide (H2O2)-induced upregulation of reactive oxygen species (ROS) free radicals and HNE in normal human skin fibroblast WS-1 cells in vitro. GTPs and caffeine were selected for evaluation because these compounds have demonstrated antioxidative properties in various skin models. Furthermore, GTPs and caffeine share a close natural botanical association as caffeine is present in green tea, as well. Hydrogen peroxide is a well-known generator of free radicals that is produced during endogenous and UV-induced oxidation processes in human skin and was used to upregulate ROS and HNE in normal human fibroblast WS-1 cells. Using a flow cytometry-based assay, the results demonstrate that at 0.001% concentration, green tea polyphenols alone, and in combination with 0.1 mM caffeine, inhibited the upregulation of H2O2-generated free radicals and HNE in human skin fibroblasts in vitro. Caffeine alone demonstrated limited anti-oxidant properties. PMID:21720657

Jagdeo, Jared; Brody, Neil

2011-07-01

71

Human skin in organ culture and human skin cells (keratinocytes and fibroblasts) in monolayer culture for assessment of chemically induced skin damage.  

Science.gov (United States)

Human skin cells (epidermal keratinocytes and dermal fibroblasts) in monolayer culture and human skin in organ culture were exposed to agents that are known to produce irritation (redness, dryness, edema and scaly crusts) when applied topically to skin. Among the agents used were three well accepted contact irritants (i.e., all-trans retinoic acid [RA], sodium lauryl sulfate [SLS] and benzalkonium chloride) as well as the corrosive organic mercury compound, aminophenyl mercuric acetate (APMA), and 5 contact sensitizers (oxazolone, nickel sulfate, eugenol, isoeugenol and ethylene glycol dimethacrylate [EGDM]). As a group, the contact irritants (including the corrosive mercuric compound) were cytotoxic for keratinocytes and fibroblasts and suppressed growth at lower concentrations than the contact sensitizers. The contact irritants also produced histological changes (hyperplasia, incomplete keratinization, loss of the granular layer, acantholysis and necrosis) in organ-cultured skin at dose levels at which the contact sensitizers appeared to be inert. Finally, the profile of secreted molecules from organ-cultured skin was different in the presence of contact irritants versus contact sensitizers. Taken together, these data suggest that the use of organ-cultured skin in conjunction with cells derived from the skin in monolayer culture may provide an initial approach to screening agents for deleterious changes in skin. PMID:17763283

Varani, James; Perone, Patricia; Spahlinger, Diana M; Singer, Lisa M; Diegel, Kelly L; Bobrowski, Walter F; Dunstan, Robert

2007-08-01

72

Assembly and alignment of fibronectin-coated gold beads into fibrils by human skin fibroblasts.  

Science.gov (United States)

The assembly of fibronectin into fibrils was monitored by high voltage electron microscopy using 18 nm colloidal gold beads bound to fibronectin (Au18-fibronectin) or an amino terminal 70 kd fragment of fibronectin (Au18-70 kd) that blocks the incorporation of fibronectin into disulfide bonded fibrils. Subconfluent cultures of human skin fibroblasts were incubated with the colloidal gold complexes for 0.25, 0.5, 1.5 and 5 h. In fibroblast cultures incubated with Au18-fibronectin and Au18-70 kd fragments for 0.25 and 0.5 h, the complexes of Au18-fibronectin and Au18-70 kd fragment were observed bound to the cell surface in clusters near bundles of intracellular microfilaments, usually along the lateral edges of the fibroblasts. Fibroblast cultures incubated with Au18-fibronectin and Au18-70 kd fragment for 1.5 and 5 h showed a more linear arrangement of the Au18-fibronectin and Au18-70 kd fragment along the lateral and retracting edges and on filopodia. This alignment of Au18-fibronectin into linear arrangements on the fibroblast cell surface suggests that the assembly of fibronectin fibrils occurs on specific regions of the fibroblast. PMID:3616572

Peters, D M; Mosher, D F

1987-06-01

73

Assembly and alignment of fibronectin-coated gold beads into fibrils by human skin fibroblasts.  

UK PubMed Central (United Kingdom)

The assembly of fibronectin into fibrils was monitored by high voltage electron microscopy using 18 nm colloidal gold beads bound to fibronectin (Au18-fibronectin) or an amino terminal 70 kd fragment of fibronectin (Au18-70 kd) that blocks the incorporation of fibronectin into disulfide bonded fibrils. Subconfluent cultures of human skin fibroblasts were incubated with the colloidal gold complexes for 0.25, 0.5, 1.5 and 5 h. In fibroblast cultures incubated with Au18-fibronectin and Au18-70 kd fragments for 0.25 and 0.5 h, the complexes of Au18-fibronectin and Au18-70 kd fragment were observed bound to the cell surface in clusters near bundles of intracellular microfilaments, usually along the lateral edges of the fibroblasts. Fibroblast cultures incubated with Au18-fibronectin and Au18-70 kd fragment for 1.5 and 5 h showed a more linear arrangement of the Au18-fibronectin and Au18-70 kd fragment along the lateral and retracting edges and on filopodia. This alignment of Au18-fibronectin into linear arrangements on the fibroblast cell surface suggests that the assembly of fibronectin fibrils occurs on specific regions of the fibroblast.

Peters DM; Mosher DF

1987-06-01

74

Chemical transformation of cultured skin fibroblasts from humans genetically predisposed to cancer.  

UK PubMed Central (United Kingdom)

Adenomatosis of the colon and rectum (ACR) is an inherited form of cancer. Assuming that phenotypic expressions that appear in cell strains reflect its biological abnormalities, the study of cultured skin fibroblasts derived from individuals with an inherited form of cancer such as ACR provides a unique study for analysis of the oncogenic process. Growth disorders and increased susceptibility to tumor promoters and to transformation by an oncogenic RNA tumor virus have been demonstrated in these skin fibroblasts. We found that human skin fibroblasts (PF) derived from ACR individuals were sensitive to a chemical carcinogen. Cells treated only with various levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large cell aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities but did not form colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (1 microgram/ml) and showed prolonged life span compared to those untreated cells. However, no tumors were produced when cells were inoculated subcutaneously into nude mice. Data suggest that neoplastic transformation of these skin cells by chemical carcinogens is a multi-phase process.

Rhim JS; Arnstein P; Huebner RJ

1981-01-01

75

Malignant transformation of human skin fibroblasts by two alternative pathways.  

UK PubMed Central (United Kingdom)

We developed a telomerase-positive, infinite life span human fibroblast cell strain (MSU-1.0) by transfection of a v-MYC oncogene and spontaneous over-expression of transcription factors SP1/SP3. Loss of expression of p14(ALT) and enhanced expression of SPRY2 gave rise to the MSU-1.1 cell strain. Unlike MSU-1.0 cells, the MSU-1.1 cells can be malignantly transformed by expression of N-RAS(LYS61) or H-Ras(v12) oncoproteins (driven by their original promoters) and expression of a SRC-family protein, v-FES. MSU-1.1 cells can also be malignantly transformed by high expression of these RAS oncogenes or the v-K-RAS oncogene. PDGF-B transformed MSU-1.1 cells give rise to benign tumors (fibromas) in athymic mice. A second route to malignant transformation of the MSU-1.1 cells involves loss of functional TP53 protein by carcinogen treatment and loss of expression of wild type p16(INK). These studies indicate 6-8 "hits" are required to activate the oncogenes and inactivate the suppressor genes we identified.

McCormick JJ; Maher VM

2011-01-01

76

Alginate oligosaccharides modulate cell morphology, cell proliferation and collagen expression in human skin fibroblasts in vitro.  

UK PubMed Central (United Kingdom)

Effects of alginate oligosaccharides on cell proliferation and expression of collagen in cultured skin fibroblasts were studied. The oligosaccharides were found to suppress fibroblast proliferation to half the level in control cultures at a dose of 10 mg/ml during a period of 5 days. The inhibition was accompanied by a change in cell shape. The inhibition of cell proliferation was reversible, since depletion of these oligosaccharides led to a recovery of cell motility. Treatment of confluent cells with 10 mg/ml oligosaccharides for 5 days resulted in a reduction in collagen synthesis to one half of that in control cultures and inhibition of steady state levels of alpha1(I), alpha2(I), alpha1(III) and alpha1(VI) collagen mRNAs. These results suggest that alginate oligosaccharides are potential modulators of dermal fibroblasts and may provide a useful tool for the treatment of disorders related to abnormal collagen metabolism.

Tajima S; Inoue H; Kawada A; Ishibashi A; Takahara H; Hiura N

1999-07-01

77

Cultured skin fibroblasts from patients with porokeratosis are hypersensitive to the lethal effects of X-radiation  

Energy Technology Data Exchange (ETDEWEB)

Porokeratosis is an autosomal dominant inherited skin disorder. The lesions are characterized by localized abnormal keratinization and may develop into malignant tumors. To determine the cellular basis of the cancer susceptibility associated with this skin condition, we examined the colony-forming ability of X-ray or ultraviolet (UV) light irradiated, cultured fibroblasts derived from porokeratosis patients' normal-appearing skin. Four fibroblast strains derived from four porokeratosis patients' skin were significantly hypersensitive to the lethal effects of X-radiation. However, they all showed a similar sensitivity to strains from normal donors to 254 nm UV light. The hypersensitivity to X-ray radiation in cultured skin fibroblasts from porokeratosis patients suggests an inherent instability of cellular DNA and may prbably be associated with the cancer-prone nature of this skin condition.

Otsuka, Fujio; Watanabe, Ryohji; Moro, Akiko; Ohkochi, Hitoshi; Ishibashi, Yasumasa

1989-01-01

78

Juglans mandshurica leaf extract protects skin fibroblasts from damage by regulating the oxidative defense system.  

UK PubMed Central (United Kingdom)

Skin is mainly damaged by genetic and environmental factors such as ultraviolet light, xenobiotics, hormonal changes, heat, and smoking. ROS production is commonly involved in the pathogenesis of skin damage induced by these factors, causing skin aging, including wrinkling, by activating the metalloproteinases (MMP-1) that break down type I collagen (COL1A1). The walnut tree Juglans mandshurica MAX. (JM) is found in China, Siberia and Korea. JM has been reported to have various pharmacological activities, such as anti-tumor, anti-oxidative, and anti-bacterial effects. In the present study, we investigated the protective effect of JM leaf extract (JME) against oxidative stress in HS68 human skin fibroblasts. JME significantly and dose-dependently protected HS68 cells against H?O?-induced damage, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay. Other assays demonstrated that JME protected HS68 cells by regulating ROS production and increasing levels of glutathione, heme oxygenase-1, and activated NF-E2-related factor 2. JME additionally prevented the elevation of MMP-1 and reduction of COL1A1 induced by H?O?. It also inhibited H?O?-induced phosphorylation of ERK, p38, and JNK. These results indicate that JME protects human skin fibroblasts from H?O?-induced damage by regulating the oxidative defense system.

Park G; Jang DS; Oh MS

2012-05-01

79

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells.

Zamansky, G.B.

1986-08-01

80

Recovery from UV-induced potentially lethal damage in systemic lupus erythematosus skin fibroblasts  

International Nuclear Information System (INIS)

[en] The repair of ultraviolet light-induced potentially lethal damage was investigated in density-inhibited skin fibroblast cell strains derived from patients with systemic lupus erythematosus. The effect of exposure to polychromatic ultraviolet light composed of environmentally relevant wavelengths or to the more commonly studied, short wavelength (254 nm) ultraviolet light was studied. Systemic lupus erythematosus cells, which are hypersensitive to ultraviolet light under growth promoting conditions, were able to repair potentially lethal damage as well as normal cells. (author)

1986-01-01

 
 
 
 
81

Oxidant stress leads to transcriptional activation of the human heme oxygenase gene in cultured skin fibroblasts  

International Nuclear Information System (INIS)

Treatment of cultured human skin fibroblasts with near-UV radiation, hydrogen peroxide, and sodium arsenite induces accumulation of heme oxygenase mRNA and protein. In this study, these treatments led to a dramatic increase in the rate of RNA transcription from the heme oxygenase gene but had no effect on mRNA stability. Transcriptional activation, therefore, appears to be the major mechanism of stimulation of expression of this gene by either oxidative stress or sulfydryl reagents.

1990-01-01

82

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

Energy Technology Data Exchange (ETDEWEB)

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.

Bredberg, A.

1981-06-01

83

The effect of topical analgesics on ex vivo skin growth and human keratinocyte and fibroblast behavior.  

Science.gov (United States)

The application of topical analgesics to the donor site of split thickness skin grafts has been proven to be an effective method of pain management but little is known about their effects on wound reepithelialization. This study compares the effect of four analgesics on human keratinocytes and fibroblasts and whole skin explants in vitro to determine whether epithelial cell behavior is affected by topical analgesics. The effect of diclofenac, bupivacaine, lidocaine, and ketorolac was studied at concentrations between 10 mM and 1 nM. The effect on epithelial growth was measured using an ex vivo skin explant model. In addition, cell proliferation, and cytotoxicity were measured in cultured primary human keratinocytes and fibroblasts. Epithelial growth from the explant model was most inhibited by diclofenac with a significant reduction at 100 microM (p=>0.001). Diclofenac also exhibited the strongest inhibitory effect on cell proliferation especially in keratinocytes. Ketorolac was the most cytotoxic. Bupivacaine showed cytotoxicity in a dose-dependent manner with only the very highest concentrations having a significant inhibitory effect. Lidocaine showed no evidence of cytotoxicity at the concentrations tested in either the in vitro cell studies or the ex vivo explant model. Topical analgesics alter keratinocyte and fibroblast behavior and such inhibition may affect wound healing. PMID:19660041

Harris, Kathryn L; Bainbridge, Natalie J; Jordan, Nigel R; Sharpe, Justin R

84

The effect of topical analgesics on ex vivo skin growth and human keratinocyte and fibroblast behavior.  

UK PubMed Central (United Kingdom)

The application of topical analgesics to the donor site of split thickness skin grafts has been proven to be an effective method of pain management but little is known about their effects on wound reepithelialization. This study compares the effect of four analgesics on human keratinocytes and fibroblasts and whole skin explants in vitro to determine whether epithelial cell behavior is affected by topical analgesics. The effect of diclofenac, bupivacaine, lidocaine, and ketorolac was studied at concentrations between 10 mM and 1 nM. The effect on epithelial growth was measured using an ex vivo skin explant model. In addition, cell proliferation, and cytotoxicity were measured in cultured primary human keratinocytes and fibroblasts. Epithelial growth from the explant model was most inhibited by diclofenac with a significant reduction at 100 microM (p=>0.001). Diclofenac also exhibited the strongest inhibitory effect on cell proliferation especially in keratinocytes. Ketorolac was the most cytotoxic. Bupivacaine showed cytotoxicity in a dose-dependent manner with only the very highest concentrations having a significant inhibitory effect. Lidocaine showed no evidence of cytotoxicity at the concentrations tested in either the in vitro cell studies or the ex vivo explant model. Topical analgesics alter keratinocyte and fibroblast behavior and such inhibition may affect wound healing.

Harris KL; Bainbridge NJ; Jordan NR; Sharpe JR

2009-05-01

85

Effects of asiaticoside on the expression of Smad protein by normal skin fibroblasts and hypertrophic scar fibroblasts.  

UK PubMed Central (United Kingdom)

BACKGROUND: The precise mechanism of asiaticoside in molecular and gene expression levels of Smad protein and mRNA still remains unknown. We hypothesised that asiaticoside might inhibit the formation of hypertrophic scarring by affecting the expression of Smad protein and interfering with the Smad signalling pathway. AIMS: To investigate the effects of asiaticoside on the expression of Smad protein by hypertrophic scar fibroblasts (HSFs) and to clarify the mechanism of asiaticoside in scar treatment. METHODS: Normal skin fibroblasts and HSFs were exposed to various concentrations of asiaticoside in serum-free medium for 72 h, then immunocytochemistry was used to examine the localization and expression of phosphorylated Smad2 and Smad7. The expression of Smad protein was studied both at the transcriptional and post-transcriptional levels, using conventional reverse transcription PCR and Western blotting. RESULTS: Asiaticoside markedly enhanced the expression of inhibitory Smad7, but it had no effect on the expression of Smad2. Further study revealed that asiaticoside could induce Smad7 to enter the cytoplasm from the nucleus. CONCLUSIONS: Asiaticoside inhibits scarring probably by enhancing the expression of inhibitory Smad7, and is a potential treatment for scarring.

Qi SH; Xie JL; Pan S; Xu YB; Li TZ; Tang JM; Liu XS; Shu B; Liu P

2008-03-01

86

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

International Nuclear Information System (INIS)

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

1990-07-15

87

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

Energy Technology Data Exchange (ETDEWEB)

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.

Hannan, M.A.; Smith, B.P.; Sigut, D.; Sackey, K. (King Faisal Specialist Hospital and Research Centre, Riyadh (Saudi Arabia))

1990-07-15

88

Upregulation of matrix metalloproteinase 1 and disruption of mitochondrial network in skin fibroblasts of patients with MERRF syndrome.  

UK PubMed Central (United Kingdom)

By using cDNA microarray and RT-PCR techniques, we investigated the genome-wide alteration of gene expression in skin fibroblasts from patients with myoclonic epilepsy and ragged-red fibers (MERRF) syndrome. By screening for the genes with altered levels of expression, we first discovered that matrix metalloproteinase 1 (MMP1) was highly induced in the primary culture of skin fibroblasts of a female patient in a four-generation family with MERRF syndrome. This phenomenon was confirmed in skin fibroblasts from three other MERRF patients harboring about 85% of mtDNA with A8344G mutation. A further study revealed that the expression of MMP1 could be further induced by treatment of the skin fibroblasts with 200 microM hydrogen peroxide (H2O2) and inhibited by 1 mM N-acetylcysteine. Moreover, the intracellular level of H2O2 in skin fibroblasts of the female MERRF patient was higher than those of the asymptomatic family members and age-matched healthy controls. These findings imply that the increase in the expression of MMP1 may represent one of the responses to the increased oxidative stress in the skin fibroblasts of MERRF patients. We suggest that in affected tissues the oxidative stress-elicited overexpression of MMP1, and probably other matrix metalloproteinases involved in cytoskeleton remodeling, may play an important role in the pathogenesis and progression of mitochondrial encephalomyopathies such as MERRF syndrome.

Ma YS; Chen YC; Lu CY; Liu CY; Wei YH

2005-05-01

89

Artificial Skin - Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres  

Science.gov (United States)

Background In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. Methodology/Principal Findings Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross- sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses. Conclusion/Significance Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration.

Reimers, Kerstin; Kuhbier, Joern W.; Schafer-Nolte, Franziska; Allmeling, Christina; Kasper, Cornelia; Vogt, Peter M.

2011-01-01

90

Artificial skin--culturing of different skin cell lines for generating an artificial skin substitute on cross-weaved spider silk fibres.  

UK PubMed Central (United Kingdom)

BACKGROUND: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. METHODOLOGY/PRINCIPAL FINDINGS: Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross-sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses. CONCLUSION/SIGNIFICANCE: Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration.

Wendt H; Hillmer A; Reimers K; Kuhbier JW; Schäfer-Nolte F; Allmeling C; Kasper C; Vogt PM

2011-01-01

91

Human skin keratinocytes, melanocytes, and fibroblasts contain distinct circadian clock machineries.  

Science.gov (United States)

Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin. PMID:22627494

Sandu, Cristina; Dumas, Marc; Malan, André; Sambakhe, Diariétou; Marteau, Clarisse; Nizard, Carine; Schnebert, Sylvianne; Perrier, Eric; Challet, Etienne; Pévet, Paul; Felder-Schmittbuhl, Marie-Paule

2012-05-25

92

Human skin keratinocytes, melanocytes, and fibroblasts contain distinct circadian clock machineries.  

UK PubMed Central (United Kingdom)

Skin acts as a barrier between the environment and internal organs and performs functions that are critical for the preservation of body homeostasis. In mammals, a complex network of circadian clocks and oscillators adapts physiology and behavior to environmental changes by generating circadian rhythms. These rhythms are induced in the central pacemaker and peripheral tissues by similar transcriptional-translational feedback loops involving clock genes. In this work, we investigated the presence of functional oscillators in the human skin by studying kinetics of clock gene expression in epidermal and dermal cells originating from the same donor and compared their characteristics. Primary cultures of fibroblasts, keratinocytes, and melanocytes were established from an abdominal biopsy and expression of clock genes following dexamethasone synchronization was assessed by qPCR. An original mathematical method was developed to analyze simultaneously up to nine clock genes. By fitting the oscillations to a common period, the phase relationships of the genes could be determined accurately. We thereby show the presence of functional circadian machinery in each cell type. These clockworks display specific periods and phase relationships between clock genes, suggesting regulatory mechanisms that are particular to each cell type. Taken together, our data demonstrate that skin has a complex circadian organization. Oscillators are present not only in fibroblasts but also in epidermal keratinocytes and melanocytes and are likely to act in coordination to drive rhythmic functions within the skin.

Sandu C; Dumas M; Malan A; Sambakhe D; Marteau C; Nizard C; Schnebert S; Perrier E; Challet E; Pévet P; Felder-Schmittbuhl MP

2012-10-01

93

Abnormal phenotype of cultured fibroblasts in human skin with chronic radiotherapy damage  

International Nuclear Information System (INIS)

[en] Purpose: The pathophysiological aspects of radiation-induced fibrosis (RIF) have not been well characterized. We therefore cultured human fibroblasts from samples of skin with RIF to investigate the long-term effects of therapeutic irradiation. Materials and methods: Biopsies of normal and RIF skin were obtained from patients previously irradiated for cancer, without recurrence. Cells were extracted from dermis samples by the outgrowth technique, seeded as monolayers and cultured at confluence. Enzyme activities and proteins were assayed, RNA was isolated and Northern blot analysis was performed on surviving cells between passages 2 and 5. Results: RIF cell cultures displayed heterogeneous fibroblasts populations. The initial outgrowth consisted of one-third small cells that floated rapidly, one-third spindle-shaped cells migrating far from the explant to form islets and one-third large pleiomorphic cells. In subsequent subcultures, surviving cells exhibited either myofibroblastic characteristics with a normal proliferative capacity or senescent morphology with a reduced proliferative capacity. These RIF cells had a brief finite lifespan, with dramatically reduced growth rate during their initial outgrowth and the following passages. Study of the antioxidant metabolism showed that Mn superoxide dismutase and catalase activities were significantly weaker in surviving RIF cells than healthy fibroblasts. These exhausted RIF cells exhibited no overexpression of transforming growth factor ? or tissue inhibitor of metalloproteinase. Conclusion: Irradiation may lead to apparently contradictory effects such as fibrosis and necrosis in clinical practice. In cell culture, we observed two main cellular phenotypes which may be related to both processes, i.e. myofibroblast-like cells and fibrocyte-like cells. These two phenotypes may represent two steps in the differentiation induced as a long-term effect of therapeutic irradiation of the skin. Cell culture probably accelerates the induction of the terminal differentiation in RIF fibroblasts. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

1998-06-01

94

Hexavalent chromium-induced alteration of proteomic landscape in human skin fibroblast cells.  

UK PubMed Central (United Kingdom)

Hexavalent chromium [Cr(VI)] generated during industrial processes is carcinogenic. Although much is known about the deleterious effects caused by reactive oxygen species generated during the reduction of Cr(VI) after its absorption by biological systems, the precise mechanisms underlying Cr(VI) cytotoxicity remain poorly defined. Here, we analyzed, at the global proteome scale, the perturbation of protein expression in GM00637 human skin fibroblast cells upon exposure to potassium dichromate (K?Cr?O?). We were able to quantify ?4600 unique proteins, among which ?400 exhibited significant alterations in expression levels upon a 24-h treatment with 0.5 ?M K?Cr?O?. Pathway analysis revealed the Cr(VI)-induced perturbation of cholesterol biosynthesis, G-protein signaling, inflammatory response, and selenoprotein pathways. In particular, we discovered that the K?Cr?O? treatment led to pronouncedly elevated expression of a large number of enzymes involved in de novo cholesterol biosynthesis. Real-time PCR analysis revealed the increased mRNA expression of selected genes involved in cholesterol biosynthesis. Consistently, K?Cr?O? treatment resulted in marked increases in cellular cholesterol level in multiple cell lines. Moreover, the Cr(VI)-induced growth inhibition of cultured human cells could be rescued by a cholesterol-lowering drug, lovastatin. Together, we demonstrated, for the first time, that Cr(VI) may exert its cytotoxic effect, at least partly, through the up-regulation of enzymes involved in de novo cholesterol biosynthesis and the resultant increase of cholesterol level in cells.

Guo L; Xiao Y; Wang Y

2013-07-01

95

Hexavalent chromium-induced alteration of proteomic landscape in human skin fibroblast cells.  

Science.gov (United States)

Hexavalent chromium [Cr(VI)] generated during industrial processes is carcinogenic. Although much is known about the deleterious effects caused by reactive oxygen species generated during the reduction of Cr(VI) after its absorption by biological systems, the precise mechanisms underlying Cr(VI) cytotoxicity remain poorly defined. Here, we analyzed, at the global proteome scale, the perturbation of protein expression in GM00637 human skin fibroblast cells upon exposure to potassium dichromate (K?Cr?O?). We were able to quantify ?4600 unique proteins, among which ?400 exhibited significant alterations in expression levels upon a 24-h treatment with 0.5 ?M K?Cr?O?. Pathway analysis revealed the Cr(VI)-induced perturbation of cholesterol biosynthesis, G-protein signaling, inflammatory response, and selenoprotein pathways. In particular, we discovered that the K?Cr?O? treatment led to pronouncedly elevated expression of a large number of enzymes involved in de novo cholesterol biosynthesis. Real-time PCR analysis revealed the increased mRNA expression of selected genes involved in cholesterol biosynthesis. Consistently, K?Cr?O? treatment resulted in marked increases in cellular cholesterol level in multiple cell lines. Moreover, the Cr(VI)-induced growth inhibition of cultured human cells could be rescued by a cholesterol-lowering drug, lovastatin. Together, we demonstrated, for the first time, that Cr(VI) may exert its cytotoxic effect, at least partly, through the up-regulation of enzymes involved in de novo cholesterol biosynthesis and the resultant increase of cholesterol level in cells. PMID:23718831

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

2013-06-11

96

The chromene sargachromanol E inhibits ultraviolet A-induced ageing of skin in human dermal fibroblasts.  

UK PubMed Central (United Kingdom)

BACKGROUND: Skin ageing is influenced by environmental factors such as ultraviolet (UV) radiation. The effects of UV radiation on skin functions should be investigated using human in vitro models to understand the mechanisms of skin ageing. Additionally, marine algae provide a valuable source for identifying and extracting biologically active substances. OBJECTIVES: In this study, sargachromanol E was isolated from a marine brown alga, Sargassum horneri, and its inhibitory effect on skin ageing was investigated using UVA-irradiated dermal fibroblasts. METHODS: Formation of intracellular reactive oxygen species (ROS), lipid peroxidation and protein oxidation induced by UVA irradiation were investigated in UVA-irradiated human dermal fibroblasts. The levels of matrix metalloproteinases (MMPs) were determined by reverse-transcriptase polymerase chain reaction and Western blot analysis. RESULTS: Sargachromanol E did not exhibit any significant cytotoxicity or phototoxicity in UVA-exposed dermal fibroblasts. Additionally, sargachromanol E suppressed intracellular formation of ROS, membrane protein oxidation, lipid peroxidation and expression of collagenases such as MMP-1, MMP-2 and MMP-9, all of which are caused by UVA exposure. It was further found that these inhibitions were related to an increase in the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP2. Moreover, we have shown that the transcriptional activation of activator protein 1 (AP-1) signalling caused by UVA irradiation was inhibited by treatment with sargachromanol E. CONCLUSIONS: This study suggests that UVA irradiation modulates MMP expression via the transcriptional activation of AP-1 signalling, whereas treatment with sargachromanol E protected cell damage caused by UVA irradiation.

Kim JA; Ahn BN; Kong CS; Kim SK

2013-05-01

97

Skin fibroblasts from a D-deletion type retinoblastoma patient are abnormally X-ray sensitive  

International Nuclear Information System (INIS)

[en] Retinoblastoma is a rare malignant eye tumour that appears either spontaneously or in genetically predisposed persons. The latter group is composed of persons who inherit the tumour with a dominant mode of transmission (the familial type) and those who have a deletion in the long arm of chromosome 13 referred to as the D-deletion type. When this deletion is present it is observed in many somatic cells and is often associated with structural defects. Survivors of the genetic forms of retinoblastoma have an increased risk of the development of cancers at other sites. A single genetic locus is unlikely to predispose many somatic cells to tumour formation unless a fundamental molecular defect, possibly related to DNA repair, is present. In order to investigate this hypothesis a study was made of the in vitro X-ray sensitivity of skin fibroblasts derived from three retinoblastoma patients, comprising a pair of twins with the familial type accompanied by no gross chromosome abnormalities, and a patient with the D-deletion type. It was found that fibroblasts derived from the D-deletion patient were significantly more radiosensitive than those from the other two patients. X-ray survival curves are shown. It is concluded that skin fibroblasts derived from a patient with the D-deletion variant of retinoblastoma are abnormally radiosensitive. Future investigations may indicate a specific defect in molecular repair of DNA that will explain the predisposition of these patients to the development of other tumours. (U.K.)

1977-04-21

98

Effects of subepithelial fibroblasts on epithelial differentiation in human skin and oral mucosa: heterotypically recombined organotypic culture model.  

UK PubMed Central (United Kingdom)

The stratified squamous epithelia differ regionally in their patterns of morphogenesis and differentiation. Although some reports suggested that the adult epithelial phenotype is an intrinsic property of the epithelium, there is increasing evidence that subepithelial connective tissue can modify the phenotypic expression of the epithelium. The aim of this study was to elucidate whether the differentiation of cutaneous and oral epithelia is influenced by underlying mesenchymal tissues. Three normal skin samples and three normal buccal mucosa samples were used for the experiments. Skin equivalents were constructed in four ways, depending on the combinations of keratinocytes (cutaneous or mucosal keratinocytes) and fibroblasts (dermal or mucosal fibroblasts), and the effects of subepithelial fibroblasts on the differentiation of oral and cutaneous keratinocytes were studied with histological examinations and immunohistochemical analyses with anti-cytokeratin (keratins 10 and 13) antibodies. For each experiment, three paired skin equivalents were constructed by using single parent keratinocyte and fibroblast sources for each group; consequently, nine (3 x 3) organotypic cultures per group were constructed and studied. The oral and cutaneous epithelial cells maintained their intrinsic keratin expression. The keratin expression patterns in oral and cutaneous epithelia of skin equivalents were generally similar to their original patterns but were partly modified exogenously by the topologically different fibroblasts. The mucosal keratinocytes were more differentiated and expressed keratin 10 when cocultured with dermal fibroblasts, and the expression patterns of keratin 13 in cutaneous keratinocytes cocultured with mucosal fibroblasts were different from those in keratinocytes cocultured with cutaneous fibroblasts. The results suggested that the epithelial phenotype and keratin expression could be extrinsically modified by mesenchymal fibroblasts. In epithelial differentiation, however, the intrinsic control by epithelial cells may still be stronger than extrinsic regulation by mesenchymal fibroblasts.

Okazaki M; Yoshimura K; Suzuki Y; Harii K

2003-09-01

99

Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

Agmon, V.; Dinur, T.; Cherbu, S.; Dagan, A.; Gatt, S. (Hebrew Univ., Jerusalem (Israel))

1991-10-01

100

The angiogenic factor Cyr61 activates a genetic program for wound healing in human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Cyr61 is a heparin-binding, extracellular matrix-associated protein of the CCN family, which also includes connective tissue growth factor, Nov, WISP-1, WISP-2, and WISP-3. Cyr61 is capable of multiple functions, including induction of angiogenesis in vivo. Purified Cyr61 mediates cell adhesion and induces adhesive signaling, stimulates cell migration, enhances cell proliferation, and promotes cell survival in both fibroblasts and endothelial cells. In this study, we have used cDNA array hybridization to identify genes regulated by Cyr61 in primary human skin fibroblasts. The Cyr61-regulated genes fall into several groups known to participate in processes important for cutaneous wound healing, including: 1) angiogenesis and lymphogenesis (VEGF-A and VEGF-C); 2) inflammation (interleukin-1beta); 3) extracellular matrix remodeling (MMP1, MMP3, TIMP1, uPA, and PAI-1); and 4) cell-matrix interactions (Col1alpha1, Col1alpha2, and integrins alpha(3) and alpha(5)). Cyr61-mediated gene expression requires heparin binding activity of Cyr61, cellular de novo transcription, and protein synthesis and is largely dependent on the activation of p42/p44 MAPKs. Cyr61 regulates gene expression not only in serum-free medium but also in fibroblasts cultured on various matrix proteins or in the presence of 10% serum. These effects of Cyr61 can be sustained for at least 5 days, consistent with the time course of wound healing in vivo. Interestingly, Cyr61 can interact with transforming growth factor-beta1 to regulate expression of specific genes in an antagonistic, additive, or synergistic manner. Furthermore, we show that the Cyr61 gene is highly induced in dermal fibroblasts of granulation tissue during cutaneous wound repair. Together, these results show that Cyr61 is inducibly expressed in granulation tissues after wounding and that Cyr61 activates a genetic program for wound repair in skin fibroblasts. We propose a model in which Cyr61 integrates its activities on endothelial cells, fibroblasts, and macrophages to regulate the processes of angiogenesis, inflammation, and matrix remodeling in the context of cutaneous wound healing.

Chen CC; Mo FE; Lau LF

2001-12-01

 
 
 
 
101

Near-ultraviolet radiation-induced lipid peroxidation and membrane effects in Escherichia coli and human skin fibroblasts  

International Nuclear Information System (INIS)

The first part of this thesis examines the response of an unsaturated fatty acid auxotroph, Escherichia coli K1060 to broad-band near-UV radiation. Sensitivity, lipid peroxidation and leakage of rubidium from irradiated cells were found to increase with increasing unsaturation of membrane fatty acids. The involvement of singlet oxygen was implicated by an increase in sensitivity, lipid peroxidation and leakage of rubidium following irradiation in deuterium oxide. Some factors influencing survival following irradiation were investigated, where lower growth rates were shown to enhance survival. In the second part, the study was extended to human fibroblasts where a normal human skin fibroblast strain, GM730 and a strain derived from an actinic reticuloid patient, AR6LO, are compared. Lipid peroxidation was measured in both cell lines following broad-band near-UV irradiation. Membrane activity, as assessed by the pinocytic uptake of 14C-sucrose and its subsequent release from the cell, was measured. Near-UV irradiation was found to increase such activity in both strains. Vitamin E and Trolox-C were found to decrease this response in AR6LO but not GM730 cells. The final part consists of preliminary investigations into the near-UV induced peroxidation of fatty acids and liposomes, and the subsequent increase in the level of hydroperoxides in the hours following irradiation. (author)

1987-01-01

102

Oxidative stress in skin fibroblasts cultures from patients with Parkinson's disease  

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Full Text Available Abstract Background In the substantia nigra of Parkinson's disease (PD) patients, increased lipid peroxidation, decreased activities of the mitochondrial complex I of the respiratory chain, catalase and glutathione-peroxidase, and decreased levels of reduced glutathione have been reported. These observations suggest that oxidative stress and mitochondrial dysfunction play a role in the neurodegeneration in PD. We assessed enzymatic activities of respiratory chain and other enzymes involved in oxidative processes in skin fibroblasts cultures of patients with PD. Methods We studied respiratory chain enzyme activities, activities of total, Cu/Zn- and Mn-superoxide-dismutase, gluthatione-peroxidase and catalase, and coenzyme Q10 levels in skin fibroblasts cultures from 20 Parkinson's disease (PD) patients and 19 age- and sex- matched healthy controls. Results When compared with controls, PD patients showed significantly lower specific activities for complex V (both corrected by citrate synthase activity and protein concentrations). Oxidized, reduced and total coenzyme Q10 levels (both corrected by citrate synthase and protein concentrations), and activities of total, Cu/Zn- and Mn-superoxide-dismutase, gluthatione-peroxidase and catalase, did not differ significantly between PD-patients and control groups. Values for enzyme activities in the PD group did not correlate with age at onset, duration, scores of the Unified Parkinson's Disease Rating scales and Hoehn-Yahr staging. Conclusions The main result of this study was the decreased activity of complex V in PD patients. This complex synthesizes ATP from ADP using an electrochemical gradient generated by complexes I-IV. These results suggest decreased energetic metabolism in fibroblasts of patients with PD.

del Hoyo Pilar; García-Redondo Alberto; de Bustos Fernando; Molina José; Sayed Youssef; Alonso-Navarro Hortensia; Caballero Luis; Arenas Joaquín; Agúndez José AG; Jiménez-Jiménez Félix

2010-01-01

103

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

International Nuclear Information System (INIS)

[en] 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

1986-01-01

104

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

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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.

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

1986-01-01

105

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

International Nuclear Information System (INIS)

[en] 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 14C-protein hydrolysate, (14C)uridine, and (14C) thymidine. Stimulation was determined by measuring incorporation of (14C)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

1985-01-01

106

Dengue virus infection of human skin fibroblasts in vitro production of IFN-beta, IL-6 and GM-CSF.  

UK PubMed Central (United Kingdom)

Dengue virus is transmitted to humans by the bite of infected mosquitos. In our efforts to understand the pathogenesis of dengue virus infection, we examined whether skin fibroblasts can be infected in vitro with dengue viruses. Fibroblasts could be infected with dengue viruses, yellow fever virus and West Nile virus. Dengue virus antigen-positive cells were detected as early as 4 h and the percentage of dengue virus antigen-positive cells reached maximum levels by 24 h after infection. High titers of infectious dengue virus were also detected in the culture supernatants at 20 h after infection. Dengue virus-infected fibroblasts produced interferon-beta (IFN-beta), and the IFN-beta protected uninfected fibroblasts from dengue virus infection. Dengue virus-infected fibroblasts also produced interleukin 6 (IL-6) and granulocyte macrophage colony stimulation factor (GM-CSF). These results suggest that skin fibroblasts may be one of the cell types which first support dengue virus and other flavivirus infections in vivo after introduction by the bite of infected mosquito, and that production of IFN-beta, IL-6, and GM-CSF by these virus-infected fibroblasts may be important host immune responses to control flavivirus infections.

Kurane I; Janus J; Ennis FA

1992-01-01

107

Interleukin (IL)-19 promoted skin wound healing by increasing fibroblast keratinocyte growth factor expression.  

UK PubMed Central (United Kingdom)

BACKGROUND: Interleukin (IL)-19, a member of the IL-10 cytokine family, is involved in keratinocyte proliferation in psoriasis. OBJECTIVES: We investigated the role of IL-19 in the wound-healing process in vivo and in vitro. METHODS: Two full-thickness circular wounds (4mm in diameter) were punched into the skin of BALB/C mice. IL-19 and keratinocyte growth factor (KGF) mRNA in wounded skin were determined using real-time PCR. The wounds were treated with PBS, vehicle, IL-19 (400ng/mL), or IL-20 (400ng/mL) (n=6 in each group) twice daily and the percentage of wound healing was measured daily for 7days. In vitro, human skin fibroblast CCD966-SK cells and keratinocyte HaCaT cells were treated with IL-19 or KGF. Cell proliferation and migration were determined using bromodeoxyuridine (BrdU) and transwell assays, respectively. The expression of IL-19 and KGF mRNA was also analyzed. RESULTS: In wounded mouse skin, IL-19 mRNA was upregulated at 12h, and KGF at 24h after the injury. Both increases in gene expression declined 72h after the skin had been wounded. The percentage of wound healing in IL-19-treated mice was higher than in control mice. In vitro, IL-19 upregulated KGF expression in the CCD966-SK cells; IL-19 was upregulated in KGF-treated HaCaT cells. KGF but not IL-19 promoted HaCaT cell proliferation. However, IL-19 significantly increased the migration of HaCaT cells. HaCaT cells treated with the cultured supernatants of IL-19-stimulated CCD966-SK cells showed significantly more proliferation than in controls. CONCLUSIONS: IL-19 is important for cutaneous wound healing because it upregulates KGF expression.

Sun DP; Yeh CH; So E; Wang LY; Wei TS; Chang MS; Hsing CH

2013-06-01

108

Responses of human skin in organ culture and human skin fibroblasts to a gadolinium-based MRI contrast agent: comparison of skin from patients with end-stage renal disease and skin from healthy subjects.  

UK PubMed Central (United Kingdom)

OBJECTIVE: Nephrogenic systemic fibrosis is a clinical syndrome occurring in a small subset of patients with end-stage renal disease (ESRD). Exposure to certain of the gadolinium-based contrast agents during magnetic resonance imaging appears to be a trigger. The pathogenesis of the disease is largely unknown. The present study addresses potential pathophysiologic mechanisms. MATERIALS AND METHODS: We have compared responses in organ-cultured skin and skin fibroblasts from individuals with ESRD to responses of healthy control subjects to Omniscan treatment. RESULTS: Treatment of skin from ESRD patients with Omniscan stimulated production of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, but not type I procollagen. The same treatment also stimulated an increase in hyaluronan production. Similar results were seen with skin from normal controls but basal levels were higher in ESRD patients. Fibroblasts in monolayer culture gave the same responses, but there were no differences based on whether the cells were isolated from the skin of healthy subjects or those with ESRD. CONCLUSION: These data indicate that Omniscan exposure alters an enzyme/inhibitor system responsible for regulating collagen turnover in the skin and directly stimulates hyaluronan production. The higher basal levels of type I procollagen, matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1, and hyaluronan in the skin from ESRD patients could contribute to the sensitivity of this patient population to fibrotic changes, which might be induced by exposure to some of the gadolinium-based contrast agents.

DaSilva M; O'Brien Deming M; Fligiel SE; Dame MK; Johnson KJ; Swartz RD; Varani J

2010-11-01

109

Cell fusion corrects the 4-nitroquinoline 1-oxide sensitivity of Werner syndrome fibroblast cell lines.  

UK PubMed Central (United Kingdom)

We have shown that Werner syndrome (WRN) fibroblast cell lines are unusually sensitive to the DNA-damaging agent 4-nitroquinoline 1-oxide (4NQO), though not to gamma radiation or to hydrogen peroxide. The fusion of 4NQO-sensitive WRN and 4NQO-resistant control fibroblast cell lines generated proliferating WRN x control cell hybrids that expressed WRN protein and were 4NQO-resistant. These results establish the recessive nature of 4NQO sensitivity in WRN cell lines and provide a cellular assay for WRN protein function.

Prince PR; Ogburn CE; Moser MJ; Emond MJ; Martin GM; Monnat RJ Jr

1999-07-01

110

Cerebrohepatorenal Syndrome (CHRS) of Zellweger: lysosomal enzyme activities, sulfation of glycosaminoglycans, and pipecolic acid levels in cultured skin fibroblasts  

International Nuclear Information System (INIS)

The defect in the cerebrohepatorenal syndrome (CHRS), a fatal hereditary disorder primarily affecting neurological development, is unknown. Three areas were studied for specific biochemical abnormalities which might aid in diagnosis and understanding of the disorder: (1) Clinico-pathological similarities to inherited degenerative neurologic disorders suggested decreased activity of certain lysosomal enzymes. Assays of ?-galactosidase, ?-hexosaminidase, ?-mannosidase, and arylsulfatase A activities in fibroblasts from four infants with CHRS indicated no deficiency of enzyme activities. (2) Undersulfation of glycosaminoglycans (GAGs) has been reported in patients with the clinically similar Lowe's syndrome. The rate and amount of incorporation of 35SO4 = into intracellular 35S-GAGs up to 48 hours was comparable in fibroblasts from six CHRS infants and controls. Loss of 35-GAGs also followed a normal pattern. (3) Because pipecolic acid (PA) has been reported to be elevated in body fluids of patients with CHRS, cultured skin fibroblasts were examined for such an abnormality. Lysosomal enzyme activities and metabolism of sulfated glycosaminoglycans appear to be normal in cultured skin fibroblasts from infants with CHRS. Despite the sensitivity of the method, examination of pipecolic acid in cultured skin fibroblasts does not seem to be useful for diagnosis of CHRS.

1985-01-01

111

Cerebrohepatorenal Syndrome (CHRS) of Zellweger: lysosomal enzyme activities, sulfation of glycosaminoglycans, and pipecolic acid levels in cultured skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

The defect in the cerebrohepatorenal syndrome (CHRS), a fatal hereditary disorder primarily affecting neurological development, is unknown. Three areas were studied for specific biochemical abnormalities which might aid in diagnosis and understanding of the disorder: (1) Clinico-pathological similarities to inherited degenerative neurologic disorders suggested decreased activity of certain lysosomal enzymes. Assays of ..beta..-galactosidase, ..beta..-hexosaminidase, ..cap alpha..-mannosidase, and arylsulfatase A activities in fibroblasts from four infants with CHRS indicated no deficiency of enzyme activities. (2) Undersulfation of glycosaminoglycans (GAGs) has been reported in patients with the clinically similar Lowe's syndrome. The rate and amount of incorporation of /sup 35/SO/sub 4/ = into intracellular /sup 35/S-GAGs up to 48 hours was comparable in fibroblasts from six CHRS infants and controls. Loss of /sup 35/-GAGs also followed a normal pattern. (3) Because pipecolic acid (PA) has been reported to be elevated in body fluids of patients with CHRS, cultured skin fibroblasts were examined for such an abnormality. Lysosomal enzyme activities and metabolism of sulfated glycosaminoglycans appear to be normal in cultured skin fibroblasts from infants with CHRS. Despite the sensitivity of the method, examination of pipecolic acid in cultured skin fibroblasts does not seem to be useful for diagnosis of CHRS.

Johnson, E.C.P.

1985-01-01

112

Regulation and inhibition of collagenase expression by long-wavelength ultraviolet radiation in cultured human skin fibroblasts  

International Nuclear Information System (INIS)

[en] The cellular mechanisms responsible for the connective tissue changes produced by chronic exposure to UV light are poorly understood. collagenase, a metalloproteinase, initiates degradation of types I and III collagen and thus plays a key role in the remodeling of dermal collagen. Collagenase synthesis by fibroblasts and keratinocytes involves the protein kinase C (PKC) second messenger system, and corticosteroids have been shown to suppress its synthesis at the level of gene transcription. Long-wavelength UV light (UVA, 320-400 nm) stimulates the synthesis of interstitial collagenase, as well as increasing PKC activity, in human skin fibroblasts in vitro. This study explores the regulation of collagenase expression by UVA in cultured human skin fibroblasts. Specifically, the time course, the effect of actinomycin D, an inhibitor of RNA synthesis, as well as the effect of PKC inhibitors and dexamethansone on expression of collagenase following UVA irradiation were examined. (Author)

1995-01-01

113

Regulation and inhibition of collagenase expression by long-wavelength ultraviolet radiation in cultured human skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

The cellular mechanisms responsible for the connective tissue changes produced by chronic exposure to UV light are poorly understood. collagenase, a metalloproteinase, initiates degradation of types I and III collagen and thus plays a key role in the remodeling of dermal collagen. Collagenase synthesis by fibroblasts and keratinocytes involves the protein kinase C (PKC) second messenger system, and corticosteroids have been shown to suppress its synthesis at the level of gene transcription. Long-wavelength UV light (UVA, 320-400 nm) stimulates the synthesis of interstitial collagenase, as well as increasing PKC activity, in human skin fibroblasts in vitro. This study explores the regulation of collagenase expression by UVA in cultured human skin fibroblasts. Specifically, the time course, the effect of actinomycin D, an inhibitor of RNA synthesis, as well as the effect of PKC inhibitors and dexamethansone on expression of collagenase following UVA irradiation were examined. (Author).

Petersen, Marta; Hamilton, Tiffani; Haili Li [Utah Univ., Salt Lake City, UT (United States). Dept. of Internal Medicine

1995-09-01

114

Human breast milk stimulates prostaglandin synthesis in cultured human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Effect of human breast milk or its fractions on prostaglandin synthesis was investigated in cultured human skin fibroblasts. Prostaglandins released into the media were measured by radioimmunoassay. Incorporation of breast milk (2% level) into 10% fetal calf serum media (for 48 hours) stimulated the synthesis of 6-keto-PGF1 alpha (stable product of prostacyclin) by 800%. This stimulating effect of milk persisted after cold acetone extraction to remove phospholipids and potentiated further after dialysis. Stimulation by one of the commercial formulas (Similac) was less than 50% of the milk effect. Milk also stimulated PGE2 synthesis, although to a much lesser degree. These studies show for the first time that a) human breast milk contains potent factor(s) capable of influencing prostaglandin synthesis and suggest that b) these factors might have a role in the development of lipid synthetic pathways during early life.

Subbiah MT; Yunker RL; Yamamoto M; Kottke BA; Bale LK

1985-06-01

115

N-Methyl-L-serine stimulates hyaluronan production in human skin fibroblasts.  

UK PubMed Central (United Kingdom)

We examined the effects of N-methyl-L-serine (NMS), an amino acid derivative, on hyaluronan (HA) synthesis in human skin fibroblasts. NMS (1-10 mM), but not L-serine, stimulated the incorporation of [(3)H]glucosamine into HA dose-dependently, with a maximum stimulation of 1.5-fold compared to the control. The effect of NMS was specific for HA production, because there was no change in sulfated glycosaminoglycan formation. Neither the N-methyl derivatives of L-glycine or L-alanine, nor N-methyl-D-serine, could stimulate HA synthesis, indicating that the beta-hydroxyl group and the L-configuration were essential for the activity. Gel filtration of the products showed that NMS stimulated the production of high-molecular-mass HA (>10(6) D) without affecting the production of low-molecular-mass HA. NMS required 24 h to stimulate HA production, and when fibroblasts were pretreated for 10-24 h with NMS (1-10 mM), membrane-associated HA synthase activity was increased dose-dependently. Thus, a second messenger is likely to be involved in the stimulation of HA production by NMS.

Sakai S; Sayo T; Kodama S; Inoue S

1999-09-01

116

[Expressions of fibroblast activation protein during skin scald burn healing in rats].  

UK PubMed Central (United Kingdom)

OBJECTIVE: To investigate the time course of changes in the expression of fibroblast activation protein (FAP) during healing of skin scald burns in rats. METHODS: Adult Wistar rats were randomized into two equal groups (n=42) and subject to superficial second degree and deep second degree scald burns on the dorsal skin groups, with 6 normal rats serving as the control group. At 6 h, 12 h, and 1, 3, 7, 14, and 21 days after burns, 6 rats from each group were sacrificed to detect FAP expression by immunohistochemistry and Western blotting. RESULTS: FAP was expressed on the cell membrane and in the cytoplasm of the fibroblasts, especially those around the neovessels. In both burn groups, FAP expression increased significantly at 6 h after burns. In superficial burn group, FAP expression was comparable between 6 and 12 h and between 1 and 3 days (P>0.05), but showed significant differences between the other time points (P<0.05). In deep burn group, FAP expression was comparable between 12 h, 1 day and 3 days (P>0.05) but differed significantly between the other time points (P<0.05). In both burn groups, FAP expression reached the peak level at 7 days followed by a gradual declination. At 21 days after the burns, FAP maintained a significantly higher expression level than the control level (P<0.05). CONCLUSION: The time course of the changes of FAP expression following scald burns suggests an important role of FAP in the healing process of scald burns.

Jing G; Chen J; Wang J

2013-04-01

117

Cultured skin keratinocytes from both normal individuals and basal cell naevus syndrome patients are more resistant to ?-rays and UV light compared with cultured skin fibroblasts  

International Nuclear Information System (INIS)

Measurement of colony-forming ability following exposure to ?-rays was performed on cultured skin keratinocytes and skin fibroblasts from normal individuals, basal cell naevus syndrome patients (BCNS) and ataxia telangiectasia (A-T) patients. The most striking observation was the radiation resistance of 8/8 keratinocyte strains compared with fibroblasts whether from BCNS patients or normals. The single A-T keratinocyte cell strain showed the same radiosensitivity as A-T fibroblast cell strains. The differential survival of keratinocytes and fibroblasts was also observed following exposure to 254 nm UV light. Survival curves of SV40 immortalized keratinocytes and retinoblasts derived from normal individuals or BCNS patients showed large shoulder regions following exposure to ?-rays or 254 nm UV light. An increased D37 rather than an increased Do was therefore the feature of such curves, contrasting with SV40 immortalized A-T keratinocytes or fibroblasts which showed a minimal shoulder effect and an increased Do. No difference in survival was observed between BCNS and normal cells following exposure to either UV or ?-rays. (author).

1989-01-01

118

Cultured skin keratinocytes from both normal individuals and basal cell naevus syndrome patients are more resistant to. gamma. -rays and UV light compared with cultured skin fibroblasts  

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Measurement of colony-forming ability following exposure to {gamma}-rays was performed on cultured skin keratinocytes and skin fibroblasts from normal individuals, basal cell naevus syndrome patients (BCNS) and ataxia telangiectasia (A-T) patients. The most striking observation was the radiation resistance of 8/8 keratinocyte strains compared with fibroblasts whether from BCNS patients or normals. The single A-T keratinocyte cell strain showed the same radiosensitivity as A-T fibroblast cell strains. The differential survival of keratinocytes and fibroblasts was also observed following exposure to 254 nm UV light. Survival curves of SV40 immortalized keratinocytes and retinoblasts derived from normal individuals or BCNS patients showed large shoulder regions following exposure to {gamma}-rays or 254 nm UV light. An increased D{sub 37} rather than an increased D{sub o} was therefore the feature of such curves, contrasting with SV40 immortalized A-T keratinocytes or fibroblasts which showed a minimal shoulder effect and an increased D{sub o}. No difference in survival was observed between BCNS and normal cells following exposure to either UV or {gamma}-rays. (author).

Stacey, M.; Thacker, S.; Taylor, A.M.R. (Birmingham Univ. (UK). Cancer Research Campaign Labs.)

1989-07-01

119

Radiation-induced comet-formation in human skin fibroblasts from radiotherapy patients with different normal tissue reactions  

International Nuclear Information System (INIS)

Background: In clinical radiotherapy most patients tolerate the applied dosage with no or moderate side effects. However, 5 to 10% of all individuals show increased acute and/or late reactions. In-vitro test systems are investigated for their suitability for predictive purposes. This paper attempts a correlation between the induction and repair of DNA damage measured in the comet assay and the clinical observed reaction in order to evaluate the suitability of the comet assay for prediction of radiation sensitivity. Patients and methods: Skin fibroblasts of 30 patients with average tissue reactions or acute and/or late increased side effects and cell lines of 4 individuals carrying the heritable disease ataxia telangiectasia (AT) were irradiated in vitro. The induction and repair of DNA damage was measured at different time points after irradiation in the comet assay (single cell gel electrophoresis). These results were compared to the acute and late clinical reactions classified according to the RTOG grading system. Results: The radiation induced DNA damage decreased over time reflecting DNA repair. Cells of the AT individuals showed and elevated damage induction and a reduced repair capacity compared to patients with average tissue reactions. Fibroblast of patients with increased acute and late side effects exhibited slower DNA repair. In addition to the known lack of cell cycle control, our results indicate that AT cells show reduced DNA repair capacity. Conclusions: The comet assay seems to be able to detect some types of increased individual radiation sensitivity. In contrast to other predictive in-vitro tests, the comet assay needs less time and fewer cells, which would be useful in a clinical setting. (orig.)

1999-01-01

120

Protective effect of chromene isolated from Sargassum horneri against UV-A-induced damage in skin dermal fibroblasts.  

Science.gov (United States)

Skin homoeostasis is interrupted during UV-A irradiation. How the UV-A-altered skin components influences photoageing of skin should be investigated using human in vitro models that are important for understanding skin ageing. In this study, chromene compound, sargachromenol, was isolated from Sargassum horneri, and its potency on inhibition of photoageing was investigated in UV-A-irradiated dermal fibroblasts. Effects of sargachromenol on the prevention of photoageing were evaluated by measuring ROS production, membrane protein oxidation, lipid peroxidation and ageing-related gene expression in UV-A-irradiated human skin dermal fibroblasts. The results indicated that treatment with sargachromenol suppressed the collagenase matrix metalloproteinases (MMPs), MMP-1, MMP-2 and MMP-9 expression without any cytotoxicity and phototoxicity. It was further found that these inhibitions were because of increase in the expression of TIMP-1 and TIMP-2 genes. Furthermore, we confirmed that the UV-A-induced transcriptions of AP-1 signalling pathway were regulated by sargachromenol treatment in UV-A-irradiated dermal fibroblasts. PMID:22775999

Kim, Jung-Ae; Ahn, Byul-Nim; Kong, Chang-Suk; Kim, Se-Kwon

2012-08-01

 
 
 
 
121

Protective effect of chromene isolated from Sargassum horneri against UV-A-induced damage in skin dermal fibroblasts.  

UK PubMed Central (United Kingdom)

Skin homoeostasis is interrupted during UV-A irradiation. How the UV-A-altered skin components influences photoageing of skin should be investigated using human in vitro models that are important for understanding skin ageing. In this study, chromene compound, sargachromenol, was isolated from Sargassum horneri, and its potency on inhibition of photoageing was investigated in UV-A-irradiated dermal fibroblasts. Effects of sargachromenol on the prevention of photoageing were evaluated by measuring ROS production, membrane protein oxidation, lipid peroxidation and ageing-related gene expression in UV-A-irradiated human skin dermal fibroblasts. The results indicated that treatment with sargachromenol suppressed the collagenase matrix metalloproteinases (MMPs), MMP-1, MMP-2 and MMP-9 expression without any cytotoxicity and phototoxicity. It was further found that these inhibitions were because of increase in the expression of TIMP-1 and TIMP-2 genes. Furthermore, we confirmed that the UV-A-induced transcriptions of AP-1 signalling pathway were regulated by sargachromenol treatment in UV-A-irradiated dermal fibroblasts.

Kim JA; Ahn BN; Kong CS; Kim SK

2012-08-01

122

Immortalization of four new Fanconi anemia fibroblast cell lines by an improved procedure.  

UK PubMed Central (United Kingdom)

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D.

Jakobs PM; Sahaayaruban P; Saito H; Reifsteck C; Olson S; Joenje H; Moses RE; Grompe M

1996-03-01

123

Immortalization of four new Fanconi anemia fibroblast cell lines by an improved procedure.  

Science.gov (United States)

Fanconi anemia (FA) is an autosomal recessive disease characterized by birth defects, progressive bone marrow failure and increased risk for leukemia. FA cells display chromosome breakage and increased cell killing in response to DNA crosslinking agents. At least 5 genes have been defined by cell complementation studies, but only one of these, FAC has been cloned to date. Efforts to map and isolate new FA genes by functional complementation have been hampered by the lack of immortalized FA fibroblast cell lines. Here we report the use of a novel immortalization strategy to create 4 new immortalized FA fibroblast lines, including one from the rare complementation group D. PMID:8782494

Jakobs, P M; Sahaayaruban, P; Saito, H; Reifsteck, C; Olson, S; Joenje, H; Moses, R E; Grompe, M

1996-03-01

124

Cytotoxicity in human skin fibroblasts induced by photoactivated polycyclic aromatic hydrocarbons  

Energy Technology Data Exchange (ETDEWEB)

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.

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

1980-01-01

125

Cell chip-based monitoring of toxic effects of cosmetic compounds on skin fibroblast cells.  

UK PubMed Central (United Kingdom)

The present study estimated the efficacy of electrochemical detection of imidazolidinyl urea-induced cell toxicity in skin human fibroblast cells (HFF cells). The gold nanopunct structures were fabricated through a nanoporous alumina mask, and the structural formations were confirmed via scanning electron microscopy. The HFF cells were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to skin cells attached to the chip surface. The HFF cells evidenced inflammation responses to allergens such as imidazolidinyl urea. The cells were subsequently treated with different concentrations of imidazolidinyl urea for 24 h in culture, which induced a change in the cyclic voltammetry (CV) current peak. Treatment with imidazolidinyl urea induced a loss of cell viability and accelerated inflammation in a concentration-dependent manner. The expression level of inflammation-related proteins such as IL-1 beta were increased in imidazolidinyl urea-treated cells. The CV results demonstrated that imidazolidinyl urea significantly reduced the current peaks in a dose-dependent manner. The results showed that the current peak was reduced in accordance with the increases in imidazolidinyl urea-induced inflammation. In conclusion, the results of this study suggest that the electrochemical-based chip provides crucial information for improvements to a cell chip system for drug screening applications.

An JH; Lee JS; Chun JR; Oh BK; Kafi MD; Choi JW

2012-07-01

126

Cell chip-based monitoring of toxic effects of cosmetic compounds on skin fibroblast cells.  

Science.gov (United States)

The present study estimated the efficacy of electrochemical detection of imidazolidinyl urea-induced cell toxicity in skin human fibroblast cells (HFF cells). The gold nanopunct structures were fabricated through a nanoporous alumina mask, and the structural formations were confirmed via scanning electron microscopy. The HFF cells were allowed to attach to RGD (Arg-Gly-Asp) peptide nanopatterned surfaces, and electrochemical tools were applied to skin cells attached to the chip surface. The HFF cells evidenced inflammation responses to allergens such as imidazolidinyl urea. The cells were subsequently treated with different concentrations of imidazolidinyl urea for 24 h in culture, which induced a change in the cyclic voltammetry (CV) current peak. Treatment with imidazolidinyl urea induced a loss of cell viability and accelerated inflammation in a concentration-dependent manner. The expression level of inflammation-related proteins such as IL-1 beta were increased in imidazolidinyl urea-treated cells. The CV results demonstrated that imidazolidinyl urea significantly reduced the current peaks in a dose-dependent manner. The results showed that the current peak was reduced in accordance with the increases in imidazolidinyl urea-induced inflammation. In conclusion, the results of this study suggest that the electrochemical-based chip provides crucial information for improvements to a cell chip system for drug screening applications. PMID:22966535

An, Jeung Hee; Lee, Jin-Sung; Chun, Je-Ran; Oh, Byung-Keun; Kafi, M D Abdul; Choi, Jeong-Woo

2012-07-01

127

Neutron skin of nuclei near the neutron drip line  

International Nuclear Information System (INIS)

[en] Performing Skyrme-type deformed Hartree-Fock calculations, the possible presence of neutron skin in nuclei towards neutron drip line is studied. The thickness of the neutron skin is found to be nearly constant in all directions if it is measured perpendicular to the surface, and in a given nucleus the number of neutrons being inside of the neutron skin is almost independent of the deformation (namely, spherical shape or normal deformation or superdeformation). In the region of medium-heavy nuclei our calculation shows the presence of a series of neutron-rich nuclei, in which a neutron skin is present and yet the neutron one-particle spectra are far from those in a harmonic oscillator (plus spin-orbit) potential

1995-01-01

128

Topical treatment with basic fibroblast growth factor promotes wound healing and barrier recovery induced by skin abrasion.  

UK PubMed Central (United Kingdom)

It has been reported that basic fibroblast growth factor (bFGF) promotes the healing of skin ulceration by inducing fibroblast proliferation, yet the role of bFGF on epidermal barrier function, especially from the perspective of scratch-induced skin abrasion, remains unknown. To this end, we initially developed an epidermal abrasion mouse model induced by scratching with a stainless-steel wire brush, and examined the effects of bFGF on the wound healing induced by skin abrasion. This procedure induced a significant elevation of transepidermal water loss (TEWL) in a scratch-count-dependent manner. This elevated TEWL was significantly decreased following topical application of bFGF to the skin. In addition, bFGF increased the expression of Ki67 in keratinocytes following mechanical scratching. These results suggest that bFGF enhances keratinocyte proliferation, which, in turn, repairs the skin barrier disruption and wounds caused by scratching in mice. Consistently, bFGF stimulated proliferation of normal human epidermal keratinocytes (NHEK). Intriguingly, the effect of bFGF and other growth factors on NHEK proliferation was additive. However, high cell density diminished the effect of bFGF on NHEK proliferation. This particular result can be explained by our observation that FGF receptor mRNA expression in NHEK was low under conditions of high cell density. Our findings suggest that bFGF stimulates keratinocyte proliferation, especially in a lower cell density environment, to repair skin wound in accord with skin barrier recovery.

Nakamizo S; Egawa G; Doi H; Natsuaki Y; Miyachi Y; Kabashima K

2013-01-01

129

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

Science.gov (United States)

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. PMID:18838164

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

2008-10-05

130

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

UK PubMed Central (United Kingdom)

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.

El Ghalbzouri A; Commandeur S; Rietveld MH; Mulder AA; Willemze R

2009-01-01

131

Up-regulation of putative hyaluronan synthase mRNA by basic fibroblast growth factor and insulin-like growth factor-1 in human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Recently, cDNAs for the three putative human hyaluronan synthase (HAS) genes, HAS1, HAS2 and HAS3, have been cloned. In this study we investigated the effects of basic fibroblast growth factor (bFGF) and insulin-like growth factor-1 (IGF-1) on the expression of HAS genes in cultured skin fibroblasts. Northern blot analyses showed that treatment of fibroblasts with bFGF enhanced the mRNA levels of all three genes. HAS2 gene expression showed the strongest up-regulation with a more than 10-fold increase at 50 ng/ml of bFGF. bFGF also increased hyaluronan production. Incubation of fibroblasts with IGF-1 increased HAS1, HAS2, and HAS3 mRNA levels, as well as hyaluronan production. Our results suggest that up-regulation of the HAS genes by bFGF and IGF-1 is closely associated with the stimulation of hyaluronan synthesis, and that effects of growth factors on HAS gene expression may have important implications for tissue remodeling, such as in development and wound healing.

Kuroda K; Utani A; Hamasaki Y; Shinkai H

2001-06-01

132

Relationship between the in vitro radiosensitivity of skin fibroblasts and the expression of subcutaneous fibrosis, telangiectasia, and skin erythema after radiotherapy  

International Nuclear Information System (INIS)

Objective: To investigate if the occurrence of subcutaneous fibrosis after radiotheraphy in an unselected group of breast cancer patients is related to cellular radiosensitivity of skin fibroblasts as measured in a clonogenic assay. Materials and methods: An in vitro colony-forming assay of normal fibroblast radiosensitivity was applied to primary skin biopsies from 31 breast cancer patients who received post-mastectomy radiotherapy with large doses per fraction (2.7-3.9 Gy) more than 10 years earlier. Three clinical normal-tissue endpoints were assessed. Two late endpoints, subcutaneous fibrosis and telangiectasia, were evaluated in three treatments fields by a single experienced clinician. In addition, skin erythema had been assessed at the end of treatment by members of the staff and junior staff. >From previous analyses of normal tissue response, individual clinical radiosensitivity could be assessed as 'excess risk' of each of the three reactions. This was defined as the difference between the actual observed response in the patient and the expected response estimated from individual treatment characteristics in a linear quadratic (LQ) mixture model and, for the two late endpoints, with correction for the follow-up time. This clinical radioresponsiveness was compared with the in vitro radiosensitivity of the skin fibroblasts. To this end, the fractions of colony-forming cells after graded single doses were fitted by an LQ survival curve using non-linear and linear regression from which the surviving fraction at 3.5 Gy (SF3.5) was estimated. Assessment at 3.5 Gy was chosen to reflect the fraction size during clinical radiotherapy. Results: A statistically significant variability of in vitro radiosensitivity between patients could be detected for both SF2 (P = 0.0095) and SF3.5 (P = 0.0008). A significant correlation was observed between SF3.5 and excess risk of fibrosis (rs -0.46, P = 0.009) while no association was found between fibroblast radiosensitivity and either the occurrence of severe skin telangiectasia or the acute endpoint skin erythema. Conclusion: These results suggest that variability in the occurrence of subcutaneous fibrosis, but not telangiectasia or erythema, after radiotherapy is partly accounted for by differences in cellular radiosensitivity of normal skin fibroblasts.

1996-01-01

133

The influence of LTS-4, a saponoside from Lysimachia thyrsiflora L., on human skin fibroblasts and human melanoma cells.  

Science.gov (United States)

We investigated the effect of a triterpene saponoside from Lysimachia thyrsiflora L. upon the viability, proliferation, morphology and cell motility of human melanoma HTB-140 cells and human skin fibroblasts (HSFs). The compound, denoted LTS-4, decreased the viability and rate of cell growth of both cell types in a time-and dose-dependent manner, and proved cytotoxic against cancer cells at significantly lower concentrations than for fibroblasts. LTS-4 also affected the morphology of the examined cells, causing vacuolisation and actin cytoskeleton disintegration, and had an inhibitory effect on the tumour cell motility. PMID:18553182

Galanty, Agnieszka; Michalik, Marta; Sedek, Lukasz; Podolak, Irma

2008-06-13

134

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

UK PubMed Central (United Kingdom)

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 ?m). 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 ?m 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.

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

2013-04-01

135

[Infection of skin fibroblasts in animals with different levels of sensitivity to Leishmania infantum and Leishmania mexicana (Kinetoplastida: Trypanosomatidae)].  

UK PubMed Central (United Kingdom)

Infection and multiplication of Leishmania infantum and L. mexicana inside of skin fibroblasts from hamsters, mice and rats was achieved. This process was demonstrated either by counting parasites inside the stained cells or by electronic microscopy studies. In addition multiplication rate differences in the cells from these rodent species were determined, for L. infantum as well as for L. mexicana. Parasite development in hamsters and mice fibroblasts was evident but there was not multiplication in rat cells showing that apparently they are refractory to Leishmania infection. These results suggest that the parasite affinity for each animal, as well as any intracellular environment resistance, could involve genetic factors in the parasite multiplication. On the other hand, presence of amastigote multiplication inside of parasitophorus vacuole, showed by electronic microscopy images, probes a true parasite transformation. Therefore it is suggested that fibroblasts could work as host cells for parasite survival and permanency in the infected animals.

Minero MA; Chinchilla M; Guerrero OM; Castro A

2004-03-01

136

Autophagic-lysosomal dysregulation downstream of cathepsin B inactivation in human skin fibroblasts exposed to UVA.  

Science.gov (United States)

Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and ?-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, ?-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage. PMID:21773629

Lamore, Sarah D; Wondrak, Georg T

2011-07-20

137

Autophagic-lysosomal dysregulation downstream of cathepsin B inactivation in human skin fibroblasts exposed to UVA.  

UK PubMed Central (United Kingdom)

Recently, using 2D-DIGE proteomics we have identified cathepsin B as a novel target of UVA in human Hs27 skin fibroblasts. In response to chronic exposure to noncytotoxic doses of UVA (9.9 J cm(-2), twice a week, 3 weeks), photooxidative impairment of cathepsin B enzymatic activity occurred with accumulation of autofluorescent aggregates colocalizing with lysosomes, an effect mimicked by pharmacological antagonism of cathepsin B using the selective inhibitor CA074Me. Here, we have further explored the mechanistic involvement of cathepsin B inactivation in UVA-induced autophagic-lysosomal alterations using autophagy-directed PCR expression array analysis as a discovery tool. Consistent with lysosomal expansion, UVA upregulated cellular protein levels of the lysosomal marker glycoprotein Lamp-1, and increased levels of the lipidated autophagosomal membrane constituent LC3-II were detected. UVA did not alter expression of beclin 1 (BECN1), an essential factor for initiation of autophagy, but upregulation of p62 (sequestosome 1, SQSTM1), a selective autophagy substrate, and ?-synuclein (SNCA), an autophagic protein substrate and aggresome component, was observed at the mRNA and protein level. Moreover, UVA downregulated transglutaminase-2 (TGM2), an essential enzyme involved in autophagolysosome maturation. Strikingly, UVA effects on Lamp-1, LC3-II, beclin 1, p62, ?-synuclein, and transglutaminase-2 were mimicked by CA074Me treatment. Taken together, our data suggest that UVA-induced autophagic-lysosomal alterations occur as a consequence of impaired autophagic flux downstream of cathepsin B inactivation, a novel molecular mechanism potentially involved in UVA-induced skin photodamage.

Lamore SD; Wondrak GT

2012-01-01

138

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

UK PubMed Central (United Kingdom)

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.

Chen SK; Hsu CH; Tsai ML; Chen RH; Drummen GP

2013-01-01

139

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

Science.gov (United States)

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. PMID:24071940

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

2013-09-25

140

Regulation of fibroblast gene expression by keratinocytes in organotypic skin culture provides possible mechanisms for the antifibrotic effect of reepithelialization.  

Science.gov (United States)

To investigate the mechanisms behind the antifibrotic effect associated with epidermal regeneration, the expression of 12 fibroblast genes important for the modulation of the extracellular matrix (ECM), as well as ?-smooth muscle actin, was studied in a keratinocyte-fibroblast organotypic skin culture model. The study was performed over time during epidermal generation and in the presence or absence of the profibrotic factor transforming growth factor-?. the Presence of epidermal differentiation markers in the model was essentially coherent with that of native skin. Fibroblast gene expression was analyzed with real-time polymerase chain reaction after removal of the epidermal layer. After 2 days of air-exposed culture, 11 out of the 13 genes studied were significantly regulated by keratinocytes in the absence or presence of transforming growth factor-?. The regulation of connective tissue growth factor, collagen I and III, fibronectin, plasmin system regulators, matrix metalloproteinases and their inhibitors as well as ?-smooth muscle actin was consistent with a suppression of ECM formation or contraction. Overall, the results support a view that keratinocytes regulate fibroblasts to act catabolically on the ECM in epithelialization processes. This provides possible mechanisms for the clinical observations that reepithelialization and epidermal wound coverage counteract excessive scar formation. PMID:20731800

Koskela, Anita; Engström, Kristina; Hakelius, Malin; Nowinski, Daniel; Ivarsson, Mikael

2010-08-19

 
 
 
 
141

Regulation of fibroblast gene expression by keratinocytes in organotypic skin culture provides possible mechanisms for the antifibrotic effect of reepithelialization.  

UK PubMed Central (United Kingdom)

To investigate the mechanisms behind the antifibrotic effect associated with epidermal regeneration, the expression of 12 fibroblast genes important for the modulation of the extracellular matrix (ECM), as well as ?-smooth muscle actin, was studied in a keratinocyte-fibroblast organotypic skin culture model. The study was performed over time during epidermal generation and in the presence or absence of the profibrotic factor transforming growth factor-?. the Presence of epidermal differentiation markers in the model was essentially coherent with that of native skin. Fibroblast gene expression was analyzed with real-time polymerase chain reaction after removal of the epidermal layer. After 2 days of air-exposed culture, 11 out of the 13 genes studied were significantly regulated by keratinocytes in the absence or presence of transforming growth factor-?. The regulation of connective tissue growth factor, collagen I and III, fibronectin, plasmin system regulators, matrix metalloproteinases and their inhibitors as well as ?-smooth muscle actin was consistent with a suppression of ECM formation or contraction. Overall, the results support a view that keratinocytes regulate fibroblasts to act catabolically on the ECM in epithelialization processes. This provides possible mechanisms for the clinical observations that reepithelialization and epidermal wound coverage counteract excessive scar formation.

Koskela A; Engström K; Hakelius M; Nowinski D; Ivarsson M

2010-09-01

142

Radiosensitivity of skin fibroblasts from atomic bomb survivors with and without breast cancer.  

UK PubMed Central (United Kingdom)

Fibroblasts were established in vitro from skin biopsies obtained from 55 women and 1 man with or without breast cancer and with or without exposure to radiation from the atomic bomb explosion in Hiroshima. The radiosensitivity of these cells was evaluated by clonogenic assays after exposure to X-rays or to fission neutrons from a 252Cf source. Data were fitted to a multitarget model, S/S0 = A [1 - (1 - ekD)N], for both X-ray and neutron dose-survival curves. A single hit model, S/S0 = AekD, fits the neutron dose-survival responses as well. There were no differences in the means or variances of radiosensitivity between exposed and nonexposed groups or between patients with or without breast cancer. Hence, although the sample is not large, it provides no support for the hypothesis that atomic bomb radiation preferentially induces breast cancer in women whose cells in vitro are sensitive to cell killing by radiation.

Ban S; Setlow RB; Bender MA; Ezaki H; Hiraoka T; Yamane M; Nishiki M; Dohi K; Awa AA; Miller RC

1990-07-01

143

Radiosensitivity of skin fibroblasts from atomic bomb survivors with and without breast cancer  

International Nuclear Information System (INIS)

Fibroblasts were established in vitro from skin biopsies obtained from 55 women and one man with or without breast cancer and with or without exposure to radiation from the atomic bomb explosion in Hiroshima. The radiosensitivity of these cells was evaluated by clonogenic assays after exposure to X rays or to fission neutrons from a 252Cf source. Data were fitted to a multitarget model, S/S0 = A[1-(1-ekD)N], for both X-ray and neutron dose-survival curves. A single-hit model, S/S0 = AekD, fits the neutron dose-survival responses as well. These was no difference in the means or variances of radiosensitivity between exposed and nonexposed groups, or between patients with or without breast cancer. Hence, although the sample is not large, it provides no support for the hypothesis that A-bomb radiation preferentially induces breast cancer in women whose cells in vitro are sensitive to cell killing by radiation. (author)

1990-01-01

144

Gene expression changes in normal human skin fibroblasts induced by HZE-particle radiation.  

Science.gov (United States)

Studies have shown that radiation exposure affects global gene expression in mammalian cells. However, little is known about the effects of HZE particles on gene expression. To study these effects, human skin fibroblasts were irradiated with HZE particles of different energies and LETs. The data obtained from these experiments indicate that changes in gene expression are dependent on the energy of the radiation source. Particles with the highest energy, i.e. iron, induced the biggest expression changes in terms of numbers of genes and magnitudes of changes. Many genes were found to undergo significant expression changes after HZE-particle irradiation, including CDKN1A/p21, MDM2, TNFRSF6/fas, PCNA and RAD52. Unlike X rays, HZE particles expose cells to two types of radiation: primary ions and delta rays. We hypothesized that the biological effects of delta rays, which are secondary electron emissions, should resemble the effects of X rays. To explore this idea, gene expression changes between cells that had been irradiated with HZE particles and X rays were compared. The results support our hypothesis since the number of genes that commonly changed after exposure to both radiations increased as a function of particle energy. PMID:16187761

Ding, Liang-Hao; Shingyoji, Masato; Chen, Fanqing; Chatterjee, Aloke; Kasai, Kiyomi-Eguchi; Chen, David J

2005-10-01

145

Gene expression changes in normal human skin fibroblasts induced by HZE-particle radiation.  

UK PubMed Central (United Kingdom)

Studies have shown that radiation exposure affects global gene expression in mammalian cells. However, little is known about the effects of HZE particles on gene expression. To study these effects, human skin fibroblasts were irradiated with HZE particles of different energies and LETs. The data obtained from these experiments indicate that changes in gene expression are dependent on the energy of the radiation source. Particles with the highest energy, i.e. iron, induced the biggest expression changes in terms of numbers of genes and magnitudes of changes. Many genes were found to undergo significant expression changes after HZE-particle irradiation, including CDKN1A/p21, MDM2, TNFRSF6/fas, PCNA and RAD52. Unlike X rays, HZE particles expose cells to two types of radiation: primary ions and delta rays. We hypothesized that the biological effects of delta rays, which are secondary electron emissions, should resemble the effects of X rays. To explore this idea, gene expression changes between cells that had been irradiated with HZE particles and X rays were compared. The results support our hypothesis since the number of genes that commonly changed after exposure to both radiations increased as a function of particle energy.

Ding LH; Shingyoji M; Chen F; Chatterjee A; Kasai KE; Chen DJ

2005-10-01

146

[Action of an extract of Vanda coerulea on the senescence of skin fibroblasts].  

UK PubMed Central (United Kingdom)

The family of the orchids to date is poorly studied as a potential source of molecules with biological activity. The phytochemical analysis of extracts of Vanda coerulea stems (Orchidaceae), the isolation and the purification of the secondary metabolites realized by CLHP followed with high-resolution mass spectrometry and mono and two-dimensional nuclear magnetic resonance made it possible to identify the joint presence in an orchid of three stilbenoïds i.e, imbricatin, methoxycoelonin and gigantol. By flow cytometry, it is shown that the replicative senescence of human normal skin fibroblasts involves a reduction in the number of cells in phase S. A proteins chips technology dedicated to cell cycle proteins makes it possible to correlate this decrease of the number of cells in phase S to a decrease in cyclin E and cyclin dependant kinase 2, cdk2. The treatment by an ethanolic extract of stems of Vanda coerulea titrated in the three stilbenoids restores the percentage at an equivalent rate to that of young cells and the rate of cyclin E and, cdk2, thus bringing a beginning of explanation of their mechanism. These activities let predict an interesting potential as active ingredients to fight against the visible signs of cutaneous ageing.

Bonté F; Simmler C; Lobstein A; Pellicier F; Cauchard JH

2011-05-01

147

Spontaneous immortalization of cultured skin fibroblasts obtained from a high-dose atomic bomb survivor  

International Nuclear Information System (INIS)

Two immortal fibroblastic cell strains (substrains) were established by culturing healthy skin cells obtained from a high-dose atomic bomb survivor (female, age 76 years, 5.14 Gy) for more than 4 years. Designated FM-U and FM-M, the two substrains share the same marker chromosome, t(5q-;6p+), but are karyotypically different, possessing hypodiploid chromosome numbers (39-43) in the former and hypertriploid (69-76) in the latter. Thus far, the two strains have passed through 117 and 156 subcultures or more than 230 and 310 cumulative population doublings, respectively, each passage requiring 4-6 days in the former and 3-4 days in the latter. In the process of immortalization, sequential rearrangement among various chromosomes presumably due to telomeric and interstitial telomeric fusions took place following the telomere shortening, particularly in the senescence and postsenescence phase cells. Of particular interest is the fact that loss of heterozygosity (LOH) of the p53 gene was demonstrated in these immortalized cell populations. In addition, the allelic patterns of the LOH of p53 differed. Further evidence indicative of infinite proliferation was demonstrated in both strains, such as the telomere elongation and the significantly low frequency of cells possessing dicentric chromosomes.

1996-01-01

148

Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dihydrate deposition disease.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition...

Ryan, L M; Wortmann, R L; Karas, B; Lynch, M P; McCarty, D J

149

Metabolism of [1-(14)C] and [2-(14)C] leucine in cultured skin fibroblasts from patients with isovaleric acidemia. Characterization of metabolic defects.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Leucine metabolism in cultured skin fibroblasts from patients with isovaleric acidemia was compared with that in normal fibroblasts and in cells from patients with maple syrup urine disease using [1-(14)C] and [2-(14)C] leucine as substrates. Inhibitory effects of methylenecyclopropylacetic acid on ...

Tanaka, K; Mandell, R; Shih, V E

150

Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans  

Energy Technology Data Exchange (ETDEWEB)

Purine nucleoside phosphorylase deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim the authors constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 /mu/mol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 /mu/mol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

Osborne, W.R.A.; Miller, A.D.

1988-09-01

151

Functional consequences of mitochondrial DNA deletions in human skin fibroblasts: increased contractile strength in collagen lattices is due to oxidative stress-induced lysyl oxidase activity.  

UK PubMed Central (United Kingdom)

Deletions within the mitochondrial DNA (mtDNA) are thought to contribute to extrinsic skin aging. To study the translation of mtDNA deletions into functional and structural changes in the skin, we seeded human skin fibroblasts into collagen gels to generate dermal equivalents. These cells were either derived from Kearns-Sayre syndrome (KSS) patients, who constitutively carry large amounts of the UV-inducible mitochondrial common deletion, or normal human volunteers. We found that KSS fibroblasts, in comparison with normal human fibroblasts, contracted the gels faster and more strongly, an effect that was dependent on reactive oxygen species. Gene expression and Western blot analysis revealed significant upregulation of lysyl oxidase (LOX) in KSS fibroblasts. Treatment with the specific LOX inhibitor beta-aminopropionitrile decreased the contraction difference between KSS and normal human fibroblast equivalents. Also, addition of the antioxidant N-tert-butyl-alpha-phenylnitrone reduced the contraction difference by inhibiting collagen gel contraction in KSS fibroblasts, and both beta-aminopropionitrile and N-tert-butyl-alpha-phenylnitrone diminished LOX activity. These data suggest a causal relationship between mtDNA deletions, reactive oxygen species production, and increased LOX activity that leads to increased contraction of collagen gels. Accordingly, increased LOX expression was also observed in vivo in photoaged human and mouse skin. Therefore, mtDNA deletions in human fibroblasts may lead to functional and structural alterations of the skin.

Majora M; Wittkampf T; Schuermann B; Schneider M; Franke S; Grether-Beck S; Wilichowski E; Bernerd F; Schroeder P; Krutmann J

2009-09-01

152

Paracrine cytokine interaction between UVB-exposed epidermal keratinocytes and dermal fibroblasts in stimulating expression of skin fibroblast-derived elastase.  

UK PubMed Central (United Kingdom)

BACKGROUND: We recently reported that over-expression of skin fibroblast-derived elastase (SFE) plays a pivotal role in the mechanism of UVB-induced skin wrinkling. Since UVB penetrates only modestly to the dermis, we hypothesized that factors secreted by UVB-exposed keratinocytes in the epidermis trigger fibroblasts in the dermis to increase their expression of SFE which then degrades the elastic fibers. OBJECTIVE: In this study, we characterized the paracrine interaction between human keratinocytes (HK) and human fibroblasts (HF) which leads to increased expression of SFE. METHODS AND RESULTS: Medium conditioned by UVB-exposed HK contained increased levels of IL-1?, GM-CSF, IL-6, TNF? and IL-8. While HF cultured with those conditioned medium slightly down-regulated the gene expression of collagen and elastin, they significantly increased their expression of SFE at the transcriptional, translational and enzymatic levels. Neutralizing antibodies to IL-1? or GM-CSF significantly abolished the increased expression of SFE at the translational and/or enzymatic levels in HF cultured with those conditioned medium, while neutralizing antibodies to IL-6, IL-8 or TNF? had no such effect. The addition of IL-1? or GM-CSF, but not TNF?, IL-6 or IL-8, at concentrations ranging from 1 to 10nm, significantly stimulated the enzymatic levels of SFE in HF. CONCLUSIONS: The sum of these findings suggests that IL-1? and GM-CSF are intrinsic cytokines secreted by UVB-exposed HK that stimulate expression of SFE by HF, leading to UVB-induced wrinkle formation.

Nakajima H; Yoshioka R; Ezaki Y; Nagai T; Imokawa G

2012-07-01

153

Skin Equivalent Tissue-Engineered Construct: Co-Cultured Fibroblasts/ Keratinocytes on 3D Matrices of Sericin Hope Cocoons  

Science.gov (United States)

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-?, 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-?, IL-1? 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.

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

2013-01-01

154

Hypersensitivity of skin fibroblasts from basal cell nevus syndrome patients to killing by ultraviolet B but not by ultraviolet C radiation  

Energy Technology Data Exchange (ETDEWEB)

Basal cell nevus syndrome (BCNS) is an autosomal dominant genetic disorder in which the afflicted individuals are extremely susceptible to sunlight-induced skin cancers, particularly basal cell carcinomas. However, the cellular and molecular basis for BCNS is unknown. To ascertain whether there is any relationship between genetic predisposition to skin cancer and increased sensitivity of somatic cells from BCNS patients to killing by UV radiation, we exposed skin fibroblasts established from unexposed skin biopsies of several BCNS and age- and sex-matched normal individuals to either UV-B (280-320 nm) or UV-C (254 nm) radiation and determined their survival. The results indicated that skin fibroblasts from BCNS patients were hypersensitive to killing by UV-B but not UV-C radiation as compared to skin fibroblasts from normal individuals. DNA repair studies indicated that the increased sensitivity of BCNS skin fibroblasts to killing by UV-B radiation was not due to a defect in the excision repair of pyrimidine dimers. These results indicate that there is an association between hypersensitivity of somatic cells to killing by UV-B radiation and the genetic predisposition to skin cancer in BCNS patients. In addition, these results suggest that DNA lesions (and repair processes) other than the pyrimidine dimer are also involved in the pathogenesis of sunlight-induced skin cancers in BCNS patients. More important, the UV-B sensitivity assay described here may be used as a diagnostic tool to identify presymptomatic individuals with BCNS.

Applegate, L.A.; Goldberg, L.H.; Ley, R.D.; Ananthaswamy, H.N. (Univ. of Texas M. D. Anderson Cancer Center, Houston (USA))

1990-02-01

155

Growth properties and growth factor responsiveness in skin fibroblasts from centenarians.  

UK PubMed Central (United Kingdom)

Human fibroblast cultures, which have a finite replicative lifespan in vitro, are the most widely used model for the study of senescence at the cellular level. An inverse relationship between replicative capability and donor age has been reported in human fibroblast strains. We studied the growth capacity of fibroblast primary cultures derived from people whose lifespan was as closer as possible to the expected maximum human lifespan, i.e. people over one hundred. Our data suggest that outgrowth of fibroblasts from biopsies, growth kinetics at different population doubling levels, capability to respond to a classical mitogenic stimulus (such as 20% serum) and a variety of growth factors, were remarkably similar in fibroblasts from centenarians and young controls. On the whole, our data challenge the tenet of a simple and strict relationship between in vivo aging and in vitro proliferative capability of human fibroblasts, at least at the individual level.

Tesco G; Vergelli M; Grassilli E; Salomoni P; Bellesia E; Sikora E; Radziszewska E; Barbieri D; Latorraca S; Fagiolo U; Santacaterina S; Amaducci L; Tiozzo R; Franceschi C; Sorbi S

1998-03-01

156

Molecular alterations of tropoelastin and proteoglycans induced by tobacco smoke extracts and ultraviolet A in cultured skin fibroblasts  

International Nuclear Information System (INIS)

Functional integrity of normal skin is dependent on the balance between the biosynthesis and degradation of extracellular matrix, primarily composed of collagen, elastin and proteoglycans. In our previous studies, we found that tobacco smoke extracts decreased expressions of type I and III procollagen and induced matrix metalloproteinase-1 (MMP-1) and MMP-3 in the cultured skin fibroblasts. We here further investigated the effects of tobacco smoke extracts or ultraviolet A (UVA) treatments on the expression of tropoelastin (soluble elastin protein), and versican and decorin (proteoglycans) in cultured skin fibroblasts. The mRNA of tropoelastin increased by tobacco smoke extracts or UVA irradiation. Versican was markedly shown to decrease after these treatments by using western blotting and the mRNA of versican V0 also significantly decreased. UVA treatment did not show remarkable change in decorin protein, but resulted in marked decrease of decorin D1 mRNA. In contrast to UVA irradiation, the treatments of tobacco smoke extracts resulted in significant increase in decorin, while mRNA of decorin D1 decreased as compared to the control. MMP-7 increased after the treatment of tobacco smoke extracts or UVA. These results indicated that common molecular features might underlie the skin premature aging induced by tobacco smoke extracts and UVA, including abnormal regulation of extracellular matrix deposition through elevated MMPs, reduced collagen production, abnormal tropoelastin accumulation, and altered proteoglycans. (author)

2002-01-01

157

Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells.  

UK PubMed Central (United Kingdom)

Assay of alpha-L-iduronidase, heparin sulphamidase, N-acetyl-alpha-D-glucosaminidase, arylsulphatase B, alpha-L-fucosidase, beta-glucuronidase, beta-galactosidase and alpha-D-mannosidase in cultured cells is described. Activities in deficient fibroblast strains are compared to control fibroblast strains. The first case of Sanfilippo B in the United Kingdom is reported. A comparison of enzyme activities in cultured fibroblasts and amniotic fluid cells is made.

Butterworth J

1978-01-01

158

Diagnosis of the mucopolysaccharidoses using cultured skin fibroblasts and amniotic fluid cells.  

Science.gov (United States)

Assay of alpha-L-iduronidase, heparin sulphamidase, N-acetyl-alpha-D-glucosaminidase, arylsulphatase B, alpha-L-fucosidase, beta-glucuronidase, beta-galactosidase and alpha-D-mannosidase in cultured cells is described. Activities in deficient fibroblast strains are compared to control fibroblast strains. The first case of Sanfilippo B in the United Kingdom is reported. A comparison of enzyme activities in cultured fibroblasts and amniotic fluid cells is made. PMID:117232

Butterworth, J

1978-01-01

159

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

Science.gov (United States)

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.

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

2005-08-01

160

Cytotoxic and mutagenic effects of high let charged particles on human skin fibroblasts  

International Nuclear Information System (INIS)

[en] Cytotoxic and mutagenic effects of high LET charged particles were quantitatively measured using primary cultures of human skin fibroblasts. The span of LETs selected were from 25 keV/?m(330 MeV/u) to 920 keV/?m (600 MeV/u). Mutations were scored at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus using 6-thioguanine (6-TG) for selection. Exposure to these high LET charged particles resulted in exponential survival curves whereas mutant induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/?m. The inactivation cross-section (?i) and the action cross-section for mutant induction (?m) ranged from 2.2 to 92.0 ?m2 and 0.09 to 5.56 x 10-3 ?m2 respectively, the maximum values were obtained by 56Fe with an LET of 200 keV/?m. The mutagenicity (?m/?i) ranged from 2.05 to 7.99 x 10-5 with the maximum value at 150 keV/?m. Furthermore, the results of multiplex polymerase chain reaction (PCR) of some of the mutants induced by charged particles indicate that higher LET beams are more likely to cause larger deletions in the hprt locus. (author)

1992-01-01

 
 
 
 
161

Influence of genistein on c-Jun, c-Fos and Fos-B of AP-1 subunits expression in skin keratinocytes, fibroblasts and keloid fibroblasts cultured in vitro.  

UK PubMed Central (United Kingdom)

Genistein is a well known flavonoid that exhibits antioxidant, antiproliferative, proapoptotic, antiangiogenic, as well as estrogenic and anti-estrogenic activity. Extensive studies in the field of dermatology and cosmetology in recent years revealed that genistein is a promising anti-aging, anti-photoaging and anti-carcinogenic agent for skin care. Essential role in skin prematuring aging and carcinogenesis play AP-1 transcription factors that are activated, among others, by environmental factors: ultraviolet light and free radicals. Genistein is a potent antioxidant and inhibitor of AP-1 activity. The aim of the study was to investigate genistein as a potential regulator of C-JUN, C-FOS and FOS-B of AP-1 subunits expression in skin keratinocytes, fibroblasts and keloid fibroblasts cultured in vitro. In presented study, genistein modulated C-JUN expression in epidermal keratinocytes and dermal fibroblasts cultured in vitro. The expression of C-JUN was higher in keratinocytes treated with 37 and 370 microM genistein. In dermal fibroblasts genistein regulated C-JUN expression in dose-dependent manner. The expression of C-JUN was lower in fibroblasts treated with 370 microM genistein and higher in fibroblasts treated with 37 microM genistein. Genistein in 370 microM concentration inhibited C-FOS expression in fibroblasts, whereas in 370 and 37 microM concentration genistein inhibited FOS-B expression in keratinocytes. Furthermore, genistein was able to modulate C-JUN and C-FOS genes expression in keloid fibroblasts cultured in vitro. In these cells, transcriptional activity of C-JUN and C-FOS expression depended on employed concentration of genistein. The expression of C-JUN and C-FOS was higher in keloid fibroblasts treated with 370 microM genistein and lower in keloid fibroblasts treated with 37 microM genistein.

Jurzak M; Adamczyk K

2013-03-01

162

Methylmalonic and propionic acidemias: lipid profiles of normal and affected human skin fibroblasts incubated with [1-14C]propionate  

International Nuclear Information System (INIS)

[en] Normal human skin fibroblasts and those from methylmalonic acidemia and propionic acidemia patients were grown in culture. Following incubation with [1-14C]propionate, the major lipid classes in the cells were separated by thin layer chromatography and isolated fractions analyzed by radio gas chromatography for the presence of odd-numbered long-chain fatty acids; the pattern of even-numbered long-chain fatty acids was obtained also. Normal fibroblasts incorporated a small percentage of propionate into odd-numbered fatty acids which were present in all lipids studied. The abnormal cells incorporated a larger amount while maintaining the characteristic ratios of odd-numbered fatty acids found in the normal line. Most of the radioactivity was associated with phospholipids which are the predominant constituents of cell membranes. A characteristic C15/C17 ratio was found for different phospholipids and the triglyceride fraction; pentadecanoic acid was the principal odd-numbered fatty acid utilized in the assembly of complex lipids. Compared to even-numbered long-chain fatty acids the absolute amount of odd-numbered fatty acids was low (1-2%), even in affected cells. An unusual polar lipid fraction was isolated in the course of the study. In the normal cell it contained several unlabeled eicosanoids which were missing from the same fraction of both affected cell lines

1986-01-01

163

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.  

Science.gov (United States)

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases. PMID:19041695

Gysin, René; Riederer, Irène M; Cuénod, Michel; Do, Kim Q; Riederer, Beat M

2008-11-28

164

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.  

UK PubMed Central (United Kingdom)

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Gysin R; Riederer IM; Cuénod M; Do KQ; Riederer BM

2009-04-01

165

Impaired colony-forming ability following ? irradiation of skin fibroblasts from tuberous scierosis patients  

International Nuclear Information System (INIS)

[en] 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 ?-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 60Co ?-ray treatment, whether administered oxically (air-saturated) or hypoxically (N2-gassed); a fourth strain (GM1644) from a third patient exhibited normal radiosensitivity under both treatment conditions. The post-?-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

1982-01-01

166

Negative modulation of alpha1(I) procollagen gene expression in human skin fibroblasts: transcriptional inhibition by interferon-gamma.  

UK PubMed Central (United Kingdom)

Interferon-gamma (IFN-gamma), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of alpha1(I) procollagen gene (COL1A1) expression by recombinant IFN-gamma. The results showed that IFN-gamma stimulated the rapid accumulation of interferon regulated factor (IRF)-1 mRNA, followed by a delayed and dose-dependent inhibition of alpha1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF-1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre-mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter-chloramphenicol acetyltransferase reporter gene plasmids, IFN-gamma selectively inhibited promoter activity and abrogated its stimulation induced by TGF-beta. The inhibition by IFN-gamma was not due to downregulation of TGF-beta receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN-alpha and IFN-beta by themselves had little effect on promoter activity, but IFN-alpha augmented the inhibitory effect of IFN-gamma. Using a series of 5' deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN-gamma response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF-1 but no homologs of previously characterized IFN-gamma response elements. The putative IFN-gamma response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN-gamma. Gel mobility shift analysis showed that two distinct and specific DNA-protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN-gamma response region. IFN-gamma did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN-gamma inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF-1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN-gamma regulation of fibroblast function.

Yuan W; Yufit T; Li L; Mori Y; Chen SJ; Varga J

1999-04-01

167

Relationship between in vitro radiosensitivity of normal human skin fibroblasts and the occurrence of late normal tissue reactions after radiotherapy  

International Nuclear Information System (INIS)

Late complications in normal tissues are limiting for the doses that can be administered during clinical radiotherapy. Awareness of these complications, and comprehension of the underlying biological mechanisms, is extremely important to improve cancer treatment. Fibrosis is one of the most critical injuries to radiotherapy. It varies significantly among patients despite of identical treatments. The large patient-to-patient variability of normal tissue sections to clinical radiation can possibly be accounted for by the considerable individual variation in cellular radiosensitivity of normal human fibroblasts, as shown in vitro. The purpose of the present investigation has been to analyze individual cellular radiosensitivity of normal human skin fibroblasts, as measured in a colony-forming assay, and the relationship to the occurrence of subcutaneous fibrosis after radiotherapy for breast cancer. (au) 97 refs.

1995-01-01

168

Fabrication of a nanofibrous scaffold with improved bioactivity for culture of human dermal fibroblasts for skin regeneration  

Energy Technology Data Exchange (ETDEWEB)

Engineering dermal substitutes with electrospun nanofibres have lately been of prime importance for skin tissue regeneration. Simple electrospinning technology served to produce nanofibrous scaffolds morphologically and structurally similar to the extracellular matrix of native tissues. The nanofibrous scaffolds of poly(l-lactic acid)-co-poly({epsilon}-caprolactone) (PLACL) and PLACL/gelatin complexes were fabricated by the electrospinning process. These nanofibres were characterized for fibre morphology, membrane porosity, wettability and chemical properties by FTIR analysis to culture human foreskin fibroblasts for skin tissue engineering. The nanofibre diameter was obtained between 282 and 761 nm for PLACL and PLACL/gelatin scaffolds; expressions of amino and carboxyl groups and porosity up to 87% were obtained for these fibres, while they also exhibited improved hydrophilic properties after plasma treatment. The results showed that fibroblasts proliferation, morphology, CMFDA dye expression and secretion of collagen were significantly increased in plasma-treated PLACL/gelatin scaffolds compared to PLACL nanofibrous scaffolds. The obtained results prove that the plasma-treated PLACL/gelatin nanofibrous scaffold is a potential biocomposite material for skin tissue regeneration.

Chandrasekaran, Arun Richard; Venugopal, J; Sundarrajan, S; Ramakrishna, S, E-mail: nnijrv@nus.edu.s [Healthcare and Energy Materials Laboratory, Nanoscience and Nanotechnology Initiative, Faculty of Engineering, National University of Singapore (Singapore)

2011-02-15

169

Epilobium angustifolium extract demonstrates multiple effects on dermal fibroblasts in vitro and skin photo-protection in vivo.  

UK PubMed Central (United Kingdom)

Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits.

Ruszová E; Cheel J; Pávek S; Moravcová M; Hermannová M; Mat?jková I; Spilková J; Velebný V; Kubala L

2013-09-01

170

Epilobium angustifolium extract demonstrates multiple effects on dermal fibroblasts in vitro and skin photo-protection in vivo.  

Science.gov (United States)

Stress-induced fibroblast senescence is thought to contribute to skin aging. Ultraviolet light (UV) radiation is the most potent environmental risk factor in these processes. An Epilobium angustifolium (EA) extract was evaluated for its capacity to reverse the senescent response of normal human dermal fibroblasts (NHDF) in vitro and to exhibit skin photo-protection in vivo. The HPLC-UV-MS analysis of the EA preparation identified three major polyphenol groups: tannins (oenothein B), phenolic acids (gallic and chlorogenic acids) and flavonoids. EA extract increased the cell viability of senescent NHDF induced by serum deprivation. It diminished connective tissue growth factor and fibronectin gene expressions in senescent NHDF. Down-regulation of the UV-induced release of both matrix metalloproteinase-1 and -3 and the tissue inhibitor of matrix metalloproteinases-1 and -2, and also down-regulation of the gene expression of hyaluronidase 2 were observed in repeatedly UV-irradiated NHDF after EA extract treatment. Interestingly, EA extract diminished the down-regulation of sirtuin 1 dampened by UV-irradiation. The application of EA extract using a sub-irritating dose protected skin against UV-induced erythema formation in vivo. In summary, EA extract diminished stress-induced effects on NHDF, particularly on connective tissue growth factor, fibronectin and matrix metalloproteinases. These results collectively suggest that EA extract may possess anti-aging properties and that the EA polyphenols might account for these benefits. PMID:23817638

Ruszová, Ema; Cheel, José; Pávek, Stanislav; Moravcová, Martina; Hermannová, Martina; Mat?jková, Ilona; Spilková, Ji?ina; Velebný, Vladimír; Kubala, Lukáš

2013-07-02

171

Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro  

DEFF Research Database (Denmark)

The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particuar proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increases the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples without any further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

JØrgensen, Peter; Milkovic, Lidija

2013-01-01

172

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

Science.gov (United States)

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.

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

2006-03-01

173

Specificity in the synergism between retinoic acid and EGF on the growth of adult human skin fibroblasts  

International Nuclear Information System (INIS)

[en] Vitamin A (retinol) and five retinoids were tested for their ability to enhance epidermal growth factor (EGF) stimulation of adult human skin fibroblast growth in vitro. The retinoids utilized in this study were RO-1-5488 (all-trans-retinoic acid), RO-4-3780 (13-cis-retinoic acid), RO-10-9359, RO-10-1670, and RO-21-6583. Retinol and each retinoid were capable of stimulating fibroblast growth alone (0-86%), while 13-cis and all-trans-retinoic acid were the most potent in potentiating the EGF promotion of fibroblast growth. Since retinoic acid might enhance the EGF stimulation of cell growth by increasing either EGF receptor number or binding affinity, the binding of 125I-labeled EGF was carried out in the presence of retinoic acid and the data were subjected to a Scatchard-type analysis. No change in EGF receptor number or affinity was seen in the presence of retinoic acid. The data indicate a specific interaction between retinoid acid and EGF which results in the potentiation of the EGF-stimulated cell growth. Furthermore, the mechanism of this interaction does not seem to involve the initial binding of EGF to its plasma membrane receptor or the available number of EGF receptors located on the cell surface

1988-01-01

174

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

Science.gov (United States)

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. PMID:9702916

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

1998-08-01

175

RAC activity in keloid disease: comparative analysis of fibroblasts from margin of keloid to its surrounding normal skin.  

UK PubMed Central (United Kingdom)

BACKGROUND: Keloids are characterized by excess collagen deposition within the dermis. Although the exact cause of the potentially overactive fibroblasts has yet to be elucidated, many etiological possibilities have been suggested. As fibroblasts originating from keloids appear to have an increased migration and proliferation rate, cell-signaling studies examining these factors may offer an opportunity to further our understanding of the pathogenesis of this disease. One of such cell-signaling messengers is the enzyme Ras-related C3 botulinum toxin substrate (RAC), which has never been investigated in keloid scars. OBJECTIVE: This study explores the role of RAC activity in keloid disease. METHOD: Primary fibroblast cell lines were established from the margin of keloid (KF) scars as well as from the surrounding normal tissue (NF) from one anatomical site of the same patient. Migration and proliferation assays were performed, comparing matching NFs and KFs, and after cell lysis, RAC activity was assessed. RESULTS: Comparing fibroblasts from 3 different patients, KFs migrated (P < .05) and proliferated (P < .05) faster than NFs. The activity levels of RAC were increased in KFs compared with NFs. CONCLUSION: KFs migrate and proliferate faster than NFs. RAC activity increases in KFs when compared with NFs. Inhibition of RAC could lead to a new therapeutic approach.

Witt E; Maliri A; McGrouther DA; Bayat A

2008-01-01

176

Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency.  

Science.gov (United States)

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway. PMID:22180458

Baresova, Veronika; Skopova, Vaclava; Sikora, Jakub; Patterson, David; Sovova, Jana; Zikanova, Marie; Kmoch, Stanislav

2011-12-16

177

Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency.  

UK PubMed Central (United Kingdom)

The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.

Baresova V; Skopova V; Sikora J; Patterson D; Sovova J; Zikanova M; Kmoch S

2012-04-01

178

One-stage, simultaneous skin grafting with artificial dermis and basic fibroblast growth factor successfully improves elasticity with maturation of scar formation.  

UK PubMed Central (United Kingdom)

The efficacy of one-stage artificial dermis and skin grafting was tested in a nude rat model. Reconstruction with artificial dermis is usually a two-stage procedure with 2- to 3-week intermission. If one-stage use of artificial dermis and split-thickness skin grafting are effective, the overall burden on patients and the medical cost will markedly decrease. The graft take rate, contraction rate, tissue elasticity, histology, morphometric analysis of the dermal thickness, fibroblast counting, immunohistochemistry of ?-smooth muscle actin, matrix metalloproteinase-2, CD31, and F4/80, as well as gelatin zymography, real-time reverse transcriptase polymerase chain reaction for matrix metalloproteinase-2, and electron microscopy, were investigated from day 3 to 3 months postoperatively. The graft take rate was good overall in one-stage artificial dermis and skin grafting groups up to 3 weeks, and the contraction rate was greater in the two-staged artificial dermis and skin grafting group than in the skin grafting alone or one stage of artificial dermis and skin grafting groups. Split-thickness skin grafting with artificial dermis and basic fibroblast growth factor at a concentration of 1??g/cm(2) showed significantly greater elasticity by Cutometer, and the dermal thickness was significantly thinner, fibroblast counting was significantly greater, and the ?-smooth muscle actin expression level was more notable with a more mature blood supply in the dermis and more organized dermal fibrils by electron microscopy at 3 weeks. Thus, one-stage artificial dermis and split-thickness skin grafting with basic fibroblast growth factor show a high graft take rate and better tissue elasticity determined by Cutometer analysis, maturity of the dermis, and increased fibroblast number and blood supply compared to a standard two-stage reconstruction.

Hamuy R; Kinoshita N; Yoshimoto H; Hayashida K; Houbara S; Nakashima M; Suzuki K; Mitsutake N; Mussazhanova Z; Kashiyama K; Hirano A; Akita S

2013-01-01

179

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

1985-08-01

180

Chemical transformation of cultured human skin fibroblasts derived from individuals with hereditary adenomatosis of the colon and rectum.  

UK PubMed Central (United Kingdom)

Chemical transformation of cultured human skin fibroblasts (PF) derived from individuals with hereditary adenomatosis of the colon and rectum is reported. Cells treated only with various levels of N-methyl-N'-nitro N-nitrosoguanidine (MNNG) underwent morphological alteration. The morphologically altered cells formed large aggregates when suspended in liquid growth medium above an agar base and grew to high saturation densities. One altered (MNNG, 1.0 microgram/ml) cell culture formed colonies in soft agar. Transformed cells were resistant to rechallenge of MNNG (l microgram/ml) and showed a more prolonged life-span compared to the untreated cells. Altered cells became heteroploid cells. However, no progressively growing tumors were produced when cells were inoculated subcutaneously into nude mice. The data suggest that chemical carcinogens alone may not induce neoplastic transformation of fibroblasts from humans genetically predisposed to cancer and that neoplastic transformation of these skin cells by chemical carcinogens might require the presence of a tumor promotor and the use of an immuno-privileged site in the nude mouse system.

Rhim JS; Huebner RJ; Arnstein P; Kopelovich L

1980-11-01

 
 
 
 
181

Inhibition of Oxidative Stress by Low-Molecular-Weight Polysaccharides with Various Functional Groups in Skin Fibroblasts  

Directory of Open Access Journals (Sweden)

Full Text Available 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.

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

2013-01-01

182

Line patterns in the mosaic electrical properties of human skin--a cross-correlation study.  

UK PubMed Central (United Kingdom)

A vehicle with 16 electrodes for the two-dimensional electrical admittance mapping of human skin is presented. Measurements on 20 test subjects have been carried out and analyzed for the possible detection of line patterns in the electrical properties of the skin, claimed to coincide with the so-called acupuncture meridian lines of ancient Chinese medicine. No such lines were found.

Martinsen OG; Grimnes S; Mørkrid L; Hareide M

2001-06-01

183

2,4,5,2',4',5'-Hexachlorobiphenyl-lipoprotein (LDL, HDL, VLDL) interaction and induced lipidosis in cultured skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

2,4,5,2',4',5'-Hexachlorobiphenyl (HCB) induced cytoplasmic inclusions and lipidosis in normal (AG1437) and hypercholesterolemic (GM488) human skin fibroblasts. Quantitative and qualitative microscopic fluorescence analysis showed that the cytoplasmic inclusions are formed as early as 3 hr after treatment with HCB. The inclusions contain lipids but no detectable nonesterified cholesterol or cholesteryl ester. High density lipoproteins (HDL), low density lipoproteins (LDL), and very low density lipoproteins (VLDL) facilitate the apparent uptake of HCB by skin fibroblasts. HDL and LDL appeared to reverse the induction of cytoplasmic inclusions and lipidosis when cells were pretreated with HCB, the HCB was removed from media, and the cells were incubated with LDL or HDL. The results suggest that lipoproteins participate in the uptake and egress of HCB from skin fibroblasts.

Kling, D.; Becker, M.M.; Kruth, H.S.; Gamble, W.

1984-06-01

184

Effect of low-dose-rate irradiation on the division potential of cells in vitro. V. Human skin fibroblasts from donors with a high risk of cancer  

International Nuclear Information System (INIS)

[en] Skin fibroblasts from normal donors, donors with ataxia-telanglectasia or Fanconi's anemia, and from 1 cancer patient were treated with repeated ? radiation at about 16 rads per hour. The remaining division potential of all fibroblasts, except for the Fanconi's anemia cells, was reduced to different extents by radiation. The growth potential of Fanconl's anemia cells was increased in all the irradiated cultures. The increase was 54% in the group that survived the longest. These results were identical to those obtained with fibroblasts from certain species that have a high probability of transformation

1979-01-01

185

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

Energy Technology Data Exchange (ETDEWEB)

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.

Cockayne, D.; Cutroneo, K.R.

1988-04-19

186

Radiation-induced comet-formation in human skin fibroblasts from radiotherapy patients with different normal tissue reactions  

Energy Technology Data Exchange (ETDEWEB)

Background: In clinical radiotherapy most patients tolerate the applied dosage with no or moderate side effects. However, 5 to 10% of all individuals show increased acute and/or late reactions. In-vitro test systems are investigated for their suitability for predictive purposes. This paper attempts a correlation between the induction and repair of DNA damage measured in the comet assay and the clinical observed reaction in order to evaluate the suitability of the comet assay for prediction of radiation sensitivity. Patients and methods: Skin fibroblasts of 30 patients with average tissue reactions or acute and/or late increased side effects and cell lines of 4 individuals carrying the heritable disease ataxia telangiectasia (AT) were irradiated in vitro. The induction and repair of DNA damage was measured at different time points after irradiation in the comet assay (single cell gel electrophoresis). These results were compared to the acute and late clinical reactions classified according to the RTOG grading system. Results: The radiation induced DNA damage decreased over time reflecting DNA repair. Cells of the AT individuals showed and elevated damage induction and a reduced repair capacity compared to patients with average tissue reactions. Fibroblast of patients with increased acute and late side effects exhibited slower DNA repair. In addition to the known lack of cell cycle control, our results indicate that AT cells show reduced DNA repair capacity. Conclusions: The comet assay seems to be able to detect some types of increased individual radiation sensitivity. In contrast to other predictive in-vitro tests, the comet assay needs less time and fewer cells, which would be useful in a clinical setting. (orig.) [Deutsch] Hintergrund: Eine strahlentherapeutische Behandlung wird von der Mehrzahl der Patienten mit keinen oder nur geringen Nebenwirkungen ueberstanden. Bei 5 bis 10% der Behandelten zeigen sich jedoch staerkere Akut- und/oder Spaetnebenwirkungen. Verschiedene In-vitro-Tests werden untersucht, um die individuelle Strahlensensibilitaet schon praetherpeutisch zu bestimmen. In dieser Arbeit wird der mit dem Comet Assay gemessene induzierte und reparierte DNS-Schaden in in vitro bestrahlten Patientenfibroblasten mit den klinisch beobachteten Nebenwirkungen korreliert und somit seine Eignung als praediktiver Test fuer Strahlensensibilitaet ueberprueft. Patienten und Methodik: Die Hautfibroblasten von insgesamt 30 Patienten mit durchschnittlichen Strahlenreaktionen oder mit erhoehten Frueh- und/oder Spaetnebenwirkungen sowie von 4 Patienten mit Ataxia teleangiectatica wurden in vitro bestrahlt. Induktion und Reparatur des DNS-Schadens wurden zu verschiedenen Zeitpunkten nach Bestrahlung mit dem Comet Assay gemessen. Die Ergebnisse wurden mit den klinischen Akut- und Spaetnebenwirkungen (klassifiziert nach der RTOG-Graduierung) dieser Patienten verglichen. Ergebnisse: Der strahleninduzierte DNS-Schaden nahm im Laufe der Zeit durch DNS-Reparatur wieder ab. Die Zellen der homozygoten AT-Traeger zeigten im Vergleich zu den uebrigen Patienten eine erhoehte Schadensinduktion und eine verminderte Reparaturkapazitaet. Zusaetzlich zum Verlust der Zellzykluskontrolle scheinen AT-Zellen eine verminderte DNS Reparaturkapazitaet zu haben. Die Fibroblasten der Patienten mit ueberdurchschnittlichen Akut- und/oder Spaetnebenwirkungen zeigten im Vergleich zu den Patienten mit durchschnittlicher Strahlenreaktion eine langsamere DNS-Reparatur. Schlussfolgerungen: Mit Hilfe des Comet Assay scheint sich die individuelle Strahlensensibilitaet bestimmen zu lassen. Im Gegensatz zu anderen praediktiven Tests braucht diese Methode weniger Zeit und Zellen und erleichtert somit den klinischen Einsatz. (orig.)

Oppitz, U.; Flentje, M. [Wuerzburg Univ. (Germany). Klinik und Poliklinik fuer Strahlentherapie; Denzinger, S.; Nachtrab, U. [Wuerzburg Univ. (Germany). Klinik und Poliklinik fuer Strahlentherapie]|[Wuerzburg Univ. (Germany). Inst. fuer Toxikologie; Stopper, H. [Wuerzburg Univ. (Germany). Inst. fuer Toxikologie

1999-07-01

187

Effect of bradykinin on prostaglandin production by human skin fibroblasts in culture  

Energy Technology Data Exchange (ETDEWEB)

In studying the effect of bradykinin on prostaglandin formation in fibroblasts, two general types of assays have been employed. Radioimmunoassays were utilized to determine specific prostaglandins, PGI/sub 2/ and 6-keto-PGF, using tritium as radiolabel. A second procedure involved initial incubation of the fibroblasts with (/sup 14/C)arachidonate and identification and quantification of the metabolites by chromatographic methods. After radiolabelling of fibroblasts with (/sup 14/C)arachidonate, bradykinin was found to release radiolabel from membrane lipids approximately 2-fold. In the presence of bradykinin, there was a close coupling of phospholipase S/sub 2/ activity, arachidonate mobilization, and synthesis of a specific prostaglandin, PGI/sub 2/.

Jelsema, C.L.; Moss, J.; Manganiello, V.C.

1985-01-01

188

The role of fibroblast growth factor receptor 2b in skin homeostasis and cancer development  

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The epithelial isoform of fibroblast growth factor receptor 2 (Fgfr2b) is essential for embryogenesis, and Fgfr2b-null mice die at birth. Using Cre-Lox transgenics to delete Fgfr2b in cells expressing keratin 5, we show that mice lacking epidermal Fgfr2b survive into adulthood but display striking a...

Grose, Richard; Fantl, Vera; Werner, Sabine; Chioni, Athina-Myrto; Jarosz, Monika; Rudling, Robert; Cross, Barbara

189

Complementation group assignments in Fanconi anemia fibroblast cell lines from North America.  

UK PubMed Central (United Kingdom)

Fanconi anemia is a rare autosomal recessive disease characterized by developmental defects of the thumb and radius, childhood onset of pancytopenic anemia and increased risk of leukemia. At least five complementation groups (A-E) have been defined but only the FAC gene has been cloned. Cells can be assigned to complementation group C by direct mutation analysis. To facilitate the search for additional FA genes and to measure the frequency of complementation groups, we have established new genetically marked immortalized FA-A and FA-D fibroblast cell lines and show their usefulness as universal fusion donors. These reference FA cell lines facilitated somatic cell fusion analysis and enabled us to assign the complementation group in 16 unrelated FA patients from North America. The majority of patients, belong to FA complementation group A (69%), followed by FA-C (18%), FA-D (4%) and FA-B or FA-E (9%).

Jakobs PM; Fiddler-Odell E; Reifsteck C; Olson S; Moses RE; Grompe M

1997-01-01

190

Complementation group assignments in Fanconi anemia fibroblast cell lines from North America.  

Science.gov (United States)

Fanconi anemia is a rare autosomal recessive disease characterized by developmental defects of the thumb and radius, childhood onset of pancytopenic anemia and increased risk of leukemia. At least five complementation groups (A-E) have been defined but only the FAC gene has been cloned. Cells can be assigned to complementation group C by direct mutation analysis. To facilitate the search for additional FA genes and to measure the frequency of complementation groups, we have established new genetically marked immortalized FA-A and FA-D fibroblast cell lines and show their usefulness as universal fusion donors. These reference FA cell lines facilitated somatic cell fusion analysis and enabled us to assign the complementation group in 16 unrelated FA patients from North America. The majority of patients, belong to FA complementation group A (69%), followed by FA-C (18%), FA-D (4%) and FA-B or FA-E (9%). PMID:9217996

Jakobs, P M; Fiddler-Odell, E; Reifsteck, C; Olson, S; Moses, R E; Grompe, M

1997-01-01

191

Novel collagen/gelatin scaffold with sustained release of basic fibroblast growth factor: clinical trial for chronic skin ulcers.  

Science.gov (United States)

Chronic skin ulcers such as diabetic ulcers and venous leg ulcers are increasing and are a costly problem in healthcare. We have developed a novel artificial dermis, collagen/gelatin sponge (CGS), which is capable of sustained release of basic fibroblast growth factor (bFGF) for more than 10 days. The objective of this study was to investigate the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. Patients with chronic skin ulcers that had not healed in at least 4 weeks were treated with CGS impregnated with bFGF at 7 or 14??g/cm(2) after debridement, and the wound bed improvement was assessed 14 days after application. Wound bed improvement was defined as a granulated and epithelialized area on day 14 with a proportion to the baseline wound area after debridement of 50% or higher. The wound area, the wound area on day 14, and the granulation area on day 14 were independently measured by blinded reviewers in a central review using digital images of wounds taken with a calibrator. Patients were followed up until 28 days after application to observe the adverse reactions related to the application of CGS. From May 2010 to June 2011, 17 patients were enrolled and, in 16 patients, the wound bed improved. Among the randomized patients in step 2, no significant difference was seen between the low-dose group and the high-dose group. No serious adverse reactions were observed. Adverse reactions with a clear causal relationship to the study treatment were mild and patients quickly recovered from them. This study is the first-in-man clinical trial of CGS and showed the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. This combination therapy could be a promising therapy for chronic skin ulcers. PMID:23541061

Morimoto, Naoki; Yoshimura, Kenichi; Niimi, Miyuki; Ito, Tatsuya; Aya, Rino; Fujitaka, Junpei; Tada, Harue; Teramukai, Satoshi; Murayama, Toshinori; Toyooka, Chikako; Miura, Kazumi; Takemoto, Satoru; Kanda, Norikazu; Kawai, Katsuya; Yokode, Masayuki; Shimizu, Akira; Suzuki, Shigehiko

2013-04-27

192

Anti-ageing effects of alpha-naphthoflavone on normal and UVB-irradiated human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Ageing is a complex and multifactorial process resulting in several functional and aesthetic changes to the skin. We found that ?-Naphthoflavone (?-NF) concentration-dependently induced pro-collagen type I protein expression and inhibited MMP-1 protein expression, in both normal and UVB-irradiated cells. SB431542 and SIS3 - inhibitors of TGF-? and Smad3, respectively - significantly alleviate ?-NF-caused response of MMP-1 and pro-collagen. LY294002 (PI3K inhibitor) can reverse ?-NF-induced ERK, Akt, Smad-3 activation, pro-collagen synthesis and ?-NF-suppressed AP-1 activation. PD (ERK inhibitor) was not involved in pro-collagen generation and MMP-1 inhibition. We concluded that ?-NF promotes pro-collagen production and inhibits MMP-1 expression via the activation of a PI3K/Akt/Smad-3 pathway in normal and UVB-irradiated human skin fibroblasts, while TGF-? may play an important role in transducing this pathway. These results suggest that ?-NF, a natural plant product, has the potential to become a novel anti-ageing skin application.

Liao PL; Li CH; Chang CY; Lu SR; Lin CH; Tse LS; Cheng YW

2012-07-01

193

Mechanisms of sulfur mustard analog 2-chloroethyl ethyl sulfide-induced DNA damage in skin epidermal cells and fibroblasts  

Science.gov (United States)

Employing mouse skin epidermal JB6 cells and dermal fibroblasts, here we examined the mechanisms of DNA damage by 2-chloroethyl ethyl sulfide (CEES), a monofunctional analog of sulfur mustard (SM). CEES exposure caused H2A.X and p53 phosphorylation as well as p53 accumulation in both cell types, starting at 1 h, that was sustained for 24 h, indicating a DNA-damaging effect of CEES, which was also confirmed and quantified by alkaline comet assay. CEES exposure also induced oxidative stress and oxidative DNA damage in both cell types, measured by an increase in mitochondrial and cellular reactive oxygen species and 8-hydroxydeoxyguanosine levels, respectively. In the studies distinguishing between oxidative and direct DNA damage, 1 h pretreatment with glutathione (GSH) or the antioxidant Trolox showed a decrease in CEES-induced oxidative stress and oxidative DNA damage. However, only GSH pretreatment decreased CEES-induced total DNA damage measured by comet assay, H2A.X and p53 phosphorylation, and total p53 levels. This was possibly due to the formation of GSH–CEES conjugates detected by LC-MS analysis. Together, our results show that CEES causes both direct and oxidative DNA damage, suggesting that to rescue SM-caused skin injuries, pleiotropic agents (or cocktails) are needed that could target multiple pathways of mustard skin toxicities.

Inturi, Swetha; Tewari-Singh, Neera; Gu, Mallikarjuna; Shrotriya, Sangeeta; Gomez, Joe; Agarwal, Chapla; White, Carl W.; Agarwal, Rajesh

2011-01-01

194

Studies on skin aging. Preparation and properties of fucose-rich oligo- and polysaccharides. Effect on fibroblast proliferation and survival.  

UK PubMed Central (United Kingdom)

Skin aging represents an important chapter of connective tissue aging and concerns an organ of vital importance. Here we describe the preparation as well as the biological properties of fucose-rich oligo- and polysaccharides (FROPs), composed of polymers of a trisaccharide containing galactose, acetyl galacturonic acid and fucose, from the original high molecular weight bacterial polysaccharide (Fucogel), Solabia, France). Using endoglycosidases, oligo- and polysaccharides were prepared and characterized by physical and chemical procedures. The non-reducing end-groups comprise equal amounts of galactose and fucose. The here-described biological properties are: stimulation of cell proliferation of cultured human skin fibroblasts, protection of cells against ascorbate-induced cytotoxicity due to the release of reactive oxygen species (ROS). Properties elsewhere described concern the inhibition of matrix metallo-proteinases 2 and 9 (MMP-2 and MMP-9), their expression and activation. Using fluorescein isothiocyanate (FITC)-labeled polysaccharides, their interaction with cell membranes and also their penetration and accumulation in cells, especially in the cell nucleus could be demonstrated, probably via cell-membrane receptor-mediated mechanisms. We describe some of the symptoms of skin aging and show, that the here-described polysaccharide preparations are susceptible to slow down some of the cellular and molecular mechanisms involved, partly by the mediation of the above-mentioned receptors, partly by acting directly on the regulation of gene expression.

Péterszegi G; Isnard N; Robert AM; Robert L

2003-07-01

195

Studies on skin aging. Preparation and properties of fucose-rich oligo- and polysaccharides. Effect on fibroblast proliferation and survival.  

Science.gov (United States)

Skin aging represents an important chapter of connective tissue aging and concerns an organ of vital importance. Here we describe the preparation as well as the biological properties of fucose-rich oligo- and polysaccharides (FROPs), composed of polymers of a trisaccharide containing galactose, acetyl galacturonic acid and fucose, from the original high molecular weight bacterial polysaccharide (Fucogel), Solabia, France). Using endoglycosidases, oligo- and polysaccharides were prepared and characterized by physical and chemical procedures. The non-reducing end-groups comprise equal amounts of galactose and fucose. The here-described biological properties are: stimulation of cell proliferation of cultured human skin fibroblasts, protection of cells against ascorbate-induced cytotoxicity due to the release of reactive oxygen species (ROS). Properties elsewhere described concern the inhibition of matrix metallo-proteinases 2 and 9 (MMP-2 and MMP-9), their expression and activation. Using fluorescein isothiocyanate (FITC)-labeled polysaccharides, their interaction with cell membranes and also their penetration and accumulation in cells, especially in the cell nucleus could be demonstrated, probably via cell-membrane receptor-mediated mechanisms. We describe some of the symptoms of skin aging and show, that the here-described polysaccharide preparations are susceptible to slow down some of the cellular and molecular mechanisms involved, partly by the mediation of the above-mentioned receptors, partly by acting directly on the regulation of gene expression. PMID:12888253

Péterszegi, G; Isnard, N; Robert, A M; Robert, L

196

Pyrophosphohydrolase activity and inorganic pyrophosphate content of cultured human skin fibroblasts. Elevated levels in some patients with calcium pyrophosphate dihydrate deposition disease.  

UK PubMed Central (United Kingdom)

In calcium pyrophosphate dihydrate (CPPD) crystal deposition disease, metabolic abnormalities favoring extracellular inorganic pyrophosphate (PPi) accumulation have been suspected. Elevations of intracellular PPi in cultured skin fibroblasts from a single French kindred with familial CPPD deposition (19) and elevated nucleoside triphosphate pyrophosphohydrolase activity (NTPPPH), which generates PPi in extracts of CPPD crystal-containing cartilages (14) favor this suspicion. To determine whether NTPPPH activity or PPi content of cells might be a disease marker expressed in extraarticular cells, human skin-derived fibroblasts were obtained from control donors and patients affected with the sporadic and familial varieties of CPPD (CPPD-S and CPPD-F) deposition. Intracellular PPi was elevated in both CPPD-S (P less than 0.05) and CPPD-F (P less than 0.01) fibroblasts compared with control fibroblasts. Ecto-NTPPPH activity was elevated in CPPD-S (P less than 0.01) but not CPPD-F. Intracellular PPi correlated with ecto-NTPPPH (P less than 0.01). Elevated PPi levels in skin fibroblasts may serve as a biochemical marker for patients with familial or sporadic CPPD crystal deposition disease; ecto-NTPPPH activity further separates the sporadic and familial disease types. Expression of these biochemical abnormalities in nonarticular cells implies a generalized metabolic abnormality.

Ryan LM; Wortmann RL; Karas B; Lynch MP; McCarty DJ

1986-05-01

197

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

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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.

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

1987-02-01

198

The Penetration of Titanium Dioxide Nanoparticles: From Dermal Fibroblasts to Skin Tissue  

Science.gov (United States)

TiO2 particles are widely used in industry; however concerns are arising about their penetration into cells and tissue. In this study, we cultured dermal fibroblasts together with different commercial formulations of TiO2 nanoparticles and observed the morphology,traction forces, proliferation, and migration of the cells as a function of nanoparticles dispersion and concentration. The location of the particles within the cell was studied with TEM. We found significant penetration after 30 minutes. In all cases damage to cell structure and function was observed. Actin fibril formation was disturbed, proliferation was severely hindered, and cell motility was impaired. The effects were more pronounced in fibroblasts from older subjects. These effects were attributed to both dimensionality, as well as UV photocatalysis of the particles. The implications for tissues will be discussed. This work is supported by NSF-MRSEC program.

Sipzner, Lauren; Stettin, Jaimie; Pan, Zhi; Fang, Xiaohua; Lee, Wilson; Pernodet, Nadine; Rafailovich, Miriam

2006-03-01

199

Cholesteatoma Fibroblasts Promote Epithelial Cell Proliferation through Overexpression of Epiregulin  

Science.gov (United States)

To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b). To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial–mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.

Yoshikawa, Mamoru; Kojima, Hiromi; Yaguchi, Yuichiro; Okada, Naoko; Saito, Hirohisa; Moriyama, Hiroshi

2013-01-01

200

Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines  

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Full Text Available Abstract Background Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice. Results Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/- iMEFs was severely reduced while Gli2-/-3-/- iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/- iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent. Conclusion This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

Lipinski Robert J; Bijlsma Maarten F; Gipp Jerry J; Podhaizer David J; Bushman Wade

2008-01-01

 
 
 
 
201

Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines.  

UK PubMed Central (United Kingdom)

BACKGROUND: Hedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice. RESULTS: Here, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/-iMEFs was severely reduced while Gli2-/-3-/-iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/-iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent. CONCLUSION: This study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

Lipinski RJ; Bijlsma MF; Gipp JJ; Podhaizer DJ; Bushman W

2008-01-01

202

c-fos/c-jun expression and AP-1 activation in skin fibroblasts from centenarians.  

UK PubMed Central (United Kingdom)

In vitro replicative senescence is characterized by an irreversible growth arrest due to the inability of the cell to induce some key regulators of cell cycle progression, such as c-fos and AP-1, in response to mitogenic stimuli. In vitro replicative senescence and in vivo aging have been assumed to be two related phenomena, likely controlled by overlapping or interacting genes. As a corollary, fibroblasts from centenarians, which have undergone a long process of senescence in vivo should have very limited proliferative capability. On the contrary, in a previous work we found that fibroblasts from centenarians exhibited the same capacity to respond to different mitogenic stimuli as fibroblasts from young donors. Here we provide evidences that the well preserved proliferative response is likely due to the fact that some pivotal regulators- c-fos, c-jun and AP-1-are still fully inducible, despite a long process of in vivo senescence. Our data therefore suggest that in vivo and in vitro aging are separate phenomena whose possible relationships, if any, have to be ascertained very carefully.

Grassilli E; Bellesia E; Salomoni P; Croce MA; Sikora E; Radziszewska E; Tesco G; Vergelli M; Latorraca S; Barbieri D; Fagiolo U; Santacaterina S; Amaducci L; Tiozzo R; Sorbi S; Franceschi C

1996-09-01

203

Molecular Species of Phospholipids with Very Long Chain Fatty Acids in Skin Fibroblasts of Zellweger Syndrome.  

UK PubMed Central (United Kingdom)

The ratio of C26:0/C22:0 fatty acids in patient lipids is widely accepted as a critical clinical criterion of peroxisomal diseases, such as Zellweger syndrome and X-linked adrenoleukodystrophy (X-ALD). However, phospholipid molecular species with very long chain fatty acids (VLCFA) have not been precisely characterized. In the present study, the structures of such molecules in fibroblasts of Zellweger syndrome and X-ALD were examined using LC-ESI-MS/MS analysis. In fibroblasts from Zellweger patients, a large number of VLCFA-containing molecular species were detected in several phospholipid classes as well as neutral lipids, including triacylglycerol and cholesteryl esters. Among these lipids, phosphatidylcholine showed the most diversity in the structures of VLCFA-containing molecular species. Some VLCFA possessed longer carbon chains and/or larger number of double bonds than C26:0-fatty acid (FA). Similar VLCFA were also found in other phospholipid classes, such as phosphatidylethanolamine and phosphatidylserine. In addition, VLCFA-containing phospholipid species showed some differences among fibroblasts from Zellweger patients. It appears that phospholipids with VLCFA, with or without double bonds, as well as C26:0-FA might affect cellular functions, thus leading to the pathogenesis of peroxisomal diseases, such as Zellweger syndrome and X-ALD.

Hama K; Nagai T; Nishizawa C; Ikeda K; Morita M; Satoh N; Nakanishi H; Imanaka T; Shimozawa N; Taguchi R; Inoue K; Yokoyama K

2013-10-01

204

Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through ?V?3 integrin in human skin fibroblasts.  

Science.gov (United States)

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 ?V?3 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 ?V?3 integrin. The interaction of VWC domain with integrin ?V?3 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 ?V?3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation. PMID:23504324

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

2013-03-15

205

Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through ?V?3 integrin in human skin fibroblasts.  

UK PubMed Central (United Kingdom)

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 ?V?3 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 ?V?3 integrin. The interaction of VWC domain with integrin ?V?3 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 ?V?3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

Qin Z; Fisher GJ; Quan T

2013-04-01

206

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site.  

UK PubMed Central (United Kingdom)

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.

Fields GB; Van Wart HE; Birkedal-Hansen H

1987-05-01

207

Cyclobutane-type pyrimidine photodimer formation and induction of ornithine decarboxylase in human skin fibroblasts after UV irradiation  

International Nuclear Information System (INIS)

[en] Cyclobutane-type pyrimidine photodimers as well as the induction of ornithine decarboxylase (ODC) may serve as biochemical markers of the mutagenic and carcinogenic effects of ultraviolet light (UV). For this reason, it is important to compare the formation of pyrimidine dimers with the induction of ODC in human skin fibroblasts after irradiation with UVC (200-290 nm) and UVB (290-320 nm). In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer yields (T-T) by high-pressure liquid chromatography in cultures of neonatal normal human foreskin-derived fibroblasts after irradiation with UVC and UVB light. It was found that the yield of dimerization and the ratio of T-T/C-T decreased from the UVC to the UVB region. Time-course studies of ODC-induction in the same cells indicated that the maximal activity after UVB irradiation was retarded compared to UVC exposure. For the UV-induced ODC-levels, however, no significant difference in maximal induction could be measured after UVC and UVB irradiation at fluences where comparable yields of thymine dimerization are produced. Similar ODC-maxima were obtained with strains from children, while cells from adults showed significantly less pronounced ODC induction, indicating that ODC-response decreases with age and may therefore be used as a marker of aging

1988-01-01

208

Wound healing morbidity in STS patients treated with preoperative radiotherapy in relation to in vitro skin fibroblast radiosensitivity, proliferative capacity and TGF-? activity  

International Nuclear Information System (INIS)

Background and purpose: In a recent study, we demonstrated that the ability of dermal fibroblasts, obtained from soft tissue sarcoma (STS) patients, to undergo initial division in vitro following radiation exposure correlated with the development of wound healing morbidity in the patients following their treatment with preoperative radiotherapy. Transforming growth factor beta (TGF-?) is thought to play an important role in fibroblast proliferation and radiosensitivity both of which may impact on wound healing. Thus, in this study we examined the interrelationship between TGF-? activity, radiosensitivity and proliferation of cultured fibroblasts and the wound healing response of STS patients after preoperative radiotherapy to provide a validation cohort for our previous study and to investigate mechanisms. Patients and methods: Skin fibroblasts were established from skin biopsies of 46 STS patients. The treatment group consisted of 28 patients who received preoperative radiotherapy. Eighteen patients constituted a control group who were either irradiated postoperatively or did not receive radiation treatment. Fibroblast cultures were subjected to the colony forming and cytokinesis-blocked binucleation assays (low dose rate: ?0.02 Gy/min) and TGF-? assays (high dose-rate: ?1.06 Gy/min) following ?-irradiation. Fibroblast radiosensitivity and initial proliferative ability were represented by the surviving fraction at 2.4 Gy (SF2.4) and binucleation index (BNI), respectively. Active and total TGF-? levels in fibroblast cultures were determined using a biological assay. Wound healing complication (WHC), defined as the requirement for further surgery or prolonged deep wound packing, was the clinical endpoint examined. Results: Of the 28 patients treated with preoperative radiotherapy, 8 (29%) had wound healing difficulties. Fibroblasts from patients who developed WHC showed a trend to retain a significantly higher initial proliferative ability after irradiation compared with those from individuals in the treatment group with normal wound healing, consistent with the results of our previous study. No link was observed between fibroblast radiosensitivity and WHC. Neither active nor total TGF-? levels in cultures were significantly affected by irradiation. Fibroblast proliferation in unirradiated and irradiated cultures, as well as radiosensitivity, was not influenced by TGF-? content. TGF-? expression in fibroblast cultures did not reflect wound healing morbidity. Conclusions: These data are consistent with our previous study and combined the results suggest that in vitro fibroblast proliferation after irradiation may be a useful predictor of wound healing morbidity in STS patients treated with preoperative radiotherapy. TGF-? levels in culture do not predict WHC, suggesting that the role of TGF-? in wound healing is likely controlled by other in vivo factors.

2006-01-01

209

Sphinganine 1-phosphate metabolism in cultured skin fibroblasts: evidence for the existence of a sphingosine phosphatase.  

UK PubMed Central (United Kingdom)

On addition of [4,5-3H]sphinganine 1-phosphate to human fibroblast monolayers, the label was efficiently removed from the culture medium. In contrast with the reported stability of phosphorylated sphingenine in 3T3 cells [Desai, Zhang, Olivera, Mattie and Spiegel (1992). J. Biol. Chem. 267, 23122-23128] and B16 melanoma cells [Sadahira, Ruan, Hakomuri and Igarashi (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9686-9690], sphinganine 1-phosphate appeared to be subjected to a fast and extensive metabolism in fibroblasts, the major pathways being cleavage and dephosphorylation. The first of these pathways, catalysed by sphingosine-phosphate lyase, resulted in the formation of labelled palmitaldehyde, which was recovered, mainly after oxidation, in glycerophospholipids in an ester bond. A smaller part of the palmitaldehyde was reduced and incorporated in alk(en)ylphospholipids. Dephosphorylation of spinganine 1-phosphate, a hitherto overlooked pathway catalysed by an unknown phosphatase(s), gave rise to sphinganine, which was converted by N-acylation into ceramide and then incorporated in spingomyelin and glycosphingolipids.

Van Veldhoven PP; Mannaerts GP

1994-05-01

210

Sulphamidase activity in leucocytes, cultured skin fibroblasts and amniotic cells: diagnosis of the Sanfilippo A syndrome with the use of radiolabelled disaccharide substrate.  

UK PubMed Central (United Kingdom)

1. Sulphamidase activity was assayed by incubation of the radiolabelled disaccharide O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)- (1 leads to 3)-L-[6-3H]idonic acid with homogenates of leucocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with sulphamidase deficiency disorder [mucopolysaccharidosis type IIIA (MPS IIIA): the Sanfilippo A syndrome], parents of such patients and patients affected with other mucopolysaccharidoses and lysosomal enzyme deficiencies. 2. The assay clearly distinguished affected homozygotes from normal controls, heterozygotes and other mucopolysaccharidoses types. 3. Sulphamidase displayed remarkable thermal stability; reaction rates were constant for at least 24 h at 60 degrees C for leucocyte and 20 h at 37 degrees C for cultured fibroblast preparations. Apparent Km values for fibroblast sulphamidase were 71 mumol/l at 37 degrees C and 100 mumol/l at 50 degrees C; the corresponding Vmax, values were 21 and 72 pmol min-1 mg-1 of protein respectively. An incubation temperature of 60 degrees C was used for the routine assay of sulphamidase activity in leucocytes, urine and amniotic supernatant preparations. The specific activities of fibroblast and amniotic cell sulphamidase, assessed at incubation temperatures of 37 degrees C, were more than 10-fold the leucocyte enzyme activity at 60 degrees C. 4. We recommend the use of radiolabelled disaccharide substrate for the assay of sulphamidase in leucocytes, skin fibroblasts and urine, for the routine enzymic detection of the sulphamidase deficiency disorder of the Sanfilippo A syndrome.

Hopwood JJ; Elliott H

1981-12-01

211

Sulphamidase activity in leucocytes, cultured skin fibroblasts and amniotic cells: diagnosis of the Sanfilippo A syndrome with the use of radiolabelled disaccharide substrate.  

Science.gov (United States)

1. Sulphamidase activity was assayed by incubation of the radiolabelled disaccharide O-(alpha-2-sulphamino-2-deoxy-D-glucopyranosyl)- (1 leads to 3)-L-[6-3H]idonic acid with homogenates of leucocytes and cultured skin fibroblasts and concentrates of urine derived from normal individuals, patients affected with sulphamidase deficiency disorder [mucopolysaccharidosis type IIIA (MPS IIIA): the Sanfilippo A syndrome], parents of such patients and patients affected with other mucopolysaccharidoses and lysosomal enzyme deficiencies. 2. The assay clearly distinguished affected homozygotes from normal controls, heterozygotes and other mucopolysaccharidoses types. 3. Sulphamidase displayed remarkable thermal stability; reaction rates were constant for at least 24 h at 60 degrees C for leucocyte and 20 h at 37 degrees C for cultured fibroblast preparations. Apparent Km values for fibroblast sulphamidase were 71 mumol/l at 37 degrees C and 100 mumol/l at 50 degrees C; the corresponding Vmax, values were 21 and 72 pmol min-1 mg-1 of protein respectively. An incubation temperature of 60 degrees C was used for the routine assay of sulphamidase activity in leucocytes, urine and amniotic supernatant preparations. The specific activities of fibroblast and amniotic cell sulphamidase, assessed at incubation temperatures of 37 degrees C, were more than 10-fold the leucocyte enzyme activity at 60 degrees C. 4. We recommend the use of radiolabelled disaccharide substrate for the assay of sulphamidase in leucocytes, skin fibroblasts and urine, for the routine enzymic detection of the sulphamidase deficiency disorder of the Sanfilippo A syndrome. PMID:6794973

Hopwood, J J; Elliott, H

1981-12-01

212

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, xp heterozygotes and normal skin fibroblasts  

International Nuclear Information System (INIS)

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 ?g N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus. (Auth.)

1978-01-01

213

Skin graft rejection in mice repopulated with marrow of the skin donor type: a Skn gene in a congenic line  

International Nuclear Information System (INIS)

Genetically anemic W/Wv mice and lethally irradiated wild-type mice were cured and populated by grafted marrow cells from donor mice of three congenic lines that differed at non-H-2 histocompatibility loci. Tail skin from mice of the same congenic lines was grafted 3-4 weeks later. In two cases, the recipients behaved as expected, no longer rejecting skin syngeneic with the marrow graft that had repopulated them. However, B6-H-24c skin was rejected by WBB6F1-W/Wv mice that were cured with B6-H-24c marrow showing a mean survival time of 9.9 weeks. It was rejected somewhat faster, with a mean survival time of 5.9 weeks, by W/Wv mice cured with marrow from other types of donors. Results were more variable in lethally irradiated WBB6F1-+/+ recipients of B6-H-24c marrow, but they also rejected B6-H-24c skin. Both types of recipients remained chimeras after the skin was rejected, showing more than 90% of the B6-H-24c hemoglobin type. This is the first report of a Skn gene in a congenic line

1984-01-01

214

Skin graft rejection in mice repopulated with marrow of the skin donor type: a Skn gene in a congenic line  

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Genetically anemic W/Wv mice and lethally irradiated wild-type mice were cured and populated by grafted marrow cells from donor mice of three congenic lines that differed at non-H-2 histocompatibility loci. Tail skin from mice of the same congenic lines was grafted 3-4 weeks later. In two cases, the recipients behaved as expected, no longer rejecting skin syngeneic with the marrow graft that had repopulated them. However, B6-H-24c skin was rejected by WBB6F1-W/Wv mice that were cured with B6-H-24c marrow showing a mean survival time of 9.9 weeks. It was rejected somewhat faster, with a mean survival time of 5.9 weeks, by W/Wv mice cured with marrow from other types of donors. Results were more variable in lethally irradiated WBB6F1-+/+ recipients of B6-H-24c marrow, but they also rejected B6-H-24c skin. Both types of recipients remained chimeras after the skin was rejected, showing more than 90% of the B6-H-24c hemoglobin type. This is the first report of a Skn gene in a congenic line.

Harrison, D.E.; Mobraaten, L.E.

1984-01-01

215

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines  

Directory of Open Access Journals (Sweden)

Full Text Available The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

ÁNGELA D ARMENDÁRIZ; FELIPE OLIVARES; RODRIGO PULGAR; ALEX LOGUINOV; VERÓNICA CAMBIAZO; CHRISTOPHER D VULPE; MAURICIO GONZÁLEZ

2006-01-01

216

Adipose tissue-derived mesenchymal cells support skin reepithelialization through secretion of KGF-1 and PDGF-BB: comparison with dermal fibroblasts.  

UK PubMed Central (United Kingdom)

Epidermal organization and homeostasis are regulated by mesenchymal influences through paracrine actions. Until today, dermal fibroblasts (DFs) are used in the "dermal" layer to support keratinocyte growth in vitro in dermal and skin substitutes. In the present work, we used human adipose tissue-derived mesenchymal cells (ADMCs) as a support of keratinocyte growth in vitro (in monolayer culture and in 3D skin cell culture models) and in vivo (mouse wound healing models) and compared our findings with those obtained using dermal fibroblasts. ADMCs induce reepithelialization during wound healing more efficiently than DFs, by enhancing keratinocyte proliferation through cell cycle progression, and migration. This effect is mediated (at least partially) by a paracrine action of KGF-1 and PDGF-BB, which are more prominently expressed in ADMCs than in DFs. Furthermore, replacement of DFs by ADMCs in the dermal compartment of organotypic skin cultures leads to an artificial epidermis resembling to that of normal skin, concerning the general histology, although with a higher expression of cytokeratins 5 and 19. In Rag1 knockout mice, ADMCs induced a more rapid reepithelialization and a more effective wound healing, compared to dermal fibroblasts. In conclusion, we provide evidence that ADMCs can serve as supportive cells for primary keratinocyte cultures. In addition, because of their abundance and the great cell yield achieved during ADMC isolation, they represent an interesting cell source, with potential aspects for clinical use.

Alexaki VI; Simantiraki D; Panayiotopoulou M; Rasouli O; Venihaki M; Castana O; Alexakis D; Kampa M; Stathopoulos EN; Castanas E

2012-01-01

217

MRI Breast Skin-line Segmentation and Removal using Integration Method of Level Set Active Contour and Morphological Thinning Algorithms  

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In the MRI breast Computer Aided Detection systems, skin-line segmentation and removal is a vital process. Similar intensity levels of the skin-line and the other parts of the breast image such as; dense tissue and tumor could possibly lead to faulty tumor segmentation if the skin-line is not rem...

Ali Qusay Al-Faris; Umi Kalthum Ngah; Nor Ashidi Mat Isa; Ibrahim Lutfi Shuaib

218

Transcriptome Comparison of Human Neurons Generated Using Induced Pluripotent Stem Cells Derived from Dental Pulp and Skin Fibroblasts  

Science.gov (United States)

Induced pluripotent stem cell (iPSC) technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD) and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs) and fibroblasts (F-iPSCs). This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (p<0.05), of which 63 remained significant after correcting for genome-wide analysis (FDR <0.05). The most striking difference was the lower level of expression detected for numerous members of the all four HOX gene families in neurons derived from T-iPSCs. In addition, an increased level of expression was seen for several transcription factors expressed in the developing forebrain (FOXP2, OTX1, and LHX2, for example). Overall, pathway analysis revealed that differentially expressed genes that showed higher levels of expression in neurons derived from T-iPSCs were enriched for genes implicated in schizophrenia (SZ). The findings suggest that neurons derived from T-iPSCs are suitable for disease-modeling neuropsychiatric disorder and may have some advantages over those derived from F-iPSCs.

Foxe, John J.; Pedrosa, Erika; Hrabovsky, Anastasia; Carroll, Reed; Zheng, Deyou; Lachman, Herbert M.

2013-01-01

219

Usefulness of Basic Fibroblast Growth Factor (bFGF) Loaded Dissolving Microneedles for Local Therapy of Skin Wounds  

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Full Text Available Purpose: The usefulness of dissolving microneedles (DMs) for local skin therapy by basic fibroblast growth factor (bFGF) was studied in rats. Methods: We prepared four kinds of bFGF-loaded DMs, approximately 500 ?m length and 300 ?m diameter at the bottom. Long-term stability and dissolution studies were performed by HPLC method. Pharmacokinetic and pharmacological evaluations were performed after administration of bFGF loaded DMs to rats. Results: The bFGF contents were 2.15 ± 0.07, 1.07 ± 0.04, 0.56 ± 0.07 and 0.12 ± 0.03 ?g. The 100.2 ± 3.4%, 100.2 ± 3.3%, 99.3 ± 1.4% and 100.4 ± 3.0% of bFGF were recovered after 1, 3 and 6 months and 1 year incubation at 40°C. The bFGF was released from DMs within 5 min. In a pharmacokinetic study using 2.0 and 1.0 ?g bFGF-loaded DMs, no systemic exposure of bFGF was detected. The initial bFGF concentrations in the rat skin tissue after administration of bFGF-loaded DMs to the hair-removed rat abdominal skin were 510.2 ± 20.1 ng/g wet weight for 2 ?g bFGF DMs and 264.2 ± 56.5 ng/g wet weight for 1 ?g DMs, declining slowly thereafter to 226.3 ± 33.5 and 105.1 ± 27.4 ng/g wet weight at 6 hr after administration. Good dose-dependency was observed. Pharmacological evaluation of bFGF-loaded DMs of 2.0, 1.0, 0.5, and 0.1 ?g, in the wound healing rat model, all used DMs, but 0.1 ?g DMs, showed good healing effects. Considered collectively, these results suggest the usefulness of bFGF-loaded DMs for local therapy of skin wound disease.

Kanji Takada; Yukako Ito; Kengo Matsumoto; Yuka Sato; Maimi Nishio; Yurie Tadano; Yuri Kamei; Yutaro Takemura; Nana Inoue; Yoshiki Akasaka; Ichiro Ono

2013-01-01

220

EFFECTS OF CIPROFLOXACIN ON GROWTH OF HUMAN RHABDOMYOSARCOMA (RD) AND RAT EMBRYO FIBROBLASTS (REF) CELL LINES: IN VITRO STUDY  

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Full Text Available The possible anti-proliferative effects of ciprofloxacin utilizing cell lines obtained from different sources [human rhabdomyosarcoma (RD) and transitional rat embryo fibroblasts (REF) – (passage89)] were studied. The present study was carried out from January 2011 to May 2011.Each of cell lines mentioned above was exposed to various concentrations of ciprofloxacin at concentrations (from 62.5 to 1000 mcg/ml) for 48hours in addition to control (zero) concentration. Four replicates were used for each data point and the results were expressed as mean ± SD. Ciprofloxacin caused significant growth inhibition on both cell lines only at 1000 mcg/ml concentration; but does not exert in vitro inhibitory effect on either human rhabdomyosarcoma (RD) or transformed rat embryo fibroblasts (REF) when assayed at concentrations of less than 1000 micrograms/ml.

Nada N. Al-Shawi

2012-01-01

 
 
 
 
221

TGF-?1-treated ADSCs-CM promotes expression of type I collagen and MMP-1, migration of human skin fibroblasts, and wound healing in vitro and in vivo.  

UK PubMed Central (United Kingdom)

Conditioned medium from adipose-derived stem cells (ADSCs) stimulates both collagen synthesis and migration of dermal fibroblasts. However, it is still unknown whether conditioned media from tumor growth factor (TGF)-?1-treated ADSCs (TGF-?1-treated ADSCs-CM) induces increased expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and migration as well as cell cycle regulatory proteins in fibroblasts, compared to non-treated ADSCs-CM. Our data showed that TGF-?1-treated ADSCs-CM promoted effectively the proliferation and migration of human skin fibroblasts, compared to non-treated ADSCs-CM. In addition the expression of MMP-1 were markedly increased by treatment of TGF-?1-treated ADSCs-CM in fibroblasts, compared to non-treated ADSCs-CM. Expression of type I collagen protein were slightly increased by treatment of TGF-?1-treated ADSCs-CM in fibroblasts. The expression of cell cycle regulators of G1/S phase transition were not markedly altered by treatment of TGF-?1-treated ADSCs-CM. Finally, artificial wounds were made using a 4-mm punch biopsy in hairless mice and TGF-?1-treated ADSCs-CM were injected into the wound area. The injection of TGF-?1-treated ADSCs-CM promoted the wound healing process in hairless mice. Taken together, our data indicated that TGF-?1-treated ADSCs-CM induced up-regulation of type I collagen and MMP-1, promoted the migration of skin fibroblasts, and thereby promoted the wound healing process in vivo. Our data indicate that TGF-?1-treated ADSCs-CM will be a component for a wound healing accelerating agent.

Cho JW; Kang MC; Lee KS

2010-12-01

222

TGF-?1-treated ADSCs-CM promotes expression of type I collagen and MMP-1, migration of human skin fibroblasts, and wound healing in vitro and in vivo.  

Science.gov (United States)

Conditioned medium from adipose-derived stem cells (ADSCs) stimulates both collagen synthesis and migration of dermal fibroblasts. However, it is still unknown whether conditioned media from tumor growth factor (TGF)-?1-treated ADSCs (TGF-?1-treated ADSCs-CM) induces increased expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and migration as well as cell cycle regulatory proteins in fibroblasts, compared to non-treated ADSCs-CM. Our data showed that TGF-?1-treated ADSCs-CM promoted effectively the proliferation and migration of human skin fibroblasts, compared to non-treated ADSCs-CM. In addition the expression of MMP-1 were markedly increased by treatment of TGF-?1-treated ADSCs-CM in fibroblasts, compared to non-treated ADSCs-CM. Expression of type I collagen protein were slightly increased by treatment of TGF-?1-treated ADSCs-CM in fibroblasts. The expression of cell cycle regulators of G1/S phase transition were not markedly altered by treatment of TGF-?1-treated ADSCs-CM. Finally, artificial wounds were made using a 4-mm punch biopsy in hairless mice and TGF-?1-treated ADSCs-CM were injected into the wound area. The injection of TGF-?1-treated ADSCs-CM promoted the wound healing process in hairless mice. Taken together, our data indicated that TGF-?1-treated ADSCs-CM induced up-regulation of type I collagen and MMP-1, promoted the migration of skin fibroblasts, and thereby promoted the wound healing process in vivo. Our data indicate that TGF-?1-treated ADSCs-CM will be a component for a wound healing accelerating agent. PMID:21042785

Cho, Jae-We; Kang, Min-Chul; Lee, Kyu-Suk

2010-12-01

223

Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite  

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We have shown that UVA (320-380 nm) radiation, hydrogen peroxide, and sodium arsenite induce a stress protein of approximately 32 kDa in human skin fibroblasts. The synthesis and cloning of cDNA from arsenite-induced mRNA populations have now allowed us to unequivocally identify the 32-kDa protein as heme oxygenase. By mRNA analysis we have shown that the heme oxygenase gene is also induced in cultured human skin fibroblasts by UVA radiation, hydrogen peroxide, cadmium chloride, iodoacetamide, and menadione. The known antioxidant properties of heme catabolites taken together with the observation of a high level of induction of the enzyme in cells from an organ not involved in hemoglobin breakdown strongly supports the proposal that the induction of heme oxygenase may be a general response to oxidant stress and constitutes an important cellular defense mechanism against oxidative damage.

Keyse, S.M.; Tyrrell, R.M.

1989-01-01

224

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

Science.gov (United States)

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.

Hawkins, Denise H.; Abrahamse, Heidi

2007-06-01

225

Ionising radiation response of mouse embryonic fibroblast cell lines lacking cohesin REC8  

International Nuclear Information System (INIS)

Full text: The cohesin complex is required for the cohesion of sister chromatids. Cohesin is loaded onto chromosomes during DNA replication and maintains sister chromatid association until anaphase. The cohesin complex is also implicated in homologous recombination, the upkeep of chromosome stability, the repair of double stranded DNA breaks (dsbs) and most recently, chromatin remodelling. REC8 is a key component of the meiotic cohesin complex that we showed was conserved from yeast to humans (Parisi, et. al., 1999). We identified a mouse ortholog of the Rec8 gene and generated Rec8 mutant mice by targeted deletion. To determine the role of REC8 in DNA repair, we derived embryonic fibroblast cell lines from Rec8 mutant mice. By clonogenic assay we are evaluating the resistance of Rec8 -/- cells to ionising (IR) radiation, an indirect measure of repair of dsbs. The study may provide insights into the involvement of cohesins in recombination and IR response. Reference: Parisi, S., McKay, M.J., Molnar, M., Thompson, M.A., van der Spek, P.J., van Drunken-Schoenmaker, E., Kanaar, R., Lehmann, E., Hoeijmakers, J.H.J. and Kohli, J., 1999. Rec8p, a meiotic recombination and sister chromatid cohesion phosphoprotein of the Rad21 family conserved from fission yeast to humans. Mol Cell Biol. 19(5):3515-3528

2003-01-01

226

Radio electric conveyed fields directly reprogram human dermal skin fibroblasts toward cardiac, neuronal, and skeletal muscle-like lineages.  

Science.gov (United States)

Somatic cells can be directly reprogrammed to alternative differentiated fates without first becoming stem/progenitor cells. Nevertheless, the initial need for viral-mediated gene delivery renders this strategy unsafe in humans. Here, we provide evidence that exposure of human skin fibroblasts to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering radio electric conveyed fields at a radiofrequency of 2.4 GHz, afforded remarkable commitment toward cardiac, neuronal, and skeletal muscle lineages. REAC induced the transcription of tissue-restricted genes, including Mef2c, Tbx5, GATA4, Nkx2.5, and prodynorphin for cardiac reprogramming, as well as myoD, and neurogenin 1 for skeletal myogenesis and neurogenesis, respectively. Conversely, REAC treatment elicited a biphasic effect on a number of stemness-related genes, leading to early transcriptional increase of Oct4, Sox2, cMyc, Nanog, and Klf4 within 6-20 h, followed by a downregulation at later times. The REAC action bypassed a persistent reprogramming toward an induced pluripotent stem cell-like state and involved the transcriptional induction of the NADPH oxidase subunit Nox4. Our results show for the first time the feasibility of using a physical stimulus to afford the expression of pluripotentiality in human adult somatic cells up to the attainment of three major target lineages for regenerative medicine. PMID:23057961

Maioli, Margherita; Rinaldi, Salvatore; Santaniello, Sara; Castagna, Alessandro; Pigliaru, Gianfranco; Gualini, Sara; Cavallini, Claudia; Fontani, Vania; Ventura, Carlo

2012-10-02

227

Radio electric conveyed fields directly reprogram human dermal skin fibroblasts toward cardiac, neuronal, and skeletal muscle-like lineages.  

UK PubMed Central (United Kingdom)

Somatic cells can be directly reprogrammed to alternative differentiated fates without first becoming stem/progenitor cells. Nevertheless, the initial need for viral-mediated gene delivery renders this strategy unsafe in humans. Here, we provide evidence that exposure of human skin fibroblasts to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering radio electric conveyed fields at a radiofrequency of 2.4 GHz, afforded remarkable commitment toward cardiac, neuronal, and skeletal muscle lineages. REAC induced the transcription of tissue-restricted genes, including Mef2c, Tbx5, GATA4, Nkx2.5, and prodynorphin for cardiac reprogramming, as well as myoD, and neurogenin 1 for skeletal myogenesis and neurogenesis, respectively. Conversely, REAC treatment elicited a biphasic effect on a number of stemness-related genes, leading to early transcriptional increase of Oct4, Sox2, cMyc, Nanog, and Klf4 within 6-20 h, followed by a downregulation at later times. The REAC action bypassed a persistent reprogramming toward an induced pluripotent stem cell-like state and involved the transcriptional induction of the NADPH oxidase subunit Nox4. Our results show for the first time the feasibility of using a physical stimulus to afford the expression of pluripotentiality in human adult somatic cells up to the attainment of three major target lineages for regenerative medicine.

Maioli M; Rinaldi S; Santaniello S; Castagna A; Pigliaru G; Gualini S; Cavallini C; Fontani V; Ventura C

2013-01-01

228

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal (more) and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

ARMENDÁRIZ, ÁNGELA D; OLIVARES, FELIPE; PULGAR, RODRIGO; LOGUINOV, ALEX; CAMBIAZO, VERÓNICA; VULPE, CHRISTOPHER D; GONZÁLEZ, MAURICIO

2006-01-01

229

The combined effect of interferon beta and radiation on five human tumor cell lines and embryonal lung fibroblasts  

International Nuclear Information System (INIS)

Purpose: The combined effect of natural Interferon-beta (n-IFN-?) and ionizing radiation was tested in vitro on 5 different tumor cell lines and 1 embryonal lung fibroblast cell line. Materials and Methods: The following cell lines were used: A549 (lung cancer), MCF-7 (breast cancer), CaSki (cervical cancer), WiDr (colon cancer), ZMK-1 (head and neck cancer), and MRC-5 (embryonal lung fibroblast line). Cells were incubated with n-IFN-? (30 I.U./ml to 3000 I.U./ml) 24 h before irradiation. Irradiation was given as single dose between 1 and 6 Gy. Cell survival was evaluated using a standard colony-forming assay. Results: Incubation with n-IFN-? enhanced the effect of radiation in all tumor cell lines tested. The maximum sensitizing enhancement ratios (SER) at the 37% survival level were: 1.66 for A549 cells, 1.47 for CaSki cells, 1.56 for MCF-7 cells, 1.40 for WiDr cells, and 1.57 for ZMK-1 cells. In the nonneoplastic MRC-5 cell line, no radiosensitizing effect of n-IFN-? could be demonstrated. The linear quadratic fit of the survival curves showed an increase of the ?-component for all tumor cell lines treated with n-IFN-?. Conclusions: IFN-? enhanced the effect of radiation in the tumor cell lines, but not in the nonmalignant lung fibroblasts. The increase of the ? component in the survival curves indicates that impaired radiation repair or the accumulation of sublethal damage might play a role for the radiosensitizing effect of n-IFN-?.

1999-01-15

230

Effect of human skin explants on the neurite growth of the PC12 cell line.  

UK PubMed Central (United Kingdom)

The skin is a densely innervated organ. After a traumatic injury, such as an amputation, burn or skin graft, nerve growth and the recovery of sensitivity take a long time and are often incomplete. The roles played by growth factors and the process of neuronal growth are crucial. We developed an in vitro model of human skin explants co-cultured with a rat pheochromocytoma cell line differentiated in neuron in presence of nerve growth factor (NGF). This model allowed the study of the influence of skin explants on nerve cells and nerve fibre growth, probably through mediators produced by the explant, in a simplified manner. The neurite length of differentiated PC12 cells co-cultured with skin explants increased after 6 days. These observations demonstrated the influence of trophic factors produced by skin explants on PC12 cells.

Lebonvallet N; Pennec JP; Le Gall C; Pereira U; Boulais N; Cheret J; Jeanmaire C; Danoux L; Pauly G; Misery L

2013-03-01

231

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

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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.

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

1987-02-01

232

Separation and cultivation of laryngeal carcinoma-associated fibroblasts and biological influence on a laryngeal carcinoma cell line.  

UK PubMed Central (United Kingdom)

CONCLUSIONS: Carcinoma-associated fibroblasts (CAFs) can influence the biological characteristics of a laryngeal carcinoma cell line. These results could lay the foundation for further studies on the role of CAFs in the laryngeal tumor-host microenvironment. OBJECTIVE: CAFs are important contributors to the microenvironment in determining the fate of tumors. The aim of this study was to separate, culture, and identify laryngeal CAFs and investigate their biological influence on the laryngeal carcinoma cell line. METHODS: The primary CAFs and normal fibroblasts (NFs) of the larynx were obtained by tissue culture. The cells were verified according to immunohistochemical and immunofluorescence staining of certain proteins. Conditioned medium (CM) from CAFs and NFs was obtained. Functional assays were performed to test the influence of each CM on laryngeal carcinoma cell lines. RESULTS: Third-passage purified laryngeal CAFs and NFs were successfully attained. The CAFs showed positive staining for vimentin, ?-smooth muscle actin (?-SMA), and fibroblast activation protein (FAP). The migration ability of the CAFs increased significantly compared with that of NFs (p < 0.05). CM from CAFs (compared with CM from NFs) stimulated proliferation, migration, and invasion to a greater extent.

Du HD; Wu CP; Zhou L; Tian J

2013-07-01

233

Cytotoxicity of endodontic irrigants containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from mouse L929 cell line.  

UK PubMed Central (United Kingdom)

The aim of this study was to evaluate the cytotoxicity of root canal irrigating solutions containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from L929 cell line. Saturated calcium hydroxide aqueous solution (CH), sodium lauryl sulphate (SLS) and SLS associated with calcium hydroxide (HCT20) were diluted with sterile distilled water at 50%, 20%, 10% and 5% concentrations. Minimum essential medium (MEM) served as the control group. The cytotoxicity of the solutions was evaluated on L929 mouse fibroblast cell line, at 4 and 24 h of contact time by the 51Cr radiotracer method. Data were compared and statistical inferences were made with the chi-square test. In all analysis, significance level was set at 5%. CH and HCT20 showed toxicity at 50% concentration, while at concentrations lower than 50% these solutions showed cell tolerance. SLS was cytotoxic at all concentrations. In conclusion, the association of calcium hydroxide and SLS (HCT20) combines the beneficial properties of these solutions and was not harmful to the fibroblast cell line, seeming to be a suitable endodontic irrigating solution.

Barbosa SV; Barroso CM; Ruiz PA

2009-01-01

234

MRI Breast Skin-line Segmentation and Removal using Integration Method of Level Set Active Contour and Morphological Thinning Algorithms  

Directory of Open Access Journals (Sweden)

Full Text Available In the MRI breast Computer Aided Detection systems, skin-line segmentation and removal is a vital process. Similar intensity levels of the skin-line and the other parts of the breast image such as; dense tissue and tumor could possibly lead to faulty tumor segmentation if the skin-line is not removed correctly. In this study, a technique for skin-line segmentation and removal is presented. The approach integrates two algorithms. Level Set Active Contour algorithm is used to segment the breast skin border; the Morphological Thinning Algorithm is used to delete the detected breast skin-line. The approach is applied and tested on the RIDER breast MRI dataset and the results are evaluated using six measures. The evaluation results show high performance for the proposed approach with accuracy of 0.9607 for the skin-line segmentation stage and 0.9099 for the removal stage.

Ali Qusay Al-Faris; Umi Kalthum Ngah; Nor Ashidi Mat Isa; Ibrahim Lutfi Shuaib

2012-01-01

235

In vitro skin equivalents  

Directory of Open Access Journals (Sweden)

Full Text Available Sun Pharmaceutical Advanced research Centre (SPARC),17/B, Off Mahakali caves road, Mahal Indl. Estate,Andheri (E), Mumbai-400 093,INDIA,email : hemantjoshee@sify.comTo assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms.Keywords: In vitro models, keratinocytes, fibroblasts, skin re-epithelialization, in situ hybridization, cadaver skin

Hemant P. Joshi

2006-01-01

236

Human fibroblast-derived cell lines have characteristics of embryonic stem cells and cells of neuro-ectodermal origin.  

UK PubMed Central (United Kingdom)

Fibroblasts are the most ubiquitous cells in complex organisms. They are the main cells of stromal tissue and play an important role in repair and healing of damaged organs. Here we report new data-initially serendipitous findings-that fibroblast-derived cell line (human fetal lung derived cells, MRC-5) have the morphology, growth rate and gene expression pattern characteristic of embryonic stem cells and cells of neuro-ectodermal origin. We have developed a serum-free culture system to maintain these cells in proliferative state. We discovered that, at proliferative state, these cells express transcription factors of pluripotent cells, OCT-3/4 and REX-1, and embryonic cell surface antigens SSEA-1, SSEA-3, and SSEA-4, as well as TRA-1-60 and TRA-1-81. In addition to embryonic cell markers, the fibroblasts expressed neuroectodermal genes: Musashi-1, nestin, medium neurofilament, and beta-III tubulin. RT-PCR data revealed that mesencephalic transcription factors, Nurr-1 and PTX-3, were also expressed in MRC-5 cells, and that these cells could be induced to express tyrosine hydroxylase (TH). Expression of TH followed down-regulation of genes associated with cell proliferation, OCT-3/4, REX-1, and beta-catenin. These data indicate that the cells commonly known as fibroblasts have some of the characteristics of stem cells, and can be induced to become neuroectodermal cells and perhaps even mature neurons.

Rieske P; Krynska B; Azizi SA

2005-12-01

237

Pallister-Killian syndrome: normal karyotype in prenatal chorionic villi, in postnatal lymphocytes, and in slowly growing epidermal cells, but mosaic tetrasomy 12p in skin fibroblasts.  

UK PubMed Central (United Kingdom)

We report on two patients with Pallister-Killian syndrome: an 18 month old male infant followed since the neonatal period and a 4 year old boy. Prenatal diagnosis by chorionic villi sampling (CVS) in the first case showed a normal karyotype without mosaicism. Chromosome analysis on peripheral lymphocytes of the newborn also showed a normal karyotype. The clinical diagnosis of Pallister-Killian syndrome was made after the first year of life because of the typical facial dysmorphism and other characteristic clinical features, such as frontotemporal alopecia, depigmented area of the skin, sensorineural hearing loss, and severe psychomotor retardation. Chromosome analysis from skin fibroblasts now showed an isochromosome 12p mosaicism. The origin of the extra chromosome was confirmed by in situ hybridisation using a chromosome 12 specific library. In the second case chromosomal analysis from peripheral lymphocytes at the age of 19 months showed a normal karyotype 46,XY. Following the clinical diagnosis of Pallister-Killian syndrome a superficial skin biopsy was performed which showed very poor and slow growth of cells and a normal karyotype. Because of the typical symptoms a larger and deeper skin biopsy was performed from which there was rapid growth of fibroblasts. Now the diagnosis was established on the basis of the presence of an i(12p) extra chromosome in 69% of the metaphases.

Horn D; Majewski F; Hildebrandt B; Körner H

1995-01-01

238

Pallister-Killian syndrome: normal karyotype in prenatal chorionic villi, in postnatal lymphocytes, and in slowly growing epidermal cells, but mosaic tetrasomy 12p in skin fibroblasts.  

Science.gov (United States)

We report on two patients with Pallister-Killian syndrome: an 18 month old male infant followed since the neonatal period and a 4 year old boy. Prenatal diagnosis by chorionic villi sampling (CVS) in the first case showed a normal karyotype without mosaicism. Chromosome analysis on peripheral lymphocytes of the newborn also showed a normal karyotype. The clinical diagnosis of Pallister-Killian syndrome was made after the first year of life because of the typical facial dysmorphism and other characteristic clinical features, such as frontotemporal alopecia, depigmented area of the skin, sensorineural hearing loss, and severe psychomotor retardation. Chromosome analysis from skin fibroblasts now showed an isochromosome 12p mosaicism. The origin of the extra chromosome was confirmed by in situ hybridisation using a chromosome 12 specific library. In the second case chromosomal analysis from peripheral lymphocytes at the age of 19 months showed a normal karyotype 46,XY. Following the clinical diagnosis of Pallister-Killian syndrome a superficial skin biopsy was performed which showed very poor and slow growth of cells and a normal karyotype. Because of the typical symptoms a larger and deeper skin biopsy was performed from which there was rapid growth of fibroblasts. Now the diagnosis was established on the basis of the presence of an i(12p) extra chromosome in 69% of the metaphases. Images

Horn, D; Majewski, F; Hildebrandt, B; Korner, H

1995-01-01

239

Hypoxic culture of human pluripotent stem cell lines is permissible using mouse embryonic fibroblasts.  

UK PubMed Central (United Kingdom)

AIM: Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate. MATERIALS & METHODS: Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A. RESULTS: Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers. CONCLUSION: The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.

Badger JL; Byrne ML; Veraitch FS; Mason C; Wall IB; Caldwell MA

2012-09-01

240

Effect of chlorhexidine gluconate on the skin integrity at PICC line sites.  

UK PubMed Central (United Kingdom)

OBJECTIVE: To determine the effects of chlorhexidine gluconate (CHG) on skin inflammation and stratum corneum barrier integrity at peripherally inserted central catheter (PICC) sites among patients in the neonatal intensive care setting. STUDY DESIGN: In a within-subject design, PICC sites with CHG plus semipermeable dressing (PICC) were compared with contralateral dressing sites and untreated controls among 40 neonates (gestational age 32.1+/-4.7) at weekly dressing changes, using quantitative measures of skin erythema, dryness and barrier integrity (transepidermal water loss, TEWL). Data were analyzed using analysis of variance and linear mixed methods. RESULTS: At week 1, all three sites differed for erythema with the highest value indicating poorer skin condition at the PICC site. Dressing-site erythema was higher than the untreated control. Dryness and TEWL were higher, indicating poorer skin integrity, for the PICC site than either the dressing or the control. After 2 weeks, erythema and dryness scores were higher for the PICC site than the dressing and control skin. By week 3, scores were comparable for PICC and dressing sites and both were higher than the control for erythema and dryness. After 3 weeks, PICC skin TEWL was higher than both dressing and control and they did not differ from each other. CONCLUSION: The dressings used to secure PICC lines contribute to the observed skin compromise at CHG-treated skin sites and may affect skin barrier development in similar populations of neonates.

Visscher M; deCastro MV; Combs L; Perkins L; Winer J; Schwegman N; Burkhart C; Bondurant P

2009-12-01

 
 
 
 
241

Quantitation of androgen receptor messenger RNA from genital skin fibroblasts by reverse transcription--competitive polymerase chain reaction.  

Science.gov (United States)

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions. PMID:9712409

Nirdé, P; Georget, V; Térouanne, B; Galifer, R B; Belon, C; Sultan, C

1998-07-01

242

Quantitation of androgen receptor messenger RNA from genital skin fibroblasts by reverse transcription--competitive polymerase chain reaction.  

UK PubMed Central (United Kingdom)

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.

Nirdé P; Georget V; Térouanne B; Galifer RB; Belon C; Sultan C

1998-07-01

243

[The study of quantitative karyotypic variability by induction of chromosomal instability in cultured cells of the Indian muntjac skin fibroblasts  

UK PubMed Central (United Kingdom)

The influence of mycoplasmal contamination and somatic cell hybridization on the character of karyotypic variability in cell cultures of Indian muntjac skin fibroblasts has been investigated. Mycoplasma arginini and Acholeplasma laidlawii, used as factors inducing chromosomal instability, do not break the main regulations peculiar to intact control. They regulations are: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous mainly single directed numeral deviations. However, mycoplasmal contamination promotes the increase in the number of deviations in the direction of a decreasing chromosomes number. There is a breach of some connections between chromosomes by simultaneous deviations. They are chromosomes with broken connections according to the number of deviations which form telomeric associations (dicentrics). The number of these associations excel essentially intact control. The formation of new MSVK in subline M2 cells of the Indian muntjac in the process of chromosomal segregation in cell hybrid (M2 x clone of JF1 rat Jensen sarcoma) depends on the presence of significant connections between chromosomes by simultaneous numerical deviations in direction of MSVK formation. They are chromosomes that take part in the formation of new MSVK which form telomeric associations. These associations can be observed till stabilization of new MSVK. Probably, the support of the balance of karyotypic structure by factors inducing chromosomal instability is connected with change of some connections between chromosomes according to the number by simultaneous deviations as well as with the formation of dicentrics.

Polianskaia GG; Samokish VA

1999-01-01

244

[The study of quantitative karyotypic variability by induction of chromosomal instability in cultured cells of the Indian muntjac skin fibroblasts].  

Science.gov (United States)

The influence of mycoplasmal contamination and somatic cell hybridization on the character of karyotypic variability in cell cultures of Indian muntjac skin fibroblasts has been investigated. Mycoplasma arginini and Acholeplasma laidlawii, used as factors inducing chromosomal instability, do not break the main regulations peculiar to intact control. They regulations are: 1) nonrandom character of cell distribution according to the number of chromosomal deviations from MSVK; 2) specific character of deviations of each chromosome from MSVK; 3) presence of significant connections between separate chromosomes by simultaneous mainly single directed numeral deviations. However, mycoplasmal contamination promotes the increase in the number of deviations in the direction of a decreasing chromosomes number. There is a breach of some connections between chromosomes by simultaneous deviations. They are chromosomes with broken connections according to the number of deviations which form telomeric associations (dicentrics). The number of these associations excel essentially intact control. The formation of new MSVK in subline M2 cells of the Indian muntjac in the process of chromosomal segregation in cell hybrid (M2 x clone of JF1 rat Jensen sarcoma) depends on the presence of significant connections between chromosomes by simultaneous numerical deviations in direction of MSVK formation. They are chromosomes that take part in the formation of new MSVK which form telomeric associations. These associations can be observed till stabilization of new MSVK. Probably, the support of the balance of karyotypic structure by factors inducing chromosomal instability is connected with change of some connections between chromosomes according to the number by simultaneous deviations as well as with the formation of dicentrics. PMID:10591121

Polianskaia, G G; Samokish, V A

1999-01-01

245

Cadmium induced changes in cell organelles: An ultrastructural study using cadmium sensitive and resistant muntjac fibroblast cell lines  

Energy Technology Data Exchange (ETDEWEB)

A detailed electron microscopy study of cadmium sensitive and resistant muntjac fibroblast cell lines has identified a wide range of intracellular damage following exposure to cadmium. Damaged organelles included cell membrane, mitochondria, Golgi cisternae and tubular network, chromatin, nucleoli, microfilaments and ribosomes. Although cell membrane damage was generally the earliest indication of adverse cadmium action, particularly with continuous cadmium exposures, cells could tolerate extensive membrane loss. Mitochondrial distortion and some damage to Golgi was also tolerated. The turning point at which cadmium became lethal was generally marked by a cascade of events which included damage to both nuclear and cytoplasmic components. These results for fibroblasts are discussed and compared with damage reported in other types of cells.

Ord, M.J.; Chibber, R.; Bouffler, S.D.

1988-09-01

246

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

1982-05-01

247

Antiprion Activity of Cholesterol Esterification Modulators: a Comparative Study Using Ex Vivo Sheep Fibroblasts and Lymphocytes and Mouse Neuroblastoma Cell Lines?  

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Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared t...

Pani, Alessandra; Norfo, Claudia; Abete, Claudia; Mulas, Claudia; Putzolu, Marirosa; Laconi, Sergio; Orrù, Christina Doriana

248

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

Science.gov (United States)

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 ? (TGF?)/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 TGF? signaling, suggesting its potential as a potent antiphotoaging agent. PMID:21544886

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

2011-05-05

249

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

UK PubMed Central (United Kingdom)

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 ? (TGF-?)/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-?/Smad signaling, suggesting its potential as an efficacious antiphotoaging agent.

Lee KE; Mun S; Pyun HB; Kim MS; Hwang JK

2012-01-01

250

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

UK PubMed Central (United Kingdom)

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 ? (TGF?)/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 TGF? signaling, suggesting its potential as a potent antiphotoaging agent.

Kwon YY; Kim D; Kim J; Hwang JK

2011-12-01

251

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

Science.gov (United States)

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 ? (TGF-?)/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-?/Smad signaling, suggesting its potential as an efficacious antiphotoaging agent. PMID:23037157

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

2012-01-01

252

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

International Nuclear Information System (INIS)

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 ? (TGF-?)/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-?/Smad signaling, suggesting its potential as an efficacious antiphotoaging agent. (author)

2012-01-01

253

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

UK PubMed Central (United Kingdom)

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.

Lee K; Kim H; Kim JM; Chung YH; Lee TY; Lim HS; Lim JH; Kim T; Bae JS; Woo CH; Kim KJ; Jeong D

2012-03-01

254

Adhesion of human skin fibroblasts to Cyr61 is mediated through integrin alpha 6beta 1 and cell surface heparan sulfate proteoglycans.  

UK PubMed Central (United Kingdom)

The angiogenic inducer Cyr61 is an extracellular matrix-associated heparin-binding protein that can mediate cell adhesion, stimulate cell migration, and enhance growth factor-stimulated DNA synthesis in both fibroblasts and endothelial cells in culture. In vivo, Cyr61 induces neovascularization and promotes tumor growth. Cyr61 is a prototypic member of a highly conserved family of secreted proteins that includes connective tissue growth factor, nephroblastoma overexpressed, Elm-1/WISP-1, Cop-1/WISP-2, and WISP-3. Encoded by an immediate early gene, Cyr61 synthesis is induced by serum growth factors in cultured fibroblasts and in dermal fibroblasts during cutaneous wound healing. We previously demonstrated that Cyr61 mediates adhesion of vascular endothelial cells and activation-dependent adhesion of blood platelets through direct interaction with integrins alpha(V)beta(3) and alpha(IIb)beta(3), respectively. In this study, we show that the adhesion of primary human skin fibroblasts to Cyr61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans (HSPGs), which most likely serve as co-receptors. Either destruction of cell surface HSPGs or prior occupancy of the Cyr61 heparin-binding site completely blocked cell adhesion to Cyr61. A heparin-binding defective mutant of Cyr61 was unable to mediate fibroblast adhesion through integrin alpha(6)beta(1) but still mediated endothelial cell adhesion through integrin alpha(V)beta(3), indicating that endothelial cell adhesion through integrin alpha(V)beta(3) is independent of the heparin-binding activity of Cyr61. These results identify Cyr61 as a novel adhesive substrate for integrin alpha(6)beta(1) and provide the first demonstration of the requirement for HSPGs in integrin-mediated cell attachment. In addition, these findings suggest that Cyr61 might elicit disparate biological effects in different cell types through interaction with distinct integrin receptors.

Chen N; Chen CC; Lau LF

2000-08-01

255

Establishment of a murine epidermal cell line suitable for in vitro and in vivo skin modelling  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes. Methods We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods. Results COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability. Conclusions We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

Segrelles Carmen; Holguín Almudena; Hernández Pilar; Ariza José M; Paramio Jesús M; Lorz Corina

2011-01-01

256

Intracellular insulin-receptor dissociation and segregation in a rat fibroblast cell line transfected with a human insulin receptor gene  

International Nuclear Information System (INIS)

The cellular processing of insulin and insulin receptors was studied using a rat fibroblast cell line that had been transfected with a normal human insulin receptor gene, expressing approximately 500 times the normal number of native fibroblasts insulin receptors. These cells bind and internalize insulin normally. Biochemically assays based on the selective precipitation by polyethylene glycol of intact insulin-receptor complexes but not of free intracellular insulin were developed to study the time course of intracellular insulin-receptor dissociation. Fibroblasts were incubated with radiolabeled insulin at 40C, and internalization of insulin-receptor complexes was initiated by warming the cells to 370C. Within 2 min, 90% of the internalized radioactivity was composed of intact insulin-receptor complexes. The dissociation of insulin from internalized insulin-receptor complexes was markedly inhibited by monensin and chloroquine. Furthermore, chloroquine markedly increased the number of cross-linkable intracellular insulin-receptor complexes, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. These findings suggest that acidification of intracellular vesicles is responsible for insulin-receptor dissociation. Physical segregation of dissociated intracellular insulin from its receptor was monitored. The results are consistent with the view that segregation of insulin and receptor occurs 5-10 min after initiation of dissociation. These studies demonstrate the intracellular itinerary of insulin-receptor complexes, including internalization, dissociation of insulin from the internalized receptor within an acidified compartment, segregation of insulin from the receptor, and subsequent ligand degradation

1988-05-05

257

Phospholipid and glyceride biosynthesis in 2,4,5,2',4',5'-hexachlorobiphenyl-treated human skin fibroblasts  

Energy Technology Data Exchange (ETDEWEB)

2,4,5,2',4',5'-Hexachlorobiphenyl (HCB) was taken up by cultured human skin fibroblasts (A61437; GM488). HCB caused enhanced incorporation of (2-/sup 14/C)acetate into phospholipids and glycerides at low concentration and reduced incorporation at high concentrations. sn-(U-/sup 14/C)Glycerol-3-phosphate incorporation into phospholipids was inhibited. No significant change in total cellular phospholipids was observed. Triglyceride cellular content was increased 29%. The observed stimulation and inhibition of phosphoglyceride synthesis are similar to results obtained with rat liver microsomes.

Beranek, S.R.; Becker, M.M.; Kling, D.; Gamble, W.

1984-06-01

258

Relaxin regulates fibrillin 2, but not fibrillin 1, mRNA and protein expression by human dermal fibroblasts and murine fetal skin.  

Science.gov (United States)

Relaxin modulates connective tissue remodeling by altering matrix molecule expression. We have found that relaxin specifically inhibits a microfibril component, fibrillin 2 (FBN2), without affecting fibrillin 1 (FBN1). Human dermal fibroblasts (HDFs) grown or stimulated to overexpress fibrillin expression were used to show that relaxin specifically down-regulated FBN2 mRNA and protein levels. Continuous exposure of HDFs to relaxin (30ng/ml) significantly (PFBN2 mRNA and protein in fetal skin from pregnant relaxin knockout mice. The regulation of FBN2 expression may be associated with functional changes in elastic tissues during development and growth. PMID:12590922

Samuel, Chrishan S; Sakai, Lynn Y; Amento, Edward P

2003-03-01

259

Establishment of immortal normal and ataxia telangiectasia fibroblast cell lines by introduction of the hTERT gene  

Energy Technology Data Exchange (ETDEWEB)

To establish immortal human cells, we introduced the human catalytic subunit of telomerase (hTERT) gene into skin fibroblast cells obtained from normal and ataxia telangiectasia (AT) individuals of Japanese origin. After hTERT introduction, these cells continue to grow beyond a population doubling number of 200 while maintaining their original radiosensitivity. Inductions of p53, phosphorylation of Serl5 in p53, and induction of p21 by X-ray irradiation in immortal cells derived from normal individual were not affected by the hTERT introduction. Both normal and AT immortal cells exhibited an apparent inhibition of growth as original primary cells when they reached confluence. Karyotype analysis has revealed that they are in a diploid range. These results suggest that cells immortalized by hTERT introduction retain their original characteristics except for immortalization, and that they may be useful for analyzing various effects of radiation on human cells. (author)

Nakamura, Hideaki; Fukami, Hiroko; Hayashi, Yuko; Kiyono, Tohru; Ishizaki, Kanji [Aichi Cancer Center, Nagoya (Japan). Research Inst.; Nakatsugawa, Shigekazu; Hamaguchi, Michinari [Nagoya Univ. (Japan). School of Medicine

2002-06-01

260

Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF  

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Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1) To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on these cells. 2) To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA) Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

Brem Harold; Golinko Michael S; Stojadinovic Olivera; Kodra Arber; Diegelmann Robert F; Vukelic Sasa; Entero Hyacinth; Coppock Donald L; Tomic-Canic Marjana

2008-01-01

 
 
 
 
261

Radiation-induced alterations of the proliferation dynamics of human skin fibroblasts after repeated irradiation in the subtherapeutic dose range  

International Nuclear Information System (INIS)

The aim of this study was to determine the effect of single and multiple irradiations on the differentiation and proliferation pattern of the stem cell system of human fibroblasts. The pattern of differentiation of fibroblast cultures was analyzed by morphological criteria using colony forming assays. Proliferation rates were assessed by cell counting and measuring the incorporation of BrdU. Ionizing radiation both in low and high dose ranges exerts differential effects on the cellular processes of differentiation and proliferation in human fibroblasts. Single irradiations of fibroblasts in the dose range of 1 to 8 Gy induced terminal differentiation into postmitotic fibrocytes at high percentage level. Irradiation of longterm cultures of fibroblasts with repeated doses of 0.2, 0.6 and 1.0 Gy revealed that only in cultures, which were irradiated repeatedly (x10) with 0.6 and 1.0 Gy a marked reduction of the proliferation capacity was apparent. Inhibition of proliferation by repeated irradiations with cumulative doses up to 10 Gy was not more pronounced as compared to single irradiations. 1. These results of radiation-induced changes in the proliferation and differentiation pattern of cells may be a basis for the understanding of the cellular processes leading to radiation-induced fibrosis and tissue aging. 2. Multiple irradiations with single doses up to 1 Gy and cumulative doses up to 10 Gy did not change the radiosensitivity of fibroblast cultures regarding effects on cell proliferation. (orig.)

1995-01-01

262

Screening of medicinal and edible plants in Okinawa, Japan, for enhanced proliferative and collagen synthesis activities in NB1RGB human skin fibroblast cells.  

UK PubMed Central (United Kingdom)

To identify plants with bioactive potential for skin care, methanol extracts of 56 plant parts from 47 medical and edible plants cultivated in Okinawa were tested for their proliferative effects on NB1RGB skin fibroblast cells. Extracts from six plants, Bischofia javanica, Colocasia esculenta, Melaleuca alternifolia, Piper angustifolia, Jasminum sambac, and Curcuma longa, showed higher NB1RGB cell proliferation activity (>10%) than the control, at various concentrations. Among the six extracts, only the C. longa extract caused an increase in collagen synthesis in NB1RGB cells, as compared to treatment with the positive control, ascorbic acid (AsA). Expression of the collagen synthesis marker, transforming growth factor-?1, was higher after treatment with the C. longa extract than with AsA.

Takahashi M; Asikin Y; Takara K; Wada K

2012-01-01

263

Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.  

UK PubMed Central (United Kingdom)

This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

Liu J; Luo Y; Zheng L; Liu Q; Yang Z; Wang Y; Su J; Quan F; Zhang Y

2013-10-01

264

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

UK PubMed Central (United Kingdom)

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.

Biesbroeck R; Oram JF; Albers JJ; Bierman EL

1983-03-01

265

Skin tension lines and preferable incisions in context of biomechanical skin characteristics. Linie napi?cia i wyboru kierunków naci?? skóry w kontek?cie jej biomechanicznych w?a?ciwo?ci.  

Directory of Open Access Journals (Sweden)

Full Text Available The proper orientation of skin incisions influence the functional and aesthetic results in dermatosurgery. Langer’s lines, dynamic wrinkle expression lines and relaxed skin tension lines are the basic marks for the planning of incisions. Fundamental biomechanical skin properies, such as skin elasticity, strain, creep and stress relaxation are disscused. The knowledge of these properties contributes to effective planning and execution of surgical skin procesures.

Adam W?odarkiewicz; Igor Michaj?owski; Micha? Sobjanek; Ryszard Kaca?a

2009-01-01

266

Cytotoxic effects of denture adhesives on primary human oral keratinocytes, fibroblasts and permanent L929 cell lines.  

UK PubMed Central (United Kingdom)

BACKGROUND: To date, there have been very little data on the cytotoxic responses of different cell lines to denture adhesives. OBJECTIVE: To determine the cytotoxicity of three denture adhesives on primary human oral keratinocytes (HOKs), fibroblasts (HOFs) and permanent mouse fibroblasts cell lines (L929). METHODS: Three commercial denture adhesives (two creams and one powder) were prepared for indirect contact using the agar diffusion test, as well as extracts in MTT assay. The results of the MTT assay were statistically analysed by one-way anova and Tukey's test (p < 0.05). RESULTS: All of the tested denture adhesives showed mild to moderate cytotoxicity to primary HOKs (p < 0.001), whereas none of three was toxic to L929 cells (p > 0.05) in both assays. For primary HOFs cultures, slight cytotoxicity was observed for one of the products from the agar diffusion test and undiluted eluates of all tested adhesives with MTT assay (p < 0.01). CONCLUSION: Denture adhesives are toxic to the primary HOKs and HOFs cultures, whereas non-toxic to L929 cells. The results suggest that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives.

Chen F; Wu T; Cheng X

2012-05-01

267

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

1983-08-01

268

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

International Nuclear Information System (INIS)

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

1983-01-01

269

Semi-Quantitative Histological Analysis of the Effect of Intense Pulsed Light (IPL) and Carbon Dioxide (CO2) Intradermic Injection on Fibroblast and Collagen Proliferation in the Skin of Wistar Rats  

Directory of Open Access Journals (Sweden)

Full Text Available Background: In recent years, so-called “non-ablative rejuvenation” has been carried out with the use of lasers or intense pulsed light (IPL) to stimulate collagen production by dermal fibroblasts. Intradermal infusion of CO2 stimulates fibroblasts and the synthesis of collagen and elastin, contributing to the retraction of the skin and tissue rejuvenation. Objectives: To evaluate the effects of IPL and the intradermal infusion of CO2 on fibroblast proliferation and collagen in the skin of female rats. Methods: Sixteen adult female Wistar rats were divided into two groups of eight animals. Group 1 underwent IPL and group 2 underwent intradermal CO2 infusion. There was a total of 8 weeks of treatment. We conducted a punch in each animal before any procedure (T0), another punch in the middle of treatment at 4 weeks post-procedure (T1) and a punch at the end of treatment at 8 weeks post-procedure (T2). The cells involved in inflammation, fibrosis and vascularization of the injured tissue by histopathology were analyzed. Results: There was statistically significant fibroblast proliferation and collagen proliferation noted when analyzing all 16 animals together and also when considering the two study groups separately. In both groups, the greatest proliferation of fibroblasts coincided with periods of increased collagen production. Conclusion: Both IPL and intradermal CO2 infusion stimulated fibroblast and collagen proliferation in the skin of the rats studied.

Tamara Lemos Maia-Figueiró; Alexandre Nakao Odashiro; Giovanna Padoa de Menezes; Lilian Rezende Coelho; Ili Breda; Bruno Areco de Souza; Ernesto Antonio Figueiró-Filho

2012-01-01

270

Studying the Activity of Fibroblast Growth Factor 18 and Urokinase Plasminogen Activator Receptor Promoters in Two Colon Cancer Cell Lines  

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Full Text Available Introduction: Wnt and k-ras are two main signaling pathways activated in colon cancer.Many genes are upregulated downstream of these signaling pathways. The aim of this studywas to assess the activity of Wnt and k-ras in HCT116 and SW480 cell lines by makingtwo reporter constructs using promoters downstream of these pathways (fibroblast growthfactor18 [FGF18] and urokinase plasminogen activator receptor [UPAR]).Materials and Methods: UPARLacZ, FGF18LacZ, negative (pUCLacZ) and positive (CMVLacZ)control plasmids and pRc/CMV2CAT were constructed. Expressions of LacZ in bothcell lines were studied by ?gal staining and ELISA after normalization with CAT expression.Results: In both cell lines, FGF18LacZ transfected cells stained more than UPARLacZ transfectedones. This difference was more prominent in SW480. Both constructs have the abilityof expression in both cell lines. It was also proven that FGF18LacZ was significantly moreactive than UPARLacZ in both cell lines. Expression of FGF18LacZ in HCT116 and SW480cell lines was respectively 1.34 and 4.4 times more than UPARLacZ.Conclusion: Despite the fact that in HCT116 the Ras pathway is activated, FGF18LacZ ismore active than UPARLacZ although the UPAR promoter is more active in HCT116 cell linethan SW480 cell line. These findings are in accordance with previous studies that in all coloncancer cell lines Wnt signaling pathway is active even though there is no mutation in anypart of it. Wnt is the main signaling pathway responsible for carcinogenesis in colon epithelialcells. These constructs can be used as reporters for studying the above mentioned signalingpathways in other cell lines.

Ladan Teimoori-Toolabi; Kayhan Azadmanesh; Somayeh Jamali; Amir Amanzadeh; Morteza Karimipoor; Sirous Zeinali

2009-01-01

271

Effect of low-level laser therapy on the release of interleukin-6 and basic fibroblast growth factor from cultured human skin fibroblasts in normal and high glucose mediums.  

UK PubMed Central (United Kingdom)

Abstract Introduction: This study evaluated the effects of low-level laser therapy (LLLT) on human skin fibroblasts (HSFs) that have been cultured in high glucose concentration media. Materials and Methods: HSFs were cultured under physiologic glucose condition medium, and then cultured in high glucose concentration medium (15 mM/L) for one or two weeks prior to LLLT. Experimental HSFs were irradiated with three energy densities (0.5, 1, and 2 J/cm(2)) once daily on three consecutive days. Release of interleukin-6 (IL-6) and basic fibroblast growth factor (bFGF) were evaluated by the enzyme-linked immunosorbent assay (ELISA) method. Results: Statistical analysis showed three doses of 0.5 (p=0.049), 1 (p=0.027), and 2 J/cm(2) (p=0.004) stimulated the release of IL-6 in HSFs cultured in high glucose concentration medium compared to non-irradiated HSFs that were cultured in the same medium. LLLT with 2 J/cm(2) induced the release of bFGF from HSFs cultured in high glucose concentration medium for one (p=0.047) or two weeks (both p=0.04). Conclusion: Our study showed LLLT stimulated the release of IL-6 and bFGF from HSFs cultured in high glucose concentration medium. LLLT was more effective in releasing IL-6 and bFGF while HSFs which were cultured in physiologic glucose concentration medium during laser irradiation.

Esmaeelinejad M; Bayat M

2013-05-01

272

Adaptive response to ionizing radiation in normal human skin fibroblasts. Enhancement of DNA repair rate and modulation of gene expression  

International Nuclear Information System (INIS)

Low doses and dose rates of ionizing radiation enhance the rate of DNA repair in human fibroblasts and protect the cells against radiation-induced micronucleus formation. Chronic exposures reduce the mRNA levels of the genes topoisomerase II and FACC-1 (Fanconi's anemia, group C). (authors). 11 refs., 1 tab., 2 figs.

1994-01-01

273

Mixture of fibroblasts and adipose tissue-derived stem cells can improve epidermal morphogenesis of tissue-engineered skin  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Many studies demonstrate that the type of adjacent mesenchymal cells can affect epidermal morphogenesis of bilayered tissue-engineered skin. However, whether a mixture of different mesenchymal cell types can improve epidermal morphogenesis of bioengineered skin remains unknown. In this study, kerati...

Lu, W; Yu, J; Zhang, Y; Ji, K; Zhou, Y; Li, Y; Deng, Z; Jin, Y

274

The effect of silymarin (Silybum marianum) on human skin fibroblasts in an in vitro wound healing model.  

UK PubMed Central (United Kingdom)

CONTEXT: Silymarin, a flavonolignan from Silybum marianum (L.) Gaertn. (Asteraceae), has been reported to have antioxidant and anti-inflammatory properties. Therefore, it may be worthwhile to study the effect of silymarin on wound healing. OBJECTIVE: To evaluate the effect of silymarin on human fibroblast cells in an in vitro model of wound healing. MATERIALS AND METHODS: Human fibroblast cells were treated with different concentrations (4.5, 9, 18, 36 µg/mL) of silymarin. The effects of silymarin on cell viability, proliferation, collagen synthesis, and expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthetase (iNOS) were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5-bromo-2'-deoxy-uridine, hydroxyproline analysis and real-time PCR, respectively. The effect of silymarin on cellular antioxidant status was determined by protection against hydrogen peroxide (H?O?)-induced cell injury and free radical scavenging activity (ABTS assay) of the cells. RESULTS: Results of the present study indicate that pretreatment of fibroblast cells with silymarin significantly protected cells against H?O?-induced injury (p < 0.05). After an 18?h treatment of cells with 36 µg/mL silymarin, total antioxidant capacity of cells significantly increased (p < 0.05). Furthermore, pretreatment of human fibroblast cells with silymarin significantly inhibited lipopolysaccharide (LPS)-induced COX-2 mRNA expression (p < 0.001). There was no significant difference in fibroblast proliferation and collagen synthesis between treatment and control groups (p > 0.05). DISCUSSION AND CONCLUSION: Silymarin may be useful as a therapeutic agent for the treatment of cutaneous wounds through its antioxidation and anti-inflammation effects.

Sharifi R; Pasalar P; Kamalinejad M; Dehpour AR; Tavangar SM; Paknejad M; Mehrabani Natanzi M; Nourbakhsh M; Ahmadi Ashtiani HR; Akbari M; Rastegar H

2013-03-01

275

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle, Chelonia mydas.  

UK PubMed Central (United Kingdom)

A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle, Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30 degrees C although replication occurred between 16 and 37 degrees C. These cells may be held as monolayers at 8 degrees C or stored frozen in growth medium containing 10% dimethyl-sulfoxide at -70 or -196 degrees C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species.

Koment RW; Haines H

1982-03-01

276

Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle, Chelonia mydas.  

Science.gov (United States)

A continuous line of epithelioid cells was established from explant skin tissues of the green sea turtle, Chelonia mydas. These cells, designated GTS, have been subcultured more than 60 times in commercially available mammalian cell culture medium supplemented with 5% bovine calf serum. Of those temperatures tested, optimal growth was achieved at 30 degrees C although replication occurred between 16 and 37 degrees C. These cells may be held as monolayers at 8 degrees C or stored frozen in growth medium containing 10% dimethyl-sulfoxide at -70 or -196 degrees C. The modal number of 55 chromosomes per cell is in agreement with the heterogametic female diploid number of this species. The GTS line represents the first established culture of normal epithelioid skin cells to be reported for a poikilothermic species. PMID:7129477

Koment, R W; Haines, H

1982-03-01

277

Cytotoxicity of endodontic irrigants containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from mouse L929 cell line  

Scientific Electronic Library Online (English)

Full Text Available Abstract in portuguese O objetivo desta pesquisa foi avaliar a citotoxicidade de soluções irrigadoras de canais radiculares contendo hidróxido de cálcio e lauril sulfato de sódio em linhagem de fibroblastos L929. Solução aquosa saturada de hidróxido de cálcio, lauril sulfato de sódio e HCT20 (lauril sulfato de sódio e hidróxido de cálcio) foram diluídos em água destilada em concentrações de 50%, 20%, 10% e 5%. O grupo controle foi representado por meio de cultura de células (M (more) EM - minimum essential medium). A citotoxicidade das soluções sobre os fibroblastos foi avaliada em 4 e 24 h de contato, pelo método do cromo radioativo. Os resultados foram analisados estatisticamente pelo teste do qui-quadrado. Em todas as análises, o intervalo de confiança referente às médias entre os grupos foi estabelecido em 95%. As soluções saturadas de hidróxido de cálcio e o HCT20 apresentaram toxicidade nas concentrações de 50%. O lauril sulfato de sódio foi tóxico em todas as concentrações. As soluções de hidróxido de cálcio em concentrações menores que 50% apresentaram tolerância celular, assim como combinadas ao lauril sulfato de sódio. Tal comportamento não foi observado na solução pura de lauril sulfato de sódio em todas as concentrações. Abstract in english The aim of this study was to evaluate the cytotoxicity of root canal irrigating solutions containing calcium hydroxide and sodium lauryl sulphate on fibroblasts derived from L929 cell line. Saturated calcium hydroxide aqueous solution (CH), sodium lauryl sulphate (SLS) and SLS associated with calcium hydroxide (HCT20) were diluted with sterile distilled water at 50%, 20%, 10% and 5% concentrations. Minimum essential medium (MEM) served as the control group. The cytotoxici (more) ty of the solutions was evaluated on L929 mouse fibroblast cell line, at 4 and 24 h of contact time by the 51Cr radiotracer method. Data were compared and statistical inferences were made with the chi-square test. In all analysis, significance level was set at 5%. CH and HCT20 showed toxicity at 50% concentration, while at concentrations lower than 50% these solutions showed cell tolerance. SLS was cytotoxic at all concentrations. In conclusion, the association of calcium hydroxide and SLS (HCT20) combines the beneficial properties of these solutions and was not harmful to the fibroblast cell line, seeming to be a suitable endodontic irrigating solution.

Barbosa, Sérgio Valmor; Barroso, Cristiane Maria Sodré; Ruiz, Patrícia Alvarez

2009-01-01

278

Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) against UV irradiation on keratinocytes and fibroblasts cell lines.  

Science.gov (United States)

Considering the belief that natural lipids are safer for topical applications and that carotenoids are able to protect cells against photooxidative damage, we have investigated whether topical creams and lotions, produced with Buriti oil and commercial surfactants, can exert photoprotective effect against UVA and UVB irradiation on keratinocytes and fibroblasts. Cell treatment was divided into two steps, prior and after exposition to 30 min of UVA plus UVB radiation or to 60 min of UVA radiation. Emulsions prepared with ethoxylated fatty alcohols as surfactants and containing alpha-tocopherol caused phototoxic damage to the cells, especially when applied prior to UV exposure. Damage reported was due to prooxidant activity and phototoxic effect of the surfactant. Emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil and containing panthenol as active ingredient, were able to reduce the damages caused by radiation when compared to non-treated cells. When the two cell lines used in the study were compared, keratinocytes showed an increase in cell viability higher than fibroblasts. The Buriti oil emulsions could be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection, especially in after sun formulations. PMID:19766688

Zanatta, C F; Mitjans, M; Urgatondo, V; Rocha-Filho, P A; Vinardell, M P

2009-09-18

279

Photoprotective potential of emulsions formulated with Buriti oil (Mauritia flexuosa) against UV irradiation on keratinocytes and fibroblasts cell lines.  

UK PubMed Central (United Kingdom)

Considering the belief that natural lipids are safer for topical applications and that carotenoids are able to protect cells against photooxidative damage, we have investigated whether topical creams and lotions, produced with Buriti oil and commercial surfactants, can exert photoprotective effect against UVA and UVB irradiation on keratinocytes and fibroblasts. Cell treatment was divided into two steps, prior and after exposition to 30 min of UVA plus UVB radiation or to 60 min of UVA radiation. Emulsions prepared with ethoxylated fatty alcohols as surfactants and containing alpha-tocopherol caused phototoxic damage to the cells, especially when applied prior to UV exposure. Damage reported was due to prooxidant activity and phototoxic effect of the surfactant. Emulsions prepared with Sorbitan Monooleate and PEG-40 castor oil and containing panthenol as active ingredient, were able to reduce the damages caused by radiation when compared to non-treated cells. When the two cell lines used in the study were compared, keratinocytes showed an increase in cell viability higher than fibroblasts. The Buriti oil emulsions could be considered potential vehicles to transport antioxidants precursors and also be used as adjuvant in sun protection, especially in after sun formulations.

Zanatta CF; Mitjans M; Urgatondo V; Rocha-Filho PA; Vinardell MP

2010-01-01

280

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.  

Science.gov (United States)

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained. PMID:18823265

Brunetti, Dario; Perota, Andrea; Lagutina, Irina; Colleoni, Silvia; Duchi, Roberto; Calabrese, Fiorella; Seveso, Michela; Cozzi, Emanuele; Lazzari, Giovanna; Lucchini, Franco; Galli, Cesare

2008-12-01

 
 
 
 
281

Transgene expression of green fluorescent protein and germ line transmission in cloned pigs derived from in vitro transfected adult fibroblasts.  

UK PubMed Central (United Kingdom)

The pig represents the xenogeneic donor of choice for future organ transplantation in humans for anatomical and physiological reasons. However, to bypass several immunological barriers, strong and stable human genes expression must occur in the pig's organs. In this study we created transgenic pigs using in vitro transfection of cultured cells combined with somatic cell nuclear transfer (SCNT) to evaluate the ubiquitous transgene expression driven by pCAGGS vector in presence of different selectors. pCAGGS confirmed to be a very effective vector for ubiquitous transgene expression, irrespective of the selector that was used. Green fluorescent protein (GFP) expression observed in transfected fibroblasts was also maintained after nuclear transfer, through pre- and postimplantation development, at birth and during adulthood. Germ line transmission without silencing of the transgene was demonstrated. The ubiquitous expression of GFP was clearly confirmed in several tissues including endothelial cells, thus making it a suitable vector for the expression of multiple genes relevant to xenotransplantation where tissue specificity is not required. Finally cotransfection of green and red fluorescence protein transgenes was performed in fibroblasts and after nuclear transfer blastocysts expressing both fluorescent proteins were obtained.

Brunetti D; Perota A; Lagutina I; Colleoni S; Duchi R; Calabrese F; Seveso M; Cozzi E; Lazzari G; Lucchini F; Galli C

2008-12-01

282

Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts  

DEFF Research Database (Denmark)

We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear transfer. PFV cells supported Flp mediated cassette exchange for transgene substitution of eGFP with dsRED, and the dsRED transgenic PFV cells generated blastocysts with transgene expression. Hence, the PFV cell line constitutes a valuable pig equivalent to transformed cell lines from other mammalian species suitable for locus-directed transgene expression in cell cultures and, in addition, for transgene analyses in the very early embryonic stages.

Jakobsen, Jannik E; Li, Juan

2011-01-01

283

Patterns of epidermal growth factor receptor, basic fibroblast growth factor and transforming growth factor-beta3 expression in skin with chronic venous insufficiency.  

UK PubMed Central (United Kingdom)

Growth factors which act as signalling peptides through specific cell surface receptors are involved in functions such as cell proliferation, migration, and differentiation. Here, we report on alterations of the epidermal growth factor receptor (EGFR), basic fibroblast growth factor (bFGF) and transforming growth factor beta3 (TGF-beta3) expression patterns in the skin at various stages of chronic venous insufficiency (CVI). Thirty punch biopsies were taken from patients with CVI and growth factors or the growth factor receptor were detected by indirect immunofluorescence and immunoperoxidase techniques. EGFR, bFGF, and TGF-beta3 expression is strongly increased in the stroma of venous eczema and in leg ulcer skin, and to a lesser extent in the dermis of patients with lipodermatosclerosis. Venous eczema and lipodermatosclerosis epidermis show an elevated EGFR and bFGF synthesis throughout all strata. In the different CVI stages, telangiectases and reticular veins and pigmentation EGFR and bFGF staining are limited to the basal layer. We conclude that the alterations in the expression of EGFR, bFGF and TGF-beta3 precede changes in the affected skin within progressing stages of CVI. The exact mechanisms of growth factor involvement in the pathogenesis of venous ulceration remain to be resolved.

Peschen M; Grenz H; Grothe C; Schöpf E; Vanscheidt W

1998-07-01

284

Repair of sublethal damage in diploid human fibroblasts: a comparison between human and rodent cell lines  

International Nuclear Information System (INIS)

[en] The repair of sublethal damage has been compared in the human cell lines GM-498 and GM 3440 with that which occurs in the rodent lines CHO and V-79. The data suggests that the human cell lines repair sublethal damage at least as well as the rodent cell lines. Another object of this investigation was to determine if the repair of sublethal damage can be observed in the initial region of the survival curve. While repair of sublethal damage can be observed in the second decade, it cannot be observed in the first, assuming no redistribution in the cell cycle. 12 references, 6 figures

1983-01-01

285

Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols.  

Science.gov (United States)

Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene. PMID:20932619

Mei, Xin; Wu, Yuan-Yuan; Mao, Xiao; Tu, You-Ying

2010-10-06

286

Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro.  

Science.gov (United States)

In vitro tests are needed to replace animal tests to screen for the skin sensitization potential of chemicals. Skin sensitizers are electrophilic molecules and the Nrf2-electrophile-sensing pathway comprising the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE) is emerging as a toxicity pathway induced by skin sensitizers. Previously, we screened a large set of chemicals in the reporter cell line AREc32, which contains an eight-fold repeat of the rat GSTA2 ARE-sequence upstream of a luciferase reporter gene in the human breast cancer cell line MCF7. This approach was now further developed to bring it closer to the conditions in the human skin and to propose a fully standardized assay. To this end, a luciferase reporter gene under control of a single copy of the ARE-element of the human AKR1C2 gene was stably inserted into HaCaT keratinocytes. A standard operating procedure was developed whereby chemicals are routinely tested at 12 concentrations in triplicate for significant induction of gene activity. We report results from this novel assay on (i) a list of reference chemicals published by ECVAM, (ii) the ICCVAM list of chemicals for validation of alternative endpoints in the LLNA and (iii) on a more general list of 67 chemicals derived from the ICCVAM database. For comparison, peptide reactivity data are presented for the same chemicals. The results indicate a good predictive value of this approach for hazard identification. Its technical simplicity, the high-throughput format and the good predictivity may make this assay a candidate for rapid validation to meet the tight deadline to replace animal tests for skin sensitization by 2013 set by the European authorities. PMID:20307559

Emter, Roger; Ellis, Graham; Natsch, Andreas

2010-03-20

287

Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro  

International Nuclear Information System (INIS)

In vitro tests are needed to replace animal tests to screen for the skin sensitization potential of chemicals. Skin sensitizers are electrophilic molecules and the Nrf2-electrophile-sensing pathway comprising the repressor protein Keap1, the transcription factor Nrf2 and the antioxidant response element (ARE) is emerging as a toxicity pathway induced by skin sensitizers. Previously, we screened a large set of chemicals in the reporter cell line AREc32, which contains an eight-fold repeat of the rat GSTA2 ARE-sequence upstream of a luciferase reporter gene in the human breast cancer cell line MCF7. This approach was now further developed to bring it closer to the conditions in the human skin and to propose a fully standardized assay. To this end, a luciferase reporter gene under control of a single copy of the ARE-element of the human AKR1C2 gene was stably inserted into HaCaT keratinocytes. A standard operating procedure was developed whereby chemicals are routinely tested at 12 concentrations in triplicate for significant induction of gene activity. We report results from this novel assay on (i) a list of reference chemicals published by ECVAM, (ii) the ICCVAM list of chemicals for validation of alternative endpoints in the LLNA and (iii) on a more general list of 67 chemicals derived from the ICCVAM database. For comparison, peptide reactivity data are presented for the same chemicals. The results indicate a good predictive value of this approach for hazard identification. Its technical simplicity, the high-throughput format and the good predictivity may make this assay a candidate for rapid validation to meet the tight deadline to replace animal tests for skin sensitization by 2013 set by the European authorities.

2010-06-15

288

The Oncolytic Effect of Respiratory Syncytial Virus (RSV) in Human Skin Cancer Cell Line, A431.  

UK PubMed Central (United Kingdom)

BACKGROUND: Oncolytic viruses have become of noticeable interest as a novel biological approach for selectively infecting cancer cells and triggering apoptosis in a number of malignant cells. Many researches are devoted to characterize more viruses with oncolytic properties. OBJECTIVES: Evidences on the oncolytic feature of respiratory syncytial virus (RSV) are conflicting; therefore, this study was designed to elucidate the possible role of RSV on the modulation of cell growth and apoptosis in the skin cancer cells. MATERIALS AND METHODS: Plaque assay was used to determine RSV titers. The cytotoxic effect of RSV in A431 (skin carcinoma cell line) was determined using MTT assay. The detection of apoptosis was performed via Annexin-V-FITC staining method and analyzed with flow cytometry. RESULTS: The results indicated that A431 cell growth was inhibited following infection by RSV in a dose- and time-dependent manner. The most growth inhibitory effect of RSV was occurred at the MOI of 3, and 48 hour after infection. The inhibitory effect of RSV on the cell growth was accompanied by the induction of apoptosis in the skin cancer cells. The percentages of early and late apoptotic cells were increased following exposure to RSV in a concentration- and time-dependent manner. CONCLUSIONS: This study delineated the beneficial role of RSV for growth regulation of skin cancer cells and highlighted the involvement of RSV in the induction of apoptosis in A431 cells. These findings might conduct evidence into the oncolytic properties of RSV in the skin cancer. Further studies are required to indicate intracellular targets for RSV-induced apoptosis in skin cancer cells.

Salimi V; Tavakoli-Yaraki M; Mahmoodi M; Shahabi S; Gharagozlou MJ; Shokri F; Mokhtari-Azad T

2013-01-01

289

Relaxin regulates fibrillin 2, but not fibrillin 1, mRNA and protein expression by human dermal fibroblasts and murine fetal skin.  

UK PubMed Central (United Kingdom)

Relaxin modulates connective tissue remodeling by altering matrix molecule expression. We have found that relaxin specifically inhibits a microfibril component, fibrillin 2 (FBN2), without affecting fibrillin 1 (FBN1). Human dermal fibroblasts (HDFs) grown or stimulated to overexpress fibrillin expression were used to show that relaxin specifically down-regulated FBN2 mRNA and protein levels. Continuous exposure of HDFs to relaxin (30ng/ml) significantly (P<0.05) decreased fibrillin 2 protein (40%) while FBN1 protein expression was unchanged. Our in vitro studies were confirmed using relaxin null mice whereby the absence of relaxin was associated with increased FBN2 mRNA and protein in fetal skin from pregnant relaxin knockout mice. The regulation of FBN2 expression may be associated with functional changes in elastic tissues during development and growth.

Samuel CS; Sakai LY; Amento EP

2003-03-01

290

Low-level laser therapy on tissue-engineered skin substitutes: effect on the proliferation rate of 3T3 mouse fibroblast cells  

Science.gov (United States)

With the rapid development of tissue engineering and gene therapy, collagen-based biomaterials are frequently used as cell transplant devices; an example is tissue-engineered skin substitutes. In this study of low level laser therapy (LLLT) we determined the influence of the irradiation and treatment parameters on the proliferation rate of 3T3 mouse fibroblast cells cultured on collagen-glycosaminoglycan (GAG) lattices and Petri dishes for up to 4 and 7 days respectively. Helium-Neon (He-Ne) laser at 1 - 4 J/cm2 was used to irradiate the cells. Using 5-carboxyfluorescein diacetate (CFDA) fluorescence, studies on the proliferation rate of irradiated cells before and after cell attachment, and on different treatment days were conducted. The viability of cells on collagen-GAG lattices were assessed using the MTT assay. It was found that in terms of cell proliferation, the cells irradiated at different fluences and treatment modes (at 3 J/cm2) showed no statistically significant difference from the control cells. Control cells on collagen-GAG lattices were found to be more viable than the irradiated cells. It was concluded that with existing experimental conditions, LLLT was found to have no statistically significant effect on the post-cell attachment proliferation and viability of 3T3 mouse fibroblast cells.

Ho, Gideon; Grant, M. Helen; Barbenel, Joseph C.; Henderson, Catherine J.

2004-09-01

291

Comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture  

Energy Technology Data Exchange (ETDEWEB)

Uptake of low density lipoprotein (LDL) and of acetyl LDL was compared in skin fibroblasts, smooth muscle cells, and peritoneal macrophages with the use of lipoproteins labeled with either /sup 125/I or the fluorescent probe 3,3'-dioctadecylindocarbocyanine (DiI). The uptake of DiI-labeled lipoproteins was assessed by quantitative spectrofluorometry and by fluorescence microscopy. The DiI was quantitatively retained by the cells, while the /sup 125/I-LDL was degraded and /sup 125/I-labeled degradation products were excreted from the cells. In smooth muscle cells and fibroblasts the uptake of LDL was virtually the same whether measured with the use of the DiI or /sup 125/I-label. The labeling of acetyl LDL with DiI enhanced its uptake in peritoneal macrophages by an average of 18%. With the DiI label, lipoprotein uptake could be determined after as little as 10 minutes of incubation at 37 C. The pattern of uptake of the DiI-labeled lipoproteins was consistent with binding to specific receptors, because no DiI could be detected in mutant cells without LDL receptors, and uptake was competitively inhibited by addition of excess unlabeled lipoprotein. When the DiI-labeled lipoproteins were removed from the medium, there was a 5-15% loss of DiI from all cell types studied over the first 24 hours.

Reynolds, G.D.; St. Clair, R.W.

1985-11-01

292

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

UK PubMed Central (United Kingdom)

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.

Manosroi A; Kumguan K; Chankhampan C; Manosroi W; Manosroi J

2012-09-01

293

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

Science.gov (United States)

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. PMID:23035451

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

2012-09-01

294

Repair of chromosome damage induced by X-irradiation during G/sub 2/ phase in a line of normal human fibroblasts and its malignant derivative  

Energy Technology Data Exchange (ETDEWEB)

A line of normal human skin fibroblasts (KD) differed from its malignant derivative (HUT-14) in the extent of cytogenetic damage induced by X-irradiation during G/sub 2/ phase. Malignant cells had significantly more chromatid breaks and gaps after exposure to 25, 50, or 100 rad. Results from alkaline elution of cellular DNA immediately after irradiation showed that the normal and malignant cells in asynchronous population were equally sensitive to DNA single-strand breakage by X-irradiation. Caffeine or ..beta..-cytosine arabinoside (ara-C), inhibitors of DNA repair, when added directly following G/sub 2/ phase exposure, significantly increased the incidence of radiation-induced chromatid damage in the normal cells. In contrast, similar treatment of the malignant cells had little influence. Ara-C differed from caffeine in its effects; whereas both agents increased the frequency of chromatid breaks and gaps, only ara-C increased the frequency of gaps to the level observed in the irradiated malignant cells. Addition of catalase, which destroys H/sub 2/O/sub 2/, or mannitol, a scavenger of the derivative free hydroxyl radical (.OH), to the cultures of malignant cells before, during, and following irradiation significantly reduced the chromatid damage; and catalase prevented formation of chromatid gaps. The DNA damage induced by X-ray during G/sub 2/ phase in the normal KD cells was apparently repaired by a caffeine- and ara-C-sensitive mechanism(s) that was deficient or absent in their malignant derivatives.

Parshad, R. (Howard Univ. College of Medicine, Washington, DC); Gantt, R.; Sanford, K.K.; Jones, G.M.; Tarone, R.E.

1982-08-01

295

Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: Geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts.  

Science.gov (United States)

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-?-D-glucoside (isoquercitrin), kaempferol-3-O-?-D-glucoside (astragalin), quercetin-3-O-D-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract. Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol). Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5 ?M, and of keratinocytes at 50-100 ?M. Furosin stimulated NHDF at about 50 ?M, keratinocytes at about 150-200 ?M. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20 ?M for NHDF and 150 ?M for keratinocytes. Toxicity of furosin--less than that of geraniin--was observed at > 400 ?M. Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50 ?M and 5-10 ?M respectively. Geraniin at 105 ?M significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified. PMID:21036574

Agyare, Christian; Lechtenberg, Matthias; Deters, Alexandra; Petereit, Frank; Hensel, Andreas

2010-10-30

296

Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: Geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts.  

UK PubMed Central (United Kingdom)

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-?-D-glucoside (isoquercitrin), kaempferol-3-O-?-D-glucoside (astragalin), quercetin-3-O-D-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid, 3,5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract. Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol). Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5 ?M, and of keratinocytes at 50-100 ?M. Furosin stimulated NHDF at about 50 ?M, keratinocytes at about 150-200 ?M. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20 ?M for NHDF and 150 ?M for keratinocytes. Toxicity of furosin--less than that of geraniin--was observed at > 400 ?M. Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50 ?M and 5-10 ?M respectively. Geraniin at 105 ?M significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.

Agyare C; Lechtenberg M; Deters A; Petereit F; Hensel A

2011-05-01

297

Repair of sublethal damage in diploid human fibroblasts: a compoarison between human and rodent cell lines  

International Nuclear Information System (INIS)

[en] Two diploid cell lines of human origin were irradiated in vitro. Repair of sublethal damage was observed in both lines when they were exposed to a single ?-ray dose and incubated at 370C prior to receiving a graded second dose. In addition cells were irradiated at a low dose rate (56 or 76 rad/hr) and survival was compared to that after an acute exposure. A dose-rate effect was demonstrated. Sublethal damage repair was also studied using CHO or V-79 Chinese hamster cells, and the data suggest that the degree of repair is similar in human and rodent cell lines at photon doses up to 500 rad. The repair of sublethal damage can be a significant factor in the survival of human cells in vitro for fractionated doses

1983-01-01

298

Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effects against H(2)O(2)-induced oxidative stress in cultured human skin fibroblasts.  

UK PubMed Central (United Kingdom)

Mannosylerythritol lipids (MELs) are biosurfactants known for their versatile interfacial and biochemical properties. To broaden their application in cosmetics, we investigated the antioxidant properties of different MEL derivatives (MEL-A, -B, and -C) by using a 1,1-diphenyl-2-picryl hydrazine (DPPH) free-radical- and superoxide anion-scavenging assay. All MEL derivatives tested showed antioxidant activity in vitro, but at lower levels than those of arbutin. Of the MELs, MEL-C, which is produced from soybean oil by Pseudozyma hubeiensis, showed the highest rates of DPPH radical scavenging (50.3% at 10 mg/mL) and superoxide anion scavenging (>50% at 1 mg/mL). The antioxidant property of MEL-C was further examined using cultured human skin fibroblasts (NB1RGB cells) under H(2)O(2) induced oxidative stress. Surprisingly, MEL-C had a higher protective activity against oxidative stress than arbutin did: 10 µg/mL of MEL-C and arbutin had protective activities of 30.3% and 13%, respectively. Expression of an oxidative stress marker, cyclooxygenase-2, in these cells was repressed by treatment with MEL-C as well as by arbutin. MEL-C was thus confirmed to have antioxidant and protective effects in cells, and we suggest that MELs have potential as anti-aging skin care ingredients.

Takahashi M; Morita T; Fukuoka T; Imura T; Kitamoto D

2012-01-01

299

Glycolipid biosurfactants, mannosylerythritol lipids, show antioxidant and protective effects against H(2)O(2)-induced oxidative stress in cultured human skin fibroblasts.  

Science.gov (United States)

Mannosylerythritol lipids (MELs) are biosurfactants known for their versatile interfacial and biochemical properties. To broaden their application in cosmetics, we investigated the antioxidant properties of different MEL derivatives (MEL-A, -B, and -C) by using a 1,1-diphenyl-2-picryl hydrazine (DPPH) free-radical- and superoxide anion-scavenging assay. All MEL derivatives tested showed antioxidant activity in vitro, but at lower levels than those of arbutin. Of the MELs, MEL-C, which is produced from soybean oil by Pseudozyma hubeiensis, showed the highest rates of DPPH radical scavenging (50.3% at 10 mg/mL) and superoxide anion scavenging (>50% at 1 mg/mL). The antioxidant property of MEL-C was further examined using cultured human skin fibroblasts (NB1RGB cells) under H(2)O(2) induced oxidative stress. Surprisingly, MEL-C had a higher protective activity against oxidative stress than arbutin did: 10 µg/mL of MEL-C and arbutin had protective activities of 30.3% and 13%, respectively. Expression of an oxidative stress marker, cyclooxygenase-2, in these cells was repressed by treatment with MEL-C as well as by arbutin. MEL-C was thus confirmed to have antioxidant and protective effects in cells, and we suggest that MELs have potential as anti-aging skin care ingredients. PMID:22864517

Takahashi, Makoto; Morita, Tomotake; Fukuoka, Tokuma; Imura, Tomohiro; Kitamoto, Dai

2012-01-01

300

Topically delivered adipose derived stem cells show an activated-fibroblast phenotype and enhance granulation tissue formation in skin wounds.  

UK PubMed Central (United Kingdom)

Multipotent mesenchymal stem cells (MSCs) are found in various tissues and can proliferate extensively in vitro. MSCs have been used in preclinical animal studies and clinical trials in many fields. Adipose derived stem cells (ASCs) have several advantages compared to other MSCs for use in cell-based treatments because they are easy to isolate with relative abundance. However, quantitative approaches for wound repair using ASCs have been limited because of lack of animal models which allow for quantification. Here, we addressed the effect of topically delivered ASCs in wound repair by quantitative analysis using the rabbit ear model. We characterized rabbit ASCs, and analyzed their multipotency in comparison to bone marrow derived-MSCs (BM-MSCs) and dermal fibroblasts (DFs) in vitro. Topically delivered ASCs increased granulation tissue formation in wounds when compared to saline controls, whereas BM-MSCs or DFs did not. These studies suggest that ASCs and BM-MSCs are not identical, though they have similar surface markers. We found that topically delivered ASCs are engrafted and proliferate in the wounds. We showed that transplanted ASCs exhibited activated fibroblast phenotype, increased endothelial cell recruitment, and enhanced macrophage recruitment in vivo.

Hong SJ; Jia SX; Xie P; Xu W; Leung KP; Mustoe TA; Galiano RD

2013-01-01

 
 
 
 
301

Morphological features of iPS cells generated from Fabry disease skin fibroblasts using Sendai virus vector (SeVdp).  

UK PubMed Central (United Kingdom)

We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.

Kawagoe S; Higuchi T; Otaka M; Shimada Y; Kobayashi H; Ida H; Ohashi T; Okano HJ; Nakanishi M; Eto Y

2013-08-01

302

Morphological features of iPS cells generated from Fabry disease skin fibroblasts using Sendai virus vector (SeVdp).  

Science.gov (United States)

We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture. PMID:23810832

Kawagoe, Shiho; Higuchi, Takashi; Otaka, Manami; Shimada, Yohta; Kobayashi, Hiroshi; Ida, Hiroyuki; Ohashi, Toya; Okano, Hirotaka J; Nakanishi, Mahito; Eto, Yoshikatsu

2013-06-12

303

Establishment of transformed swine fibroblast cell lines using SV40 large T antigen.  

UK PubMed Central (United Kingdom)

Swine testicle cell lines were established by transformation of primary swine testicle (PST) cells with an SV40 plasmid (pSV3-neo), which contains genes conferring resistance to neomycin and expressing SV40 large T antigen. Plasmid DNA was transfected into PST cells using a lipofection system. Two related plasmids, pSV2-neo and pSV5-neo, failed to induce transformed cells. Cells transformed with pSV3-neo formed single colonies that were resistant to the antibiotic, G418, and expressed large T antigen. Upon two cycles of cloning by endpoint dilution method, three transformed clones, designated transformed swine testicle (tST)-3, tST-14 and tST-18, were selected and characterized in regards to cell replication and susceptibility to swine viruses. The resultant clones were compared with a counterpart non-transformed ST cell line (ATCC-ST). The three tST cell lines showed longer or the same doubling times and higher saturation densities compared to ATCC-ST cells. These cells were free from a range of adventitious agents and supported the replication of porcine parvovirus (PPV), pseudorabies virus (PRV) and transmissible gastroenteritis virus (TGEV), comparable to ATCC-ST cells. All three cell lines have been maintained in continuous cultures for over 60 passages with no changes in growth characteristics. These findings indicate that lipofection with pSV3-neo is an efficient means for the introduction of exogenous DNA into porcine cells and for establishment of transformed immortalized cell lines.

Choi CS; Murtaugh MP; Molitor TW

1990-01-01

304

Expression of two different forms of fibroblast growth factor receptor 1 in different mouse tissues and cell lines.  

UK PubMed Central (United Kingdom)

The fibroblast growth factor receptors (FGFRs) form a multigene family of at least four members, all having extracellular regions consisting of either two or three immunoglobin-like (Ig-like) domains. By RNase protection analysis we have analyzed the expression of FGFR-1 mRNA in various tissues and cell lines and demonstrated that all of the cell lines studied expressed at least two different forms of the FGFR-1 at similar levels. Although muscle and heart express forms having either two [FGFR-1 short (FGFR-1S)] or three [FGFR-1 long (FGFR-1L)] Ig-like domains, the developing brain and adult brain express only mRNA encoding the longer form. The two forms of the receptor were characterized further by stably introducing expression vectors expressing them into Rat-2 fibroblasts and FDC-P1 myeloid cells. Treatment of the transfected Rat-2 cells with acidic FGF (aFGF) or basic FGF (bFGF) resulted in focus formation. The transformed phenotype was observed even without addition of ligand after growth in culture for greater than 2 months. Cross-linking of 125I-labeled bFGF (125I-bFGF) to Rat-2 cells expressing either FGFR-1L or FGFR-1S yielded two similar complexes of 150 and 110 kDa. Although Rat-2 cells expressing FGFR-1L yielded similar complexes with 125I-labeled aFGF (125I-aFGF), only the 150-kDa complex was formed with cells expressing FGFR-1S. The 150-kDa complex was also observed when 125I-aFGF or 125I-bFGF was cross-linked to FDC-P1 cells expressing FGFR-1L. Significantly, these complexes were only observed when heparin was present in the cross-linking reaction. FDC-P1 cells expressing FGFR-1 bound aFGF and bFGF with high affinity but only in the presence of heparin. The factor dependence of these cells could be switched from interleukin 3 to FGF in the presence of heparin.

Bernard O; Li M; Reid HH

1991-09-01

305

Mechanism of increased susceptibility to 4-nitroquinoline-1-oxide in cultured skin fibroblasts from patients with familial polyposis coli.  

UK PubMed Central (United Kingdom)

About 50% of the strains of cultured fibroblasts from patients with familial polyposis coli (FPC) exhibited increased susceptibility to cytotoxicity of 4-nitroquinoline-1-oxide (4NQO) compared with cells from normal individuals. The FPC cells that showed hyper-sensitivity to 4NQO were also hyper-sensitive to mitomycin C (MMC), but susceptibilities of these cells to UV radiation, methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were within the normal range. The extent of single-strand scission of DNA in the 4NQO-sensitive FPC cells was greater than in normal cells, and the amount of [14C]4NQO bound to DNA in the FPC cells was twice as high as in normal cells. The rate of release of [14C]4NQO from DNA by the post-culture was the same as in both FPC and normal cells. The 4NQO-sensitive FPC cells exhibited increased 4NQO-reductase activity; the level of this activity was consistent with the extent of the decrease in colony formation by 4NQO. These results suggest that the enhanced ability to activate 4NQO might be an important factor in the mechanism of susceptibility of FPC cells to 4NQO rather than the reduced ability to repair DNA.

Akamatsu N; Miyaki M; Suzuki K; Ono T; Sasaki MS

1983-05-01

306

Microflora of the penile skin-lined neovagina of transsexual women  

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Full Text Available Abstract Background The microflora of the penile skin-lined neovagina in male-to-female transsexuals is a recently created microbial niche which thus far has been characterized only to a very limited extent. Yet the knowledge of this microflora can be considered as essential to the follow-up of transsexual women. The primary objective of this study was to map the neo-vaginal microflora in a group of 50 transsexual women for whom a neovagina was constructed by means of the inverted penile skin flap technique. Secondary objectives were to describe possible correlations of this microflora with multiple patients' characteristics, such as sexual orientation, the incidence of vaginal irritation and malodorous vaginal discharge. Results Based on Gram stain the majority of smears revealed a mixed microflora that had some similarity with bacterial vaginosis (BV) microflora and that contained various amounts of cocci, polymorphous Gram-negative and Gram-positive rods, often with fusiform and comma-shaped rods, and sometimes even with spirochetes. Candida cells were not seen in any of the smears. On average 8.6 species were cultured per woman. The species most often found were: Staphylococcus epidermidis, Streptococcus anginosus group spp., Enterococcus faecalis, Corynebacterium sp., Mobiluncus curtisii and Bacteroides ureolyticus. Lactobacilli were found in only one of 30 women There was no correlation between dilatation habits, having coitus, rinsing habits and malodorous vaginal discharge on the one hand and the presence of a particular species on the other. There was however a highly significant correlation between the presence of E. faecalis on the one hand and sexual orientation and coitus on the other (p = 0.003 and p = 0.027 respectively). Respectively 82%, 58% and 30% of the samples showed an amplicon after amplification with M. curtisii, Atopobium vaginae and Gardnerella vaginalis primer sets. Conclusion Our study is the first to describe the microflora of the penile skin-lined neovagina of transsexual women. It reveals a mixed microflora of aerobe and anaerobe species usually found either on the skin, in the intestinal microflora or in a BV microflora.

Weyers Steven; Verstraelen Hans; Gerris Jan; Monstrey Stan; dos Santos Lopes Santiago Guido; Saerens Bart; De Backer Ellen; Claeys Geert; Vaneechoutte Mario; Verhelst Rita

2009-01-01

307

The role of p53 in growth of mouse fibroblast lines.  

Science.gov (United States)

To examine the hypothesis that p53 protein may play a central role in regulating reproduction of mammalian cells, we compared the absolute amounts and relative rates of synthesis of p53 protein in two pseudonormal cell lines, 3T3 and C3H 10T1/2, during quiescence, during log proliferation, and in quiescent cells stimulated with serum. The absolute amount of p53 protein per cell was found to be severalfold lower in quiescent cells than in log-phase cells. The ratio of the rate of synthesis of p53 protein to the rate of synthesis of total protein was slightly higher in quiescent cells than the same ratio in log-phase cells. Thus, entry into quiescence is not accompanied by a differential switch-off of synthesis of p53 protein. In quiescent cells stimulated with serum the amount of p53 protein per cell and its rate of synthesis increase, but only in proportion to the increase in total protein per cell and the increase in rate of total protein synthesis. Similarly, 12-14 h after serum stimulation, the time of the G1 to S transition, the accumulated increase in p53 protein per cell is about what would be expected for a short-lived protein whose rate of synthesis has increased in proportion to the increase in rate of synthesis of total protein. The results are not those expected for a protein that functions specifically in release from quiescence or in transition from G1 to S. PMID:3896824

Wynford-Thomas, D; LaMontagne, A; Marin, G; Prescott, D M

1985-07-01

308

The role of p53 in growth of mouse fibroblast lines.  

UK PubMed Central (United Kingdom)

To examine the hypothesis that p53 protein may play a central role in regulating reproduction of mammalian cells, we compared the absolute amounts and relative rates of synthesis of p53 protein in two pseudonormal cell lines, 3T3 and C3H 10T1/2, during quiescence, during log proliferation, and in quiescent cells stimulated with serum. The absolute amount of p53 protein per cell was found to be severalfold lower in quiescent cells than in log-phase cells. The ratio of the rate of synthesis of p53 protein to the rate of synthesis of total protein was slightly higher in quiescent cells than the same ratio in log-phase cells. Thus, entry into quiescence is not accompanied by a differential switch-off of synthesis of p53 protein. In quiescent cells stimulated with serum the amount of p53 protein per cell and its rate of synthesis increase, but only in proportion to the increase in total protein per cell and the increase in rate of total protein synthesis. Similarly, 12-14 h after serum stimulation, the time of the G1 to S transition, the accumulated increase in p53 protein per cell is about what would be expected for a short-lived protein whose rate of synthesis has increased in proportion to the increase in rate of synthesis of total protein. The results are not those expected for a protein that functions specifically in release from quiescence or in transition from G1 to S.

Wynford-Thomas D; LaMontagne A; Marin G; Prescott DM

1985-07-01

309

Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat.  

UK PubMed Central (United Kingdom)

Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h.

Hu PF; Guan WJ; Li XC; Zhang WX; Li CL; Ma YH

2013-01-01

310

Study on characteristics of in vitro culture and intracellular transduction of exogenous proteins in fibroblast cell line of Liaoning cashmere goat.  

Science.gov (United States)

Establishment of fibroblast cell lines of endangered goat breeds and research on the gene or protein functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. In this study, a fibroblast cell line of Liaoning cashmere goat, frozen in 174 cryovials with 5 × 10(6) cells each, was successfully established from 60 goats ear marginal tissues using explant culture and cryopreservation techniques. Biological analysis of in vitro cultured cell line showed that, the cells were morphologically consistent with fibroblasts; the average viability of the cells was 94.9 % before freezing and 90.1 % after thawing; the growth process of cells was consisted of a lag phase, a logarithmic phase and a plateau phase; cell population doubling time was 65.5 h; more than 90 % of cells were diploid prior to the 6th generation; Neither microbial contamination nor cross-contamination was detected. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, a TAT protein transduction domain was fused to the C-terminus of enhanced green fluorescent protein, the established fibroblast cell line was treated with the purified exogenous proteins at various concentrations by adding them to the cell culture media for 1-24 h and assayed cell morphology and protein presence, it was found that the purified exogenous proteins readily entered cells at a concentration of 0.1 mg/ml within 1.5 h and some of them could translocate into nucleus, moreover, the exogenous proteins appeared to be stable inside cells for up to 24 h. PMID:23065271

Hu, P F; Guan, W J; Li, X C; Zhang, W X; Li, C L; Ma, Y H

2012-10-14

311

NF-?B accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a -112/-61-bp region of the COL1A1 promoter in human skin fibroblasts.  

UK PubMed Central (United Kingdom)

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-?B (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-?B performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

Bigot N; Beauchef G; Hervieu M; Oddos T; Demoor M; Boumediene K; Galéra P

2012-10-01

312

NF-?B accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a -112/-61-bp region of the COL1A1 promoter in human skin fibroblasts.  

Science.gov (United States)

The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-?B (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-?B performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts. PMID:22673730

Bigot, Nicolas; Beauchef, Gallic; Hervieu, Magalie; Oddos, Thierry; Demoor, Magali; Boumediene, Karim; Galéra, Philippe

2012-06-07

313

Low-dose radiation activates Nrf1/2 through reactive species and the Ca2+/ERK1/2 signaling pathway in human skin fibroblast cells  

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Full Text Available In the current study, we explored the effect of LDR on theactivation of Nrfs transcription factor involved in cellular redoxevents. Experiments were carried out utilizing 0.05 and 0.5 GyX-ray irradiated normal human skin fibroblast HS27 cells. Theresults showed LDR induced Nrf1 and Nrf2 activation andexpression of antioxidant genes HO-1, Mn-SOD, and NQO1.In particular, 0.05 Gy-irradiation increased only Nrf1 activation,but 0.5 Gy induced both Nrf1 and Nrf2 activation.LDR-mediated Nrf1/2 activation was accompanied by reactivespecies (RS) generation and Ca2+ flux. This effect was abolishedin the presence of N-acetyl-cysteine and BAPTA- AM.Furthermore, Nrf1/2 activation by LDR was suppressed byPD98059, an inhibitor of ERK1/2. In conclusion, LDR inducesNrf1 and Nrf2 activation and expression of Nrf-regulatedantioxidant defense genes through RS and Ca2+/ERK1/2pathways, suggesting new insights into the molecularmechanism underlying the beneficial role of LDR in HS27cells. [BMB Reports 2013; 46(5): 258-263

Eun Kyeong Lee; Jin-Ah Kim; Seong Joon Park; Jeung Ki Kim; Kyu Heo; Kwang Mo Yang; Tae Gen Son

2013-01-01

314

Low-dose radiation activates Nrf1/2 through reactive species and the Ca(2+)/ERK1/2 signaling pathway in human skin fibroblast cells.  

UK PubMed Central (United Kingdom)

In the current study, we explored the effect of LDR on the activation of Nrfs transcription factor involved in cellular redox events. Experiments were carried out utilizing 0.05 and 0.5 Gy X-ray irradiated normal human skin fibroblast HS27 cells. The results showed LDR induced Nrf1 and Nrf2 activation and expression of antioxidant genes HO-1, Mn-SOD, and NQO1. In particular, 0.05 Gy-irradiation increased only Nrf1 activation, but 0.5 Gy induced both Nrf1 and Nrf2 activation. LDR-mediated Nrf1/2 activation was accompanied by reactive species (RS) generation and Ca(2+) flux. This effect was abolished in the presence of N-acetyl-cysteine and BAPTA- AM. Furthermore, Nrf1/2 activation by LDR was suppressed by PD98059, an inhibitor of ERK1/2. In conclusion, LDR induces Nrf1 and Nrf2 activation and expression of Nrf-regulated antioxidant defense genes through RS and Ca(2+)/ERK1/2 pathways, suggesting new insights into the molecular mechanism underlying the beneficial role of LDR in HS27 cells.

Lee EK; Kim JA; Park SJ; Kim JK; Heo K; Yang KM; Son TG

2013-05-01

315

Low-dose radiation activates Nrf1/2 through reactive species and the Ca(2+)/ERK1/2 signaling pathway in human skin fibroblast cells.  

Science.gov (United States)

In the current study, we explored the effect of LDR on the activation of Nrfs transcription factor involved in cellular redox events. Experiments were carried out utilizing 0.05 and 0.5 Gy X-ray irradiated normal human skin fibroblast HS27 cells. The results showed LDR induced Nrf1 and Nrf2 activation and expression of antioxidant genes HO-1, Mn-SOD, and NQO1. In particular, 0.05 Gy-irradiation increased only Nrf1 activation, but 0.5 Gy induced both Nrf1 and Nrf2 activation. LDR-mediated Nrf1/2 activation was accompanied by reactive species (RS) generation and Ca(2+) flux. This effect was abolished in the presence of N-acetyl-cysteine and BAPTA- AM. Furthermore, Nrf1/2 activation by LDR was suppressed by PD98059, an inhibitor of ERK1/2. In conclusion, LDR induces Nrf1 and Nrf2 activation and expression of Nrf-regulated antioxidant defense genes through RS and Ca(2+)/ERK1/2 pathways, suggesting new insights into the molecular mechanism underlying the beneficial role of LDR in HS27 cells. PMID:23710636

Lee, Eun Kyeong; Kim, Jin-Ah; Park, Seong Joon; Kim, Jeung Ki; Heo, Kyu; Yang, Kwang Mo; Son, Tae Gen

2013-05-01

316

Impaired dynamics of the late endosome/lysosome compartment in human Niemann-Pick type C skin fibroblasts carrying mutation in NPC1 gene.  

UK PubMed Central (United Kingdom)

The Niemann-Pick type C (NPC) disease is characterized by accumulation of lipids within the late endosome/lysosome (LE/LY) compartment as a result of dysfunctions of the NPC1 or NPC2 proteins and an altered distribution and/or functioning of proteins involved in the regulation of membrane dynamics. In our previous report we isolated membranes of the LE/LY compartment from NPC L1 skin fibroblasts with a mutation in the NPC1 gene (exon 8, R348X) and showed that annexin A6 (AnxA6) may contribute to the impaired dynamics of these membranes in a cholesterol-dependent manner and therefore to the overnormative storage of cholesterol. In this report we show that the LE/LY fraction isolated from NPC L1 cells is characterized by a 4-fold enrichment in cholesterol, 2.5-fold in sphingomyelin and 2-fold in saturated fatty acids. As a result, the fluidity of LE/LY membranes isolated from NPC L1 cells is greatly reduced in comparison to control ones. We conclude that modified lipid composition and properties of this compartment may affect distribution and function of proteins implicated in cellular membrane dynamics. As a consequence, the backward vesicular transport of cholesterol from the LE/LY compartment to the Golgi apparatus, endoplasmic reticulum and finally to plasma membrane is impaired.

Sztolsztener ME; Dobrzyn A; Pikula S; Tylki-Szymanska A; Bandorowicz-Pikula J

2012-04-01

317

Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts  

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We have analyzed the pattern of protein synthesis in solar near ultraviolet (334 nm, 365 nm) and near visible (405 nm) irradiated normal human skin fibroblasts. Two hours after irradiation we find that one major stress protein of approximately 32 kDa is induced in irradiated cells. This protein is not induced by ultraviolet radiation at wavelengths shorter than 334 nm and is not inducible by heat shock treatment of these cells. Although sodium arsenite, diamide, and menadione all induced a 32-kDa protein, they also induced the major heat shock proteins. In contrast, the oxidizing agent, hydrogen peroxide, induced the low molecular weight stress protein without causing induction of the major heat shock proteins. A comparison of the 32-kDa proteins induced by sodium arsenite, H/sub 2/O/sub 2/, and solar near ultraviolet radiation using chemical peptide mapping shows that they are closely related. These results imply that the pathways for induction of the heat shock response and the 32-kDa protein are not identical and suggest that, at least in the case of radiation and treatment with H/sub 2/O/sub 2/, the 32-kDa protein might be induced in response to cellular oxidative stress. This conclusion is supported by the observation that depletion of endogenous cellular glutathione prior to solar near ultraviolet irradiation lowers the fluence threshold for induction of the 32-kDa stress protein.

Keyse, S.M.; Tyrrell, R.M.

1987-10-25

318

Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids  

International Nuclear Information System (INIS)

It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independently of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.

2008-04-10

319

IFN-? Is Constitutively Expressed by Cells of the Reproductive Tract and Is Inefficiently Secreted by Fibroblasts and Cell Lines  

Science.gov (United States)

Type-I interferons (IFNs) form a large family of cytokines that primarily act to control the early development of viral infections. Typical type-I IFN genes, such as those encoding IFN-? or IFN-? are upregulated by viral infection in many cell types. In contrast, the gene encoding IFN-? was reported to be constitutively expressed by cells of the female reproductive tract and to contribute to the protection against vaginal infections with herpes simplex virus 2 and Chlamydia muridarum. Our data confirm the lack of induction of IFN-? expression after viral infection and the constitutive expression of IFN-? by cells of the female but also of the male reproductive organs. Interestingly, when expressed from transfected expression plasmids in 293T, HeLa or Neuro2A cells, the mouse and human IFN-? precursors were inefficiently processed and secretion of IFN-? was minimal. Analysis of chimeric constructs produced between IFN-? and limitin (IFN-?) showed that both the signal peptide and the mature moiety of IFN-? contribute to poor processing of the precursor. Immunofluorescent detection of FLAG-tagged IFN-? in transfected cells suggested that IFN-? and chimeric proteins were defective for progression through the secretory pathway. IFN-? did not, however, act intracellularly and impart an antiviral state to producing cells. Given the constitutive expression of IFN-? in specialized cells and the poor processing of IFN-? precursor in fibroblasts and cell lines, we hypothesize that IFN-? secretion may require a co-factor specifically expressed in cells of the reproductive organs, that might secure the system against aberrant release of this IFN.

Hermant, Pascale; Francius, Cedric; Clotman, Frederic; Michiels, Thomas

2013-01-01

320

Cyclic AMP-mediated inhibition of cell growth by prostaglandin D2 in a fibroblastic cell line (EBTr).  

UK PubMed Central (United Kingdom)

We have recently reported that prostaglandin D2 (PGD2) specifically elevates the intracellular cyclic AMP (cAMP) level in a fibroblastic cell line derived from bovine embryonic trachea (EBTr) (K. Sugama, T. Tanaka, H. Yokohama, M. Negishi, H. Hayashi, S. Ito, and O. Hayaishi, Biochim. Biophys. Acta, 1011: 75-80, 1989). Since the in vitro and in vivo inhibitory effects of PGD2 on cell growth are attributed to the dehydrate product 9-deoxy-delta 9,12-13,14-dihydro-PGD2 (delta 12-PGJ2), the role of cAMP as a mediator of PGD2-induced cell growth has been questioned. In this study we investigated the effects of PGD2 on cAMP level, DNA synthesis, and c-myc expression using EBTr cells growth-arrested by serum starvation. Quiescent EBTr cells synchronously resumed [3H]thymidine incorporation at 12 h after the addition of serum, an incorporation which ended at 21 h. PGD2 at 1 microM inhibited approximately 30% incorporation without any delay of initiation, and it also caused a rapid increase in the cAMP level at a maximum of 10 min. The inhibition of [3H]thymidine incorporation was increased 40-45% by 5 microM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The expression of c-myc mRNA induced by serum at 1-4 h was also inhibited by PGD2. Time-course studies support the association between the reduction of c-myc expression and the successive inhibition of DNA synthesis by PGD2. The dose dependency of PGD2 for these three processes correlated well with each other, and the effects were evident at as low as 10 nM and specific for PGD2.(ABSTRACT TRUNCATED AT 250 WORDS)

Okuda-Ashitaka E; Negishi M; Sugama K; Hatanaka M; Ito S

1990-01-01

 
 
 
 
321

Cyclic AMP-mediated inhibition of cell growth by prostaglandin D2 in a fibroblastic cell line (EBTr).  

Science.gov (United States)

We have recently reported that prostaglandin D2 (PGD2) specifically elevates the intracellular cyclic AMP (cAMP) level in a fibroblastic cell line derived from bovine embryonic trachea (EBTr) (K. Sugama, T. Tanaka, H. Yokohama, M. Negishi, H. Hayashi, S. Ito, and O. Hayaishi, Biochim. Biophys. Acta, 1011: 75-80, 1989). Since the in vitro and in vivo inhibitory effects of PGD2 on cell growth are attributed to the dehydrate product 9-deoxy-delta 9,12-13,14-dihydro-PGD2 (delta 12-PGJ2), the role of cAMP as a mediator of PGD2-induced cell growth has been questioned. In this study we investigated the effects of PGD2 on cAMP level, DNA synthesis, and c-myc expression using EBTr cells growth-arrested by serum starvation. Quiescent EBTr cells synchronously resumed [3H]thymidine incorporation at 12 h after the addition of serum, an incorporation which ended at 21 h. PGD2 at 1 microM inhibited approximately 30% incorporation without any delay of initiation, and it also caused a rapid increase in the cAMP level at a maximum of 10 min. The inhibition of [3H]thymidine incorporation was increased 40-45% by 5 microM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The expression of c-myc mRNA induced by serum at 1-4 h was also inhibited by PGD2. Time-course studies support the association between the reduction of c-myc expression and the successive inhibition of DNA synthesis by PGD2. The dose dependency of PGD2 for these three processes correlated well with each other, and the effects were evident at as low as 10 nM and specific for PGD2.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1705811

Okuda-Ashitaka, E; Negishi, M; Sugama, K; Hatanaka, M; Ito, S

1990-01-01

322

The effect of tetraethylammonium on intracellular calcium concentration in Alzheimer's disease fibroblasts with APP, S182 and E5-1 missense mutations.  

UK PubMed Central (United Kingdom)

It has been proposed that the lack of intracellular calcium concentration ([Ca2+]i) increase induced by the potassium channel blocker tetraethylammonium (TEA) in skin fibroblast cell lines identifies patients with both sporadic and familial Alzheimer's disease (AD). In order to verify this hypothesis, the effect of TEA on [Ca2+]i was studied in single fura-2-loaded skin fibroblast cell lines available in the Tissue Bank of the Italian Research Council. Four out of eight familial AD patients (one patient with S182 mutation, one patient with E5-1 mutation and two patients with 717 Val-->Ile APP mutation) and two out of five sporadic AD patients showed a positive response to TEA, whereas five out of 11 control lines were unresponsive. Our data suggest that the absence of the TEA-induced increase in [Ca2+]i in skin fibroblast cell lines does not identify all AD patients.

Failli P; Tesco G; Ruocco C; Ginestroni A; Amaducci L; Giotti A; Sorbi S

1996-04-01

323

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines  

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Full Text Available Abstract Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1?, IL-2, IL-6, IL-8 and TGF-? for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1?, IL-2, TGF-?, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

Selvey Saxon; Haupt Larisa M; Thompson Erik W; Matthaei Klaus I; Irving Michael G; Griffiths Lyn R

2004-01-01

324

The sensitizer 2,4-dinitrofluorobenzene activates caspase-3 and induces cell death in a skin dendritic cell line  

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In this work, a dendritic cell line derived from mouse skin (FSDC) was used, as an in vitro experimental model, to evaluate the cytotoxic effect of two chemical sensitizers, a strong sensitizer (2,4-dinitrofluorobenzene, DNFB) and a weak sensitizer (2,4-dichloronitrobenzene, DCNB). The results indic...

Cruz, MT; Duarte, CB; Gonçalo, Margarida; Figueiredo, A; Carvalho, AP; Lopes, MC

325

The effects of low-level laser irradiation on cellular viability and proliferation of human skin fibroblasts cultured in high glucose mediums.  

UK PubMed Central (United Kingdom)

Delayed wound healing is one of the most challenging complications of diabetes mellitus (DM) in clinical medicine. This study has aimed to evaluate the effects of low-level laser therapy (LLLT) on human skin fibroblasts (HSFs) cultured in a high glucose concentration. HSFs were cultured either in a concentration of physiologic glucose (5.5 mM/l) or high glucose media (11.1 and15?mM/l) for either 1 or 2 weeks after which they were subsequently cultured in either the physiologic glucose or high concentration glucose media during laser irradiation. LLLT was carried out with a helium-neon (He-Ne) laser unit at energy densities of 0.5, 1, and 2 J/cm(2), and power density of 0.66 mW/cm(2) on 3 consecutive days. HSFs' viability and proliferation rate were evaluated with the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay. The LLLT at densities of 0.5 and 1 J/cm(2) had stimulatory effects on the viability and proliferation rate of HSFs cultured in physiologic glucose (5.5 mM/l) medium compared to their control cultures (p?=?0.002 and p?=?0.046, respectively). All three doses of 0.5, 1, and 2 J/cm(2) had stimulatory effects on the proliferation rate of HSFs cultured in high glucose concentrations when compared to their control cultures (p?=?0.042, p?=?0.000, and p?=?0.000, respectively). This study showed that HSFs originally cultured for 2 weeks in high glucose concentration followed by culture in physiologic glucose during laser irradiation showed enhanced cell viability and proliferation. Thus, LLLT had a stimulatory effect on these HSFs.

Esmaeelinejad M; Bayat M; Darbandi H; Bayat M; Mosaffa N

2013-03-01

326

Insulin stimulates the biosynthesis of chiro-inositol-containing phospholipids in a rat fibroblast line expressing the human insulin receptor.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

HIRc-B cells (rat fibroblasts expressing the human insulin receptor) were incubated with myo-[3H]inositol for 48 hr, and the biosynthesis of chiro-[3H]inositol was investigated in the absence and presence of insulin following a time course up to 60 min. After phase separation, treatment with insulin...

Pak, Y; Paule, C R; Bao, Y D; Huang, L C; Larner, J

327

Liposomes encapsulating Aloe vera leaf gel extract significantly enhance proliferation and collagen synthesis in human skin cell lines.  

Science.gov (United States)

Aloe vela leaf gel extract (AGE) are widely used as cosmetic and pharmaceutical ingredients because of its versatile skin care properties. In order to enhance the bioavailability of AGE, liposomes encapsulating AGE were prepared and examined for their interfacial and biochemical properties. The liposomes prepared from a soybean lecithin (SLP-WHITE, 1.0 wt%) by the Bangham method gave relatively a good trapping efficiency up to the AGE concentration of 0.5 wt%. The stable liposomes were then prepared from 1.0 wt% of SLP-WHITE and different concentrations of AGE by the mechanochemical method using a homogenizer and microfluidizer. The liposomes obtained from 0.25 wt% of AGE were confirmed to be small unilamellar vesicles with a diameter of less than 200 nm, and remained well dispersed for at least two weeks. The obtained liposomes encapsulating AGE were further examined for the effects on proliferation and type I collagen synthesis in normal human neonatal skin fibroblasts, NB1RGB cells. Liposomal AGE clearly showed higher proliferation rate than that of AGE alone. In addition, compared to the control, liposomal AGE significantly increased the collagen synthesis by 23%, while AGE alone showed a small effect. Liposomal AGE was also assayed for the effect on proliferation in normal human epidermal keratinocytes, NHEK(F) cells. Interestingly, liposomal AGE fractions containing 4 and 20 microg/mL of the extract considerably increased the proliferation rate by 77% and 101%, respectively. In contrast, AGE alone fractions containing 4 and 20 microg/mL of the extract increased the rate by 41% and 60%, respectively. Accordingly, the bioavailability and skin care properties of AGE will be significantly enhanced by liposome encapsulation, and the present liposomal AGE should have a great potential as an effective skin care formulation. PMID:19915322

Takahashi, Makoto; Kitamoto, Dai; Asikin, Yonathan; Takara, Kensaku; Wada, Koji

2009-01-01

328

Liposomes encapsulating Aloe vera leaf gel extract significantly enhance proliferation and collagen synthesis in human skin cell lines.  

UK PubMed Central (United Kingdom)

Aloe vela leaf gel extract (AGE) are widely used as cosmetic and pharmaceutical ingredients because of its versatile skin care properties. In order to enhance the bioavailability of AGE, liposomes encapsulating AGE were prepared and examined for their interfacial and biochemical properties. The liposomes prepared from a soybean lecithin (SLP-WHITE, 1.0 wt%) by the Bangham method gave relatively a good trapping efficiency up to the AGE concentration of 0.5 wt%. The stable liposomes were then prepared from 1.0 wt% of SLP-WHITE and different concentrations of AGE by the mechanochemical method using a homogenizer and microfluidizer. The liposomes obtained from 0.25 wt% of AGE were confirmed to be small unilamellar vesicles with a diameter of less than 200 nm, and remained well dispersed for at least two weeks. The obtained liposomes encapsulating AGE were further examined for the effects on proliferation and type I collagen synthesis in normal human neonatal skin fibroblasts, NB1RGB cells. Liposomal AGE clearly showed higher proliferation rate than that of AGE alone. In addition, compared to the control, liposomal AGE significantly increased the collagen synthesis by 23%, while AGE alone showed a small effect. Liposomal AGE was also assayed for the effect on proliferation in normal human epidermal keratinocytes, NHEK(F) cells. Interestingly, liposomal AGE fractions containing 4 and 20 microg/mL of the extract considerably increased the proliferation rate by 77% and 101%, respectively. In contrast, AGE alone fractions containing 4 and 20 microg/mL of the extract increased the rate by 41% and 60%, respectively. Accordingly, the bioavailability and skin care properties of AGE will be significantly enhanced by liposome encapsulation, and the present liposomal AGE should have a great potential as an effective skin care formulation.

Takahashi M; Kitamoto D; Asikin Y; Takara K; Wada K

2009-01-01

329

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum  

Energy Technology Data Exchange (ETDEWEB)

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.

Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.; Kraemer, K.H.

1987-12-01

330

Effect of Atropa belladonna L. on skin wound healing: biomechanical and histological study in rats and in vitro study in keratinocytes, 3T3 fibroblasts, and human umbilical vein endothelial cells.  

Science.gov (United States)

The effect of Atropa belladonna L. (AB) aqueous extract on skin wound healing was studied in male Sprague-Dawley rats subjected to two parallel full-thickness skin incisions on the back. Specimens for histological evaluation were collected on days 2 and 5 whereas for biomechanical testing, they were collected on day 5. In the in vitro study, a different concentration of AB extract was used to test the differentiation of keratinocytes using a panel of selected antibodies, proliferation, and cell survival of 3T3 fibroblasts and human umbilical vein endothelial cells using the MTT-assay. Results of the in vivo experiments showed in AB-treated wounds a shortened process of inflammation and accelerated collagen formation, as well as significantly increased wound stiffness as compared with control tissues. The in vitro examination showed that control keratinocytes were cytokeratin 19 free, while samples exposed to the highest AB extract concentration expressed CK19. Moreover, all concentrations were stimulatory to human umbilical vein endothelial cell proliferation. In addition, only the AB extract at the lowest tested concentration increased fibroblast growth, but higher concentrations decreased cell survival. In conclusion, our results indicate that the AB water extract positively affects early phases of skin wound healing in rats. However, the in vitro results on the inverse relation between the concentration of the AB extract and its effects on cell proliferation may be important for future research. PMID:19660046

Gál, Peter; Toporcer, Tomás; Grendel, Tomás; Vidová, Zuzana; Smetana, Karel; Dvoránková, Barbora; Gál, Tomás; Mozes, Stefan; Lenhardt, L'udovít; Longauer, Frantisek; Sabol, Marián; Sabo, Ján; Backor, Martin

331

Efficacy and Tolerability of a Facial Serum for Fine Lines, Wrinkles, and Photodamaged Skin  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Background: Dermatology visits for the prevention and treatment of aging skin are rapidly increasing. The clinical sequelae including wrinkling, pigmentary changes, roughness, laxity, and telangiectasia can all result in the appearance of aging skin, impacting quality of life. A facial serum was dev...

Mccall-Perez, Fred; Stephens, Thomas J.; Herndon, James H.

332

Cytotoxicity and Proliferation Studies with Arsenic in Established Human Cell Lines: Keratinocytes, Melanocytes, Dendritic Cells, Dermal Fibroblasts, Microvascular Endothelial Cells, Monocytes and T-Cells  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract: Based on the hypothesis that arsenic exposure results in toxicity and mitogenecity, this study examined the dose-response of arsenic in established human cell lines of keratinocytes (HaCaT), melanocytes (1675), dendritic cells (THP-1/A23187), dermal fibroblasts (CRL1904), microvascular endothelial cells (HMEC), monocytes (THP-1), and T cells (Jurkat). Cytotoxicity was determined by incubating THP-1, THP-1+ A23187 and JKT cells in RPMI 1640, 1675 in Vitacell, HMEC in EBM, and dermal fibroblasts and HaCaT in DMEM with 10% fetal bovine serum, 1% streptomycin and penicillin for 72 hrs in 96-well microtiter plates, at 37oC in a 5% CO2 incubator with different concentrations of arsenic using fluorescein diacetate (FDA). Cell proliferation in 96-well plates was determined in cultured cells starved by prior incubation for 24 hrs in 1% FBS and exposed for 72 hours, using the 96 cell titer proliferation solution (Promega) assay. Cytotoxicity assays yielded LD50s of 9 μg/mL for HaCaT, 1.5 μg/mL for CRL 1675, 1.5 μg/mL for dendritic cells, 37 μg/mL for dermal fibroblasts, 0.48 μg/mL for HMEC, 50 μg/mL for THP-1 cells and 50 μg/mL for JKT-T cells. The peak proliferation was observed at 6 μg/mL for HaCaT and THP-1 cells, 0.19 μg/mL for CRL 1675, dendritic cells, and HMEC, and 1.5 μg/mL for dermal fibroblasts and Jurkat T cells. These results show that arsenic is toxic at high doses to keratinocytes, fibroblasts, monocytes and T cells, and toxic at lower doses to melanocytes, microvascular endothelial cells and dendritic cells. Proliferation studies showed sub-lethal doses of arsenic to be mitogenic.

Barbara Graham-Evans; Paul B. Tchounwou; Hari H. P. Cohly

2003-01-01

333

[Pharmacoeconomic assessment of daptomycin as first-line therapy for bacteraemia and complicated skin and skin structure infections caused by gram-positive pathogens in Spain].  

UK PubMed Central (United Kingdom)

OBJECTIVE: To assess the efficiency of daptomycin as firstline therapy (D) versus daptomycin as salvage therapy after vancomycin (V?D ) or linezolid (L?D) failure in gram-positive bacteraemia and complicated skin and skin-structure infections (cSSTIs). METHODS: Cost-effectiveness analysis of 161 bacteraemia and 84 cSSTIs patients comparing the above mentioned therapeutic alternatives was performed using the data from 27 Spanish hospitals involved in the EUCORE study. Direct medical costs were considered. Patients were observed from the first antibiotic dose for infection until either the end of daptomycin therapy or exitus. A multivariate Monte Carlo probabilistic sensitivity analysis was applied for costs (lognormal distribution) and effectiveness (normal distribution). RESULTS: In terms of effectiveness there were no statistical differences between groups but referring total costs per patient, there were significant differences. Sensitivity analysis confirmed that D dominates over L?D between 44.2%-62.1% of simulations in bacteraemia and between 48.2%-67.5% in cSSTIs. In comparison to V?D, D dominance was detected in 29.2%-33.2% of simulations in bacteraemia and between 48.2%-59.3% in cSSTIs. CONCLUSIONS: Daptomycin as first-line therapy dominates over daptomycin as salvage therapy after linezolid failure both in bacteraemia and cSSTIs. Comparing daptomycin as first-line therapy with its use after vancomycin failure, in cSSTIs the former is dominant. In bacteremia daptomycin as first line therapy is as effective as daptomycin as salvage therapy after vancomycin failure and implies lower costs.

Grau S; Rebollo P; Cuervo J; Gil-Parrado S

2011-09-01

334

An integrative framework of the skin receptors activation: mechanoreceptors activity patterns versus "labeled lines".  

UK PubMed Central (United Kingdom)

The paper presents a review of electrophysiological data which indicate the integrative mechanisms of information coded in the human and animal peripheral skin receptors. The activity of the skin sensory receptors was examined by applying various natural stimuli. It was revealed that numerous identical receptors respond to various stimuli (mechanical, temperature, and pain ones), but the spike patterns of these receptors were found to be specific for each stimulus. The description of characteristic structures of spike patterns in the cutaneous nerve fibers in response to five major modalities, namely: "touch", "pain", "vibration/breath", "cold", and "heat", is being presented. The recordings of the cutaneous physical state revealed a correlation between the patterns of spatiotemporal skin deformation and the receptors activity. A rheological state of the skin can be changed either in response to external temperature variation or by the sympathetic pilomotor activation. These results indicate that the skin sensory receptors activity may be considered as an integrative process. It depends not only on the receptors themselves, but also on the changes in the surrounding tissue and on the adaptive influence of the central nervous system. A new framework for the sensory channel system related to the skin is proposed on the basis of experimental results.

Zeveke AV; Efes ED; Polevaya SA

2013-03-01

335

Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency.  

UK PubMed Central (United Kingdom)

The human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test, is based on the augmentation of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. The h-CLAT was found to be capable of determining the hazard of skin sensitization. In contrast, the local lymph node assay (LLNA), widely used as a stand-alone method in Europe and US, identifies the same hazard, but also classifies the potency by using the estimated concentration of SI=3 (EC3). In this study, several values calculated from the h-CLAT data were evaluated for its correlation to the LLNA EC3 determination. A statistically significant correlation was observed between h-CLAT concentration providing a cell viability of 75% (CV75), h-CLAT estimated concentration of RFI=150 for CD86 (EC150), and for CD54 (EC200) with LLNA's EC3. From EC150 and EC200, a minimum induction threshold (MIT) was determined as the smaller of either EC150 or EC200. MIT showed a correlation with EC3 (R=0.638). Also, MIT had an approximate 80% accuracy for sub-categories of the globally harmonized system (GHS) when a tentative threshold of 13 ?g/mL was used. From these data, the h-CLAT values may be one of the useful tools to predict the allergic potency of chemicals.

Nukada Y; Ashikaga T; Miyazawa M; Hirota M; Sakaguchi H; Sasa H; Nishiyama N

2012-10-01

336

PAMPs and DAMPs stimulate the expression of pro-inflammatory cytokines in vitro in a fibroblast cell-line from rainbow trout (Oncorhynchus mykiss)  

DEFF Research Database (Denmark)

The recognition of PAMPs by immune cells relies on conserved PRRs such as TLRs, NLRs and RLRs leading to activation of NF?B signalling pathways. These receptors are activated upon stimulation by different ligands such as bacterial or viral components. The binding of ligands to the receptors activates downstream signalling pathways, which subsequently leads to expression of pro-inflammatory cytokines and chemokines. DAMPs released from necrotic cells may also bind to and activate similar downstream signalling events. In telosts was found that mechanical damage of the muscle tissue using sterile needles induced a very rapid expression of the pro-inflammatory cytokines IL-1?, IL-8 and IL-10 as measured by real-time PCR. The results imply that cells located in the muscular tissue in addition to recruited cells are involved in the observed increased cytokine / chemokine expression. It is believed that this expression to a large extend is mediated by fibroblasts in the musculature. To investigate this, a fibroblastcell-line (RTHDF1) from the rainbow trout was stimulated with either LPS from E. coli, cell debris or supernatant from sonicated fibroblasts. Whereas LPS stimulation resulted in a significant up-regulation of the expression of IL-1?, IL-8 and IL-10 and stimulation with supernatant from sonicated cells led to a significant up-regulation of IL-1? and IL-10, while debris only stimulated the expression of IL-1?. TLR-2 and -4 are not described from salmonid fishes, however TLR-3, -5 and -9 are described in this evolutionary lineage of the bony fishes. The expression of TLR-3 and -9 receptors were significantly up-regulated following physical damage of muscle tissue as well as in stimulated fibroblasts, where LPS induced both TLR-3 and -9, supernatant from sonicated cells only TLR-9 while debris caused no induction. The present study reinforce the idea that fibroblasts are able to react to PAMPs and DAMPs and that non-immune cell-types play an important role in the inflammatory reaction per se. From an evolutionary perspective the facilitation of an inflammatory response through recognition of PAMPs and DAMPs by non immune cells seems plausible.

Ingerslev, Hans-Christian; Ossum, C.G.

337

Differentiation of normal and transformed human fibroblasts in vitro is influenced by electromagnetic fields  

Energy Technology Data Exchange (ETDEWEB)

We studied the effect of symmetric, biphasic sinusoidal electromagnetic fields (EMF) (20 Hz, 6 mT) on the differentiation of normal human skin fibroblasts (HH-8), normal human lung fibroblasts (WI38), and SV40-transformed human lung fibroblasts (WI38SV40) in in vitro cultures. Cells were exposed up to 21 days for 2 x 6 h per day to EMF. Normal mitotic human skin and lung fibroblasts could be induced to differentiate into postmitotic cells upon exposure to EMF. Concomitantly, the synthesis of total collagen as well as total cellular protein increased significantly by a factor of 5-13 in EMF-induced postmitotic cells. As analyzed by two-dimensional gel electrophoresis of (/sup 35/S)methionine-labeled polypeptides, EMF-induced postmitotic cells express the same differentiation-dependent and cell type-specific marker proteins as their spontaneously arising counterparts. In SV40-transformed human lung fibroblasts (cell line WI38SV40) the exposure to EMF induced the differentiation of mitotic WI38SV40 cells into postmitotic and degenerating cells in subpopulations of WI38SV40 cell cultures. Other subpopulations of WI38SV40 cells did not show any effect of EMF on cell proliferation and differentiation. These results indicate that long-term EMF exposure of fibroblasts in vitro induces the differentiation of mitotic to postmitotic cells that are characterized by differentiation-specific proteins and differentiation-dependent enhanced metabolic activities.

Rodemann, H.P.; Bayreuther, K.; Pfleiderer, G.

1989-06-01

338

Cytotoxic Evaluation of Elastomeric Dental Impression Materials on a Permanent Mouse Cell Line and on a Primary Human Gingival Fibroblast Culture  

Directory of Open Access Journals (Sweden)

Full Text Available The need for clinically relevant in vitro tests of dental materials is widely recognized. Nearly all dental impression materials are introduced into the mouth just after mixing and allowed to set in contact with the oral tissues. Under these conditions, the materials may be toxic to cells or may sensitize the tissues. The aim of the present study is to evaluate the potential cytotoxicity of new preparations of elastomeric dental impression materials: A) four vinylpolysiloxanes: Elite H-D Putty and Elite H-D Light Body (Zhermack, Badia Polesine, Rovigo, Italy); Express Putty and Express Light Body (3M ESPE AG Seefeld, Germany) and B) two polyethers: Impregum Penta and Permadyne Penta L (3M ESPE AG Seefeld, Germany). The cytotoxicity of these impression materials were examined using two different cell lines: Balb/c 3T3 (permanent cell line) and human gingival fibroblasts (primary cell line) and their effects were studied by indirect and direct tests. The direct tests are performed by placing one sample of the impression materials in the centre of the Petri dishes at the time of the seeding of cells. The cell growth was evaluated at the 12th and 24th hours by cell number. The indirect tests were performed by incubating a square of 1 cm diameter impression material in 5 mL of medium at 37 °C for 24 hours (“eluates”). Subconfluent cultures are incubated with “eluates” for 24 hours. The MTT-formazan production is the method used for measuring the cell viability. The results indicate that: a) polyether materials are cytotoxic under both experimental conditions; b) among vinylpolysiloxanes, only Express Light Body (3M ESPE AG Seefeld, Germany) induces clear inhibition of cellular viability of Balb/c 3T3 evaluated by direct and indirect tests and c) the primary cell line is less sensitive to the toxic effect than the permanent cell line.

Federica Boraldi; Chiara Coppi; Sergio Bortolini; Ugo Consolo; Roberta Tiozzo

2009-01-01

339

Transcription of canine toll-like receptor 2, ?-defensin 1 and ?-defensin 103 in infected atopic skin, non-infected atopic skin, healthy skin and the CPEK cell line.  

UK PubMed Central (United Kingdom)

Dogs and humans with atopic dermatitis (AD) have a high prevalence of recurrent staphylococcal pyoderma. ?-Defensins (BDs) and toll-like receptors (TLRs) are important in innate immunity against bacterial skin infections, and decreased BD and TLR2 expression has been associated with human AD. However, findings from recent studies of BD expression in human and canine AD have been variable and contradictory. The aim of this study was to further our understanding of the role of antimicrobial proteins in canine AD by quantifying mRNA for canine (c) BD1, cBD103 and TLR2 in healthy skin (n=17 dogs), matched samples of atopic skin with and without active infection by Staphylococcus pseudintermedius (n=13 dogs), and the canine keratinocyte cell line CPEK cultured with 5 ng/ml tumour necrosis factor alpha (TNF?) and 10 ?g/ml lipopolysaccharide (LPS). mRNA for cBD1, CB103 and TLR2 were detected in all samples. TNF? significantly increased transcription of cBD1, cBD103 and TLR2 in the CPEK cells. mRNA for cBD103 was also significantly increased after stimulation with LPS. There were no significant differences in mRNA levels for cBD1, cBD103 or TLR2 in healthy, non-infected atopic or infected atopic skin. Canine AD did not appear to be associated with altered expression of cBD1, cBD103 and TLR2 in these dogs. Other studies have reported both increased and decreased expression of these antimicrobial peptides in canine AD and pyoderma, and therefore further investigation of the clinical significance of these mediators is required.

Mullin J; Carter S; Williams N; McEwan N; Nuttall T

2013-03-01

340

Transcription of canine toll-like receptor 2, ?-defensin 1 and ?-defensin 103 in infected atopic skin, non-infected atopic skin, healthy skin and the CPEK cell line.  

Science.gov (United States)

Dogs and humans with atopic dermatitis (AD) have a high prevalence of recurrent staphylococcal pyoderma. ?-Defensins (BDs) and toll-like receptors (TLRs) are important in innate immunity against bacterial skin infections, and decreased BD and TLR2 expression has been associated with human AD. However, findings from recent studies of BD expression in human and canine AD have been variable and contradictory. The aim of this study was to further our understanding of the role of antimicrobial proteins in canine AD by quantifying mRNA for canine (c) BD1, cBD103 and TLR2 in healthy skin (n=17 dogs), matched samples of atopic skin with and without active infection by Staphylococcus pseudintermedius (n=13 dogs), and the canine keratinocyte cell line CPEK cultured with 5 ng/ml tumour necrosis factor alpha (TNF?) and 10 ?g/ml lipopolysaccharide (LPS). mRNA for cBD1, CB103 and TLR2 were detected in all samples. TNF? significantly increased transcription of cBD1, cBD103 and TLR2 in the CPEK cells. mRNA for cBD103 was also significantly increased after stimulation with LPS. There were no significant differences in mRNA levels for cBD1, cBD103 or TLR2 in healthy, non-infected atopic or infected atopic skin. Canine AD did not appear to be associated with altered expression of cBD1, cBD103 and TLR2 in these dogs. Other studies have reported both increased and decreased expression of these antimicrobial peptides in canine AD and pyoderma, and therefore further investigation of the clinical significance of these mediators is required. PMID:23067725

Mullin, Jenny; Carter, Stuart; Williams, Nicola; McEwan, Neil; Nuttall, Tim

2012-09-26

 
 
 
 
341

Liposomal formulation of chondroitin sulfate enhances its antioxidant and anti-inflammatory potential in L929 fibroblast cell line.  

UK PubMed Central (United Kingdom)

Liposomes have the capacity to be used as efficient, biodegradable and nontoxic carriers of bioactive molecules and are able to better control their delivery at the site of interest. The objective of this study was to obtain and characterize an appropriate liposomal formulation of the bioactive molecule chondroitin sulfate (CS) for its use in the local treatment of inflammatory and degenerative disorders, specifically osteoarthritis (OA). Empty liposomes (L) and CS-entrapping liposomes (L-CS) were prepared by thin film hydration method followed by sonication and extrusion. They were characterized in terms of size, polydispersity index and ?-potential by dynamic light scattering (DLS) and morphology by transmission electron microscopy. The effect of L-CS formulation on viability and morphology of mouse fibroblast cells and its biologic activity in hydrogen peroxide-stimulated cells were compared to those of L, non-encapsulated CS and a mixture of L and CS (L?+?CS). Our results demonstrated a high biocompatibility of L-CS and a more efficient cell protection against oxidative damage using L-CS treatment than CS or L?+?CS treatment. Also, L-CS exhibited a higher anti-inflammatory activity than CS in stimulated cells by reducing the level of IL-8 and TNF-? proinflammatory cytokines. The overall results suggest that the delivery of CS in liposomal formulation could improve its therapeutic potential in intra-articular treatment of OA.

Craciunescu O; Moldovan L; Moisei M; Trif M

2013-06-01

342

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms  

Scientific Electronic Library Online (English)

Full Text Available Abstract in english Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D (more) breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.

Ramezanpour, M; Burke da Silva, K; Sanderson, BJ

2012-01-01

343

Differential susceptibilities of human lung, breast and skin cancer cell lines to killing by five sea anemone venoms  

Directory of Open Access Journals (Sweden)

Full Text Available Although sea anemones are well known for being rich sources of toxins, including cytolysins and neurotoxins, their venoms and toxins have been poorly studied. In the present study, the venoms from five sea anemones (Heteractis crispa, Heteractis magnifica, Heteractis malu, Cryptodendrum adhaesivum and Entacmaea quadricolor) were obtained by the milking technique, and the potential of these venoms to kill cancer cells was tested on three cell lines (A549 lung cancer, T47D breast cancer and A431 skin cancer). The total protein level in the crude extract was determined by the bicinchoninic acid (BCA) protein assay. The cytotoxicity on different cell lines was assayed using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay which measures survival based on the detection of mitochondrial activity and by the crystal violet assay, which measures survival based on the ability of cells to remain adherent to microplates. The results indicate that the sea anemone venom is cytotoxic to human cancer cells. The A549 cell line was the most sensitive of the cell lines tested with a significant reduction in viability observed at 40 µg/mL. H. malu, C. adhaesivum and E. quadricolor had a significant inhibitory effect on A431 cells. Furthermore, H. malu and C. adhaesivum had a significant inhibitory effect on T47D cell line at 40 µg/mL. In conclusion, the sea anemone venoms tested have the potential to be developed as anticancer agents.

M Ramezanpour; K Burke da Silva; BJ Sanderson

2012-01-01

344

Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.  

UK PubMed Central (United Kingdom)

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

Abyzov A; Mariani J; Palejev D; Zhang Y; Haney MS; Tomasini L; Ferrandino AF; Rosenberg Belmaker LA; Szekely A; Wilson M; Kocabas A; Calixto NE; Grigorenko EL; Huttner A; Chawarska K; Weissman S; Urban AE; Gerstein M; Vaccarino FM

2012-12-01

345

Organotypic cocultures as skin equivalents: A complex and sophisticated in vitro system  

Directory of Open Access Journals (Sweden)

Full Text Available To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms.

Stark Hans-Jürgen; Szabowski Axel; Fusenig Norbert E.; Maas-Szabowski Nicole

2004-01-01

346

The repair of sublethal damage in diploid human fibroblasts: a comparison between human and rodent cell lines  

International Nuclear Information System (INIS)

[en] Two diploid cell lines of human origin were irradiated in vitro. Repair of sublethal damage was observed in both lines when they were exposed to a single gamma-ray dose and incubated at 37 degrees C prior to receiving a graded second dose. In addition cells were irradiated at a low dose rate (56 or 76 rad/hr) and survival was compared to that after an acute exposure. A dose-rate effect was demonstrated. Sublethal damage repair was also studied using CHO or V-79 Chinese hamster cells, and the data suggest that the degree of repair is similar in human and rodent cell lines at photon doses up to 500 rad. The repair of sublethal damage can be a significant factor in the survival of human cells in vitro for fractionated doses

1983-01-01

347

Effect of phosphatidylserine on free radical susceptibility in human diploid fibroblasts.  

UK PubMed Central (United Kingdom)

We studied the effect of phosphatidylserine (PdtSER) on oxygen metabolite toxicity in skin fibroblast cell lines from apparently normal subjects. Fibroblast damage was produced by the generation of oxygen metabolites during the enzymatic oxidation of acetaldehyde by xanthine-oxidase (Xo). In order to quantify cell damage, we measured lactate dehydrogenase (LDH) activity in culture medium and cell viability in fibroblast cultures, with and without preincubation for 4 days with PdtSER 13 microM, after Xo incubation. We found a significant increase of LDH activity in culture medium of cells without preincubation with PdtSER. No significant increase of LDH activity was observed in the same cell lines after preincubation with PdtSER.

Latorraca S; Piersanti P; Tesco G; Piacentini S; Amaducci L; Sorbi S

1993-01-01

348

Analysis of the tumor suppressor gene p53 in xeroderma pigmentosum fibroblasts.  

UK PubMed Central (United Kingdom)

Genetic risk factor(s) for skin cancers have been described in patients with xeroderma pigmentosum (XP). The tumor suppressor gene, p53, is one of the most frequently mutated genes found in human tumors. To evaluate the role of XP-related genetic defects in the p53 gene, skin fibroblast cell lines derived from XP donors were analysed for mutations in exons 5-9 (the regions of gene highly susceptible for mutations) by single-strand conformation polymorphism (SSCP) and nucleotide sequencing. Of the five XP-derived fibroblasts (complementation group A) and two control fibroblast cell lines, only one XP cell line showed an aberrant SSCP banding pattern in the region of the p53 gene (comprising the 7th exon and neighbouring intronic sequences). Nucleotide sequencing of this region confirmed a mutation in the 7th intron adjacent to the 7th exon, which did not affect the RNA splice site. These results suggest that constitutional/germ-line mutations in the p53 gene may not play a role in the occurrence of skin carcinomas in XP patients.

Lavu S; Srivastava M; Srivastava SK

1994-09-01

349

Analysis of the tumor suppressor gene p53 in xeroderma pigmentosum fibroblasts.  

Science.gov (United States)

Genetic risk factor(s) for skin cancers have been described in patients with xeroderma pigmentosum (XP). The tumor suppressor gene, p53, is one of the most frequently mutated genes found in human tumors. To evaluate the role of XP-related genetic defects in the p53 gene, skin fibroblast cell lines derived from XP donors were analysed for mutations in exons 5-9 (the regions of gene highly susceptible for mutations) by single-strand conformation polymorphism (SSCP) and nucleotide sequencing. Of the five XP-derived fibroblasts (complementation group A) and two control fibroblast cell lines, only one XP cell line showed an aberrant SSCP banding pattern in the region of the p53 gene (comprising the 7th exon and neighbouring intronic sequences). Nucleotide sequencing of this region confirmed a mutation in the 7th intron adjacent to the 7th exon, which did not affect the RNA splice site. These results suggest that constitutional/germ-line mutations in the p53 gene may not play a role in the occurrence of skin carcinomas in XP patients. PMID:7923108

Lavu, S; Srivastava, M; Srivastava, S K

1994-09-30

350

Quantification and topographical description of Ki-67 antibody labelling during the cell cycle of normal fibroblastic (MRC-5) and mammary tumour cell lines (MCF-7).  

Science.gov (United States)

The expression of a human nuclear antigen associated with cell proliferation and recognized by monoclonal antibody Ki-67 was investigated during the cell cycle in a breast cancer cell line (MCF-7) and in a normal human fibroblastic cell line (MRC-5). Image cytometry was used to determine Ki-67 antigen and DNA amounts at the cell level. Results confirm Ki-67 antibody specificity for proliferating cells. The antigen expression increases with cell progression through the cycle. Image cytometry also permits the description of the Ki-67 topographic distribution at the various phases of the cell cycle. Ki-67 antigen is essentially localized in the nucleoli at G1 phase. A nucleoplasmic distribution appears later in the cell cycle. The parameters measured on the labelling allow the calculation of the growth fraction and an evaluation of the phase fractions. Other results in this study strongly suggest that nucleolar rRNA synthesis is required for Ki-67 antigen expression. Hypotheses on Ki-67 functions are proposed. PMID:2488698

Guillaud, P; du Manoir, S; Seigneurin, D

1989-02-01

351

Quantification and topographical description of Ki-67 antibody labelling during the cell cycle of normal fibroblastic (MRC-5) and mammary tumour cell lines (MCF-7).  

UK PubMed Central (United Kingdom)

The expression of a human nuclear antigen associated with cell proliferation and recognized by monoclonal antibody Ki-67 was investigated during the cell cycle in a breast cancer cell line (MCF-7) and in a normal human fibroblastic cell line (MRC-5). Image cytometry was used to determine Ki-67 antigen and DNA amounts at the cell level. Results confirm Ki-67 antibody specificity for proliferating cells. The antigen expression increases with cell progression through the cycle. Image cytometry also permits the description of the Ki-67 topographic distribution at the various phases of the cell cycle. Ki-67 antigen is essentially localized in the nucleoli at G1 phase. A nucleoplasmic distribution appears later in the cell cycle. The parameters measured on the labelling allow the calculation of the growth fraction and an evaluation of the phase fractions. Other results in this study strongly suggest that nucleolar rRNA synthesis is required for Ki-67 antigen expression. Hypotheses on Ki-67 functions are proposed.

Guillaud P; du Manoir S; Seigneurin D

1989-02-01

352

Construção de substituto da pele composto por matriz de colágeno porcino povoada por fibroblastos dérmicos e queratinócitos humanos: avaliação histológica Construction of a skin substitute composed of porcine collagen matrix populated with human dermal fibroblasts and keratinocytes: histological evaluation  

Directory of Open Access Journals (Sweden)

Full Text Available INTRODUÇÃO: O uso de enxertos autólogos é limitado pela extensão da área doadora e pelo estado clínico dos pacientes, no caso de lesões extensas. Alotransplantes coletados de cadáveres ou voluntários são rejeitados após uma ou duas semanas, servindo apenas como cobertura temporária para essas lesões. O tratamento de grandes lesões cutâneas com tegumento autólogo reconstruído constitui alternativa atraente, já que, a partir de um pequeno fragmento de pele do paciente, pode-se obter culturas de células que se multiplicam rapidamente e podem ser criopreservadas, permitindo, assim, sua utilização em novos tratamentos por tempo indeterminado. Este estudo pretendeu avaliar o comportamento histológico de queratinócitos e fibroblastos humanos cultivados sobre uma matriz de colágeno porcino derivada da submucosa intestinal. MÉTODO: Células da epiderme e derme humana foram cultivadas separadamente e semeadas sobre matriz de colágeno porcino, onde permaneceram em ambiente controlado por 21 dias, antes de serem submetidas a análise histológica. RESULTADOS: Observou-se que os fibroblastos invadem e colonizam a matriz de colágeno, enquanto os queratinócitos se organizam de forma laminar e estratificada sobre a superfície em que foram semeados. CONCLUSÕES: A utilização da matriz de colágeno porcino como carreador de células da pele humana é possível e a organização dessas células se assemelha à arquitetura da pele humana.BACKGROUND: In the case of extensive lesions, the use of autologous grafts is limited by the extent of the donor area and the clinical condition of patients. Allografts collected from cadavers or volunteers are usually rejected after 1 to 2 weeks, thus serving only as temporary cover for these lesions. Treating major cutaneous lesions with reconstructed autologous skin is an attractive alternative, because it is possible to obtain cultures of cells that multiply rapidly and can be cryopreserved from a small fragment of the patient's skin, thereby facilitating its indefinite use in new treatments. This study evaluated the histological behavior of cultured human keratinocytes and fibroblasts on a collagen matrix derived from porcine small intestinal submucosa. METHODS: Cells from human epidermis and dermis were grown separately and seeded on porcine collagen matrix, which was maintained in a controlled environment for 21 days before being subjected to histological analysis. RESULTS: Fibroblasts invaded and colonized the collagen matrix, whereas keratinocytes were organized in laminated and stratified layers on the surface on which they were seeded. CONCLUSIONS: The use of porcine collagen matrix as a support for human skin cells is feasible, and the organization of these cells resembles the architecture of human skin.

Cesar Isaac; Francinni M. P. Rego; Pedro Ribeiro Soares de Ladeir; Silvana C. Altram; Renata C. de Oliveira; Johnny L. C. B. Aldunate; Andr