WorldWideScience

Sample records for b-irradiated premalignant keratinocytes

  1. Infliximab inhibits DNA repair in ultraviolet B-irradiated premalignant keratinocytes

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.; Wulf, Hans Chr.

    2008-01-01

    Anti-tumor necrosis factor-alpha (TNF alpha) approaches are increasingly used in the therapy of autoimmune diseases. One of the safety concerns is the potential enhancement of skin carcinogenesis. The aim of this study was to investigate if the TNF alpha neutralizing antibody, infliximab, directly...... affects the cell cycle and DNA repair in premalignant human keratinocytes after ultraviolet-B (UVB) irradiation. We found that infliximab-treated cells exhibited an enhanced G2/M cell cycle arrest and increased apoptosis after 10-20 mJ/cm(2) UVB. In spite of this, the level of cyclobutane pyrimidine...... dimers (CPD) in infliximab-treated cells was significantly increased at both 24 and 48 h after irradiation with 10 mJ/cm(2) UVB. As we have recently shown that protein kinase B/Akt is involved in the TNF alpha signalling pathway and promotes cell survival and skin carcinogenesis, we measured activatory...

  2. Vanillin protects human keratinocyte stem cells against ultraviolet B irradiation.

    Science.gov (United States)

    Lee, Jienny; Cho, Jae Youl; Lee, Sang Yeol; Lee, Kyung-Woo; Lee, Jongsung; Song, Jae-Young

    2014-01-01

    Ultraviolet-B (UVB) irradiation is one of major factors which induce cellular damages in the epidermis. We investigated protective effects and mechanisms of vanillin, a main constituent of vanilla beans, against UVB-induced cellular damages in keratinocyte stem cells (KSC). Here, vanillin significantly attenuated UVB irradiation-induced cytotoxicity. The vanillin effects were also demonstrated by the results of the senescence-associated β-galactosidase and alkaline comet assays. In addition, vanillin induced production of pro-inflammatory cytokines. Attempts to elucidate a possible mechanism underlying the vanillin-mediated effects revealed that vanillin significantly reduced UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), p38/mitogen-activated protein kinase (p38), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), S6 ribosomal protein (S6RP), and histone 2A family member X (H2A.X). UVB-induced activation of p53 luciferase reporter was also significantly inhibited by vanillin. In addition, while ATM inhibitor had no effect on the vanillin effects, mouse double minute 2 homolog (MDM2) inhibitor significantly attenuated suppressive effects of vanillin on UVB-induced activation of p53 reporter in KSC. Taken together, these findings suggest that vanillin protects KSC from UVB irradiation and its effects may occur through the suppression of downstream step of MDM2 in UVB irradiation-induced p53 activation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Molecular mechanism(s) for UV-B irradiation-induced glutathione depletion in cultured human keratinocytes.

    Science.gov (United States)

    Zhu, Ming; Bowden, G Tim

    2004-01-01

    Glutathione (GSH) plays a central role in maintenance of cellular redox homeostasis and protection against oxidative injury. Ultraviolet B (UV-B) irradiation-induced GSH depletion is believed to be involved in the pathogenesis of several cutaneous disorders. In this study, the molecular mechanism(s) of UV-B-induced GSH depletion was investigated in cultured human keratinocytes, HaCaT cells. We found that UV-B irradiation caused GSH depletion in a dose- and time-dependent manner in HaCaT cells. The mechanistic studies showed that UV-B-induced GSH depletion did not result from the GSH efflux. UV-B irradiation appeared to cause a slight decrease in enzymatic activity of gamma-glutamate cysteine ligase (GCL), a rate-limiting enzyme in GSH biosynthesis. UV-B irradiation resulted in the GCL cleavage through the activation of a caspase cascade. Inhibition of total caspase activity by the general caspase inhibitor, zVAD-fmk, partially reversed the UV-B-induced GSH depletion. More importantly, we found that UV-B irradiation could dramatically decrease the cystine uptake through the functional inhibition of the system Xc(-), a cystine transporter on the cell membrane. The results suggest that the inactivation of cystine transporter system Xc(-) was a major contributor to the UV-B-mediated decrease of GSH levels in human keratinocytes.

  4. Protective effect of silk lutein on ultraviolet B-irradiated human keratinocytes

    Directory of Open Access Journals (Sweden)

    Sutatip Pongcharoen

    2013-01-01

    Full Text Available Carotenoids are efficient antioxidants that are of great importance for human health. Lutein and zeaxanthin are carotinoids present in high concentrations in the human retina which are involved in the photoprotection of the human eye. Lutein may also protect the skin from ultraviolet (UV-induced damage. The present study investigated the protective effect of lutein extracted from yellow silk cocoons of Bombyx mori on human keratinocytes against UVB irradiation. A human keratinocyte cell line and primary human keratinocytes were used to investigate the UVB protection effects of silk lutein and plant lutein. Silk lutein showed no cytotoxicity to keratinocytes. Treatment with silk lutein prior to UVB irradiation enhanced cell viability and cell proliferation, and reduced cell apoptosis. The protective effects of silk lutein may be superior to those of plant lutein. Silk lutein may have a benefit for protection of keratinocytes against UVB-irradiation.

  5. The Effect of Lycopene Preexposure on UV-B-Irradiated Human Keratinocytes

    Science.gov (United States)

    Ascenso, Andreia; Pedrosa, Tiago; Pinho, Sónia; Pinho, Francisco; de Oliveira, José Miguel P. Ferreira; Cabral Marques, Helena; Oliveira, Helena; Simões, Sandra; Santos, Conceição

    2016-01-01

    Lycopene has been reported as the antioxidant most quickly depleted in skin upon UV irradiation, and thus it might play a protective role. Our goal was to investigate the effects of preexposure to lycopene on UV-B-irradiated skin cells. Cells were exposed for 24 h to 10 M lycopene, and subsequently irradiated and left to recover for another 24 h period. Thereafter, several parameters were analyzed by FCM and RT-PCR: genotoxicity/clastogenicity by assessing the cell cycle distribution; apoptosis by performing the Annexin-V assay and analyzing gene expression of apoptosis biomarkers; and oxidative stress by ROS quantification. Lycopene did not significantly affect the profile of apoptotic, necrotic and viable cells in nonirradiated cells neither showed cytostatic effects. However, irradiated cells previously treated with lycopene showed an increase in both dead and viable subpopulations compared to nonexposed irradiated cells. In irradiated cells, lycopene preexposure resulted in overexpression of BAX gene compared to nonexposed irradiated cells. This was accompanied by a cell cycle delay at S-phase transition and consequent decrease of cells in G0/G1 phase. Thus, lycopene seems to play a corrective role in irradiated cells depending on the level of photodamage. Thus, our findings may have implications for the management of skin cancer. PMID:26664697

  6. The induction of apoptosis in pre-malignant keratinocytes by omega-3 polyunsaturated fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) is inhibited by albumin.

    Science.gov (United States)

    Nikolakopoulou, Zacharoula; Shaikh, Mushfiq Hassan; Dehlawi, Hebah; Michael-Titus, Adina Teodora; Parkinson, Eric Kenneth

    2013-04-12

    The long chain omega-3 polyunsaturated fatty acids (PUFA) have been reported to exert anti-cancer effects. At this study we tested the effect of the omega-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on pre-malignant keratinocytes growth in the well-characterised human pre-malignant epidermal cell line, HaCaT and attempted to identify a PUFA serum antagonist. Both EPA and DHA inhibited HaCaT growth and induced apoptosis. At the 10% (v/v) foetal bovine serum (FBS) medium, limited growth inhibition (3-20% for 50μM DHA and EPA respectively) and negligible apoptosis were observed with PUFA use. However, at 3% (v/v) FBS medium, 30-50μM of PUFA caused impressive levels of growth inhibition (82-83% for 50μM DHA and EPA respectively) and increase of apoptosis (8-19% increase in 72h). None of the numerous serum growth factors present in FBS or the antioxidant n-tert-butyl-α-phenylnitrone could inhibit the PUFA-induced cytotoxicity. In contrast, bovine and human albumin (0.1-0.3%, w/v) significantly antagonized the growth inhibitory and apoptosis-inducing effects of PUFA. In conclusion, we have shown for the first time that omega-3 PUFA inhibit the growth and induce apoptosis of pre-malignant keratinocytes and identified albumin as a major antagonistic factor in serum that could limit their effectiveness at pharmacologically-achievable doses. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Ultraviolet B irradiation induces changes in the distribution and release of arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture

    Energy Technology Data Exchange (ETDEWEB)

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1987-05-01

    There is increasing evidence that derivatives of 20-carbon polyunsaturated fatty acids, the eicosanoids, play an important role in the inflammatory responses of the human skin. To better understand the metabolic fate of fatty acids in the skin, the effect of ultraviolet B (UVB) irradiation (280-320 nm) on the distribution and release of /sup 14/C-labeled arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture was investigated. Ultraviolet B irradiation induced the release of all three /sup 14/C-labeled fatty acids from the phospholipids, especially from phosphatidylethanolamine, and this was accompanied by increased labeling of the nonphosphorus lipids. This finding suggests that UVB induces a significant liberation of eicosanoid precursor fatty acids from cellular phospholipids, but the liberated fatty acids are largely reincorporated into the nonphosphorus lipids. In conclusion, the present study suggests that not only arachidonic acid but also dihomo-gamma-linolenic acid, and eicosapentaenoic acid might be involved in the UVB irradiation-induced inflammatory reactions of human skin.

  8. Ultraviolet B irradiation induces changes in the distribution and release of arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture

    International Nuclear Information System (INIS)

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1987-01-01

    There is increasing evidence that derivatives of 20-carbon polyunsaturated fatty acids, the eicosanoids, play an important role in the inflammatory responses of the human skin. To better understand the metabolic fate of fatty acids in the skin, the effect of ultraviolet B (UVB) irradiation (280-320 nm) on the distribution and release of 14 C-labeled arachidonic acid, dihomo-gamma-linolenic acid, and eicosapentaenoic acid in human keratinocytes in culture was investigated. Ultraviolet B irradiation induced the release of all three 14 C-labeled fatty acids from the phospholipids, especially from phosphatidylethanolamine, and this was accompanied by increased labeling of the nonphosphorus lipids. This finding suggests that UVB induces a significant liberation of eicosanoid precursor fatty acids from cellular phospholipids, but the liberated fatty acids are largely reincorporated into the nonphosphorus lipids. In conclusion, the present study suggests that not only arachidonic acid but also dihomo-gamma-linolenic acid, and eicosapentaenoic acid might be involved in the UVB irradiation-induced inflammatory reactions of human skin

  9. Myricetin inhibits Akt survival signaling and induces Bad-mediated apoptosis in a low dose ultraviolet (UV)-B-irradiated HaCaT human immortalized keratinocytes.

    Science.gov (United States)

    Kim, Wanyeon; Yang, Hee Jung; Youn, HyeSook; Yun, Young Ju; Seong, Ki Moon; Youn, BuHyun

    2010-01-01

    Deregulation of cell survival pathways and resistance to apoptosis are generally accepted as crucial aspects of tumorigenesis. As in many tumors, increasing occurrence of human skin cancer and other conflicting effects of solar ultraviolet (UV) radiation enhance the demand for novel chemoprevention agents. Myricetin, a naturally occurring phytochemical, is potent in anti-cancer promoting activity and affords to the chemopreventive potential of several healthy-foods, including fruits and vegetables. We demonstrate here that myricetin inhibits Akt activity to induce apoptosis in a low dose ('repairable dose') UVB-irradiated keratinocytes. Treatment of UVB-irradiated HaCaT cells with an apoptosis-inducing concentration of myricetin (20 microM) resulted in a decrease in phosphorylation of Akt leading to inhibition of its kinase activity. Myricetin treatment also caused a decrease in phosphorylation of Bad (a pro-apoptotic protein), a direct target of Akt in signaling pathway. Interaction between Bad and 14-3-3beta was reduced markedly in UVB-irradiated cells upon a treatment with myricetin. Comparable to these results, myricetin treatment promoted mitochondrial translocation of Bad, loss of the mitochondrial membrane potential, and release of the mitochondrial apoptotic proteins including cytochrome c, Smac, and AIF. Ectopic expression of constitutively active Akt granted statistically significant protection against myricetin-induced apoptosis. In addition, myricetin-induced apoptosis in UVB-irradiated cells was notably attenuated in the presence of caspase inhibitors. Together, these results indicate that myricetin might take on potent chemopreventive activity by inhibiting the Akt-mediated survival signaling axis in UVB-induced skin carcinogenesis.

  10. Aneuploidy and proliferation in keratinocytic intraepidermal neoplasias.

    NARCIS (Netherlands)

    Smits, T.; Olthuis, D.; Blokx, W.A.M.; Kleinpenning, M.M.; Kerkhof, P.C.M. van de; Erp, P.E.J. van; Gerritsen, M.J.P.

    2007-01-01

    Cutaneous squamous (pre)malignancies can be classified according to the keratinocytic intraepidermal neoplasia (KIN) classification. Aneuploidy can be seen as the result of chromosomal aberrations leading to altered DNA content and has been strongly associated with malignancy. Hyperproliferation is

  11. Expression profiling of human melanocytes in response to UV-B irradiation

    Directory of Open Access Journals (Sweden)

    Saioa López

    2015-12-01

    Full Text Available A comprehensive gene expression analysis of human melanocytes was performed assessing the transcriptional profile of dark melanocytes (DM and light melanocytes (LM at basal conditions and after UV-B irradiation at different time points (6, 12 and 24 h, and in culture with different keratinocyte-conditioned media (KCM+ and KCM−. The data, previously published in [1], have been deposited in NCBI's Gene Expression Omnibus (GEO accession number: GSE70280.

  12. Multiple Cutaneous (pre)-Malignancies

    NARCIS (Netherlands)

    R.J.T. van der Leest (Robert)

    2015-01-01

    markdownabstract__Abstract__ The three most common cutaneous malignancies are derived from melanocytes and keratinocytes (ordered in decreasing aggressiveness): melanoma, squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). This thesis focuses only on these three types of cancer and

  13. Premalignant Lesions in the Kidney

    Directory of Open Access Journals (Sweden)

    Ziva Kirkali

    2001-01-01

    Full Text Available Renal cell carcinoma (RCC is the most malignant urologic disease. Different lesions, such as dysplasia in the tubules adjacent to RCC, atypical hyperplasia in the cyst epithelium of von Hippel-Lindau syndrome, and adenoma have been described for a number of years as possible premalignant changes or precursor lesions of RCC. In two recent papers, kidneys adjacent to RCC or removed from other causes were analyzed, and dysplastic lesions were identified and defined in detail. Currently renal intraepithelial neoplasia (RIN is the proposed term for classification. The criteria for a lesion to be defined as premalignant are (1 morphological similarity; (2 spatial association; (3 development of microinvasive carcinoma; (4 higher frequency, severity, and extent then invasive carcinoma; (5 progression to invasive cancer; and (6 similar genetic alterations. RIN resembles the neoplastic cells of RCC. There is spatial association. Progression to invasive carcinoma is described in experimental cancer models, and in some human renal tumors. Similar molecular alterations are found in some putative premalignant changes. The treatment for RCC is radical or partial nephrectomy. Preneoplastic lesions may remain in the renal remnant in patients treated by partial nephrectomy and may be the source of local recurrences. RIN seems to be a biologic precursor of some RCCs and warrants further investigation. Interpretation and reporting of these lesions would reveal important resources for the biological nature and clinical significance. The management of RIN diagnosed in a renal biopsy and partial nephrectomy needs to be answered.

  14. Cytoprotective function of sAppalpha in human keratinocytes.

    Science.gov (United States)

    Wehner, Sven; Siemes, Christina; Kirfel, Gregor; Herzog, Volker

    2004-12-01

    sAPPalpha, the soluble form of the beta-amyloid precursor protein, has been shown to act as a potent epidermal growth factor by stimulating keratinocyte proliferation and migration. In this report we provide evidence for a cytoprotective role of sAPPalpha. As a model we used HaCaT cells and normal human keratinocytes (NHK) cultured in the absence of fetal calf serum and bovine pituitary extract. Under these conditions keratinocytes began to undergo apoptosis at increasing rates after 96 h of culture. Surprisingly, keratinocytes were protected from apoptosis by the addition of 50 nM recombinant sAPPalpha. Subsequent experiments were performed to elucidate the regulatory basis of the cytoprotective role of sAPPalpha. We found that recombinant sAPPalpha facilitated the substrate adhesion of keratinocytes in the first 30 minutes after seeding. The basis for this adhesion-promoting function was shown by the ability of recombinant sAPPalpha to continuously coat the culture dish thereby promoting the ability to bind keratinocytes. A second mechanism explaining the cytoprotective role was found in the significant inhibition of apoptosis by recombinant sAPPalpha. In HaCaT cells moderate UV-B irradiation was sufficient to induce apoptosis. In contrast, induction of apoptosis in NHK required additionally the depletion of endogenous sAPPalpha suggesting that sAPPalpha mediates protection against UV-B irradiation. Staurosporine-induced apoptosis rates were significantly reduced by about 59% after addition of recombinant sAPPalpha. These results show that sAPPalpha exerts a pronounced cytoprotective effect and that this effect is mediated by facilitated cell adhesion and by the antiapoptotic function of sAPPalpha.

  15. Paeonol attenuates aging MRC-5 cells and inhibits epithelial-mesenchymal transition of premalignant HaCaT cells induced by aging MRC-5 cell-conditioned medium.

    Science.gov (United States)

    Yang, Lihua; Xing, Shangping; Wang, Kun; Yi, Hua; Du, Biaoyan

    2018-02-01

    Senescence-associated secretory phenotype (SASP) factors, such as IL-6 and IL-8, are extremely critical in tissue microenvironment. Senescent human fibroblasts facilitate epithelial-mesenchymal transition (EMT) in premalignant epithelial cells mainly through the secretion of SASP factors. Meanwhile, premalignant human HaCaT Keratinocyte (HaCaT) cells as immortal epithelial cells are susceptible to malignant transformation. Paeonol, an herbal phenolic component found in peonies, exerts anti-aging and anti-tumor efficacies, while the molecular mechanisms of paeonol on EMT in premalignant HaCaT cells induced by SASP factors are unclear. In this study, we first established a senescent human fetal lung fibroblast MRC-5 cell model using hydrogen peroxide evaluated by senescence-associated β-galactosidase assay. Upon paeonol treatment, intracellular reactive oxygen species levels in aging MRC-5 cells were significantly decreased via regulation of nuclear translocation of Nrf2. Then we curiously studied whether the aging MRC-5 cell-conditioned medium could induce EMT in premalignant HaCaT cells, and the results showed that paeonol significantly reduced the clonogenic, migratory, and invasive capacities of premalignant HaCaT cells potentially induced by IL-6 and IL-8. Moreover, we found that paeonol notably altered pluripotency of EMT-associated markers via the modulation of ERK and TGF-β1/Smad pathway in premalignant HaCaT cells. These findings suggest that paeonol may be used as an adjuvant therapy for SASP factor-mediated EMT in premalignant lesion.

  16. Autocrine abscisic acid mediates the UV-B-induced inflammatory response in human granulocytes and keratinocytes.

    Science.gov (United States)

    Bruzzone, Santina; Basile, Giovanna; Mannino, Elena; Sturla, Laura; Magnone, Mirko; Grozio, Alessia; Salis, Annalisa; Fresia, Chiara; Vigliarolo, Tiziana; Guida, Lucrezia; De Flora, Antonio; Tossi, Vanesa; Cassia, Raul; Lamattina, Lorenzo; Zocchi, Elena

    2012-06-01

    UV-B is an abiotic environmental stress in both plants and animals. Abscisic acid (ABA) is a phytohormone regulating fundamental physiological functions in plants, including response to abiotic stress. We previously demonstrated that ABA is an endogenous stress hormone also in animal cells. Here, we investigated whether autocrine ABA regulates the response to UV-B of human granulocytes and keratinocytes, the cells involved in UV-triggered skin inflammation. The intracellular ABA concentration increased in UV-B-exposed granulocytes and keratinocytes and ABA was released into the supernatant. The UV-B-induced production of NO and of reactive oxygen species (ROS), phagocytosis, and cell migration were strongly inhibited in granulocytes irradiated in the presence of a monoclonal antibody against ABA. Moreover, presence of the same antibody strongly inhibited release of NO, prostaglandin E2 (PGE(2)), and tumor necrosis factor-α (TNF-α) by UV-B irradiated keratinocytes. Lanthionine synthetase C-like protein 2 (LANCL2) is required for the activation of the ABA signaling pathway in human granulocytes. Silencing of LANCL2 in human keratinocytes by siRNA was accompanied by abrogation of the UV-B-triggered release of PGE(2), TNF-α, and NO and ROS production. These results indicate that UV-B irradiation induces ABA release from human granulocytes and keratinocytes and that autocrine ABA stimulates cell functions involved in skin inflammation. Copyright © 2011 Wiley Periodicals, Inc.

  17. Sialyl Lewis x expression in cervical scrapes of premalignant lesions

    Indian Academy of Sciences (India)

    Sialylated oligosaccharides of glycoproteins and glycolipids have been implicated in tumour progression and metastases. Altered expression of glycosidic antigens has been reported in cervical cancer. In cervix premalignant lesions, an increased expression of sialic acid has been reported. In the present study we ...

  18. Is endoscopic nodular gastritis associated with premalignant lesions?

    Science.gov (United States)

    Niknam, R; Manafi, A; Maghbool, M; Kouhpayeh, A; Mahmoudi, L

    2015-06-01

    Nodularity on the gastric mucosa is occasionally seen in general practice. There is no consensus about the association of nodular gastritis and histological premalignant lesions. This study is designed to investigate the prevalence of histological premalignant lesions in dyspeptic patients with endoscopic nodular gastritis. Consecutive patients with endoscopic nodular gastritis were compared with an age- and sex-matched control group. Endoscopic nodular gastritis was defined as a miliary nodular appearance of the gastric mucosa on endoscopy. Biopsy samples of stomach tissue were examined for the presence of atrophic gastritis, intestinal metaplasia, and dysplasia. The presence of Helicobacter pylori infection was determined by histology. From 5366 evaluated patients, a total of 273 patients with endoscopic nodular gastritis and 1103 participants as control group were enrolled. H. pylori infection was detected in 87.5% of the patients with endoscopic nodular gastritis, whereas 73.8% of the control group were positive for H. pylori (p gastritis were significantly higher than in the control group. Prevalence of atrophic gastritis and complete intestinal metaplasia were also more frequent in patients with endoscopic nodular gastritis than in the control group. Dysplasia, incomplete intestinal metaplasia and H. pylori infection are significantly more frequent in patients with endoscopic nodular gastritis. Although further studies are needed before a clear conclusion can be reached, we suggest that endoscopic nodular gastritis might serve as a premalignant lesion and could be biopsied in all patients for the possibility of histological premalignancy, in addition to H. pylori infection.

  19. Keratinocyte growth factor promotes melanosome transfer to keratinocytes.

    Science.gov (United States)

    Cardinali, Giorgia; Ceccarelli, Simona; Kovacs, Daniela; Aspite, Nicaela; Lotti, Lavinia Vittoria; Torrisi, Maria Rosaria; Picardo, Mauro

    2005-12-01

    Melanogenesis and melanosome transfer from the melanocytes to the neighboring keratinocytes are induced by ultraviolet radiation and modulated by autocrine and paracrine factors. Keratinocyte growth factor (KGF/fibroblast growth factor (FGF)7) is a paracrine mediator of human keratinocyte growth and differentiation. We evaluated the influence of KGF on melanosome transfer in co-cultures of keratinocytes and melanocytes. Immunofluorescence analysis using anti-tyrosinase and anti-human cytokeratin antibodies, phagocytic assays using fluorescent latex beads, and ultrastructural analysis indicated that KGF is able to induce melanosome transfer acting only on the recipient keratinocytes and as a consequence of a general role of KGF in the promotion of the phagocytic process. Inhibition of proteinase-activated receptor-2, to block the Rho-dependent phagocytic pathway, or of the Src family tyrosine kinases, to inhibit the Rac-dependent pathway, showed that KGF promotes phagocytosis through both mechanisms. Increased expression of the KGF receptor (KGFR) on the keratinocytes by transfection led to increased phagocytosis of latex beads following KGF treatment, suggesting that the KGF effect is directly mediated by KGFR expression and activation. Moreover, confocal microscopic analysis revealed that KGFR localize in phagosomes during KGF-induced phagocytosis, suggesting a direct role of the receptor in regulating both the early steps of uptake and the intracellular traffic of the phagosomes.

  20. Betel nut chewing, oral premalignant lesions, and the oral microbiome.

    Directory of Open Access Journals (Sweden)

    Brenda Y Hernandez

    Full Text Available Oral cancers are attributed to a number of causal agents including tobacco, alcohol, human papillomavirus (HPV, and areca (betel nut. Although betel nut chewing has been established as an independent cause of oral cancer, the mechanisms of carcinogenesis are poorly understood. An investigation was undertaken to evaluate the influence of betel nut chewing on the oral microbiome and oral premalignant lesions. Study participants were recruited from a dental clinic in Guam. Structured interviews and oral examinations were performed. Oral swabbing and saliva samples were evaluated by 454 pyrosequencing of the V3- V5 region of the 16S rRNA bacterial gene and genotyped for HPV. One hundred twenty-two adults were enrolled including 64 current betel nut chewers, 37 former chewers, and 21 with no history of betel nut use. Oral premalignant lesions, including leukoplakia and submucous fibrosis, were observed in 10 chewers. Within-sample bacterial diversity was significantly lower in long-term (≥10 years chewers vs. never chewers and in current chewers with oral lesions vs. individuals without lesions. Between-sample bacterial diversity based on Unifrac distances significantly differed by chewing status and oral lesion status. Current chewers had significantly elevated levels of Streptococcus infantis and higher and lower levels of distinct taxa of the Actinomyces and Streptococcus genera. Long-term chewers had reduced levels of Parascardovia and Streptococcus. Chewers with oral lesions had significantly elevated levels of Oribacterium, Actinomyces, and Streptococcus, including Streptococcus anginosus. In multivariate analyses, controlling for smoking, oral HPV, S.anginosus, and S. infantis levels, current betel nut chewing remained the only predictor of oral premalignant lesions. Our study provides evidence that betel nut chewing alters the oral bacterial microbiome including that of chewers who develop oral premalignant lesions. Nonetheless, whether

  1. Effect of ultraviolet B irradiation on accumulation of catechins in tea ...

    African Journals Online (AJOL)

    Effect of UV-B irradiation time on accumulation of foliar catechins in two tea cultivars was investigated. Low influence rate and short term irradiation of UV-B stimulated accumulation of major tea catechins, resulting in an increase in level of total catechins. Excessive irradiation of UV-B supressed the accumulation of tea ...

  2. Targeting Premalignant Lesions: Implications for Early Breast Cancer Detection and Intervention

    Science.gov (United States)

    2016-04-01

    1 AWARD NUMBER: W81XWH-14-1-0032 TITLE: Targeting Premalignant Lesions: Implications for Early Breast Cancer Detection and Intervention ...therapeutically intervene to successfully inhibit or even reverse tumor progression. 15. SUBJECT TERMS Breast cancer, Premalignant lesions, early intervention ...INTRODUCTION: Difficulty in managing treatment of advanced stage breast cancer has led to the goal for detection and intervention of early -stage disease

  3. The somatic mutation landscape of premalignant colorectal adenoma.

    Science.gov (United States)

    Lin, Shu-Hong; Raju, Gottumukkala S; Huff, Chad; Ye, Yuanqing; Gu, Jian; Chen, Jiun-Sheng; Hildebrandt, Michelle A T; Liang, Han; Menter, David G; Morris, Jeffery; Hawk, Ernest; Stroehlein, John R; Futreal, Andrew; Kopetz, Scott; Mishra, Lopa; Wu, Xifeng

    2017-06-12

    There are few studies which characterised the molecular alterations in premalignant colorectal adenomas. Our major goal was to establish colorectal adenoma genome atlas and identify molecular markers of progression from colorectal adenoma to adenocarcinoma. Whole-exome sequencing and targeted sequencing were carried out in 149 adenoma samples and paired blood from patients with conventional adenoma or sessile serrated adenoma to characterise the somatic mutation landscape for premalignant colorectal lesions. The identified somatic mutations were compared with those in colorectal cancer (CRC) samples from The Cancer Genome Atlas. A supervised random forest model was employed to identify gene panels differentiating adenoma from CRC. Similar somatic mutation frequencies, but distinctive driver mutations, were observed in sessile serrated adenomas and conventional adenomas. The final model included 20 genes and was able to separate the somatic mutation profile of colorectal adenoma and adenocarcinoma with an area under the curve of 0.941. The findings of this project hold potential to better identify patients with adenoma who may be candidates for targeted surveillance programmes and preventive interventions to reduce the incidence of CRC. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  4. Screening of oral premalignant lesions in smokers using toluidine blue

    Directory of Open Access Journals (Sweden)

    Yanti Leosari

    2009-06-01

    Full Text Available Background: A smoker is associated with the risk of developing oral premalignant lesions due to the cacinogenic contents in cigarette. Toluidine blue is a basic chromatic dye used in screening the presence of premalignant lesions due to its ability to detect acidic components in cells and tissues. Purpose: This study was purposed to observe the outcomes of toluidine blue staining on oral mucosa of smokers and non smokers and to find out whether quantity and duration of smoking affect the final results of toluidine blue staining. Methods: Forty male subjects, aged 20-60 years old were involved in this study, consisted of 10 heavy smokers, 10 moderate smokers, 10 light smokers and 10 non smokers. Subjects were instructed to rinse their mouths with mineral water for 20 seconds followed by acetic acid 1% for another 20 seconds. Toluidine blue stain was applied in excess and left on site for 1 minute. Subjects were instructed to rinse with acetic acid 1% and sufficient water consecutively for 20 seconds each. The areas of oral mucosa that stained blue were captured with intraoral camera and transferred to the computer unit. The staining procedure was repeated after 14 days. Results: Chi-square test showed that toluidine blue positive staining dominates the smokers group. Regression and correlation test indicate that Toluidine blue staining is more obvious in subjects who consume more cigarettes. Conclusion: It was concluded that oral mucosa of smokers absorbed more toluidine blue than that of non smokers and retention of toluidine blue is affected by quantity and duration of smoking.

  5. Effects of UV-B irradiation on photomovement in the desmid, Cosmarium cucumis

    International Nuclear Information System (INIS)

    Haeder, D.-P.

    1987-01-01

    Monochromatic UV-B irradiation affects neither the absorption nor the fluorescence of the bulk pigments in the desmid Cosmarium cucumis but it impairs photomovement of these organisms at fluence rates which are not higher than the ambient level of solar UV-B irradiation. Photoaccumulations and phototaxis are strongly inhibited especially at wavelengths <= 300 nm while photodispersal at higher white light fluence rates is hardly affected by supplementary UV-B. This effect has important consequences for the growth and survival of populations in their natural environment: these photosynthetic organisms utilize photomovement to find and stay in areas of suitable visible light fluence rates. The UV-B component of solar irradiation both impairs the strategy of the organisms to find a suitable position and the escape mechanism by which the cells move out of areas with too strong white illuminances which photooxidize the bulk pigments and bleach the population within a few days. (author)

  6. Keratinocyte cytokine and chemokine receptors.

    Science.gov (United States)

    Tüzün, Yalçin; Antonov, Meltem; Dolar, Neslihan; Wolf, Ronni

    2007-10-01

    Chemokines are a superfamily of small, secreted proteins that regulate cell traffic in homeostatic and inflammatory conditions. Keratinocytes synthesize many chemokines, including members of the CC and CXC subfamilies, such as regulated on activation of normal T-cell expressed and secreted, gamma-interferon inducible protein-10, monokine induced by gamma-interferon, and thymus- and activation-regulated chemokine. They also express some chemokine receptors that mediate the inflammatory or immune response by attracting various kinds of leukocytes.

  7. Thalidomide increases human keratinocyte migration and proliferation.

    Science.gov (United States)

    Nasca, M R; O'Toole, E A; Palicharla, P; West, D P; Woodley, D T

    1999-11-01

    Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behçet's disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 microM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2. 5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2-4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway.

  8. Clinician's Update on the Benign, Premalignant, and Malignant Skin Tumours of the Vulva

    DEFF Research Database (Denmark)

    Sand, Freja Lærke; Thomsen, Simon Francis

    2017-01-01

    Correct and rapid diagnosis of skin tumours often requires biopsy and histopathological examination to differentiate benign lesions such as seborrhoeic keratoses or melanocytic naevi from premalignant and malignant lesions such as malignant melanoma. Particularly, to the untrained eye, any benign...

  9. Biological and Genetic Analysis of a New Model for Breast Premalignancy

    National Research Council Canada - National Science Library

    MacLeod, Carol L

    2004-01-01

    ... microenvironment of the mammary gland. These cell lines were ideally suited to DNA micro array to identity changes in gene expression between premalignant cells growing in the mammary gland and tumors that arise from these tissues...

  10. Photo-oxidative stress by ultraviolet-B radiation and antioxidative defense of eckstolonol in human keratinocytes.

    Science.gov (United States)

    Jang, Jiyi; Ye, Bo-Ram; Heo, Soo-Jin; Oh, Chulhong; Kang, Do-Hyung; Kim, Ji Hyung; Affan, Abu; Yoon, Kon-Tak; Choi, Young-Ung; Park, Se Chang; Han, Seunghee; Qian, Zhong-Ji; Jung, Won-Kyo; Choi, Il-Whan

    2012-11-01

    Ultraviolet-B (UV-B) irradiation has been known to generate oxidative stress by increasing reactive oxygen species (ROS) in skin cells. Several naturally occurring antioxidant compounds isolated from marine algae are believed to protect against ROS. In this study, we assessed the antioxidative effect of eckstolonol isolated from Ecklonia cava against UV-B-induced ROS in human keratinocytes (HaCaTs). We investigated the effects of photo-oxidative stress by UV-B (50 mJ/cm(2)) and the antioxidative effects of eckstolonol using fluorometry, flow cytometry, microscopy, and cell viability and comet assays. UV-B irradiation decreased cell viability, which was restored in a dose-dependent manner with eckstolonol treatment (0, 5, 50, 100, and 200 μM). Moreover, eckstolonol reduced UV-B-induced ROS, lipid peroxidation, damaged DNA levels, and cell death. These antioxidative effects seem to be due to the enzymatic activities of catalase (CAT) and superoxide dismutase (SOD). Collectively, these results indicate that eckstolonol is capable of protecting keratinocytes from photo-oxidative stress. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. TNF-alpha impairs the S-G2/M cell cycle checkpoint and cyclobutane pyrimidine dimer repair in premalignant skin cells: Role of the PI3K-Akt pathway

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.; Calay, D.

    2008-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is induced by UVB radiation and has been implicated in the early stages of skin carcinogenesis. Here, we show that in normal keratinocytes and the transformed keratinocyte cell lines, HaCaT and A431, TNF-alpha stimulates protein kinase B/Akt, which results...... cycling. TNF-alpha enhanced apoptosis less potently and did not increase the level of CPD or stimulate cell cycle progression in normal keratinocytes. Our data suggest that TNF-alpha overrides the G2/M checkpoint in premalignant skin cells and allows for some cells containing unrepaired CPD to enter...... in activation of the survival complex mTORC1 (mammalian target of rapamycin complex 1) and inhibition of the proapoptotic proteins Bad and Fox03a. In UVB-irradiated HaCaT cells (10-20 mJ cm(-2)), TNF-alpha increased the proportion of cycling cells and enhanced the rate of apoptosis. A significantly higher...

  12. Keratinocyte-melanocyte interactions during melanosome transfer.

    Science.gov (United States)

    Seiberg, M

    2001-08-01

    The epidermal-melanin unit is composed of one melanocyte and approximately 36 neighboring keratinocytes, working in synchrony to produce and distribute melanin. Melanin is synthesized in melanosomes, transferred to the dendrite tips, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms involved in melanosome transfer and the keratinocyte-melanocyte interactions required for this process are not yet completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation ('injection'), keratinocyte-melanocyte membrane fusion, and phagocytosis. Studies of the keratinocyte receptor protease-activated receptor-2 (PAR-2) support the phagocytosis theory. PAR-2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin pigmentation. Modulation of PAR-2 activity can enhance or decrease melanosome transfer and affects pigmentation only when there is keratinocyte-melanocyte contact. Moreover, PAR-2 is induced by UV irradiation and inhibition of PAR-2 activation results in the prevention of UVB-induced tanning. The role of PAR-2 in mediating UV-induced responses remains to be elucidated.

  13. Treatment of burn injuries with keratinocyte cultures

    International Nuclear Information System (INIS)

    Syring, C.; Maenig, H.J.; Von Versen, R.; Bruck, J.

    1999-01-01

    The German Institute for Cell and Tissue Replacement (DIZG) provides burned patients with skin and amnion for a temporary wound closure. Severely burned patients (>60% BSA for adults, >40% BSA for children) were supplied with autologous and allogenic grafts from cultured keratinocytes. The keratinocyte culture is done under GMP-conditions using the method of Rheinwald and Green. The 3T3 fibroblasts were irradiated with 60 Gy and used as feeder cells to produce keratinocyte sheets within 3 weeks. In this time up to 6.000 cm are available. The sheets were harvested by detachment with dispase (1,2 U/ml), fixed to gauze and transported to the hospital. The DIZG has a 3 years experience in the treatment of burns with keratinocyte sheets. The sheets were transplanted to patients in different hospitals, the total transplanted area is about 30.000 cm. This paper describes the experiences with ten severely burned patients treated with keratinocyte sheet

  14. Prevention of HLA alloimmunization: Role of leukocyte depletion and UV-B irradiation

    International Nuclear Information System (INIS)

    Snyder, E.L.

    1990-01-01

    HLA alloimmunization is a major cause of the platelet refractory state. The stimulus for HLA alloimmunization is believed to derive from incompatibility between the recipient's lymphocytes and the passenger donor lymphocytes contained in transfused red cells or platelet concentrates. Two techniques to prevent post-transfusion HLA alloimmunization include filtration, which physically removes the donor lymphocytes, and UV-B irradiation, which renders the donor leukocytes biologically inactive. The role of these two techniques in the prevention of HLA alloimmunization is the focus of this review.42 references

  15. Sensitivity of two ecotypes of Arabidopsis Thaliana (Cvi and Te) towards UV-B irradiation

    International Nuclear Information System (INIS)

    Velichkova, M.; Stanoeva, D.; Popova, A.

    2013-01-01

    he susceptibility of Arabidopsis thaliana towards the detrimental effect of UV-B irradiation was investigated using two ecotypes, Cvi and Te. The effect of UV-B treatment on primary photosynthetic reactions - energy interaction between the main pigment-protein complexes and oxygen evolution, was evaluated at low (4 0 C) and at room (22 0 C) temperature. UV-B-induced alterations of investigated photosynthetic reactions are better expressed at 22 0 C than at 4 0 C for Cvi. For Te ecotype the energy interaction was suppressed to higher extent at 22 0 C, while oxygen evolving activity was affected similarly at both temperatures. At low and room temperature, the energy interaction in the complex PSII-core antenna is affected stronger by UV-B treatment than the energy distribution between both photosystems, as revealed by fluorescence ratios of 77 K spectra. The results presented indicate that the Arabidopsis thaliana ecotype Cvi (Cape Verde Islands) is less affected by UV-B irradiation in respect to the investigated primary photosynthetic reactions than the ecotype Te (Finland)

  16. Protective Effect of HemoHIM on Epidermal Melanocytes in Ultraviolet-B irradiated Mice

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hae June [Korea Institute of Radiological and Medical Science, Seoul (Korea, Republic of); Kim, Jong Choon; Moon, Chang Jong; Kim, Sung Ho [Chonnam National University, Gwangju (Korea, Republic of); Jung, U Hee; Park, Hae Ran; Jo, Sung Kee [Jeongeup Campus of Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of); Jang, Jong Sik; Kim, Tae Hwan [Kyungpook National University, Daegu (Korea, Republic of)

    2011-06-15

    We induced the activation of melanocytes in the epidermis of C57BL/6 mice by ultraviolet-B (UV-B) irradiation, and observed the effect of an herbal preparation (HemoHIM, HH) on the formation, and decrease of UV-B-induced epidermal melanocytes. C57BL/6 mice were irradiated by UV-B 80 mJ:cm{sup -2} (0.5 mW:sec{sup -1}) daily for 7 days, and HH was intraperitoneally, orally or topically applied pre- or post-irradiation. For the estimation of change of epidermal melanocytes, light microscopic observation with dihydroxyphenylalanine (DOPA) stain was performed. Split epidermal sheets prepared from the ear of untreated mice exhibited 13∼15 melanocytes:mm{sup -2}, and one week after UV irradiation, the applied areas showed an increased number of strongly DOPA-positive melanocytes with stout dendrites. But intraperitoneal, oral or topical treatment with HH before each irradiation interrupted UV-B-induced pigmentation and resulted in a marked reduction in the number of epidermal melanocytes as compared to the number found in UV-B-irradiated, untreated control skin. The number and size of DOPA-positive epidermal melanocytes were also significantly decreased in intraperitoneally injected or topically applicated group after irradiation with HH at 3rd and 6th weeks after irradiation. The present study suggests the HH as inhibitor of UV-B-induced pigmentation, and depigmenting agent.

  17. In vivo Raman spectroscopic identification of premalignant lesions in oral buccal mucosa

    Science.gov (United States)

    Singh, S. P.; Deshmukh, Atul; Chaturvedi, Pankaj; Murali Krishna, C.

    2012-10-01

    Cancers of oral cavities are one of the most common malignancies in India and other south-Asian countries. Tobacco habits are the main etiological factors for oral cancer. Identification of premalignant lesions is required for improving survival rates related to oral cancer. Optical spectroscopy methods are projected as alternative/adjunct for cancer diagnosis. Earlier studies have demonstrated the feasibility of classifying normal, premalignant, and malignant oral ex-vivo tissues. We intend to evaluate potentials of Raman spectroscopy in detecting premalignant conditions. Spectra were recorded from premalignant patches, contralateral normal (opposite to tumor site), and cancerous sites of subjects with oral cancers and also from age-matched healthy subjects with and without tobacco habits. A total of 861 spectra from 104 subjects were recorded using a fiber-optic probe-coupled HE-785 Raman spectrometer. Spectral differences in the 1200- to 1800-cm-1 region were subjected to unsupervised principal component analysis and supervised linear discriminant analysis followed by validation with leave-one-out and an independent test data set. Results suggest that premalignant conditions can be objectively discriminated with both normal and cancerous sites as well as from healthy controls with and without tobacco habits. Findings of the study further support efficacy of Raman spectroscopic approaches in oral-cancer applications.

  18. Proposed Terminology and Classification of Pre-Malignant Neoplastic Conditions: A Consensus Proposal

    Directory of Open Access Journals (Sweden)

    Peter Valent

    2017-12-01

    Full Text Available Cancer evolution is a step-wise non-linear process that may start early in life or later in adulthood, and includes pre-malignant (indolent and malignant phases. Early somatic changes may not be detectable or are found by chance in apparently healthy individuals. The same lesions may be detected in pre-malignant clonal conditions. In some patients, these lesions may never become relevant clinically whereas in others, they act together with additional pro-oncogenic hits and thereby contribute to the formation of an overt malignancy. Although some pre-malignant stages of a malignancy have been characterized, no global system to define and to classify these conditions is available. To discuss open issues related to pre-malignant phases of neoplastic disorders, a working conference was organized in Vienna in August 2015. The outcomes of this conference are summarized herein and include a basic proposal for a nomenclature and classification of pre-malignant conditions. This proposal should assist in the communication among patients, physicians and scientists, which is critical as genome-sequencing will soon be offered widely for early cancer-detection.

  19. Direct immunofluorescence testing results in cases of premalignant and malignant oral lesions.

    Science.gov (United States)

    Montague, L J; Bhattacharyya, I; Islam, M N; Cohen, D M; Fitzpatrick, S G

    2015-06-01

    Oral premalignant and malignant lesions may mimic oral lichen planus (OLP) clinically and microscopically. OLP often shows basement membrane fibrinogen positivity on direct immunofluorescence testing (DIF). This study examined fibrinogen positivity in oral premalignant lesions and squamous cell carcinoma. The University of Florida Oral Pathology Biopsy Service records were searched for the years 2003 to 2013 for oral premalignant lesions and squamous cell carcinoma with concurrent DIF testing. Demographic, clinical, and DIF or histologic information was collected and analyzed. Sixty-eight fibrinogen positive lesions were identified within a total of 164 cases. Low-grade dysplasia and premalignant verrucous lesions made up the majority of the fibrinogen positive lesions (combined n = 43; 63.2%), and the most common locations in positive cases were the buccal mucosa, tongue, and gingiva. A lichenoid distribution of the inflammatory infiltrate significantly predicted fibrinogen positivity (P < .0005). Fibrinogen positivity may be seen in premalignant and malignant oral lesions increasing the risk of misdiagnosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Melanosome transfer promoted by keratinocyte growth factor in light and dark skin-derived keratinocytes.

    Science.gov (United States)

    Cardinali, Giorgia; Bolasco, Giulia; Aspite, Nicaela; Lucania, Giuseppe; Lotti, Lavinia V; Torrisi, Maria R; Picardo, Mauro

    2008-03-01

    The transfer of melanin from melanocytes to keratinocytes is upregulated by UV radiation and modulated by autocrine and paracrine factors. Among them, the keratinocyte growth factor (KGF/FGF7) promotes melanosome transfer acting on the recipient keratinocytes through stimulation of the phagocytic process. To search for possible differences in the melanosome uptake of keratinocytes from different skin color, we analyzed the uptake kinetics and distribution pattern of fluorescent latex beads in primary cultures of light and dark skin-derived keratinocytes stimulated with KGF and we compared the direct effect of KGF on the melanosome transfer in co-cultures of human primary melanocytes with light and dark keratinocytes. KGF-promoted melanosome transfer was more significant in light keratinocytes compared to dark, due to an increased expression of KGF receptor in light skin keratinocytes. Colocalization studies performed by confocal microscopy using FITC-dextran as a phagocytic marker and fluorescent beads as well as inhibition of particle uptake by cytochalasin D, revealed that beads internalization induced by KGF occurs via actin-dependent phagocytosis. 3D image reconstruction by fluorescence microscopy and ultrastructural analysis through transmission electron microscopy showed differences in the distribution pattern of the beads in light and dark keratinocytes, consistent with the different melanosome distribution in human skin.

  1. Effects of UV-B irradiation on isoforms of antioxidant enzymes and their activities in red alga Grateloupia filicina (Rhodophyta)

    Science.gov (United States)

    Zhao, Jiqiang; Li, Lixia

    2014-11-01

    Macroalgae in a littoral zone are inevitably exposed to UV-B irradiance. We analyzed the effects of UV-B on isoenzyme patterns and activities of superoxide dismutase (SOD), peroxidase (POX), catalase (CAT), and ascorbate peroxidase (APX) of red algae Grateloupia filicina (Lamour.) C. Agardh. The activities of SOD, CAT, and APX changed in response to UV-B in a time- and dose-dependent manner. POX activity increased significantly under all three UV-B treatments. The enzymatic assay showed three distinct bands of SODI (Mn-SOD), SODII (Fe-SOD), and SODIII (CuZn-SOD) under a low (Luv) and medium (Muv) dose of UV-B irradiation, while SODI and SODIII activities decreased significantly when exposed to a high dose of UV-B irradiation (Huv). The activity of POX isoenzymes increased significantly after exposure to UV-B, which is consistent with the total activity. In addition, a clear decrease in activity of CATIV was detected in response to all the three doses of UV treatments. Some bands of APX isoenzyme were also clearly influenced by UV-B irradiation. Correspondingly, the daily growth rate declined under all the three exposure doses, and was especially significant under Muv and Huv treatments. These data suggest that, although the protection mechanisms of antioxidant defense system are partly inducible by UV-B to prevent the damage, G. filicina has incomplete tolerance to higher UV-B irradiation stress.

  2. [Allelopathic effect of Corallina pilulifera on Heterosigma akashiwo and its responses to UV-B irradiation].

    Science.gov (United States)

    Zhao, Yan; Yu, Qing-Yun; Zhou, Bin; Ju, Qing; Tang, Xue-Xi

    2009-10-01

    By the method of co-culture and using cell density as the main indicator, this paper studied the allelopathic effect of Corallina pilulifera on Heterosigma akashiwo and its responses to UV-B irradiation. Under normal condition, the fresh tissue and aqueous extracts of C. pilulifera had significant inhibitory effects on the growth of H. akashiwo (P 0.05). After pre-treated with different dose UV-B radiation and then co-cultured with H. akashiwo, C. pilulifera had some changes in the allelopathic activity of its fresh tissue, dry powder, and aqueous extracts. High-dose UV-B radiation (3.0 J x m(-2)) induced the decrease of the allelopathic effect, whereas low-dose UV-B radiation (0.9 J x m(-2)) was in adverse (P < 0.05).

  3. The influence of urban area opacity on biologically active UV-B irradiance

    Science.gov (United States)

    Chubarova, Nataly; Rozental', Victor

    2013-04-01

    The study of UV irradiance changes in urban area is an essential problem due to the significant effect of UV irradiance on human health which can be positive (vitamin D synthesis) and negative (erythema, skin cancer, eye damage). According to the results of several experiments within the Moscow megacity we studied the effects of urban area opacity on the different types of biologically active UV-B irradiance on the base of a specially developed mobile photometric complex snd additional measurements of the urban opacity by Nikon Fisheye Converter FC-E8. We analyzed both the level of erythemally-active irradiance and the UV eye damaging radiation using the broadband UVB-1 YES pyranometer calibrated against ultraviolet spectroradiometer Bentham DTM-300 of the Medical University of Innsbruck (courtesy of Dr. M.Blumthaler). In order to estimate the effects of the urban opacity the measurements were normalized on similar measurements at the Meteorological Observatory of Moscow State University with zero opacity. This ratio is defined as an urban radiative transmittance (URT). Different atmospheric conditions were considered. In cloudy conditions the effect of opacity on URT is much less than that in conditions when the sun disk is open from clouds. We revealed some spectral features in transmittance of biologically active UV-B irradiance which is characterized by higher URT variations in overcast cloudy conditions due to more intensive scattering and smaller direct solar radiation component. In the absence of cloudiness the effect of opacity was studied for open and screening solar disk conditions. We obtained much higher URT in UVB spectral region compared with that for total solar irradiance for screening solar disk conditions with a significant URT dependence on the opacity only in UVB spectral region. No URT dependence was obtained for total solar irradiance in these conditions. Some model calculations were fulfilled to match the experimental results.

  4. Effect of the Premalignant and Tumor Microenvironment on Immune Cell Cytokine Production in Head and Neck Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Sara D. [Department of Microbiology and Immunology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425 (United States); De Costa, Anna-Maria A. [Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, 135 Rutledge Avenue, Charleston, SC 29425 (United States); Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425 (United States); Young, M. Rita I., E-mail: rita.young@va.gov [Department of Otolaryngology, Head and Neck Surgery, Medical University of South Carolina, 135 Rutledge Avenue, Charleston, SC 29425 (United States); Department of Medicine, Medical University of South Carolina, 96 Jonathan Lucas Street, Charleston, SC 29425 (United States); Medical Research Service (151), Ralph H. Johnson Veterans Affairs Medical Center, 109 Bee Street, Charleston, SC 29401 (United States)

    2014-04-02

    Head and neck squamous cell carcinoma (HNSCC) is marked by immunosuppression, a state in which the established tumor escapes immune attack. However, the impact of the premalignant and tumor microenvironments on immune reactivity has yet to be elucidated. The purpose of this study was to determine how soluble mediators from cells established from carcinogen-induced oral premalignant lesions and HNSCC modulate immune cell cytokine production. It was found that premalignant cells secrete significantly increased levels of G-CSF, RANTES, MCP-1, and PGE{sub 2} compared to HNSCC cells. Splenocytes incubated with premalignant supernatant secreted significantly increased levels of Th1-, Th2-, and Th17-associated cytokines compared to splenocytes incubated with HNSCC supernatant. These studies demonstrate that whereas the premalignant microenvironment elicits proinflammatory cytokine production, the tumor microenvironment is significantly less immune stimulatory and may contribute to immunosuppression in established HNSCC.

  5. Expression of filaggrin and its degradation products in human skin following erythemal doses of ultraviolet B irradiation

    DEFF Research Database (Denmark)

    Simonsen, Stine; Thyssen, Jacob P.; Heegaard, Steffen

    2017-01-01

    Epidermal filaggrin level is affected by ultraviolet B irradiation in animal and experimental models. This study examined the effect of ultraviolet B irradiation on epidermal filaggrin and natural moisturizing factors in vivo in healthy adults (n = 22). Participants were irradiated with 2 minimal...... moisturizing factor levels after irradiation, but mean trans-urocanic acid was significantly reduced, as expected (n = 8). In conclusion, erythemal doses of ultraviolet B exert acute effects on profilaggrin mRNA and filaggrin protein in human skin in vivo.......Epidermal filaggrin level is affected by ultraviolet B irradiation in animal and experimental models. This study examined the effect of ultraviolet B irradiation on epidermal filaggrin and natural moisturizing factors in vivo in healthy adults (n = 22). Participants were irradiated with 2 minimal...... erythema doses of ultraviolet B on the skin. Biopsies and tape strips were collected from skin irradiated 24 and 72 h earlier and from nonirradiated skin (control). Real-time quantitative PCR on skin biopsies showed significantly reduced profilaggrin mRNA expression 24 h after irradiation (mean relative m...

  6. Impact of enhanced ultraviolet-B irradiance on cotton growth, development, yield, and qualities under field conditions

    Science.gov (United States)

    Wei Gao; Youfei Zheng; James R. Slusser; Gordon M. Heisler

    2003-01-01

    The stratospheric ozone depletion and enhanced solar ultraviolet-B (UV-B) irradiance may have adverse impacts on the productivity of agricultural crops. The effect of UV-B enhancements on agricultural crops includes reduction in yield, alteration in species competition, decrease in photosynthetic activity, susceptibility to disease, and changes in structure and...

  7. Expression of podoplanin in oral premalignant and malignant lesions and its potential as a biomarker

    Directory of Open Access Journals (Sweden)

    J Logeswari

    2014-01-01

    Conclusion: The podoplanin may be considered as a predictor marker in assessing malignant transformation of premalignancies and prognosis of oral malignancy. Indeed it is believed that podoplanin might play a role in tumor progression though exact mechanism is not fully elucidated. Further research is required to understand its exact pathophysiology.

  8. The role of HPV in diagnosis and management of cervical premalignancies

    NARCIS (Netherlands)

    Hamont, D. van

    2008-01-01

    Cervical cytological pathology is not uncommon. Prevention of cervical cancer by detection of the disease in an early and pre-malignant stage is practised globally either through population-based screening programmes or more optimistically non-organised ones. High-grade cervical intraepithelial

  9. HPV-related (pre)malignancies of the female anogenital tract in renal transplant recipients.

    NARCIS (Netherlands)

    Hinten, F.; Meeuwis, K.A.P.; Rossum, M.M. van; Hullu, J.A. de

    2012-01-01

    Renal transplantations (RTs) are performed routinely in many countries. After RT, the administration of lifelong immunosuppressive therapy is required. As a consequence, renal transplant recipients (RTRs) have a high risk to develop virus-associated (pre)malignancies, such as Human papillomavirus

  10. Clinical experiences with optical coherence tomography in epithelial (pre)malignancies

    NARCIS (Netherlands)

    Wessels, R.

    2015-01-01

    This thesis describes the potential of optical coherence tomography (OCT) to differentiate between normal tissue and (pre)malignant tissue in epithelial cancers. It can be divided in research performed in the genital area and the field of melanoma. Chapter 2 describes the principles of the

  11. Optical detection of (pre-)malignant lesions of the oral mucosa : autofluorescence characteristics of healthy mucosa

    NARCIS (Netherlands)

    de Veld, DCG; Witjes, MJH; Roodenburg, JLN; Sterenborg, HJCM; Papazoglou, TG; Wagnieres, GA

    2001-01-01

    Previous clinical results demonstrate the potential of in vivo autofluorescence spectroscopy for early detection of (pre-)malignant lesions of the oral mucosa. For reliable diagnosis, it is necessary to study auto fluorescence spectra of healthy mucosa first. We measured excitation-emission maps in

  12. Diagnostic efficiency of toluidine blue with Lugol's iodine in oral premalignant and malignant lesions.

    Science.gov (United States)

    Nagaraju, Kamarthi; Prasad, Shiva; Ashok, L

    2010-01-01

    In vivo stains are prompt resources, which have emerged, in the recent years, to aid as clinical diagnostic tools in detecting early premalignant and malignant lesions. The aim of the study was to determine the diagnostic efficiency of toluidine blue with Lugol's iodine in oral premalignancies and malignancies and to evaluate the reliability of in vivo staining with toluidine blue and Lugol's iodine in the lesions at risk of malignancy. The study group comprised 30 subjects with clinically suspicious premalignant lesions and 30 subjects with clinically suspicious malignant lesions. All the lesions were stained consecutively with toluidine blue and Lugol's iodine and the dye retention were recorded with photographs. Depending on the retention of the dyes, the biopsy site was determined. The biopsy specimens were sent for histological confirmation and results were statistically analyzed. The overall diagnostic accuracy of Lugol's iodine when used consecutively with toluidine blue stain in distinguishing premalignant lesions and malignant lesions was 90%. As the degree of differentiation of malignant lesions progressed toward more severity, they failed to show the retention of Lugol's iodine and the result was highly significant statistically, with a P value Lugol's iodine when used with toluidine blue helped in delineating the inflammatory lesions and was the mean source in determining clinically the degrees of differentiation of malignant lesions as the poorly differentiated malignant lesions without glycogen content failed to show Lugol's iodine retention. Toluidine blue with Lugol's iodine can be used as a pretherapeutic assessment of the biologic aggressiveness of the disease.

  13. The co-application effects of fullerene and ascorbic acid on UV-B irradiated mouse skin

    International Nuclear Information System (INIS)

    Ito, Shinobu; Itoga, Kazuyoshi; Yamato, Masayuki; Akamatsu, Hirohiko; Okano, Teruo

    2010-01-01

    The role of fullerene as a pro-oxidant or anti-oxidant in Ultraviolet B ray (UV-B)-induced disorders in mouse skin was investigated. Fullerene gave no photo-toxic effect to UV-B-irradiated mouse skin. Since erythema was concentrated at the pore circumference in a UV-B irradiation experiment in mouse skin, the sebaceous gland pairs was strongly implicated as a site for the generation of reactive oxygen species (ROS). In a histological evaluation of the skin stained with CH 3 MDFDA (ROS index) and YO-Pro-1 (apoptosis index), the fluorescence intensity of a sebaceous gland significantly increased with UV-B irradiation. With the application of fullerene to UV-irradiated mouse skin, no toxicity was recognized in comparison with the control, and erythema, the ROS index, and the apoptosis index decrease with the application of fullerene. Ascorbyl radical (AA·) increased with the application of ascorbate (AA) to UV-B-irradiated mouse skin, and AA· decreased with the application of fullerene. The co-application of AA and fullerene, which suppressed AA· in vitro, significantly suppressed erythema, and also suppressed both the ROS index and apoptosis index in mouse skin after UV-B irradiation. In both mouse skin at 48 h after UV-B irradiation and in an attempt to reproduce this phenomenon artificially in vitro, a similar high AA· peak (AA·/H· > 4) was observed in electron spin resonance (ESR) charts. The binding of fullerene with AA impairs the Fenton reaction between AA and Fe-protein based on the observation of ascorbate-specific UV absorption and a linear equation for the calibration curve. Therefore, fullerene may impair the intercalation of AA to a heme pocket by binding with AA. These results suggest that the co-application of AA and fullerene is effective against oxidative skin damage caused by UV-B irradiation, and the development of an AA· inhibitor such as fullerene should be useful for reducing organ damage associated with Fe-protein oxidation.

  14. Discrimination of premalignant conditions of oral cancer using Raman spectroscopy of urinary metabolites

    Science.gov (United States)

    Elumalai, Brindha; Rajasekaran, Ramu; Aruna, Prakasarao; Koteeswaran, Dornadula; Ganesan, Singaravelu

    2015-03-01

    Oral cancers are considered to be one of the most commonly occurring malignancy worldwide. Over 70% of the cases report to the doctor only in advanced stages of the disease, resulting in poor survival rates. Hence it is necessary to detect the disease at the earliest which may increase the five year survival rate up to 90%. Among various optical spectroscopic techniques, Raman spectroscopy has been emerged as a tool in identifying several diseased conditions, including oral cancers. Around 30 - 80% of the malignancies of the oral cavity arise from premalignant lesions. Hence, understanding the molecular/spectral differences at the premalignant stage may help in identifying the cancer at the earliest and increase patient's survival rate. Among various bio-fluids such as blood, urine and saliva, urine is considered as one of the diagnostically potential bio-fluids, as it has many metabolites. The distribution and the physiochemical properties of the urinary metabolites may vary due to the changes associated with the pathologic conditions. The present study is aimed to characterize the urine of 70 healthy subjects and 51 pre-malignant patients using Raman spectroscopy under 785nm excitation, to know the molecular/spectral differences between healthy subjects and premalignant conditions of oral malignancy. Principal component analysis based Linear discriminant analysis were also made to find the statistical significance and the present technique yields the sensitivity and specificity of 86.3% and 92.9% with an overall accuracy of 90.9% in the discrimination of premalignant conditions from healthy subjects urine.

  15. Estimation of salivary sialic acid in oral premalignancy and oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Vishakha Chaudhari

    2016-01-01

    Full Text Available Aims: Oral cancer is the most life-threatening disease of oral tissues. In societies where the incidence of oral cancer is high, clinically recognizable premalignant lesions are particularly common. Diagnosing oral cancers at an early stage is critical in improving the survival rate and reducing the morbidity associated with the disease. Alterations in the sialic acid levels in cancer patients have stimulated interest in this sugar residue as a possible tumor marker. Settings and Design: The purpose of this study was to estimate the salivary sialic acid levels in patients with oral premalignancy and squamous cell carcinoma and to correlate it with their grades to develop a cost-effective and noninvasive diagnostic parameter. Materials and Methods: Unstimulated whole saliva was collected from the groups under study and subjected to biochemical analysis for determination of sialic acid levels. Statistical Analysis Used: The salivary sialic acid levels were correlated with the clinical stage and histological grade by one-way ANOVA (SPSS software version 15. Results: Salivary sialic acid was elevated in oral squamous cell carcinoma (OSCC compared to oral premalignancy and control group. A statistically significant correlation was observed between the grades of squamous cell carcinoma, grades of dysplasia in premalignancy, and sialic acid level. Conclusion and Clinical Significance: Evaluation of salivary sialic acid levels in premalignant and malignant lesions can serve as a screening tool. The mortality and morbidity of OSCC can be reduced if the lesions are diagnosed in early precancerous states using such noninvasive diagnostic methods for screening and monitoring of the population.

  16. Topical Administration of Manuka Oil Prevents UV-B Irradiation-Induced Cutaneous Photoaging in Mice

    Directory of Open Access Journals (Sweden)

    Oh Sook Kwon

    2013-01-01

    Full Text Available Manuka tree is indigenous to New Zealand, and its essential oil has been used as a traditional medicine to treat wounds, fever, and pain. Although there is a growing interest in the use of manuka oil for antiaging skin care products, little is known about its bioactivity. Solar ultraviolet (UV radiation is the primary environmental factor causing skin damage and consequently premature aging. Therefore, we evaluated manuka oil for its effects against photoaging in UV-B-irradiated hairless mice. Topical application of manuka oil suppressed the UV-B-induced increase in skin thickness and wrinkle grading in a dose-dependent manner. Application of 10% manuka oil reduced the average length, depth, and % area of wrinkles significantly, and this was correlated with inhibition of loss of collagen fiber content and epidermal hyperplasia. Furthermore, we observed that manuka oil could suppress UV-B-induced skin inflammation by inhibiting the production of inflammatory cytokines. Taken together, this study provides evidence that manuka oil indeed possesses antiphotoaging activity, and this is associated with its inhibitory activity against skin inflammation induced by UV irradiation.

  17. Probing behaviors of Sitobion avenae (Hemiptera: Aphididae on enhanced UV-B irradiated plants

    Directory of Open Access Journals (Sweden)

    Hu Zu-Qing

    2013-01-01

    Full Text Available UV-B induced changes in plants can influence sap-feeding insects through mechanisms that have not been studied. Herein the grain aphid, Sitobion avenae (Fabricius (Hemiptera: Aphididae, was monitored on barley plants under the treatments of control [0 kJ/ (m2.d], ambient UV-B [60 kJ/ (m2.d], and enhanced UV-B [120 kJ/ (m2.d] irradiation. Electrical penetration graph (EPG techniques were used to record aphid probing behaviors. Enhanced UV-B irradiated plants negatively affected probing behaviors of S. avenae compared with control plants. In particular, phloem factors that could diminish sieve element acceptance appeared to be involved, as reflected by smaller number of phloem phase, shorter phloem ingestion, and fewer aphids reaching the sustained phloem ingestion phase (E2>10min. On the other hand, factors from leaf surface, epidermis, and mesophyll cannot be excluded, as reflected by higher number of non-probing, longer non-probing and pathway phase, and later the time to first probe.

  18. Is pollen morphology of Salix polaris affected by enhanced UV-B irradiation? Results from a field experiment in high arctic tundra.

    NARCIS (Netherlands)

    Yeloff, D.; Blokker, P.; Boelen, P.; Rozema, J.

    2008-01-01

    This study tested the hypothesis that the thickness of the pollen wall will increase in response to enhanced UV-B irradiation, by examining the effect of enhanced UV-B irradiance on the pollen morphology of Salix polaris Wahlem. grown in a field experiment on the Arctic tundra of Svalbard.

  19. Is pollen morphology of Salix polaris affected by enhanced UV-B irradiation? Results from a field experiment in High Arctic tundra

    NARCIS (Netherlands)

    Yeloff, D.; Blokker, P.; Boelen, P.; Rozema, J.

    2008-01-01

    This study tested the hypothesis that the thickness of the pollen wall will increase in response to enhanced UV-B irradiation, by examining the effect of enhanced UV-B irradiance on the pollen morphology of Salix polaris Wahlem. grown in a field experiment on the Arctic tundra of Svalbard.

  20. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.

    Science.gov (United States)

    Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2013-05-01

    Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

  1. Studies of early effects of ultraviolet B irradiation on hairless mouse epidermis

    International Nuclear Information System (INIS)

    Olsen, W.M.

    1990-01-01

    The present study describes various early biochemical and cell kinetic aspects of the acute response of hairless epidermis with irradiation of narrow-banded wavelengths in the ultraviolet B region of the spectrum (280-320 nm), and their possible relationship to ultraviolet carcinogenicity. In vivo exposure of hairless mouse skin to a single dose of various narrow-banded wavelengths of ultraviolet B light demonstrated that 280, 290, 297 and 302 nm had a carcinogenic potency according to the tetrazolium test. No induction of DT-diaphorase was observed, which may signify that the actions of ultraviolet B light and chemical skin carcinogens differ at the cellular level, even though the nuclear effect on DNA may in principle be the same, e.g. mutation events, activation or amplication of oncogens, inhibition of anti-oncogens, etc. The early epidermal cell kinetic after a biologically relevant dose of ultraviolet B irradiation at a wavelength of 297 nm could be divided into two periods: the initial inhibition in the uptake of tritiated thymidine and the mitotic rate were followed by a long-lasting depression in the DNA synthesis rate combined with rapid cell proliferation. This shows that the acute vascular response (erythema and edema) to ultraviolet B lights is also associated with epidermal perturbations similar to the carcinogen-associated delay in cell cycle passage seen after chemical skin carcinogens like 7,12-dimethylbenz(α)anthracene and methylnitrosourea, as well as to the regenerative proliferation observed after chemical skin irritants like cantharidin. 93 refs., 6 figs

  2. Chlorophyll degradation in aqueous mediums induced by light and UV-B irradiation: An UHPLC-ESI-MS study

    Science.gov (United States)

    Petrović, Sanja; Zvezdanović, Jelena; Marković, Dejan

    2017-12-01

    Irreversible chlorophyll degradation induced by continuous white light illumination and UV-B irradiation in the aqueous mediums (with 10%, 30% and 50% of methanol) was investigated using the ultrahigh liquid chromatography coupled with diode array and electrospray ionization mass spectrometry detectors (UHPLC-DAD-ESIMS). The degradation was governed by energy input of photons: higher energy of UV-B irradiation induced faster chlorophyll degradation and accordingly faster products formation in comparison to the white light treatment. Main light- or/and UV-B-induced products of chlorophyll in the aqueous mediums were hydroxy-pheophytin a, pheophytin a and hydroxy-lactone-pheophytin a, accompanied with the corresponding epimers. Chlorophylls aggregation dominant in the aqueous medium with the highest methanol content (50%) play a protective role against the UV-B radiation and white light illumination.

  3. Effect of UV-B irradiance on the ATP content of microorganisms of the Weddell Sea (Antartica)

    Energy Technology Data Exchange (ETDEWEB)

    Vosjan, J.H.; Nieuwland, G. (Netherlands Inst. for Sea Research, Den Burg (Netherlands)); Doehler, G. (Frankfurt Universitaet (Federal Republic of Germany). Botanisches Institut)

    1990-06-01

    The effect of UV-B irradiation on the ATP content of natural assemblages of planktonic microorganisms in the upper 30-m water layer of the Weddell Sea (Antartica) was studied. After five hours of irradiation with UV (290-320 nm) of 1.35 W.m{sup -2} a 75% decrease in the ATP content of the microorganisms was observed. (author). 11 refs.; 3 figs.

  4. Effect of UV-B irradiance on the ATP content of microorganisms of the Weddell Sea (Antartica)

    International Nuclear Information System (INIS)

    Vosjan, J.H.; Nieuwland, G.; Doehler, G.

    1990-01-01

    The effect of UV-B irradiation on the ATP content of natural assemblages of planktonic microorganisms in the upper 30-m water layer of the Weddell Sea (Antartica) was studied. After five hours of irradiation with UV (290-320 nm) of 1.35 W.m -2 a 75% decrease in the ATP content of the microorganisms was observed. (author). 11 refs.; 3 figs

  5. Influence of uvA on the erythematogenic and therapeutic effects of uvB irradiation in psoriasis; photoaugmentation effects

    International Nuclear Information System (INIS)

    Boer, J.; Schothorst, A.A.; Suurmond, D.

    1981-01-01

    The effect of repeated exposure to an additive dose of long ultraviolet (uvA) radiation on the erythemogenic and therapeutic effects of middle ultraviolet (uvB) irradiation was investigated in 8 patients with psoriasis. The surface of the backs of these patients was divided into 2 parts, 1 of which received only uvB irradiation 4 times a week and the other uvA + uvB. uvB was provided by Philips TL-12 lamps and uvA by glass-filtered Philips TL-09 lamps. uvA was held constantly at 10 J/cm2, whereas uvB alone were evaluated by 4 tests during the treatment to determine the minimal erythema dose (MED). Test I (at the start of the therapy) showed a photoaugmentative effect which was no longer apparent in Test III (third week). Test III showed a reversal of the ratios of the MEDs of the sites irradiated with the uvA + uvB and uvB (MED A + B/MED B). This is ascribed to the marked pigmentation which appeared after repeated irradiation with the uvA + uvB combination. Comparison showed for the improvement of the psoriasis no distinct differences between uvA + uvB irradiation and uvB alone, but the former had the cosmetic advantage of giving pleasing tan

  6. Targeting Premalignant Lesions - Implications for Early Breast Cancer Detection and Intervention

    Science.gov (United States)

    2017-04-01

    AWARD NUMBER: W81XWH-14-1-0032 TITLE: Targeting Premalignant Lesions - Implications for Early Breast Cancer Detection and Intervention PRINCIPAL...INTRODUCTION: Difficulty in managing treatment of advanced stage breast cancer has led to the goal for detection and intervention of early -stage...Furthermore, these probes will be used to develop targeted therapeutic nanoparticles for early intervention in breast cancer. 2. KEYWORDS

  7. Human Papillomavirus as a Potential Risk Factor for Oral Premalignant Lesions.

    Science.gov (United States)

    Zendeli-Bedjeti, Lindita; Popovska, Mirjana; Atanasovska-Stojanovska, Aneta; Duvlis, Sotirija

    2017-09-01

    Oral premalignant lesions (OPLs) and numerous alterations of oral mucosa remain unsolved due to their complex etiopathogenesis. Human papillomaviruses (HPVs), in particular, have been reported as the possible risk factors or cofactors. The aim of the study was to determine the association of different HPV types with oral premalignant lesions, and the potential role of smoking and alcohol use. Eighty patients (mean age ± SD, 52.45±5.56) of both genders, 19 (23.75%) male and 61 (76.25%) female, were enrolled in the study. Study group included 40 patients diagnosed with OPLs (leukoplakia, erythroplakia, actinic keratosis and lichen planus), while control group included another 40 patients with healthy oral mucosa. Genotyping of the HPV types was performed by qualitative real-time HPV typing polymerase chain reaction test. HPV DNA was detected in 30% (12/40) of study group patients and 2.5% (1/40) of control group patients. The results revealed the presence of HPV16 in 15% (6/40), HPV56 in 10% (4/40), and HPV18 in 5% (2/40) of study group cases, and HPV31 in 1 (2.5%) control group patient. Th e association of oral HPV positivity and smoking/alcohol use in the study group was not statistically significant (pHPV types are associated with oral premalignant disorders. However, it remains unknown whether HPV acts as an innocent bystander or it has a role in initiating development of premalignant lesions. Smoking and alcohol use were not associated with the existing oral HPV infection.

  8. Relationship between cancer survival and ambient ultraviolet B irradiance in China.

    Science.gov (United States)

    Chen, Wanqing; Armstrong, Bruce K; Rahman, Bayzidur; Zheng, Rongshou; Zhang, Siwei; Clements, Mark

    2013-07-01

    Ecological studies in predominantly European populations have reported higher cancer survival in areas of higher solar ultraviolet (UV) B irradiation, perhaps due to a cancer protective effect of vitamin D synthesized photochemically in the skin. Such studies have not been done in developing countries, perhaps because of lack of cancer registries that can do outcome follow-up. One minus the mortality-to-incidence ratio (1-MIR), however, can be used as a measure of survival, and MIR as a measure of fatality, in developing country cancer registries. We analyzed the association between ambient solar UVB and MIR in China. National cancer registration data in 32 counties of China in 2004-2005 were used to estimate MIR by age, sex, and area. The accuracy of 1-MIR as a measure of survival was assessed in the Cixian County cancer registry. Contemporary satellite measurements of cloud-adjusted ambient UVB intensity at 305 nm were taken from an NASA database and spatial Kriging methods used to estimate the average daily irradiance in each county. We estimated mortality hazard ratios (HRs) per 10 mW/m(2) of UVB for all cancers together, and the ten commonest cancer types by fitting a generalized linear model assuming mortality had a binomial distribution conditional on the sum of mortality and incidence, adjusted for sex, age, and location. The 5-year survival proportions for the main cancer types were in good agreement with 1-MIR in Cixian County. MIR ratios for all cancers combined were inversely associated with ambient UVB in men (HR = 0.96, 95% CI 0.93-0.99) and women (HR = 0.91, 95% CI 0.88-0.94) and in urban (HR = 0.95, 95% CI 0.94-0.96) and rural areas (HR = 0.90, 95% CI 0.87-0.93). Similar inverse associations were present for cancers of esophagus, stomach, and bladder in both sexes together and breast cancer in women. They were present in urban residents for all major cancers except liver cancer, bladder cancer, and breast cancer in women. For rural residents, most

  9. Death penalty for keratinocytes: apoptosis versus cornification.

    Science.gov (United States)

    Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

    2005-11-01

    Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

  10. Culture technique of rabbit primary epidermal keratinocytes

    Directory of Open Access Journals (Sweden)

    Marini M

    2012-10-01

    Full Text Available The epidermis is the protective covering outer layer of the mammalian skin. The epidermal cells are stratified squamous epithelia which undergo continuous differentiation of loss and replacement of cells. Ninety per cent of epidermal cells consist of keratinocytes that are found in the basal layer of the stratified epithelium called epidermis. Keratinocytes are responsible for forming tight junctions with the nerves of the skin as well as in the process of wound healing. This article highlights the method of isolation and culture of rabbit primary epidermal keratinocytes in vitro. Approximately 2cm x 2cm oval shaped line was drawn on the dorsum of the rabbit to mark the surgical area. Then, the skin was carefully excised using a surgical blade and the target skin specimens harvested from the rabbits were placed in transport medium comprising of Dulbecco’s Modified Eagle Medium (DMEM and 1% of antibiotic-antimycotic solution. The specimens were transferred into a petri dish containing 70% ethanol and washed for 5 min followed by a wash in 1 x Dulbecco’s Phosphate Buffered Saline (DBPS. Then, the skin specimens were placed in DMEM and minced into small pieces using a scalpel. The minced pieces were placed in a centrifuge tube containing 0.6% Dispase and 1% antibiotic-antimycotic solution overnight at 4°C in a horizontal orientation. The epidermis layer (whitish, semi-transparent was separated from the dermis (pink, opaque, gooey with the aid of curved forceps by fixing the dermis with one pair of forceps while detaching the epidermis with the second pair. The cells were cultured at a density of 4 x 104 cells/cm2 in culture flask at 37°C and 5% CO2. The cell morphology of the keratinocytes was analyzed using inverted microscope.

  11. Automated identification of epidermal keratinocytes in reflectance confocal microscopy

    Science.gov (United States)

    Gareau, Dan

    2011-03-01

    Keratinocytes in skin epidermis, which have bright cytoplasmic contrast and dark nuclear contrast in reflectance confocal microscopy (RCM), were modeled with a simple error function reflectance profile: erf( ). Forty-two example keratinocytes were identified as a training set which characterized the nuclear size a = 8.6+/-2.8 μm and reflectance gradient b = 3.6+/-2.1 μm at the nuclear/cytoplasmic boundary. These mean a and b parameters were used to create a rotationally symmetric erf( ) mask that approximated the mean keratinocyte image. A computer vision algorithm used an erf( ) mask to scan RCM images, identifying the coordinates of keratinocytes. Applying the mask to the confocal data identified the positions of keratinocytes in the epidermis. This simple model may be used to noninvasively evaluate keratinocyte populations as a quantitative morphometric diagnostic in skin cancer detection and evaluation of dermatological cosmetics.

  12. 11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Itoi, Saori; Terao, Mika, E-mail: mterao@derma.med.osaka-u.ac.jp; Murota, Hiroyuki; Katayama, Ichiro

    2013-10-18

    Highlights: •We investigate the role of 11β-HSD1 in skin inflammation. •Various stimuli increase expression of 11β-HSD1 in keratinocytes. •11β-HSD1 knockdown by siRNA decreases cortisol levels in media. •11β-HSD1 knockdown abrogates the response to pro-inflammatory cytokines. •Low-dose versus high-dose cortisol has opposing effects on keratinocyte inflammation. -- Abstract: The endogenous glucocorticoid, cortisol, is released from the adrenal gland in response to various stress stimuli. Extra-adrenal cortisol production has recently been reported to occur in various tissues. Skin is known to synthesize cortisol through a de novo pathway and through an activating enzyme. The enzyme that catalyzes the intracellular conversion of hormonally-inactive cortisone into active cortisol is 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). We recently reported that 11β-HSD1 is expressed in normal human epidermal keratinocytes (NHEKs) and negatively regulates proliferation of NHEKs. In this study, we investigated the role of 11β-HSD1 in skin inflammation. Expression of 11β-HSD1 was induced by UV-B irradiation and in response to the pro-inflammatory cytokines, IL-1β and TNFα. Increased cortisol concentrations in culture media also increased in response to these stimuli. To investigate the function of increased 11β-HSD1 in response to pro-inflammatory cytokines, we knocked down 11β-HSD1 by transfecting siRNA. Production of IL-6 and IL-8 in response to IL-1β or TNFα stimulation was attenuated in NHEKs transfected with si11β-HSD1 compared with control cells. In addition, IL-1β-induced IL-6 production was enhanced in cultures containing 1 × 10{sup −13} M cortisol, whereas 1 × 10{sup −5} M cortisol attenuated production of IL-6. Thus, cortisol showed immunostimulatory and immunosuppressive activities depending on its concentration. Our results indicate that 11β-HSD1 expression is increased by various stimuli. Thus, regulation of cytosolic cortisol

  13. [Use of keratinocytes in therapy of ulcera crurum].

    Science.gov (United States)

    Moll, I; Schönfeld, M; Jung, E G

    1995-08-01

    Freshly isolated keratinocytes were used for the treatment of non-healing leg ulcers (21 cases). For application, keratinocytes were suspended in fibrin (Tissucol Duo S). One or more applications of keratinocytes initiated granulation and reepithelialization of nearly all leg ulcers (20 cases). The method is compared with the established therapy entailing transplantation of allogenic keratinocyte sheets onto leg ulcers. Our method shows the advantages: autologous cells that are immediately and easily available, freedom from pain, and short-time immobilization (48 h) after application.

  14. Decorin gene expression and its regulation in human keratinocytes

    International Nuclear Information System (INIS)

    Highlights: → We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. → Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. → Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. → Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  15. Decorin gene expression and its regulation in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico); Kuri-Harcuch, Walid, E-mail: walidkuri@gmail.com [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico)

    2011-07-22

    Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  16. Differentiation of human scalp hair follicle keratinocytes in culture.

    Science.gov (United States)

    Weterings, P J; Verhagen, H; Wirtz, P; Vermorken, A J

    1984-01-01

    The morphology of human scalp hair follicle keratinocytes, cultured on the bovine eye lens capsule, is studied by light and electron microscopy. The hair follicle keratinocytes in the stratified cultures are characterized by the presence of numerous tonofilaments, desmosomes and lysosomes and by the presence of glycogen accumulations. The cells in the upper layers develop a cornified envelope. Moreover, an incomplete basal lamina is found between the capsule and the basal cells. However, some features of epidermal keratinocytes in vivo, such as keratohyalin granules and stratum corneum formation, are absent. Analysis of the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis also reveals differences between the cultured hair follicle cells and epidermis, whilst the patterns of cultured cells and hair follicle sheaths are similar. The morphological and protein biosynthetic aspects of terminal differentiation of the keratinocytes in vitro are correlated. These results are discussed in the light of the findings with cultured epidermal keratinocytes, reported in the literature.

  17. Benzophenone Suppression of Quercetin Antioxidant Activity towards Lipids under UV-B Irradiation Regime: Detection by HPLC Chromatography

    Directory of Open Access Journals (Sweden)

    Jelena S. Stanojević

    2013-01-01

    Full Text Available Quercetin, a well-known flavonoid antioxidant, has been employed to control benzophenone-sensitized peroxidation of the lipid mixture in methanol solution, induced by continuous UV-B irradiation. Surprisingly, the detected quercetin antioxidant activity was almost negligible. The presented data suggests that the reason is not in its own UV-B-induced degradation but rather in its interrelationship with benzophenone during UV-B stress. On the other side of this relationship, benzophenone anticipated sensitizing role towards lipids; that is, the initiation of lipid peroxidation has been affected as well. These results, obtained by HPLC chromatography, partly confirm but partly relativize to some extent recent results obtained with the same system by spectrophotometric method.

  18. Changes of cell growth and magnetosome biomineralization in Magnetospirillum magneticum AMB-1 after ultraviolet-B irradiation

    Directory of Open Access Journals (Sweden)

    Yinzhao eWang

    2013-12-01

    Full Text Available Effects of ultraviolet radiation on microorganisms are of great interest in field of microbiology and planetary sciences. In the present study, we used Magnetospirillum magneticum AMB-1 as a model organism to examine the influence of ultraviolet-B (UV-B radiation on cell growth and magnetite biomineralization of magnetotactic bacteria. Live AMB-1 cells were exposed to UV-B radiation for 60 s, 300 s and 900 s, which correspond to radiation doses of 120 J/m2, 600 J/m2 and 1800 J/m2, respectively. After irradiation, the amounts of cyclobutane pyrimidine dimers and reactive oxygen species of the cells were increased, and cell growth was stunted up to ~170 h, depending on the UV-B radiation doses. The UV-B irradiated cells also produced on average more magnetite crystals with larger grain sizes and longer chains, which results in changes of their magnetic properties.

  19. Melanosome transfer to and translocation in the keratinocyte.

    Science.gov (United States)

    Boissy, Raymond E

    2003-01-01

    Complexion coloration in humans is primarily regulated by the amount and type of melanin synthesized by the epidermal melanocyte. However, additional and equally contributing factors consist of (1) efficient transfer of melanin from the melanocytes to the neighboring keratinocytes and (2) distribution and degradation of the transferred melanosomes by the recipient keratinocytes. Once synthesized in the cell body of the epidermal melanocyte, pigmented melanosomes are translocated down the dendrites and captured at the dendritic tips via various cytoskeletal elements. Molecules recently identified that participate in this process consist of Rab27a, myosin-Va and melanophilin. Eventually, these peripherally localized melanosomes are transferred to keratinocytes by a presently undefined mechanism. The protease-activated receptor-2 (PAR-2) and unidentified surface lectins and glycoproteins facilitate this transfer process. Once incorporated into the keratinocytes, melanosomes are distributed individually or as clusters, aggregated towards the apical pole of the nucleus, and degraded as the keratinocytes undergo terminal differentiation and desquamation. Ultraviolet irradiation (UVR) can modulate the process of melanosome transfer from the melanocytes to the keratinocytes. UVR can upregulate expression of PAR-2 and lectin-binding receptors and increase phagocytic activity of cultured keratinocytes. Therefore, many cellular and molecular events that occur after melanogenesis contribute to skin color.

  20. Melanin Transferred to Keratinocytes Resides in Nondegradative Endocytic Compartments.

    Science.gov (United States)

    Correia, Maria S; Moreiras, Hugo; Pereira, Francisco J C; Neto, Matilde V; Festas, Tiago C; Tarafder, Abul K; Ramalho, José S; Seabra, Miguel C; Barral, Duarte C

    2018-03-01

    Melanin transfer from melanocytes to keratinocytes and subsequent accumulation in the supranuclear region is a critical process in skin pigmentation and protection against UVR. We have previously proposed that the main mode of transfer between melanocytes and keratinocytes is through exo/endocytosis of the melanosome core, termed melanocore. In this study, we developed an in vitro uptake assay using melanocores secreted by melanocytes. We show that the uptake of melanocores, but not melanosomes, by keratinocytes is protease-activated receptor-2-dependent. Furthermore, we found that the silencing of the early endocytic regulator Rab5b, but not the late endocytic regulators Rab7a or Rab9a, significantly impairs melanocore uptake by keratinocytes. After uptake, we observed that melanin accumulates in compartments that are positive for both early and late endocytic markers. We found that melanin does not localize to either highly degradative or acidic organelles, as assessed by LysoTracker and DQ-BSA staining, despite the abundance of these types of organelles within keratinocytes. Therefore, we propose that melanocore uptake leads to storage of melanin within keratinocytes in hybrid endocytic compartments that are not highly acidic or degradative. By avoiding lysosomal degradation, these specialized endosomes may allow melanin to persist within keratinocytes for long periods. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. Cytomorphometric Analysis of Oral Premalignant and Malignant Lesions Using Feulgen Stain and Exfoliative Brush Cytology.

    Directory of Open Access Journals (Sweden)

    Priya Shirish Joshi

    2013-08-01

    Full Text Available Introduction: Oral squamous cell carcinoma (OSCC is the sixth most common cancer worldwide and accounting for 90% of cancers of oral cavity. Tobacco abuse has been proved to be the major risk factor in the development of OSCC. Despite advances in surgery, radiation and chemotherapy, the five year survival rate for oral cancer has not improved significantly over the past several decades and it remains at about 50 to 55%. Cytobrush sampling is more frequently used nowadays for exfoliative cytology, since it maximizes the number of cells obtained, and facilitates their uniform distribution onto the microscope slide, thus probably improving sensitivity.Our study was therefore carried out to analyze the cytomorphometric features of cells obtained by cytobrush and stained with Feulgen stain from oral premalignant and malignant lesions and to find out whether these features could be used to detect dysplasia and malignancy in their early stages. Aims: To analyze the cytomorphological features of cells in smears of oral premalignant and malignant lesions obtained from exfoliative brush cytology using Feulgen stain and to assess the efficacy of the same in detecting dysplasia and malignancy. Methods: Our study comprised of clinically and histopathologically diagnosed sixty cases which were grouped into twenty cases each of tobacco users with lesions (Leukoplakia and Erythroplakia (Group I; tobacco users without lesions (Group II; Oral squamous cell carcinoma (OSCC lesions (Group III; and normal mucosa (Group IV. The epithelial cells from the lesion were collected with a cytobrush and smears were stained with Feulgen stain. The cells were measured using software for their nuclear area, nuclear diameter, cellular area, cellular diameter and nuclear to cellular area ratio (N:C. Results: The exfoliated cells showed similar alterations as those occuring in histopathological sections of premalignant and malignant lesions. The N:C ratio, mean nuclear area and

  2. Scanning Ion Conductance Microscopy of Live Keratinocytes

    International Nuclear Information System (INIS)

    Hegde, V; Mason, A; Saliev, T; Smith, F J D; McLean, W H I; Campbell, P A

    2012-01-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (< nN), could not routinely image microvilli: rather, an apparently convolved image of the underlying cytoskeleton was instead prevalent. We note that the present incarnation of the commercial instrument falls some way behind the market leading SPMs in terms of technical prowess and scanning speed, however, the intrinsic non-obtrusive nature of

  3. BRAF mutations in conjunctival melanoma: investigation of incidence, clinicopathological features, prognosis and paired premalignant lesions

    DEFF Research Database (Denmark)

    Larsen, Ann-Cathrine; Dahl, Christina; Dahmcke, Christina M.

    2016-01-01

    with atypia. BRAF mutations were identified in 39 of 111 (35%) cases. The rate ratio of BRAF-mutated versus BRAF-wild-type melanoma did not change over time. BRAF mutations were associated with T1 stage (p = 0.007), young age (p = 0.001), male gender (p = 0.02), sun-exposed location (p = 0.01), mixed....../non-pigmented tumour colour (p = 0.02) and nevus origin (p = 0.005), but did not associate with prognosis. BRAF status in conjunctival melanoma and paired premalignant lesions corresponded in 19 of 20 cases. Immunohistochemistry detected BRAF V600E mutations with a sensitivity of 0.94 and a specificity of 1...

  4. Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions

    Science.gov (United States)

    Mehrotra, Ravi; Gupta, Anurag; Singh, Mamta; Ibrahim, Rahela

    2006-01-01

    Early detection of a premalignant or cancerous oral lesion promises to improve the survival and the morbidity of patients suffering from these conditions. Cytological study of oral cells is a non-aggressive technique that is well accepted by the patient, and is therefore an attractive option for the early diagnosis of oral cancer, including epithelial atypia and squamous cell carcinoma. However its usage has been limited so far due to poor sensitivity and specificity in diagnosing oral malignancies. Lately it has re-emerged due to improved methods and it's application in oral precancer and cancer as a diagnostic and predictive method as well as for monitoring patients. Newer diagnostic techniques such as "brush biopsy" and molecular studies have been developed. Recent advances in cytological techniques and novel aspects of applications of scraped or exfoliative cytology for detecting these lesions and predicting their progression or recurrence are reviewed here. PMID:16556320

  5. Premalignant quiescent melanocytic nevi do not express the MHC class I chain-related protein A

    Directory of Open Access Journals (Sweden)

    Mercedes B. Fuertes

    2011-08-01

    Full Text Available The MHC class I chain-related protein A (MICA is an inducible molecule almost not expressed by normal cells but strongly up-regulated in tumor cells. MICA-expressing cells are recognized by natural killer (NK cells, CD8+ aßTCR and ?dTCR T lymphocytes through the NKG2D receptor. Engagement of NKG2D by MICA triggers IFN-? secretion and cytotoxicity against malignant cells. Although most solid tumors express MICA and this molecule is a target during immune surveillance against tumors, it has been observed that high grade tumors from different histotypes express low amounts of cell surface MICA due to a metalloprotease- induced shedding. Also, melanomas develop after a complex process of neotransformation of normal melanocytes. However, the expression of MICA in premalignant stages (primary human quiescent melanocytic nevi remains unknown. Here, we assessed expression of MICA by flow cytometry using cell suspensions from 15 primary nevi isolated from 11 patients. When collected material was abundant, cell lysates were prepared and MICA expression was also analyzed by Western blot. We observed that MICA was undetectable in the 15 primary nevi (intradermic, junction, mixed, lentigo and congenital samples as well as in normal skin, benign lesions (seborrheic keratosis, premalignant lesions (actinic keratosis and benign basocellular cancer. Conversely, a primary recently diagnosed melanoma showed intense cell surface MICA. We conclude that the onset of MICA expression is a tightly regulated process that occurs after melanocytes trespass the stage of malignant transformation. Thus, analysis of MICA expression in tissue sections of skin samples may constitute a useful marker to differentiate between benign and malignant nevi.

  6. Seminal epithelium in prostate biopsy can mimic malignant and premalignant prostatic lesions.

    Science.gov (United States)

    Arista-Nasr, J; Trolle-Silva, A; Aguilar-Ayala, E; Martínez-Benítez, B

    2016-01-01

    In most prostate biopsies, the seminal epithelium is easily recognised because it meets characteristic histological criteria. However, some biopsies can mimic malignant or premalignant prostatic lesions. The aims of this study were to analyse the histological appearance of the biopsies that mimic adenocarcinomas or preneoplastic prostatic lesions, discuss the differential diagnosis and determine the frequency of seminal epithelia in prostate biopsies. We consecutively reviewed 500 prostate puncture biopsies obtained using the sextant method and selected those cases in which we observed seminal vesicle or ejaculatory duct epithelium. In the biopsies in which the seminal epithelium resembled malignant or premalignant lesions, immunohistochemical studies were conducted that included prostate-specific antigen and MUC6. The most important clinical data were recorded. Thirty-six (7.2%) biopsies showed seminal epithelium, and 7 of them (1.4%) resembled various prostate lesions, including high-grade prostatic intraepithelial neoplasia, atypical acinar proliferations, adenocarcinomas with papillary patterns and poorly differentiated carcinoma. The seminal epithelium resembled prostate lesions when the lipofuscin deposit, the perinuclear vacuoles or the nuclear pseudoinclusions were inconspicuous or missing. Five of the 7 biopsies showed mild to moderate cellular atypia with small and hyperchromatic nuclei, and only 2 showed cellular pleomorphism. The patients were alive and asymptomatic after an average of 6 years of progression. The seminal epithelium resembles prostatic intraepithelial neoplasia, atypical acinar proliferations and various types of prostatic adenocarcinomas in approximately 1.4% of prostate biopsies. Copyright © 2015 AEU. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. Prognostic value of molecular markers of oral pre-malignant and malignant lesions

    Directory of Open Access Journals (Sweden)

    Peter Agus

    2009-06-01

    Full Text Available Background: The representation of oral cancer and precancerous lesions is often undetected until at later stage and the survival rate of oral cancer has remained essentially unchanged over the past three decades. Over 90% of these tumors are squamous cell carcinoma. The American Cancer Society estimates that among 28,900 new cases of oral diagnosis in 2002, nearly 7,400 people will die from this disease. Oral pre-malignant and malignant lesions have multi-step process both at phenotype and genetic levels that influence tumor behavior and genetic mutations. Purpose: The aim of this presentation was to review the current knowledge of prognostic value of tumor marker in order to achieve early detection, prognostic value, proper and accurate treatment of oral cancer. Reviews: Technological advances in molecular biology have greatly increased the number of new molecular markers that can be detected by molecular analysis such as immunohistochemistry (IHC, polymerase chain reaction (PCR and surgical margin analysis that may increase prognosis and treatment of oral cancer. The result of most valuable tumor markers is twenty nine divided into four groups according to their function such as enhancement of tumor growth, tumor suppression and anti tumor defense, including immune response and apoptosis, angiogenesis, tumor invasion and metastatic potential, including adhesion molecules and matrix degradation. Conclusion: In general the conclusion is that the location of markers within the tumor and not the quantitative assessment is as same as emphasized. Especially, the analysis of new molecular markers have been used to be of great importance for early detection, surgical margin analysis, prognostication and treatment of oral pre-malignant and cancerous lesion.

  8. Interleukin 7 is produced by murine and human keratinocytes.

    Science.gov (United States)

    Heufler, C; Topar, G; Grasseger, A; Stanzl, U; Koch, F; Romani, N; Namen, A E; Schuler, G

    1993-09-01

    Interleukin 7 (IL-7) was originally identified as a growth factor for B cell progenitors, and subsequently has been shown to exert proliferative effects on T cell progenitors and mature peripheral T cells as well. Constitutive IL-7 mRNA expression so far had been demonstrated in bone marrow stromal cell lines, thymus, spleen, and among nonlymphoid tissues in liver and kidney. Here we show that both murine and human keratinocytes express IL-7 mRNA and release IL-7 protein in biologically relevant amounts. The physiological or pathological relevance of keratinocyte-derived IL-7 is presently unknown. Our finding that keratinocytes can produce IL-7 in concert with reports that IL-7 is a growth factor for in vivo primed antigen-specific T cells, as well as for T lymphoma cells suggests, however, that keratinocyte-derived IL-7 is important in the pathogenesis of inflammatory skin diseases and cutaneous T cell lymphoma.

  9. Serial cultivation of human scalp hair follicle keratinocytes.

    Science.gov (United States)

    Weterings, P J; Roelofs, H M; Vermorken, A J; Bloemendal, H

    1983-01-01

    A method is described for the serial cultivation of adult human hair follicle keratinocytes. Plucked scalp hair follicles, placed on bovine eye lens capsules as a growth substrate, give rise to quickly expanding colonies within a few days. After trypsinization, the cells are replated with irradiated 3T3 cells as 'feeders'. Using this combination of techniques the keratinocytes can be subcultured up to four times. In this way about 10(7) keratinocytes can be generated from one single hair follicle. Moreover, the technique enables cryogenic storage of the cells, allowing for instance, convenient transportation. Subcultured hair follicle keratinocytes can be plated on glass coverslips. This allows immunofluorescence studies. The keratin cytoskeletons visualized using an antiserum against human keratin.

  10. Shining a Light on Black Holes in Keratinocytes.

    Science.gov (United States)

    Bowman, Shanna L; Marks, Michael S

    2018-03-01

    The mechanisms by which melanins are transferred from melanocytes and stored within keratinocytes to generate skin pigmentation are hotly debated. Correia et al. and Hurbain et al. provide evidence that melanin cores of melanosomes are secreted from melanocytes and taken up and stored within non-degradative membranous organelles in keratinocytes in the basal epidermis of human skin. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Effect of UV-B Irradiation on Physiologically Active Substance Content and Antioxidant Properties of the Medicinal Caterpillar Fungus Cordyceps militaris (Ascomycetes).

    Science.gov (United States)

    Huang, Shih-Jeng; Lin, Chun-Ping; Mau, Jeng-Leun; Li, Yu-Shan; Tsai, Shu-Yao

    2015-01-01

    Ultraviolet-B (UV-B) light irradiation is a well-known technique for converting vitamin D2 from ergosterol in mushroom fruit bodies. Mushrooms are a natural and nonanimal food source of vitamin D2. We studied the effect of UV-B light irradiation on the amount of vitamin D2 and physiologically active substances in Cordyceps militaris and their antioxidant properties. After UV-B irradiation for 2 hours, the vitamin D2 content of freshly harvested C. militaris fruiting bodies, mycelia, whole submerged culture (WSC), and homogenized submerged culture (HSC) increased from 0 to 0.03 to 0.22 to 1.11 mg/g, but the ergosterol content was reduced from 1.36 to 2.50 to 1.24 to 2.06 mg/g, respectively. After UV-B irradiation, the amount of adenosine, cordycepin, and ergothioneine of fruiting bodies dramatically increased 32-128%, but the polysaccharide content slightly decreased 36%. The reverse trends were observed in mycelia, WSC, and HSC. UV-B irradiation could reduce the effective concentrations at 50% of fruiting bodies for ethanolic and hot water extracts in reducing power, scavenging, and chelating abilities, whereas mycelia, WSC, and HSC of ethanolic extracts increased effective concentrations at 50% in reducing power, scavenging, and chelating abilities. UV-B irradiation slightly increased flavonoid content (10-56%) and slightly affected total phenol content.

  12. Uv - b irradiation effects on biological activities and cytological behavior of sainfoin (onobrychis viciifolia scop.) grown in vivo and in vitro

    International Nuclear Information System (INIS)

    Mohajer, S.; Taha, R. M.; Mohajer, M.; Javan, I. Y.

    2015-01-01

    To investigate the feasibility of UV-B irradiation (312 nm), seeds of Onobrychis viciifolia were exposed to five different intensities for determining the effectiveness of cellular behavior, nutritional constituents and biological activities in In vivo and In vitro growth cultures. The atomic spectroscopy analysis confirmed that concentrations of two macronutrients (P and N) improved after UV-B exposure as compared with control plants. Near infrared radiation conducted on both In vivo and In vitro plants showed significant differences on dry matter digestibility (DMD) and crude fiber (CF). Flavonoid and phenolic compounds were increased in both growth cultures by 40 percentage intensity of UV-B irradiation, although In vitro plants had the higher compounds than intact plants. Increasing the UV-B irradiation intensity was also found to yield positive effect on anthocyanin. Observations on cellular behavior such as determination of nuclear and cell areas, mitotic index and chromosomal aberrations were proven to be essential in deducing the effectiveness of UV-B irradiation to induce somaclonal variation in sainfoin. (author)

  13. Stereospecificity of ginsenoside Rg2 epimers in the protective response against UV-B radiation-induced oxidative stress in human epidermal keratinocytes.

    Science.gov (United States)

    Kang, Hyun-Ji; Huang, Yu-Hua; Lim, Hye-Won; Shin, Daehyun; Jang, Kyounghee; Lee, Yoonjin; Kim, Kyunghoon; Lim, Chang-Jin

    2016-12-01

    Ginseng, referring to the dried roots of Panax ginseng C.A. Meyer, has been known as a famous traditional folkloric medicine in East Asian countries for a long time. In recent years, it has been gaining a worldwide popularity as a dietary herbal supplement. Ginsenosides are bioactive ingredients that are responsible for most pharmacological efficacies of ginseng. Ginsenoside Rg2 (Rg2), one of minor protopanaxatriol (PPT)-type ginsenosides, exists in two epimeric forms, 20(S)-ginsenoside Rg2 [20(S)-Rg2] and 20(R)-ginsenoside Rg2 [20(R)-Rg2]. This work was undertaken to assess and compare their skin anti-photoaging properties. When they were applied to HaCaT keratinocytes prior to the irradiation, 20(S)-Rg2 only could attenuate the UV-B-induced intracellular reactive oxygen species (ROS) elevation, which were detected using three fluorescent ROS dyes, such as 2',7'-dichlorodihydrofluorescein diacetate, dihydroethidium and dihydrorhodamine 123. 20(S)-Rg2 but not 20(R)-Rg2 significantly attenuated the UV-B-induced promatrix metalloproteinase-2 (proMMP-2) gelatinolytic activity and protein levels. Likewise, 20(S)-Rg2 only augmented the UV-B-reduced total glutathione (GSH) and superoxide dismutase (SOD) activity levels in a concentration-dependent manner. Neither of the two Rg2 epimers was cytotoxic to HaCaT keratinocytes, regardless of UV-B irradiation. Taken together, of the two Rg2 epimers, 20(S)-Rg2 only possesses the stereospecific protective properties against the UV-B-induced skin photoaging in HaCaT keratinocytes. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Ultraviolet B irradiation of skin induces mast cell degranulation and release of tumour necrosis factor-{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Walsh, L.J. [University of Queensland, Brisbane, QLD (Australia). Dept. of Dentistry, Immunopathology Unit

    1995-06-01

    In the `sunburn` response in skin, dermal blood vessels are activated and traffic of dendritic Langerhans` cells altered. While these changes have been attributed to the cytokine TNF-{alpha}, the source of this acutely released TNF has not been identified. This report demonstrates that the `sunburn` response, both in vivo and in vitro, is accompanied by rapid degranulation of cutaneous mast cells, with consequential release of intracellular stores of TNF. Epidermal keratinocytes were only minor contributors to local TNF production. Expression of the TNF-inducible CD62E (E-selectin/ELAM-1) and CD54 adhesion molecules on cutaneous endothelium occurred 2 hours following mast cell degranulation, and this event was sensitive to blockade of mast cells with disodium cromoglycate. These results indicate that TNF release in skin in the acute sunburn response can largely be attributed to mast cells. 47 refs., 5 tabs., 2 figs.

  15. Human Keratinocytes Radioprotection with Mentha Longifolia

    Science.gov (United States)

    Rizzo, Angela Maria; Berselli, P.; Zava, S.; Negroni, M.; Corsetto, P.; Montorfano, G.; Bertolotti, A.; Ranza, E.; Ottolenghi, A.; Berra, B.

    Antioxidants are suggested to act as radioprotectors, and dietary supplements based on antiox-idants have been proposed for astronauts involved in long-term space missions. Plant extracts with antioxidant properties may be used in dietetic supplements for astronauts; in fact recent nutritional guidelines suggest that "fruits and vegetables may become as important on space-going vessels as limes were on the sea-going vessels of old". Mint presents a large variety of biological properties, such as antiallergenic, antibacterial, anti-inflammatory, antitumor, an-tiviral, gastrointestinal protective, hepatoprotective, chemopreventive activities, most of which are attributable to its antioxidant activity. The aim of the present study is to evaluate the antioxidant properties and protective bio-efficacy of a phenol enriched Mentha longifolia ex-tract on gamma rays stressed human keratinocytes (NCTC2544). We assessed first the in vitro antioxidant activity (ABTS and DPPH), and then evaluated different stress markers in order to investigate various oxidative stress targets: cell viability (MTT); retained proliferating ca-pability (CA); DNA damage (histone H2AX) and protein damage (HSP70 induction). Results indicate that this Mint extract has a higher antioxidant activity respect to fresh extracts, that could be responsible of its really interesting radio-protective effects.

  16. Roles of sonography and hysteroscopy in the detection of premalignant and malignant polyps in women presenting with postmenopausal bleeding and thickened endometrium.

    Science.gov (United States)

    Cavkaytar, Sabri; Kokanali, Mahmut Kuntay; Ceran, Ufuk; Topcu, Hasan Onur; Sirvan, Levent; Doganay, Melike

    2014-01-01

    To assess the role of sonographic endometrial thickness and hysteroscopic polyp size in predicting premalignant and malignant polyps in postmenopausal women. A total of 328 postmenopausal women with abnormal uterine bleeding and thickened endometrium underwent operative hysteroscopy due to detection of endometrial polyps were included in this retrospective study. Preoperative endometrial thickness measured by transvaginal ultrasonography and polyp size on hysteroscopy were noted. Hysteroscopic resection with histology was performed for endometrial polyps. Endometrial thickness and polyp size were evaluated on the basis of final diagnosis established by histologic examination. Receiver operator characteristic curves were calculated to assess the sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of endometrial thickness and polyp size for detecting premalignant and malignant polyps. Premalignant and malignant polyps were identified in 26 (7.9%) of cases. Sonographic measurement showed a greater endometrial thickness in cases of premalignant and malignant polyps when compared to benign polyps. On surgical hysteroscopy, premalignant and malignant polyps were also larger. Endometrial thickness demonstrated a sensitivity of 53.8%, specificity of 85.8%, PPV of 24.6% and NPV of 95.6% at a cut-off limit of 11.5 mm with diagnostic accuracy of 83.2%. Polyp size has a diagnostic accuracy of 94.8% with a sensitivity of 92.3%, specificity of 95.0%, PPV of 61.5% and NPV of 99.3% at a cut-off point of 19.5mm. Endometrial thickness measured by transvaginal ultrasonography is not sufficient in predicting premalignant and malignant endometrial polyps in postmenopausal women with abnormal uterine bleeding and thickened endometrium. Polyp size on hysteroscopy is a more accurate parameter, because of better sensitivity and specificity. However, while polyp size ≥ 19.5mm seems to have a great accuracy for predicting premalignancy and

  17. AMPK regulation of the growth of cultured human keratinocytes

    International Nuclear Information System (INIS)

    Saha, Asish K.; Persons, Kelly; Safer, Joshua D.; Luo Zhijun; Holick, Michael F.; Ruderman, Neil B.

    2006-01-01

    AMP kinase (AMPK) is a fuel sensing enzyme that responds to cellular energy depletion by increasing processes that generate ATP and inhibiting others that require ATP but are not acutely necessary for survival. In the present study, we examined the relationship between AMPK activation and the growth (proliferation) of cultured human keratinocytes and assessed whether the inhibition of keratinocyte growth by vitamin D involves AMPK activation. In addition, we explored whether the inhibition of keratinocyte proliferation as they approach confluence could be AMPK-related. Keratinocytes were incubated for 12 h with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). At concentrations of 10 -4 and 10 -3 M, AICAR inhibited keratinocyte growth by 50% and 95%, respectively, based on measurements of thymidine incorporation into DNA. It also increased AMPK and acetyl CoA carboxylase phosphorylation (P-AMPK and P-ACC) and decreased the concentration of malonyl CoA confirming that AMPK activation had occurred. Incubation with the thiazolidinedione, troglitazone (10 -6 M) caused similar alterations in P-AMPK, P-ACC, and cell growth. In contrast, the well known inhibition of keratinocyte growth by 1,25-dihydroxyvitamin D 3 (10 -7 and 10 -6 M) was not associated with changes in P-AMPK or P-ACC. Like most cells, the growth of keratinocytes diminished as they approached confluence. Thus, it was of note that we found a progressive increase in P-AMPK (1.5- to 2-fold, p 3 is AMPK-independent

  18. Tumor-associated macrophages in oral premalignant lesions coexpress CD163 and STAT1 in a Th1-dominated microenvironment

    International Nuclear Information System (INIS)

    Mori, Kazumasa; Haraguchi, Shigeki; Hiori, Miki; Shimada, Jun; Ohmori, Yoshihiro

    2015-01-01

    Tumor-associated macrophages (TAMs) are implicated in the growth, invasion and metastasis of various solid tumors. However, the phenotype of TAMs in premalignant lesions of solid tumors has not been clarified. In the present study, we identify the phenotype of TAMs in leukoplakia, an oral premalignant lesion, by immunohistochemical analysis and investigate the involvement of infiltrated T cells that participate in the polarization of TAMs. The subjects included 30 patients with oral leukoplakia and 10 individuals with normal mucosa. Hematoxylin and eosin slides were examined for the histological grades, and immunohistochemical analysis was carried out using antibodies against CD68 (pan-MΦ), CD80 (M1 MΦ), CD163 (M2 MΦ), CD4 (helper T cells: Th), CD8 (cytotoxic T cells), CXCR3, CCR5 (Th1), CCR4 (Th2), signal transducer and activator of transcription (STAT1), phosphorylated STAT1 (pSTAT1) and chemokine CXCL9. The differences in the numbers of positively stained cells among the different histological grades were tested for statistical significance using the Kruskal-Wallis test. Correlations between different types of immune cells were determined using Spearman’s rank analysis. An increase in the rate of CD163 + TAM infiltration was observed in mild and moderate epithelial dysplasia, which positively correlated with the rate of intraepithelial CD4 + Th cell infiltration. Although CCR4 + cells rarely infiltrated, CXCR3 + and CCR5 + cells were observed in these lesions. Cells positive for STAT1 and chemokine CXCL9, interferon- (IFN)-induced gene products, and pSTAT1 were also observed in the same lesions. Double immunofluorescence staining demonstrated that the cells that were positive for CD163 were also positive for STAT1. CD163 + TAMs in oral premalignant lesions coexpress CD163 and STAT1, suggesting that the TAMs in oral premalignant lesions possess an M1 phenotype in a Th1-dominated micromilieu

  19. Improvement in Flavonoids and Phenolic Acids Production and Pharmaceutical Quality of Sweet Basil (Ocimum basilicum L. by Ultraviolet-B Irradiation

    Directory of Open Access Journals (Sweden)

    Ali Ghasemzadeh

    2016-09-01

    Full Text Available Sweet basil (Ocimum basilicum Linnaeus is aromatic herb that has been utilized in traditional medicine. To improve the phytochemical constituents and pharmaceutical quality of sweet basil leaves, ultraviolet (UV-B irradiation at different intensities (2.30, 3.60, and 4.80 W/m2 and durations (4, 6, 8, and 10-h was applied at the post-harvest stage. Total flavonoid content (TFC and total phenolic content (TPC were measured using spectrophotometric method, and individual flavonoids and phenolic acids were identified using ultra-high performance liquid chromatography. As a key enzyme for the metabolism of flavonoids, chalcone synthase (CHS activity, was measured using a CHS assay. Antioxidant activity and antiproliferative activity of extracts against a breast cancer cell line (MCF-7 were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH assays and MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assays, respectively. UV-B irradiation at an intensity of 3.60 W/m2 increased TFC approximately 0.85-fold and also increased quercetin (0.41-fold, catechin (0.85-fold, kaempferol (0.65-fold rutin (0.68-fold and luteolin (1.00-fold content. The highest TPC and individual phenolic acid (gallic acid, cinnamic acid and ferulic acid was observed in the 3.60 W/m2 of UV-B treatment. Cinnamic acid and luteolin were not detected in the control plants, production being induced by UV-B irradiation. Production of these secondary metabolites was also significantly influenced by the duration of UV-B irradiation. Irradiation for 8-h led to higher TFC, TPC and individual flavonoids and phenolic acids than for the other durations (4, 8, and 10-h except for cinnamic acid, which was detected at higher concentration when irradiated for 6-h. Irradiation for 10-h significantly decreased the secondary metabolite production in sweet basil leaves. CHS activity was induced by UV-B irradiation and highest activity was observed at 3.60 W/m2 of UV-B irradiation. UV

  20. Improvement in Flavonoids and Phenolic Acids Production and Pharmaceutical Quality of Sweet Basil (Ocimum basilicum L.) by Ultraviolet-B Irradiation.

    Science.gov (United States)

    Ghasemzadeh, Ali; Ashkani, Sadegh; Baghdadi, Ali; Pazoki, Alireza; Jaafar, Hawa Z E; Rahmat, Asmah

    2016-09-09

    Sweet basil (Ocimum basilicum Linnaeus) is aromatic herb that has been utilized in traditional medicine. To improve the phytochemical constituents and pharmaceutical quality of sweet basil leaves, ultraviolet (UV)-B irradiation at different intensities (2.30, 3.60, and 4.80 W/m²) and durations (4, 6, 8, and 10-h) was applied at the post-harvest stage. Total flavonoid content (TFC) and total phenolic content (TPC) were measured using spectrophotometric method, and individual flavonoids and phenolic acids were identified using ultra-high performance liquid chromatography. As a key enzyme for the metabolism of flavonoids, chalcone synthase (CHS) activity, was measured using a CHS assay. Antioxidant activity and antiproliferative activity of extracts against a breast cancer cell line (MCF-7) were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. UV-B irradiation at an intensity of 3.60 W/m² increased TFC approximately 0.85-fold and also increased quercetin (0.41-fold), catechin (0.85-fold), kaempferol (0.65-fold) rutin (0.68-fold) and luteolin (1.00-fold) content. The highest TPC and individual phenolic acid (gallic acid, cinnamic acid and ferulic acid) was observed in the 3.60 W/m² of UV-B treatment. Cinnamic acid and luteolin were not detected in the control plants, production being induced by UV-B irradiation. Production of these secondary metabolites was also significantly influenced by the duration of UV-B irradiation. Irradiation for 8-h led to higher TFC, TPC and individual flavonoids and phenolic acids than for the other durations (4, 8, and 10-h) except for cinnamic acid, which was detected at higher concentration when irradiated for 6-h. Irradiation for 10-h significantly decreased the secondary metabolite production in sweet basil leaves. CHS activity was induced by UV-B irradiation and highest activity was observed at 3.60 W/m² of UV-B irradiation. UV

  1. Growth analysis of UV-B-irradiated cucumber seedlings as influenced by photosynthetic photon flux source and cultivar

    International Nuclear Information System (INIS)

    Krizek, D.T.; Mirecki, R.M.; Kramer, G.F.

    1994-01-01

    A growth analysis was made of ultraviolet-B (UV-B)-sensitive (Poinsett) and insensitive (Ashley) cultivars of Cucuumis satives L. grown in growth chambers at 600 μmol m −2 s −1 of photosynthetic photon flux (PPF) provided by red- and far-red-deficient metal halide (MH) or blue- and UV-A-deficient high pressure sodium/deluxe f HPS/DX) lamps. Plants were irradiated 6 h daily with 0.2 f-UV-B) or 18.2 C+UV-B) kJ m −2 day −1 of biologically effective UV-B for 8 or 15 days from time of seeding. In general, plants given supplemental UV-B for 15 days showed lower leaf area ratio (LARs, and higher specific leaf mass (SLM) mean relative growth rate (MRGR) and net assimilation rate (NAR) than that of control plants, but they showed no difference in leaf mass ratio (LMR), Plants grown under HPS/DX lamps vs MH lamps showed higher SLM and NAR. lower LAR and LMR. hut no difference in MRGR. LMR was the only growth parameter affected by cultivar: at 15 days, it was slightly greater in Poinsett than in Ashley. There were no interactive effects of UV-B. PPF source or cultivar on any of the growth parameters determined, indicating that the choice of either HPS/DX or MH lamps should not affect growth response to UV-B radiation. This was true even though leaves of UV-B-irradiated plants grown under HPS/DX lamps have been shown to have greater chlorosis than those grown under MH lamps. (author)

  2. Ultraviolet-B irradiation of seeds affects photochemical and reproductive performance of the arid-environment ephemeral Dimorphotheca pluvialis

    International Nuclear Information System (INIS)

    Musil, C.F.

    1994-01-01

    A higher polyphenolic content and thicker sclerenchymatous cylinder in the pericarp of ray than of disc seed morphs (diaspores) of Dimorphotheca pluvialis (L.) Moench (Asteraceae) could limit possible damage to the embryo during long-term seed exposure to solar UV-B radiation. This hypothesis was tested by irradiating sun-dried disc and ray diaspores continuously for 6 weeks with four different doses of biologically effective UV radiation, viz 0.0, 0.2, 9.46 and 11.97 kj m −2 8 hr −1 of visible (> 400 nm), UV-A (320–400 nm), ambient and enhanced UV-B (280–320 nm) radiation, respectively. Total effective UV-B doses approximated those received over an 18-week period following seed dispersal at this species' northerly distribution limit (26° 38′ S), at normal ozone levels (ambient UV-B) and anticipated 20% ozone depletion (enhanced UV-B). Irradiation of diaspores with enhanced UV-B improved germination in both seed morphs. However, disc diaspores exhibited a greater fractional increase in germination than ray diaspores. Disc and ray plants grown from diaspores irradiated with enhanced UV-B exhibited decreased photochemical efficiency (reduced variable to maximal fluorescence, F v /F m ), but only disc plants showed decreased potential photosynthetic activity (reduced areas over fluorescence curves, A fd ). This was accompanied by increased diaspore production and reduced diaspore mass. Irradiation of diaspores with photoreactivating UV-A produced a contrasting response (increased F v /F m and A fd , accompanied by decreased diaspore production) in disc plants only. Altered photochemical and reproductive performance, and visibly diminished leaf pigmentation, in disc plants indicated increased sensitivity to photoinhibition, a possible consequence of UV-B induced cellular (membrane and DNA) damage in seeds. (author)

  3. Cold activity and tolerance of the entomopathogenic fungus Tolypocladium spp. to UV-B irradiation and heat.

    Science.gov (United States)

    Santos, Maiara P; Dias, Luciana P; Ferreira, Paulo C; Pasin, Liliana A A P; Rangel, Drauzio E N

    2011-11-01

    Studies on the stress resistance of insect-pathogenic fungi are very important to better understand the survival of these organisms in the environment. In this study, we examined the cold activity (8 ± 1°C for 7 days), UV-B tolerance (Quaite-weighted UV-B irradiance at 847.90 mW m(-2) for 1, 2, 3, and 4 h), and wet-heat tolerance (45°C for 1, 2, 3, and 4 h) of two isolates of Tolypocladiumcylindrosporum (ARSEF 3392 and 5558), one isolate of Tolypocladium geodes (ARSEF 3275), and two isolates of Tolypocladium inflatum (ARSEF 4772 and 4877) based on their germination, compared with Metarhizium robertsii (ARSEF 2575). After 3 h of UV-B exposure, T. cylindrosporum germinated at a greater rate than the other Tolypocladium species and had similar viability to that of the M. robertsii. Most Tolypocladium isolates, however, were less UV-B tolerant than M. robertsii. The T.cylindrosporum isolates were also the most thermotolerant, with similar tolerance to the M. robertsii. The isolates of T. inflatum and T. geodes, which had similar heat tolerance, were the least heat tolerant compared with the isolates of T. cylindrosporum and M. robertsii. After 4h of heat exposure, the germination of T. inflatum and T. geodes isolates was not significantly different. For cold activity, both T.cylindrosporum isolates germinated to ca. 100% in only 3 days. Approximately 50% of the two T. inflatum isolates germinated, and less than 5% of T. geodes germinated after 3 days. All fungal isolates, however, completely germinated by the seventh day, except M.robertsii. The isolates of T. cylindrosporum, therefore, were the most heat and UV-B tolerant, and had the highest cold activity compared to the other species. The tolerance of M. robertsii to UV-B radiation and heat was similar to that of T.cylindrosporum. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity.

    Directory of Open Access Journals (Sweden)

    Alvar Grönberg

    Full Text Available BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS. We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+, completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.

  5. Filopodia are conduits for melanosome transfer to keratinocytes.

    Science.gov (United States)

    Scott, Glynis; Leopardi, Sonya; Printup, Stacey; Madden, Brian C

    2002-04-01

    Melanosomes are specialized melanin-synthesizing organelles critical for photoprotection in the skin. Melanosome transfer to keratinocytes, which involves whole organelle donation to another cell, is a unique biological process and is poorly understood. Time-lapse digital movies and electron microscopy show that filopodia from melanocyte dendrites serve as conduits for melanosome transfer to keratinocytes. Cdc42, a small GTP-binding protein, is known to mediate filopodia formation. Melanosome-enriched fractions isolated from human melanocytes expressed the Cdc42 effector proteins PAK1 and N-WASP by western blotting. Expression of constitutively active Cdc42 (Cdc42(V12)) in melanocytes co-cultured with keratinocytes induced a highly dendritic phenotype with extensive contacts between melanocytes and keratinocytes through filopodia, many of which contained melanosomes. These results suggest a unique role for filopodia in organelle transport and, in combination with our previous work showing the presence of SNARE proteins and rab3a on melanosomes, suggest a novel model system for melanosome transfer to keratinocytes.

  6. Induction of differentiation in psoriatic keratinocytes by propylthiouracil and fructose

    Directory of Open Access Journals (Sweden)

    Santhosh Arul

    2016-12-01

    Full Text Available Psoriasis is characterized by uncontrolled proliferation and poor differentiation. Sirtuin1 (SIRT1 a class III deacetylase, crucial for differentiation in normal keratinocytes, is reduced in psoriasis. Down regulated SIRT1 levels may contribute to poor differentiation in psoriasis. In addition, the levels of early differentiation factors Keratin1 (K1 and Keratin10 (K10 are depleted in psoriasis. We attempted to study a possible effect of fructose, a SIRT1 upregulator and Propylthiouracil (PTU to augment differentiation in psoriatic keratinocytes. Keratinocytes were cultured from lesional biopsies obtained from psoriatic patients and control cells were obtained from patients undergoing abdominoplasty. Cells were treated with fructose and PTU individually. K1 and K10 transcript levels were measured to evaluate early differentiation; SIRT1 protein expression was also studied to decipher its role in the mechanism of differentiation. The K1, K10 transcript levels, SIRT1 protein and transcript levels in fructose treated psoriatic keratinocytes were improved. This suggests keratinocyte differentiation was induced by fructose through SIRT1 upregulation. Whereas PTU induced differentiation, as confirmed by improved K1, K10 transcript levels followed a non-SIRT1 mechanism. We conclude that the use of fructose and PTU may be an adjunct to the existing therapies for psoriasis.

  7. Histamine effect on melanocyte proliferation and vitiliginous keratinocyte survival.

    Science.gov (United States)

    Kim, Nan-Hyung; Lee, Ai-Young

    2010-12-01

    Repigmention of vitiligo requires melanocyte proliferation and migration. Keratinocytes have been shown to play a role in this process. Data from this laboratory showed that bee venom (BV) stimulated melanocyte proliferation and migration as well as melanogenesis. As histamine release is associated with BV, its effect on melanocyte proliferation and migration was examined. Cultured normal human melanocytes treated with histamine were studied with and without receptor-specific antagonists or agonists. The effect of histamine on vitiliginous keratinocytes, in cultured cells treated with a PI3K inhibitor in the presence of TNF-α, was also examined. Histamine exerted a more significant effect on melanocyte proliferation than on melanogenesis. This occurred through the H2 receptor with complex signalling to ERK, CREB, and Akt activation, which stimulated melanocyte migration. Histamine and the H2 receptor agonist also increased survival of vitiliginous, but not normal, keratinocytes, with NF-κB activation. Because expression levels of the H2 receptor was significantly decreased in depigmented compared to normally pigmented epidermis, in patients with vitiligo, histamine may increase the survival of vitiliginous keratinocytes. Overall, histamine stimulated the proliferation and migration of melanocytes and the vitiliginous keratinocyte survival, providing the basis for novel therapeutic approaches to vitiligo repigmentation. © 2010 John Wiley & Sons A/S.

  8. Amarogentin Displays Immunomodulatory Effects in Human Mast Cells and Keratinocytes

    Directory of Open Access Journals (Sweden)

    Ute Wölfle

    2015-01-01

    Full Text Available Keratinocytes express the bitter taste receptors TAS2R1 and TAS2R38. Amarogentin as an agonist for TAS2R1 and other TAS2Rs promotes keratinocyte differentiation. Similarly, mast cells are known to express bitter taste receptors. The aim of this study was to assess whether bitter compounds display immunomodulatory effects on these immunocompetent cells in the skin, so that they might be a target in chronic inflammatory diseases such as atopic dermatitis and psoriasis. Here, we investigated the impact of amarogentin on substance P-induced release of histamine and TNF-α from the human mast cell line LAD-2. Furthermore, the effect of amarogentin on HaCaT keratinocytes costimulated with TNF-α and histamine was investigated. Amarogentin inhibited in LAD-2 cells substance P-induced production of newly synthesized TNF-α, but the degranulation and release of stored histamine were not affected. In HaCaT keratinocytes histamine and TNF-α induced IL-8 and MMP-1 expression was reduced by amarogentin to a similar extent as with azelastine. In conclusion amarogentin displays immunomodulatory effects in the skin by interacting with mast cells and keratinocytes.

  9. Photodynamic detection in visualisation of cutaneous and oral mucosa premalignant and malignant lesions: two clinical cases

    Science.gov (United States)

    Jurczyszyn, Kamil; Ziólkowski, Piotr; Osiecka, Beata; Gerber, Hanna; Dziedzic, Magdalena

    2008-11-01

    Photodynamic diagnosis (PDD) is promising method of visualisation of premalignant and malignant lesions. PDD is consisted of two main agents: special chemical compound which is called photosensitizer and light. Photosensitizer has affinity to fast proliferating cells such as pre- or malignant. During light irradiation (with proper wavelength - corresponding to absorption peak of photosensitizer) photosensitizer gains energy and passes into excited singlet state S1. Returning to basic singlet state Sn, leads to fluorescence. Due to difference between concentration of photosensitizer in lesion and normal tissue it is possible to obtain high contrast image of lesion. Case #1: 53 years old woman with basal cell carcinoma (BCC) in nasal region; 20% delta-aminolevulinic acid as a precursor of photosensitizer on eucerin base was used. Case #2: 57 years old woman with multifocal oral leukoplakia on cheek mucosa and tongue; 2% chlorophyll gel as photosesitizer was used. All photographs were taken in white light without any filter and in blue and UV light with orange filter: in both cases the total area of the lesions appeared to be larger than it has been clinically observed. Thus, the PDD might be helpful in evaluation of margins of surgical excision of such lesions.

  10. Glucose transporter-1 (GLUT-1) immunoreactivity in benign, premalignant and malignant lesions of the gallbladder.

    Science.gov (United States)

    Legan, Mateja; Tevžič, Spela; Tolar, Ana; Luzar, Boštjan; Marolt, Vera Ferlan

    2011-03-01

    GLUT-1 is a transmembrane glucose transport protein that allows the facilitated transport of glucose into cells, normally expressed in tissues which depend mainly on glucose metabolism. Enhanced expression of GLUT-1 can also be found in a large spectrum of carcinomas. This study aimed to investigate GLUT-1 expression in gallbladder tissue: from normal tissue samples, hyperplasias, low-grade and high-grade dysplasias to gallbladder carcinomas. In all, 115 archived samples of gallbladder tissue from 68 patients, presented after cholecystectomy, were immunohistochemically stained for GLUT-1. According to the intensity of GLUT-1 immunoreactivity, samples were divided into negative (stained 0-10% of cells stained), positive with weak to moderate (10-50%) and positive with strong (>50%) GLUT-1 expression. The GLUT-1 immunoreactivity of the samples showed a characteristic increase from premalignant lesions to carcinomas. Normal gallbladder tissue samples did not express GLUT-1 (100%). Weak expression was shown only focally in hyperplasias, but to a greater extent with low-grade dysplasias (20%), high-grade dysplasias (40%) and carcinomas (51.8%). Normal gallbladder tissue is GLUT-1 negative. GLUT-1 expression in carcinoma tissue is significantly higher than in dysplastic lesions. Strong GLUT-1 expression indicates 100% specificity for detecting gallbladder carcinomas. Therefore, GLUT-1 is a candidate as a diagnostic as well as a tissue prognostic marker in gallbladder carcinoma patients.

  11. Transition of Immunohistochemical Expression of E-Cadherin and Vimentin from Premalignant to Malignant Lesions of Oral Cavity and Oropharynx

    Directory of Open Access Journals (Sweden)

    Kafil Akhtar

    2016-05-01

    Full Text Available Objectives: We sought to study the expression of epithelial-to-mesenchymal transition markers E-cadherin and vimentin in precancerous lesions of the oral cavity and oropharynx and to use the specific pattern of expression to predict invasiveness. Methods: This cross-sectional study looked at 87 cases of oral and oropharyngeal lesions obtained between December 2012 and November 2014 in the Department of Pathology, Jawaharlal Nehru Medical College, Aligarh Muslim University, India. Fifty-three biopsies from the buccal mucosa, tongue, and pharynx and 34 resected oral specimens were evaluated for premalignant and malignant lesions using hematoxylin and eosin and immunohistochemical stains. Immunohistochemical expression of epithelial marker E-cadherin and mesenchymal marker vimentin was evaluated wherever possible. Slides were examined for staining pattern (cytoplasmic or membrane, proportion, and intensity of staining of tumor cells. Patients follow-up and therapy related changes were also studied. Results: There were 64 premalignant and 23 malignant cases in our study with 65 (74.7% cases seen in males and 22 (25.3% cases seen in females. The majority of malignant cases, (n = 15; 64.2% were seen in the fifth and sixth decades of life while most of the premalignant lesions (n = 36; 56.4% were seen in the fourth and fifth decade. Amongst the 64 premalignant oral lesions, leukoplakia comprised of 14 cases (21.9%, of which three cases had associated mild to moderate dysplasia. The majority of premalignant lesions showed strong E-cadherin expression and decreased expression of vimentin with negative and weak expression in both dysplasias and carcinoma in situ (p = 0.013. E-cadherin expression was significantly reduced in invasive carcinomas compared to dysplasias and carcinoma in situ and the difference in immunoreactivity was statistically significant (p < 0.050. Vimentin expression increased as the tumor progressed from dysplasias to carcinoma in

  12. Micronucleus formation in cultured human keratinocytes: Involvement of intercellular bioactivation.

    Science.gov (United States)

    van Pelt, F N; Haring, R M; Weterings, P J

    1991-01-01

    Micronucleus formation in cultured human keratinocytes was studied after exposure to benzo[a]pyrene, cyclophosphamide and 12-O-tetradecanoylphorbol-13-acetate without the addition of an exogenous metabolizing system. The first two agents need bioactivation by specific isoenzymes of cytochrome P-450 to form genotoxic intermediates. Benzo[a]pyrene induced the micronucleus formation in both uninduced and Aroclor 1254-pretreated cultures. Clastogenic effects of cyclophosphamide were observed only in Aroclor 1254-pretreated cells. The tumour promotor 12-O-tetradecanoylphorbol-13-acetate did not affect the frequency of micronuclei in human keratinocytes. The data indicate that cultured human keratinocytes can be used to study the tissue-specific response to genotoxic agents as well as interindividual variation in biotransformation capacity.

  13. Migration patterns of dendritic cells in the rat: comparison of the effects of gamma and UV-B irradiation on the migration of dendritic cells and lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Oluwole, S.F.; Engelstad, K.; De Rosa, C.; Wang, T.S.; Fawwaz, R.A.; Reemtsma, K.; Hardy, M.A. (Columbia Univ., New York, NY (USA))

    1991-04-01

    To further define the underlying mechanisms of immune suppression induced by UV-B irradiation, we have examined the kinetics of homing patterns of in vitro UV-B-irradiated and gamma-irradiated-thoracic duct lymphocytes (TDL) compared to dendritic cells (DC). Our findings show that {sup 111}In-oxine-labeled TDL specifically home to the spleen, liver, lymph nodes, and bone marrow with subsequent recirculation of a large number of cells from the spleen to lymph nodes. In contrast, DC preferentially migrate to the spleen and liver with a relatively insignificant distribution to lymph nodes and an absence of subsequent recirculation. Splenectomy prior to cell injection significantly diverts the spleen-seeking DC to the liver but not to the lymph nodes, while the homing of TDL to lymph nodes is significantly increased. In vitro exposure of 111In-oxine labeled TDL to gamma irradiation does not significantly impair immediate homing to lymphoid tissues but inhibits cell recirculation between 3 and 24 hr. In contrast, gamma irradiation does not affect the tissue distribution of labeled DC, suggesting that DC are more radioresistant to gamma irradiation than TDL. Unlike the findings in animals injected with gamma-irradiated cells, UV-B irradiation virtually abolished the homing of TDL to lymph nodes and significantly reduced the homing of the spleen-seeking DC to the splenic compartment while a large number of cells were sequestered in the liver. The results of in vitro cell binding assay show that TDL, unlike DC, have the capacity to bind to high endothelial venules (HEV) within lymph node frozen sections while gamma and UV-B irradiation significantly inhibit the binding of TDL to lymph node HEV.

  14. Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes

    Directory of Open Access Journals (Sweden)

    Lee Suk-Keun

    2007-09-01

    Full Text Available Abstract Background Interleukin-8 (IL-8 is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK and malignant human oral keratinocytes (HN12 cells with deferoxamine (DFO. Methods IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. Electrophoretic mobility shift assays was used to determine NF-κB binding activity. Phosphorylation and degradation of the I-κB were analyized by Western blot. Results IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1β to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IκB, and activation of NF-κB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione, and by pyrrolidine dithiocarbamate, an inhibitor of NF-κB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(PH oxidase. Conclusion This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions.

  15. Intermittent pressure decreases human keratinocyte proliferation in vitro.

    Science.gov (United States)

    Nasca, Maria R; Shih, Alan T; West, Dennis P; Martinez, Wanda M; Micali, Giuseppe; Landsman, Adam S

    2007-01-01

    The aim of this study was to investigate the correlation between pressure changes and keratinocyte proliferation by determining whether keratinocytes exposed to altered mechanical pressures would proliferate at different rates compared to control cells not subjected to pressure changes. Tissue culture flasks of human keratinocytes plated at an approximate density of 15,000 cells/cm(2) undergoing an intermittent cyclic pressure of 362 mm Hg at a frequency of 2.28 or 5.16 cycles/min (0.038 or 0.086 Hz) for 8 h were compared to control flasks grown at ambient room pressure. An in-line pressure transducer was used to monitor and adjust pressure within the cell chambers, using a solenoid valve. A thymidine incorporation assay assessed the amount of cell proliferation in each set of experiments. Differences in proliferation between keratinocytes subjected to cyclic pressure changes and control cells were found to be statistically significant (p < 0.05) in 4 out of 5 proliferation assays. Also, a higher frequency of pressure changes consistently generated a reduced proliferation rate compared to that seen in cells exposed to a lower frequency of pressure changes. These data indicate that keratinocytes undergoing intermittent pressure changes exhibit decreased proliferation rates compared to controls. Furthermore, an increased frequency rate seems to have a greater effect on proliferation than low-frequency rate pressure changes, suggesting that the stress caused by frequently changed pressure may play a greater role in reducing keratinocyte proliferation than the actual magnitude of load applied to the cells. Our results support the current treatment protocol of reducing speed and duration of walking on the site of the wound to promote healing of foot ulcers. (c) 2007 S. Karger AG, Basel.

  16. The caspase-1 inhibitor CARD18 is specifically expressed during late differentiation of keratinocytes and its expression is lost in lichen planus.

    Science.gov (United States)

    Qin, Haihong; Jin, Jiang; Fischer, Heinz; Mildner, Michael; Gschwandtner, Maria; Mlitz, Veronika; Eckhart, Leopold; Tschachler, Erwin

    2017-08-01

    CARD18 contains a caspase recruitment domain (CARD) via which it binds to caspase-1 and thereby inhibits caspase-1-mediated activation of the pro-inflammatory cytokine interleukin (IL)-1β. To determine the expression profile and the role of CARD18 during differentiation of keratinocytes and to compare the expression of CARD18 in normal skin and in inflammatory skin diseases. Human keratinocytes were induced to differentiate in monolayer and in 3D skin equivalent cultures. In some experiments, CARD18-specific siRNAs were used to knock down expression of CARD18. CARD18 mRNA levels were determined by quantitative real-time PCR, and CARD18 protein was detected by Western blot and immunofluorescence analyses. In situ expression was analyzed in skin biopsies obtained from healthy donors and patients with psoriasis and lichen planus. CARD18 mRNA was expressed in the epidermis at more than 100-fold higher levels than in any other human tissue. Within the epidermis, CARD18 was specifically expressed in the granular layer. In vitro CARD18 was strongly upregulated at both mRNA and protein levels in keratinocytes undergoing terminal differentiation. In skin equivalent cultures the expression of CARD18 was efficiently suppressed by siRNAs without impairing stratum corneum formation. Epidermal expression of CARD18 was increased after ultraviolet (UV)B irradiation of skin explants. In skin biopsies of patients with psoriasis no consistent regulation of CARD18 expression was observed, however, in lesional epidermis of patients with lichen planus, CARD18 expression was either greatly diminished or entirely absent whereas in non-lesional areas expression was comparable to normal skin. Our results identify CARD18 as a differentiation-associated keratinocyte protein that is altered in abundance by UV stress. Its downregulation in lichen planus indicates a potential role in inflammatory reactions of the epidermis in this disease. Copyright © 2017 Japanese Society for Investigative

  17. Novel approach in the management of an oral premalignant condition - A case report

    Directory of Open Access Journals (Sweden)

    Sankaranarayanan S

    2007-01-01

    Full Text Available Oral submucous fibrosis is a progressive oral disease first described by Pindborg and Sirsat 3 decades ago. It is a premalignant condition. The signs and symptoms depend on the involvement of the different sites in the oral cavity. The patient feels burning sensation for normal diet and trismus which may be so severe. If not properly treated the risk of malignant change in advanced cases of OSMF is relatively high. Wide ranges of treatment such as medical management, surgical therapy and physiotherapy have been attempted in the past, but none of them has proved to be a cure for this chronic fibrotic disease.Histopathologically as the disease progresses, (i change in the morphology of collagen, (ii increased accumulation of amorphous collagen, and (iii decreased collagen degradation results in decrease in number of blood vessels are observed in the affected area compared to the normal area. With an aim of bringing more blood supply to the affected area which is expected to bring ?more nutrients and help in collagen degradation, earlier application of vasodilators and studies with curcumin have been done, but still with - no significant outcome.As an alternative approach to improve the blood circulation, we have tried Autologous bone marrow stem cells which have been earlier applied in several diseases such as ischemic peripheral vascular diseases, ischemic heart diseases etc with proven improvement in angiogenesis. A 38 year old patient with oral submucosal fibrosis, proven by histopathology, and endothelial markers was injected 175 million BMMNCs into the area affected. The paramaters such as blanching, fibrous band have significantly improved, 4 weeks after the injection. We could observe positive changes clinically to prove the improvement. The mouth opening has improved to 35 mm from the previous 30.0 mm. Further histopathology and SEM studies with larger samples are done for establishing stem cell therapy’s safety and efficacy.

  18. Optimized endoscopic autofluorescence spectroscopy for the identification of premalignant lesions in Barrett's oesophagus.

    Science.gov (United States)

    Holz, Jasmin A; Boerwinkel, David F; Meijer, Sybren L; Visser, Mike; van Leeuwen, Ton G; Aalders, Maurice C G; Bergman, Jacques J G H M

    2013-12-01

    Fluorescence spectroscopy has the potential to detect early cellular changes in Barrett's oesophagus before these become visible. As the technique is based on varying concentrations of intrinsic fluorophores, each with its own optimal excitation wavelength, it is important to assess the optimal excitation wavelength(s) for identification of premalignant lesions in patients with Barrett's oesophagus. The endoscopic spectroscopy system used contained five (ultra)violet light sources (λexc=369-416 nm) to generate autofluorescence during routine endoscopic surveillance. Autofluorescence spectroscopy was followed by a biopsy for histological assessment and spectra correlation. Three intensity ratios (r1, r2, r3) were calculated by dividing the area, A, under the spectral curve of selected emission wavelength ranges for each spectrum generated by each excitation wavelength λexc as follows (Equation is included in full-text article.). Double intensity ratios were calculated using two excitation wavelengths. Fifty-eight tissue areas from 22 patients were used for autofluorescence spectra analysis. Excitation with 395, 405 or 410 nm showed a significant (P≤0.0006) differentiation between intestinal metaplasia and grouped high-grade dysplasia/early carcinoma for intensity ratios r2 and r3. A sensitivity of 80.0% and specificity of 89.5% with an area under the ROC curve of 0.85 was achieved using 395 nm excitation and intensity ratio r3. Double excitation showed no additional value over single excitation. The combination of 395 nm excitation and intensity ratio r3 showed optimal conditions to discriminate nondysplastic from early neoplasia in Barrett's oesophagus.

  19. Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies

    Directory of Open Access Journals (Sweden)

    Karin Hellner

    2016-08-01

    Full Text Available Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC. The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE cells. We identified frequent mutations involving a 40 kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p < 2−16, which was not found in patients without cancer (n = 108. Importantly, we show that SOX2 overexpression in FTE is nearly ubiquitous in patients with HGSOCs (n = 100, and common in BRCA1-BRCA2 mutation carriers (n = 71 who underwent prophylactic salpingo-oophorectomy. We propose that the finding of SOX2 overexpression in FTE could be exploited to develop biomarkers for detecting disease at a premalignant stage, which would reduce mortality from this devastating disease.

  20. Nuclear expression of Rac1 in cervical premalignant lesions and cervical cancer cells

    International Nuclear Information System (INIS)

    Mendoza-Catalán, Miguel A; Castañeda-Saucedo, Eduardo; Cristóbal-Mondragón, Gema R; Adame-Gómez, Jesús; Valle-Flores, Heidi N del; Coppe, José Fco; Sierra-López, Laura; Romero-Hernández, Mirna A; Carmen Alarcón-Romero, Luz del; Illades-Aguiar, Berenice

    2012-01-01

    Abnormal expression of Rho-GTPases has been reported in several human cancers. However, the expression of these proteins in cervical cancer has been poorly investigated. In this study we analyzed the expression of the GTPases Rac1, RhoA, Cdc42, and the Rho-GEFs, Tiam1 and beta-Pix, in cervical pre-malignant lesions and cervical cancer cell lines. Protein expression was analyzed by immunochemistry on 102 cervical paraffin-embedded biopsies: 20 without Squamous Intraepithelial Lesions (SIL), 51 Low- grade SIL, and 31 High-grade SIL; and in cervical cancer cell lines C33A and SiHa, and non-tumorigenic HaCat cells. Nuclear localization of Rac1 in HaCat, C33A and SiHa cells was assessed by cellular fractionation and Western blotting, in the presence or not of a chemical Rac1 inhibitor (NSC23766). Immunoreacivity for Rac1, RhoA, Tiam1 and beta-Pix was stronger in L-SIL and H-SIL, compared to samples without SIL, and it was significantly associated with the histological diagnosis. Nuclear expression of Rac1 was observed in 52.9% L-SIL and 48.4% H-SIL, but not in samples without SIL. Rac1 was found in the nucleus of C33A and SiHa cells but not in HaCat cells. Chemical inhibition of Rac1 resulted in reduced cell proliferation in HaCat, C33A and SiHa cells. Rac1 is expressed in the nucleus of epithelial cells in SILs and cervical cancer cell lines, and chemical inhibition of Rac1 reduces cellular proliferation. Further studies are needed to better understand the role of Rho-GTPases in cervical cancer progression

  1. Classification of premalignant pancreatic cancer mass-spectrometry data using decision tree ensembles

    Directory of Open Access Journals (Sweden)

    Wong G William

    2008-06-01

    Full Text Available Abstract Background Pancreatic cancer is the fourth leading cause of cancer death in the United States. Consequently, identification of clinically relevant biomarkers for the early detection of this cancer type is urgently needed. In recent years, proteomics profiling techniques combined with various data analysis methods have been successfully used to gain critical insights into processes and mechanisms underlying pathologic conditions, particularly as they relate to cancer. However, the high dimensionality of proteomics data combined with their relatively small sample sizes poses a significant challenge to current data mining methodology where many of the standard methods cannot be applied directly. Here, we propose a novel methodological framework using machine learning method, in which decision tree based classifier ensembles coupled with feature selection methods, is applied to proteomics data generated from premalignant pancreatic cancer. Results This study explores the utility of three different feature selection schemas (Student t test, Wilcoxon rank sum test and genetic algorithm to reduce the high dimensionality of a pancreatic cancer proteomic dataset. Using the top features selected from each method, we compared the prediction performances of a single decision tree algorithm C4.5 with six different decision-tree based classifier ensembles (Random forest, Stacked generalization, Bagging, Adaboost, Logitboost and Multiboost. We show that ensemble classifiers always outperform single decision tree classifier in having greater accuracies and smaller prediction errors when applied to a pancreatic cancer proteomics dataset. Conclusion In our cross validation framework, classifier ensembles generally have better classification accuracies compared to that of a single decision tree when applied to a pancreatic cancer proteomic dataset, thus suggesting its utility in future proteomics data analysis. Additionally, the use of feature selection

  2. Hyaluronan minimizes effects of UV irradiation on human keratinocytes

    Czech Academy of Sciences Publication Activity Database

    Hašová, M.; Crhák, Tomáš; Šafaříková, Barbora; Dvořáková, J.; Muthný, T.; Velebný, V.; Kubala, Lukáš

    2011-01-01

    Roč. 303, č. 4 (2011), s. 277-284 ISSN 0340-3696 R&D Projects: GA ČR(CZ) GA305/08/1704 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : hyaluronan * keratinocyte * ultraviolet light Subject RIV: BO - Biophysics Impact factor: 2.279, year: 2011

  3. N-acetyltransferase in human skin and keratinocytes

    NARCIS (Netherlands)

    Vogel, Tanja; Bonifas, Jutta; Wiegman, Marjon; Pas, Hendrikus; Blömeke, Brunhilde; Coenraads, Pieter Jan; Schuttelaar, Marie-Louise

    Background: N-acetyltransferase 1 (NAT1) mediated Nacetylation in human skin and keratinocytes is an important detoxification pathway for aromatic amines including the strong sensitizer para-phenylenediamine (PPD), an important component of oxidative hair dyes. Objectives: Human skin and

  4. Dental metal-induced innate reactivity in keratinocytes

    NARCIS (Netherlands)

    Rachmawati, D.; Buskermolen, J.K.; Scheper, R.J.; Gibbs, S.; von Blomberg, B.M.E.; van Hoogstraten, I.M.W.

    2015-01-01

    Gold, nickel, copper and mercury, i.e. four metals frequently used in dental applications, were explored for their capacity to induce innate immune activation in keratinocytes (KC). Due to their anatomical location the latter epithelial cells are key in primary local irritative responses of skin and

  5. Is it photosensitization or photodegradation when UV-B irradiation is combined with BDE-47? Evidence from the growth and reproduction changes of rotifer Brachionus plicatilis.

    Science.gov (United States)

    Liu, Chunchen; Tang, Xuexi; Zhou, Bin; Jiang, Yongshun; Lv, Mengchen; Zang, Yu; Wang, You

    2018-07-01

    Ecotoxicological methods were applied in the present study, and the marine rotifer Brachionus plicatilis was used as the toxic endpoint to depict what occurred when 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) was combined with solar ultraviolet-B radiation (UV-B). B. plicatilis was exposed to three different combination methods of BDE-47 and UV-B at an equal toxicity ratio, including normal rotifer co-cultured with UV-B-irradiated BDE-47 (known as Method I), UV-B-irradiated rotifer co-cultured with BDE-47 exposure (known as Method II) and normal rotifer co-cultured with the simultaneous addition of BDE-47 and UV-B irradiation (known as Method III). Acute and chronic experiments were preformed to determine the toxicity differentiation according to the growth and reproduction changes in the rotifer. Twenty-four-hour acute experiments showed that the modes of three combined methods changed from antagonism to additive, to synergistic with the concentration/dose increment, and the contribution rates of Method I and Method II to Method III were calculated by approximately 40.4% and 59.6%, respectively. Chronic exposure to either the single stressor or the combination of stressors inhibited the growth and reproduction of the rotifer, demonstrating the inhibition of the population growth rate and the decrease in the larvae production. Three combined groups presented more serious damages compared to groups with single stress exposure, and the ascending sequence of toxicity was Method IB was mainly due to photosensitization and photodegradation, and the photosensitization might be more noxious to the growth and reproduction of the rotifer. Copyright © 2018. Published by Elsevier B.V.

  6. Effect of Mild Water Stress and Enhanced Ultraviolet-B Irradiation on Leaf Growth of Rumex obtusifolius L. and Rumex patientia L. (Polygonaceae).

    OpenAIRE

    Holman, Steven R.

    1981-01-01

    Leaves of Rumex obtusifolius L. and R. patientia L.were exposed to combinations of mild water stress and enhanced ultraviolet-B irradiation during their ontogeny. Two UV-B treatments (enhanced UV-B and control) and three water stress treatments (-0.0 MPa, -0.2 MPa and -0.4 MPa rooting medium matric potentials) were employed. The impact of the stress interaction was assessed on the basis of changes in leaf area, average adaxial epidermal cell size, and total number of adaxial epidermal cells p...

  7. Exploring type II microcalcifications in benign and premalignant breast lesions by shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS)

    Science.gov (United States)

    Liang, Lijia; Zheng, Chao; Zhang, Haipeng; Xu, Shuping; Zhang, Zhe; Hu, Chengxu; Bi, Lirong; Fan, Zhimin; Han, Bing; Xu, Weiqing

    2014-11-01

    The characteristics of type II microcalcifications in fibroadenoma (FB), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) breast tissues has been analyzed by the fingerprint features of Raman spectroscopy. Fresh breast tissues were first handled to frozen sections and then they were measured by normal Raman spectroscopy. Due to inherently low sensitivity of Raman scattering, Au@SiO2 shell-isolated nanoparticle-enhanced Raman spectroscopy (SHINERS) technique was utilized. A total number of 71 Raman spectra and 70 SHINERS spectra were obtained from the microcalcifications in benign and premalignant breast tissues. Principal component analysis (PCA) was used to distinguish the type II microcalcifications between these tissues. This is the first time to detect type II microcalcifications in premalignant (ADH and DCIS) breast tissue frozen sections, and also the first time SHINERS has been utilized for breast cancer detection. Conclusions demonstrated in this paper confirm that SHINERS has great potentials to be applied to the identification of breast lesions as an auxiliary method to mammography in the early diagnosis of breast cancer.

  8. Associated factors with cervical pre-malignant lesions among the married fisher women community at Sadras, Tamil Nadu

    Directory of Open Access Journals (Sweden)

    Sornam Ganesan

    2015-01-01

    Full Text Available Objective: To identify the associated factors of cervical pre-malignant lesions among the married fisher women residing in the coastal areas of Sadras, Tamil Nadu. Methods: The study was conducted in five fishermen communities under Sadras, a coastal area in Tamil Nadu, India. Two hundred and fifty married fisher women residing in the area. Quantitative descriptive approach with a cross-sectional study design was used. Data were collected using a structured interview schedule for identifying the associated factors and Pap smear test was performed for identifying the pre-malignant cervical lesions among the married fisher women. Data were analyzed using descriptive and inferential statistics. Results: Among 250 women, about six (2.4% of them presented with pre-cancerous lesions such as atypical squamous cell of undifferentiated significance (ASCUS - five (2% and mild dysplasia one (0.4%. Majority of the women, about 178 (71.2% women, had abnormal cervical findings. Statistical analysis showed a significant association of risk factors such as advanced age, lack of education, low socioeconomic status, using tobacco, multiparity, premarital sex, extramarital relationship, using cloth as sanitary napkin, etc. Conclusion: The study findings clearly show the increased vulnerable state of the fisher women for acquiring cervical cancer as they had many risk factors contributing to the same.

  9. Vascular endothelial growth factor is up-regulated in the early pre-malignant stage of colorectal tumour progression.

    Science.gov (United States)

    Wong, M P; Cheung, N; Yuen, S T; Leung, S Y; Chung, L P

    1999-06-11

    Angiogenesis is an essential requirement for the development, progression and metastasis of malignant tumours. Studies on transgenic mouse models have shown that angiogenesis begins in the pre-malignant phase of oncogenesis, when dysplastic lesions acquire an increased microvasculature. To investigate the relationship between the expression of vascular endothelial growth factor (VEGF) and colorectal tumour progression, we have studied VEGF expression level and splice variant pattern by semi-quantitative RT-PCR and the cellular source of VEGF expression by in situ hybrization (ISH) in a range of lesions that modelled the tumour-development pathway from normal colon to invasive colorectal adenocarcinomas. Colonic adenomas showed a statistically significant up-regulation of VEGF expression over normal tissues, with a further increase during the development of adenocarcinomas. Tumour cells formed the major source of VEGF expression, with a minor contribution from mononuclear cells in the tumour stroma and enhanced expression in tumour cells around necrotic regions. The comparable expression level in both the in situ and invasive components in the same tumours indicated that a high VEGF expression capacity had been acquired prior to establishment of the invasive phenotype. Our findings support activation of VEGF as the molecular basis for the discrete induction of angiogenesis in the pre-malignant phase of colorectal tumour development.

  10. Astaxanthin induces migration in human skin keratinocytes via Rac1 activation and RhoA inhibition.

    Science.gov (United States)

    Ritto, Dakanda; Tanasawet, Supita; Singkhorn, Sawana; Klaypradit, Wanwimol; Hutamekalin, Pilaiwanwadee; Tipmanee, Varomyalin; Sukketsiri, Wanida

    2017-08-01

    Re-epithelialization has an important role in skin wound healing. Astaxanthin (ASX), a carotenoid found in crustaceans including shrimp, crab, and salmon, has been widely used for skin protection. Therefore, we investigated the effects of ASX on proliferation and migration of human skin keratinocyte cells and explored the mechanism associated with that migration. HaCaT keratinocyte cells were exposed to 0.25-1 µg/mL of ASX. Proliferation of keratinocytes was analyzed by using MTT assays and flow cytometry. Keratinocyte migration was determined by using a scratch wound-healing assay. A mechanism for regulation of migration was explored via immunocytochemistry and western blot analysis. Our results suggest that ASX produces no significant toxicity in human keratinocyte cells. Cell-cycle analysis on ASX-treated keratinocytes demonstrated a significant increase in keratinocyte cell proliferation at the S phase. In addition, ASX increased keratinocyte motility across the wound space in a time-dependent manner. The mechanism by which ASX increased keratinocyte migration was associated with induction of filopodia and formation of lamellipodia, as well as with increased Cdc42 and Rac1 activation and decreased RhoA activation. ASX stimulates the migration of keratinocytes through Cdc42, Rac1 activation and RhoA inhibition. ASX has a positive role in the re-epithelialization of wounds. Our results may encourage further in vivo and clinical study into the development of ASX as a potential agent for wound repair.

  11. Toxicity of silver nanoparticles in monocytes and keratinocytes

    DEFF Research Database (Denmark)

    Orłowski, Piotr; Krzyzowska, Malgorzata; Winnicka, Anna

    2012-01-01

    Silver nanoparticles are of interest to be used as antimicrobial agents in wound dressings and coatings in medical devices, but potential adverse effects have been reported in the literature. The possible local inflammatory response to silver nanoparticles and the role of cell death in determining...... these effects are largely unknown. Effects of the mixture of silver nanoparticles of different sizes were compared in in vitro assays for cytotoxicity, caspase-1 and caspase-9 activity and bax expression. In all tested concentrations, silver nanoparticles were more toxic for RAW 264.7 monocytes than for 291.03C...... keratinocytes and induced significant caspase-1 activity and necrotic cell death. In keratinocytes, more significantly than in macrophages, silver nanoparticles led to increase of caspase-9 activity and apoptosis. These results indicate that effects of silver nanoparticles depend on the type of exposed cells...

  12. Enhancement of the tolerance to oxidative stress in cucumber (Cucumis sativus L.) seedlings by UV-B irradiation: Possible involvement of phenolic compounds and antioxidative enzymes

    International Nuclear Information System (INIS)

    Kondo, N.; Kawashima, M.

    2000-01-01

    L.) seedlings were irradiated or not irradiated with UV-B for several days in environment-controlled growth chambers. The first leaves irradiated with UV-B were retarded in growth but simultaneously acquired a remarkably high tolerance to oxidative stress, as induced by paraquat treatment, compared with the non-irradiated leaves. This enhanced tolerance was observed within 1d after the start of UV-B irradiation and was maintained during the 12 d period of UV-B treatment. The effects of UV-B on several antioxidative enzymes were examined, and activities of superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase, but not of glutathione reductase, were found to be enhanced. However, activation of these enzymes occurred only from 6 d after the start of irradiation. In contrast, accumulation of phenolic compounds was observed within 1d after the start of UV-B irradiation. HPLC analysis of phenolic compounds showed the distinct enhancement of a substance, which may have antioxidative properties in cucumber seedlings irradiated with UV-B. On the basis of these results, we conclude that not only antioxidative enzymes but also other factors in cucumber seedlings irradiated with UV-B, such as phenolic compounds, may participate in the enhanced tolerance to oxidative stress

  13. The burden of keratinocyte cancer: occurrence, multiplicity and predicting risk

    OpenAIRE

    Whiteman, David C; Pandeya, Nirmala; Thompson, Bridie; Subramaniam, Padmini; Dusingize, Jean Claude; Neale, Rachel E; Green, Adele C; Olsen, Catherine M

    2016-01-01

    The costs of diagnosing and treating basal cell carcinomas (BCC) and squamous cell carcinomas (SCC) are the highest of all cancers in Australia. However, information regarding incidence, multiplicity and risk are scarce as keratinocyte cancers (KCs) are not captured by most registries. To address these gaps, we initiated the QSKIN Study in 2010 recruiting 43,794 Queensland residents from a population register (participation 24%). Participants self-completed a baseline survey recording informa...

  14. SIRT1 Promotes Differentiation of Normal Human Keratinocytes

    OpenAIRE

    Blander, Gil; Bhimavarapu, Anupama; Mammone, Thomas; Maes, Daniel; Elliston, Keith; Reich, Christian; Matsui, Mary Steidl; Guarente, Leonard; Loureiro, Joseph Jorge

    2008-01-01

    Sir2 regulates lifespan in model organisms, which has stimulated interest in understanding human Sir2 homolog functions. The human Sir2 gene family comprises seven members (SIRT1–SIRT7). SIRT1, the human ortholog of the yeast Sir2 by closest sequence similarity, is a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase with enzymatic properties indistinguishable from the yeast enzyme. We studied the involvement of SIRT1 in normal human keratinocyte physiology by a transcriptional mi...

  15. Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

    Science.gov (United States)

    Kodet, Ondřej; Lacina, Lukáš; Krejčí, Eliška; Dvořánková, Barbora; Grim, Miloš; Štork, Jiří; Kodetová, Daniela; Vlček, Čestmír; Šáchová, Jana; Kolář, Michal; Strnad, Hynek; Smetana, Karel

    2015-01-05

    Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

  16. Oral keratinocyte immune responses in HIV-associated candidiasis.

    Science.gov (United States)

    Eversole, L R; Reichart, P A; Ficarra, G; Schmidt-Westhausen, A; Romagnoli, P; Pimpinelli, N

    1997-10-01

    Candidiasis is the most commonly encountered opportunistic infection among HIV-positive subjects. The purpose of this study was to assess specific keratinocyte immune parameters in the pseudomembranous and erythematous forms of HIV-associated oral candidiasis. This collaborative study from three centers analyzed 25 HIV-positive and 10 HIV-negative subjects with either pseudomembranous or erythematous candidiasis. Oral biopsy specimens from lesional tissues were procured, and histopathologic features were correlated with immunohistochemical and in situ hybridization investigations for the expression of interleukin 1 alpha, interleukin 8, antimicrobial calprotectin, lymphocyte populations, and Candida antigen. Both pseudomembranous and erythematous candidiasis among HIV-infected subjects showed a mild interface lymphocytic mucositis with the presence of neutrophilic subcorneal abscesses in the latter. Erythematous candidiasis cases that failed to show surface mycelia, did yield positive results for Candida antigens in the parakeratinized layer. The expression of inflammatory chemokines were positive in all groups and calprotectin appeared to serve as a keratinocyte barrier to hyphal penetration. The erythematous form of candidiasis is often devoid of hyphae yet the presence of Candida antigens in the surface epithelium implicates an immune or allergic process. The intactness of chemokines and antimicrobial calprotectin in keratinocytes may explain why disseminated candidiasis is rarely encountered in HIV-infected patients.

  17. Propionibacterium acnes induces autophagy in keratinocytes: involvement of multiple mechanisms.

    Science.gov (United States)

    Megyeri, Klára; Orosz, László; Bolla, Szilvia; Erdei, Lilla; Rázga, Zsolt; Seprényi, György; Urbán, Edit; Szabó, Kornélia; Kemény, Lajos

    2017-11-27

    Propionibacterium acnes is a dominant member of the cutaneous microbiota. Herein, we evaluate the effects of different P. acnes strains and propionic acid on autophagy in keratinocytes. Our results showed that P. acnes strain 889 altered the architecture of the mitochondrial network, elevated the levels of LC3B-II, Beclin-1 and phospho-AMPKα, stimulated autophagic flux, facilitated intracellular redistribution of LC3B, increased average number of autophagosomes per cell, and enhanced development of acidic vesicular organelles in the HPV-KER cell line. Propionic acid increased the level of phospho-AMPKα, enhanced lipidation of LC3B, stimulated autophagic flux, as well as facilitated translocation of LC3B into autophagosomes in HPV-KER cells. P. acnes strains 889, 6609 and heat-killed strain 889 also stimulated autophagosome formation in primary keratinocytes to varying degrees. These results indicate that cell wall components and secreted propionic acid metabolite of P. acnes evoke mitochondrial damage successively, thereby trigger AMPK-associated activation of autophagy, which in turn facilitates the removal of dysfunctional mitochondria and promotes survival of keratinocytes. Thus, we suggest that low-level colonization of hair follicles with non-invasive P. acnes strains, by triggering a local increase in autophagic activity, might exert a profound effect on several physiological processes responsible for the maintenance of skin tissue homeostasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Response of human epidermal keratinocytes to UV light

    International Nuclear Information System (INIS)

    Kartasova, A.A.

    1987-01-01

    This thesis presents a study on the response of human epidermal keratinocytes to UV light as well as to other agents like 4-NQO and TPA. The effects of ultraviolet (UV) light on the protein synthesis in cultured keratinocytes are presented in ch. III. The next chapter describes the construction of a cDNA library using mRNA isolated from UV irradiated kernatinocytes. This library was differentially screened with cDNA probes synthesized on mRNA from either UV irradiated or nonirradiated cells. Several groups of cDNA clones corresponding to transcripts whose level in the cytoplasm seem to be affected by exposure to UV light have been isolated and characterized by cross-hybridization, sequencing and Northern blot analysis. More detailed analysis of some of the cDNA clones is presented in the two chapters following ch. IV. The complete cDNA sequence of the proteinase inhibitor cystatin A and the modulation of its expression by UV light and the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in keratinocytes are described in ch. V. Two other groups of cDNA clones have been isolated which do not cross-hybridize with each other on Southern blots. However, the primary structures of the proteins deduced from the nucleotide sequences of these two groups of cDNA clones are very similar. 212 refs.; 33 figs.; 2 tabs

  19. High molecular weight plant heteropolysaccharides stimulate fibroblasts but inhibit keratinocytes.

    Science.gov (United States)

    Shahbuddin, Munira; Shahbuddin, Dahlia; Bullock, Anthony J; Ibrahim, Halijah; Rimmer, Stephen; MacNeil, Sheila

    2013-06-28

    Konjac glucomannan (KGM) is a natural polysaccharide of β(1-4)-D-glucomannopyranosyl backbone of D-mannose and D-glucose derived from the tuber of Amorphophallus konjac C. Koch. KGM has been reported to have a wide range of activities including wound healing. In this study we examined KGM extracts prepared from five plant species, (Amorphophallus konjac Koch, Amorphophallus oncophyllus, Amorphophallus prainii, Amorphophallus paeoniifolius and Amorphophallus elegans) for their effects on cultured human keratinocytes and fibroblasts. Extracts from A. konjac Koch, A. oncophyllus and A. prainii (but not from A. paeoniifolius or A. elegans) stimulated fibroblast proliferation both in the absence and presence of serum. However, these materials inhibited keratinocyte proliferation. The fibroblast stimulatory activity was associated with high molecular weight fractions of KGM and was lost following ethanol extraction or enzyme digestion with β-mannanase. It was also reduced by the addition of concanavalin A but not mannose suggesting that these heteropolysaccharides are acting on lectins but not via receptors specific to mannose. The most dramatic effect of KGM was seen in its ability to support fibroblasts for 3weeks under conditions of deliberate media starvation. This effect did not extend to supporting keratinocytes under conditions of media starvation but KGM did significantly help support adipose derived stem cells under media starvation conditions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Methotrexate treatment provokes apoptosis of proliferating keratinocyte in psoriasis patients.

    Science.gov (United States)

    Elango, Tamilselvi; Thirupathi, Anand; Subramanian, Swapna; Ethiraj, Purushoth; Dayalan, Haripriya; Gnanaraj, Pushpa

    2017-08-01

    Psoriasis is a chronic inflammatory skin disease characterized by hyper proliferation of keratinocytes. Recent data show that the epidermis thickening in psoriasis may be related to imbalance of homeostasis caused by abnormal apoptotic process. Maintenance of keratinocyte apoptotic process is very important in psoriasis. Methotrexate (MTX) has been used for many years to restore the normal skin in psoriasis condition. However, the exact mechanism of MTX in psoriasis condition is poorly understood. The aim of this study was to examine the role of MTX on keratinocyte apoptosis pathway in psoriasis patients. A total of 58 psoriasis vulgaris patients were recruited for this study. Nonlesional skin biopsies served as control. Skin biopsies of psoriatic patients were collected and analyzed for cytosolic, mitochondria and total cytochrome c by ELISA. Expression of caspase-9, NFκBp65, pAkt1 by western blot, real-time PCR and immunohistochemical analysis of c-FLIP protein was analyzed in nonlesional and lesional skin biopsies before (day 0) and after (at the end of 6 and 12 weeks) MTX treatment. After MTX treatment, a significant increase in cytochrome c was observed when compared with before MTX treatment in psoriasis patients (p psoriasis by controlling the acanthosis.

  1. Keratinocytes at the uppermost layer of epidermis might act as sensors of atmospheric pressure change.

    Science.gov (United States)

    Denda, Mitsuhiro

    2016-01-01

    It has long been suggested that climate, especially atmospheric pressure change, can cause health problems ranging from migraine to myocardial infarction. Here, I hypothesize that the sensory system of epidermal keratinocytes mediates the influence of atmospheric pressure change on the human physiological condition. We previously demonstrated that even subtle changes of atmospheric pressure (5-20 hPa) induce elevation of intracellular calcium level in cultured human keratinocytes (excitation of keratinocytes). It is also established that communication occurs between epidermal keratinocytes and peripheral nerve systems. Moreover, various neurotransmitters and hormones that influence multiple systems (nervous, cardiovascular, endocrine, and immune systems) are generated and released from epidermal keratinocytes in response to various external stimuli. Thus, I suggest that pathophysiological phenomena induced by atmospheric pressure changes might be triggered by epidermal keratinocytes.

  2. Enhanced Keratinocyte Proliferation and Migration in Co-culture with Fibroblasts

    Science.gov (United States)

    Wang, Zhenxiang; Wang, Ying; Farhangfar, Farhang; Zimmer, Monica; Zhang, Yongxin

    2012-01-01

    Wound healing is primarily controlled by the proliferation and migration of keratinocytes and fibroblasts as well as the complex interactions between these two cell types. To investigate the interactions between keratinocytes and fibroblasts and the effects of direct cell-to-cell contact on the proliferation and migration of keratinocytes, keratinocytes and fibroblasts were stained with different fluorescence dyes and co-cultured with or without transwells. During the early stage (first 5 days) of the culture, the keratinocytes in contact with fibroblasts proliferated significantly faster than those not in contact with fibroblasts, but in the late stage (11th to 15th day), keratinocyte growth slowed down in all cultures unless EGF was added. In addition, keratinocyte migration was enhanced in co-cultures with fibroblasts in direct contact, but not in the transwells. Furthermore, the effects of the fibroblasts on keratinocyte migration and growth at early culture stage correlated with heparin-binding EGF-like growth factor (HB-EGF), IL-1α and TGF-β1 levels in the cultures where the cells were grown in direct contact. These effects were inhibited by anti-HB-EGF, anti-IL-1α and anti-TGF-β1 antibodies and anti-HB-EGF showed the greatest inhibition. Co-culture of keratinocytes and IL-1α and TGF-β1 siRNA-transfected fibroblasts exhibited a significant reduction in HB-EGF production and keratinocyte proliferation. These results suggest that contact with fibroblasts stimulates the migration and proliferation of keratinocytes during wound healing, and that HB-EGF plays a central role in this process and can be up-regulated by IL-1α and TGF-β1, which also regulate keratinocyte proliferation differently during the early and late stage. PMID:22911722

  3. Astaxanthin induces migration in human skin keratinocytes via Rac1 activation and RhoA inhibition

    OpenAIRE

    Ritto, Dakanda; Tanasawet, Supita; Singkhorn, Sawana; Klaypradit, Wanwimol; Hutamekalin, Pilaiwanwadee; Tipmanee, Varomyalin; Sukketsiri, Wanida

    2017-01-01

    BACKGROUND/OBJECTIVES Re-epithelialization has an important role in skin wound healing. Astaxanthin (ASX), a carotenoid found in crustaceans including shrimp, crab, and salmon, has been widely used for skin protection. Therefore, we investigated the effects of ASX on proliferation and migration of human skin keratinocyte cells and explored the mechanism associated with that migration. MATERIAL/METHOD HaCaT keratinocyte cells were exposed to 0.25-1 ?g/mL of ASX. Proliferation of keratinocytes ...

  4. Effectiveness of systematic chromoendoscopy for diagnosis of early cancer and gastric premalignant lesions. Results of two consecutive screening campaigns in Colombia

    International Nuclear Information System (INIS)

    Emura, Fabian; Mejia, Juan; Mejia, Marcela; Osorio, Camilo

    2010-01-01

    Gastric cancer is the most common malignancy in South America and East Asia. In addition to the high mortality, in Colombia a great disvantage is the lack of data regarding premalignant lesions and early cancer. Aim: To evaluate the usefulness of systematic chromoendoscopy in the prevalence of early cancer and gastric premalignant lesions. A total of 950 were invited to participate, 800 fulfilled the inclusion criteria and finally 650 were analyzed. Results: None of participants had normal gastric mucosa. Mild antrum gastritis was found in 21.8% (142/650), meanwhile moderate or severe antrum gastritis in 77.4% (508/650). Atrophy and metaplasia was found in 14.5% (94/650) and 15.5% (101/650) respectively. H Pilory infection was found in 7.3%, 79.3% 75.5% 57.4% y 0% of subjects with mild, moderate and severe, atrophy, metaplasia and dysplasia respectively. Gastric premalignant lesion was found in 30% (195/650). Two subjects were diagnosed as early gastric cancer and treated by endoscopic submucosal dissection (ESD) with curability as final result. Conclusions: By systematic chromoendoscopy this series has demonstrated that 1/325 healthy volunteers had early gastric cancer and that 1/33 had a premalignant lesion explaining in part the high prevalence of gastric cancer in the region. Bases on this series, gastric cancer is diagnosable and curable among healthy volunteers in Colombia.

  5. Promoter polymorphisms of ST3GAL4 and ST6GAL1 genes and associations with risk of premalignant and malignant lesions of the cervix.

    Science.gov (United States)

    Rivera-Juarez, Maria de Los Angeles; Rosas-Murrieta, Nora Hilda; Mendieta-Carmona, Victoriano; Hernandez-Pacheco, Raquel Esneidy; Zamora-Ginez, Irma; Rodea-Avila, Carlos; Apresa-Garcia, Teresa; Garay-Villar, Onix; Aguilar-Lemarroy, Adriana; Jave-Suarez, Luis Felipe; Diaz-Orea, Maria Alicia; Milflores-Flores, Lorena; Reyes-Salinas, Juan Salvador; Ceja-Utrera, Francisco Javier; Vazquez-Zamora, Victor Javier; Vargas-Maldonado, Tomas; Reyes-Carmona, Sandra; Sosa-Jurado, Francisca; Santos-Lopez, Gerardo; Reyes-Leyva, Julio; Vallejo-Ruiz, Veronica

    2014-01-01

    Sialyltransferase gene expression is altered in several cancers, including examples in the cervix. Transcriptional regulation of the responsible genes depends on different promoters. We aimed to determine the association of single-nucleotide polymorphisms in the B3 promoter of the ST3GAL4 gene and the P1 promoter of the ST6GAL1 gene with cervical premalignant lesions or cervical cancer. A blood sample and/or cervical scrapes were obtained from 104 women with normal cytology, 154 with premalignant lesions and 100 with cervical cancer. We also included 119 blood samples of random donors. The polymorphisms were identified by sequencing from PCR products. For the B3 promoter, a fragment of 506 bp (from nucleotide -408 to +98) was analyzed, and for the P1 promoter a 490 bp (-326 to +164) fragment. The polymorphism analysis showed that at SNP rs10893506, genotypes CC and CT of the ST3GAL4 B3 promoter were associated with the presence of premalignant lesions (OR=2.89; 95%CI 1.72-4.85) and cervical cancer (OR=2.23; 95%CI 1.27-3.91). We detected only one allele of each polymorphism in the ST6GAL1 P1 promoter. We did not detect any genetic variability in the P1 promoter region in our study population. Our results suggest that the rs10893506 polymorphism -22C/T may increase susceptibility to premalignant and malignant lesions of the cervix.

  6. Enhancement of human papilloma virus type 16 E7 specific T cell responses by local invasive procedures in patients with (pre)malignant cervical neoplasia

    NARCIS (Netherlands)

    Visser, Jeroen; van Baarle, D; Hoogeboom, BN; Reesink, N; Klip, H; Schuuring, E; Nijhuis, E; Pawlita, M; Bungener, L; de Vries-Idema, J; Nijman, H; Miedema, F; Daemen, T; van der Zee, A

    2006-01-01

    It has been suggested that local invasive procedures may alter the natural course of (pre)malignant cervical disease. This could be due to partial excision of the lesions, or via induction of cellular immunity against human papillomavirus (HPV) by the local invasive procedures. We studied the

  7. Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis

    Directory of Open Access Journals (Sweden)

    Ju-Suk Nam

    2014-03-01

    Full Text Available Wingless-type (Wnt signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα. Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers.

  8. Study of proliferation and 3D epidermal reconstruction from foreskin, auricular and trunk keratinocytes in children.

    Science.gov (United States)

    Mcheik, Jiad N; Barrault, Christine; Pedretti, Nathalie; Garnier, Julien; Juchaux, Franck; Levard, Guillaume; Morel, Frank; Bernard, François-Xavier; Lecron, Jean-Claude

    2015-03-01

    Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. An alternative approach is to graft isolated keratinocytes. We evaluated foreskin and other anatomic sites as donor sources for autologous keratinocyte graft in children. We studied in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from foreskin, auricular skin, chest and abdominal skin by enzymatic digestion. Living cell recovery, in vitro proliferation, epidermal reconstruction capacities and differentiation status were analyzed. In vitro studies revealed the higher yield of living keratinocyte recovery from foreskin and higher potential in terms of proliferative capacity, regeneration and differentiation. Cultured keratinocytes from foreskin express lower amounts of differentiation markers than those isolated from trunk and ear. Histological analysis of reconstituted human epidermis derived from foreskin and inguinal keratinocytes showed a structured multilayered epithelium, whereas those obtained from ear pinna-derived keratinocytes were unstructured. Our studies highlight the potential of foreskin tissue for autograft applications in boys. A suitable alternative donor site for autologous cell transplantation in female paediatric burn patients remains an open question in our department. We tested the hypothesis that in vitro studies and RHE reconstructive capacities of cells from different body sites can be helpful to select an optimal site for keratinocyte isolation before considering graft protocols for girls. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

  9. Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

    Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

  10. Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion

    Science.gov (United States)

    Prince, Tessa; McBain, Andrew J.

    2012-01-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

  11. Arsenite suppression of BMP signaling in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, Marjorie A.; Qin, Qin [Department of Environmental Toxicology, University of California, Davis, CA 95616-8588 (United States); Hu, Qin; Zhao, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Rice, Robert H., E-mail: rhrice@ucdavis.edu [Department of Environmental Toxicology, University of California, Davis, CA 95616-8588 (United States)

    2013-06-15

    Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of keratinocyte

  12. Steroid synthesis by primary human keratinocytes; implications for skin disease

    International Nuclear Information System (INIS)

    Research highlights: → Primary keratinocytes express the steroid enzymes required for cortisol synthesis. → Normal primary human keratinocytes can synthesise cortisol. → Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. → StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3βHSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7- 3 H]-pregnenolone through each steroid intermediate to [7- 3 H]-cortisol in cultured PHK. Trilostane (a 3βHSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data show that PHK are capable of extra

  13. Steroid synthesis by primary human keratinocytes; implications for skin disease

    Energy Technology Data Exchange (ETDEWEB)

    Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Michael, Anthony E. [Centre for Developmental and Endocrine Signalling, Academic Section of Obstetrics and Gynaecology, Division of Clinical Developmental Sciences, 3rd Floor, Lanesborough Wing, St. George' s, University of London, Cranmer Terrace, Tooting, London SW17 0RE (United Kingdom); Jaulim, Adil [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Bhogal, Ranjit [Life Science, Unilever R and D Colworth House, Sharnbrook, Bedfordshire MK44 1LQ (United Kingdom); Burrin, Jacky M. [Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ (United Kingdom); Philpott, Michael P. [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom)

    2011-01-07

    Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

  14. Histamine enhances keratinocyte-mediated resolution of inflammation by promoting wound healing and response to infection.

    Science.gov (United States)

    Gutowska-Owsiak, D; Selvakumar, T A; Salimi, M; Taylor, S; Ogg, G S

    2014-03-01

    The role of the epidermis in the immune response is well known. While multiple cytokines are implicated in keratinocyte-mediated infection clearance and wound healing, little is known about the involvement of keratinocytes in promoting resolution of inflammation. To assess effects of histamine stimulation on keratinocyte function. We performed a combined microarray/Gene Ontology analysis of histamine-stimulated keratinocytes. Functional changes were tested by apoptosis assessment and scratch assays. Histamine receptor involvement was also assessed by blocking wound closure with specific antagonists. Histamine treatment had extensive effects on keratinocytes, including effects on proinflammatory responses and cellular functions promoting wound healing. At the functional level, there was reduced apoptosis and enhancement of wound healing in vitro. At the receptor level, we identified involvement of all keratinocyte-expressed histamine receptors (HRHs), with HRH1 blockage resulting in the most prominent effect. Histamine activates wound healing and infection clearance-related functions of keratinocytes. While enhancement of histamine-mediated wound healing is mediated predominantly via the HRH1 receptor, other keratinocyte-expressed receptors are also involved. These effects could promote resolution of skin inflammation caused by infection or superficial injury. © 2014 British Association of Dermatologists.

  15. Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes

    DEFF Research Database (Denmark)

    Shahbazi, Marta N; Peña-Jimenez, Daniel; Antonucci, Francesca

    2017-01-01

    Epidermal homeostasis is tightly controlled by a balancing act of self-renewal or terminal differentiation of proliferating basal keratinocytes. An increase in DNA content as a consequence of a mitotic block is a recognized mechanism underlying keratinocyte differentiation, but the molecular mech...

  16. Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte-melanocyte co-culture system.

    Science.gov (United States)

    Li, Jian; Tang, Lu-Yan; Fu, Wen-Wen; Yuan, Jin; Sheng, You-Yu; Yang, Qin-Ping

    2016-12-01

    Hydrogen peroxide (H 2 O 2 ) may have a biphasic effect on melanin synthesis and melanosome transfer. High H 2 O 2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H 2 O 2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H 2 O 2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H 2 O 2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H 2 O 2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H 2 O 2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H 2 O 2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H 2 O 2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H 2 O 2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

  17. Effect of Nanodiamond and Nanoplatinum Liquid, DPV576, on Human Primary Keratinocytes.

    Science.gov (United States)

    Ghoneum, Mamdooh H; Katano, Hideki; Agrawal, Sudhanshu; Ganguly, Sreerupa; Agrawal, Anshu

    2017-01-01

    Nanofabrics are now being used in a wide range of products that come into direct contact with skin, including carpet, clothing, and medical fabrics. In the current study, we examined the effect of a dispersed aqueous mixture of nanodiamond (ND) and nanoplatinum (NP) (DPV576) on human primary keratinocytes with respect to transient receptor potential vanilloid (TRPV) channel expression, secretion of cytokines and production of nerve growth factor (NGF). Keratinocytes were treated with DPV576 at concentrations of 1:10 and 1:100 dilutions for 24 hours in vitro, and their activation of was determined by production of cytokines TNF-α, IL-1β, and prostaglandin (PGE2), and by production of NGF. Inhibitor experiments were carried out by incubating keratinocytes with the TRPV4-selective antagonist HC-067047. TRPV receptor expression (TRPV1, TRPV3 and TRPV4) on keratinocytes as well as changes in Ca2+ potential were examined by flow cytometry. DPV576 treatment of keratinocytes resulted in the following effects: (1) stimulation of keratinocytes as indicated by the significant secretion of cytokines IL-1β, TNF-α, and PGE2, an effect noted only at higher concentration (1:10); (2) significant decrease in the expression of TRPV4 on keratinocytes with a spike in the calcium flux, but no change in the expression of TRPV1 and TRPV3; (3) induction of cytokine secretion independent of TRPV4, as the addition of TRPV4 inhibitor had no significant effect on the cytokine production from keratinocytes; (4) induction of NGF secretion by keratinocytes. These results demonstrate that DPV576 activates keratinocytes via multiple signaling pathways which may reduce stress associated with inflammation, pain, and circadian rhythms. ND/NP-coated fabrics that target the modulation of local inflammation, pain, and circadian rhythms could potentially be of benefit to humans.

  18. Clinical Features of Cutaneous Premalignant Lesions in Busan City and the Eastern Gyeongnam Province, Korea: A Retrospective Review of 1,292 Cases over 19 Years (1995~2013).

    Science.gov (United States)

    Choi, Seung-Hwan; Kim, Ki-Ho; Song, Ki-Hoon

    2016-04-01

    The global prevalence of premalignant lesions has been continuously increasing in recent years, but there has been little research regarding the distribution and incidence of cutaneous premalignant lesions in Korean populations. We conducted this retrospective study to analyze recent trends in the incidence and clinical patterns of cutaneous premalignant lesions in the Korean population. We reviewed 1,292 cases (3,651 lesions) of patients with cutaneous premalignant lesions, including actinic keratosis (AK) and Bowen's disease (BD), from the Department of Dermatology at Dong-A University Hospital (January 1995 to December 2013). The average cutaneous premalignant lesion annual incidence was 1.82%, and the incidence continuously increased from 0.70% to 4.25% over the study period. The most common cutaneous premalignant lesion was AK (75.85%), followed by BD (24.15%). The mean age of onset was 68.76 years (men, 70.89 years; women, 65.56 years), and the male:female ratio of patients was 1:1.52. Major skin cancers, including squamous cell carcinoma (SCC, 8.90%), basal cell carcinoma (BCC, 6.42%), and malignant melanoma (MM, 0.70%), were detected in 15.79% of patients with cutaneous premalignant lesions. Three patients (0.23%) were previously diagnosed with both SCC and BCC. In addition, 59.13% of patients had a single lesion, while 40.87% had multiple lesions. Patient age, history of previous skin cancers, and occupation-related exposure to ultraviolet radiation were more common in patients with multiple lesions. Cutaneous premalignant lesion incidence has gradually increased in the Korean population.

  19. Presence of histopathological premalignant lesions and infection caused by high-risk genotypes of human papillomavirus in patients with suspicious cytological and colposcopy results: A prospective study

    Directory of Open Access Journals (Sweden)

    Golubović Mileta

    2017-01-01

    Full Text Available Background/Aim. In patients with premalignant cervical lesions, human papillomavirus (HPV infection, at any moment, may be spontaneously eliminated, or may persist or transform cervical epithelium from a lower to a higher degree. Due to that, it is necessary to wisely select the patients who are at high risk of cancer development. The aim of the study was to establish the interdependence between a suspicious Papanicolaou (Pap test and colposcopy with the infection caused by high-risk genotypes of human papillomavirus and the presence of premalignant cervical lesions. Methods. This prospective study used cytological, colposcopy, real-time polymerase chain reaction (PCR of high-risk genotypes of human papillomavirus and histopathological analysis of cervical biopsy specimen. Out of 2,578 female patients sent to cytological analyses in Clinical Center of Montenegro, during 2012, 2013 and 2014, the study included 80 women who had to submit their biopsy specimens due to a suspicious Pap test and atypical colposcopy results. Results. In the group of 80 (3.1%; n = 80/2,578 of the selected female patients with suspicious Pap test and colposcopy, 2/3 or 56 (70% of them had cervicitis, and 1/3 or 24 (30% had cervical intraepithelial neoplasia. The most common type in cervical intraepithelial neoplasia was HPV16 in 8 female patients, ie 61.53% out of the number of infected, or 33.33% out of the total number of premalignant lesions. Conclusion. Patients with suspicious Papanicolaou test, colposcopy results and infection which is caused by high-risk HPV infection (HPV 16 in particular often have premalignant cervical lesions. In these cases, histopathological confirmation of lesions is mandatory, since it serves as a definitive diagnostic procedure.

  20. To Study the Prevalence of Premalignancies in Teenagers having Betel, Gutkha, Khaini, Tobacco Chewing, Beedi and Ganja Smoking Habit and Their Association with Social Class and Education Status.

    Science.gov (United States)

    Kumar Srivastava, Vinay

    2014-05-01

    Premalignant oral lesions are usually associated with noxious oral addiction habits. These habits are common in both, high as well as low socioeconomic status but education status of parent and patients significantly affects the development of noxious oral addictions. A total of 872 patients (cases and controls) were included in the study. Social class was determined as per modified Prasad's classification (1970) with price index correction of 2004. Prevalence of lichen planus, to be only 0.4 and 2.6% present in groups III and IV of cases, and submucous fibrosis (SMF) - stromal one lanocytic foci - was 2.4% in male (group III) whereas it was not found in female cases (group IV). Teenagers having higher frequency and longer duration of noxious habits were more prone for development of premalignant lesions. 0.6% of leukoplakia, 0.3% erythroplakia, 0.7% lichen planus and 0.7% submucous fibrosis were present in 872 observed patients of control and cases. How to cite this article: Srivastava VK. To Study the Prevalence of Premalignancies in Teenagers having Betel, Gutkha, Khaini, Tobacco Chewing, Beedi and Ganja Smoking Habit and Their Association with Social Class and Education Status. Int J Clin Pediatr Dent 2014;7(2):86-92.

  1. Possible role of epidermal keratinocytes in the construction of acupuncture meridians.

    Science.gov (United States)

    Denda, Mitsuhiro; Tsutsumi, Moe

    2014-04-01

    Acupuncture meridians consist of a network of acupuncture points on the skin, stimulation of which is well established to have a variety of physiological effects. We have previously demonstrated that epidermal keratinocytes contain multiple sensory systems for temperature, mechanical stimuli, electric potentials and other stimuli. These sensory systems generate changes in the calcium-ion concentration in the epidermis, so epidermal keratinocytes can generate spatially-localized electro-physiological patterns in the skin. We have previously demonstrated signaling between epidermal keratinocytes and peripheral nerve systems. Therefore, stimuli sensed by epidermal keratinocytes might be transferred to the unmyelinated nerve fibers that are known to exist in the epidermis and, thence, to the spinal cord and brain. We propose that epidermal keratinocytes form an information-gathering network in the skin and that this network plays a key role in whole-body homeostasis in response to the changing environment. We also hypothesize that this network corresponds to the acupuncture meridians. As supporting examples, we present some striking calcium propagation patterns observed in cultured human keratinocytes after adenosine-triphosphate (ATP) stimulation. These results support the ideas that keratinocytes can generate spatially-restricted signaling patterns after environmental stimulation and that the cultures might be in-vitro models of meridians as an information-gathering network in skin. Copyright © 2014. Published by Elsevier B.V.

  2. Altered expression of keratinocyte growth factor and its receptor in psoriasis.

    Science.gov (United States)

    Finch, P W; Murphy, F; Cardinale, I; Krueger, J G

    1997-12-01

    One of the biological characteristics of psoriasis is excessive flaking of the skin. This is directly related to the marked hyperplasia of epidermal keratinocytes and to incomplete epidermal differentiation. Keratinocyte growth factor (KGF), a potent mitogen for human keratinocytes, is expressed by stromal cells. Alterations in the KGF signaling pathway might account for the epidermal hyperplasia associated with psoriasis. To test this hypothesis, we investigated the expression of KGF and its receptor (KGFR) in psoriasis tissue. KGF and KGFR mRNA levels were found to be frequently elevated in psoriatic skin specimens as compared with normal skin. Increased KGF transcript expression was localized to the dermal layer of the involved skin specimen using in situ hybridization. In contrast, KGFR transcript and protein expression was localized to the basal layer of keratinocytes in normal skin and to the basal and suprabasal layers of the psoriatic epidermis, coincident with the expanded proliferative keratinocyte pool. To identify molecules that might regulate KGFR expression we investigated the effects of various pharmacological agents and cytokines on KGFR synthesis by keratinocytes. Phorbol ester, interleukin-6, interferon-gamma, and ultraviolet B (UVB) treatment all led to substantial down-regulation of KGFR expression. The down-regulation of KGFR synthesis by UVB suggests a possible mechanism for the antiproliferative action of this agent in the treatment of psoriasis. Taken together, these results suggest that increased KGFR-mediated signaling in keratinocytes in the lesional epidermis might account in part for the epidermal hyperplasia in psoriasis.

  3. A method for quantifying melanosome transfer efficacy from melanocytes to keratinocytes in vitro.

    Science.gov (United States)

    Lin, Hwang-Chi; Shieh, Bin-Han; Lu, Mei-Hua; Chen, Jang-Yi; Chang, Li-Tze; Chao, Chung-Faye

    2008-10-01

    Several different in vivo and in vitro bioassays are used to evaluate melanosome transfer efficacy from melanocytes to keratinocytes. However, these methods are complicated and time consuming. Here, we report on a simple, rapid, direct, and reliable in vitro method for observing the process of melanosome transfer from melanocytes to keratinocytes. First, we selected and tested a melanoma cell line RPMI-7951 that can normally synthesize melanin and transfer from mature melanosomes to keratinocytes in vitro. We cocultured these cells with a human ovarian teratoma transformed epidermal carcinoma cell line, which is also capable of accepting melanosomes transferred from melanocytes, as in normal keratinocytes. The cells were cocultured for 24-72 h and double labeled with FITC-conjugated antibody against the melanosome-associated protein TRP-1, and with Cy5-conjugated antibody against the keratinocyte-specific marker keratin 14. The cells were examined by fluorescence microscope and flow cytometry. Melanosome transfer from melanocytes to keratinocytes increased in a time-dependent manner. To verify the accessibility of this method, the melanosome transfer inhibitor, a serine protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and a melanosome transfer stimulator, alpha-melanocyte-stimulating hormone, were added. The serine protease inhibitor decreased melanosome transfer, and alpha-melanocyte-stimulating hormone increased melanosome transfer, in a dose-dependent manner. In conclusion, this is a simple, rapid, and effective model system to quantify the melanosome transfer efficacy from melanocytes to keratinocytes in vitro.

  4. Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

    Science.gov (United States)

    Mohammedsaeed, Walaa; Cruickshank, Sheena; McBain, Andrew J; O'Neill, Catherine A

    2015-11-05

    A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

  5. Separation of Normal and Premalignant Cervical Epithelial Cells Using Confocal Light Absorption and Scattering Spectroscopic Microscopy Ex Vivo

    Directory of Open Access Journals (Sweden)

    Ling Yang

    2011-01-01

    Full Text Available Confocal light absorption and scattering spectroscopic (CLASS microscopy can detect changes in biochemicals and the morphology of cells. It is therefore used to detect high-grade cervical squamous intraepithelial lesion (HSIL cells in the diagnosis of premalignant cervical lesions. Forty cervical samples from women with abnormal Pap smear test results were collected, and twenty cases were diagnosed as HSIL; the rest were normal or low-grade cervical squamous intraepithelial lesion (LSIL. The enlarged and condensed nuclei of HSIL cells as viewed under CLASS microscopy were much brighter and bigger than those of non-HSIL cells. Cytological elastic scattered light data was then collected at wavelengths between 400 and 1000 nm. Between 600 nm to 800 nm, the relative elastic scattered light intensity of HSIL cells was higher than that of the non-HSIL. Relative intensity peaks occurred at 700 nm and 800 nm. CLASS sensitivity and specificity results for HSIL and non-HSIL compared to cytology diagnoses were 80% and 90%, respectively. This study demonstrated that CLASS microscopy could effectively detect cervical precancerous lesions. Further study will verify this conclusion before the method is used in clinic for early detection of cervical cancer.

  6. Prevention of MHC-alloimmunization by UV-B irradiation in a murine model: effects of UV dose and number of transfused cells

    International Nuclear Information System (INIS)

    Grijzenhout, M.A.; Claas, F.H.J.

    1994-01-01

    The optimal dose of UV-B radiation for prevention of in vivo alloimmunization (AI) against major histocompatibility complex (MHC) antigens was investigated in a murine transfusion model. Two groups with five C57BL/6 mice (H-2 b ) each were transfused at weekly intervals with 1 x 10 5 or 1 x 10 6 DBA/2 (H-2 d ) leucocytes. Both suspensions induced anti-H-2 d antibodies in all mice after the second transfusion. The minimal UV-B dose required for abolition of alloreactivity in the mixed leucocyte reaction (MLR) was 0.6 J/cm 2 . This dose completely prevented the onset of MHC-AI in all five mice transfused with six suspensions containing 1 x 10 5 leucocytes. In contrast, suspensions with 1 x 10 6 leucocytes and exposed to 0.6 J/cm 2 induced immunization in 4/5 mice. Further increase of the dose to 1.8 or 5.4 J/cm 2 did not prevent the onset of MHC-AI. We conclude that the number of leucocytes per transfusion determines the efficacy of UV irradiation for the prevention of MHC-AI. For UV irradiation of human platelet concentrates (PCs) we propose to reduce the number of leucocytes by centrifugation prior to UV exposure. UV-B irradiation of PCs with high numbers of leucocytes may not be effective for prevention of alloimmunization. (Author)

  7. Effects of a turmeric extract (Curcuma longa) on chronic ultraviolet B irradiation-induced skin damage in melanin-possessing hairless mice.

    Science.gov (United States)

    Sumiyoshi, Maho; Kimura, Yoshiyuki

    2009-12-01

    Turmeric (the rhizomes of Curcuma longa L., Zingiberacease) is widely used as a dietary pigment and spice, and has been traditionally used for the treatment of inflammation, skin wounds and hepatic disorders in Ayurvedic, Unani and Chinese medicine. Although the topical application or oral administration of turmeric is used to improve skin trouble, there is no evidence to support this effect. The aim of this study was to clarify whether turmeric prevents chronic ultraviolet B (UVB)-irradiated skin damage. We examined the effects of a turmeric extract on skin damage including changes in skin thickness and elasticity, pigmentation and wrinkling caused by long-term, low-dose ultraviolet B irradiation in melanin-possessing hairless mice. The extract (at 300 or 1000 mg/kg, twice daily) prevented an increase in skin thickness and a reduction in skin elasticity induced by chronic UVB exposure. It also prevented the formation of wrinkles and melanin (at 1000 mg/kg, twice daily) as well as increases in the diameter and length of skin blood vessels and in the expression of matrix metalloproteinase-2 (MMP-2). Prevention of UVB-induced skin aging by turmeric may be due to the inhibition of increases in MMP-2 expression caused by chronic irradiation.

  8. T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

    Directory of Open Access Journals (Sweden)

    Cécilia Brun

    Full Text Available Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and β6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte

  9. Hyaluronan minimizes effects of UV irradiation on human keratinocytes.

    Science.gov (United States)

    Hašová, Martina; Crhák, Tomáš; Safránková, Barbora; Dvořáková, Jana; Muthný, Tomáš; Velebný, Vladimír; Kubala, Lukáš

    2011-05-01

    Exposure to ultraviolet (UV) irradiation has detrimental effects on skin accompanied by the increased metabolism of hyaluronan (HA), a linear polysaccharide important for the normal physiological functions of skin. In this study, the modulation of human keratinocyte response to UVB irradiation by HA (970 kDa) was investigated. Immortalized human keratinocytes (HaCaT) were irradiated by a single dose of UVB and immediately treated with HA for 6 and 24 h. The irradiation induced a significant decrease in the gene expression of CD44 and toll-like receptor 2 6 h after irradiation. The expressions of other HA receptors, including toll-like receptor 4 and the receptor for HA-mediated motility, were not detected in either the control or UVB-irradiated or HA-treated HaCaT cells. UVB irradiation induced a significant decrease in the gene expression of HA synthase-2 and hyaluronidase-2 6 h after irradiation. The expressions of HA synthase-3 and hyaluronidase-3 were not significantly modulated by UV irradiation. Interestingly, HA treatment did not significantly modulate any of these effects. In contrast, HA significantly suppressed UVB-induced pro-inflammatory cytokine release including interleukin-6 and interleukin-8. Similarly, HA treatment reduced the UVB-mediated production of transforming growth factor β1. HA treatment also significantly reduced the UV irradiation-mediated release of soluble CD44 into the media. Finally, HA partially, but significantly, suppressed the UVB-induced decrease in cell viability. Data indicate that HA had significant protective effects for HaCaT cells against UVB irradiation.

  10. Chrysin attenuates atopic dermatitis by suppressing inflammation of keratinocytes.

    Science.gov (United States)

    Choi, Jin Kyeong; Jang, Yong Hyun; Lee, Soyoung; Lee, Sang-Rae; Choi, Young-Ae; Jin, Meiling; Choi, Jung Ho; Park, Jee Hun; Park, Pil-Hoon; Choi, Hyukjae; Kwon, Taeg Kyu; Khang, Dongwoo; Kim, Sang-Hyun

    2017-12-01

    We previously reported the inhibitory effect of chrysin, a natural flavonoid plentifully contained in propolis, vegetables and fruits, on the mast cell-mediated allergic reaction. In this study, we evaluated the effect of chrysin on atopic dermatitis (AD) and defined underlying mechanisms of action. We used an AD model in BALB/c mice by the repeated local exposure of 2,4-dinitrochlorobenzene (DNCB) and house dust mite (Dermatophagoides farinae extract, DFE) to the ears. Repeated alternative treatment of DNCB/DFE caused AD-like skin lesions. Oral administration of chrysin diminished AD symptoms such as ear thickness and histopathological analysis, in addition to serum IgE and IgG2a levels. Chrysin decreased infiltration of mast cells, and reduced serum histamine level. Chrysin also suppressed AD by inhibiting the inflammatory responses of Th1, Th2, and Th17 cells in mouse lymph node and ear. Interestingly, chrysin significantly inhibited the production of cytokines, Th2 chemokines, CCL17 and CCL22 by the down-regulation of p38 MAPK, NF-κB, and STAT1 in tumor necrosis factor (TNF)-α/interferon (IFN)-γ-stimulated human keratinocytes (HaCaT). Chrysin also inhibited TNF-α/IFN-γ-stimulated IL-33 expression in HaCaT cells and mouse primary keratinocytes. Taken together, the results indicate that chrysin suppressed AD symptoms, suggesting that chrysin might be a candidate for the treatment of AD and skin allergic diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Up-modulation of the expression of functional keratinocyte growth factor receptors induced by high cell density in the human keratinocyte HaCaT cell line.

    Science.gov (United States)

    Capone, A; Visco, V; Belleudi, F; Marchese, C; Cardinali, G; Bellocci, M; Picardo, M; Frati, L; Torrisi, M R

    2000-11-01

    Keratinocyte growth factor (KGF) is involved in the control of proliferation and differentiation of human keratinocytes. It binds to, and activates, the tyrosine kinase KGF receptor (KGFR), a splicing transcript variant of the fibroblast growth factor receptor 2. We have previously shown (C. Marchese et al., Cell Growth Differ., 8: 989-997, 1997) that differentiation of primary cultured keratinocytes triggered by high Ca2+ concentrations in the growing medium induced up-regulation of KGFR expression, which suggested that KGFR may play a crucial role in the control of the proliferative/differentiative program during transition from basal to suprabasal cells. Here we analyzed the process of modulation of the expression of KGFRs in the human keratinocyte cell line HaCaT, widely used as a model to study keratinocyte differentiation. Western blot and double immunofluorescence for KGFR and the K1 differentiation marker showed that cell differentiation and stratification induced by confluence and high cell density correlated with an increase in KGFR expression. KGFRs, present on suprabasal differentiated cells, appeared to be efficiently tyrosine-phosphorylated by KGF, which indicated that the receptors up-regulated by differentiation can be functionally activated by ligand binding. Bromodeoxyuridine incorporation assay revealed that a significant portion of suprabasal differentiated cells expressing KGFR seemed to be still able to synthesize DNA and to proliferate in response to KGF, which suggested that increased KGFR expression may be required for retention of the proliferative activity.

  12. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

    International Nuclear Information System (INIS)

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-ichi; Kotani, Eiji; Hirano, Tomoko; Nakajima, Yumiko; Matsumoto, Goichi; Mori, Hajime

    2014-01-01

    Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase–Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. - Highlights: • 3D cultures using FGF-2 and FGF-7 microcrystals as a human skin model • Cytoprotection of keratinocytes against ROS by FGF-7 microcrystals • Overexpression of SOD and Prdx6 in keratinocytes by FGF-7 microcrystals

  13. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

    Energy Technology Data Exchange (ETDEWEB)

    Shimabukuro, Junji; Yamaoka, Ayako; Murata, Ken-ichi [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Kotani, Eiji [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Kyoto (Japan); Hirano, Tomoko [Venture Laboratory, Kyoto Institute of Technology, Kyoto (Japan); Nakajima, Yumiko [Functional Genomics Group, COMB, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa (Japan); Matsumoto, Goichi [Division of Oral Surgery, Yokohama Clinical Education Center of Kanagawa Dental University, Yokohama (Japan); Mori, Hajime, E-mail: hmori@kit.ac.jp [Department of Applied Biology, Kyoto Institute of Technology, Kyoto (Japan); Insect Biomedical Research Center, Kyoto Institute of Technology, Kyoto (Japan)

    2014-09-01

    Stable protein microcrystals called polyhedra are produced by certain insect viruses. Cytokines, such as fibroblast growth factors (FGFs), can be immobilized within polyhedra. Here, we investigated three-dimensional (3D) co-cultures of keratinocytes and melanocytes on collagen gel containing FGF-2 and FGF-7 polyhedra. Melanocytes were observed to reside at the base of the 3D cell culture and melanin was also typically observed in the lower layer. The 3D cell culture model with FGF-2 and FGF-7 polyhedra was a useful in vitro model of the epidermis due to effective melanogenesis, proliferation and differentiation of keratinocytes. FGF-7 polyhedra showed a potent cytoprotective effect when keratinocytes were treated with menadione, which is a generator of reactive oxygen species. The cytoprotective effect was activated by the inositol triphosphate kinase–Akt pathway leading to upregulation of the antioxidant enzymes superoxide dismutase and peroxiredoxin 6. - Highlights: • 3D cultures using FGF-2 and FGF-7 microcrystals as a human skin model • Cytoprotection of keratinocytes against ROS by FGF-7 microcrystals • Overexpression of SOD and Prdx6 in keratinocytes by FGF-7 microcrystals.

  14. Effects of low-energy gallium-aluminum-arsenide laser irradiation on cultured fibroblasts and keratinocytes.

    Science.gov (United States)

    Pogrel, M A; Chen, J W; Zhang, K

    1997-01-01

    To assess whether the gallium-aluminum-arsenide low energy laser will increase cell proliferation, cell attachment, or cell migration in cultured fibroblasts and keratinocyte models. Monolayer cultures of fibroblasts and keratinocytes were subjected to gallium-aluminum-arsenide laser irradiation at varying power densities for varying time intervals. Cell proliferation was assessed by absorbent spectrophotometry while cell adhesion was assessed by a microcolorimetric assay for cells attached to bovine dermis collagen. Cell migration was assessed through a filter utilizing high power microscopic fields. There were no differences in cell proliferation, adhesion, or migration in either the fibroblasts or keratinocyte culture treated with the gallium-aluminum-arsenide laser at any power density or time compared with nontreated controls. The gallium-aluminum-arsenide laser, when utilized at powers 5-100 milliwatts and times of between 10-120 seconds has no biostimulatory effects on fibroblasts or keratinocyte cultures as assessed by cell proliferation, adhesion, or migration.

  15. The Cytoskeleton and ATP in Sulfur Mustard-Medicated Injury to Endothelial Cells and Keratinocytes

    National Research Council Canada - National Science Library

    Hinshaw, Daniel

    1998-01-01

    The major goal of this project is to test the hypothesis that sulfur mustard (SM)-mediated cell death in keratinocytes and endothelial cells is primarily apoptotic in nature, and that several factors...

  16. Prevention of burn wound conversion by allogeneic keratinocytes cultured on acellular xenodermis

    Czech Academy of Sciences Publication Activity Database

    Matoušková, Eva; Brož, L.; Pokorná, Eva; Königová, R.

    2002-01-01

    Roč. 3, č. 1 (2002), s. 29-35 ISSN 1389-9333 Institutional research plan: CEZ:AV0Z5052915 Keywords : human keratinocytes * tissue engineered skin * dried porcine dermis Subject RIV: EB - Genetics ; Molecular Biology

  17. Acellular porcine xenodermis as a temporary wound cover and substratum for cultured keratinocytes

    Czech Academy of Sciences Publication Activity Database

    Matoušková, Eva; Stehlíček, P.; Veselý, Pavel

    2002-01-01

    Roč. 4, - (2002), s. 83-85 ISSN 1473-2262 Institutional research plan: CEZ:AV0Z5052915 Keywords : wound healing * cultured keratinocytes * dried porcine dermis Subject RIV: EB - Genetics ; Molecular Biology

  18. Modulation of Keratinocyte Gene Expression and Differentiation by PPAR-Selective Ligands and Tetradecylthioacetic Acid

    DEFF Research Database (Denmark)

    Westergaard, M; Henningsen, J; Svendsen, M L

    2001-01-01

    Peroxisome proliferator-activated receptors (PPARs) are pleiotropic regulators of growth and differentiation of many cell types. We have performed a comprehensive analysis of the expression of PPARs, transcriptional cofactors, and marker genes during differentiation of normal human keratinocytes ...

  19. Keratinocyte-derived growth factors play a role in the formation of hypertrophic scars

    NARCIS (Netherlands)

    Niessen, FB; Andriessen, MP; Schalkwijk, J; Visser, L; Timens, W

    In predisposed individuals, wound healing can lead to hypertrophic scar or keloid formation, characterized by an overabundant extracellular matrix. It has recently been shown that hypertrophic scars are accompanied by abnormal keratinocyte differentiation and proliferation, and significantly

  20. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Ruijin [Pharmacology and Toxicology and Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Environmental Health Science, New York Medical College, Valhalla, NY (United States); Mishin, Vladimir; Black, Adrienne T. [Pharmacology and Toxicology and Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ (United States); Shakarjian, Michael P. [Environmental Health Science, New York Medical College, Valhalla, NY (United States); Kong, Ah-Ng Tony; Laskin, Debra L. [Pharmacology and Toxicology and Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Environmental and Occupational Medicine, Rutgers University-Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

  1. Interleukin-8 production in response to tumor necrosis factor-alpha by cholesteatoma keratinocytes in cell culture.

    Science.gov (United States)

    Hilton, Christopher W; Ondrey, Frank G; Wuertz, Beverley R; Levine, Samuel C

    2011-02-01

    Keratinocytes harvested from acquired cholesteatoma and grown in cell culture will demonstrate increased interleukin-8 (IL-8) production in response to tumor necrosis factor (TNF)-alpha as compared with a control keratinocyte cell line. Immunohistochemical studies have identified IL-8 and TNF-alpha, mediators of bony destruction, in tissue samples of cholesteatoma. TNF-alpha stimulates IL-8 production in healthy epidermal keratinocyte cell lines. It is not known whether TNF-alpha stimulates IL-8 production in cultured cholesteatoma keratinocytes. Prospective controlled tissue culture experiment. Tissue from an acquired cholesteatoma was dissociated and grown in keratinocyte serum-free media for 8 weeks. Cholesteatoma keratinocytes and a control cell line of skin epidermal keratinocytes were treated with TNF-alpha. Conditioned media were harvested; production of IL-8 was measured by enzyme-linked immunosorbent assay, and cell counts were performed. At a zero concentration of TNF-alpha, mean production of IL-8 by cholesteatoma keratinocytes was 39,809 pg/mL/24hr/1 × 10(6) cells versus 1,907 pg/mL/24hr/1 × 10(6) cells from skin epidermal keratinocytes, a statistically significant difference (P alpha and a 2.44-fold increase in response to 20 pg/mL of TNF-alpha. The skin epidermal keratinocyte cell line demonstrated a 1.07- and 1.13-fold increase to respective concentrations of TNF-alpha. Cholesteatoma keratinocytes appear to retain cell signaling characteristics in vitro that distinguish them from skin epidermal keratinocytes. This finding may indicate that cholesteatoma keratinocytes undergo a change in behavior in vivo that is preserved after the cells are removed from the inflammatory environment of the middle ear. Copyright © 2011 The American Laryngological, Rhinological, and Otological Society, Inc.

  2. Effect of Absolute From Hibiscus syriacus L. Flower on Wound Healing in Keratinocytes

    OpenAIRE

    Yoon, Seok Won; Lee, Kang Pa; Kim, Do-Yoon; Hwang, Dae Il; Won, Kyung-Jong; Lee, Dae Won; Lee, Hwan Myung

    2017-01-01

    Background: Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. Objective: To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. Materials and Methods: We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysi...

  3. Adherence of human oral keratinocytes and gingival fibroblasts to nano-structured titanium surfaces.

    Science.gov (United States)

    Dorkhan, Marjan; Yücel-Lindberg, Tülay; Hall, Jan; Svensäter, Gunnel; Davies, Julia R

    2014-06-21

    A key element for long-term success of dental implants is integration of the implant surface with the surrounding host tissues. Modification of titanium implant surfaces can enhance osteoblast activity but their effects on soft-tissue cells are unclear. Adherence of human keratinocytes and gingival fibroblasts to control commercially pure titanium (CpTi) and two surfaces prepared by anodic oxidation was therefore investigated. Since implant abutments are exposed to a bacteria-rich environment in vivo, the effect of oral bacteria on keratinocyte adhesion was also evaluated. The surfaces were characterized using scanning electron microscopy (SEM). The number of adhered cells and binding strength, as well as vitality of fibroblasts and keratinocytes were evaluated using confocal scanning laser microscopy after staining with Live/Dead Baclight. To evaluate the effect of bacteria on adherence and vitality, keratinocytes were co-cultured with a four-species streptococcal consortium. SEM analysis showed the two anodically oxidized surfaces to be nano-structured with differing degrees of pore-density. Over 24 hours, both fibroblasts and keratinocytes adhered well to the nano-structured surfaces, although to a somewhat lesser degree than to CpTi (range 42-89% of the levels on CpTi). The strength of keratinocyte adhesion was greater than that of the fibroblasts but no differences in adhesion strength could be observed between the two nano-structured surfaces and the CpTi. The consortium of commensal streptococci markedly reduced keratinocyte adherence on all the surfaces as well as compromising membrane integrity of the adhered cells. Both the vitality and level of adherence of soft-tissue cells to the nano-structured surfaces was similar to that on CpTi. Co-culture with streptococci reduced the number of keratinocytes on all the surfaces to approximately the same level and caused cell damage, suggesting that commensal bacteria could affect adherence of soft-tissue cells to

  4. Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

    Science.gov (United States)

    Choi, Eun-Jung; Kang, Young-Gyu; Kim, Jaekyung; Hwang, Jae-Kwan

    2011-01-01

    Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

  5. Comparative studies of types 1 and 2 herpes simplex virus infection of cultured normal keratinocytes.

    OpenAIRE

    Su, S J; Wu, H H; Lin, Y H; Lin, H Y

    1995-01-01

    AIMS--To investigate the differences in biological properties, multiplication patterns, and cytopathic effects between type 1 and type 2 herpes simplex virus (HSV) through the replication of HSV in cultured normal human keratinocytes. METHODS--Keratinocytes were obtained from surgical specimens of normal gingiva, cervix, trunk skin, and newborn foreskin. They were cultured in serum free, chemically defined, culture medium and infected with a pool of HSV collected from clinical specimens. RESU...

  6. Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

    Science.gov (United States)

    Ma, Dongrui; Chua, Alvin Wen Choong; Yang, Ennan; Teo, Peiyun; Ting, Yixin; Song, Colin; Lane, Ellen Birgitte; Lee, Seng Teik

    2015-03-24

    There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, β1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

  7. Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.

    Science.gov (United States)

    Khafif, A; Schantz, S P; Chou, T C; Edelstein, D; Sacks, P G

    1998-03-01

    An in vitro model for oral cancer was used to examine the growth inhibitory effects of chemopreventive agents when used singly and in combination. The model consists of primary cultures of normal oral epithelial cells, newly established cell lines derived from dysplastic leukoplakia and squamous cell carcinoma. Two naturally occurring substances, (-)-epigallocatechin-3-gallate (EGCG) from green tea and curcumin from the spice turmeric were tested. Cells were treated singly and in combination and effects on growth determined in 5-day growth assays and by cell cycle analysis. Effective dose 50s and the combination index were calculated with the computerized Chou-Talalay method which is based on the median-effect principle. Agents were shown to differ in their inhibitory potency. EGCG was less effective with cell progression; the cancer cells were more resistant than normal or dysplastic cells. In contrast, curcumin was equally effective regardless of the cell type tested. Cell cycle analysis indicated that EGCG blocked cells in G1, whereas curcumin blocked cells in S/G2M. The combination of both agents showed synergistic interactions in growth inhibition and increased sigmoidicity (steepness) of the dose-effect curves, a response that was dose and cell type dependent. Combinations allowed for a dose reduction of 4.4-8.5-fold for EGCG and 2.2-2.8-fold for curcumin at ED50s as indicated by the dose reduction index (DRI). Even greater DRI values were observed above ED50 levels. Our results demonstrate that this model which includes normal, premalignant and malignant oral cells can be used to analyse the relative potential of various chemopreventive agents. Two such naturally-occurring agents, EGCG and curcumin, were noted to inhibit growth by different mechanisms, a factor which may account for their demonstrable interactive synergistic effect.

  8. Vitamin D Repletion Reduces the Progression of Premalignant Squamous Lesions in the NTCU Lung Squamous Cell Carcinoma Mouse Model

    Science.gov (United States)

    Mazzilli, Sarah A.; Hershberger, Pamela A.; Reid, Mary E.; Bogner, Paul N.; Atwood, Kristopher; Trump, Donald L.; Johnson, Candace S.

    2015-01-01

    The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C) (80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the % of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D-deficient +NTCU (DN), 22.7 % (p<0.05 compared to vitamin D-sufficient +NTCU (SN)); DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared to SN mice, DN mice exhibited a 3-fold increase (p <0.005) in circulating white blood cells (WBC), a 20% (p <0.05) increase in IL-6 levels, and a 4 -fold (p <0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. PMID:26276745

  9. Microscopic analysis of the chromium content in the chromium-induced malignant and premalignant bronchial lesions of the rat

    International Nuclear Information System (INIS)

    Takahashi, Yuji; Kondo, Kazuya; Ishikawa, Sumiyo; Uchihara, Hiroshi; Fujino, Haruhiko; Sawada, Naruhiko; Miyoshi, Takanori; Sakiyama, Shoji; Izumi, Keisuke; Monden, Yasumasa

    2005-01-01

    Objective: Our previous studies demonstrated that the frequency of gene instability in lung cancer of chromate workers was very high, but the frequencies of the p53 and ras gene mutations were low. To clarify the carcinogenesis of chromate in the lung, we established a chromate-induced cancer model in the rat proximal airway and examined the relationship between chromium accumulations and the chromium-induced cancer and premalignant bronchial lesions of the rat. Methods: Fifteen male, bred, 12-week-old Jcl-Wister rats were used. A pellet of strontium chromate were inserted into the bronchus of the rats. The rats were sacrificed 9 months after the pellet was inserted. We pathologically examined the region of the bronchi to which the pellet was attached. We quantified the amount of chromium accumulation in the bronchial lesions using a microscopic X-ray fluorescence analyzer. Results: Of the 15 rats, 1 rat had a lesion of squamous cell carcinoma (SCC), 7 rats had carcinoma in situ (CIS) or dysplasia, 8 rats had squamous metaplasia, and 5 rats had goblet cell hyperplasia. The amounts of chromium accumulation in normal epithelium (n=24), goblet cell hyperplasia (n=14), squamous metaplasia (n=8), and dysplasia plus CIS plus SCC (n=9) were 500±1354, 713±1062, 941±1328, and 3511±4473 (mean±SD) counts/s/mA, respectively. The amount of chromium accumulation was significantly increased according to the progression of malignant change of the bronchial epithelium (Spearman's correlation coefficient by ranks, rs=0.454, P<0.01). Conclusions: The amount of chromium accumulation was significantly increased according to the progression of malignant change of the bronchial epithelium. Examining the genetic alterations of histologic changes in this model was helpful in elucidating the process of carcinogenesis of chromium in the lung

  10. Effects of ultraviolet-B irradiance on intraspecific competition and facilitation of plants: self-thinning, size inequality, and phenotypic plasticity.

    Science.gov (United States)

    Zhang, Rui-Chang; Lin, Yue; Yue, Ming; Li, Qian; Zhang, Xiao-Fei; Liu, Xiao; Chi, Hong; Chai, Yong-Fu; Wang, Mao

    2012-01-01

    (1) The effects of facilitation on the structure and dynamics of plant populations have not been studied so widely as competition. The UV-B radiation, as a typical environmental factor causing stress, may result in direct stress and facilitation. (2) The effects of UV-B radiation on intraspecific competition and facilitation were investigated based on the following three predictions on self-thinning, size inequality, and phenotypic plasticity: i) Self-thinning is the reduction in density that results from the increase in the mean biomass of individuals in crowded populations, and is driven by competition. In this study, the mortality rate of the population is predicted to decrease from UV-B irradiance. ii) The size inequality of a population increases with competition intensity because larger individuals receive a disproportionate share of resources, thereby leaving limited resources for smaller individuals. The second hypothesis assumes that direct stress decreases the size inequality of the population. iii) Phenotypic plasticity is the ability to alter one's morphology in response to environmental changes. The third hypothesis assumes that certain morphological indices can change among the trade-offs between competition, facilitation, and stress. These predictions were tested by conducting a field pot experiment using mung beans, and were supported by the following results: (3) UV-B radiation increased the survival rate of the population at the end of self-thinning. However, this result was mainly due to direct stress rather than facilitation. (4) Just as competitor, facilitation was also asymmetric. It increased the size inequality of populations during self-thinning, whereas stress decreased the size inequality. (5) Direct stress and facilitation influence plants differently on various scales. Stress inhibited plant growth, whereas facilitation showed the opposite on an individual scale. Stress increased survival rate, whereas facilitation increased individual

  11. Effects of ultraviolet-B irradiance on intraspecific competition and facilitation of plants: self-thinning, size inequality, and phenotypic plasticity.

    Directory of Open Access Journals (Sweden)

    Rui-Chang Zhang

    Full Text Available (1 The effects of facilitation on the structure and dynamics of plant populations have not been studied so widely as competition. The UV-B radiation, as a typical environmental factor causing stress, may result in direct stress and facilitation. (2 The effects of UV-B radiation on intraspecific competition and facilitation were investigated based on the following three predictions on self-thinning, size inequality, and phenotypic plasticity: i Self-thinning is the reduction in density that results from the increase in the mean biomass of individuals in crowded populations, and is driven by competition. In this study, the mortality rate of the population is predicted to decrease from UV-B irradiance. ii The size inequality of a population increases with competition intensity because larger individuals receive a disproportionate share of resources, thereby leaving limited resources for smaller individuals. The second hypothesis assumes that direct stress decreases the size inequality of the population. iii Phenotypic plasticity is the ability to alter one's morphology in response to environmental changes. The third hypothesis assumes that certain morphological indices can change among the trade-offs between competition, facilitation, and stress. These predictions were tested by conducting a field pot experiment using mung beans, and were supported by the following results: (3 UV-B radiation increased the survival rate of the population at the end of self-thinning. However, this result was mainly due to direct stress rather than facilitation. (4 Just as competitor, facilitation was also asymmetric. It increased the size inequality of populations during self-thinning, whereas stress decreased the size inequality. (5 Direct stress and facilitation influence plants differently on various scales. Stress inhibited plant growth, whereas facilitation showed the opposite on an individual scale. Stress increased survival rate, whereas facilitation increased

  12. Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

    International Nuclear Information System (INIS)

    Matsumoto, K.; Hashimoto, K.; Yoshikawa, K.; Nakamura, T.

    1991-01-01

    Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes

  13. Derivation of keratinocytes from chicken embryonic stem cells: Establishment and characterization of differentiated proliferative cell populations

    Directory of Open Access Journals (Sweden)

    Mathilde Couteaudier

    2015-03-01

    Full Text Available A common challenge in avian cell biology is the generation of differentiated cell-lines, especially in the keratinocyte lineage. Only a few avian cell-lines are available and very few of them show an interesting differentiation profile. During the last decade, mammalian embryonic stem cell-lines were shown to differentiate into almost all lineages, including keratinocytes. Although chicken embryonic stem cells had been obtained in the 1990s, few differentiation studies toward the ectodermal lineage were reported. Consequently, we explored the differentiation of chicken embryonic stem cells toward the keratinocyte lineage by using a combination of stromal induction, ascorbic acid, BMP4 and chicken serum. During the induction period, we observed a downregulation of pluripotency markers and an upregulation of epidermal markers. Three homogenous cell populations were derived, which were morphologically similar to chicken primary keratinocytes, displaying intracellular lipid droplets in almost every pavimentous cell. These cells could be serially passaged without alteration of their morphology and showed gene and protein expression profiles of epidermal markers similar to chicken primary keratinocytes. These cells represent an alternative to the isolation of chicken primary keratinocytes, being less cumbersome to handle and reducing the number of experimental animals used for the preparation of primary cells.

  14. Fos and jun proteins are specifically expressed during differentiation of human keratinocytes.

    Science.gov (United States)

    Mehic, Denis; Bakiri, Latifa; Ghannadan, Minoo; Wagner, Erwin F; Tschachler, Erwin

    2005-01-01

    Activator protein 1 (AP-1) proteins play key roles in the regulation of cell proliferation and differentiation. In this study we investigated the expression of Fos and Jun proteins in different models of terminal differentiation of human keratinocytes and in skin from psoriasis patients. All Jun and Fos proteins, with the exception of FosB, were efficiently expressed in keratinocytes in monolayer cultures. In contrast, in normal epidermis as well as in organotypic epidermal cultures, the expression pattern of AP-1 proteins was dependent on the differentiation stage. Fos proteins were readily detected in nuclei of keratinocytes of basal and suprabasal layers. JunB and JunD were expressed in all layers of normal epidermis. Interestingly, expression of c-Jun started suprabasally, then disappeared and became detectable again in distinct cells of the outermost granular layer directly at the transition zone to the stratum corneum. In psoriatic epidermis, c-Jun expression was prominent in both hyperproliferating basal and suprabasal keratinocytes, whereas c-Fos expression was unchanged. These data indicate that AP-1 proteins are expressed in a highly specific manner during terminal differentiation of keratinocytes and that the enhanced expression of c-Jun in basal and suprabasal keratinocytes might contribute to the pathogenesis of psoriasis.

  15. Enhanced secretion of TIMP-1 by human hypertrophic scar keratinocytes could contribute to fibrosis.

    Science.gov (United States)

    Simon, Franck; Bergeron, Daniele; Larochelle, Sébastien; Lopez-Vallé, Carlos A; Genest, Hervé; Armour, Alexis; Moulin, Véronique J

    2012-05-01

    Hypertrophic scars are a pathological process characterized by an excessive deposition of extracellular matrix components. Using a tissue-engineered reconstructed human skin (RHS) method, we previously reported that pathological keratinocytes induce formation of a fibrotic dermal matrix. We further investigated keratinocyte action using conditioned media. Results showed that conditioned media induce a similar action on dermal thickness similar to when an epidermis is present. Using a two-dimensional electrophoresis technique, we then compared conditioned media from normal or hypertrophic scar keratinocytes and determined that TIMP-1 was increased in conditioned media from hypertrophic scar keratinocytes. This differential profile was confirmed using ELISA, assaying TIMP-1 presence on media from monolayer cultured keratinocytes and from RHS. The dermal matrix of these RHS was recreated using mesenchymal cells from three different origins (skin, wound and hypertrophic scar). The effect of increased TIMP-1 levels on dermal fibrosis was also validated independently from the mesenchymal cell origin. Immunodetection of TIMP-1 showed that this protein was increased in the epidermis of hypertrophic scar biopsies. The findings of this study represent an important advance in understanding the role of keratinocytes as a direct potent modulator for matrix degradation and scar tissue remodeling, possibly through inactivation of MMPs. Copyright © 2011 Elsevier Ltd and ISBI. All rights reserved.

  16. Podoplanin expression in peritumoral keratinocytes predicts aggressive behavior in extramammary Paget's disease.

    Science.gov (United States)

    Cho, Zaigen; Konishi, Eiichi; Kanemaru, Mai; Isohisa, Taro; Arita, Takahiro; Kawai, Minako; Tsutsumi, Miho; Mizutani, Hiromi; Takenaka, Hideya; Ozawa, Toshiyuki; Tsuruta, Daisuke; Katoh, Norito; Asai, Jun

    2017-07-01

    Recent studies have demonstrated podoplanin expression in several tumors, which has been associated with lymph node metastasis and poor prognosis. Podoplanin expression in peritumoral cells such as cancer-associated fibroblasts also correlates with tumor progression in several cancers. However, podoplanin expression and its association with extramammary Paget's disease (EMPD) remain unclear. In this study, we examined whether the presence of podoplanin expression in tumor cells or peritumoral basal keratinocytes correlated with aggressive behavior in patients with EMPD and investigated the mechanisms of podoplanin-mediated tumor invasion in this disorder. Skin samples of 37 patients with EMPD were investigated by immunohistochemical analysis. The functions of podoplanin in keratinocytes were examined in vitro by RT-PCR and with invadopodia gelatin-degradation assays using HaCaT cells. Podoplanin was not identified in tumor cells in all cases. Podoplanin expression in peritumoral basal keratinocytes was observed in 25 patients (67.6%). In in situ EMPD, 50% of cases (9 in 18) exhibited podoplanin-positive keratinocytes, whereas 84.2% (16 in 19) demonstrated positive staining in invasive EMPD (P<0.05). Podoplanin expression in peritumoral keratinocytes was also associated with tumor thickness (P<0.005). By immunohistochemical analysis, podoplanin-positive peritumoral keratinocytes were found to be negative for E-cadherin, one of the major adhesion molecules of keratinocytes, which might contribute to tumor invasion into the dermis through a crack in the basal cell layer induced by down-regulation of cell adhesion therein. We further found that podoplanin-positive keratinocytes exhibited invadopodia, which are thought to function in the migration of cancer cells through tissue barriers, indicating that podoplanin-positive peritumoral basal keratinocytes might assist tumor invasion by degrading the extracellular matrix. The presence of podoplanin expression in

  17. Low power millimeter wave irradiation exerts no harmful effect on human keratinocytes in vitro.

    Science.gov (United States)

    Szabo, Imre; Manning, Michael R; Radzievsky, Alexander A; Wetzel, Michele A; Rogers, Thomas J; Ziskin, Marvin C

    2003-04-01

    Low power millimeter wave (LP-MW) irradiation has been successfully used in clinical practice as an independent and/or supplemental therapy in patients with various diseases. It is still not clear, however, whether exposed skin is directly affected by repeated LP-MW irradiation and whether cells of the epidermis can be activated by the absorbed energy. Keratinocytes, the most numerous component of the epidermis are believed to manifest functional responses to physical stimuli. In this study we analyzed whether LP-MW irradiation modulated the production of chemokines, including RANTES and IP-10 of keratinocytes in vitro. We also investigated whether LP-MW irradiation induces a heat stress reaction in keratinocytes, and stimulates heat shock protein 70 (Hsp70) production. Vital staining of keratinocytes with carboxyfluorescein succinimidyl ester and ethidium bromide was used to analyze the MW effect on the viability of adherent cells. In addition, we studied the effect of LP-MW irradiation on intercellular gap junctional communication in keratinocyte monolayers by Lucifer yellow dye transfer. We found no significant changes in constitutive RANTES and inducible IP-10 production following LP-MW irradiation. LP-MW exposure of keratinocyte monolayers did not alter Hsp70 production, unlike exposure to higher power MWs (HP-MW) or hyperthermia (43 degrees C; 1 h). LP-MW irradiation and hyperthermia did not alter the viability of adherent keratinocytes, while HP-MW irradiation induced cellular damage within the beam area. Finally, we found no alteration in the gap junctional intercellular communication of keratinocytes following LP-MW irradiation, which on the other hand, was significantly increased by hyperthermia. In summary, we detected no harmful effect of LP-MW irradiation on both keratinocyte function and structure in vitro, although these cells were sensitive to higher MW power that developed heat stress reaction and cellular damage. Our results provide further

  18. Longitudinal study of mammary epithelial and fibroblast co-cultures using optical coherence tomography reveals morphological hallmarks of pre-malignancy.

    Directory of Open Access Journals (Sweden)

    Raghav K Chhetri

    Full Text Available The human mammary gland is a complex and heterogeneous organ, where the interactions between mammary epithelial cells (MEC and stromal fibroblasts are known to regulate normal biology and tumorigenesis. We aimed to longitudinally evaluate morphology and size of organoids in 3D co-cultures of normal (MCF10A or pre-malignant (MCF10DCIS.com MEC and hTERT-immortalized fibroblasts from reduction mammoplasty (RMF. This co-culture model, based on an isogenic panel of cell lines, can yield insights to understand breast cancer progression. However, 3D cultures pose challenges for quantitative assessment and imaging, especially when the goal is to measure the same organoid structures over time. Using optical coherence tomography (OCT as a non-invasive method to longitudinally quantify morphological changes, we found that OCT provides excellent visualization of MEC-fibroblast co-cultures as they form ductal acini and remodel over time. Different concentrations of fibroblasts and MEC reflecting reported physiological ratios [1] were evaluated, and we found that larger, hollower, and more aspherical acini were formed only by pre-malignant MEC (MCF10DCIS.com in the presence of fibroblasts, whereas in comparable conditions, normal MEC (MCF10A acini remained smaller and less aspherical. The ratio of fibroblast to MEC was also influential in determining organoid phenotypes, with higher concentrations of fibroblasts producing more aspherical structures in MCF10DCIS.com. These findings suggest that stromal-epithelial interactions between fibroblasts and MEC can be modeled in vitro, with OCT imaging as a convenient means of assaying time dependent changes, with the potential for yielding important biological insights about the differences between benign and pre-malignant cells.

  19. H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

    Energy Technology Data Exchange (ETDEWEB)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan); Akagawa, Mitsugu [Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531 (Japan); Tsuji-Naito, Kentaro, E-mail: knaito@dhc.co.jp [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan)

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.

  20. A review of the pattern of AIDS defining, HIV associated neoplasms and premalignant lesions diagnosed from 2000?2011 at Kenyatta National Hospital, Kenya

    OpenAIRE

    Rogena, Emily A.; Simbiri, Kenneth O.; De Falco, G.; Leoncini, L.; Ayers, Leona; Nyagol, J.

    2015-01-01

    Background Sub-Sahara Africa hosts up to 71?% of all HIV infected people in the world. With this high incidence of Human immunodeficiency virus ( HIV) comes the burden of co-morbidities such as malignant and premalignant lesions. Aids defining malignancies have been listed as Kaposi?s sarcoma, Non-Hodgkin?s lymphoma and invasive squamous cell carcinoma of the cervix. People with HIV/AIDS(PLWAS) have a higher risk of developing these neoplasms than the rest of the population. The pathogenesis ...

  1. Lithospermum erythrorhizon extract protects keratinocytes and fibroblasts against oxidative stress.

    Science.gov (United States)

    Yoo, Hee Geun; Lee, Bong Han; Kim, Wooki; Lee, Jong Suk; Kim, Gun Hee; Chun, Ock K; Koo, Sung I; Kim, Dae-Ok

    2014-11-01

    Oxidative stress damages dermal and epidermal cells and degrades extracellular matrix proteins, such as collagen, ultimately leading to skin aging. The present study evaluated the potential protective effect of the aqueous methanolic extract obtained from Lithospermum erythrorhizon (LE) against oxidative stress, induced by H2O2 and ultraviolet (UV) irradiation, on human keratinocyte (HaCaT) and human dermal fibroblast-neonatal (HDF-n) cells. Exposure of cells to H2O2 or UVB irradiation markedly increased oxidative stress and reduced cell viability. However, pretreatment of cells with the LE extract not only increased cell viability (up to 84.5%), but also significantly decreased oxidative stress. Further, the LE extract downregulated the expression of matrix metalloproteinase-1, an endopeptidase that degrades extracellular matrix collagen. In contrast, treatment with the LE extract did not affect the expression of procollagen type 1 in HDF-n cells exposed to UVA irradiation. Thirteen phenolic compounds, including derivatives of shikonin and caffeic acid, were identified by ultrahigh-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. These results suggest that LE-derived extracts may protect oxidative-stress-induced skin aging by inhibiting degradation of skin collagen, and that this protection may derive at least in part from the antioxidant phenolics present in these extracts. Further studies are warranted to determine the potential utility of LE-derived extracts in both therapeutic and cosmetic applications.

  2. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xin [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Dai, Hui [Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang Province (China); Zhuang, Binyu [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Chai, Li; Xie, Yanguang [Institute of Dermatology of Heilongjiang Province, Harbin, 150001, Heilongjiang Province (China); Li, Yuzhen, E-mail: liyuzhen@medmail.com.cn [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China)

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.

  3. Lactobacillus rhamnosus GG Inhibits the Toxic Effects of Staphylococcus aureus on Epidermal Keratinocytes

    Science.gov (United States)

    Mohammedsaeed, Walaa; McBain, Andrew J.; Cruickshank, Sheena M.

    2014-01-01

    Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 108 CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion. PMID:25015889

  4. Geraniin supplementation increases human keratinocyte proliferation in serum-free culture

    Directory of Open Access Journals (Sweden)

    Indra Kusuma

    2013-04-01

    Full Text Available Background Various products used in cellular therapy utilize tissue culture techniques requiring keratinocyte culture. An efficient and clinically acceptable keratinocyte culture system requires supplements with mitogenic activity. Geraniin is a phytochemical with the potential as a supplement for expansion culture of keratinocytes. The objective of the present study was to verify the mitogenic activity of geraniin on human keratinocytes. Methods This was an experimental study using two samples of human foreskin obtained by circumcision of a male child. Epidermal keratinocytes were isolated from the foreskin samples and were divided into paired groups, comprising intervention and control groups. The intervention groups were cultured with geraniin supplementation, whereas the control groups with standard supplements, without the addition of geraniin. Mitochondrial activity of the cells was evaluated by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide (MTT proliferation assay. Absorbance values in each of the groups was measured at 450 nm. Data analysis was performed with the paired t-test. Results Geraniin supplementation significantly increased the keratinocyte proliferation rates at dosages of 0.8 to 3.1 µM. An increase of 57% in the proliferation rate was obtained at a dosage of 1.6 µM, while at a dosage of 12.5 µM toxic effects were starting to appear. Geraniin presumably causes increased cellular energy status, resulting in increased proliferation rates. Conclusion The findings in this study provide evidence in support of the utilization of geraniin as a supplement for expansion culture of keratinocytes. Further studies may presumably identify the molecules acting as geraniin receptors and the intracellular mechanisms underlying the increase in proliferation rates.

  5. Lactobacillus rhamnosus GG inhibits the toxic effects of Staphylococcus aureus on epidermal keratinocytes.

    Science.gov (United States)

    Mohammedsaeed, Walaa; McBain, Andrew J; Cruickshank, Sheena M; O'Neill, Catherine A

    2014-09-01

    Few studies have evaluated the potential benefits of the topical application of probiotic bacteria or material derived from them. We have investigated whether a probiotic bacterium, Lactobacillus rhamnosus GG, can inhibit Staphylococcus aureus infection of human primary keratinocytes in culture. When primary human keratinocytes were exposed to S. aureus, only 25% of the keratinocytes remained viable following 24 h of incubation. However, in the presence of 10(8) CFU/ml of live L. rhamnosus GG, the viability of the infected keratinocytes increased to 57% (P = 0.01). L. rhamnosus GG lysates and spent culture fluid also provided significant protection to keratinocytes, with 65% (P = 0.006) and 57% (P = 0.01) of cells, respectively, being viable following 24 h of incubation. Keratinocyte survival was significantly enhanced regardless of whether the probiotic was applied in the viable form or as cell lysates 2 h before or simultaneously with (P = 0.005) or 12 h after (P = 0.01) S. aureus infection. However, spent culture fluid was protective only if added before or simultaneously with S. aureus. With respect to mechanism, both L. rhamnosus GG lysate and spent culture fluid apparently inhibited adherence of S. aureus to keratinocytes by competitive exclusion, but only viable bacteria or the lysate could displace S. aureus (P = 0.04 and 0.01, respectively). Furthermore, growth of S. aureus was inhibited by either live bacteria or lysate but not spent culture fluid. Together, these data suggest at least two separate activities involved in the protective effects of L. rhamnosus GG against S. aureus, growth inhibition and reduction of bacterial adhesion. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  6. Matrix metalloproteinase 7 restrains Helicobacter pylori-induced gastric inflammation and premalignant lesions in the stomach by altering macrophage polarization.

    Science.gov (United States)

    Krakowiak, M S; Noto, J M; Piazuelo, M B; Hardbower, D M; Romero-Gallo, J; Delgado, A; Chaturvedi, R; Correa, P; Wilson, K T; Peek, R M

    2015-04-02

    results suggest that MMP7 exerts a restrictive role on H. pylori-induced gastric injury and the development of premalignant lesions by suppressing M1 macrophage polarization.

  7. Dysregulation of mitotic machinery genes precedes genome instability during spontaneous pre-malignant transformation of mouse ovarian surface epithelial cells

    Directory of Open Access Journals (Sweden)

    Ulises Urzúa

    2016-10-01

    Full Text Available Abstract Background Based in epidemiological evidence, repetitive ovulation has been proposed to play a role in the origin of ovarian cancer by inducing an aberrant wound rupture-repair process of the ovarian surface epithelium (OSE. Accordingly, long term cultures of isolated OSE cells undergo in vitro spontaneous transformation thus developing tumorigenic capacity upon extensive subcultivation. In this work, C57BL/6 mouse OSE (MOSE cells were cultured up to passage 28 and their RNA and DNA copy number profiles obtained at passages 2, 5, 7, 10, 14, 18, 23, 25 and 28 by means of DNA microarrays. Gene ontology, pathway and network analyses were focused in passages earlier than 20, which is a hallmark of malignancy in this model. Results At passage 14, 101 genes were up-regulated in absence of significant DNA copy number changes. Among these, the top-3 enriched functions (>30 fold, adj p < 0.05 comprised 7 genes coding for centralspindlin, chromosome passenger and minichromosome maintenance protein complexes. The genes Ccnb1 (Cyclin B1, Birc5 (Survivin, Nusap1 and Kif23 were the most recurrent in over a dozen GO terms related to the mitotic process. On the other hand, Pten plus the large non-coding RNAs Malat1 and Neat1 were among the 80 down-regulated genes with mRNA processing, nuclear bodies, ER-stress response and tumor suppression as relevant terms. Interestingly, the earliest discrete segmental aneuploidies arose by passage 18 in chromosomes 7, 10, 11, 13, 15, 17 and 19. By passage 23, when MOSE cells express the malignant phenotype, the dysregulated gene expression repertoire expanded, DNA imbalances enlarged in size and covered additional loci. Conclusion Prior to early aneuploidies, overexpression of genes coding for the mitotic apparatus in passage-14 pre-malignant MOSE cells indicate an increased proliferation rate suggestive of replicative stress. Concomitant down-regulation of nuclear bodies and RNA processing related genes

  8. Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

    Directory of Open Access Journals (Sweden)

    Stefanie Löffek

    Full Text Available The hemidesmosomal transmembrane component collagen XVII (ColXVII plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1⁻/⁻ keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1⁻/⁻ keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

  9. Effective inhibition of melanosome transfer to keratinocytes by lectins and niacinamide is reversible.

    Science.gov (United States)

    Greatens, Amanda; Hakozaki, Tomohiro; Koshoffer, Amy; Epstein, Howard; Schwemberger, Sandy; Babcock, George; Bissett, Donald; Takiwaki, Hirotsugu; Arase, Seiji; Wickett, R Randall; Boissy, Raymond E

    2005-07-01

    Skin pigmentation results in part from the transfer of melanized melanosomes synthesized by melanocytes to neighboring keratinocytes. Plasma membrane lectins and their glycoconjugates expressed by these epidermal cells are critical molecules involved in this transfer process. In addition, the derivative of vitamin B(3), niacinamide, can inhibit melanosome transfer and induce skin lightening. We investigated the effects of these molecules on the viability of melanocytes and keratinocytes and on the reversibility of melanosome-transfer inhibition induced by these agents using an in vitro melanocyte-keratinocyte coculture model system. While lectins and neoglycoproteins could induce apoptosis in a dose-dependent manner to melanocytes or keratinocytes in monoculture, similar dosages of the lectins, as opposed to neoglycoproteins, did not induce apoptosis to either cell type when treated in coculture. The dosages of lectins and niacinamide not affecting cell viability produced an inhibitory effect on melanosome transfer, when used either alone or together in cocultures of melanocytes-keratinocytes. Cocultures treated with lectins or niacinamide resumed normal melanosome transfer in 3 days after removal of the inhibitor, while cocultures treated with a combination of lectins and niacinamide demonstrated a lag in this recovery. Subsequently, we assessed the effect of niacinamide on facial hyperpigmented spots using a vehicle-controlled, split-faced design human clinical trial. Topical application of niacinamide resulted in a dose-dependent and reversible reduction in hyperpigmented lesions. These results suggest that lectins and niacinamide at concentrations that do not affect cell viability are reversible inhibitors of melanosome transfer.

  10. Grading keratinocyte atypia in actinic keratosis: a correlation of reflectance confocal microscopy and histopathology.

    Science.gov (United States)

    Pellacani, G; Ulrich, M; Casari, A; Prow, T W; Cannillo, F; Benati, E; Losi, A; Cesinaro, A M; Longo, C; Argenziano, G; Soyer, H P

    2015-11-01

    Actinic Keratosis (AK) is the clinical manifestation of cutaneous dysplasia of epidermal keratinocytes, with progressive trend towards squamous cell carcinoma. To evaluate the strength of the correlation between keratinocyte atypia, as detected by Reflectance Confocal Microscopy (RCM) and histopathology, and to develop a more objective atypia grading scale for RCM quantification, through a discrete ranking. A total of 48 AKs and two control areas (photodamaged and non-photodamaged skin) were selected for this study. All these areas were documented by RCM and biopsied for histopathology. One representative image of the epidermis was selected for RCM and for histopathology and used for side-by-side comparison with purpose written software. The assessor chose which of two images displayed more keratinocyte atypia, and an ordered list from the image showing the least to the most keratinocyte atypia was generated. Three evaluations were obtained for RCM and two for histopathology. Good interobserver correlation was obtained for RCM and histopathology grading, with high concordance between RCM and histopathology grading. Expert rater scan consistently distinguish different grades of cytological atypia. Non-invasive RCM data from in vivo imaging can be graded for keratinocyte atypia, comparable to histopathological grading. © 2015 European Academy of Dermatology and Venereology.

  11. Production of superoxide anions by keratinocytes initiates P. acnes-induced inflammation of the skin.

    Directory of Open Access Journals (Sweden)

    Philippe A Grange

    2009-07-01

    Full Text Available Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes, a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O2(*-, were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O2(*- was produced by NAD(PH oxidase through activation of the scavenger receptor CD36. O2(*- was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O2(*- abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O2(*- with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O2(*- production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.

  12. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    International Nuclear Information System (INIS)

    Bernard, Eric; Hamel, Rodolphe; Neyret, Aymeric; Ekchariyawat, Peeraya; Molès, Jean-Pierre; Simmons, Graham; Chazal, Nathalie; Desprès, Philippe

    2015-01-01

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV

  13. GRHL3 binding and enhancers rearrange as epidermal keratinocytes transition between functional states.

    Directory of Open Access Journals (Sweden)

    Rachel Herndon Klein

    2017-04-01

    Full Text Available Transcription factor binding, chromatin modifications and large scale chromatin re-organization underlie progressive, irreversible cell lineage commitments and differentiation. We know little, however, about chromatin changes as cells enter transient, reversible states such as migration. Here we demonstrate that when human progenitor keratinocytes either differentiate or migrate they form complements of typical enhancers and super-enhancers that are unique for each state. Unique super-enhancers for each cellular state link to gene expression that confers functions associated with the respective cell state. These super-enhancers are also enriched for skin disease sequence variants. GRHL3, a transcription factor that promotes both differentiation and migration, binds preferentially to super-enhancers in differentiating keratinocytes, while during migration, it binds preferentially to promoters along with REST, repressing the expression of migration inhibitors. Key epidermal differentiation transcription factor genes, including GRHL3, are located within super-enhancers, and many of these transcription factors in turn bind to and regulate super-enhancers. Furthermore, GRHL3 represses the formation of a number of progenitor and non-keratinocyte super-enhancers in differentiating keratinocytes. Hence, chromatin relocates GRHL3 binding and enhancers to regulate both the irreversible commitment of progenitor keratinocytes to differentiation and their reversible transition to migration.

  14. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  15. Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes.

    Science.gov (United States)

    Sancilio, Silvia; Di Staso, Silvio; Sebastiani, Stefano; Centurione, Lucia; Di Girolamo, Nick; Ciancaglini, Marco; Di Pietro, Roberta

    2017-01-01

    Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody) and CD140 (anti-fibroblast transmembrane glycoprotein antibody) expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

  16. Curcuma longa Is Able to Induce Apoptotic Cell Death of Pterygium-Derived Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Silvia Sancilio

    2017-01-01

    Full Text Available Pterygium is a relatively common eye disease that can display an aggressive clinical behaviour. To evaluate the in vitro effects of Curcuma longa on human pterygium-derived keratinocytes, specimens of pterygium from 20 patients undergoing pterygium surgical excision were collected. Pterygium explants were put into culture and derived keratinocytes were treated with an alcoholic extract of 1.3% Curcuma longa in 0.001% Benzalkonium Chloride for 3, 6, and 24 h. Cultured cells were examined for CAM5.2 (anti-cytokeratin antibody and CD140 (anti-fibroblast transmembrane glycoprotein antibody expression between 3th and 16th passage to assess cell homogeneity. TUNEL technique and Annexin-V/PI staining in flow cytometry were used to detect keratinocyte apoptosis. We showed that Curcuma longa exerts a proapoptotic effect on pterygium-derived keratinocytes already after 3 h treatment. Moreover, after 24 h treatment, Curcuma longa induces a significant increase in TUNEL as well as Annexin-V/PI positive cells in comparison to untreated samples. Our study confirms previous observations highlighting the expression, in pterygium keratinocytes, of nuclear VEGF and gives evidence for the first time to the expression of nuclear and cytoplasmic VEGF-R1. All in all, these findings suggest that Curcuma longa could have some therapeutic potential in the treatment and prevention of human pterygium.

  17. Activation of caspase-9 is required for UV-induced apoptosis of human keratinocytes.

    Science.gov (United States)

    Sitailo, Leonid A; Tibudan, Shalini S; Denning, Mitchell F

    2002-05-31

    UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH(3))-His-Asp(OCH(3))-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen.

  18. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, Eric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Hamel, Rodolphe [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Neyret, Aymeric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Ekchariyawat, Peeraya [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Molès, Jean-Pierre [INSERM U1058, UM1, CHU Montpellier (France); Simmons, Graham [Blood Systems Research Institute, San Francisco, CA 94118 (United States); Chazal, Nathalie [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Desprès, Philippe [Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris (France); and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  19. Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

    Science.gov (United States)

    Barr, Travis P.; Albrecht, Phillip J.; Hou, Quanzhi; Mongin, Alexander A.; Strichartz, Gary R.; Rice, Frank L.

    2013-01-01

    Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease. PMID:23457608

  20. DNA repair response in human epidermal keratinocytes from donors of different age

    Energy Technology Data Exchange (ETDEWEB)

    Liu, S.C.; Parsons, C.S.; Hanawalt, P.C.

    1982-11-01

    We have compared the excision-repair and growth properties of epidermal keratinocytes from humans of different ages. Keratinocytes isolated from newborn and adult abdominal skin at autopsy were cultured on collagen gels. Repair replication was assayed by the 5-bromodeoxyuridine density-labeling method following ultraviolet (UV) irradiation (254 nm) of the cultures. The keratinocytes from newborn donors proliferated more rapidly and attained a higher concentration at confluence than did those from aged donors. Semiconservative DNA replication was inhibited by UV radiation to an equal extent in cell cultures from newborns and adults. After a UV dose of 13 J/m2, the time course of DNA repair was similar for the respective cultures. Furthermore, there were no significant differences in the time course of repair for keratinocytes in the proliferative or the plateau phase of growth. The dose-response curves for repair replication in cells from both young and old donors maximized at about 50 J/m2 but the attenuation in repair at higher doses appeared somewhat greater in cells from older donors. We conclude that no significant age-related differences exist in the rate and extent of the repair-replication response of human epidermal keratinocytes to UV-radiation damage in DNA. However, it remains to be determined whether other cellular recovery responses to damaged DNA are also relatively unrelated to age.

  1. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    Science.gov (United States)

    Ramos-Jerz, Maria del R.; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M.

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes. PMID:24371457

  2. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Maria del R. Ramos-Jerz

    2013-01-01

    Full Text Available Methanolic avocado (Persea americana Mill., Lauraceae seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK and normal human dermal fibroblasts (NHDF. The methanol-water partition (M from avocado seeds and HSCCC fraction 3 (M.3 were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  3. Reproductive Tract infections and Premalignant Lesions of Cervix: Evidence from Women Presenting at the Cancer Detection Centre of the Indian Cancer Society, Delhi, 2000-2012.

    Science.gov (United States)

    Dey, Subhojit; Pahwa, Parika; Mishra, Arti; Govil, Jyotsna; Dhillon, Preet K

    2016-10-01

    Burden of cervical cancer (CC) is highest for women in low- and middle-income countries (LMICs). Human papillomavirus (HPV) is implicated as the necessary cause of CC although a number of other factors aid the long process of CC development. One among them is the presence of reproductive tract infections (RTIs). This study investigated the associations between RTIs and CC from India. This study utilized secondary data from the Cancer Detection Centre of the ICS, Delhi. Data were accessed from MS access database and were analyzed using MS Excel and SPSS 16.0. Multivariate analysis using unconditional logistic regression produced odds ratios (ORs) and 95 % confidence intervals (CIs). This study used data from 11,427 women over a period of 2000-2012. Women with RTIs had Candida, Trichomonas vaginalis (TV) or coccoid infections with all having similar prevalence (~4-5 %). 9.4 % of women had premalignant lesions of cervix; ASCUS was most common (7.9 %) followed by LSIL (1.3 %). TV was significantly associated with ASCUS, LSIL and all premalignant lesions of cervix (P access to gynecologists in LMICs lead to frequent and persistent RTIs which aid and abet HPV infection and CC occurrence. These also need to be addressed to reduce CC and RTIs among women in LMICs.

  4. CtBP1 overexpression in keratinocytes perturbs skin homeostasis.

    Science.gov (United States)

    Deng, Hui; Li, Fulun; Li, Hong; Deng, Yu; Liu, Jing; Wang, Donna; Han, Gangwen; Wang, Xiao-Jing; Zhang, Qinghong

    2014-05-01

    Carboxyl-terminal-binding protein-1 (CtBP1) is a transcriptional corepressor with multiple in vitro targets, but its in vivo functions are largely unknown. We generated keratinocyte-specific CtBP1 transgenic mice with a keratin-5 promoter (K5.CtBP1) to probe the pathological roles of CtBP1. At transgene expression levels comparable to endogenous CtBP1 in acute skin wounds, the K5.CtBP1 epidermis displayed hyperproliferation, loss of E-cadherin, and failed terminal differentiation. Known CtBP1 target genes associated with these processes, e.g., p21, Brca1, and E-cadherin, were downregulated in K5.CtBP1 skin. Surprisingly, K5.CtBP1 pups also exhibited a hair loss phenotype. We found that expression of the Distal-less 3 (Dlx3), a critical regulator of hair follicle differentiation and cycling, was decreased in K5.CtBP1 mice. Molecular studies revealed that CtBP1 directly suppressed Dlx3 transcription. Consistently, K5.CtBP1 mice displayed abnormal hair follicles with decreased expression of Dlx3 downstream targets Gata3, Hoxc13, and hair keratins. In summary, this CtBP1 transgenic model provides in vivo evidence for certain CtBP1 functions predicted from in vitro studies, reveals--to our knowledge--previously unreported functions and transcriptional activities of CtBP1 in the context of epithelial-mesenchymal interplay, and suggests that CtBP1 has a pathogenic role in hair follicle morphogenesis and differentiation.

  5. Escherichia coli ghosts promote innate immune responses in human keratinocytes.

    Science.gov (United States)

    Abtin, Arby; Kudela, Pavol; Mayr, Ulrike Beate; Koller, Verena Juliana; Mildner, Michael; Tschachler, Erwin; Lubitz, Werner

    2010-09-10

    Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin. Copyright © 2010 Elsevier Inc. All rights reserved.

  6. Mechanisms of Mitochondrial Damage in Keratinocytes by Pemphigus Vulgaris Antibodies*

    Science.gov (United States)

    Kalantari-Dehaghi, Mina; Chen, Yumay; Deng, Wu; Chernyavsky, Alex; Marchenko, Steve; Wang, Ping H.; Grando, Sergei A.

    2013-01-01

    The development of nonhormonal treatment of pemphigus vulgaris (PV) has been hampered by a lack of clear understanding of the mechanisms leading to keratinocyte (KC) detachment and death in pemphigus. In this study, we sought to identify changes in the vital mitochondrial functions in KCs treated with the sera from PV patients and healthy donors. PV sera significantly increased proton leakage from KCs, suggesting that PV IgGs increase production of reactive oxygen species. Indeed, measurement of intracellular reactive oxygen species production showed a drastic increase of cell staining in response to treatment by PV sera, which was confirmed by FACS analysis. Exposure of KCs to PV sera also caused dramatic changes in the mitochondrial membrane potential detected with the JC-1 dye. These changes can trigger the mitochondria-mediated intrinsic apoptosis. Although sera from different PV patients elicited unique patterns of mitochondrial damage, the mitochondria-protecting drugs nicotinamide (also called niacinamide), minocycline, and cyclosporine A exhibited a uniform protective effect. Their therapeutic activity was validated in the passive transfer model of PV in neonatal BALB/c mice. The highest efficacy of mitochondrial protection of the combination of these drugs found in mitochondrial assay was consistent with the ability of the same drug combination to abolish acantholysis in mouse skin. These findings provide a theoretical background for clinical reports of the efficacy of mitochondria-protecting drugs in PV patients. Pharmacological protection of mitochondria and/or compensation of an altered mitochondrial function may therefore become a novel approach to development of personalized nonhormonal therapies of patients with this potentially lethal autoimmune blistering disease. PMID:23599429

  7. Effects of titanium dioxide nanoparticles on human keratinocytes.

    Science.gov (United States)

    Wright, Clayton; Iyer, Anand Krishnan V; Wang, Liying; Wu, Nianqiang; Yakisich, Juan S; Rojanasakul, Yon; Azad, Neelam

    2017-01-01

    Titanium dioxide (TiO 2 ) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO 2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO 2 nanoparticles (H 2 TiO 7 ) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO 2 particles varying in size (Fine, Ultrafine and H 2 TiO 7 ) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO 2 ; however, there is no consistent effect on cell viability and proliferation with either of these TiO 2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO 2 , we did not observe any significant effect of UV-C exposure combined with TiO 2 treatment on HaCaTs. Furthermore, TiO 2 -treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H 2 TiO 7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO 2 .

  8. Transplantation of autologous keratinocyte suspension in fibrin matrix to chronic venous leg ulcers: improved long-term healing after removal of the fibrin carrier.

    NARCIS (Netherlands)

    Hartmann, A.; Quist, J.; Hamm, H.; Brocker, E.B.; Friedl, P.H.A.

    2008-01-01

    BACKGROUND: The transplantation of keratinocytes suspended in fibrin carrier represents a candidate regimen for chronic ulcer treatment in an outpatient setting. We evaluated the integration and survival of autologous individualized keratinocytes applied within fibrin matrix onto chronic venous leg

  9. RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

    International Nuclear Information System (INIS)

    Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara

    2010-01-01

    We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-α-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.

  10. Testosterone Stimulates Duox1 Activity through GPRC6A in Skin Keratinocytes*

    Science.gov (United States)

    Ko, Eunbi; Choi, Hyun; Kim, Borim; Kim, Minsun; Park, Kkot-Nara; Bae, Il-Hong; Sung, Young Kwan; Lee, Tae Ryong; Shin, Dong Wook; Bae, Yun Soo

    2014-01-01

    Testosterone is an endocrine hormone with functions in reproductive organs, anabolic events, and skin homeostasis. We report here that GPRC6A serves as a sensor and mediator of the rapid action of testosterone in epidermal keratinocytes. The silencing of GPRC6A inhibited testosterone-induced intracellular calcium ([Ca2+]i) mobilization and H2O2 generation. These results indicated that a testosterone-GPRC6A complex is required for activation of Gq protein, IP3 generation, and [Ca2+]i mobilization, leading to Duox1 activation. H2O2 generation by testosterone stimulated the apoptosis of keratinocytes through the activation of caspase-3. The application of testosterone into three-dimensional skin equivalents increased the apoptosis of keratinocytes between the granular and stratified corneum layers. These results support an understanding of the molecular mechanism of testosterone-dependent apoptosis in which testosterone stimulates H2O2 generation through the activation of Duox1. PMID:25164816

  11. Micronucleus formation in cultured human keratinocytes following exposure to mitomycin C and cyclophosphamide.

    Science.gov (United States)

    van Pelt, F N; Haring, R M; Overkamp, M J; Weterings, P J

    1991-02-01

    A method is described to investigate the induction of micronuclei in cultured human keratinocytes after short-term exposure to known clastogenic agents. The cytokinesis-block method was applied to facilitate the scoring of micronucleated cells. Mitomycin C, a direct-acting compound, caused a 5-20-fold increase in micronuclei over the controls at the highest concentration tested (1 microgram/ml). Cyclophosphamide, an agent requiring metabolic activation, did not induce the formation of micronuclei in cultured keratinocytes. However, after pretreatment of the keratinocyte cultures with Aroclor 1254 for 72 h, exposure to cyclophosphamide resulted in a 3-fold increase in micronucleus frequency over the controls. No cytogenetic effect of Aroclor 1254 was observed in control experiments.

  12. MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

    International Nuclear Information System (INIS)

    MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.

  13. H-Ras activation promotes cytoplasmic accumulation and phosphoinositide 3-OH kinase association of beta-catenin in epidermal keratinocytes

    DEFF Research Database (Denmark)

    Espada, J; Pérez-Moreno, M; Braga, V M

    1999-01-01

    keratinocytes. Microinjection or stable expression of V12Ras into keratinocytes promotes the loss of E-cadherin and alpha-catenin and relocalization of beta-catenin to the cytoplasm and nucleus. Moreover, these effects are dependent on PI3K (phosphoinositide 3-OH kinase) activity. Interestingly, a strong...

  14. MiR-217 is down-regulated in psoriasis and promotes keratinocyte differentiation via targeting GRHL2

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Haigang; Hou, Liyue; Liu, Jingjing; Li, Zhiming, E-mail: lizm_1001@sina.com

    2016-02-26

    MiR-217 is a well-known tumor suppressor, and its down-regulation has been shown in a wide range of solid and leukaemic cancers. However, the biological role of miR-217 in psoriasis pathogenesis, especially in keratinocyte hyperproliferation and differentiation, is not clearly understood. In this study, we found the expression of miR-217 was markedly down-regulated in psoriasis keratinocytes of psoriatic patients. In addition, overexpression of miR-217 inhibited the proliferation and promoted the differentiation of primary human keratinocytes. On the contrary, inhibition of endogenous miR-217 increased cell proliferation and delayed differentiation. Furthermore, Grainyhead-like 2 (GRHL2) was identified as a direct target of miR-217 by luciferase reporter assay. The expression of miR-217 and GRHL2 was inversely correlated in both transfected keratinocytes and in psoriasis lesional skin. Moreover, knocking down GRHL2 expression by siRNA enhanced keratinocyte differentiation. Taken together, our results demonstrate a role for miR-217 in the regulation of keratinocyte differentiation, partially through the regulation of GRHL2. - Highlights: • miR-217 is down-regulated in psoriasis skin lesions. • miR-217 inhibits the proliferation and promotes differentiation of keratinocytes. • GRHL2 is a novel target of miR-217 in keratinocytes. • GRHL2 is up-regulated and inversely correlated with miR-217 in psoriasis skin lesions.

  15. Concentration-dependent effect of platelet-rich plasma on keratinocyte and fibroblast wound healing.

    Science.gov (United States)

    Xian, Law Jia; Chowdhury, Shiplu Roy; Bin Saim, Aminuddin; Idrus, Ruszymah Bt Hj

    2015-03-01

    Platelet-rich plasma (PRP) has been found to contain a high concentration of growth factors that are present during the process of healing. Studies conducted found that application of PRP accelerates wound healing. In this study, we characterized the skin cell suspension harvested using the co-isolation technique and evaluated the effects of PRP (10% and 20%, v/v) on co-cultured keratinocytes and fibroblasts in terms of wound healing. Human keratinocytes and fibroblasts were harvested via co-isolation technique and separated via differential trypsinization. These cells were then indirectly co-cultured in medium supplemented with 10% or 20% PRP for 3 days without medium change for analysis of wound-healing potential. The wound-healing potential of keratinocytes and fibroblasts was evaluated in terms of growth property, migratory property, extracellular matrix gene expression and soluble factor secretion. The co-isolation technique yielded a skin cell population dominated by fibroblasts and keratinocytes, with a small amount of melanocytes. Comparison between the 10% and 20% PRP cultures showed that the 10% PRP culture exhibited higher keratinocyte apparent specific growth rate, and secretion of hepatocyte growth factor, monocyte chemoattractant protein-1, epithelial-derived neutrophil-activating protein 78 and vascular endothelial growth factor A, whereas the 20% PRP culture has significantly higher collagen type 1 and collagen type 3 expressions and produced more granulocyte-macrophage colony-stimulating factor. PRP concentration modulates keratinocyte and fibroblast wound healing potential, whereby the 10% PRP promotes wound remodeling, whereas the 20% PRP enhances inflammation and collagen deposition. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  16. Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

    Science.gov (United States)

    Hau, Carren S; Kanda, Naoko; Makimura, Koichi; Watanabe, Shinichi

    2014-02-01

    Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12,14) -prostaglandin J2 (15d-PGJ2 ), a prostaglandin D2 metabolite. Antimycotics increased the release of 15d-PGJ2 from keratinocytes. The antimycotic-induced suppression of CXCL10 production and STAT1 activity was counteracted by a lipocalin-type prostaglandin D synthase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine may suppress the M. restricta-induced production of CXCL10 by inhibiting STAT1 through an increase in 15d-PGJ2 production in keratinocytes. These antimycotics may block the Th1-mediated inflammation triggered by Malassezia in the chronic phase of atopic dermatitis. © 2014

  17. The Effects of Antifungal Azoles on Inflammatory Cytokine Production in Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    K Zomorodian

    2008-04-01

    Full Text Available ABSTRACT: Introduction & Objective: Azoles drugs are being used successfully in treatment of fungal infections. Recently, immunosuppressive effects of some of these agents have been reported. Keratinocytes, as the major cells of the skin, have an important role in innate immunity against pathogenic agents. Considering the scanty of information about the effects of azoles on immune responces, this study was conducted to assess the expression and secretion of inflammatory cytokines in keratinocytes following treatment with azole drugs. Materials & Methods: This is an exprimental study conducted in in molecular biology division in Tehran University of Medical Sciences and Immunodermatology Department in Vienna Medical University. Primery keratinocytes were cultured and treated with different concentrations of fluconazole, itraconazole, ketoconazole and griseofulvin. Secreted IL1, IL6 and TNF-α by keratinocytes in culture supernatant were measured by quantitative enzyme immunoassay technique. Moreover, expression of the genes encoding IL1 and IL8 was evaluated by Real Time-PCR. Results: Treatment of keratinocytes with different concentrations of fluconazole and low concentration of ketoconazole resulted in decrease in IL1 secretion, but Itraconazole and griseofulvin did not show such an effect at the same concentrations. In addition, none of the examined drugs had an effect on secretion level of IL6 and TNF-α. Quantitative analysis of IL1 and IL8 encoding genes revealed that transcription on these genes might be suppressed following treatment with fluconazole or ketoconazole. Conclusion: Fluconazole and ketoconazole might modulate the expression and secretion of IL1 and IL8 and affect the direction of immune responses induced by keratinocytes

  18. The stress caused by nitrite with titanium dioxide nanoparticles under UVA irradiation in human keratinocyte cell

    International Nuclear Information System (INIS)

    Tu, Min; Huang, Yi; Li, Hai-Ling; Gao, Zhong-Hong

    2012-01-01

    Highlights: ► Nitrite increased photo-toxicity of nano-TiO 2 on human keratinocyte cells in a dose-dependant manner. ► Morphological study suggested the cell death may be mediated by apoptosis inducing factor. ► Protein nitration was generated in the cells, and the most abundant nitrated protein was identified as cystatin-A. ► Tyr35 was the most likely site to be nitrated in cystatin-A. -- Abstract: Our previous work found that in the presence of nitrite, titanium dioxide nanoparticles can cause protein tyrosine nitration under UVA irradiation in vivo. In this paper, the human keratinocyte cells was used as a skin cell model to further study the photo-toxicity of titanium dioxide nanoparticles when nitrite was present. The results showed that nitrite increased the photo-toxicity of titanium dioxide in a dose-dependant manner, and generated protein tyrosine nitration in keratinocyte cells. Morphological study of keratinocyte cells suggested a specific apoptosis mediated by apoptosis inducing factor. It was also found the main target nitrated in cells was cystatin-A, which expressed abundantly in cytoplasm and functioned as a cysteine protease inhibitor. The stress induced by titanium dioxide with nitrite under UVA irradiation in human keratinocyte cells appeared to trigger the apoptosis inducing factor mediated cell death and lose the inhibition of active caspase by cystatin-A. We conclude that nitrite can bring new damage and stress to human keratinocyte cells with titanium dioxide nanoparticles under UVA irradiation.

  19. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    Energy Technology Data Exchange (ETDEWEB)

    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of ' Tor Vergata' , Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133 (Italy); Istituto Dermopatico dell' Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Laboratory of Biochemistry c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly/Seine (France); Melino, G., E-mail: gerry.melino@uniroma2.it [University of ' Tor Vergata' , Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133 (Italy); Istituto Dermopatico dell' Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Laboratory of Biochemistry c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); Association Cell Death and Differentiation c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  20. Vitamin D derivatives enhance cytotoxic effects of H2O2 or cisplatin on human keratinocytes

    Science.gov (United States)

    Anna, Piotrowska; Justyna, Wierzbicka; Tomasz, Ślebioda; Michał, Woźniak; Robert, C. Tuckey; Andrzej, T. Slominski; Michał, A. Żmijewski

    2016-01-01

    Although the skin production of vitamin D is initiated by ultraviolet radiation type B (UVB), the role vitamin D plays in antioxidative or pro-oxidative responses remains to be elucidated.. We have used immortalized human HaCaT keratinocytes as a model of proliferating epidermal cells to test the influence of vitamin D on cellular response to H2O2 or the anti-cancer drug, cisplatin. Incubation of keratinocytes with 1,25(OH)2D3 or its low calcemic analogues, 20(OH)D3, 21(OH)pD or calcipotriol, sensitized cells to ROS resulting in more potent inhibition of keratinocyte proliferation by H2O2 in the presence of vitamin D compounds. These results were supported by cell cycle and apoptosis analyses, and measurement of the mitochondrial transmembrane potentials (MMP), however some unique properties of individual secosteroids were observed. Furthermore, in HaCaT keratinocytes treated with H2O2, 1,25(OH)2D3, 21(OH)pD and calcipotriol stimulated the expression of SOD1 and CAT genes, but not SOD2, indicating a possible role of mitochondria in ROS-modulated cell death. 1,25(OH)2D3 also showed a short-term, protective effect on HaCaT keratinocytes, as exemplified by the inhibition of apoptosis and the maintenance of MMP. However, with prolonged incubation with H2O2 or cisplatin, 1,25(OH)2D3 caused an acceleration in the death of the keratinocytes. Therefore, we propose that lead vitamin D derivatives can protect the epidermis against neoplastic transformation secondary to oxidative or UV-induced stress through activation of vitamin D-signaling. Furthermore, our data suggest that treatment with low calcemic vitamin D analogs or the maintenance of optimal level of vitamin D by proper supplementation, can enhance the anticancer efficacy of cisplatin PMID:27083311

  1. Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

    DEFF Research Database (Denmark)

    Dabelsteen, S; Wandall, H H; Grøn, B

    1997-01-01

    Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expres......Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF m...

  2. Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

    Science.gov (United States)

    Noguchi, Fumihito; Nakajima, Takeshi; Inui, Shigeki; Reddy, Janardan K; Itami, Satoshi

    2014-01-01

    MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair

  3. Alteration of skin wound healing in keratinocyte-specific mediator complex subunit 1 null mice.

    Directory of Open Access Journals (Sweden)

    Fumihito Noguchi

    Full Text Available MED1 (Mediator complex subunit 1 is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/- that develop epidermal hyperplasia. Herein, to investigate the function(s of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/- and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/- mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/- mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/- keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/- keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/- keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/- keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/- mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/- mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/- mice, indicating a decreased contribution of hair

  4. [Evaluation of the cytotoxicity of antiseptics used in current practice on cultures of fibroblasts and keratinocytes].

    Science.gov (United States)

    Fabreguette, A; Zhi Hua, S; Lasne, F; Damour, O

    1994-11-01

    Infection is the greatest problem in burn patients and topical antiseptics must be chosen with great care especially when cultured skin is grafted. We examined the cytotoxicity of 6 antiseptics commonly used on cultured human fibroblasts and keratinocytes. Cultured cells were exposed for 15 min to Hibitane (chlorhexidine), Biseptine (chlorhexidine + benzalkonium chloride + benzylic alcool), dermic Betadine (polvidone iodine + nonoxinol), scrub Betadine (polyvidone iodine + quaternary ammonium) and gynecologic Betadine (polyvidone iodine). The cell viability was determined using the MTT test. At therapeutic concentration all the antiseptics were cytotoxic for fibroblasts and keratinocytes. The data suggest that the antiseptics must be used in function of the time of the grafting of the cultured epithelium.

  5. Vitamin D protects keratinocytes from apoptosis induced by osmotic shock, oxidative stress, and tumor necrosis factor.

    Science.gov (United States)

    Diker-Cohen, Talia; Koren, Ruth; Liberman, Uri A; Ravid, Amiram

    2003-12-01

    Calcitriol, the hormonal form of vitamin D, inhibited caspase-3-like activation in HaCaT keratinocytes exposed to hyperosmotic and oxidative stresses, heat shock, and the inflammatory cytokine TNF. The hormone also protected the cells from caspase-independent cell death induced by hyperosmotic and oxidative stresses. The protection against hyperosmotic stress is not affected by inhibitors of the EGF receptor, ERK or PI13 kinase pathways, neither is it due to reduced activity of the proapoptotic p38 MAP kinase. These results are in accordance with previous in vivo findings that vitamin D protects epidermal keratinocytes from apoptosis due to UV radiation or chemotherapy.

  6. Serially cultured keratinocytes from human scalp hair follicles: a tool for cytogenetic studies.

    Science.gov (United States)

    Weterings, P J; Roelofs, H M; Jansen, B A; Vermorken, A J

    1983-01-01

    Keratinocytes originating from adult human hair follicles, the most convenient biopsy tissue, can be serially cultured using a combination of two techniques. Primary cultures are established using plucked scalp hair follicles and the bovine eye lens capsule as a growth substrate. Subsequently, cells from these cultures are serially cultivated in the presence of irradiated 3T3 cells as feeders. By this combination of techniques many keratinocytes can be generated from one single hair follicle. These cultures, appropriately treated with colchicine, can provide an adequate number of metaphases suitable for chromosome studies.

  7. Keratinocyte Growth Factor Causes Cystic Dilation of the Mammary Glands of Mice: Interactions of Keratinocyte Growth Factor, Estrogen, and Progesterone In Vivo

    OpenAIRE

    Yi, Eunhee S.; Bedoya, Adriana A.; Lee, Hyesun; Kim, Seokhyun; Housley, Regina M.; Aukerman, Sharon L.; Tarpley, John E.; Starnes, Charles; Yin, Songmei; Pierce, Glenn F.; Ulich, Thomas R.

    1994-01-01

    Keratinocyte growth factor (KGF) is a paracrine mediator of epithelial cell proliferation that has been reported to induce marked proliferation of mammary epithelium in rats. In this study, systemic administration of KGF into naive and oophorectomized mice causes mammary gland proliferation, as evidenced histologically by the appearance of cysts lined by a single layer of epithelium and by hyperplastic epithelium. Whole mount preparations of the mammary glands reveal that the histologically n...

  8. Transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papillomaviruses alters human keratinocyte growth and differentiation in organotypic cultures

    NARCIS (Netherlands)

    Boxman, I. L.; Mulder, L. H.; Noya, F.; de Waard, V.; Gibbs, S.; Broker, T. R.; ten Kate, F.; Chow, L. T.; ter Schegget, J.

    2001-01-01

    Epidermodysplasia-verruciformis-associated human papilloma virus DNA has been detected in skin cancers, in premalignant and benign skin lesions, and in plucked hairs from immunocompetent and immunosuppressed patients. The role of epidermodysplasia-verruciformis-associated human papilloma virus in

  9. Inhibitory mechanism of Korean Red Ginseng on GM-CSF expression in UVB-irradiated keratinocytes

    Directory of Open Access Journals (Sweden)

    Ira Chung

    2015-10-01

    Conclusion: Taken together, we found that treatment with SKRG decreased the phosphorylation of EGFR and ERK in UVB-irradiated SP-1 keratinocytes and subsequently inhibited the expression of GM-CSF. Furthermore, we identified ginsenoside-Rh3 as the active saponin in Korean Red Ginseng.

  10. Expression of microRNA-184 in keratinocytes represses argonaute 2.

    Science.gov (United States)

    Roberts, Julian C; Warren, Richard B; Griffiths, Christopher E M; Ross, Kehinde

    2013-12-01

    Interleukin-22 (IL-22) is a proinflammatory cytokine that has been associated with the pathogenesis of inflammatory skin disorders. However, the impact of IL-22 on microRNA (miRNA) expression in epidermal keratinocytes is unknown. Here we show that IL-22 induces miR-184 in reconstituted human epidermis (RHE) and in the HaCaT keratinocyte cell line. Exposure to IL-22 increased miR-184 expression 8- and 15-fold in RHE and HaCaT cells, respectively. Oncostatin M, an unrelated proinflammatory cytokine, also raised miR-184 expression in RHE and HaCaT keratinocytes. Pharmacologic and genetic inhibition demonstrated that cytokine-induced expression of miR-184 was mediated by signal transducer and activation of transcription 3 (STAT3). Argonaute 2 (AGO2), a member of the RNA-induced silencing complex (RISC), is a predicted miR-184 target. Using protein, messenger RNA and reporter analyses, we found that miR-184 regulates the expression of AGO2. We conclude that cytokine-induced miR-184 attenuates AGO2 expression in keratinocytes. Copyright © 2013 Wiley Periodicals, Inc.

  11. Identification of extra- and intracellular alanyl aminopeptidases as new targets to modulate keratinocyte growth and differentiation

    International Nuclear Information System (INIS)

    Aminopeptidase inhibitors strongly affect proliferation, differentiation, and function of immune cells and show therapeutic potential in inflammatory disorders. In psoriatic lesions, keratinocytes display increased cellular turnover and disturbed differentiation, leading to epidermal hyperplasia accompanied by the loss of stratum granulosum. Here, we report in the HaCaT hyperproliferative keratinocyte cell line as well as in two primary keratinocyte strains in vitro a molecular and biochemical analysis of the expression of both membrane and cytosol alanyl aminopeptidase (cAAP) on the mRNA, protein, and enzymatic activity level. We found a clear dose-dependent suppression of DNA synthesis in vitro in the presence of the inhibitors actinonin, bestatin, and the cAAP-specific inhibitor PAC-22 correlating well with the simultaneous decrease in enzyme activity. In vivo, actinonin dose-dependently restored the stratum granulosum and ameliorated the impaired keratinocyte differentiation in the mouse tail model of psoriasis. Taken together, these data suggest that targeting alanyl aminopeptidases may be beneficial for psoriasis and other inflammatory skin disorders

  12. A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus

    DEFF Research Database (Denmark)

    Jensen, Uffe Birk; Yan, Xiaohong; Triel, Charlotte

    2008-01-01

    lineages. We have identified a distinct population of murine hair follicle keratinocytes residing in the upper isthmus (UI) between the infundibulum and bulge regions that are distinguished by low alpha6 integrin levels and are negative for CD34 and Sca-1. Purified UI cells give rise to long-term, stable...

  13. ZNF750 is expressed in differentiated keratinocytes and regulates epidermal late differentiation genes.

    Directory of Open Access Journals (Sweden)

    Idan Cohen

    Full Text Available Disrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype reminiscent of psoriasis and seborrheic dermatitis. Here we show that ZNF750 is a nuclear protein bearing a functional C-terminal nuclear localization signal. ZNF750 was specifically expressed in the epidermal suprabasal layers and its expression was augmented during differentiation, both in human skin and in-vitro, peaking in the granular layer. Silencing of ZNF750 in Ca2+-induced HaCaT keratinocytes led to morphologically apparent arrest in the progression of late differentiation, as well as diminished apoptosis and sustained proliferation. ZNF750 knockdown cells presented with markedly reduced expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, overexpression of ZNF750 in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation and with its downstream targets can serve in future elucidation of therapeutics for common diseases of skin barrier.

  14. Reconstruction of epidermis by grafting of keratinocytes cultured on polymer support - clinical study

    Czech Academy of Sciences Publication Activity Database

    Dvořánková, B.; Holíková, Z.; Vacík, Jiří; Königová, R.; Kapounková, Z.; Michálek, Jiří; Přádný, Martin; Smetana, Karel

    2003-01-01

    Roč. 42, č. 3 (2003), s. 219-223 ISSN 0011-9059 R&D Projects: GA MŠk LN00A065; GA MZd ND6340; GA AV ČR IBS4050005 Institutional research plan: CEZ:AV0Z4050913 Keywords : keratinocytes * graft * polymer support Subject RIV: FH - Neurology Impact factor: 0.736, year: 2003

  15. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-01-01

    Highlights: → Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. → The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. → Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  16. SPINK9 Stimulates Metalloprotease/EGFR-Dependent Keratinocyte Migration via Purinergic Receptor Activation

    Czech Academy of Sciences Publication Activity Database

    Sperrhacke, M.; Fischer, J.; Wu, Z.H.; Klunder, S.; Sedláček, Radislav; Schroeder, J.M.; Meyer-Hoffert, U.; Reiss, K.

    2014-01-01

    Roč. 134, č. 6 (2014), s. 1645-1654 ISSN 0022-202X R&D Projects: GA ČR GAP303/10/2044 Institutional support: RVO:68378050 Keywords : SPINK * ADAM * keratinocyte Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.216, year: 2014

  17. Skin and hair follicle integrity is crucially dependent on beta 1 integrin expression on keratinocytes

    DEFF Research Database (Denmark)

    Brakebusch, C; Grose, R; Quondamatteo, F

    2000-01-01

    beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice...

  18. Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes

    Science.gov (United States)

    Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes Nanoparticle uptake in cells may be an important determinant of their potential cytotoxic and inflammatory effects. Six commercial TiO2 NP (A=Alfa Aesar,10nm, A*=Alfa Aesar 32nm, B=P25 27...

  19. Analysis of the response of human keratinocytes to Malassezia globosa and restricta strains.

    Science.gov (United States)

    Donnarumma, Giovanna; Perfetto, Brunella; Paoletti, Iole; Oliviero, Giovanni; Clavaud, Cécile; Del Bufalo, Aurelia; Guéniche, Audrey; Jourdain, Roland; Tufano, Maria Antonietta; Breton, Lionel

    2014-10-01

    Malassezia spp. are saprophyte yeasts involved in skin diseases with different degrees of severity. The aim of our study was to analyze the response of human epidermal keratinocytes to Malassezia globosa and restricta strains evaluating the host defence mechanisms induced by Malassezia spp. colonization. Our results showed a different modulation of the inflammatory and immunomodulatory cytokine pathways obtained with the different strains of Malassezia tested. In addition, this expression is altered by blocking the TLR2 receptor. In comparison with M. furfur, M. globosa and restricta displayed an unexpected and striking cytotoxicity on keratinocytes. The differences observed could be related to the different modalities of interaction between keratinocytes and Malassezia strains, but also to their growth condition. Taken together, these results indicate that M. globosa or M. restricta colonization exert a different control on the cytokine inflammatory response activated in the human keratinocyte in which TLR2 might be involved. M. globosa and M. restricta may play a synergistic role in the exacerbation of skin diseases in which both are found.

  20. Long-term Genoprotection Effect of Sechium edule Fruit Extract Against UVA Irradiation in Keratinocytes.

    Science.gov (United States)

    Metral, Elodie; Rachidi, Walid; Damour, Odile; Demarne, Frédéric; Bechetoille, Nicolas

    2018-03-01

    Photoprotection is essential to prevent the long-term deleterious effects of ultraviolet (UV), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA-induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA-irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE-treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities. © 2017 The American Society of Photobiology.

  1. Knockdown of filaggrin in a three-dimensional reconstructed human epidermis impairs keratinocyte differentiation

    NARCIS (Netherlands)

    Pendaries, Valérie; Malaisse, Jeremy; Pellerin, Laurence; Le Lamer, Marina; Nachat, Rachida; Kezic, Sanja; Schmitt, Anne-Marie; Paul, Carle; Poumay, Yves; Serre, Guy; Simon, Michel

    2014-01-01

    Atopic dermatitis is a chronic inflammatory skin disorder characterized by defects in the epidermal barrier and keratinocyte differentiation. The expression of filaggrin, a protein thought to have a major role in the function of the epidermis, is downregulated. However, the impact of this deficiency

  2. Inhibition of melanosome transfer from melanocytes to keratinocytes by lectins and neoglycoproteins in an in vitro model system.

    Science.gov (United States)

    Minwalla, L; Zhao, Y; Cornelius, J; Babcock, G F; Wickett, R R; Le Poole, I C; Boissy, R E

    2001-06-01

    We propose that some of the critical molecules involved in the transfer of melanosomes from melanocytes to keratinocytes include plasma membrane lectins and their glycoconjugates. To investigate this mechanism, co-cultures of human melanocytes and keratinocytes derived from neonatal foreskins were established. The process of melanosome transfer was assessed by two experimental procedures. The first involved labeling melanocyte cultures with the fluorochrome CFDA. Labeled melanocytes were subsequently co-cultured with keratinocytes, and the transfer of fluorochrome assessed visually by confocal microscopy and quantitatively by flow cytometry. The second investigative approach involved co-culturing melanocytes with keratinocytes, and processing the co-cultures after 3 days for electron microscopy to quantitate the numbers of melanosomes in keratinocytes. Results from these experimental approaches indicate significant transfer of dye or melanosomes from melanocytes to keratinocytes that increased with time of co-culturing. Using these model systems, we subsequently tested a battery of lectins and neoglycoproteins for their effect in melanosome transfer. Addition of these selected molecules to co-cultures inhibited transfer of fluorochrome by approximately 15-44% as assessed by flow cytometry, and of melanosomes by 67-93% as assessed by electron microscopy. Therefore, our results suggest the roles of selected lectins and glycoproteins in melanosome transfer to keratinocytes in the skin.

  3. Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

    Science.gov (United States)

    Thang, Nguyen D; Yajima, Ichiro; Ohnuma, Shoko; Ohgami, Nobutaka; Kumasaka, Mayuko Y; Ichihara, Gaku; Kato, Masashi

    2015-02-01

    We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion. © 2013 Wiley Periodicals, Inc.

  4. RAC1 in keratinocytes regulates crosstalk to immune cells by Arp2/3-dependent control of STAT1

    DEFF Research Database (Denmark)

    Pedersen, Esben Ditlev Kølle; Wang, Zhipeng; Stanley, Alanna

    2012-01-01

    Crosstalk between keratinocytes and immune cells is crucial for the immunological barrier function of the skin, and aberrant crosstalk contributes to inflammatory skin diseases. Using mice with a keratinocyte-restricted deletion of the RAC1 gene we found that RAC1 in keratinocytes plays an import...... hypersensitive to inflammatory stimuli both in vitro and in vivo, suggesting a major role for RAC1 in regulating the crosstalk between the epidermis and the immune system....... an important role in modulating the interferon (IFN) response in skin. These RAC1 mutant mice showed increased sensitivity in an irritant contact dermatitis model, abnormal keratinocyte differentiation, and increased expression of immune response genes including the IFN signal transducer STAT1. Loss of RAC1......Crosstalk between keratinocytes and immune cells is crucial for the immunological barrier function of the skin, and aberrant crosstalk contributes to inflammatory skin diseases. Using mice with a keratinocyte-restricted deletion of the RAC1 gene we found that RAC1 in keratinocytes plays...

  5. Calmodulin mediates sulfur mustard toxicity in human keratinocytes

    International Nuclear Information System (INIS)

    Simbulan-Rosenthal, Cynthia M.; Ray, Radharaman; Benton, Betty; Soeda, Emiko; Daher, Ahmad; Anderson, Dana; Smith, William J.; Rosenthal, Dean S.

    2006-01-01

    Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48 h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural

  6. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

    Science.gov (United States)

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes.

    Science.gov (United States)

    Wu, Xufeng S; Masedunskas, Andreas; Weigert, Roberto; Copeland, Neal G; Jenkins, Nancy A; Hammer, John A

    2012-07-31

    Mammalian pigmentation is driven by the intercellular transfer of pigment-containing melanosomes from the tips of melanocyte dendrites to surrounding keratinocytes. Tip accumulation of melanosomes requires myosin Va, because melanosomes concentrate in the center of melanocytes from myosin Va-null (dilute) mice. This distribution defect results in inefficient melanosome transfer and a dilution of coat color. Dilute mice that simultaneously lack melanoregulin, the product of the dilute suppressor locus, exhibit a nearly complete restoration of coat color, but, surprisingly, melanosomes remain concentrated in the center of their melanocytes. Here we show that dilute/dsu melanocytes, but not dilute melanocytes, readily transfer the melanosomes concentrated in their center to surrounding keratinocytes in situ. Using time-lapse imaging of WT melanocyte/keratinocyte cocultures in which the plasma membranes of the two cells are marked with different colors, we define an intercellular melanosome transfer pathway that involves the shedding by the melanocyte of melanosome-rich packages, which subsequently are phagocytosed by the keratinocyte. Shedding, which occurs primarily at dendritic tips but also from more central regions, involves adhesion to the keratinocyte, thinning behind the forming package, and apparent self-abscission. Finally, we show that shedding from the cell center is sixfold more frequent in cultured dilute/dsu melanocytes than in dilute melanocytes, consistent with the in situ data. Together, these results explain how dsu restores the coat color of dilute mice without restoring intracellular melanosome distribution, indicate that melanoregulin is a negative regulator of melanosome transfer, and provide insight into the mechanism of intercellular melanosome transfer.

  8. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  9. Human papillomavirus (HPV upregulates the cellular deubiquitinase UCHL1 to suppress the keratinocyte's innate immune response.

    Directory of Open Access Journals (Sweden)

    Rezaul Karim

    Full Text Available Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1 in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3 K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

  10. Eccrine sweat contains IL-1α, IL-1β and IL-31 and activates epidermal keratinocytes as a danger signal.

    Directory of Open Access Journals (Sweden)

    Xiuju Dai

    Full Text Available Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal.

  11. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

    Energy Technology Data Exchange (ETDEWEB)

    De Abrew, K. Nadira [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Thomas-Virnig, Christina L.; Rasmussen, Cathy A. [Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States); Bolterstein, Elyse A. [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Schlosser, Sandy J. [Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States); Allen-Hoffmann, B. Lynn, E-mail: blallenh@wisc.edu [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States)

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures

  12. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

    DEFF Research Database (Denmark)

    Christensen, Rikke; Güttler, Flemming; Jensen, Thomas G

    2002-01-01

    gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin......, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed...

  13. Synthesis and biological activity of M6-P and M6-P analogs on fibroblast and keratinocyte proliferation.

    Science.gov (United States)

    Clavel, Caroline; Barragan-Montero, Véronique; Garric, Xavier; Molès, Jean-Pierre; Montero, Jean-Louis

    2005-09-01

    A new synthetic route to obtain the carboxylate analog of mannose 6-phosphate (M6-P) is presented. The effects of the M6-P, the carboxylate and two other analogs (the phosphonate and the alpha,beta ethylenic carboxylate) on the proliferation of human keratinocytes and dermal fibroblasts as well as on the proliferation of a murine fibroblast cell line, 3T3-J2 are tested. We observed that M6-P is a potent inhibitor of proliferation of both fibroblasts and keratinocytes. Among its analogs, the phosphonate showed a similar effect on human dermal fibroblasts but not on keratinocytes.

  14. Effect of 1,24R-dihydroxyvitamin D3 on the growth of human keratinocytes.

    LENUS (Irish Health Repository)

    Matsumoto, K

    1990-02-01

    The effect of 1,24R-dihydroxyvitamin D3 (1,24R(OH)2D3), a synthetic analogue of a biologically active form of vitamin D3 (1,25-dihydroxyvitamin D3, 1,25(OH)2D3), on the growth of human keratinocytes cultured in serum-free medium was investigated. The growth of cultured normal human keratinocytes was inhibited by 65% by 10(-8)M 1,24R(OH)2D3 and by 90% by 10(-7)M 1,24(OH)2D3. It inhibited cell growth almost completely at 10(-6)M. The DNA synthesis of keratinocytes was also inhibited with 1,24R(OH)2D3 by 27% at 10(-8)M, 59% at 10(-7)M, and 92% at 10(-6)M. The inhibition of cell growth and DNA synthesis were more remarkable by 1,24R(OH)2D3 than by 1,25(OH)2D3. 1,24R(OH)2D3 also inhibited the growth of keratinocytes derived from patients with psoriasis vulgaris; the growth inhibitory effect was again more remarkable with 1,24R(OH)2D3 than with 1,25(OH)2D3. The viability and protein synthesis of keratinocytes were not affected by 1,24R(OH)2D3, suggesting that the growth inhibitory effect is due to its biological activity, not to cytotoxicity. The binding of [3H]-labeled 1,25(OH)2D3 to its receptor in the cytosolic fraction of cultured keratinocytes was competitively substituted by unlabeled 1,24R(OH)2D3 as well as 1,25(OH)2D3, suggesting that 1,24R(OH)2D3 binds to the 1,25(OH)2D3 receptor. It was found that the affinity of 1,24R(OH)2D3 for the receptor was slightly higher than that of 1,25(OH)2D3. These results demonstrate that 1,24R(OH)2D3 functions as a potent growth inhibitor in vitro in human keratinocytes from both normal and psoriatic epidermis, and it possesses a higher affinity for the 1,25(OH)2D3 receptor in cultured human keratinocytes. The difference in affinity of 1,24R(OH)2D3 for the 1,25(OH)2D3 receptor correlates with its greater inhibition of keratinocyte growth than 1,25(OH)2D3. 1,24R(OH)2D3 may be useful in the treatment of psoriasis.

  15. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    International Nuclear Information System (INIS)

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-01-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  16. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Ok-Nam [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Eun-Sun [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Jeong, Tae Cheon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Chun, Young-Jin [College of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Ai-Young, E-mail: leeay@duih.org [Department of Dermatology, Dongguk University Ilsan Hospital, Goyang 410-773 (Korea, Republic of); Noh, Minsoo, E-mail: minsoo@alum.mit.edu [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  17. Centella asiatica protects against UVB-induced HaCaT keratinocyte damage through microRNA expression changes.

    Science.gov (United States)

    An, In-Sook; An, Sungkwan; Choe, Tae-Βoo; Kang, Sang-Μo; Lee, Jae Ho; Park, In-Chul; Jin, Young-Woo; Lee, Su-Jae; Bae, Seunghee

    2012-12-01

    This study aimed to evaluate the protective effects of Centella asiatica (C. asiatica) against ultraviolet B (UVB) damage in human keratinocytes using microRNA (miRNA) expression profiling analysis. Titrated extract of C. asiatica (TECA) demonstrated low cytotoxicity in normal human HaCaT keratinocytes only at low doses (<5 µg/ml). UVB (50 mJ/cm2) irradiation significantly decreased cell viability, and TECA treatment decreased the UVB toxicity. By using miRNA microarrays, we determined that 72 miRNAs had an altered expression following TECA treatment in UVB-irradiated keratinocytes (46 upregulated and 26 downregulated). Using an miRNA target gene prediction tool and Gene Ontology (GO) analysis, we determined that miRNAs with altered expression were functionally related with the inhibition of apoptosis and cell proliferation. Overall, these results provide meaningful information to facilitate the understanding of TECA-mediated UVB protection in human keratinocytes.

  18. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

    2002-01-01

    The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...

  19. Cyclic stretch induces upregulation of endothelin-1 with keratinocytes in vitro: Possible role in mechanical stress-induced hyperpigmentation

    Energy Technology Data Exchange (ETDEWEB)

    Kurita, Masakazu, E-mail: masakazukurita@gmail.com [Department of Plastic Surgery, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611 (Japan); Okazaki, Mutsumi [Department of Plastic and Reconstructive Surgery, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Fujino, Takashi [Department of Pathology, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611 (Japan); Takushima, Akihiko; Harii, Kiyonori [Department of Plastic Surgery, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611 (Japan)

    2011-05-27

    Highlights: {yields} Influence of cyclic stretch on melanogenetic paracrine cytokines was investigated. {yields} Keratinocyte-derived endothelin-1 was upregulated with cyclic stretch. {yields} Degree of upregulation increases dose-dependently. {yields} This upregulation possibly plays a role in the pathogenesis of pigmented disorders. -- Abstract: The aim of this study was to investigate the possible pathological relation between mechanical stress and hyperpigmentation. We did this by investigating the influence of cyclic stretch on the expression of keratinocyte- and fibroblast-derived melanogenetic paracrine cytokines in vitro. Using primary human keratinocytes and fibroblasts, alterations of mRNA expression of melanogenetic paracrine cytokines due to cyclic stretch were investigated using a real-time polymerase chain reaction (PCR). The cytokines included basic fibroblast growth factor (bFGF), stem cell factor (SCF), granulocyte/macrophage colony-stimulating factor, interleukin-1{alpha}, and endothelin-1 (ET-1) for keratinocytes and bFGF, SCF, and hepatocyte growth factor for fibroblasts. The dose dependence of keratinocyte-derived ET-1 upregulation was further investigated using real-time PCR and an enzyme-linked immunosorbent assay. We also investigated the effects of cyclic stretch on the proliferation and differentiation of keratinocytes. Among the melanogenetic paracrine cytokines investigated, keratinocyte-derived ET-1 was consistently upregulated in all four cell lines. The degree of upregulation increased with the degree of the length and frequency of the stretch; in contrast, cell number and differentiation markers showed no obvious alterations with cyclic stretch. Keratinocyte-derived ET-1 upregulation possibly plays a significant role in the pathogenesis of pigmented disorders, such as friction melanosis, caused by mechanical stress.

  20. Nrf2 Regulates the Sensitivity of Mouse Keratinocytes to Nitrogen Mustard via Multidrug Resistance-Associated Protein 1 (Mrp1)

    Science.gov (United States)

    Udasin, Ronald G.; Wen, Xia; Bircsak, Kristin M.; Aleksunes, Lauren M.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2016-01-01

    Sulfur mustard and nitrogen mustard (mechlorethamine, HN2) are potent vesicants developed as chemical warfare agents. These electrophilic, bifunctional alkylating agents cause skin injury, including inflammation, edema, and blistering. HN2 covalently modifies macromolecules such as DNA, RNA, and proteins or is scavenged by glutathione, forming adducts that can contribute to toxicity. Multidrug resistance-associated protein 1 (Mrp1/MRP1) is a transmembrane ATPase known to efflux glutathione-conjugated electrophiles. In the present studies, we examined the effects of modulating Mrp1-mediated transport activity on the sensitivity of primary and PAM212 mouse keratinocytes to HN2. Primary keratinocytes, and to a lesser extent, PAM212 cells, express Mrp1 mRNA and protein and possess Mrp1 functional activity, as measured by calcein efflux. Sulforaphane, an activator of Nrf2, increased Mrp1 mRNA, protein, and functional activity in primary keratinocytes and PAM212 cells and decreased their sensitivity to HN2-induced growth inhibition (IC50 = 1.4 and 4.8 µM in primary keratinocytes and 1 and 13 µM in PAM212 cells, in the absence and presence of sulforaphane, respectively). The Mrp1 inhibitor, MK-571, reversed the effects of sulforaphane on HN2-induced growth inhibition in both primary keratinocytes and PAM212 cells. In primary keratinocytes from Nrf2−/− mice, sulforaphane had no impact on Mrp1 expression or activity, or on sensitivity to HN2, demonstrating that its effects depend on Nrf2. These data suggest that Mrp1-mediated efflux is important in regulating HN2-induced keratinocyte growth inhibition. Enhancing HN2 efflux from keratinocytes may represent a novel strategy for mitigating vesicant-induced cytotoxicity. PMID:26454883

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

    Science.gov (United States)

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

    2014-12-01

    Fibrosis affects most organs, it results in replacement of normal parenchymal tissue with collagen-rich extracellular matrix, which compromises tissue architecture and ultimately causes loss of function of the affected organ. Biochemical pathways that contribute to fibrosis have been extensively studied, but the role of biomechanical signaling in fibrosis is not clearly understood. In this study, we assessed the effect keratinocytes have on the biomechanical characteristics and pore microstructure of tissue engineered skin made with superficial or deep dermal fibroblasts in order to determine any biomaterial-mediated anti-fibrotic influences on tissue engineered skin. Tissue engineered skin with deep dermal fibroblasts and keratinocytes were found to be less stiff and contracted and had reduced number of myofibroblasts and lower expression of matrix crosslinking factors compared to matrices with deep fibroblasts alone. However, there were no such differences between tissue engineered skin with superficial fibroblasts and keratinocytes and matrices with superficial fibroblasts alone. Also, tissue engineered skin with deep fibroblasts and keratinocytes had smaller pores compared to those with superficial fibroblasts and keratinocytes; pore size of tissue engineered skin with deep fibroblasts and keratinocytes were not different from those matrices with deep fibroblasts alone. A better understanding of biomechanical characteristics and pore microstructure of tissue engineered skin may prove beneficial in promoting normal wound healing over pathologic healing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. The Herbal Bitter Drug Gentiana lutea Modulates Lipid Synthesis in Human Keratinocytes In Vitro and In Vivo

    Science.gov (United States)

    Haarhaus, Birgit; Seiwerth, Jasmin; Cawelius, Anja; Schwabe, Kay; Quirin, Karl-Werner; Schempp, Christoph M.

    2017-01-01

    Gentiana lutea is a herbal bitter drug that is used to enhance gastrointestinal motility and secretion. Recently we have shown that amarogentin, a characteristic bitter compound of Gentiana lutea extract (GE), binds to the bitter taste receptors TAS2R1 and TAS2R38 in human keratinocytes, and stimulates the synthesis of epidermal barrier proteins. Here, we wondered if GE also modulates lipid synthesis in human keratinocytes. To address this issue, human primary keratinocytes were incubated for 6 days with GE. Nile Red labeling revealed that GE significantly increased lipid synthesis in keratinocytes. Similarly, gas chromatography with flame ionization detector indicated that GE increases the amount of triglycerides in keratinocytes. GE induced the expression of epidermal ceramide synthase 3, but not sphingomyelinase. Lipid synthesis, as well as ceramide synthase 3 expression, could be specifically blocked by inhibitors of the p38 MAPK and PPARγ signaling pathway. To assess if GE also modulates lipid synthesis in vivo, we performed a proof of concept half side comparison on the volar forearms of 33 volunteers. In comparison to placebo, GE significantly increased the lipid content of the treated skin areas, as measured with a sebumeter. Thus, GE enhances lipid synthesis in human keratinocytes that is essential for building an intact epidermal barrier. Therefore, GE might be used to improve skin disorders with an impaired epidermal barrier, e.g., very dry skin and atopic eczema. PMID:28829355

  3. Changes in dermal matrix in the absence of Rac1 in keratinocytes

    DEFF Research Database (Denmark)

    Stanley, Alanna; Pedersen, Esben Ditlev Kølle; Brakebusch, Cord

    2016-01-01

    that the deletion of Rac1 in keratinocytes causes heightened inflammation due to aberrant crosstalk with immune cells. Indeed, the skin of these mice shows a higher inflammatory response to the induction of irritant contact dermatitis (ICD), and also even to treatment with a vehicle alone, compared with controls....... As inflammation is intimately linked with fibrotic disease in the skin, this raised the question as to whether this deletion may also affect the deposition and arrangement of the dermal ECM. This study assessed the effects of Rac1 deletion in keratinocytes and of the heightened inflammatory status by induction...... of ICD on the tissue localisation and arrangements of dermal collagen. Qualitative analysis did not reveal evidence for the formation of pathologies in the dermis. However, quantitative analysis did reveal some perturbations in the dermal matrix, namely that only the combination of the lack of Rac1...

  4. A Patient with Multiple Keratinocytic Cancers (MKC: Uncommon Presentation in a Bulgarian Patient

    Directory of Open Access Journals (Sweden)

    Georgi Tchernev

    2018-01-01

    Full Text Available Keratinocyte skin cancers, including basal cell carcinoma (BCC and squamous cell carcinoma (SCC, are the most common cancer occurring in people with fair skin, worldwide. Despite all known triggers, several suggested contributors are still investigated. We will focus our attention on the personal history of previous cancers and radiation exposure as occupational risk factors, as in the presented case. We report a patient, with multiple BCCs, and subsequent occurrence of a SCC on photo-exposed area of the face, as we want to emphasize the importance of strict following up of these patients, regarding the risk for developing new tumors in short periods of time, no matter if the triggering exposure factor is known from the history, or not.  Although keratinocytes tumours are associated with the low mortality rate, we focus the attention on the fact, that the history of non-melanoma skin cancer is associated with increased mortality.

  5. A Patient with Multiple Keratinocytic Cancers (MKC): Uncommon Presentation in a Bulgarian Patient.

    Science.gov (United States)

    Tchernev, Georgi; Philipov, Stanislav; Chokoeva, Anastasiya Atanasova; Wollina, Uwe; Lotti, Torello; Lozev, Ilia; Yungareva, Irina; Maximov, Georgi Konstantinov

    2018-01-25

    Keratinocyte skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common cancer occurring in people with fair skin, worldwide. Despite all known triggers, several suggested contributors are still investigated. We will focus our attention on the personal history of previous cancers and radiation exposure as occupational risk factors, as in the presented case. We report a patient, with multiple BCCs, and subsequent occurrence of a SCC on photo-exposed area of the face, as we want to emphasize the importance of strict following up of these patients, regarding the risk for developing new tumors in short periods of time, no matter if the triggering exposure factor is known from the history, or not. Although keratinocytes tumours are associated with the low mortality rate, we focus the attention on the fact, that the history of non-melanoma skin cancer is associated with increased mortality.

  6. Panx1 regulates cellular properties of keratinocytes and dermal fibroblasts in skin development and wound healing.

    Science.gov (United States)

    Penuela, Silvia; Kelly, John J; Churko, Jared M; Barr, Kevin J; Berger, Amy C; Laird, Dale W

    2014-07-01

    Pannexin1 (Panx1), a channel-forming glycoprotein is expressed in neonatal but not in aged mouse skin. Histological staining of Panx1 knockout (KO) mouse skin revealed a reduction in epidermal and dermal thickness and an increase in hypodermal adipose tissue. Following dorsal skin punch biopsies, mutant mice exhibited a significant delay in wound healing. Scratch wound and proliferation assays revealed that cultured keratinocytes from KO mice were more migratory, whereas dermal fibroblasts were more proliferative compared with controls. In addition, collagen gels populated with fibroblasts from KO mice exhibited significantly reduced contraction, comparable to WT fibroblasts treated with the Panx1 blocker, probenecid. KO fibroblasts did not increase α-smooth muscle actin expression in response to TGF-β, as is the case for differentiating WT myofibroblasts during wound contraction. We conclude that Panx1 controls cellular properties of keratinocytes and dermal fibroblasts during early stages of skin development and modulates wound repair upon injury.

  7. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes.

    Science.gov (United States)

    Qian, Wei; Wang, Yong; Li, Rui-Fu; Zhou, Xin; Liu, Jing; Peng, Dai-Zhi

    2017-03-03

    BACKGROUND Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. MATERIAL AND METHODS In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. RESULTS The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. CONCLUSIONS This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.

  8. TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

    Science.gov (United States)

    Yamasaki, Kenshi; Kanada, Kimberly; Macleod, Daniel T; Borkowski, Andrew W; Morizane, Shin; Nakatsuji, Teruaki; Cogen, Anna L; Gallo, Richard L

    2011-03-01

    A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea.

  9. UVB-Protective Effects of Isoflavone Extracts from Soybean Cake in Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Chi-Feng Hung

    2007-07-01

    Full Text Available It has been shown by chromatography that aglycone, glucoside, acetylglucosideand malonylglucoside isoflavone extracts prepared from soybean cake showed betterantioxidant activities than isoflavone standards. Consequently, the aim of this study was toevaluate the protective effects of these isoflavone extracts against ultraviolet B (UVB-induced keratinocyte damage. Our results demonstrated that these soybean cake isoflavoneextracts could inhibit UVB-induced keratinocyte death. Moreover, they could inhibit UVB-induced intracellular release of hydrogen peroxide (H2O2 Furthermore, these isoflavoneextracts differentially inhibited UVB-induced MAPK phosphorylation. The ERK1/2 andp38 phosphorylation was not inhibited by all tested isoflavone extracts, whereas JNKphosphorylation was inhibited by group I to group III isoflavone extracts. Since theseisoflavone extracts are relative stable and easily obtained than the isoflavone standards, wesuggest that soybean cake may be a useful potential source for developing effective skincare agents in against photoaging.

  10. Mathematical modeling of calcium waves induced by mechanical stimulation in keratinocytes.

    Directory of Open Access Journals (Sweden)

    Yasuaki Kobayashi

    Full Text Available Recent studies have shown that the behavior of calcium in the epidermis is closely related to the conditions of the skin, especially the differentiation of the epidermal keratinocytes and the permeability barrier function, and therefore a correct understanding of the calcium dynamics is important in explaining epidermal homeostasis. Here we report on experimental observations of in vitro calcium waves in keratinocytes induced by mechanical stimulation, and present a mathematical model that can describe the experimentally observed wave behavior that includes finite-range wave propagation and a ring-shaped pattern. A mechanism of the ring formation hypothesized by our model may be related to similar calcium propagation patterns observed during the wound healing process in the epidermis. We discuss a possible extension of our model that may serve as a tool for investigating the mechanisms of various skin diseases.

  11. A review of the pattern of AIDS defining, HIV associated neoplasms and premalignant lesions diagnosed from 2000-2011 at Kenyatta National Hospital, Kenya.

    Science.gov (United States)

    Rogena, Emily A; Simbiri, Kenneth O; De Falco, G; Leoncini, L; Ayers, Leona; Nyagol, J

    2015-01-01

    Sub-Sahara Africa hosts up to 71 % of all HIV infected people in the world. With this high incidence of Human immunodeficiency virus ( HIV) comes the burden of co-morbidities such as malignant and premalignant lesions. Aids defining malignancies have been listed as Kaposi's sarcoma, Non-Hodgkin's lymphoma and invasive squamous cell carcinoma of the cervix. People with HIV/AIDS(PLWAS) have a higher risk of developing these neoplasms than the rest of the population. The pathogenesis of these neoplasms in people with HIV has been linked to immune suppression, persistent antigenic stimulation and cytokine dysregulation. Current study analyzes and presents the patterns and trends in the presentation of HIV related malignancies in patients diagnosed through histopathology at Kenyatta National Hospital. To describe the patterns of AIDS- defining and non-AIDS- defining malignancies and premalignant lesions 10 years pre- and post HAART period at Kenyatta National hospital, Kenya. This was a hospital based descriptive cross sectional study. The Formalin fixed paraffin embedded (FFPE) blocks and histological reports of patients diagnosed between 2000 and 2011 were traced from archives. The patients' demographic data and clinical presentation was entered in an excel spreadsheet and the diagnosis and coding confirmed by a histopathologist. The data was then cleaned and analyzed using SSPS version 17.0 Ink. A total of 173 lesions were reviewed and analyzed. Of these 118 (68 %) were from females and 55 from males (32 %). The male to female ratio was 1:2. The age range was from two to 56 years with a median of 36 years. Kaposi sarcoma is the leading AIDS defining malignancy in Kenya while invasive squamous cell carcinoma of the conjunctiva is the leading non-AIDS defining malignancy. This is closely followed by invasive squamous cell carcinoma of the cervix and NHL. Kaposi sarcoma is the leading AIDS associated neoplasm in Kenya. Physicians and caretakers managing and

  12. Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model

    Science.gov (United States)

    Gschwandtner, M; Mildner, M; Mlitz, V; Gruber, F; Eckhart, L; Werfel, T; Gutzmer, R; Elias, P M; Tschachler, E

    2013-01-01

    Background Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis. Methods Human primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule. Results The addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80–95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls. Conclusions Our findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases. PMID:23157658

  13. Melanoregulin regulates a shedding mechanism that drives melanosome transfer from melanocytes to keratinocytes

    OpenAIRE

    Wu, Xufeng S.; Masedunskas, Andreas; Weigert, Roberto; Copeland, Neal G.; Jenkins, Nancy A.; Hammer, John A.

    2012-01-01

    Mammalian pigmentation is driven by the intercellular transfer of pigment-containing melanosomes from the tips of melanocyte dendrites to surrounding keratinocytes. Tip accumulation of melanosomes requires myosin Va, because melanosomes concentrate in the center of melanocytes from myosin Va-null (dilute) mice. This distribution defect results in inefficient melanosome transfer and a dilution of coat color. Dilute mice that simultaneously lack melanoregulin, the product of the dilute suppress...

  14. Basal keratinocytes contribute to all strata of the adult zebrafish epidermis.

    Directory of Open Access Journals (Sweden)

    Raymond T H Lee

    Full Text Available The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.

  15. Mitochondrial and lipogenic effects of vitamin D on differentiating and proliferating human keratinocytes.

    Science.gov (United States)

    Consiglio, Marco; Viano, Marta; Casarin, Stefania; Castagnoli, Carlotta; Pescarmona, Gianpiero; Silvagno, Francesca

    2015-10-01

    Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Interleukin 1 gene expression in cultured human keratinocytes is augmented by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Kupper, T.S.; Chua, A.O.; Flood, P.; McGuire, J.; Gubler, U.

    1987-01-01

    Interleukin 1 (IL-1) is a family of polypeptides initially found to be produced by activated monocytes and macrophages that mediate a wide variety of cellular responses to injury and infection. Epidermal epithelial cells (keratinocytes) produce ''epidermal cell-derived thymocyte activating factor'' or ETAF, which has been recently shown to be identical to IL-1. Human epidermis is normally exposed to significant amounts of solar ultraviolet radiation. Certain ultraviolet wavelengths (UVB, 290-320 nm) are thought to be responsible for most of the immediate and long-term pathological consequences of excessive exposure to sunlight. In this study, we asked whether exposure to UVB irradiation induced IL-1 gene expression in cultured human keratinocytes. Cultured human keratinocytes contain detectable amounts of IL-1 alpha and beta mRNA and protein in the absence of apparent stimulation; these levels could be significantly enhanced 6 h after exposure to 10 ng/ml of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Exposure to UVB irradiation with an emission spectrum comparable to that of sunlight (as opposed to that of an unfiltered artificial UV light source) significantly increased the steady state levels IL-1 alpha and beta mRNA in identical populations of human keratinocytes. This was reflected in the production of increased IL-1 activity by these cultures in vitro. In the same cell population, exposures to UVB irradiation did not alter the level of actin mRNA; therefore, the effect of UV irradiation on IL-1 represents a specific enhancement of IL-1 gene expression. Local increases of IL-1 may mediate the inflammation and vasodilation characteristic of acute UVB-injured skin, and systemic release of this epidermal IL-1 may account for fever, leukocytosis, and the acute phase response seen after excessive sun exposure

  17. Multiple biological effects of inhibitors of arachidonic acid metabolism on human keratinocytes

    Czech Academy of Sciences Publication Activity Database

    Pacherník, Jiří; Hampl, Aleš; Souček, Karel; Kovaříková, Martina; Andrysík, Zdeněk; Hofmanová, Jiřina; Kozubík, Alois

    2002-01-01

    Roč. 293, č. 12 (2002), s. 626-633 ISSN 0340-3696 R&D Projects: GA ČR GA524/99/0694; GA AV ČR IBS5004009; GA ČR GA524/98/0190 Institutional research plan: CEZ:AV0Z5004920 Keywords : lipoxygenase * keratinocytes * cell cycle Subject RIV: BO - Biophysics Impact factor: 1.452, year: 2002

  18. SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

    International Nuclear Information System (INIS)

    Liesenfeld, Melanie; Mosig, Sandy; Funke, Harald; Jansen, Lars; Runnebaum, Ingo B; Dürst, Matthias; Backsch, Claudia

    2013-01-01

    Genetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer. Microarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction. Overall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro. The relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence

  19. Basal keratinocytes contribute to all strata of the adult zebrafish epidermis.

    Science.gov (United States)

    Lee, Raymond T H; Asharani, P V; Carney, Thomas J

    2014-01-01

    The epidermis of terrestrial vertebrates is a stratified epithelium and forms an essential protective barrier. It is continually renewed, with dead corneocytes shed from the surface and replaced from a basal keratinocyte stem cell population. Whilst mouse is the prime model system used for epidermal studies, there is increasing employment of the zebrafish to analyse epidermis development and homeostasis, however the architecture and ontogeny of the epidermis in this system are incompletely described. In particular, it is unclear if adult zebrafish epidermis is derived entirely from the basal epidermal stem cell layer, as in the mouse, or if the most superficial keratinocyte layer is a remnant of the embryonic periderm. Furthermore, a relative paucity of cellular markers and genetic reagents to label and manipulate the basal epidermal stem cell compartment has hampered research. Here we show that the type I keratin, krtt1c19e, is a suitable marker of the basal epidermal layer and identify a krtt1c19e promoter fragment able to drive strong and specific expression in this cell type. Use of this promoter to express an inducible Cre recombinase allowed permanent labelling of basal cells during embryogenesis, and demonstrated that these cells do indeed generate keratinocytes of all strata in the adult epidermis. Further deployment of the Cre-Lox system highlighted the transient nature of the embryonic periderm. We thus show that the epidermis of adult zebrafish, as in the mouse, derives from basal stem cells, further expanding the similarities of epidermal ontogeny across vertebrates. Future use of this promoter will assist genetic analysis of basal keratinocyte biology in zebrafish.

  20. Niacin protects against UVB radiation-induced apoptosis in cultured human skin keratinocytes

    OpenAIRE

    LIN, FUQUAN; XU, WEN; GUAN, CUIPING; ZHOU, MIAONI; HONG, WEISONG; FU, LIFANG; LIU, DONGYIN; XU, AIE

    2012-01-01

    Niacin and its related derivatives have been shown to have effects on cellular activities. However, the molecular mechanism of its reduced immunosuppressive effects and photoprotective effects remains unclear. In this study, we investigated the molecular mechanism of the photoprotective effect of niacin in ultraviolet (UV)-irradiated human skin keratinocytes (HaCaT cells). We found that niacin effectively suppressed the UV-induced cell death and cell apoptosis of HaCaT cells. Existing data ha...

  1. In vitro human keratinocyte migration rates are associated with SNPs in the KRT1 interval.

    Directory of Open Access Journals (Sweden)

    Heng Tao

    Full Text Available Efforts to develop effective therapeutic treatments for promoting fast wound healing after injury to the epidermis are hindered by a lack of understanding of the factors involved. Re-epithelialization is an essential step of wound healing involving the migration of epidermal keratinocytes over the wound site. Here, we examine genetic variants in the keratin-1 (KRT1 locus for association with migration rates of human epidermal keratinocytes (HEK isolated from different individuals. Although the role of intermediate filament genes, including KRT1, in wound activated keratinocytes is well established, this is the first study to examine if genetic variants in humans contribute to differences in the migration rates of these cells. Using an in vitro scratch wound assay we observe quantifiable variation in HEK migration rates in two independent sets of samples; 24 samples in the first set and 17 samples in the second set. We analyze genetic variants in the KRT1 interval and identify SNPs significantly associated with HEK migration rates in both samples sets. Additionally, we show in the first set of samples that the average migration rate of HEK cells homozygous for one common haplotype pattern in the KRT1 interval is significantly faster than that of HEK cells homozygous for a second common haplotype pattern. Our study demonstrates that genetic variants in the KRT1 interval contribute to quantifiable differences in the migration rates of keratinocytes isolated from different individuals. Furthermore we show that in vitro cell assays can successfully be used to deconstruct complex traits into simple biological model systems for genetic association studies.

  2. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Ute Hofmann

    2014-06-01

    Full Text Available In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells expressing green fluorescent protein (GFP under the control of the stress-inducible HSP70B’ promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B’. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B’ gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B’ promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  3. Mannosides as crucial part of bioactive supports for cultivation of human epidermal keratinocytes without feeder cells

    Czech Academy of Sciences Publication Activity Database

    Labský, Jiří; Dvořánková, B.; Smetana, Karel; Holíková, Z.; Brož, L.; Gabius, H.J.

    2003-01-01

    Roč. 24, č. 5 (2003), s. 863-872 ISSN 0142-9612 R&D Projects: GA ČR GA203/00/1310; GA MŠk LN00A065; GA MZd ND6340 Institutional research plan: CEZ:AV0Z4050913 Keywords : cell therapy * keratinocyte * mannose Subject RIV: CD - Macromolecular Chemistry Impact factor: 2.903, year: 2003

  4. The comparison of two methods to obtain human oral keratinocytes in primary culture

    International Nuclear Information System (INIS)

    Klingbeil, Maria Fatima Guarizo

    2006-01-01

    The therapeutic procedures frequently used in oral treatments for the pathological diseases are surgical, resulting in failures of the mucosal continuity.The possibility to obtain transplantable oral epithelia from an in vitro cell culture opens new utilization perspectives not only to where it comes from, but also as a reconstructive material for other parts of the human body, such as: urethra, epithelia corneo-limbal, cornea, ocular surface. Many researchers still use controversial methods for obtaining cells. It was therefore evaluated and compared the efficiency in both methods: enzymatic and direct explant to obtain oral keratinocytes from human oral mucosa. Fragments of intra oral epithelial tissues from healthy human subjects, undergoing dental surgeries, were donated to the research project. The keratinocytes were cultivated over a feeder-layer from a previously irradiated 3T3 Swiss albino fibroblasts. In this study it was compared the time needed in the cell obtention, the best cell amount between both methods, the life-span, the cell capacity to form an in vitro epithelia and its morphologic structure. The results in the assessment of both methods have shown the possibility to obtain keratinocytes from a small oral fragment, but at the same time we may verify the advantages and peculiar restrictions for each one of both analyzed methods. (author)

  5. Valproic acid induces cutaneous wound healing in vivo and enhances keratinocyte motility.

    Directory of Open Access Journals (Sweden)

    Soung-Hoon Lee

    Full Text Available BACKGROUND: Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK signaling pathways. Valproic acid (VPA is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown. METHODS AND FINDINGS: We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA, collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase/Akt signaling pathways. CONCLUSIONS: VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.

  6. Expression of paired-like homeodomain transcription factor 2c (PITX2c) in epidermal keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Ge [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China); Sohn, Kyung-Cheol; Choi, Tae-Young; Choi, Dae-Kyoung; Lee, Sang-Sin [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Ou, Bai-sheng [Department of Dermatology, The First Affiliated Hospital, Guangxi Traditional Chinese Medical University, Guangxi, Nanning, 530023 (China); Kim, Sooil; Lee, Young Ho [Department of Anatomy, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Yoon, Tae-Jin [Department of Dermatology, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of); Institute of Health Sciences, School of Medicine, Gyeongsang National University, Jinju, 660-702 (Korea, Republic of); Kim, Seong-Jin [Department of Dermatology, Chonnam National University Medical School, Gwangju, 501-757 (Korea, Republic of); Lee, Young; Seo, Young-Joon; Lee, Jeung-Hoon [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Kim, Chang Deok, E-mail: cdkimd@cnu.ac.kr [Department of Dermatology, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of); Research Institute for Medical Sciences, School of Medicine, Chungnam National University, Daejeon, 301-747 (Korea, Republic of)

    2010-11-15

    Paired-like homeodomain transcription factor 2 (PITX2) has been implicated as one of the genes responsible for Rieger syndrome. It has been also shown to play a central role during development. In this study, we investigated the functional role of PITX2 in keratinocyte differentiation. RT-PCR analysis showed that PITX2c isoform was predominantly expressed in a differentiation-dependent manner. Consistent with, immunohistochemical staining showed that PITX2 expression was increased in the upper layer of epidermis. When PITX2c was overexpressed in cultured keratinocytes by a recombinant adenovirus, the differentiation markers such as involucrin and loricrin were significantly increased at both mRNA and protein levels. In addition, PITX2c overexpression led to the decrease of cell growth, concomitantly with the upregulation of cell cycle-related genes p21. To investigate the effect of PITX2c in vivo, we microinjected PITX2c expression vector into zebrafish embryo. Interestingly, overexpression of PITX2c in zebrafish embryo led to the formation of horn-like structure and thickening of epidermis, together with the increase of keratin 8 (K8) expression. These results suggest that PITX2c has a role in proliferation and differentiation of epidermal keratinocytes.

  7. In Vitro Cytotoxicity and Phototoxicity Assessment of Acylglutamate Surfactants Using a Human Keratinocyte Cell Line

    Directory of Open Access Journals (Sweden)

    Abhay Kyadarkunte

    2014-07-01

    Full Text Available In the current study, human keratinocyte cell line was used as in vitro cell culture model to elucidate the effects of the fatty acid chain length of acylglutamate (amino acid-based surfactant namely, sodium cocoyl glutamate, sodium lauroyl glutamate, and sodium myristoyl glutamate on their cytotoxicity and the ultraviolet B induced phototoxicity. The endpoint used to assess toxicity was a tetrazolium-based assay whereas, the phototoxic potential of acylglutamate surfactants was predicted using two models namely, the Photo-Irritation Factor and Mean Photo Effect. The results of this study showed that the fatty acid chain length of acylglutamate greatly influences toxic effects on human keratinocyte cells. In addition, all the acylglutamate surfactants tested on human keratinocyte cells demonstrated significantly less cytotoxicity (when irradiated and non-irradiated with ultraviolet B light; p < 0.05 and no phototoxic potential was observed in any of the acylglutamate surfactants, when compared with the positive control chlorpromazine. In conclusion, the in vitro studies confirm the suitability of sodium lauroyl glutamate destined for the synthesis and stabilization of lipid nanoparticles.

  8. Activation of the low molecular weight protein tyrosine phosphatase in keratinocytes exposed to hyperosmotic stress.

    Directory of Open Access Journals (Sweden)

    Rodrigo A Silva

    Full Text Available Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. Of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.

  9. Experimental model of cultured keratinocytes Modelo experimental de cultura de queratinócitos

    Directory of Open Access Journals (Sweden)

    Alfredo Gragnani

    2003-01-01

    Full Text Available The bioengineering research is essential in the development of ideal combination of biomaterials and cultured cells to produce the permanent wound coverage. The experimental model of cultured keratinocytes presents all steps of the culture, since the isolation of the keratinocytes, preparation of the human acellular dermis, preparation of the composite skin graft and their elevation to the air-liquid interface. The research in cultured keratinocytes model advances in two main ways: 1. optimization of the methods in vitro to the skin cells culture and proliferation and 2. developing biomaterials that present similar skin properties.A pesquisa em bioengenharia é primordial no desenvolvimento da combinação ideal de biomateriais e células cultivadas para produzir a cobertura definitiva das lesões. O modelo experimental da cultura de queratinócitos apresenta toda as etapas do cultivo, desde o isolamento dos queratinócitos, preparação da derme acelular humana, do enxerto composto e da sua elevação à interface ar-líquido. A pesquisa em modelo de cultura de queratinócitos desenvolve-se em duas vias principais: 1. otimização dos métodos in vitro para cultivo e proliferação de células da pele e 2. desenvolvimento de biomateriais que mimetizem as propriedades da pele.

  10. Glucocorticoid receptor localizes to adherens junctions at the plasma membrane of keratinocytes.

    Directory of Open Access Journals (Sweden)

    Olivera Stojadinovic

    Full Text Available Glucocorticoids are important regulators of epidermal tissue homeostasis. As such, their clinical applications are widespread, ranging from inflammatory skin disorders to keloids and cancer. Glucocorticoids exert their effect by binding to glucocorticoid receptor (GR which translocates to the nucleus and regulates gene expression (genomic effect. In addition, GR has rapid non- genomic effects that are mediated by cell signaling proteins and do not involve gene transcription. Although genomic effects of GR in the epidermis are well documented, the non-genomic effects are not completely understood. Therefore, we utilized immunostaining and immunoprecipitations to determine specific localization of the GR in human keratinocytes that may contribute to non-genomic effects of glucocorticoid action. Here we describe a novel finding of GR localization to the plasma membrane of keratinocytes. Immunocytochemistry showed co-localization of GR with α-catenin. Immunoprecipitation of the membranous fraction revealed an association of GR with α-catenin, confirming its localization to adherens junctions. We conclude that GR localization to adherens junctions of keratinocytes provides a new mechanism of non-genomic signaling by glucocorticoids which may have significant biological and clinical impact.

  11. MnSOD upregulation induces autophagic programmed cell death in senescent keratinocytes.

    Directory of Open Access Journals (Sweden)

    Emeric Deruy

    Full Text Available Senescence is a state of growth arrest resulting mainly from telomere attrition and oxidative stress. It ultimately leads to cell death. We have previously shown that, in keratinocytes, senescence is induced by NF-kappaB activation, MnSOD upregulation and H(2O(2 overproduction. We have also shown that senescent keratinocytes do not die by apoptosis but as a result of high macroautophagic activity that targets the primary vital cell components. Here, we investigated the mechanisms that activate this autophagic cell death program. We show that corpses occurring at the senescence plateau display oxidatively-damaged mitochondria and nucleus that colocalize with autophagic vacuoles. The occurrence of such corpses was decreased by specifically reducing the H(2O(2 level with catalase, and, conversely, reproduced by overexpressing MnSOD or applying subtoxic doses of H(2O(2. This H(2O(2-induced cell death did occur through autophagy since it was accompanied by an accumulation of autophagic vesicles as evidenced by Lysotracker staining, LC3 vesiculation and transmission electron microscopy. Most importantly, it was partly abolished by 3-methyladenine, the specific inhibitor of autophagosome formation, and by anti-Atg5 siRNAs. Taken together these results suggest that autophagic cell death is activated in senescent keratinocytes because of the upregulation of MnSOD and the resulting accumulation of oxidative damages to nucleus and mitochondria.

  12. Study of transplantation of melanocyte keratinocyte suspension in treatment of vitiligo

    Directory of Open Access Journals (Sweden)

    Rastogi Swati

    2006-01-01

    Full Text Available Background: Vitiligo is a common skin disease; however it still remains a difficult disease to treat. Not all patients respond to current forms of treatment. Surgical techniques offer a potential solution for patients with vitiligo who fail to respond to medical treatments. Aims: Aims of the study were to evaluate response of transplantation of melanocyte keratinocyte cell suspension in patients of stable vitiligo. Materials and Methods: Total 10 patients of stable localized vitiligo were included in the study and were treated with transplantation of autologous melanocyte keratinocyte suspension after motor/manual dermabrasion. Results: Out of total 10 patients, 40% [4 patients] had excellent [95 to 100%] response, 30% [3 patients] had good [65 to 94%] response, 20% [2 patients] had fair [20 to 64%] response and 10% [1 patient] had poor response [0 to 19%]. Age and sex of the patients and size and location of lesions, did not show significant influence on results of transplantation. Conclusion: Autologus melanocyte keratinocyte suspension combined with motor/manual dermabrasion is an effective affordable treatment for patients with stable vitiligo who fail to respond to medical treatments.

  13. Anti-Inflammatory Effect of Melittin on Porphyromonas Gingivalis LPS-Stimulated Human Keratinocytes.

    Science.gov (United States)

    Kim, Woon-Hae; An, Hyun-Jin; Kim, Jung-Yeon; Gwon, Mi-Gyeong; Gu, Hyemin; Jeon, Minji; Kim, Min-Kyung; Han, Sang-Mi; Park, Kwan-Kyu

    2018-02-05

    Periodontitis is a chronic inflammatory disease that contributes to the destruction of the gingiva. Porphyromonas gingivalis ( P. gingivalis ) can cause periodontitis via its pathogenic lipopolysaccharides (LPS). Melittin, a major component of bee venom, is known to have anti-inflammatory and antibacterial effects. However, the role of melittin in the inflammatory response has not been elucidated in periodontitis-like human keratinocytes. Therefore, we investigated the anti-inflammatory effects of melittin on a P. gingivalis LPS (PgLPS)-treated HaCaT human keratinocyte cell line. The cytotoxicity of melittin was measured using a human keratinocyte cell line, HaCaT, and a Cell Counting Kit-8. The effect of melittin on PgLPS-induced inflammation was determined with Western blot, real-time quantitative PCT, and immunofluorescence. PgLPS increased the expression of toll-like receptor (TLR) 4 and proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-8, and interferon-γ (IFN-γ). Moreover, PgLPS induced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), extracellular signal-regulated kinase (ERK), and protein kinase B/Akt. Melittin also inhibited the expression of proinflammatory cytokines by suppressing the activation of the NF-κB signaling pathway, ERK, and Akt. Melittin attenuates the PgLPS-induced inflammatory response and could therefore be applied in the treatment of periodontitis for anti-inflammatory effects.

  14. Beneficial Effects of the Genus Aloe on Wound Healing, Cell Proliferation, and Differentiation of Epidermal Keratinocytes.

    Science.gov (United States)

    Moriyama, Mariko; Moriyama, Hiroyuki; Uda, Junki; Kubo, Hirokazu; Nakajima, Yuka; Goto, Arisa; Akaki, Junji; Yoshida, Ikuyo; Matsuoka, Nobuya; Hayakawa, Takao

    2016-01-01

    Aloe has been used as a folk medicine because it has several important therapeutic properties. These include wound and burn healing, and Aloe is now used in a variety of commercially available topical medications for wound healing and skin care. However, its effects on epidermal keratinocytes remain largely unclear. Our data indicated that both Aloe vera gel (AVG) and Cape aloe extract (CAE) significantly improved wound healing in human primary epidermal keratinocytes (HPEKs) and a human skin equivalent model. In addition, flow cytometry analysis revealed that cell surface expressions of β1-, α6-, β4-integrin, and E-cadherin increased in HPEKs treated with AVG and CAE. These increases may contribute to cell migration and wound healing. Treatment with Aloe also resulted in significant changes in cell-cycle progression and in increases in cell number. Aloe increased gene expression of differentiation markers in HPEKs, suggesting roles for AVG and CAE in the improvement of keratinocyte function. Furthermore, human skin epidermal equivalents developed from HPEKs with medium containing Aloe were thicker than control equivalents, indicating the effectiveness of Aloe on enhancing epidermal development. Based on these results, both AVG and CAE have benefits in wound healing and in treatment of rough skin.

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

    Science.gov (United States)

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

    2010-03-01

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

  16. Increased hydrophobicity in Malassezia species correlates with increased proinflammatory cytokine expression in human keratinocytes.

    Science.gov (United States)

    Akaza, Narifumi; Akamatsu, Hirohiko; Takeoka, Shiori; Mizutani, Hiroshi; Nakata, Satoru; Matsunaga, Kayoko

    2012-11-01

    Malassezia cells stimulate cytokine production by keratinocytes, although this ability differs among Malassezia species for unknown reasons. The aim of this study was to clarify the factors determining the ability to induce cytokine production by human keratinocytes in response to Malassezia species. M. furfur NBRC 0656, M. sympodialis CBS 7222, M. dermatis JCM 11348, M. globosa CBS 7966, M. restricta CBS 7877, and three strains each of M. globosa, M. restricta, M. dermatis, M. sympodialis, and M. furfur maintained under various culture conditions were used. Normal human epidermal keratinocytes (NHEKs) (1 × 10(5) cells) and the Malassezia species (1 × 10(6) cells) were co-cultured, and IL-1α, IL-6, and IL-8 mRNA levels were determined. Moreover, the hydrophobicity and β-1,3-glucan expression at the surface of Malassezia cells were analyzed. The ability of Malassezia cells to trigger the mRNA expression of proinflammatory cytokines in NHEKs differed with the species and conditions and was dependent upon the hydrophobicity of Malassezia cells not β-1,3-glucan expression.

  17. Adiponectin Suppresses UVB-Induced Premature Senescence and hBD2 Overexpression in Human Keratinocytes.

    Directory of Open Access Journals (Sweden)

    MinJeong Kim

    Full Text Available Recent studies have revealed that adiponectin can suppress cellular inflammatory signaling pathways. This study aimed to elucidate the effect of adiponectin on the unregulated production of hBD2 in UVB-induced premature senescent keratinocytes. We constructed an in vitro model of premature senescent keratinocytes through repeated exposure to low energy UVB. After repeated low energy UVB exposure, there was significant generation of reactive oxygen species (ROS and induction of senescence-associated markers, including senescence associated beta-galactosidase activity and expression of p16INK4a and histone H2AX. In addition, the present clinical study showed higher expression of hBD2 in sun-exposed skin of elderly group, and the overexpression of hBD2 was observed by c-Fos activation in vitro. Adiponectin has the ability to scavenge ROS and consequently inhibit MAPKs and SA-markers in UVB-exposed keratinocytes. An inhibitor study demonstrated that adiponectin downregulated hBD2 mRNA expression through suppression of the AP-1 transcription factor components c-Fos via inactivation of p38 MAPK. Collectively, the dysregulated production of hBD2 by the induction of oxidative stress was attenuated by adiponectin through the suppression of p38 and JNK/SAPK MAPK signaling in UVB-mediated premature senescent inducible conditions. These results suggest the feasibility of adiponectin as an anti-photoaging and anti-inflammatory agent in the skin.

  18. Treatment of therapy-refractive ulcera cruris of various origins with autologous keratinocytes in fibrin sealant.

    Science.gov (United States)

    Johnsen, S; Ermuth, T; Tanczos, E; Bannasch, H; Horch, R E; Zschocke, I; Peschen, M; Schöpf, E; Vanscheidt, W; Augustin, M

    2005-02-01

    Evaluation of the effects of cultivated, subconfluent, autologous keratinocytes in fibrin sealant (BioSeed-S) on the healing of therapy-refractive chronic wounds. Open observational study in 60 patients with chronic leg ulcers and impaired wound healing of various origins. After whole-skin excision and cultivation of the autologous keratinocytes, the suspended cells were applied to the preconditioned wound in fibrin sealant. Wound epithelization and wound size were recorded at defined times. Fifty-two of the 60 participating patients could be evaluated. After 6 weeks, 29 ulcers (55.8%) were healed. The mean epithelization increased between the 8th and 42nd postoperative day from 23% to 62.5%. In 50.0% of the patients, global assessment of the wound showed a high degree of epithelization or healing after 42 days. In 32.6% of treated patients, improvement was observed, while no healing tendency was to be found in 17.4%. The present observational study indicates that the transplantation of autologous keratinocytes suspended in fibrin sealant could be of advantage in the treatment of refractive leg ulcers.

  19. Melatonin increases survival of HaCaT keratinocytes by suppressing UV-induced apoptosis.

    Science.gov (United States)

    Fischer, T W; Zbytek, B; Sayre, R M; Apostolov, E O; Basnakian, A G; Sweatman, T W; Wortsman, J; Elsner, P; Slominski, A

    2006-01-01

    Melatonin is a potent antioxidant and direct radical scavenger. As keratinocytes represent the major population in the skin and UV light causes damage to these cells, the possible protective effects of melatonin against UV-induced cell damage in HaCaT keratinocytes were investigated in vitro. Cells were preincubated with melatonin at graded concentrations from 10(-9) to 10(-3) m for 30 min prior to UV irradiation at doses of 25 and 50 mJ/cm2. Biological markers of cellular viability such as DNA synthesis and colony-forming efficiency as well as molecular markers of apoptosis were measured. DNA synthesis was determined by [3H]-thymidine incorporation into insoluble cellular fraction, clonogenicity through plating efficiency experiments and apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. DNA synthesis experiments showed a strong protective effect by preincubation with melatonin at concentrations of 10(-4) m (P UV incubation protective effect. These results indicate that preincubation is a requirement for melatonin to exert its protective effects. The mechanism of melatonin's protective effect (10(-6) to 10(-3) m) includes inhibition of apoptosis as measured by TUNEL assay. Moreover, the biological significance of these effects is supported by clonogenic studies showing a significantly higher number of colonies in cultures treated with melatonin compared to controls. Thus, pretreatment with melatonin led to strong protection against UVB-induced damage in keratinocytes.

  20. Effect of dihydrocaffeic acid on UV irradiation of human keratinocyte HaCaT cells.

    Science.gov (United States)

    Poquet, Laure; Clifford, Michael N; Williamson, Gary

    2008-08-15

    Dihydrocaffeic acid, a dietary constituent and a microbial metabolite of flavonoids, is an antioxidant, but few biological effects have been examined. After its production by microflora in the colon, dihydrocaffeic acid is absorbed and found in plasma as a combination of free and metabolized forms. Excess solar UV radiation provokes damage and initiates immune response and inflammation in skin, sometimes leading to cancer. Dihydrocaffeic acid reduced the cytotoxicity and pro-inflammatory cytokine production (interleukin-6 and -8) in HaCaT cells, a keratinocyte model, following UV radiation. The effect of dihydrocaffeic acid may result from a combination of direct radical scavenging of the reactive oxygen species formed or reinforcement of the antioxidant potential of the keratinocytes, as well as a direct interference with the pathway involved in cytokine stimulation. The minimum structure required for such an effect appears to consist of a propionate side chain attached to a catechol moiety, as indicated by the efficacy of caffeic acid, but not of the methyl and glucuronide conjugates of dihydrocaffeic acid. The data obtained suggest that dihydrocaffeic acid is a potential candidate for photo-protection by interfering with the events initiated after UV exposure in keratinocytes.

  1. Synergistic Cytotoxic Effects of Ganoderma lucidum and Bacillus Calmette Guérin on Premalignant Urothelial HUC-PC Cells and Its Regulation on Proinflammatory Cytokine Secretion

    Directory of Open Access Journals (Sweden)

    John Wai-man Yuen

    2012-01-01

    Full Text Available Bacillus Calmette-Guérin (BCG is conventionally used as an adjuvant immunotherapy to reduce the recurrence of bladder cancer. To address the issues of efficacy and safety, an ethanol extract of Ganoderma lucidum (GLe was evaluated for its interaction with BCG. In a model of premalignant human uroepithelial cells (HUC-PC, GLe exerted immediate cytotoxic effects while BCG showed a delayed response, given that both were immunological active in inducing the secretion of interleukin (IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1. Synergistic cytotoxic effects were observed when cells were either coincubated with both drugs or firstly preincubated with GLe. Synergism between GLe and BCG was demonstrated to achieve a complete cytostasis in 24 hours, and such effects were progressed in the subsequent 5 days. However, the pretreatment of GLe resulted in suppression of IL-6, IL-8, and MCP-1 secretions without affecting the cytotoxicity. Given that numerous proinflammatory cytokines are associated with the high side effects toll of BCG, results herein suggested the potential implications of GL to supplement the BCG immunotherapy in bladder cancer, for better efficacy and reducing side effects.

  2. Prevalence of High-Risk Genotypes of Human Papillomavirus: Women Diagnosed with Premalignant and Malignant Pap Smear Tests in Southern Ecuador

    Directory of Open Access Journals (Sweden)

    Paola Dalgo Aguilar

    2017-01-01

    Full Text Available Human papillomavirus (HPV is the primary infectious agent for the development of cervical cancer, although the presence of the virus alone is insufficient for viral development and proliferation; this can be attributed to the increase in potential oncogenic risk, along with other risk factors. In the present investigation, the prevalence of high-risk HPV was determined from samples of premalignant or malignant cervical cytology in women from the southern region of Ecuador. The kit we used was able to detect genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. In addition, 64.5% of the analyzed samples were positive for HPV, with genotypes 16 and 18 being the most prevalent (16 was detected in 148 samples and 18 in 108. Genotypes 58 and 51 were the third most frequent simple and multiple infections, respectively. The data are very similar to those obtained worldwide, suggesting that the strategy of sex education, and the use of vaccines as primary prevention agents, could significantly decrease the incidence and mortality rate of cervical cancer in the southern region of Ecuador.

  3. Plasma carotenoids and the risk of premalignant breast disease in women aged 50 and younger: a nested case-control study.

    Science.gov (United States)

    Cohen, Kevin; Liu, Ying; Luo, Jingqin; Appleton, Catherine M; Colditz, Graham A

    2017-04-01

    To examine the association of plasma carotenoids, micronutrients in fruits, and vegetables, with risk of premalignant breast disease (PBD) in younger women. Blood samples were collected at the Siteman Cancer Center between 2008 and 2012 from 3537 women aged 50 or younger with no history of cancer or PBD. The analysis included 147 participants diagnosed with benign breast disease or breast carcinoma in situ during a 27-month follow-up and 293 controls. Cases and controls were matched on age, race/ethnicity, and date of and fasting status at blood draw. Plasma carotenoids were quantified. We used logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs) and linear regression to assess racial differences in plasma carotenoids. The risk reduction between the highest and lowest tertiles varied by carotenoid, with β-cryptoxanthin having the greatest reduction (OR 0.62; 95% CI, 0.62-1.09; P trend  = 0.056) and total carotenoids the least (OR 0.83; 95% CI, 0.48-1.44; P trend  = 0.12). We observed an inverse association between plasma carotenoids and risk of PBD in obese women (BMI ≥ 30 kg/m 2 ; 61 cases and 115 controls) but not lean women (BMI carotenoids and risk of PBD in younger women, consistent with inverse associations reported for invasive breast cancer. Carotenoids may play a role early in breast cancer development.

  4. The configuration of clinics and the use of biopsy and photography in oral premalignancy: a survey of consultants of the British Association of Oral and Maxillofacial Surgeons.

    Science.gov (United States)

    Kanatas, A N; Fisher, S E; Lowe, D; Ong, T K; Mitchell, D A; Rogers, S N

    2011-03-01

    Oral dysplastic lesions may have an increased chance of becoming oral squamous cell carcinoma, but to date their management remains controversial. The aim of this survey was to explore the current practical aspects of the management of patients with dysplasia by oral and maxillofacial consultants in the UK. In the survey we asked consultants about the numbers of patients they see with oral premalignant lesions, the frequency and specialty of designated hospital clinics, their use of photographs and biopsy, factors that influence their decision whether to biopsy a lesion at the first appointment, the procedure for treatment and follow-up and their use (if any) of chemopreventive agents. We found a wide variation in the practical aspects of managing patients with dysplasia, and the lack of consistency among clinicians supports the idea of an initiative to establish more robust national guidelines to use as a gold standard in the future. Copyright © 2010 The British Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

  5. Induction of PDGF-B in TCA-treated epidermal keratinocytes.

    Science.gov (United States)

    Yonei, Nozomi; Kanazawa, Nobuo; Ohtani, Toshio; Furukawa, Fukumi; Yamamoto, Yuki

    2007-11-01

    Trichloroacetic acid (TCA) is one of the most widely used peeling agents, and induces full necrosis of the whole epidermis, followed by reconstitution of the epidermis and the matrix of the papillary dermis. The cytotoxic effects of TCA, such as suppressing proliferation of keratinocytes and fibroblasts and protein synthesis by fibroblasts, have already been reported. However, the entire biological mechanism responsible for TCA peeling has yet to be determined. Hypothetical activation effects of TCA treatment on epidermal cells to induce production of growth factors and cytokines are examined, and are compared with its cytotoxic effects in terms of time course and applied TCA concentrations. After various periods of incubation with TCA, viability of Pam212 murine keratinocytes was investigated with MTT assay and dye exclusion assay, and production of growth factors and cytokines with reverse transcription-polymerase chain reaction (RT-PCR). Changes in platelet-derived growth factor (PDGF)-B mRNA expression and protein production in the human skin specimens after TCA application were then examined by RT-PCR and immunohistochemistry, respectively. Incubation with TCA showed cytotoxicity and induced death of Pam212 cells, depending on the incubation period and the TCA concentration. In addition, expressions of PDGF-B, tumor growth factor (TGF)-alpha, TGF- beta1 and vascular endothelial growth factor, which are the growth factors reportedly secreted from keratinocytes during wound healing, were all detected in Pam212 cells after short-term treatment with TCA. Expressions of inflammatory cytokines such as interleukin (IL)-1 and IL-10 were also induced. In TCA-treated NIH-3T3 fibroblasts, in contrast, observed was upregulation of only keratinocyte growth factor, which is reportedly secreted from fibroblasts, as well as the similar cytotoxic effect. In human skin, PDGF-B mRNA expression became significantly upregulated after TCA application, and then immediately

  6. Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

    Directory of Open Access Journals (Sweden)

    Ami Oizumi

    Full Text Available Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD. A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes", which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii S1P induces the production

  7. The vitamin D receptor is required for activation of cWnt and hedgehog signaling in keratinocytes.

    Science.gov (United States)

    Lisse, Thomas S; Saini, Vaibhav; Zhao, Hengguang; Luderer, Hilary F; Gori, Francesca; Demay, Marie B

    2014-10-01

    Alopecia (hair loss) in vitamin D receptor (VDR)-null mice is due to absence of ligand-independent actions of the VDR that are required for initiation of postmorphogenic hair cycles. Investigations were undertaken to determine whether the VDR is required for the induction of signaling pathways that play an important role in this process. The induction of cWnt and hedgehog target genes that characterizes early anagen was found to be dramatically attenuated in VDR(-/-) mice, relative to wild-type (WT) mice. To determine whether this reflects impaired responsiveness to cWnt ligands, in vitro studies were performed in primary keratinocytes. These studies demonstrated impaired induction of cWnt target genes in response to Wnt3a in VDR(-/-) keratinocytes, relative to wild-type keratinocytes. Chromatin immunoprecipitation analyses revealed that the VDR was recruited to the regulatory regions of cWnt and hedgehog target genes in WT keratinocytes but not in VDR(-/-) or Lef1(-/-) keratinocytes. Lef1 was enriched on these same regulatory regions in WT keratinocytes but not in VDR(-/-) keratinocytes. In vivo studies were performed to determine whether activation of the hedgehog pathway could bypass the defect in cWnt signaling observed in the absence of the unliganded VDR. In WT, but not VDR(-/-), mice, hedgehog agonist treatment resulted in an induction of cWnt and hedgehog target genes and the generation of mature anagen hair follicles. Thus, these studies demonstrate that the unliganded VDR interacts with regulatory regions in the cWnt and hedgehog target genes and is required for the induction of these pathways during the postnatal hair cycle.

  8. Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes

    Directory of Open Access Journals (Sweden)

    Olerud John E

    2011-06-01

    Full Text Available Abstract Background Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. Results The impact of S. aureus soluble products in biofilm-conditioned medium (BCM or in planktonic-conditioned medium (PCM on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. Conclusions Collectively the results indicate that S

  9. Cyclin D1 localizes in the cytoplasm of keratinocytes during skin differentiation and regulates cell–matrix adhesion

    Science.gov (United States)

    Fernández-Hernández, Rita; Rafel, Marta; Fusté, Noel P; Aguayo, Rafael S; Casanova, Josep M; Egea, Joaquim; Ferrezuelo, Francisco; Garí, Eloi

    2013-01-01

    The function of Cyclin D1 (CycD1) has been widely studied in the cell nucleus as a regulatory subunit of the cyclin-dependent kinases Cdk4/6 involved in the control of proliferation and development in mammals. CycD1 has been also localized in the cytoplasm, where its function nevertheless is poorly characterized. In this work we have observed that in normal skin as well as in primary cultures of human keratinocytes, cytoplasmic localization of CycD1 correlated with the degree of differentiation of the keratinocyte. In these conditions, CycD1 co-localized in cytoplasmic foci with exocyst components (Sec6) and regulators (RalA), and with β1 integrin, suggesting a role for CycD1 in the regulation of keratinocyte adhesion during differentiation. Consistent with this hypothesis, CycD1 overexpression increased β1 integrin recycling and drastically reduced the ability of keratinocytes to adhere to the extracellular matrix. We propose that localization of CycD1 in the cytoplasm during skin differentiation could be related to the changes in detachment ability of keratinocytes committed to differentiation. PMID:23839032

  10. Characterization of Fetal Keratinocytes, Showing Enhanced Stem Cell-Like Properties: A Potential Source of Cells for Skin Reconstruction

    Directory of Open Access Journals (Sweden)

    Kenneth K.B. Tan

    2014-08-01

    Full Text Available Epidermal stem cells have been in clinical application as a source of culture-generated grafts. Although applications for such cells are increasing due to aging populations and the greater incidence of diabetes, current keratinocyte grafting technology is limited by immunological barriers and the time needed for culture amplification. We studied the feasibility of using human fetal skin cells for allogeneic transplantation and showed that fetal keratinocytes have faster expansion times, longer telomeres, lower immunogenicity indicators, and greater clonogenicity with more stem cell indicators than adult keratinocytes. The fetal cells did not induce proliferation of T cells in coculture and were able to suppress the proliferation of stimulated T cells. Nevertheless, fetal keratinocytes could stratify normally in vitro. Experimental transplantation of fetal keratinocytes in vivo seeded on an engineered plasma scaffold yielded a well-stratified epidermal architecture and showed stable skin regeneration. These results support the possibility of using fetal skin cells for cell-based therapeutic grafting.

  11. Superoxide anions produced by Streptococcus pyogenes group A-stimulated keratinocytes are responsible for cellular necrosis and bacterial growth inhibition.

    Science.gov (United States)

    Regnier, Elodie; Grange, Philippe A; Ollagnier, Guillaume; Crickx, Etienne; Elie, Laetitia; Chouzenoux, Sandrine; Weill, Bernard; Plainvert, Céline; Poyart, Claire; Batteux, Frédéric; Dupin, Nicolas

    2016-02-01

    Gram-positive Streptococcus pyogenes (group A Streptococcus or GAS) is a major skin pathogen and interacts with keratinocytes in cutaneous tissues. GAS can cause diverse suppurative and inflammatory infections, such as cellulitis, a common acute bacterial dermo-hypodermitis with a high morbidity. Bacterial isolation yields from the lesions are low despite the strong local inflammation observed, raising numerous questions about the pathogenesis of the infection. Using an in vitro model of GAS-infected keratinocytes, we show that the major ROS produced is the superoxide anion ([Formula: see text]), and that its production is time- and dose-dependent. Using specific modulators of ROS production, we show that [Formula: see text] is mainly synthesized by the cytoplasmic NADPH oxidase. Superoxide anion production leads to keratinocyte necrosis but incomplete inhibition of GAS growth, suggesting that GAS may be partially resistant to the oxidative burst. In conclusion, GAS-stimulated keratinocytes are able to develop an innate immune response based on the production of ROS. This local immune response limits GAS development and induces keratinocyte cell death, resulting in the skin lesions observed in patients with cellulitis. © The Author(s) 2015.

  12. Interaction of urokinase A chain with the receptor of human keratinocytes stimulates release of urokinase-like plasminogen activator

    Energy Technology Data Exchange (ETDEWEB)

    Fibbi, G.; Magnelli, L.; Pucci, M.; Del Rosso, M. (Florence Univ. (Italy))

    1990-03-01

    On the basis of a fibrinolytic assay with {sup 125}I-fibrin, zymography, and immunoprobing with anti-human urokinase antibody, the authors have observed that the in vitro established NCTC human keratinocyte cell line releases into the culture medium a 54,000-Da plasminogen activator which is indistinguishable from human urokinase. Only the early release following the washing of keratinocyte monolayers is accounted for by secretion of preformed enzyme, while late secretory events require the de novo synthesis of urokinase. The released enzyme can interact by autocriny with its own receptor present on keratinocytes. The addition to the keratinocyte culture medium of the urokinase A chain can stimulate a concentration-dependent urokinase oversecretion, which is not paralleled by oversecretion of plasminogen activator inhibitor-1. Since stimulation of urokinase production can be obtained by an A chain concentration which was previously shown to be efficient in inducing keratinocyte mobilization in an in vitro migration model system, they hypothesize that this mechanism may be important in vivo during the process of wound repair.

  13. HaCaT Keratinocytes and Primary Epidermal Keratinocytes Have Different Transcriptional Profiles of Cornified Envelope-Associated Genes to T Helper Cell Cytokines

    Science.gov (United States)

    Seo, Min-Duk; Kang, Tae Jin; Lee, Chang Hoon; Lee, Ai-Young; Noh, Minsoo

    2012-01-01

    HaCaT cells are the immortalized human keratinocytes and have been extensively used to study the epidermal homeostasis and its pathophysiology. T helper cells play a role in various chronic dermatological conditions and they can affect skin barrier homeostasis. To evaluate whether HaCaT cells can be used as a model cell system to study abnormal skin barrier development in various dermatologic diseases, we analyzed the gene expression profile of epidermal differentiation markers of HaCaT cells in response to major T helper (Th) cell cytokines, such as IFNγ, IL-4, IL-17A and IL-22. The gene transcriptional profile of cornified envelope-associated proteins, such as filaggrin, loricrin, involucrin and keratin 10 (KRT10), in HaCaT cells was generally different from that in normal human keratinocytes (NHKs). This suggests that HaCaT cells have a limitation as a model system to study the pathophysiological mechanism associated with the Th cell cytokine-dependent changes in cornified envelope-associated proteins which are essential for normal skin barrier development. In contrast, the gene transcription profile change of human β2-defensin (HBD2) in response to IFNγ, IL-4 or IL-17A in HaCaT cells was consistent with the expression pattern of NHKs. IFNγ also up-regulated transglutaminase 2 (TGM2) gene transcription in both HaCaT cells and NHKs. As an alternative cell culture system for NHKs, HaCaT cells can be used to study molecular mechanisms associated with abnormal HBD2 and TGM2 expression in response to IFNγ, IL-4 or IL-17A. PMID:24116291

  14. UVA and UVB irradiation differentially regulate microRNA expression in human primary keratinocytes.

    Directory of Open Access Journals (Sweden)

    Anne Kraemer

    Full Text Available MicroRNA (miRNA-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2, which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

  15. Rat embryonic fibroblasts improve reprogramming of human keratinocytes into induced pluripotent stem cells.

    Science.gov (United States)

    Linta, Leonhard; Stockmann, Marianne; Kleinhans, Karin N; Böckers, Anja; Storch, Alexander; Zaehres, Holm; Lin, Qiong; Barbi, Gotthold; Böckers, Tobias M; Kleger, Alexander; Liebau, Stefan

    2012-04-10

    Patient-specific human induced pluripotent stem (hiPS) cells not only provide a promising tool for cellular disease models in general, but also open up the opportunity to establish cell-type-specific systems for personalized medicine. One of the crucial prerequisites for these strategies, however, is a fast and efficient reprogramming strategy from easy accessible somatic cell populations. Keratinocytes from plucked human hair had been introduced as a superior cell source for reprogramming purposes compared with the widely used skin fibroblasts. The starting cell population is, however, limited and thereby further optimization in terms of time, efficiency, and quality is inevitable. Here we show that rat embryonic fibroblasts (REFs) should replace mouse embryonic fibroblasts as feeder cells in the reprogramming process. REFs enable a significantly more efficient reprogramming procedure as shown by colony number and total amount of SSEA4-positive cells. We successfully produced keratinocyte-derived hiPS (k-hiPS) cells from various donors. The arising k-hiPS cells display the hallmarks of pluripotency such as expression of stem cell markers and differentiation into all 3 germ layers. The increased reprogramming efficiency using REFs as a feeder layer occurred independent of the proliferation rate in the parental keratinocytes and acts, at least in part, in a non-cell autonomous way by secreting factors known to facilitate pluripotency such as Tgfb1, Inhba and Grem1. Hence, we provide an easy to use and highly efficient reprogramming system that could be very useful for a broad application to generate human iPS cells. © Mary Ann Liebert, Inc.

  16. Voriconazole N-oxide and its UVB photoproduct sensitize keratinocytes to UVA

    Science.gov (United States)

    Ona-Vu, K.; Oh, D.H.

    2015-01-01

    Background The antifungal agent, voriconazole, is associated with phototoxicity and photocarcinogenicity. Prior work has indicated that voriconazole and its hepatic N-oxide metabolite do not sensitize keratinocytes to ultraviolet B (UVB). Clinical observations have suggested ultraviolet A (UVA) may be involved. Objectives To determine the photochemistry and photobiology of voriconazole and its major hepatic metabolite, voriconazole N-oxide. Methods Voriconazole and voriconazole N-oxide were spectrophotometrically monitored following various doses of UVB. Cultured human keratinocytes were treated with parental drugs or with their UVB photoproducts, and survival following UVA irradiation was measured by thiazolyl blue metabolism. Reactive oxygen species and 8-oxoguanine were monitored by fluorescence microscopy. Results Voriconazole and voriconazole N-oxide have varying ultraviolet B (UVB) absorption but do not acutely sensitize cultured human keratinocytes following UVB exposure. However, sustained UVB exposures produced notable dose- and solvent-dependent changes in the absorption spectra of voriconazole N-oxide which in aqueous solution acquires a prominent ultraviolet A (UVA) absorption band, suggesting formation of a discrete photoproduct. Neither the parental drugs nor their photoproducts sensitized cells to UVB though all but voriconazole N-oxide were moderately toxic to cells in the dark. Notably, both voriconazole N-oxide and its UVB photoproduct, but not voriconazole or its photoproduct, additionally sensitized cells to UVA by >3-fold relative to controls in association with UVA-induced reactive oxygen species and 8-oxoguanine levels. Conclusions Voriconazole N-oxide and its UVB-photoproduct act as UVA-sensitizers that generate reactive oxygen species and that produce oxidative DNA damage. These results suggest a mechanism for the phototoxicity and photocarcinogenicity observed with voriconazole treatment. PMID:25919127

  17. Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

    International Nuclear Information System (INIS)

    Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

    2003-01-01

    We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

  18. Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

    Science.gov (United States)

    Sun, Baihui; Ding, Ruoting; Yu, Wenlin; Wu, Yanhong; Wang, Bulin; Li, Qin

    2016-07-01

    Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.

  19. Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

    International Nuclear Information System (INIS)

    Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.; Laskin, Debra L.; Heck, Diane E.; Laskin, Jeffrey D.

    2008-01-01

    Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione-S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We found that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity

  20. The A/G Allele of Rs16906252 Predicts for MGMT Methylation and Is Selectively Silenced in Premalignant Lesions from Smokers and in Lung Adenocarcinomas

    Science.gov (United States)

    Leng, Shuguang; Bernauer, Amanda M.; Hong, Chibo; Do, Kieu C.; Yingling, Christin M.; Flores, Kristina G.; Tessema, Mathewos; Tellez, Carmen S.; Willink, Randall P.; Burki, Elizabeth A.; Picchi, Maria A.; Stidley, Christine A.; Prados, Michael D.; Costello, Joseph F.; Gilliland, Frank D.; Crowell, Richard E.; Belinsky, Steven A.

    2011-01-01

    Purpose To address the association between sequence variants within the MGMT promoter-enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy. Experimental Design SNPs identified through sequencing a 1.9kb fragment 5' of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide was assessed. Results The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20–41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to temozolamide was strongly correlated with levels of MGMT methylation and expression. Conclusions These studies provide strong evidence that the A allele of a MGMT promoter-enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, temozolamide treatment may benefit a subset of lung cancer patients methylated for MGMT. PMID:21355081

  1. Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder.

    Science.gov (United States)

    Roma-Rodrigues, Catarina; Alves-Barroco, Cynthia; Raposo, Luís R; Costa, Mafalda N; Fortunato, Elvira; Baptista, Pedro Viana; Fernandes, Alexandra R; Santos-Sanches, Ilda

    2016-04-01

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates to human cells, suggesting for the first time the ability of bovine isolates to infect human cells. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  2. Portulaca oleracea extracts protect human keratinocytes and fibroblasts from UV-induced apoptosis.

    Science.gov (United States)

    Lee, Suyeon; Kim, Ki Ho; Park, Changhoon; Lee, Jong-Suk; Kim, Young Heui

    2014-10-01

    Portulaca oleracea extracts, known as Ma Chi Hyun in the traditional Korean medicine, show a variety of biomedical efficacies including those in anti-inflammation and anti-allergy. In this study, we investigate the protective activity of the P. oleracea extracts against UVB-induced damage in human epithelial keratinocytes and fibroblasts by several apoptosis-related tests. The results suggest that P. oleracea extracts have protective effects from UVB-induced apoptosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  3. Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

    DEFF Research Database (Denmark)

    Gibson, W T; Couchman, J R; Badley, R A

    1983-01-01

    immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact...... of overlap and colinearity of actin and fibronectin filaments. The ability of keratinocytes to produce fibronectin suggests that these cells can contribute to the formation of the basement membrane in skin. The localisation of fibronectin and its close association with actin also suggests that it is involved...

  4. Expression of a constitutively active calcineurin encoded by an intron-retaining mRNA in follicular keratinocytes.

    Directory of Open Access Journals (Sweden)

    Atsushi Fujimura

    Full Text Available Hair growth is a highly regulated cyclical process. Immunosuppressive immunophilin ligands such as cyclosporin A (CsA and FK506 are known as potent hair growth modulatory agents in rodents and humans that induce active hair growth and inhibit hair follicle regression. The immunosuppressive effectiveness of these drugs has been generally attributed to inhibition of T cell activation through well-characterized pathways. Specifically, CsA and FK506 bind to intracellular proteins, principally cyclophilin A and FKBP12, respectively, and thereby inhibit the phosphatase calcineurin (Cn. The calcineurin (Cn/NFAT pathway has an important, but poorly understood, role in the regulation of hair follicle development. Here we show that a novel-splicing variant of calcineurin Aß CnAß-FK, which is encoded by an intron-retaining mRNA and is deficient in the autoinhibitory domain, is predominantly expressed in mature follicular keratinocytes but not in the proliferating keratinocytes of rodents. CnAß-FK was weakly sensitive to Ca(2+ and dephosphorylated NFATc2 under low Ca(2+ levels in keratinocytes. Inhibition of Cn/NFAT induced hair growth in nude mice. Cyclin G2 was identified as a novel target of the Cn/NFATc2 pathway and its expression in follicular keratinocytes was reduced by inhibition of Cn/NFAT. Overexpression of cyclin G2 arrested the cell cycle in follicular keratinocytes in vitro and the Cn inhibitor, cyclosporin A, inhibited nuclear localization of NFATc2, resulting in decreased cyclin G2 expression in follicular keratinocytes of rats in vivo. We therefore suggest that the calcineurin/NFAT pathway has a unique regulatory role in hair follicle development.

  5. Cytotoxicity and terminal differentiation of human oral keratinocyte by indium ions from a silver-palladium-gold-indium dental alloy.

    Science.gov (United States)

    Lee, Jung-Hwan; Seo, Sang-Hee; Lee, Sang-Bae; Om, Ji-Yeon; Kim, Kwang-Mahn; Kim, Kyoung-Nam

    2015-02-01

    Dental alloys containing indium (In) have been used in dental restoration for two decades; however, no study has investigated the biological effects of In ions, which may be released in the oral cavity, on human oral keratinocytes. The objective of the present study was to investigate the biological effects of In ions on human oral keratinocyte after confirming their release from a silver-palladium-gold-indium (Ag-Pd-Au-In) dental alloy. As a corrosion assay, a static immersion tests were performed by detecting the released ions in the corrosion solution from the Ag-Pd-Au-In dental alloy using inductively coupled plasma atomic emission spectroscopy. The cytotoxicity and biological effects of In ions were then studied with In compounds in three human oral keratinocyte cell lines: immortalized human oral keratinocyte (IHOK), HSC-2, and SCC-15. Higher concentrations of In and Cu ions were detected in Ag-Pd-Au-In (PAg-Pd-Au, and AgCl deposition occurred on the surface of Ag-Pd-Au-In after a 7-day corrosion test due to its low corrosion resistance. At high concentrations, In ions induced cytotoxicity; however, at low concentrations (∼0.8In(3+)mM), terminal differentiation was observed in human oral keratinocytes. Intracellular ROS was revealed to be a key component of In-induced terminal differentiation. In ions were released from dental alloys containing In, and high concentrations of In ions resulted in cytotoxicity, whereas low concentrations induced the terminal differentiation of human oral keratinocytes via increased intracellular ROS. Therefore, dental alloys containing In must be biologically evaluated for their safe use. Copyright © 2014 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  6. Staphylococcus aureus keratinocyte invasion is dependent upon multiple high-affinity fibronectin-binding repeats within FnBPA.

    Directory of Open Access Journals (Sweden)

    Andrew M Edwards

    2011-04-01

    Full Text Available Staphylococcus aureus is a commensal organism and a frequent cause of skin and soft tissue infections, which can progress to serious invasive disease. This bacterium uses its fibronectin binding proteins (FnBPs to invade host cells and it has been hypothesised that this provides a protected niche from host antimicrobial defences, allows access to deeper tissues and provides a reservoir for persistent or recurring infections. FnBPs contain multiple tandem fibronectin-binding repeats (FnBRs which bind fibronectin with varying affinity but it is unclear what selects for this configuration. Since both colonisation and skin infection are dependent upon the interaction of S. aureus with keratinocytes we hypothesised that this might select for FnBP function and thus composition of the FnBR region. Initial experiments revealed that S. aureus attachment to keratinocytes is rapid but does not require FnBRs. By contrast, invasion of keratinocytes was dependent upon the FnBR region and occurred via similar cellular processes to those described for endothelial cells. Despite this, keratinocyte invasion was relatively inefficient and appeared to include a lag phase, most likely due to very weak expression of α(5β(1 integrins. Molecular dissection of the role of the FnBR region revealed that efficient invasion of keratinocytes was dependent on the presence of at least three high-affinity (but not low-affinity FnBRs. Over-expression of a single high-affinity or three low-affinity repeats promoted invasion but not to the same levels as S. aureus expressing an FnBPA variant containing three high-affinity repeats. In summary, invasion of keratinocytes by S. aureus requires multiple high-affinity FnBRs within FnBPA, and given the importance of the interaction between these cell types and S. aureus for both colonisation and infection, may have provided the selective pressure for the multiple binding repeats within FnBPA.

  7. Tissue-specific regulation of CXCL9/10/11 chemokines in keratinocytes: Implications for oral inflammatory disease.

    Directory of Open Access Journals (Sweden)

    Alison Marshall

    Full Text Available The IFN-γ-inducible chemokines CXCL9, CXCL10, and CXCL11 play a key role in many inflammatory conditions, particularly those mediated by T cells. Therefore, the production of these chemokines in peripheral tissues could be instrumental in the pathophysiology of tissue-specific immunological diseases such as oral lichen planus (OLP. In the present study, we assessed the production of keratinocyte-derived CXCL9/10/11 under basal and inflammatory conditions and investigated whether these chemokines were involved in the pathogenesis of OLP. We used semi-quantitative PCR, ELISA, chemotaxis assays, and fluorescence-activated cell sorting (FACS to assess the expression and functional role of CXCL9/10/11 in oral keratinocytes (three strains of normal human oral keratinocytes (NHOK, and the H357 oral cancer cell line in the presence or absence of IFN-γ. CXCL9/10/11 were also assessed in tissues from normal patients and those with oral lichen planus (OLP. The time course study in oral keratinocytes treated with IFN-γ showed that expression of CXCL9/10/11 chemokines was significantly enhanced by IFN-γ in a time-dependent manner. In particular, CXCL10, a prominent chemokine that was overexpressed by IFN-γ-stimulated NHOK, was able to effectively recruit CD4 lymphocytes, mainly CD4+CD45RA- cells. Significantly higher levels of CXCL9/10/11 were found in tissues from patients with OLP compared to normal oral mucosa. Taken together, the results demonstrate that normal oral keratinocytes produce chemotactic molecules that mediate T cell recruitment. This study furthers understanding of chemokine production in oral keratinocytes and their role in the pathophysiology of oral mucosa, with particular relevance to OLP.

  8. Transcription factors and stress response gene alterations in human keratinocytes following Solar Simulated Ultra Violet Radiation.

    Science.gov (United States)

    Marais, Thomas L Des; Kluz, Thomas; Xu, Dazhong; Zhang, Xiaoru; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

    2017-10-19

    Ultraviolet radiation (UVR) from sunlight is the major effector for skin aging and carcinogenesis. However, genes and pathways altered by solar-simulated UVR (ssUVR), a mixture of UVA and UVB, are not well characterized. Here we report global changes in gene expression as well as associated pathways and upstream transcription factors in human keratinocytes exposed to ssUVR. Human HaCaT keratinocytes were exposed to either a single dose or 5 repetitive doses of ssUVR. Comprehensive analyses of gene expression profiles as well as functional annotation were performed at 24 hours post irradiation. Our results revealed that ssUVR modulated genes with diverse cellular functions changed in a dose-dependent manner. Gene expression in cells exposed to a single dose of ssUVR differed significantly from those that underwent repetitive exposures. While single ssUVR caused a significant inhibition in genes involved in cell cycle progression, especially G2/M checkpoint and mitotic regulation, repetitive ssUVR led to extensive changes in genes related to cell signaling and metabolism. We have also identified a panel of ssUVR target genes that exhibited persistent changes in gene expression even at 1 week after irradiation. These results revealed a complex network of transcriptional regulators and pathways that orchestrate the cellular response to ssUVR.

  9. Coriandrum sativum L. protects human keratinocytes from oxidative stress by regulating oxidative defense systems.

    Science.gov (United States)

    Park, G; Kim, H G; Kim, Y O; Park, S H; Kim, S Y; Oh, M S

    2012-01-01

    Oxidative radicals are major environmental causes of human skin damage. Oxidative defense factors, including nuclear factor erythroid-derived 2-related factor 2 (Nrf2), are centrally involved in repairing skin cells or protecting them from oxidative damage. Coriandrum sativum L. (coriander; CS) is a commonly consumed food and a traditional phytomedicine in Asia and Europe. In this study, we examined the protective effects of a standardized CS leaf extract against oxidative stress in human HaCaT keratinocytes. CS significantly and dose-dependently protected cells against reduced cell viability caused by H2O2-induced damage, as assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Other assays demonstrated that CS protected HaCaT cells by increasing the levels of glutathione and activities of oxidative defense enzymes, such as superoxide dismutase and catalase. Moreover, it increased the expression of activated Nrf2, which plays a crucial role in protecting skin cells against oxidative stress. These results suggest that CS protects human keratinocytes from H2O2-induced oxidative stress through antioxidant effects. Copyright © 2012 S. Karger AG, Basel.

  10. Non-thermal Plasma Activates Human Keratinocytes by Stimulation of Antioxidant and Phase II Pathways

    Science.gov (United States)

    Schmidt, Anke; Dietrich, Stephan; Steuer, Anna; Weltmann, Klaus-Dieter; von Woedtke, Thomas; Masur, Kai; Wende, Kristian

    2015-01-01

    Non-thermal atmospheric pressure plasma provides a novel therapeutic opportunity to control redox-based processes, e.g. wound healing, cancer, and inflammatory diseases. By spatial and time-resolved delivery of reactive oxygen and nitrogen species, it allows stimulation or inhibition of cellular processes in biological systems. Our data show that both gene and protein expression is highly affected by non-thermal plasma. Nuclear factor erythroid-related factor 2 (NRF2) and phase II enzyme pathway components were found to act as key controllers orchestrating the cellular response in keratinocytes. Additionally, glutathione metabolism, which is a marker for NRF2-related signaling events, was affected. Among the most robustly increased genes and proteins, heme oxygenase 1, NADPH-quinone oxidoreductase 1, and growth factors were found. The roles of NRF2 targets, investigated by siRNA silencing, revealed that NRF2 acts as an important switch for sensing oxidative stress events. Moreover, the influence of non-thermal plasma on the NRF2 pathway prepares cells against exogenic noxae and increases their resilience against oxidative species. Via paracrine mechanisms, distant cells benefit from cell-cell communication. The finding that non-thermal plasma triggers hormesis-like processes in keratinocytes facilitates the understanding of plasma-tissue interaction and its clinical application. PMID:25589789

  11. Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

    Science.gov (United States)

    Zhang, Xiaoru; Kluz, Thomas; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

    2016-01-01

    Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

  12. Sulfur mustard induces the formation of keratin aggregates in human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Dillman, James F.; McGary, Kriston L.; Schlager, John J.

    2003-01-01

    The vesicant sulfur mustard is an alkylating agent that has the capacity to cross-link biological molecules. We are interested in identifying specific proteins that are altered upon sulfur mustard exposure. Keratins are particularly important for the structural integrity of skin, and several genetically inherited blistering diseases have been linked to mutations in keratin 5 and keratin 14. We examined whether sulfur mustard exposure alters keratin biochemistry in cultured human epidermal keratinocytes. Western blotting with specific monoclonal antibodies revealed the formation of stable high-molecular-weight 'aggregates' containing keratin 14 and/or keratin 5. These aggregates begin to form within 15 min after sulfur mustard exposure. These aggregates display a complex gel electrophoresis pattern between ∼100 and ∼200 kDa. Purification and analysis of these aggregates by one- and two-dimensional gel electrophoresis and mass spectrometry confirmed the presence of keratin 14 and keratin 5 and indicate that at least some of the aggregates are composed of keratin 14-keratin 14, keratin 14-keratin 5, or keratin 5-keratin 5 dimers. These studies demonstrate that sulfur mustard induces keratin aggregation in keratinocytes and support further investigation into the role of keratin aggregation in sulfur mustard-induced vesication

  13. Development of the sulfur mustard resistant keratinocyte cell line HaCaT/SM.

    Science.gov (United States)

    Schmidt, Annette; Steinritz, Dirk; Thiermann, Horst

    2016-02-26

    Pairs of corresponding cytotoxic drug sensitive and resistant cell lines are powerful tools to develop treatment strategies. Developing cytotoxic drug resistant cell lines is a well-established method in cancer research. In more than fifty years of sulfur mustard (SM) resistant research such a cell pair has never been produced. Hereinafter we describe the first successful approach to develop a SM resistant keratinocyte cell line. Starting with the SM sensitive keratinocyte cell line HaCaT we used a strategy of continuous exposure with gradually increased concentrations. Cells were cultured in total for more than 40 months starting with an initial concentration of 0.07μM SM twice a week up to a final concentration of 7.2μM SM. The achieved cell line HaCaT/SM had an LC50 resistance increase of 4.7-fold and an LC90 increase of 8.2-fold. Hereinafter we demonstrate the production of the first sulfur mustard (SM) resistant cell line. The new achieved cell line called HaCaT/SM is able to tolerate a continuous exposure of an SM concentration, which is associated with an inhibitory effect of 93% within the original HaCaT cells, which were used as starting point. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  14. No evidence for follicular keratinocyte hyperproliferation in acne lesions as compared to autologous healthy hair follicles.

    Science.gov (United States)

    Persson, G; Johansson-Jänkänpää, E; Ganceviciene, R; Karadag, A S; Bilgili, S G; Omer, H; Alexeyev, O A

    2018-03-27

    Abnormal hyperkeratinization in sebaceous hair follicles has long been believed to play an important role in acne pathogenesis. Several early reports purported to provide histological evidence for hyperproliferation of keratinocytes in acne lesions by showing a higher expression of the Ki67 as well as certain keratins. The evidence is, however, not robust and a number of methodological and technical pitfalls can be identified in these studies. In this study we looked at the expression of proliferation, mitosis and apoptosis markers directly at acne skin lesions in 66 patients with acne vulgaris. Ki67 was assessed using immunohistochemistry and ⍺-tubulin, phospho-histone H3 and cleaved-PARP with immunofluorescence microscopy. Allogenic unaffected hair follicles from the same acne patients were used as an internal control. In both acne and control hair follicles the α-tubulin staining was universal, approaching 100% cells and showed no signs of changed assembly. Expression of cleaved-PARP - the apoptosis marker was a rare event. Cell proliferation rate measured by the expression of Ki67 and phospho-histone H3 was virtually identical between acne and the two control groups. Our findings show the absence of increased keratinocyte proliferation in acne vulgaris. Alternative mechanisms are likely responsible for infundibular hyperkeratinization in acne pathogenesis. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  15. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Ana Mafalda Baptista Tadeu

    Full Text Available In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

  16. Analyses of the secondary particle radiation and the DNA damage it causes to human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lebel E.; Rusek A.; Sivertz, M.; Yip, K.; Thompson, K.; Tafrov, S.

    2011-11-22

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  17. Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients

    Directory of Open Access Journals (Sweden)

    Magomet Aushev

    2017-09-01

    Full Text Available Epidermolysis bullosa simplex (EBS is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the KRT5 locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

  18. Protective effect of different antioxidant agents in UVB-irradiated keratinocytes

    Directory of Open Access Journals (Sweden)

    Sara Salucci

    2017-09-01

    Full Text Available Skin cells can respond to UVB-induced damage either by tolerating it, or restoring it through antioxidant activation and DNA repair mechanisms or, ultimately, undergoing programmed cell death, when damage is massive. Nutritional factors, in particular, food antioxidants, have attracted much interest because of their potential use in new preventive, protective, and therapeutic strategies for chronic degenerative diseases, including skin inflammation and cancer. Some polyphenols, present in virgin olive oil, well tolerated by organism after oral administration, show a variety of pharmacological and clinical benefits such as anti-oxidant, anti-cancer, anti-inflammatory, and neuro-protective activities. Here, the protective effects of antioxidant compounds against UV-induced apoptosis have been described in HaCat cell line. Human keratinocytes were pre-treated with antioxidants before UVB exposure and their effects have been evaluated by means of ultrastructural analyses. After UVB radiation, a known cell death trigger, typical apoptotic features, absent in control condition and in antioxidant alone-treated cells, appear. An evident numerical decrease of ultrastructural apoptotic patterns and TUNEL positive nuclei can be observed when natural antioxidants were supplied before cell death induction. These data have been confirmed by molecular investigation of caspase activity. In conclusion, this paper highlights antioxidant compound ability to prevent apoptotic cell death in human keratinocytes exposed to UVB, suggesting, for these molecules, a potential role in preventing skin damage. 

  19. Increased permeability of reconstructed human epidermis from UVB-irradiated keratinocytes.

    Science.gov (United States)

    Löwenau, Lilian Julia; Zoschke, Christian; Brodwolf, Robert; Volz, Pierre; Hausmann, Christian; Wattanapitayakul, Suvara; Boreham, Alexander; Alexiev, Ulrike; Schäfer-Korting, Monika

    2017-07-01

    Extrinsic (photo) aging accelerates chronologically aging in the skin due to cumulative UV irradiation. Despite recent insights into the molecular mechanisms of fibroblast aging, age-related changes of the skin barrier function have been understudied. In contrast, the constantly increasing subpopulation of aged patients causes a clinical need for effective and safe (dermatological) treatment. Herein, we reconstructed human epidermis from UVB-irradiated keratinocytes (UVB-RHE). UVB-irradiated keratinocytes show higher activity of senescence associated β-galactosidase, less cell proliferation, and reduced viability. Higher amounts of β-galactosidase are also detectable in UVB-RHE. Moreover, UVB-RHE release more interleukin-1α and -8 into the culture medium and present altered differentiation with a thinner stratum corneum compared to normal RHE. For the first time, the permeation of testosterone and caffeine through UVB-irradiated RHE indicate a clear influence of the UVB stress on the skin barrier function. Impaired barrier function was confirmed by the increased permeation of testosterone and caffeine as well as by the increased penetration of dendritic core-multishell nanocarriers into the constructs. Taken together, UVB-RHE emulate hallmarks of skin aging and might contribute to an improved non-clinical development of medicinal or cosmetic products. Copyright © 2016. Published by Elsevier B.V.

  20. Fibroblast and keratinocyte gene expression following exposure to extracts of neem plant (Azadirachta indica

    Directory of Open Access Journals (Sweden)

    Takao Someya

    2018-02-01

    Full Text Available This data article provides gene expression profiles, determined by using real-time PCR, of fibroblasts and keratinocytes treated with 0.01% and 0.001% extracts of neem plant (Azadirachta indica, local name “Kohomba” in Sri Lanka, harvested in Sri Lanka. For fibroblasts, the dataset includes expression profiles for genes encoding hyaluronan synthase 1 (HAS1, hyaluronan synthase 2 (HAS2, hyaluronidase-1 (HYAL1, hyaluronidase-2 (HYAL2, versican, aggrecan, CD44, collagen, type I, alpha 1 (COL1A1, collagen, type III, alpha 1 (COL3A1, collagen, type VII, alpha 1 (COL7A1, matrix metalloproteinase 1 (MMP1, acid ceramidase, basic fibroblast growth factor (bFGF, fibroblast growth factor-7 (FGF7, vascular endothelial growth factor (VEGF, interleukin-1 alpha (IL-1α, cyclooxygenase-2 (cox2, transforming growth factor beta (TGF-β, and aquaporin 3 (AQP3. For keratinocytes, the expression profiles are for genes encoding HAS1, HAS2, HYAL1, HYAL2, versican, CD44, IL-1α, cox2, TGF-β, AQP3, Laminin5, collagen, type XVII, alpha 1 (COL17A1, integrin alpha-6 (ITGA6, ceramide synthase 3 (CERS3, elongation of very long chain fatty acids protein 1 (ELOVL1, elongation of very long chain fatty acids protein 4 (ELOVL4, filaggrin (FLG, transglutaminase 1 (TGM1, and keratin 1 (KRT1. The expression profiles are provided as bar graphs.

  1. Atypical Diabetic Foot Ulcer Keratinocyte Protein Signaling Correlates with Impaired Wound Healing

    Directory of Open Access Journals (Sweden)

    Glenn D. Hoke

    2016-01-01

    Full Text Available Diabetes mellitus is associated with chronic diabetic foot ulcers (DFUs and wound infections often resulting in lower extremity amputations. The protein signaling architecture of the mechanisms responsible for impaired DFU healing has not been characterized. In this preliminary clinical study, the intracellular levels of proteins involved in signal transduction networks relevant to wound healing were non-biasedly measured using reverse-phase protein arrays (RPPA in keratinocytes isolated from DFU wound biopsies. RPPA allows for the simultaneous documentation and assessment of the signaling pathways active in each DFU. Thus, RPPA provides for the accurate mapping of wound healing pathways associated with apoptosis, proliferation, senescence, survival, and angiogenesis. From the study data, we have identified potential diagnostic, or predictive, biomarkers for DFU wound healing derived from the ratios of quantified signaling protein expressions within interconnected pathways. These biomarkers may allow physicians to personalize therapeutic strategies for DFU management on an individual basis based upon the signaling architecture present in each wound. Additionally, we have identified altered, interconnected signaling pathways within DFU keratinocytes that may help guide the development of therapeutics to modulate these dysregulated pathways, many of which parallel the therapeutic targets which are the hallmarks of molecular therapies for treating cancer.

  2. Hyperthermia-induced micronucleus formation in a human keratinocyte cell line

    International Nuclear Information System (INIS)

    Hintzsche, Henning; Riese, Thorsten; Stopper, Helga

    2012-01-01

    Elevated temperature can cause biological effects in vitro and in vivo. Many studies on effects of hypo- and hyperthermia have been conducted, but only few studies systematically investigated the formation of genomic damage in the micronucleus test in human cells in vitro as a consequence of different temperatures. In the present study, HaCaT human keratinocytes were exposed to different temperatures from 37 °C to 42 °C for 24 h in a regular cell culture incubator. Micronucleus frequency as a marker of genomic damage was elevated in a temperature-dependent and statistically significant manner. Apoptosis occurred at temperatures of 39 °C or higher. Cell proliferation was unaffected up to 40 °C and decreased at 41 °C and 42 °C. Expression of the heat shock protein Hsp70 was elevated, particularly at temperatures of 40 °C and higher. These findings are in agreement with several in vivo studies and some in vitro studies looking at single, specific temperatures, but a systematically investigated temperature-dependent increase of genomic damage in human keratinocytes in vitro is demonstrated for the first time here.

  3. Protection from UVB Toxicity in Human Keratinocytes by Thailand Native Herbs Extracts.

    Science.gov (United States)

    Thongrakard, Visa; Ruangrungsi, Nijsiri; Ekkapongpisit, Maneerat; Isidoro, Ciro; Tencomnao, Tewin

    2014-01-01

    Thai traditional medicine employs a wide range of indigenous herbs in the forms of tincture or tea for the cure of skin and systemic inflammatory diseases. The protection by Thai plants extracts against UVB DNA damage and cytotoxicity was investigated in human keratinocytes. Petroleum ether, dichloromethane and ethanol extracts were prepared from 15 Thai herb species, and the total phenolic and flavonoid contents, the antioxidant and UV-absorbing properties were assessed by standard procedures. Cytoprotective effects were evaluated on the basis of cell survival, caspase-3 activity and pyrimidine dimers determination. High total phenolic and flavonoid contents were found in the ethanol and dichloromethane fractions. Dichloromethane extract of turmeric was shown to possess the highest antioxidant activity. The maximum UV absorptions were found in the ethanol extract of turmeric and in the dichloromethane extract of ginger. These extracts stimulated the synthesis of Thioredoxin 1, an antioxidant protein, and could protect human HaCaT keratinocytes from UV-induced DNA damage and cytotoxicity. The present data support the utilization of turmeric and ginger extracts in anti-UV cosmetic pharmaceuticals. © 2013 The American Society of Photobiology.

  4. IKKα regulates human keratinocyte migration through surveillance of the redox environment.

    Science.gov (United States)

    Lisse, Thomas S; Rieger, Sandra

    2017-03-01

    Although the functions of H 2 O 2 in epidermal wound repair are conserved throughout evolution, the underlying signaling mechanisms are largely unknown. In this study we used human keratinocytes (HEK001) to investigate H 2 O 2 -dependent wound repair mechanisms. Scratch wounding led to H 2 O 2 production in two or three cell layers at the wound margin within ∼30 min and subsequent cysteine modification of proteins via sulfenylation. Intriguingly, exogenous H 2 O 2 treatment resulted in preferential sulfenylation of keratinocytes that adopted a migratory phenotype and detached from neighboring cells, suggesting that one of the primary functions of H 2 O 2 is to stimulate signaling factors involved in cell migration. Based on previous findings that revealed epidermal growth factor receptor (EGFR) involvement in H 2 O 2 -dependent cell migration, we analyzed oxidation of a candidate upstream target, the inhibitor of κB kinase α (IKKα; encoded by CHUK ), as a mechanism of action. We show that IKKα is sulfenylated at a conserved cysteine residue in the kinase domain, which correlates with de-repression of EGF promoter activity and increased EGF expression. Thus, this indicates that IKKα promotes migration through dynamic interactions with the EGF promoter depending on the redox state within cells. © 2017. Published by The Company of Biologists Ltd.

  5. FK506 regulates pigmentation by maturing the melanosome and facilitating their transfer to keratinocytes.

    Science.gov (United States)

    Jung, Hyejung; Chung, Heesung; Chang, Sung Eun; Kang, Duk-Hee; Oh, Eok-Soo

    2016-03-01

    Despite the clinical ability of topical tacrolimus (FK506) to effectively promote repigmentation in vitiligo, the underlying mechanism through which FK506 regulates melanogenesis was previously unclear. We found that FK506 treatment increased the melanin contents (especially that of eumelanin) in both melanocytes and melanoma cells. This treatment did not affect the transcription levels of tyrosinase, suggesting that FK506 increases melanin synthesis by regulating cellular levels of tyrosinase. Interestingly, FK506 promoted melanosome maturation by increasing melanosomal pH (a marker of melanosome maturation), thereby enhancing the stability of melanosome-localized tyrosinase. In addition, FK506 enhanced UVB-mediated melanosome secretion, the uptake of melanosomes by HaCaT cells, and the transfer of melanosomes to keratinocytes co-cultured with melanocytes. Together, these findings suggest that FK506 contributes to melanin synthesis by regulating the maturation of melanosomes and their transfer to keratinocytes. This offers a novel regulatory mechanism through which FK506 and UVB can have a combined effect on melanogenesis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Phevalin (aureusimine B production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

    Directory of Open Access Journals (Sweden)

    Patrick R Secor

    Full Text Available Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively. The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

  7. The ultrastructural surface morphology of oral cancer cells and keratinocytes after exposure to chitosan

    Science.gov (United States)

    Fatimah; Sarsito, A. S.; Wimardhani, Y. S.

    2017-08-01

    Low-molecular-weight chitosan (LMWC) has the same selective cytotoxic effects on oral cancer cells as cisplatin. The cell deaths caused by the anticancer characteristics of chitosan show that apoptosis is not the death pathway of the primary cells involved. The interactions between LMWC and the cells need to be explored. The objective of this study was to compare the ultrastructural morphology of oral Squamous Cell Carcinoma (SCC Ca)-922 and noncancer keratinocyte HaCaT cell lines after exposure to LMWC and cisplatin. The cells were treated with LMWC and cisplatin, and their ultrastructural morphology was analyzed using scanning electron micrographs. Features of early apoptosis, seen as the loss of microvilli, were detected in the LMWC-exposed Ca9-22 cells, and there was a material surrounding the cells. In contrast, the LMWC-exposed HaCaT cells showed no changes related to apoptosis. The results were the opposite when cisplatin was used. This study confirms that there are differences in the ultrastructural surface morphology of LMWC-exposed and cisplatin-exposed oral cancer cells and keratinocytes that could be correlated with their biological activity.

  8. Saponins from Tribulus terrestris L. protect human keratinocytes from UVB-induced damage.

    Science.gov (United States)

    Sisto, Margherita; Lisi, Sabrina; D'Amore, Massimo; De Lucro, Raffaella; Carati, Davide; Castellana, Donatello; La Pesa, Velia; Zuccarello, Vincenzo; Lofrumento, Dario D

    2012-12-05

    Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents

    Directory of Open Access Journals (Sweden)

    Tatiana Takahasi Komoto

    2015-01-01

    Full Text Available Trichophyton rubrum is the most common causative agent of dermatomycoses worldwide, causing infection in the stratum corneum, nails, and hair. Despite the high prevalence of these infections, little is known about the molecular mechanisms involved in the fungal-host interaction, particularly during antifungal treatment. The aim of this work was to evaluate the gene expression of T. rubrum cocultured with keratinocytes and treated with the flavonoid trans-chalcone and the glycoalkaloid α-solanine. Both substances showed a marked antifungal activity against T. rubrum strain CBS (MIC = 1.15 and 17.8 µg/mL, resp.. Cytotoxicity assay against HaCaT cells produced IC50 values of 44.18 to trans-chalcone and 61.60 µM to α-solanine. The interaction of keratinocytes with T. rubrum conidia upregulated the expression of genes involved in the glyoxylate cycle, ergosterol synthesis, and genes encoding proteases but downregulated the ABC transporter TruMDR2 gene. However, both antifungals downregulated the ERG1 and ERG11, metalloprotease 4, serine proteinase, and TruMDR2 genes. Furthermore, the trans-chalcone downregulated the genes involved in the glyoxylate pathway, isocitrate lyase, and citrate synthase. Considering the urgent need for more efficient and safer antifungals, these results contribute to a better understanding of fungal-host interactions and to the discovery of new antifungal targets.

  10. Mean Annual UV-B Irradiance

    Data.gov (United States)

    U.S. Environmental Protection Agency — Ultraviolet-B (UV-B) radiation is the most energetic part of sunlight reaching the Earth's surface (wavelength region is 280 to 315 nm), and it has been shown to...

  11. Antipsoriatic anthrones with modulated redox properties. 5. Potent inhibition of human keratinocyte growth, induction of keratinocyte differentiation, and reduced membrane damage by novel 10-arylacetyl-1,8-dihydroxy-9(10H)-anthracenones.

    Science.gov (United States)

    Müller, K; Reindl, H; Breu, K

    2001-03-01

    The synthesis and structure-activity relationships (SARs) of a series of novel 10-arylacetyl-1,8-dihydroxy-9(10H)-anthracenones are described. Acylation of anthralin with either the appropriate arylacetyl chlorides or arylacetic acids in the presence of pyridine or via the coupling agent dicyclohexylcarbodiimide (DCC), respectively, furnished this structural class of antipsoriatic agents. Potential antipsoriatic activity was evaluated in complementary assays specifically addressed to three important aspects of psoriasis. First, several compounds were identified which are equally potent as inhibitors of human keratinocyte growth as the antipsoriatic agent anthralin. Furthermore, improved ratio of antiproliferative activity to cytotoxicity is demonstrated by the reduced potential of the novel analogues to induce membrane damage, which is a benefit of their reduced ability to generate oxygen radicals as documented by deoxyribose degradation. Second, analogue 3o bearing a hydroxamate functional group was also a highly potent inhibitor of LTB(4) biosynthesis in addition to its excellent antiproliferative activity. SARs of these inhibitors of both keratinocyte growth and LTB(4) biosynthesis with respect to the nature of the para-substitution in the 10-phenylacetyl side chain are discussed. Third, the compounds were also evaluated for their ability to induce the formation of cornified envelope protein in keratinocytes. Cross-linking of cellular protein as a marker of terminal differentiation of keratinocytes was observed for many 10-arylacetyl analogues at concentrations required to arrest cell growth. This newly uncovered activity of the novel anthracenones suggests antipsoriatic potential with respect to disturbance of keratinocyte differentiation, in addition to hyperproliferative and inflammatory aspects of psoriasis.

  12. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, A.; Galosy, S.; Muschler, J.; Freedman, N.; Werb, Z.; Bissell, M.J.

    1997-08-11

    Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

  13. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  14. Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells.

    Science.gov (United States)

    Igawa, Ken; Kokubu, Chikara; Yusa, Kosuke; Horie, Kyoji; Yoshimura, Yasuhide; Yamauchi, Kaori; Suemori, Hirofumi; Yokozeki, Hiroo; Toyoda, Masashi; Kiyokawa, Nobutaka; Okita, Hajime; Miyagawa, Yoshitaka; Akutsu, Hidenori; Umezawa, Akihiro; Katayama, Ichiro; Takeda, Junji

    2014-09-01

    Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes. ©AlphaMed Press.

  15. Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines.

    Directory of Open Access Journals (Sweden)

    Ayako Uto-Konomi

    Full Text Available Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS, a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.

  16. Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration.

    Science.gov (United States)

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

    2016-08-11

    The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections.

  17. Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

    Science.gov (United States)

    While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

  18. Human allogeneic keratinocytes cultured on acellular xenodermis: the use in healing of burns and other skin defects

    Czech Academy of Sciences Publication Activity Database

    Matoušková, Eva; Brož, L.; Štolbová, V.; Klein, L.; Konigová, R.; Veselý, Pavel

    2006-01-01

    Roč. 16, č. 4 (2006), s. 63-71 ISSN 0959-2989 R&D Projects: GA AV ČR(CZ) IBS5052312 Institutional research plan: CEZ:AV0Z50520514 Keywords : acellular xenodermis * human keratinocytes * wound grafting Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.922, year: 2006

  19. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    International Nuclear Information System (INIS)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

    2011-01-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  20. Tumor necrosis factor related apoptosis inducing ligand triggers apoptosis in dividing but not in differentiating human epidermal keratinocytes

    NARCIS (Netherlands)

    Jansen, Bastiaan J. H.; van Ruissen, Fred; Cerneus, Stefanie; Cloin, Wendy; Bergers, Mieke; van Erp, Piet E. J.; Schalkwijk, Joost

    2003-01-01

    Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis

  1. HDAC Activity Is Required for p65/RelA-Dependent Repression of PPARdelta-Mediated Transactivation in Human Keratinocytes

    DEFF Research Database (Denmark)

    Aarenstrup, Lene; Flindt, Esben Noerregaard; Otkjaer, Kristian

    2008-01-01

    Peroxisome proliferator-activated receptors (PPARs) play a key role in differentiation, inflammation, migration, and survival of epidermal keratinocytes. The NF-kappaB has long been known to play pivotal roles in immune and inflammatory responses, and furthermore NF-kappaB has been implicated in ...

  2. Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, Tatsuya, E-mail: tatsuya.hasegawa@to.shiseido.co.jp; Nakashima, Masaya; Suzuki, Yoshiharu

    2016-08-26

    Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA, which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.

  3. In vitro evaluation of the effects of human umbilical cord extracts on human fibroblasts, keratinocytes, and melanocytes.

    Science.gov (United States)

    Van Pham, Phuc; Dang, Loan Thi-Tung; Dinh, Uyen Thanh; Truong, Huyen Thi-Thu; Huynh, Ba Ngoc; Van Le, Dong; Phan, Ngoc Kim

    2014-04-01

    Skin aging is the result of internal and external factors. So-called photoaging has been identified as the major factor in skin aging. Effects of photoaging include inhibition of fibroblast and keratinocyte proliferation as well as collagen and fibronectin expression, while activating expression of collagenases such as matrix metalloproteinase-1. Previous studies have shown that extracts or products from human placenta significantly improve skin aging and chronic wound healing. However, there are few studies of umbilical cord extracts. Therefore, this study aimed to evaluate the effects of umbilical cord extract-derived formulae on three kinds of skin cells including fibroblasts, keratinocytes, and melanocytes. We prepared 20 formulae from intracellular umbilical cord extracts, extracellular umbilical cord extracts, and umbilical cord-derived stem cell extracts, as well as five control formulae. We evaluated the effects of the 25 formulae on fibroblast and keratinocyte proliferation, and expression of collagen I, fibronectin, and matrix metalloproteinase-1 in fibroblasts and tyrosinase in melanocytes. The results showed that 7.5% formula 35 was the most effective formula for promotion of fibroblast and keratinocyte proliferation. At this concentration, formula 35 also induced collagen expression and inhibited matrix metalloproteinase-1 expression at the transcriptional level. However, this formula had no effect on tyrosinase expression in melanocytes. These results demonstrate that umbilical cord extracts can serve as an attractive source of proteins for skincare and chronic wound healing products.

  4. Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

    International Nuclear Information System (INIS)

    Spirito, Flavia; Capt, Annabelle; Rio, Marcela Del; Larcher, Fernando; Guaguere, Eric; Danos, Olivier; Meneguzzi, Guerrino

    2006-01-01

    Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (α3β3γ2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the α3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials

  5. Astragaloside IV Downregulates β-Catenin in Rat Keratinocytes to Counter LiCl-Induced Inhibition of Proliferation and Migration

    Directory of Open Access Journals (Sweden)

    Fu-Lun Li

    2012-01-01

    Full Text Available Re-epithelialization is a crucial step towards wound healing. The traditional Chinese medicine, Astragalus membranaceus (Fisch Bge, has been used for hundreds of years for many kinds of ulcerated wounds. Recent research has identified the active compound in this drug as astragaloside IV (AS-IV, but the underlying molecular mechanisms of its therapeutic action on keratinocytes remain poorly understood. In this study, we used an in vitro model of ulcer-like wound processes, lithium chloride (LiCl-induced cultured mouse keratinocytes, to investigate the effects of AS-IV treatment. The effects on cell proliferation were evaluated by the MTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on the cell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immunofluorescence. LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulated proliferating cell nuclear antigen (PCNA expression. AS-IV treatment attenuat the inhibition of proliferation and migration, significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression. Thus, AS-IV mediates mouse keratinocyte proliferation and migration via regulation of the Wnt signaling pathway. Down-regulating β-catenin to increase keratinocyte migration and proliferation is one mechanism by which AS-IV can promote ulcerated wound healing.

  6. The cytotoxic effect of neonatal lupus erythematosus and maternal sera on keratinocyte cultures is complement-dependent and can be augmented by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Yu, H.-S.; Chang, C.-H.; Kang, J.-W.; Chiang, L.-C.; Yu, C.-L.

    1996-01-01

    To elucidate the role of autoantibodies and ultraviolet (UV) exposure in the pathogenesis of the skin lesions in neonatal lupus erythematosus (NLE), keratinocytes were cultured, as the target cells, from a patient with NLE and from a normal neonate. We demonstrated that the expression of nuclear/cytoplasma Ro/SSA and La/SSB molecules on to the surface of NLE keratinocytes occurred to a much greater extent than that on normal keratinocytes. A dose of 200 mJ/cm 2 UVB irradiation on NLE keratinocytes induced a 2.5-3-fold increase in Ro/SSA and La/SSB expression compared to non-irradiated cells. Sera derived from both the NLE patient and from his mother exhibited a cytotoxic effect on NLE keratinocytes, but not on control cells, in the presence of complement. Furthermore, the cytotoxicity of the sera was enhanced in UVB-irradiated NLE keratinocytes, whereas it had no cytotoxic effects on UVB-irradiated control cells. This suggests that the abnormal expression of both Ro/SSA and La/SSB on the surface membrane of NLE keratinocytes induces the autoantibodies and complements to injure the cells. This complement-mediated cytotoxic effect can be augmented by UV irradiation, a concept not incompatible with the exacerbation of the skin eruption in sun-exposed skin sites. (author)

  7. Keratinocyte antiviral response to Poly(dA:dT stimulation and papillomavirus infection in a canine model of X-linked severe combined immunodeficiency.

    Directory of Open Access Journals (Sweden)

    Jennifer A Luff

    Full Text Available X-linked severe combined immunodeficiency (XSCID is caused by a genetic mutation within the common gamma chain (γc, an essential component of the cytokine receptors for interleukin (IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. XSCID patients are most commonly treated with bone marrow transplants (BMT to restore systemic immune function. However, BMT-XSCID humans and dogs remain at an increased risk for development of cutaneous papillomavirus (PV infections and their associated neoplasms, most typically cutaneous papillomas. Since basal keratinocytes are the target cell for the initial PV infection, we wanted to determine if canine XSCID keratinocytes have a diminished antiviral cytokine response to poly(dA:dT and canine papillomavirus-2 (CPV-2 upon initial infection. We performed quantitative RT-PCR for antiviral cytokines and downstream interferon stimulated genes (ISG on poly(dA:dT stimulated and CPV-2 infected monolayer keratinocyte cultures derived from XSCID and normal control dogs. We found that XSCID keratinocytes responded similarly to poly(dA:dT as normal keratinocytes by upregulating antiviral cytokines and ISGs. CPV-2 infection of both XSCID and normal keratinocytes did not result in upregulation of antiviral cytokines or ISGs at 2, 4, or 6 days post infection. These data suggest that the antiviral response to initial PV infection of basal keratinocytes is similar between XSCID and normal patients, and is not the likely source for the remaining immunodeficiency in XSCID patients.

  8. Keratinocyte antiviral response to Poly(dA:dT) stimulation and papillomavirus infection in a canine model of X-linked severe combined immunodeficiency.

    Science.gov (United States)

    Luff, Jennifer A; Yuan, Hang; Kennedy, Douglas; Schlegel, Richard; Felsburg, Peter; Moore, Peter F

    2014-01-01

    X-linked severe combined immunodeficiency (XSCID) is caused by a genetic mutation within the common gamma chain (γc), an essential component of the cytokine receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. XSCID patients are most commonly treated with bone marrow transplants (BMT) to restore systemic immune function. However, BMT-XSCID humans and dogs remain at an increased risk for development of cutaneous papillomavirus (PV) infections and their associated neoplasms, most typically cutaneous papillomas. Since basal keratinocytes are the target cell for the initial PV infection, we wanted to determine if canine XSCID keratinocytes have a diminished antiviral cytokine response to poly(dA:dT) and canine papillomavirus-2 (CPV-2) upon initial infection. We performed quantitative RT-PCR for antiviral cytokines and downstream interferon stimulated genes (ISG) on poly(dA:dT) stimulated and CPV-2 infected monolayer keratinocyte cultures derived from XSCID and normal control dogs. We found that XSCID keratinocytes responded similarly to poly(dA:dT) as normal keratinocytes by upregulating antiviral cytokines and ISGs. CPV-2 infection of both XSCID and normal keratinocytes did not result in upregulation of antiviral cytokines or ISGs at 2, 4, or 6 days post infection. These data suggest that the antiviral response to initial PV infection of basal keratinocytes is similar between XSCID and normal patients, and is not the likely source for the remaining immunodeficiency in XSCID patients.

  9. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    Science.gov (United States)

    Mauro, Marcella; Crosera, Matteo; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Piero; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Larese Filon, Francesca

    2015-01-01

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm−2), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm−2) and those with intact skin (1.08 ± 0.20 ng·cm−2). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue® and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10−4 M, 95% CL = 0.8–1.9 × 10−4 M, MTT essay; 3.7 × 10−5 M, 95% CI = 2.2–6.1 × 10−5 M, AlamarBlue® assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10−4 M, 95% CL = 0.9–1.9 × 10−4 M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure. PMID:26193294

  10. Effect of Absolute From Hibiscus syriacus L. Flower on Wound Healing in Keratinocytes.

    Science.gov (United States)

    Yoon, Seok Won; Lee, Kang Pa; Kim, Do-Yoon; Hwang, Dae Il; Won, Kyung-Jong; Lee, Dae Won; Lee, Hwan Myung

    2017-01-01

    Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysis. We also examined the effect of HSF absolute in HaCaT cells using the XTT assay, Boyden chamber assay, sprout-out growth assay, and western blotting. We conducted an in-vivo wound healing assay in rat tail-skin. Ten major active compounds were identified from HSF absolute. As determined by the XTT assay, Boyden chamber assay, and sprout-out growth assay results, HSF absolute exhibited similar effects as that of epidermal growth factor on the proliferation and migration patterns of keratinocytes (HaCaT cells), which were significantly increased after HSF absolute treatment. The expression levels of the phosphorylated signaling proteins relevant to proliferation, including extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, were also determined by western blot analysis. These results of our in-vitro and ex-vivo studies indicate that HSF absolute induced cell growth and migration of HaCaT cells by phosphorylating both Erk 1/2 and Akt. Moreover, we confirmed the wound-healing effect of HSF on injury of the rat tail-skin. Therefore, our results suggest that HSF absolute is promising for use in cosmetics and alternative medicine. Hisbiscus syriacus L. flower absolute increases HaCaT cell migration and proliferation. Hisbiscus syriacus L. flower absolute regulates phosphorylation of ERK 1/2 and Akt in HaCaT cell.Treatment with Hisbiscus syriacus L. flower induced sprout outgrowth.The wound in the tail-skin of rat was reduced by Hisbiscus syriacus L. flower absolute Abbreviations used: HSF: Hibiscus syriacus L. flower

  11. Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

    Directory of Open Access Journals (Sweden)

    Philpott Michael P

    2010-02-01

    Full Text Available Abstract Background The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. Results The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH and copy number variations (CNV. FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3 and segmental LOH (6q25.1-6q25.3. Conclusion We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant

  12. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

    International Nuclear Information System (INIS)

    Herbert, Katharine J.; Holloway, Adele; Cook, Anthony L.; Chin, Suyin P.; Snow, Elizabeth T.

    2014-01-01

    Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylation of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5 μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24 days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5 μM; > 5 weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24 h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity. - Highlights: • Submicromolar arsenic concentrations disrupt SIRT1 activity and expression in human keratinocytes. • Arsenic-induced chromatin remodelling at the miR-34a gene promoter is associated with hyperacetylation

  13. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    Directory of Open Access Journals (Sweden)

    Marcella Mauro

    2015-07-01

    Full Text Available Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1 and needle-abraded human skin (experiment 2. Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm−2, while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm−2 and those with intact skin (1.08 ± 0.20 ng·cm−2. To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue® and propidium iodide (PI uptake assays. The results indicate that a long exposure time (i.e., seven days was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10−4 M, 95% CL = 0.8–1.9 × 10−4 M, MTT essay; 3.7 × 10−5 M, 95% CI = 2.2–6.1 × 10−5 M, AlamarBlue® assay that seems to be associated to necrotic events (EC50 value: 1.3 × 10−4 M, 95% CL = 0.9–1.9 × 10−4 M, PI assay. This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

  14. Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

    Science.gov (United States)

    Wong, Victor W; Garg, Ravi K; Sorkin, Michael; Rustad, Kristine C; Akaishi, Satoshi; Levi, Kemal; Nelson, Emily R; Tran, Misha; Rennert, Robert; Liu, Wei; Longaker, Michael T; Dauskardt, Reinhold H; Gurtner, Geoffrey C

    2014-12-01

    To investigate how epithelial mechanotransduction pathways impact wound repair. Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

  15. Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis

    Directory of Open Access Journals (Sweden)

    Jussara Rehder

    Full Text Available CONTEXT: Recent progress in the field of epithelial culture techniques has allowed the development of culture systems in which the reconstructed epidermis presents characteristics of morphological differentiation similar to those seen in vivo. Human epidermis reconstructed in vitro may be used as the best alternative for the in vitro testing of the toxicology and efficiency of products for topical use, as well as in the treatment of skin burns and chronic skin ulcers. OBJECTIVE: To demonstrate a method for obtaining human epidermis reconstructed in vitro, using keratinocytes and melanocytes cultivated on dead de-epidermized human dermis. TYPE OF STUDY: Experimental/laboratory. SETTING: Skin Cell Culture Laboratory of the Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. PROCEDURE: Human keratinocytes and melanocytes cultured in vitro were grown on a biological matrix (dead de-epidermized human dermis and the system was kept at an air-liquid interface, in a suitable culturing medium, until a stratified human epidermis was formed, maintaining the histological characteristics of the epidermis in vivo. RESULTS: It was histologically demonstrated that it is possible to reproduce a differentiated epidermis through keratinocytes and melanocytes cultured on dead de-epidermized human dermis, thus obtaining a correctly positioned human epidermis reconstructed in vitro with functional keratinocytes and melanocytes that is similar to in vivo epidermis. CONCLUSIONS: It is possible to obtain a completely differentiated human epidermis reconstructed in vitro from keratinocyte and melanocyte cultures on a dead de-epidermized human dermis.

  16. Inhibition of inflammatory and proliferative responses of human keratinocytes exposed to the sesquiterpene lactones dehydrocostuslactone and costunolide.

    Directory of Open Access Journals (Sweden)

    Claudia Scarponi

    Full Text Available The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE and costunolide (CS, two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sunita Nayak

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

  19. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, Declan J., E-mail: dj.mckenna@ulster.ac.uk [Biomedical Sciences Research Institute, University of Ulster, Coleraine, Co. Derry BT52 1SA (United Kingdom); Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom); Patel, Daksha, E-mail: d.patel@qub.ac.uk [Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom); McCance, Dennis J., E-mail: d.mccance@qub.ac.uk [Centre for Cancer Research and Cell Biology, School of Medicine, Dentistry and Biomedical Science, Queen' s University Belfast, Belfast BT9 7BL (United Kingdom)

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  20. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    International Nuclear Information System (INIS)

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-01

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes

  1. The silver locus product (Silv/gp100/Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte-keratinocyte co-culture.

    Science.gov (United States)

    Singh, Suman K; Nizard, Carine; Kurfurst, Robin; Bonte, Frederic; Schnebert, Sylvianne; Tobin, Desmond J

    2008-05-01

    Melanosomes are melanocyte-specific lysosome-related organelles that are transferred to keratinocytes of the epidermis and anagen hair bulb. Transferred melanin forms supra-nuclear caps that protect epidermal keratinocytes against UV irradiation. The mechanism(s) responsible for melanosome transfer into keratinocytes and their subsequent intra-keratinocyte distribution has long remained one of the most enigmatic of heterotypic cell interactions. Although there have been many attempts to study this process, significant progress has been hindered by the absence of an adequate in vitro model. During our ongoing study of melanocyte-keratinocyte interactions in skin and hair follicle, we have developed a novel in vitro assay that exploits the specificity of Silv/Pmel17/gp100 expression for melanosome/melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, we have determined that gp100 is a surprisingly useful tracker of transferred melanin. Moreover, transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. This assay further confirmed that cytophagocytosis of melanocyte components (e.g. dendrite tips) by keratinocytes is one route for melanin incorporation into keratinocytes. Lastly, a role for the recently proposed filopodium as a direct conduit for melanin transfer was substantiated using this novel approach. In conclusion, this assay promises to significantly aid our investigations of the molecular basis of melanosome transfer and offers a new tool for the clinical evaluation of melanocyte modulators.

  2. Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Vikram Bhatia

    2014-01-01

    Full Text Available Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs and oral squamous cell carcinoma (OSCC. Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P=0.0010 and 57% (P=0.0016 of tissue samples, respectively, and 39% (P=0.0135 and 33% (P=0.0074 of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P=0.0001 and 82% (P=0.0001 in tissue and 57% (P=0.0002 and 70% (P=0.0001 in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

  3. An oral keratinocyte life cycle model identifies novel host genome regulation by human papillomavirus 16 relevant to HPV positive head and neck cancer

    OpenAIRE

    Evans, Michael R.; James, Claire D.; Loughran, Oonagh; Nulton, Tara J.; Wang, Xu; Bristol, Molly L.; Windle, Brad; Morgan, Iain M.

    2017-01-01

    Many aspects of the HPV life cycle have been characterized in cervical cell lines (W12, CIN612) and in HPV immortalized primary foreskin keratinocytes. There is now an epidemic of HPV positive oropharyngeal cancers (HPV16 is responsible for 80-90% of these); therefore increased understanding of the HPV16 life cycle in oral keratinocytes is a priority. To date there have been limited reports characterizing the HPV16 life cycle in oral keratinocytes. Using TERT immortalized “normal” oral kerati...

  4. Essential role of Nrf2 in keratinocyte protection from UVA by quercetin

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, Shintarou [Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki (Japan); Warabi, Eiji, E-mail: warabi-e@md.tsukuba.ac.jp [Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki (Japan); Yanagawa, Toru; Ma, Dongmei [Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki (Japan); Itoh, Ken [Department of Stress Response Science, Hirosaki University Graduate School of Medicine, Hirosaki (Japan); Ishii, Yoshiyuki; Kawachi, Yasuhiro; Ishii, Tetsuro [Majors of Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Ibaraki (Japan)

    2009-09-11

    Much of the cell injury caused by ultraviolet A (UVA) irradiation is associated with oxidative stress. Quercetin is a major natural polyphenol that is known to protect cells from UVA-induced damage. Here, we investigated the molecular mechanism of this protection. Quercetin pretreatment strongly suppressed UVA-induced apoptosis in human keratinocyte HaCaT cells, markedly increased protein levels of the transcription factor Nrf2, induced the expression of antioxidative genes, and dramatically reduced the production of reactive oxygen species following UVA irradiation. Importantly, these beneficial effects were greatly attenuated by downregulating Nrf2 expression. Thus, quercetin protects cells from UVA damage mainly by elevating intracellular antioxidative activity via the enhanced accumulation of a transcription factor for antioxidant genes, Nrf2.

  5. [14C]mechlorethamine binding to proteins of the human keratinocyte

    International Nuclear Information System (INIS)

    Deaton, M.A.; Jones, G.P.; Bowman, P.D.

    1990-01-01

    Much mustard agent research has focused on mustard/DNA interactions. Mustard also interacts with proteins, however, and to reach the DNA any agent must first pass through the cytoplasm. We hypothesized that the cell's proteins would covalently bind mustard, and thereby limit its access to the DNA. Keratinocyte proteins were radiolabeled with [ 14 C]mechlorethamine and separated by electrophoresis. The banding patterns that resulted were made visible on x-ray films, then compared with control patterns. A correspondence of almost one-to-one was observed, which supports the hypothesis that many cellular proteins are susceptible to mustard alkylation. It follows that some mustard symptoms probably result from effects on existing proteins

  6. UVB induces IL-12 transcription in human keratinocytes in vivo and in vitro

    International Nuclear Information System (INIS)

    Enk, C.D.; Blauvet, A.; Katz, S.I.; Mahanty, S.

    1996-01-01

    Human epidermal cells produce a wide range of cytokines, including those characteristic of Th2-like responses such as interleukin (IL)-4 and IL-10. As well, keratinocytes have recently been shown to produce Th1-like cytokines such as IL-12. Exposure to UVB has profound effects on the skin and systemic immune system, which is in part mediated by secretion of tumor necrosis factor (TNF)-α by epidermal cells. Because IL-12 induces production of TNF-α by certain cells of the immune system, we sought to determine whether UVB is an inducer of IL-12 gene expression in epidermal cells. Human epidermal cells were exposed to UVB radiation in vivo, isolated by suction blister technique and trypsinization and transcription of the IL-12 p35 and p40 chains was examined by RT-PCR. (Author)

  7. Platelet-rich plasma with keratinocytes and fibroblasts enhance healing of full-thickness wounds.

    Science.gov (United States)

    Law, Jia Xian; Chowdhury, Shiplu Roy; Saim, Aminuddin Bin; Idrus, Ruszymah Bt Hj

    2017-08-01

    Advances in tissue engineering led to the development of various tissue-engineered skin substitutes (TESS) for the treatment of skin injuries. The majority of the autologous TESS required lengthy and costly cell expansion process to fabricate. In this study, we determine the possibility of using a low density of human skin cells suspended in platelet-rich plasma (PRP)-enriched medium to promote the healing of full-thickness skin wounds. To achieve this, full-thickness wounds of size 1.767 cm 2 were created at the dorsum part of nude mice and treated with keratinocytes (2 × 10 4  cells/cm 2 ) and fibroblasts (3 × 10 4  cells/cm 2 ) suspended in 10% PRP-enriched medium. Wound examination was conducted weekly and the animals were euthanized after 2 weeks. Gross examination showed that re-epithelialization was fastest in the PRP+cells group at both day 7 and 14, followed by the PRP group and NT group receiving no treatment. Only the PRP+cells group achieved complete wound closure by 2 weeks. Epidermal layer was presence in the central region of the wound of the PRP+cells and PRP groups but absence in the NT group. Comparison between the PRP+cells and PRP groups showed that the PRP+cells-treated wound was more mature as indicated by the presence of thinner epidermis with single cell layer thick basal keratinocytes and less cellular dermis. In summary, the combination of low cell density and diluted PRP creates a synergistic effect which expedites the healing of full-thickness wounds. This combination has the potential to be developed as a rapid wound therapy via the direct application of freshly harvested skin cells in diluted PRP. Copyright © 2017 Tissue Viability Society. Published by Elsevier Ltd. All rights reserved.

  8. Forearm hair density and risk of keratinocyte cancers in Australian adults.

    Science.gov (United States)

    von Schuckmann, L A; Hughes, M C; Green, A C; van der Pols, J C

    2016-11-01

    Evidence suggests that progenitor cells of keratinocyte cancers (basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)) may originate from epidermal stem cells including hair follicle stem cells. We hypothesised that, therefore, a relatively higher density of hair follicles on human skin may increase keratinocyte cancer risk. To evaluate this, we assessed density of mid-forearm hair in Australian adults who were randomly selected participants in a community-based cohort study of skin cancer. Hair density was assessed clinically against a set of four standard photographs showing grades of hair density, and incidence data on histologically confirmed BCC and SCC across a 20-year period were collected. Incidence rate ratios were calculated for categories of forearm hair density using multivariable regression analysis with adjustment for age, sex, phenotypic characteristics and markers of chronic sun exposure. Among the 715 participants (43 % male, average age 61 years), 237 developed at least one BCC and 115 persons developed at least one SCC. Participants with dense forearm hair (n = 169, all male) had a higher incidence of BCC (IRR = 2.24, 95 % CI 1.20, 4.18, P = 0.01) and SCC (IRR = 2.80, 95 % CI 1.20, 6.57, P = 0.02) compared to individuals with sparse forearm hair after multivariable adjustment. Stratified analyses showed that among men, those with dense versus sparse hair developed SCC more commonly (IRR = 3.01, 95 % CI 1.03, 8.78, P = 0.04). Women with moderate versus sparse hair density were more likely affected by BCC (IRR = 2.29, 95 % CI 1.05, 5.00, P = 0.038). Thus, our study suggests that in both men and women, a higher density of body hair may be associated with increased BCC and SCC risk.

  9. Evaluation of gene expression profile of keratinocytes in response to JP-8 jet fuel

    International Nuclear Information System (INIS)

    Espinoza, Luis A.; Li Peijun; Lee, Richard Y.; Wang Yue; Boulares, A. Hamid; Clarke, Robert; Smulson, Mark E.

    2004-01-01

    The skin is the principal barrier against any environmental insult. Therefore, there is a high risk for a large number of military and civilian personnel exposed to jet fuel JP-8 to suffer percutaneous absorption of this fuel. This paper reports the use of cDNA microarray to identify the gene expression profile in normal human epidermal keratinocytes exposed to JP-8 for 24-h and 7-day periods. The effects of JP-8 exposure on keratinocytes at these two different periods induced a set of genes with altered expression in response to this type of insult. Microarray data were visualized using a novel algorithm based on simple statistical analyses to reduce data dimensionality and identify subsets of discriminant genes. Predictive neural networks were built using a multiplayer perceptron to carry out a proper classification task in microarray data in the untreated versus JP-8-treated samples. The pattern of expressions in response to JP-8 provides evidences that detoxificant-related and cell growth regulator genes with the most variability in the level of expression may be useful genetic markers in adverse health effects of personnel exposed to JP-8. The approaches in our analysis provide a simple, safe, novel, and effective method that is reliable in identifying and analyzing gene expression in samples treated with JP-8 or over potential toxic agents. Gene expression data from these studies can be used to build accurate predictive models that separate different molecular profiles. The data establish the use and effectiveness of these approaches for future prospective studies

  10. Cytostatic resistance profile of the sulfur mustard resistant keratinocyte cell line HaCaT/SM.

    Science.gov (United States)

    Schmidt, Annette; Wolf, Markus; Rothmiller, Simone; Worek, Franz; Steinritz, Dirk; Thiermann, Horst

    2018-03-15

    The cell line HaCaT/SM was developed as a sulfur mustard (SM) resistant cell line from the human keratinocyte cell line HaCaT. This cell line was established to learn more about the effect of SM and possible therapeutic approaches to counteract the cytotoxic effects of SM. The aim of this study was to clarify whether the SM-resistant cell line HaCaT/SM exhibit also resistance to other alkylating agents or cytotoxic drugs with different mechanism of action. The chemosensitivity of SM-resistant human keratinocyte cell line HaCaT/SM and the original cell line HaCaT were tested using the XTT assay. Nine cytotoxic drugs from five different substance groups were investigated. HaCaT/SM showed a significant increase in resistance against all tested drugs. From the substance class of the alkylating agents, HaCaT/SM showed the strongest resistance increase against chlorambucil (1.7 fold increase). Whereas over all substances strongest increase was observed against cisplatin (5.1 fold increase). The highest resistance was observed for cisplatin. The SM resistant cells revealed changes in the miRNA profile as described before. The resistance to cisplatin is also connected to a specific miRNA profile. Interestingly, changes of miRNA-203 and miRNA-21 levels were found in HaCaT/SM as well as in cisplatin resistant cells. It is therefore conceivable that the same resistance pathways are involved for both substances. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. [Promoter effect of mercury chloride and methyl-mercury on human keratinocytes in culture].

    Science.gov (United States)

    Zefferino, R; Elia, G; Petrozzi, M T; Leone, A; Corsi, P; Ambrosi, L

    2002-01-01

    Mercury has received considerable media focus because it is present in dental amalgams and seafood. There is potential exposure in gas meters, thermometers and fluorescent lamps workers. To evaluate its possible epigenetic carcinogen effect, cultures of human keratinocytes were treated with increasing doses of HgCl2 for 30 min, 24 h and of CH3HgCl for 24 h, respectively. The red neutral method was used to evaluate the doses of HgCl2 and CH3HgCl which had no cytotoxic effect. Then, the dye transfer method was used to investigate the gap junctions-mediated intercellular communication (GJIC). Cells were microinjected with Lucifer Yellow CH by using the Eppendorf Apparatus and the Leica inverted microscope. After 30 min incubation at the concentration of 10 microM, HgCl2 did not exert inhibition of GJIC. Conversely, after 24 h at the concentration of 10 nM, HgCl2 inhibited GJIC. Incubation with CH3HgCl at the concentration of 250 nM for 24 h reduced the number of fluorescent cells, thus denoting a inhibition of GJIC. Taken together our data demonstrated that: i) HgCl2 and CH3HgCl exerted an inhibitory effect upon GJIC; ii) HgCl2 resulted to inhibit GJIC at concentrations 25 folds lower than CH3HgCl. Further studies will be addressed to evaluate whether the reversal of GJIC inhibition could be obtained by withdrawal of toxic substance, or by the addition of a GJIC activator like the retinoic acid. Finally to shed light on the possible effect of mercury derivates at the transcriptional or translational levels, the expression profile of the connexin 43 gene after HgCl2 and CH3HgCl exposure of cultured human keratinocytes will be investigated.

  12. Human T-Lymphotropic virus (HTLV type I in vivo integration in oral keratinocytes

    Directory of Open Access Journals (Sweden)

    Martha C Domínguez

    2011-03-01

    Full Text Available Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP patients and 11 asymptomatic carrier individuals (AC coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14% (8/14 HAM/TSP patients and 27.28% (3/11 AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

  13. Effects of bee venom against Propionibacterium acnes-induced inflammation in human keratinocytes and monocytes.

    Science.gov (United States)

    Kim, Jung-Yeon; Lee, Woo-Ram; Kim, Kyung-Hyun; An, Hyun-Jin; Chang, Young-Chae; Han, Sang-Mi; Park, Yoon-Yub; Pak, Sok Cheon; Park, Kwan-Kyu

    2015-06-01

    Propionibacterium acnes (P. acnes) cause inflammatory acne and play an important role in the pathogenesis of acne by inducing inflammatory mediators. P. acnes contributes to the inflammatory responses of acne by activating inflammatory cells, keratinocytes and sebocytes to secrete pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-8. Bee venom has traditionally been used in the treatment of certain immune-related diseases. However, there has not yet been a robust trial to prove the therapeutic effect of bee venom in skin inflammation. The aim of the present study was to investigate anti-inflammatory properties of bee venom in skin inflammation induced by P. acnes using keratinocytes (HaCaT) and monocytes (THP-1). P. acnes is known to stimulate the production of pro-inflammatory cytokines such as IL-1, IL-8, IL-12 and TNF-α. In the present study, the production of interferon-γ (IFN-γ), IL-1β, IL-8 and TNF-α was increased by P. acnes treatment in HaCaT and THP-1 cells. By contrast, bee venom effectively inhibited the secretion of IFN-γ, IL-1β, IL-8 and TNF-α. Furthermore, P. acnes treatment activated the expression of IL-8 and toll-like receptor 2 (TLR2) in HaCaT cells. However, bee venom inhibited the expression of IL-8 and TLR2 in heat-killed P. acnes. Based on these results, it is concluded that bee venom has an effective anti-inflammatory activity against P. acnes in HaCaT and THP-1 cells. Therefore, we suggest that bee venom is an alternative treatment to antibiotic therapy of acne.

  14. Stimulation of the histamine 4 receptor upregulates thymic stromal lymphopoietin (TSLP) in human and murine keratinocytes.

    Science.gov (United States)

    Schaper, Katrin; Rossbach, Kristine; Köther, Brigitta; Stark, Holger; Kietzmann, Manfred; Werfel, Thomas; Gutzmer, Ralf

    2016-11-01

    The cytokine thymic stromal lymphopoietin (TSLP) is involved in the development and the progression of allergic diseases. It is mainly released by epithelial cells at barriers such as skin and gut in response to danger signals. Overexpression of TSLP in keratinocytes (KC) can provoke the development of a type 2 inflammatory response. Additionally, TSLP directly acts on sensory neurons and thereby triggers itch. Since histamine is also increased in lesions of inflammatory skin diseases, the aim of this study was to investigate possible effects of histamine as well as different histamine receptor subtype agonists and antagonists on TSLP production in KC. We therefore stimulated human KC with histamine in the presence or absence of the known TSLP-inductor poly I:C and measured TSLP production at protein as well as mRNA level. Histamine alone did not induce TSLP production in human KC, but pre-incubation with histamine prior to challenge with poly I:C resulted in a significant increase of TSLP production compared to stimulation with poly I:C alone. Experiments with different histamine receptor agonists (H1R: 2-pyridylethylamine; H2R: amthamine; H2R/H4R: 4-methylhistamine (4MH)) revealed a dominant role for the H4R receptor, as 4-MH in combination with poly I:C displayed a significant increase of TSLP secretion, while the other agonists did not show any effect. The increase in TSLP production by 4MH was blocked with the H4R antagonist JNJ7777120. This effect was reproducible also in the murine KC cell line MSC. Taken together, our study indicates a new role for the H4 receptor in the regulation of TSLP in keratinocytes. Therefore, blocking of the H4R receptor in allergic diseases might be promising to alleviate inflammation and pruritus via TSLP. Copyright © 2016. Published by Elsevier Ltd.

  15. Assessment of radiation induced cytogenetic damage in human keratinocytes by comet assay

    International Nuclear Information System (INIS)

    Joseph, Praveen; Sanjeev Ganesh; Narayana, Y.; Puthali, Abhay; Bhat, N.N.

    2010-01-01

    In the present study the effect of gamma radiation on normal human keratinocytes (HaCaT) cells has been analyzed using alkaline comet assay and a comparative study over the sensitivity of different comet parameters such as tail length (TL), olive tail moment (OTM) and percentage tail DNA (TDNA) has also been made. Human keratinocytes (HaCaT) cells were grown in Dulbecco's modified essential medium (DMEM) (10% FCS) at 37 °C in a humidified atmosphere containing 5% CO 2 . Cultured cells were harvested with 0.025 % trypsin EDTA. The sample (2 X 10 cells/ml) was exposed to gamma radiation of different dose using a 60 Co gamma source at dose rate of 2 Gy min -1 and the dosimetry has been carried out using Fricke and FBX dosimeters. After irradiation, to quantify the DNA damage the comet assay (single cell gel electrophoresis) was carried out under alkaline conditions, by the methods outlined by Singh et al. The quantification of the DNA strand breaks in each cells were performed using CASP software. The DNA damage quantification can be accomplished by measuring those comet parameters which exhibit a linear dependence on the amount of DNA damage. In the present study, comet parameters such as OTM, TL and TDNA were recorded and the variation of these parameters and their correlation coefficients for different doses of gamma radiation is plotted. The OTM value is normalized with control value and control for TL and TDNA is adjusted to zero to avoid initial variations in different experiments

  16. Photoprotective properties of Prunella vulgaris and rosmarinic acid on human keratinocytes.

    Science.gov (United States)

    Psotova, Jitka; Svobodova, Alena; Kolarova, Hana; Walterova, Daniela

    2006-09-01

    UVA radiation provokes the generation of reactive oxygen species (ROS), which induce oxidative stress in the exposed cells leading to extensive cellular damage and cell death either by apoptosis or necrosis. One approach to protecting human skin against the harmful effects of UV radiation is by using herbal compounds as photoprotectants. This study evaluated the protective effects of Prunella vulgaris L. (Labiatae) and its main phenolic acid component, rosmarinic acid (RA), against UVA-induced changes in a human keratinocyte cell line (HaCaT). Human keratinocytes exposed to UVA (10-30 J/cm(2)) were treated with an extract of P. vulgaris (1-75 mg/l) or RA (0.9-18 mg/l) for 4h. P. vulgaris and RA exhibited ability to reduce the UVA-caused decrease in a cell viability monitored by neutral red retention and by LDH release into medium. The P. vulgaris extract and RA significantly suppressed UVA-induced ROS production, which manifests as a decrease in intracellular lipid peroxidation, elevation of ATP and reduced glutathione. Post-treatment with P. vulgaris extract and RA also significantly reduced DNA damage. In addition, UVA-induced activation of caspase-3 was inhibited by treatment with P. vulgaris and RA. The P. vulgaris extract and RA demonstrated a concentration-dependent photoprotection (maximum at 25-50 mg/l and 9 mg/l, respectively). These results suggest that P. vulgaris and RA, used in skin care cosmetics, may offer protection against UVA-induced oxidative stress and may be beneficial as a supplement in photoprotective dermatological preparations.

  17. Evaluation of gene expression profile of keratinocytes in response to JP-8 jet fuel.

    Science.gov (United States)

    Espinoza, Luis A; Li, Peijun; Lee, Richard Y; Wang, Yue; Boulares, A Hamid; Clarke, Robert; Smulson, Mark E

    2004-10-15

    The skin is the principal barrier against any environmental insult. Therefore, there is a high risk for a large number of military and civilian personnel exposed to jet fuel JP-8 to suffer percutaneous absorption of this fuel. This paper reports the use of cDNA microarray to identify the gene expression profile in normal human epidermal keratinocytes exposed to JP-8 for 24-h and 7-day periods. The effects of JP-8 exposure on keratinocytes at these two different periods induced a set of genes with altered expression in response to this type of insult. Microarray data were visualized using a novel algorithm based on simple statistical analyses to reduce data dimensionality and identify subsets of discriminant genes. Predictive neural networks were built using a multiplayer perceptron to carry out a proper classification task in microarray data in the untreated versus JP-8-treated samples. The pattern of expressions in response to JP-8 provides evidences that detoxificant-related and cell growth regulator genes with the most variability in the level of expression may be useful genetic markers in adverse health effects of personnel exposed to JP-8. The approaches in our analysis provide a simple, safe, novel, and effective method that is reliable in identifying and analyzing gene expression in samples treated with JP-8 or over potential toxic agents. Gene expression data from these studies can be used to build accurate predictive models that separate different molecular profiles. The data establish the use and effectiveness of these approaches for future prospective studies.

  18. Sacha Inchi Oil (Plukenetia volubilis L.), effect on adherence of Staphylococus aureus to human skin explant and keratinocytes in vitro.

    Science.gov (United States)

    Gonzalez-Aspajo, German; Belkhelfa, Haouaria; Haddioui-Hbabi, Laïla; Bourdy, Geneviève; Deharo, Eric

    2015-08-02

    Plukenetia volubilis L. (Euphorbiaceae) is a domesticated vine distributed from the high-altitude Andean rain forest to the lowlands of the Peruvian Amazon. Oil from the cold-pressed seeds, sold under the commercial name of Sacha Inchi Oil (SIO) is actually much in favour because it contains a high percentage of omega 3 and omega 6, and is hence used as a dietary supplement. SIO is also used traditionally for skin care, in order to maintain skin softness, and for the treatment of wounds, insect bites and skin infections, in a tropical context where the skin is frequently damaged. This study was designed in order to verify whether the traditional use of SIO for skin care would have any impact on Staphylococcus aureus growth and skin adherence, as S. aureus is involved in many skin pathologies (impetigo, folliculitis, furuncles and subcutaneous abscesses) being one if the main pathogens that can be found on the skin. Therefore, our objective was to assess SIO bactericidal activity and interference with adherence to human skin explants and the keratinocyte cell line. Cytotoxicity on that cells was also determined. The activity of SIO was compared to coconut oil (CocO), which is widely used for skin care but has different unsaturated fatty acids contents. Laboratory testing with certified oil, determined antibacterial activity against radio labelled S. aureus. Cytotoxic effects were measured with XTT on keratinocyte cells and with neutral red on human skin explants; phenol was used as cytotoxic control. Adherence assays were carried out by mixing H3-labelled S. aureus bacteria with keratinocyte cells and human skin explants, incubated with oils 2h before (to determine the inhibition of adherence, assimilated to a preventive effect) or 2h after the contact of the biological material with S. aureus (to assess the detachment of the bacteria, assimilated to a curative effect). Residual radioactivity measured after washings made it possible to determine the adherence

  19. Spectrum of Immune-Related Conditions Associated with Risk of Keratinocyte Cancers among Elderly Adults in the United States.

    Science.gov (United States)

    Yanik, Elizabeth L; Pfeiffer, Ruth M; Freedman, D Michal; Weinstock, Martin A; Cahoon, Elizabeth K; Arron, Sarah T; Chaloux, Matthew; Connolly, M Kari; Nagarajan, Priyadharsini; Engels, Eric A

    2017-07-01

    Background: Elevated keratinocyte carcinoma risk is present with several immune-related conditions, e.g., solid organ transplantation and non-Hodgkin lymphoma. Because many immune-related conditions are rare, their relationships with keratinocyte carcinoma have not been studied. Methods: We used Medicare claims to identify cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) cases in 2012, and controls matched on sex and age. All subjects were aged 65 to 95 years, of white race, and had attended ≥1 dermatologist visit in 2010-2011. Immune-related conditions were identified during 1999-2011 using Medicare claims. Associations were estimated with logistic regression, with statistical significance determined after Bonferroni correction for multiple comparisons. Results: We included 258,683 SCC and 304,903 BCC cases. Of 47 immune-related conditions, 21 and 9 were associated with increased SCC and BCC risk, respectively. We identified strongly elevated keratinocyte carcinoma risk with solid organ transplantation (SCC OR = 5.35; BCC OR = 1.94) and non-Hodgkin lymphoma (SCC OR = 1.62; BCC OR = 1.25). We identified associations with common conditions, e.g., rheumatoid arthritis [SCC OR = 1.06, 95% confidence interval (95% CI), 1.04-1.09] and Crohn's disease (SCC OR = 1.33, 95% CI, 1.27-1.39; BCC OR = 1.10, 95% CI, 1.05-1.15), and rare or poorly characterized conditions, e.g., granulomatosis with polyangiitis (SCC OR = 1.88; 95% CI, 1.61-2.19), autoimmune hepatitis (SCC OR = 1.81; 95% CI, 1.52-2.16), and deficiency of humoral immunity (SCC OR = 1.51, 95% CI, 1.41-1.61; BCC OR = 1.22, 95% CI, 1.14-1.31). Most conditions were more positively associated with SCC than BCC. Associations were generally consistent regardless of prior keratinocyte carcinoma history. Conclusions: Many immune-related conditions are associated with elevated keratinocyte carcinoma risk and appear more tightly linked to SCC. Impact: Immunosuppression or immunosuppressive treatment

  20. Polypeptide from Chlamys farreri suppresses ultraviolet-B irradiation-induced apoptosis through restoring ER redox homeostasis, scavenging ROS generation, and suppressing the PERK-eIF2a-CHOP pathway in HaCaT cells.

    Science.gov (United States)

    Zhong, Feng; Xie, Jing; Zhang, Di; Han, Yantao; Wang, Chunbo

    2015-10-01

    The aim of this study was to investigate the effect of polypeptide from Chlamys farreri (PCF) on ultraviolet B (UVB) irradiation-induced apoptosis in human keratinocyte HaCaT cells. HaCaT cells were treated with 20 mJ/cm(2) UVB irradiation for 18 h. The cell viability was measured by MTT assay, and apoptosis was detected with Hoechst 33258 staining and caspase-3 activity detection. Protein expression levels were assessed by Western blot analysis, and the intracellular ROS levels were also measured. Our results from the MTT assay showed that UVB irradiation significantly declined the viability of HaCaT cells, which could be restored by PCF treatment. PCF decreased the apoptosis rate in HaCaT cells treated with UVB irradiation. Moreover, PCF increased the expression levels of PDI and Ero-1a, and scavenged the intracellular ROS. Furthermore, PCF inhibited the expressions of GRP78, p-PERK, p-eIF2a, and CHOP, and suppressed the ER stress-induced apoptosis, in UVB-irradiated HaCaT cells. In addition, the ROS scavenging effect of 4-PBA was less potent than PCF, indicating that ER stress-related ROS production contribute partially to the total ROS level, and ER was not the only target of PCF treatment. Our results indicate that PCF inhibits UVB irradiation-induced apoptosis through restoring ER redox homeostasis and suppressing the PERK-eIF2a-CHOP pathway. These findings provide evidence for the application of PCF in the protection of skin from UV irradiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. UVB-mediated activation of p38 mitogen-activated protein kinase enhances resistance of normal human keratinocytes to apoptosis by stabilizing cytoplasmic p53.

    OpenAIRE

    Chouinard, Nadine; Valerie, Kristoffer; Rouabhia, Mahmoud; Huot, Jacques

    2002-01-01

    Human keratinocytes respond to UV rays by developing a fast adaptive response that contributes to maintaining their functions and survival. We investigated the role of the mitogen-activated protein kinase pathways in transducing the UV signals in normal human keratinocytes. We found that UVA, UVB or UVC induced a marked and persistent activation of p38, whereas c-Jun N-terminal kinase or extracellular signal-regulated kinase were less or not activated respectively. Inhibition of p38 activity ...

  2. Internalization of EGF receptor following lipid rafts disruption in keratinocytes is delayed and dependent on p38 MAPK activation

    DEFF Research Database (Denmark)

    Lambert, S.; Ameels, H.; Gniadecki, R.

    2008-01-01

    internalization without participation of the ligand under the control of p38 MAPK during stress conditions. Since cholesterol depletion using methyl-beta-cyclodextrin is known to induce ligand-independent activation of EGFR in keratinocytes, we investigated by confocal microscopy and ligand-binding tests...... the processing and localization of EGFR following lipid raft disruption. Here, we report the dimerization and the slow internalization of the receptor accompanied by the delayed phosphorylation of tyrosine 1068 and its degradation by the proteasome. We also demonstrate the involvement of p38 MAPK during...... the process of internalization, which can be considered as a protective response to stress. Moreover, cholesterol-depleted keratinocytes recover their ability to proliferate during the recovery period that follows lipid raft disruption Udgivelsesdato: 2008/12...

  3. Gene expression signatures affected by ethanol and/or nicotine in normal human normal oral keratinocytes (NHOKs

    Directory of Open Access Journals (Sweden)

    Jeffrey J. Kim

    2014-12-01

    Full Text Available It has been reported that nicotine/alcohol alters epigenetic control and leads to abrogated DNA methylation and histone modifications, which could subsequently perturb transcriptional regulation critically important in cellular transformation. The aim of this study is to determine the molecular mechanisms of nicotine/alcohol-induced epigenetic alterations and their mechanistic roles in transcriptional regulation in human adult stem cells. We hypothesized that nicotine/alcohol induces deregulation of epigenetic machinery and leads to epigenetic alterations, which subsequently affect transcriptional regulation in oral epithelial stem cells. As an initiating step we have profiled transcriptomic alterations induced by the combinatory administration of EtOH and nicotine in primary normal human oral keratinocytes. Here we provide detailed experimental methods, analysis and information associated with our data deposited into Gene Expression Omnibus (GEO under GSE57634. Our data provide comprehensive transcriptomic map describing molecular changes induced by EtOH and nicotine on normal human oral keratinocytes.

  4. The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Gromov, P

    1995-01-01

    )vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full-length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2-D gel immunoblotting of crude keratinocyte lysates in combination with enhanced...... chemoluminescence (ECL) detection. Identified proteins are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Ultimately, the aim of the comprehensive database is to gather--through......The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post-translational modifications. 1082 polypeptides have been...

  5. RhoB promotes cancer initiation by protecting keratinocytes from UVB-induced apoptosis but limits tumor aggressiveness.

    Science.gov (United States)

    Meyer, Nicolas; Peyret-Lacombe, Alexis; Canguilhem, Bruno; Médale-Giamarchi, Claire; Mamouni, Kenza; Cristini, Agnese; Monferran, Sylvie; Lamant, Laurence; Filleron, Thomas; Pradines, Anne; Sordet, Olivier; Favre, Gilles

    2014-01-01

    The role of UVB-induced apoptosis in the formation of squamous cell carcinoma (SCC) is recognized. We previously identified the small RhoB (Ras homolog gene family, member B) GTPase, an early response gene to cellular stress, as a critical protein controlling apoptosis of human keratinocytes after UVB exposure. Here we generated SKH1 (hairless immunocompetent mouse) mice invalidated for RhoB to evaluate its role in UVB-induced skin carcinogenesis in vivo. We show that rhob-/- mice have a lower risk of developing UVB-induced keratotic tumors and actinic keratosis that is associated with a higher sensitivity of UVB-exposed keratinocytes to apoptosis. We extend this observation to primary cultures of normal human keratinocytes in which RhoB was downregulated with small interfering RNA (siRNA) and further show that the hypersensitivity to apoptosis depends on B-cell lymphoma 2 (Bcl-2) downregulation. In rhob-/- mice, the UVB-induced tumors were preferentially undifferentiated and highly proliferative. Finally, we show in humans an almost constant loss of RhoB expression in undifferentiated SCCs. These undifferentiated and RhoB-deficient tumors have elevated phosphorylated histone H2AX (γH2AX) and 53BP1, two markers of DNA double-strand breaks. Together, our results indicate that UVB-induced RhoB expression participates in in vivo SCC initiation by increasing keratinocyte survival. Conversely, RhoB may limit tumor aggressiveness as loss of RhoB expression in tumor cells is associated with tumor progression.

  6. Epidermal Rac1 regulates the DNA damage response and protects from UV-light-induced keratinocyte apoptosis and skin carcinogenesis

    Science.gov (United States)

    Deshmukh, Jayesh; Pofahl, Ruth; Haase, Ingo

    2017-01-01

    Non-melanoma skin cancer (NMSC) is the most common type of cancer. Increased expression and activity of Rac1, a small Rho GTPase, has been shown previously in NMSC and other human cancers; suggesting that Rac1 may function as an oncogene in skin. DMBA/TPA skin carcinogenesis studies in mice have shown that Rac1 is required for chemically induced skin papilloma formation. However, UVB radiation by the sun, which causes DNA damage, is the most relevant cause for NMSC. A potential role of Rac1 in UV-light-induced skin carcinogenesis has not been investigated so far. To investigate this, we irradiated mice with epidermal Rac1 deficiency (Rac1-EKO) and their controls using a well-established protocol for long-term UV-irradiation. Most of the Rac1-EKO mice developed severe skin erosions upon long-term UV-irradiation, unlike their controls. These skin erosions in Rac1-EKO mice healed subsequently. Surprisingly, we observed development of squamous cell carcinomas (SCCs) within the UV-irradiation fields. This shows that the presence of Rac1 in the epidermis protects from UV-light-induced skin carcinogenesis. Short-term UV-irradiation experiments revealed increased UV-light-induced apoptosis of Rac1-deficient epidermal keratinocytes in vitro as well as in vivo. Further investigations using cyclobutane pyrimidine dimer photolyase transgenic mice revealed that the observed increase in UV-light-induced keratinocyte apoptosis in Rac1-EKO mice is DNA damage dependent and correlates with caspase-8 activation. Furthermore, Rac1-deficient keratinocytes showed reduced levels of p53, γ-H2AX and p-Chk1 suggesting an attenuated DNA damage response upon UV-irradiation. Taken together, our data provide direct evidence for a protective role of Rac1 in UV-light-induced skin carcinogenesis and keratinocyte apoptosis probably through regulating mechanisms of the DNA damage response and repair pathways. PMID:28277539

  7. Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma

    DEFF Research Database (Denmark)

    Pedersen, Tanja Xenia; Leethanakul, Chidchanop; Patel, Vyomesh

    2003-01-01

    we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound...... microdissection and cDNA array analysis provides a powerful new tool to unravel the complex changes in gene expression that underlie physiological and pathological remodeling of keratinized epithelium....

  8. Protective effects of an extract from Citrus bergamia against inflammatory injury in interferon-γ and histamine exposed human keratinocytes.

    Science.gov (United States)

    Graziano, Adriana C E; Cardile, Venera; Crascì, Lucia; Caggia, Sivia; Dugo, Paola; Bonina, Francesco; Panico, Annamaria

    2012-06-27

    The present work evaluated the anti-inflammatory/antioxidant activity of a well characterized extract from Citrus bergamia Risso and Poiteau (CBE), containing neoeriocitrin, naringin, neohesperidin and other flavonoids, on human NCTC 2544 keratinocytes treated with interferon-gamma (IFN-γ) and histamine (H). High performance liquid chromatography (HPLC) coupled with diode array detectors was used to characterize and quantify phenolic compounds in CBE. Anti-inflammatory/antioxidant ability on keratinocytes was determined through evaluation of inter-cellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) expression by Western blot, production of nitric oxide (NO) with Griess reagent and concentration of reactive oxygen species (ROS) by fluorescent quantitative analysis with 2',7'-dichlorfluorescein-diacetate (DCFH-DA). Cell viability was assessed using 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Antioxidant activity was also measured by oxygen radical absorbance capacity (ORAC) assay. Glycosaminoglycans (GAGs) were quantified using 1.9-dimethyl methylene blue (DMB). CBE exhibited high antioxidant activity confirmed by elevated ORAC values related to high capacity in oxygen radical scavenging. The assays on keratinocytes demonstrated that CBE does not inhibit cell proliferation and is shown to significantly reduce dose-dependently ICAM-1, iNOS, NO, ROS and GAG production in cells exposed to IFN-γ and H. Our study demonstrates that the pools of compounds of an extract from C. bergamia efficiently block the proinflammatory actions induced by IFN-γ and H on human keratinocytes. CBE may be used for topic employment in some inflammatory diseases of the skin and to represent an important opportunity for the essential oil processing industries. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Deletion of the N-terminus of IKKγ induces apoptosis in keratinocytes and impairs the AKT/PTEN signaling pathway

    International Nuclear Information System (INIS)

    Leis, Hugo; Sanchis, Ana; Perez, Paloma

    2007-01-01

    The regulatory subunit IKKγ/NEMO is crucial for skin development and function and although devoid of kinase activity, loss of IKKγ function completely abolishes the activation of NF-κB by all pro-inflammatory cytokines. To inhibit the IκB kinase (IKK) complex in keratinocytes, we have used a dominant negative approach by generating stable transfectants of an N-terminal deletion of IKKγ (IKKγ-DN97) that uncouples formation of the IKK complex. Expression of this mutant in PB keratinocytes (PB-IKKγ-DN97) delayed growth kinetics, caused morphological changes and dramatically augmented apoptosis even in the absence of pro-apoptotic stimuli, as determined by cell morphology, TUNEL and caspase-3 cleavage. Moreover, in PB-IKKγ-DN97 cells, TNF-α and IL-1 treatment failed to induce degradation of IκBα, phosphorylation of p65 on Ser 536 and nuclear translocation which, consequently, reduced κB-binding activity. In PB-IKKγ-DN97 cells, accumulation of IκBα correlated with a downregulation of AKT activity and an increase of PTEN protein levels whereas pro-apoptotic p53 target genes Bax and Puma were upregulated. These effects were most likely mediated through IKK since coexpression of the wild-type form of IKKγ in keratinocytes partially reversed apoptosis and reduced PTEN expression. Thus, our data suggest a negative cross-talk mechanism involving PTEN and NF-κB, critical for the anti-apoptotic role of NF-κB in keratinocytes

  10. Amniotic Membrane Modifies the Genetic Program Induced by TGFß, Stimulating Keratinocyte Proliferation and Migration in Chronic Wounds.

    Directory of Open Access Journals (Sweden)

    Antonia Alcaraz

    Full Text Available Post-traumatic large-surface or deep wounds often cannot progress to reepithelialisation because they become irresponsive in the inflammatory stage, so intervention is necessary to provide the final sealing epidermis. Previously we have shown that Amniotic Membrane (AM induced a robust epithelialisation in deep traumatic wounds.To better understand this phenomenon, we used keratinocytes to investigate the effect of AM on chronic wounds. Using keratinocytes, we saw that AM treatment is able to exert an attenuating effect upon Smad2 and Smad3 TGFß-induced phosphorylation while triggering the activation of several MAPK signalling pathways, including ERK and JNK1, 2. This also has a consequence for TGFß-induced regulation on cell cycle control key players CDK1A (p21 and CDK2B (p15. The study of a wider set of TGFß regulated genes showed that the effect of AM was not wide but very concrete for some genes. TGFß exerted a powerful cell cycle arrest; the presence of AM however prevented TGFß-induced cell cycle arrest. Moreover, AM induced a powerful cell migration response that correlates well with the expression of c-Jun protein at the border of the healing assay. Consistently, the treatment with AM of human chronic wounds induced a robust expression of c-Jun at the wound border.The effect of AM on the modulation of TGFß responses in keratinocytes that favours proliferation together with AM-induced keratinocyte migration is the perfect match that allows chronic wounds to move on from their non-healing state and progress into epithelialization. Our results may explain why the application of AM on chronic wounds is able to promote epithelialisation.

  11. Protection against 2-chloroethyl ethyl sulfide (CEES) - induced cytotoxicity in human keratinocytes by an inducer of the glutathione detoxification pathway

    International Nuclear Information System (INIS)

    Abel, Erika L.; Bubel, Jennifer D.; Simper, Melissa S.; Powell, Leslie; McClellan, S. Alex; Andreeff, Michael; MacLeod, Michael C.; DiGiovanni, John

    2011-01-01

    Sulfur mustard (SM or mustard gas) was first used as a chemical warfare agent almost 100 years ago. Due to its toxic effects on the eyes, lungs, and skin, and the relative ease with which it may be synthesized, mustard gas remains a potential chemical threat to the present day. SM exposed skin develops fluid filled bullae resulting from potent cytotoxicity of cells lining the basement membrane of the epidermis. Currently, there are no antidotes for SM exposure; therefore, chemopreventive measures for first responders following an SM attack are needed. Glutathione (GSH) is known to have a protective effect against SM toxicity, and detoxification of SM is believed to occur, in part, via GSH conjugation. Therefore, we screened 6 potential chemopreventive agents for ability to induce GSH synthesis and protect cultured human keratinocytes against the SM analog, 2-chloroethyl ethyl sulfide (CEES). Using NCTC2544 human keratinocytes, we found that both sulforaphane and methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) stimulated nuclear localization of Nrf2 and induced expression of the GSH synthesis gene, GCLM. Additionally, we found that treatment with CDDO-Me elevated reduced GSH content of NCTC2544 cells and preserved their viability by ∼ 3-fold following exposure to CEES. Our data also suggested that CDDO-Me may act additively with 2,6-dithiopurine (DTP), a nucleophilic scavenging agent, to increase the viability of keratinocytes exposed to CEES. These results suggest that CDDO-Me is a promising chemopreventive agent for SM toxicity in the skin. - Highlights: → CDDO-Me treatment increased intracellular GSH in human keratinocytes. → CDDO-Me increased cell viability following exposure to the half-mustard, CEES. → The cytoprotective effect of CDDO-Me was likely due to scavenging with endogenous GSH.

  12. Size is an essential parameter in governing the UVB-protective efficacy of silver nanoparticles in human keratinocytes

    OpenAIRE

    Palanki, Rohan; Arora, Sumit; Tyagi, Nikhil; Rusu, Lilia; Singh, Ajay P.; Palanki, Srinivas; Carter, James E.; Singh, Seema

    2015-01-01

    Background Ultraviolet (UV) radiation from sun, particularly its UVB component (290–320 nm), is considered the major etiological cause of skin cancer that impacts over 2 million lives in the United States alone. Recently, we reported that polydisperse colloidal suspension of silver nanoparticles (AgNPs) protected the human keratinocytes (HaCaT) against UVB-induced damage, thus indicating their potential for prevention of skin carcinogenesis. Here we sought out to investigate if size controlle...

  13. Influence of acidic pH on keratinocyte function and re-epithelialisation of human in vitro wounds.

    Science.gov (United States)

    Lönnqvist, Susanna; Emanuelsson, Peter; Kratz, Gunnar

    2015-01-01

    Chronic wounds are one of the greatest challenges for the healthcare system. Today, a plethora of dressings are used in the treatment of these wounds, each with specific influence on the wound environment. Due to differences in the permeability of the dressings the use will result in differences in the pH balance in the wound bed. However, little is known about how changes in the pH in the wound environment affect the different phases of the healing process. The aim of the present study was to investigate the effects of acidic pH on the regeneration phase by studying keratinocyte function in vitro and re-epithelialisation in an in vitro model of human skin. In vitro assays showed reduced viability and migration rates in human keratinocytes when pH was lowered. Real time PCR revealed differential expression of genes related to wound healing and environmental impairment. Tissue culture showed no re-epithelialisation of wounds subjected to pH 5.0 and moderate re-epithelialisation at pH 6.0, compared to controls at pH 7.4. The results indicate that lowering pH down to pH 5.0 in wounds is counterproductive in aspect of keratinocyte function which is crucial for successful wound healing.

  14. Repair of the three main types of bipyrimidine DNA photoproducts in human keratinocytes exposed to UVB and UVA radiations.

    Science.gov (United States)

    Courdavault, Sophie; Baudouin, Caroline; Charveron, Marie; Canguilhem, Bruno; Favier, Alain; Cadet, Jean; Douki, Thierry

    2005-07-12

    Induction of DNA damage by solar UV radiation is a key event in the development of skin cancers. Bipyrimidine photoproducts, including cyclobutane pyrimidine dimers (CPDs), (6-4) photoproducts (64 PPs) and their Dewar valence isomers, have been identified as major UV-induced DNA lesions. In order to identify the predominant and most persistent lesions, we studied the repair of the three types of photolesions in primary cultures of human keratinocytes. Specific and quantitative data were obtained using HPLC associated with tandem mass spectrometry. As shown in other cell types, 64 PPs are removed from UVB-irradiated keratinocytes much more efficiently than CPDs. In contrast, CPDs are still present in high amounts when cells recover their proliferation capacities after cell cycle arrest and elimination of a part of the population by apoptosis. The predominance of CPDs is still maintained when keratinocytes are exposed to a combination of UVB and UVA. Under these conditions, 64 PPs are converted into their Dewar valence isomers that are as efficiently repaired as their (6-4) precursors. Exposure of cells to pure UVA radiation generates thymine cyclobutane dimers that are slightly less efficiently repaired than CPDs produced upon UVB irradiation. Altogether, our results show that CPDs are the most frequent and the less efficiently repaired bipyrimidine photoproducts irrespectively of the applied UV treatment.

  15. Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22.

    Science.gov (United States)

    Yuan, Shukai; Li, Feifei; Meng, Qingyong; Zhao, Yiqiang; Chen, Lei; Zhang, Hongquan; Xue, Lixiang; Zhang, Xiuqing; Lengner, Christopher; Yu, Zhengquan

    2015-05-01

    Hair follicles (HF) undergo precisely regulated recurrent cycles of growth, cessation, and rest. The transitions from anagen (growth), to catagen (regression), to telogen (rest) involve a physiological involution of the HF. This process is likely coordinated by a variety of mechanisms including apoptosis and loss of growth factor signaling. However, the precise molecular mechanisms underlying follicle involution after hair keratinocyte differentiation and hair shaft assembly remain poorly understood. Here we demonstrate that a highly conserved microRNA, miR-22 is markedly upregulated during catagen and peaks in telogen. Using gain- and loss-of-function approaches in vivo, we find that miR-22 overexpression leads to hair loss by promoting anagen-to-catagen transition of the HF, and that deletion of miR-22 delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic activation of miR-22 results in hair loss due to the repression a hair keratinocyte differentiation program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the molecular level, we demonstrate that miR-22 directly represses numerous transcription factors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We conclude that miR-22 is a critical post-transcriptional regulator of the hair cycle and may represent a novel target for therapeutic modulation of hair growth.

  16. Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22.

    Directory of Open Access Journals (Sweden)

    Shukai Yuan

    2015-05-01

    Full Text Available Hair follicles (HF undergo precisely regulated recurrent cycles of growth, cessation, and rest. The transitions from anagen (growth, to catagen (regression, to telogen (rest involve a physiological involution of the HF. This process is likely coordinated by a variety of mechanisms including apoptosis and loss of growth factor signaling. However, the precise molecular mechanisms underlying follicle involution after hair keratinocyte differentiation and hair shaft assembly remain poorly understood. Here we demonstrate that a highly conserved microRNA, miR-22 is markedly upregulated during catagen and peaks in telogen. Using gain- and loss-of-function approaches in vivo, we find that miR-22 overexpression leads to hair loss by promoting anagen-to-catagen transition of the HF, and that deletion of miR-22 delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic activation of miR-22 results in hair loss due to the repression a hair keratinocyte differentiation program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the molecular level, we demonstrate that miR-22 directly represses numerous transcription factors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We conclude that miR-22 is a critical post-transcriptional regulator of the hair cycle and may represent a novel target for therapeutic modulation of hair growth.

  17. Keratinocyte stem cells but not melanocyte stem cells are the primary target for radiation-induced hair graying.

    Science.gov (United States)

    Aoki, Hitomi; Hara, Akira; Motohashi, Tsutomu; Kunisada, Takahiro

    2013-09-01

    Ionizing radiation (IR)-induced hair graying is caused by the ectopic differentiation of melanocyte stem cells (MSCs) in their niche located at the bulge region of the hair follicle. Keratinocyte stem cells (KSCs) in the bulge region are an important component of that niche. However, little is known about the relationship between MSC differentiation and the KSC niche during IR-induced hair graying. We found that both follicular MSCs and KSCs were affected by IR by using immunohistochemical detection of γH2AX as a genotoxicity marker. We also found that KSCs prepared from irradiated mice were functionally affected by IR as indicated by their reduced colony-forming activity in culture and the delayed hair cycle in vivo. However, these effects of IR on KSCs were temporal. The MSC population, which proliferated and differentiated to melanocytes, was persistently maintained after irradiation. In addition to the loss of colony-forming activity, irradiated keratinocytes including KSCs suppressed the colony formation of MSCs in vitro. Furthermore, pigmented hairs were not reconstituted in vivo in the presence of irradiated KSCs or keratinocytes. These results provide a previously unreported insight that the primary target of IR during the induction of hair graying is follicular KSCs rather than MSCs.

  18. Keratinocyte-derived chemokines orchestrate T cell positioning in the epidermis during vitiligo and may serve as biomarkers of disease

    Science.gov (United States)

    Richmond, Jillian M.; Bangari, Dinesh S.; Essien, Kingsley I.; Currimbhoy, Sharif D.; Groom, Joanna R.; Pandya, Amit G.; Youd, Michele E.; Luster, Andrew D.; Harris, John E.

    2016-01-01

    Vitiligo is an autoimmune disease of the skin that results in the destruction of melanocytes and the clinical appearance of white spots. Disease pathogenesis depends on IFN-γ and IFN-γ-induced chemokines to promote T cell recruitment to the epidermis where melanocytes reside. The skin is a complex organ, with a variety of resident cell types. We sought to better define the microenvironment and distinct cellular contributions during autoimmunity in vitiligo, and found that the epidermis is a chemokine-high niche in both a mouse model and human vitiligo. Analysis of chemokine expression in mouse skin revealed that CXCL9 and CXCL10 expression strongly correlate with disease activity, whereas CXCL10 alone correlates with severity, supporting them as potential biomarkers for following disease progression. Further studies in both our mouse model and human patients revealed that keratinocytes were the major chemokine-producers throughout the course of disease, and functional studies using a conditional STAT1 knockout mouse revealed that IFN-γ signaling in keratinocytes was critical for disease progression and proper autoreactive T cell homing to the epidermis. In contrast, epidermal immune cell populations including endogenous T cells, Langerhans cells, and γδ T cells were not required. These results have important clinical implications, as topical therapies that target IFN-γ signaling in keratinocytes could be safe and effective new treatments, and skin expression of these chemokines could be used to monitor disease activity and treatment responses. PMID:27686391

  19. Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1986-02-01

    Human keratinocytes in culture were labelled with /sup 14/C-dihomo-gamma-linolenic acid, /sup 14/C-arachidonic acid or /sup 14/C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. Most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

  20. 3-Amino-1,2,4-triazole Limits the Oxidative Damage in UVA-Irradiated Dysplastic Keratinocytes

    Directory of Open Access Journals (Sweden)

    Marina Tamara Nechifor

    2017-01-01

    Full Text Available Reactive oxygen species (ROS generated by UVA irradiation affect the keratinocyte cell membrane, DNA, and proteins and may cause serious injury to the skin. Treating human dysplastic keratinocytes (DOK with 3-amino-1,2,4-triazole (AMT, a common catalase inhibitor, induced a compensatory mechanism for the hydrogen peroxide detoxification, which included a rise in glutathione peroxidase and glutathione reductase activities. Here, we examined a possible role of AMT in protecting a human DOK cell line against UVA-induced damage. In DOK cells exposed to UVA irradiation, we observed a substantial decrease in antioxidant enzymatic activities, such as catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase and an increase in lipid peroxidation and protein oxidation levels. Treating DOK cells with AMT prior to UVA exposure enhanced the activities of glutathione peroxidase, glutathione reductase, and glutathione-S-transferase, relative to nontreated cells. The enhanced antioxidant activities were correlated with decreased protein oxidation levels. Based on these results, we suggest that AMT may protect dysplastic keratinocytes against the harmful effects of UVA radiation.

  1. N-Acetyl-D-glucosamine oligosaccharides induce mucin secretion from colonic tissue and induce differentiation of human keratinocytes.

    Science.gov (United States)

    Deters, Alexandra; Petereit, Frank; Schmidgall, Jörg; Hensel, Andreas

    2008-02-01

    Chitin oligosaccharides (DP2, DP3, DP4, DP5 and DP7) were investigated for their effects on epithelial cells and tissue (skin keratinocytes in-vitro and ex-vivo, and gastrointestinal epithelial membranes exvivo). Oligomers DP2, DP3 and DP5 at 10microg mL(-1) significantly stimulated the mitochondrial activity of cultured keratinocytes in-vitro (primary cells and HaCaT cell line), with highest activity observed for the pentamer (150% of untreated control). The effects were dose dependent. This higher energy status of primary cells was triggered into a higher differentiation status, as determined by the early and late differentiation markers keratins K1/K10 and involucrin, respectively. In contrast, increased mitogenic cell proliferation was not induced by the oligosaccharides. Toxic effects on keratinocytes were absent. Additionally for the first time a mucin-stimulating effect of chitin oligosaccharides DP3 and DP5 was observed in an ex-vivo model based on intestinal epithelial mucosa tissue. Mucin secretion was time dependent, leading to the secretion of polymers comparable to those normally secreted under physiological conditions. Mucin induction was observed from colonic tissue isolated from humans and pigs. Also, porcine stomach mucosa was stimulated by DP5, while ileum tissue reacted to only a minor extent. Potential developments towards products with wound-healing capacity and activity against chronic bowel disease are discussed.

  2. The effect of anthocyanins from red wine and blackberry on the integrity of a keratinocyte model using ECIS.

    Science.gov (United States)

    Évora, Ana; de Freitas, Victor; Mateus, Nuno; Fernandes, Iva

    2017-11-15

    There is a growing market demand for the incorporation of plant-derived ingredients into new products for the cosmetic industry. Anthocyanins are polyphenols arising from plant secondary metabolism that have been shown to possess many bioactive properties such as free radical scavenging, antimicrobial, and chemopreventive activities. In this work, the biological activities of red wine and blackberry anthocyanins were assessed by developing a new keratinocyte barrier model using the HaCat cell line and a microelectrode-based biosensor device, referred to as Electric Cell-Substrate Impedance Sensing (ECIS). Cells were seeded at the optimal cellular density of 1.6 × 10 6 cells per mL and the half-time was calculated to be 3.55 ± 0.67 hours. The compounds' cytotoxicity was assessed and anthocyanin pigments showed no cytotoxicity towards keratinocyte cells. Wound healing assays were also performed using ECIS and it was observed that the tested pigments enhanced the healing rate of keratinocyte cells by reducing the healing time more than 50%. Cyanidin-3-glucoside presented the best results recovering 50% of the injured area in 1.48(±0.15) hours, followed by the blackberry anthocyanins (2.01 ± 0.18 hours), malvidin-3-glucoside (2.03 ± 0.09 hours) and red wine anthocyanins (2.36 ± 0.76 hours). All presented significant differences from the control 4.91(±1.11) hours.

  3. Disruption of Spectrin-Like Cytoskeleton in Differentiating Keratinocytes by PKCδ Activation Is Associated with Phosphorylated Adducin

    Science.gov (United States)

    Zhao, Kong-Nan; Masci, Paul P.; Lavin, Martin F.

    2011-01-01

    Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505) activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505) phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex. PMID:22163289

  4. Adenovirus-mediated expression of keratinocyte growth factor promotes secondary flap necrotic wound healing in an extended animal model.

    Science.gov (United States)

    Wang, Xinhua; Yu, Mengfei; Zhu, Wenyuan; Bao, Tingwei; Zhu, Liqin; Zhao, Wenquan; Zhao, Fuyan; Wang, Huiming

    2013-10-01

    No effective treatments have been found for flap necrosis. Animal models that focus on the initial flap viability are inappropriate for necrotic wound studies. Keratinocyte growth factor (KGF) promotes keratinocyte proliferation with stronger activity and fewer complications and thus may be useful for necrotic flap wound healing. Rats with modified flap necrosis were randomly divided into four groups. An adenoviral vector expressing KGF was injected subdermally in the back of the animals after necrosis began. The expression and effect of KGF was assessed by real-time polymerase chain reaction, enzyme-linked immunoassay, and transwell, and wound healing was monitored. The plasmid and adenovirus were able to express KGF and stimulate epithelial cell growth (p = 0.029). Histology showed that the necrosis healed fastest in the KGF administration group than in the control groups (p < 0.01). The adenovirus-mediated KGF (Ad-KGF) group had the thickest epithelium on days 15 (p = 0.044) and 25 (p = 0.014). The KGF level in the blood serum soared 10 and 15 days postoperatively (p < 0.01) but returned to baseline by day 25 (p = 0.561). The KGF mRNA levels in vivo increased dramatically in the Ad-KGF group (p = 0.037). The extended flap model is applicable in necrotic wound study. Keratinocyte growth factor can promote secondary necrotic flap wound healing, and administration of KGF can be achieved by an adenoviral vector.

  5. An ascorbic acid-enriched tomato genotype to fight UVA-induced oxidative stress in normal human keratinocytes.

    Science.gov (United States)

    Petruk, Ganna; Raiola, Assunta; Del Giudice, Rita; Barone, Amalia; Frusciante, Luigi; Rigano, Maria Manuela; Monti, Daria Maria

    2016-10-01

    UVA radiations contribute up to 95% of the total UV exposure and are known to induce cell damage, leading to apoptosis. Since the benefic effects of ascorbic acid on human health are well known, a new tomato genotype (named DHO4), highly rich in ascorbic acid, has been recently obtained. Here, we compared the effects of ascorbic acid and hydrophilic DHO4 extracts in protecting human keratinocytes exposed to UVA stress. Keratinocytes were pre-incubated with ascorbic acid or with extracts from the ascorbic acid enriched tomato genotype and irradiated with UVA light. Then, ROS production, intracellular GSH and lipid peroxidation levels were quantified. Western blots were carried out to evaluate mitogen-activated protein kinases cascade, activation of caspase-3 and inflammation levels. We demonstrated that ROS, GSH and lipid peroxidation levels were not altered in cell exposed to UVA stress when cells were pre-treated with ascorbic acid or with tomato extracts. In addition, no evidence of apoptosis and inflammation were observed in irradiated pre-treated cells. Altogether, we demonstrated the ability of an ascorbic acid enriched tomato genotype to counteract UVA-oxidative stress on human keratinocytes. This protective effect is due to the high concentration of vitamin C that acts as free radical scavenger. This novel tomato genotype may be used as genetic material in breeding schemes to produce improved varieties with higher antioxidant levels. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

    International Nuclear Information System (INIS)

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1986-01-01

    Human keratinocytes in culture were labelled with 14 C-dihomo-gamma-linolenic acid, 14 C-arachidonic acid or 14 C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. Most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes

  7. Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract

    Directory of Open Access Journals (Sweden)

    Sridar Chittur

    2011-01-01

    Full Text Available Chronic inflammation of the hair follicle (HF is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA. Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4 associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

  8. Toll-like receptor 2 (TLR2) mediates intracellular signalling in human keratinocytes in response to Malassezia furfur.

    Science.gov (United States)

    Baroni, Adone; Orlando, Manuela; Donnarumma, Giovanna; Farro, Pietro; Iovene, Maria Rosaria; Tufano, Maria Antonietta; Buommino, Elisabetta

    2006-01-01

    Toll-like receptors (TLRs) are crucial players in the innate immune response to microbial invaders. The lipophilic yeast Malassezia furfur has been implicated in the triggering of scalp lesions in psoriasis. The aim of the present study was to assess the role of TLRs in the defence against M. furfur infection. The expression of the myeloid differentiation factor 88 (MyD88) gene, which is involved in the signalling pathway of many TLRs, was also analysed. In addition, a possible correlation of antimicrobial peptides of the beta-defensin family to TLRs was tested. Human keratinocytes infected with M. furfur and a variety of M. furfur-positive psoriatic skin biopsies were analysed by RT-PCR, for TLRs, MyD88, human beta-defensin 2 (HBD-2), HBD-3 and interleukin-8 (IL-8) mRNA expression. When keratinocytes were infected with M. furfur, an up-regulation for TLR2, MyD88, HBD-2, HBD-3 and IL-8 mRNA was demonstrated, compared to the untreated cells. The same results were obtained when psoriatic skin biopsies were analysed. The M. furfur-induced increase in HBD-2 and IL-8 gene expression is inhibited by anti-TLR2 neutralising antibodies, suggesting that TLR2 is involved in the M. furfur-induced expression of these molecules. These findings suggest the importance of TLRs in skin protection against fungi and the importance of keratinocytes as a component of innate immunity.

  9. The flavonoid luteolin increases the resistance of normal, but not malignant keratinocytes, against UVB-induced apoptosis.

    Science.gov (United States)

    Verschooten, Lien; Smaers, Katrien; Van Kelst, Sofie; Proby, Charlotte; Maes, Daniel; Declercq, Lieve; Agostinis, Patrizia; Garmyn, Marjan

    2010-09-01

    Adequate protection of skin against the carcinogenic effects of UVB irradiation is essential. Flavonoids may have a conspicuous role in cancer prevention because of their antioxidant, anti-inflammatory, and growth-inhibitory effects. Therefore, we tested the effects of the flavone luteolin (LUT) on selected parameters of the sunburn response in normal human keratinocytes, exposed to physiological doses of UVB. LUT attenuated UVB-induced cell death through delay and inhibition of intrinsic apoptotic signaling. Moreover, LUT not only predominantly affected the mitochondrial apoptosis pathway through its antioxidant capacity, but also changed the balance of Bcl2 (B-cell leukemia/lymphoma 2)-family members. Furthermore, LUT had inhibitory effects on the UVB-induced release of the inflammatory mediators, IL-1alpha and prostaglandin-E(2). Using different cell lines derived from squamous cell carcinomas, we showed that LUT did not increase the resistance of malignant keratinocytes to UVB. Our data suggest that LUT inhibits different aspects of the sunburn response, which results ultimately in an increased survival of normal keratinocytes, whereas the sensitivity of malignant cells to UVB remain unchanged. Hence, LUT might have value in new photoprotective applications or improve existing ones.

  10. Membrane cofactor protein (CD46) is a keratinocyte receptor for the M protein of the group A streptococcus.

    Science.gov (United States)

    Okada, N; Liszewski, M K; Atkinson, J P; Caparon, M

    1995-03-28

    The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) is the causative agent of numerous suppurative diseases of human skin. The M protein of S. pyogenes mediates the adherence of the bacterium to keratinocytes, the most numerous cell type in the epidermis. In this study, we have constructed and analyzed a series of mutant M proteins and have shown that the C repeat domain of the M molecule is responsible for cell recognition. The binding of factor H, a serum regulator of complement activation, to the C repeat region of M protein blocked bacterial adherence. Factor H is a member of a large family of complement regulatory proteins that share a homologous structural motif termed the short consensus repeat. Membrane cofactor protein (MCP), or CD46, is a short consensus repeat-containing protein found on the surface of keratinocytes, and purified MCP could competitively inhibit the adherence of S. pyogenes to these cells. Furthermore, the M protein was found to bind directly to MCP, whereas mutant M proteins that lacked the C repeat domain did not bind MCP, suggesting that recognition of MCP plays an important role in the ability of the streptococcus to adhere to keratinocytes.

  11. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    International Nuclear Information System (INIS)

    Xu, Hanwen; Pirisi, Lucia; Creek, Kim E.

    2015-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling

  12. Cellular interactions of a lipid-based nanocarrier model with human keratinocytes: Unravelling transport mechanisms.

    Science.gov (United States)

    Silva, Elisabete; Barreiros, Luísa; Segundo, Marcela A; Costa Lima, Sofia A; Reis, Salette

    2017-04-15

    Knowledge of delivery system transport through epidermal cell monolayer is vital to improve skin permeation and bioavailability. Recently, nanostructured lipid carriers (NLCs) have gained great attention for transdermal delivery due to their biocompatibility, high drug payload, occlusive properties and skin hydration effect. However, the nanocarriers transport related mechanisms in epidermal epithelial cells are not yet understood. In this research, the internalization and transport pathways of the NLCs across the epidermal epithelial cell monolayer (HaCaT cells) were investigated. The 250nm sized witepsol/miglyol NLCs, prepared by hot homogenization had reduced cytotoxicity and no effect on the integrity of cell membrane in human HaCaT keratinocytes. The internalization was time-, concentration- and energy-dependent, and the uptake of NLCs was a vesicle-mediated process by macropinocytosis and clathrin-mediated pathways. 3% of NLCs were found at the apical membrane side of the HaCaT monolayer through exocytosis mechanism. Additionally, the endoplasmic reticulum, Golgi apparatus and microtubules played crucial roles in the transport of NLCs out of HaCaT cells. NLCs were transported intact across the human keratinocytes monolayer, without disturbing the tight junction's structure. From the transcytosis data only approximately 12% of the internalized NLCs were passed from the apical to the basolateral side. The transcytosis of NLCs throughout the HaCaT cell monolayer towards the basolateral membrane side requires the involvement of the endoplasmic reticulum, Golgi apparatus and microtubules. Our findings may contribute to a systematic understanding of NLCs transport across epidermal epithelial cell monolayers and their optimization for clinical transdermal application. Transdermal drug delivery is a challenging and growing area of clinical application. Lipid nanoparticles such as nanostructured lipid carriers (NLCs) have gained wide interest for transdermal drug

  13. Calcipotriol inhibits the proliferation of hyperproliferative CD29 positive keratinocytes in psoriatic epidermis in the absence of an effect on the function and number of antigen-presenting cells

    DEFF Research Database (Denmark)

    Jensen, A.M.; Llado, Minna Fyhn Lykke; Skov, L.

    1998-01-01

    -presenting cells in psoriatic epidermis. In contrast, we found that calcipotriol significantly inhibited the proliferation of epidermal cells isolated from psoriatic skin after in vivo treatment, as determined by propidium iodide staining and flow cytometry. More specifically, we stained for CD29+ keratinocytes...... and found an even more significant reduction in proliferative capacity. This cell type contains the population of hyperproliferative keratinocytes in psoriatic epidermis. In conclusion, calcipotriol seems to act via an inhibitory effect on hyperproliferative basal keratinocytes of psoriatic epidermis...

  14. Regulation of hypoxia-inducible factor-1α and related genes in equine digital lamellae and in cultured keratinocytes.

    Science.gov (United States)

    Pawlak, E A; Geor, R J; Watts, M R; Black, S J; Johnson, P J; Belknap, J K

    2014-03-01

    Hypoxia-inducible factor-1α (HIF-1A) is an important protein in the regulation/induction of many genes in the cellular and tissue response to hypoxia and a central mediator in inflammatory signalling. As both hypoxia and inflammatory events are purported to occur in the lamellar epidermis in sepsis-related laminitis in the equid, HIF-1A may play a central role in this disease process. To assess the regulation of HIF-1A and HIF-1A-related genes in the equine keratinocyte in vitro and in the lamellar tissue of horses with sepsis-related laminitis. In vivo and in vitro experiments. Real-time quantitative PCR (RT-qPCR) and immunoblotting were performed to assess the mRNA and protein concentrations of HIF-1A and the mRNA concentrations of HIF-1A-related genes in cultured equine keratinocytes and in lamellar samples from black walnut extract (BWE)- and carbohydrate overload (CHO)-induced laminitis. Hypoxia-inducible factor-1α was further localised via indirect immunofluorescence in frozen lamellar tissue sections. Hypoxia-inducible factor-1α appears to be regulated primarily at the post transcriptional level in the cultured equine keratinocyte, resulting in increased HIF-1A in response to hypoxia but not to lipopolysaccharide exposure. Hypoxia-inducible factor-1α is present at high concentrations in the normal equine lamina, and is increased in Obel grade 1 (OG1) stage laminitis in the CHO model of laminitis. Equine lamellar mRNA concentrations of cyclo-oxygenase-2 and inducible nitric oxide synthase, but not glucose transporter 1, are increased in the BWE and CHO models of laminitis. These data indicate that the normal equine lamellae are profoundly hypoxic in comparison with other tissues. The increased mRNA concentrations of cyclo-oxygenase-2 and inducible nitric oxide synthase 2 in equine keratinocytes exposed to hypoxia and lipopolysaccharide, and in lamellar tissue from BWE and CHO models of sepsis-related laminitis, suggest that the marked lamellar

  15. A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

    Directory of Open Access Journals (Sweden)

    Lu Ying

    2004-04-01

    Full Text Available Abstract Background TGM1(transglutaminase 1 is an enzyme that crosslinks the cornified envelope of mature keratinocytes. Appropriate expression of the TGM1 gene is crucial for proper keratinocyte function as inactivating mutations lead to the debilitating skin disease, lamellar ichthyosis. TGM1 is also expressed in squamous metaplasia, a consequence in some epithelia of vitamin A deficiency or toxic insult that can lead to neoplasia. An understanding of the regulation of this gene in normal and abnormal differentiation states may contribute to better disease diagnosis and treatment. Methods In vivo requirements for expression of the TGM1 gene were studied by fusing various lengths of promoter DNA to a reporter and injecting the DNA into mouse embryos to generate transgenic animals. Expression of the reporter was ascertained by Western blotting and immunohistochemistry. Further delineation of a transcriptionally important distal region was determined by transfections of progressively shortened or mutated promoter DNA into cultured keratinocytes. Results In vivo analysis of a reporter transgene driven by the TGM1 promoter revealed that 1.6 kilobases, but not 1.1 kilobases, of DNA was sufficient to confer tissue-specific and cell layer-specific expression. This same region was responsible for reporter expression in tissues undergoing squamous metaplasia as a response to vitamin A deprivation. Mutation of a distal promoter AP1 site or proximal promoter CRE site, both identified as important transcriptional elements in transfection assays, did not prevent appropriate expression. Further searching for transcriptional elements using electrophoretic mobility shift (EMSA and transfection assays in cultured keratinocytes identified two Sp1 elements in a transcriptionally active region between -1.6 and -1.4 kilobases. While mutation of either Sp1 site or the AP1 site singly had only a small effect, mutation of all three sites eliminated nearly all the

  16. Cobalt toxicity: Chemical and radiological combined effects on HaCaT keratinocyte cell line

    Energy Technology Data Exchange (ETDEWEB)

    Gault, N. [CEA Fontenay aux Roses, DSV/IRCM/SCSR/LRTS, 92265 Fontenay aux Rose (France); Sandre, C.; Moulin, B.; Bresson, C. [CEA, DEN, SECR, Laboratoire de Speciation des Radionucleides et des Molecules, F-91191 Gif-sur-Yvette (France); Poncy, J.L. [CEA Bruyeres Le Chatel, DSV/IRCM/SREIT/LRT, 91680 Bruyeres Le Chatel (France); Lefaix, J.L. [CEA Caen, DSV/IRCM/SRO/LARIA, 14070 Caen (France)

    2010-07-01

    Cobalt (Co) is an essential trace element well known as a constituent of vitamin B12, but different compounds of Co are also described as highly toxic and/or radio-toxic for individuals or the environment. In nuclear power plants, {sup 58}Co and {sup 60}Co are radioactive isotopes of cobalt present as activation products of stable Co and Ni used in alloys. Skin exposure is a current occupational risk in the hard metal and nuclear industries. As biochemical and molecular cobalt-induced toxicological mechanisms are not fully identified, we investigated cobalt toxicity in a model human keratinocyte cell line, HaCaT. In this study, we propose a model to determine the in vitro chemical impact on cell viability of a soluble form of cobalt (CoCl{sub 2}) with or without {gamma}-ray doses to mimic contamination by {sup 60}Co, to elucidate the mechanisms of cobalt intracellular chemical and radiological toxicity. Intracellular cobalt concentration was determined after HaCaT cell contamination and chemical toxicity was evaluated in terms of cellular viability and clonogenic survival. We investigated damage to DNA in HaCaT cells by combined treatment with chemical cobalt and a moderate {gamma}-ray dose. Additive effects of cobalt and irradiation were demonstrated. The underlying mechanism of cobalt toxicity is not clearly established, but our results seem to indicate that the toxicity of Co(II) and of irradiation arises from production of reactive oxygen species. (authors)

  17. Biocompatibility Evaluation of Dental Luting Cements Using Cytokine Released from Human Oral Fibroblasts and Keratinocytes

    Directory of Open Access Journals (Sweden)

    Jae-Sung Kwon

    2015-10-01

    Full Text Available Dental luting cements are commonly used in dentistry for cementation of prosthetic restoration. Many previous studies focused on the measurement of the cell viability as the method of cytotoxicity evaluation during biocompatibility study for the material. In this study, the biocompatibility of various dental luting cements were evaluated using the new method of cytokine release measurement in order to better simulate inflammatory reactions in animal or clinical model using two different oral cells; immortalized human gingival fibroblast and immortalized human oral keratinocytes. Cells were exposed to extractions of various commercially available dental luting cements for different durations. Cytokines of IL-1α and IL-8 were measured from the supernatants of the cells and the results were then compared to the conventional MTT viability test. The result from the conventional cell viability study showed a relatively simple and straight forward indication that only one of the dental luting cements tested in this study was cytotoxic with increasing duration of exposure for both cells. Meanwhile, the result from the cytokine measurement study was much more complex at the time point they were measured, type of cells used for the study and the type of cytokines measured, all of which influenced the interpretation of the results. Hence, the better understanding of the cytokine release would be required for the application in biocompatibility evaluation.

  18. Blockage of JNK pathway enhances arsenic trioxide-induced apoptosis in human keratinocytes

    International Nuclear Information System (INIS)

    Arsenic is well known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21 WAF1/CIP1 (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431 cells could inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF, critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21 were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry.

  19. Langerhans Cells Prevent Autoimmunity via Expansion of Keratinocyte Antigen-Specific Regulatory T Cells

    Directory of Open Access Journals (Sweden)

    Daniela Y. Kitashima

    2018-01-01

    Full Text Available Langerhans cells (LCs are antigen-presenting cells in the epidermis whose roles in antigen-specific immune regulation remain incompletely understood. Desmoglein 3 (Dsg3 is a keratinocyte cell-cell adhesion molecule critical for epidermal integrity and an autoantigen in the autoimmune blistering disease pemphigus. Although antibody-mediated disease mechanisms in pemphigus are extensively characterized, the T cell aspect of this autoimmune disease still remains poorly understood. Herein, we utilized a mouse model of CD4+ T cell-mediated autoimmunity against Dsg3 to show that acquisition of Dsg3 and subsequent presentation to T cells by LCs depended on the C-type lectin langerin. The lack of LCs led to enhanced autoimmunity with impaired Dsg3-specific regulatory T cell expansion. LCs expressed the IL-2 receptor complex and the disruption of IL-2 signaling in LCs attenuated LC-mediated regulatory T cell expansion in vitro, demonstrating that direct IL-2 signaling shapes LC function. These data establish that LCs mediate peripheral tolerance against an epidermal autoantigen and point to langerin and IL-2 signaling pathways as attractive targets for achieving tolerogenic responses particularly in autoimmune blistering diseases such as pemphigus.

  20. Cultured allogenic keratinocytes for extensive burns: a retrospective study over 15 years.

    Science.gov (United States)

    Auxenfans, Celine; Shipkov, Hristo; Bach, Christine; Catherine, Zulma; Lacroix, Pierre; Bertin-Maghit, Marc; Damour, Odile; Braye, Fabienne

    2014-02-01

    The aim was to review the use and indications of cultured allogenic keratinocytes (CAlloK) in extensive burns and their efficiency. This retrospective study comprised 15 years (1997-2012). all patients who received CAlloK. patients who died before complete healing. Evaluation criteria were clinical. Time and success of wound healing after CAlloK use were evaluated. The CAlloK were used for 2 indications - STSG donor sites and deep 2nd degree burns in extensively burned patients. A total of 70 patients were included with severity Baux score of 99.2 (from 51 to 144) and mean percentage of TBSA of 63.49% (from 21 to 96%). Fifty nine patients received CAlloK for STSG donor sites with a mean number of applications of 4 and mean surface of 3800 cm(2) per patient. Treated donor sites were re-harvested 2.5 times. The mean time of complete epithelialization was 7 days. In 11 patients, CAlloK were used for deep 2nd degree burns. The mean percentage of burned surface was 73.7%. The mean surface of CAlloK per patient was 2545 cm(2). Complete healing was achieved in 6.4 days. The CAlloK allow rapid healing of STSG donor-sites and deep 2nd second degree burns in extensively burned patients. Copyright © 2013 Elsevier Ltd and ISBI. All rights reserved.

  1. Characterization of sulfur mustard resistant keratinocyte cell line HaCaT/SM.

    Science.gov (United States)

    Wolf, Markus; Siegert, Markus; Rothmiller, Simone; Scheithauer, Nina; Strobelt, Romano; Steinritz, Dirk; Worek, Franz; Thiermann, Horst; Schmidt, Annette

    2016-02-26

    The cell line HaCaT/SM was derived from the human keratinocyte cell line HaCaT. HaCaT/SM cells display a high resistance against sulfur mustard (SM). Intention of the presented study was to determine the cellular and molecular differences between HaCaT/SM and HaCaT so as to evaluate which changes might be responsible for being resistant against SM. Both cell lines HaCaT and HaCaT/SM were analyzed with respect to their cell growth, nuclei perimeter, clonogenicity and secretion profile. Moreover DNA alkylation pattern under presence of SM was investigated. In comparison to HaCaT, the HaCaT/SM showed a significant smaller nuclei perimeter. For DNA alkylation a significant difference was observed over time. The clonogenicity of HaCaT/SM was increased to 150%. The secretion profile of these cells demonstrated a strong increase of ANG, PDGF-AA, TIMP1, TIMP2, and a decrease of AREG, CCL5, CXC1, CXC2/3, CXCL6, CXCL7, CXCL8, CXCL10, MIF, Trappin-1. The sulfur mustard (SM) resistant cell line HaCaT/SM demonstrates a wide range of significant differences to their origin cell line HaCaT. These differences might be responsible to provide resistance against SM and might also be useful to establish treatment concepts for humans after SM exposure. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  2. Macromolecular metabolism of a differentiated rat keratinocyte culture system following exposure to sulfur mustard

    International Nuclear Information System (INIS)

    Vaughan, F.L.; Zaman, S.; Scavarelli, R.; Bernstein, I.A.

    1988-01-01

    A method for producing a stratified, squamous epithelium in vitro by cultivating rat keratinocytes on nylon membranes has been developed in this laboratory. This epidermal-like culture is being used to obtain a better understanding of the mechanism of skin vesication after topical exposure to the sulfur mustard bis(beta-chloroethyl) sulfide (BCES) dissolved in a selected solvent. Radiolabeled macromolecular precursors (thymidine, uridine, and leucine) have been used to study the effect of BCES on the synthesis of DNA, RNA, and protein, respectively, after topical exposure to the mustard at concentrations of 0.01-500 nmol/cm2 dissolved in 70% dimethyl sulfoxide (DMSO). From these and other studies it has been determined that exposure to even the low concentration of 0.01 nmol BCES/cm2 for 30 min results in significant inhibition of [ 3 H]thymidine incorporation, although complete recovery occurs by 24 h. Significant inhibition of [ 3 H]uridine and [ 14 C]leucine incorporation is observed only after exposure to much higher concentrations of BCES (10-500 nmol/cm2). This suggests a very early lesion in macromolecular metabolism with DNA being the primary target

  3. Imaging sulfur mustard lesions in human epidermal tissues and keratinocytes by confocal and multiphoton microscopy

    Science.gov (United States)

    Werrlein, Robert; Madren-Whalley, Janna S.

    2002-06-01

    Topical exposure to sulfur mustard (HD), a known theat agent, produces persistent and debilitating cutaneous blisters. The blisters occur at the dermal-epidermal junction following a dose-dependent latent period of 8-24 h, however, the primary lesions causing vesication remain uncertain. Immunofluorescent images reveal that a 5-min exposure to 400 (mu) M HD disrupts molecules that are also disrupted by epidermolysis bullosa-type blistering diseases of the skin. Using keratinocyte cultures and fluorochomes conjugated to two different keratin-14 (K14) antibodies (clones CKB1 and LL002), results have shown a statistically significant (p<0.1) 1-h decrease of 29.2% in expression of the CKB1 epitope, a nearly complete loss of CKB1 expression within 2 h, and progressive cytoskeletal (K14) collapse without loss in expression of the LL002 epitope. With human epidermal tissues, multi-photon images of (alpha) 6 integrin and laminin 5 showed disruptive changes in the cell-surface organization and integrity of these adhesion molecules. At 1 H postexposure, analyses showed a statistically significant (p<0.1) decrease of 27.3% in (alpha) 6 integrin emissions, and a 32% decrease in laminin 5 volume. Multi-photon imaging indicates that molecules essential for epidermal-dermal attachment are early targets in the alkylating events leading to HD-induced vesication.