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Sample records for b-irradiated premalignant keratinocytes

  1. 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.

  2. TNF-alpha stimulates Akt by a distinct aPKC-dependent pathway in premalignant keratinocytes

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.

    2008-01-01

    kappaB inhibition and in the presence of p38 blockers. Akt/ERK signalling but not p38 activation was abolished in the presence of the iron chelator desferroxamine that blocks formation of hydroxyl ( OH) radicals. Thus, the TNF-alpha signalling in keratinocytes seems to bifurcate into an aPKC-, NFk......Tumor necrosis factor-alpha (TNF-alpha) is an important proinflammatory cytokine involved in the pathogenesis of inflammatory skin diseases and cutaneous squamous cell carcinoma. Some of these effects are mediated by the stimulatory effect of this cytokine on the Akt signalling pathway, which...... renders keratinocytes less susceptible to proapoptotic stimuli and enhances cell growth. We have recently shown that TNF-alpha-induced Akt activation may promote the early stages of skin cancer. In this work, we demonstrate that in the premalignant keratinocyte cell line HaCaT, TNF-alpha activates Akt...

  3. 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...... 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...... phosphorylations of Akt (Ser-473 and Thr-308) and the signalling via related pathways Erk 1/2, p38 and p70-S6K. Infliximab inhibited Akt and its downstream targets p70-S6K and Erk 1/2, and stimulated p38 both in sham-irradiated and UVB-irradiated cells. In conclusion, despite the fact that infliximab blocks Akt...

  4. Increase of integrin α6+p63+ cells after ultraviolet B irradiation in normal human keratinocytes

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    Gyeong-hun Park

    2009-12-01

    Full Text Available Epidermal stem cells (SC are believed to be resistant to environmental damage for the purpose of self renewal. Most promising SC markers include integrin a6 and p63. The aim of our study was to determine whether the integrin a6+p63+ cell fraction representative of the epidermal progenitor or SC is increased after ultraviolet B (UVB irradiation and to clarify the hypothesis that epidermal SC are resistant to high-dose UVB damage. We irradiated early passage normal human keratinocytes (NHK with 0, 25, 50, and 100 mJ/cm2 UVB. The percentage of cell death was calculated. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR and western blotting analyses were performed to identify integrin a6 and p63, and flow cytometry analysis with integrin a6 and p63 antibodies was done. After 50 and 100 mJ/cm2 UVB, integrin a6+p63+cells were found to be much increased by fluorescence-activated cell sorting. Expression of integrin a6 and p63 was increased in NHK after UVB irradiation, which was shown with real-time RT-PCR and western blotting analyses. We concluded that an increase of integrin a6+p63+ cells after high-dose UVB may suggest that the putative progenitor or SC are resistant to UVB irradiation.

  5. Skin anti-photoaging properties of ginsenoside Rh2 epimers in UV-B-irradiated human keratinocyte cells

    Indian Academy of Sciences (India)

    Sun-Joo Oh; Sihyeong Lee; Woo-Yong Choi; Chang-Jin Lim

    2014-09-01

    Ginseng, one of the most widely used herbal medicines, has a wide range of therapeutic and pharmacological applications. Ginsenosides are the major bioactive ingredients of ginseng, which are responsible for various pharmacological activities of ginseng. Ginsenoside Rh2, known as an antitumour ginsenoside, exists as two different stereoisomeric forms, 20()-ginsenoside Rh2 [20()-Rh2] and 20()-ginsenoside Rh2 [20()-Rh2]. This work aimed to assess and compare skin anti-photoaging activities of 20()-Rh2 and 20()-Rh2 in UV-B-irradiated HaCat cells. 20()-Rh2, but not 20()-Rh2, was able to suppress UV-B-induced ROS production in HaCat cells. Both stereoisomeric forms could not modulate cellular survival and NO level in UV-B-irradiated HaCat cells. Both 20()-Rh2 and 20()-Rh2 exhibited suppressive effects on UV-B-induced MMP-2 activity and expression in HaCat cells. In brief, the two stereoisomers of ginsenoside Rh2, 20()-Rh2 and 20()-Rh2, possess skin anti-photoaging effects but possibly in different fashions.

  6. Extract from Periostracum cicadae Inhibits Oxidative Stress and Inflammation Induced by Ultraviolet B Irradiation on HaCaT Keratinocytes

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    Tsong-Min Chang

    2017-01-01

    Full Text Available Periostracum cicadae is widely used for the treatment of skin diseases such as eczema, pruritus, and itching. The current study sought to evaluate the effect of P. cicadae extract on ultraviolet B (UVB irradiation and identify the mechanisms involved. Photodamage-protective activity of P. cicadae extracts against oxidative challenge was screened using HaCaT keratinocytes. P. cicadae extracts did not affect cell viability but decreased reactive oxygen species (ROS production. The extract attenuates the expression of interleukin-6 (IL-6, matrix metalloproteinase-2 (MMP-2, and MMP-9 in UVB-treated HaCaT cells. Also, P. cicadae abrogated UVB-induced activation of NF-κB, p53, and activator protein-1 (AP-1. The downmodulation of IL-6 by P. cicadae was inhibited by the p38 inhibitor (SB203580 or JNK inhibitor (SP600125. Moreover, the extract attenuated the expression of NF-κB and induced thrombomodulin in keratinocytes and thereby effectively downregulated inflammatory responses in the skin. The nuclear accumulation and expression of NF-E2-related factor (Nrf2 were increased by P. cicadae treatment. Furthermore, treatment with P. cicadae remarkably ameliorated the skin’s structural damage induced by irradiation. This study demonstrates that P. cicadae may protect skin cells against oxidative insult by modulating ROS concentration, IL-6, MMPs generation, antioxidant enzymes activity, and cell signaling pathways.

  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

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    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. Role of gp91phox Homolog Nox1 in Induction of Premalignant Spindle Phenotypes of HPV 16 E6/E7—Immortalized Human Keratinocytes

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    Walee Chamulitrat

    2010-01-01

    Full Text Available The NADPH oxidase (Nox family of superoxide- and hydrogen peroxide—producing proteins has been recognized as important for signal transduction that regulates receptor-mediated functions, including cytoskeleton remodeling, cell proliferation, migration, differentiation, and cell death. Nox1 was the first of the Nox catalytic subunits to be cloned and shown to induce tumorigenic conversion of mouse fibroblasts. While Nox1 has been shown to be expressed in human colon and prostate cancers, however, limited studies have demonstrated the involvement of Nox1 in an early step of cell transformation. The aim of this review is to provide an overview on the role of Nox1 in cancer, as well as the contribution of our studies to demonstrate the involvement of Nox1 on neoplastic progression of human keratinocytes beyond the immortalization step. The generation of spindle phenotypes concomitant with anchorage-independent growth and invasiveness will be highlighted and discussed in relation to the possible role of Nox1 in epithelial-mesenchymal transition. Understanding these mechanisms may provide insight into Nox1 and redox signaling components as potential therapeutic targets to inhibit tumor progression.

  9. Nicotinamide downregulates gene expression of interleukin-6, interleukin-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α gene expression in HaCaT keratinocytes after ultraviolet B irradiation.

    Science.gov (United States)

    Monfrecola, G; Gaudiello, F; Cirillo, T; Fabbrocini, G; Balato, A; Lembo, S

    2013-03-01

    Ultraviolet (UV) radiation has profound effects on human skin, causing sunburn, inflammation, cellular-tissue injury, cell death, and skin cancer. Most of these effects are mediated by a number of cytokines produced by keratinocytes. In this study we investigated whether nicotinamide (NCT), the amide form of vitamin B3, might have a protective function in reducing the expression of interleukin (IL)-1β, IL-6, IL-8, IL-10, monocyte chemoattractant protein (MCP)-1 and tumour necrosis factor (TNF)-α in UV-irradiated keratinocytes. HaCaT cells were treated with UVB in the presence or absence of NCT, and cytokine mRNA levels were examined by quantitative real-time PCR. NCT significantly downregulated IL-6, IL-10, MCP-1 and TNF-α mRNA expression, whereas it did not exert any significant effect on IL-1β or IL-8 expression. Because of its ability to decrease these cytokine mediators after UV exposure, NCT is a possible therapy to improve or prevent conditions induced or aggravated by UV light.

  10. Anti-wrinkle effects of Sargassum muticum ethyl acetate fraction on ultraviolet B-irradiated hairless mouse skin and mechanistic evaluation in the human HaCaT keratinocyte cell line.

    Science.gov (United States)

    Song, Jae Hyoung; Piao, Mei Jing; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Yoon, Weon Jong; Ko, Mi Hee; Lee, Nam Ho; Lee, Mi Young; Chae, Sungwook; Hyun, Jin Won

    2016-10-01

    The present study investigated the photoprotective properties of the ethyl acetate fraction of Sargassum muticum (SME) against ultraviolet B (UVB)‑induced skin damage and photoaging in a mouse model. HR‑1 strain hairless male mice were divided into three groups: An untreated control group, a UVB‑irradiated vehicle group and a UVB‑irradiated SME group. The UVB‑irradiated mice in the SME group were orally administered with SME (100 mg/kg body weight in 0.1 ml water per day) and then exposed to radiation at a dose of 60‑120 mJ/cm2. Wrinkle formation and skin damage were evaluated by analysis of skin replicas, epidermal thickness and collagen fiber integrity in the dermal connective tissue. The mechanism underlying the action of SME was also investigated in the human HaCaT keratinocyte cell line following exposure of the cells to UVB at a dose of 30 mJ/cm2. The protein expression levels and activity of matrix metalloproteinase‑1 (MMP‑1), and the binding of activator protein‑1 (AP‑1) to the MMP‑1 promoter were assessed in the HaCaT cells using western blot analysis, an MMP‑1 fluorescent assay and a chromatin immune‑precipitation assay, respectively. The results showed that the mean length and depth of the wrinkles in the UVB‑exposed hairless mice were significantly improved by oral administration of SME, which also prevented the increase in epidermal thickness triggered by UVB irradiation. Furthermore, a marked increase in collagen bundle formation was observed in the UVB‑treated mice with SME administration. SME pretreatment also significantly inhibited the UVB‑induced upregulation in the expression and activity of MMP‑1 in the cultured HaCaT keratinocytes, and the UVB‑enhanced association of AP‑1 with the MMP‑1 promoter. These results suggested that SME may be useful as an anti-photoaging resource for the skin.

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

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    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. Premalignant Lesions in the Kidney

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    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.

  13. 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 their

  14. Review of squamous premalignant vulvar lesions.

    NARCIS (Netherlands)

    Nieuwenhof, H.P. van de; Avoort, I.A.M. van der; Hullu, J.A. de

    2008-01-01

    Vulvar squamous cell carcinoma (SCC) develops following two different pathways, which have their own premalignant lesions. In the absence of human papilloma virus (HPV), vulvar SCC can develop in a background of lichen sclerosus (LS), differentiated vulvar intraepithelial neoplasia (VIN) or both. Th

  15. Premalignant cystic neoplasms of the pancreas.

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    Dudeja, Vikas; Allen, Peter J

    2015-02-01

    Due to increasing utilization of cross-sectional imaging, asymptomatic pancreatic cysts are frequently being diagnosed. Many of these cysts have premalignant potential and offer a unique opportunity for cancer prevention. Mucinous cystic neoplasm and intraductal papillary mucinous neoplasm are the major premalignant cystic neoplasms of pancreas. The prediction of the risk of malignancy (incidental and future risk of malignant transformation) and balancing the risks of watchful waiting with that of operative management with associated mortality and morbidity is the key to the management of these lesions. We review the literature that has contributed to the development of our approach to the management of these cystic neoplasms. We provide an overview of the key features used in diagnosis and in predicting malignancy. Particular attention is given to the natural history and management decision making.

  16. Xanthogranulomatous cholecystitis:a premalignant condition?

    Institute of Scientific and Technical Information of China (English)

    Mila Ghosh; Puja Sakhuja; Anil KAgarwal

    2011-01-01

    BACKGROUND: Xanthogranulomatous cholecystitis (XGC) is an uncommon variant of chronic cholecystitis, characterized by marked thickening of the gallbladder wall and dense local adhesions. It often mimics a gallbladder carcinoma (GBC), and may coexist with GBC, leading to a diagnostic dilemma. Furthermore, the premalignant nature of this entity is not known. This study was undertaken to assess the p53, PCNA and beta-catenin expression in XGC in comparison to GBC and chronic inflammation. METHODS: Sections from paraffin-embedded blocks of surgically resected specimens of GBC (69 cases), XGC (65), chronic cholecystitis (18) and control gallbladder (10) were stained with the monoclonal antibodies to p53 and PCNA, and a polyclonal antibody to beta-catenin. p53 expression was scored as the percentage of nuclei stained. PCNA expression was scored as the product of the percentage of nuclei stained and the intensity of the staining (1-3). A cut-off value of 80 for this score was taken as a positive result. Beta-catenin expression was scored as type of expression-membranous, cytoplasmic or nuclear staining. RESULTS: p53 mutation was positive in 52% of GBC cases and 3% of XGC, but was not expressed in chronic cholecystitis and control gallbladders. p53 expression was lower in XGC than in GBC (P CONCLUSION: The expression of p53, PCNA and beta-catenin in XGC was significantly different from GBC and similar to chronic cholecystitis, thus indicating the inflammatory nature of XGC and may not support a premalignant nature of the lesion.

  17. Histochemical identification of malignant and premalignant lesions

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    Liebow, Charles; Maloney, M. J.

    1991-06-01

    Malignant and transforming cells can be identified by biochemical parameters which can be used to localize lesions in situ for laser surgery. These cells express unique proteins, proteins in unusual quantities, or other biochemical alterations which can be utilized to image lesions of such cells. Several methods have been identified, both in vitro and in vivo, to identify such lesions. Several antibodies were examined for their properties of tissue identification, including CEA, F36/22, and AE1/AE3. F36/22, an antibody developed by M. T. Chu against human breast cancer cells, associated with two lines of oral cancer (KB and HCPC), and against two naturally occurring human oral squamous cell cancers. CEA, an antibody developed against human colon cancer, also reacted against both cell lines and both pathological samples. AE1/AE3, developed against normal fibrous components, also reacted against the samples, but in a much less regular manner. F36/22 associated with the histologically identifiably most dedifferentiated cells at the leading edge of the invading cancer. CEA, on the other hand, associated with more quiescent, older, established cancer cells. This demonstrates that antibodies developed against cancers of different organs can be used to identify a wide variety of cancers, and may have prognostic value. F36/22 coupled to fluorescein was used to identify oral cancer cells. Other properties of cancers and developing cancers can also be exploited to identify cancers, including their over-expression of tyrosine kinase and tyrosine kinase stimulating hormones such as Epidermal Growth Factor (EGF). A model of premalignant lesion produced in the hamster buccal cheek pouch with 6 week application of DMBA over-expresses constitutive tyrosine kinase which can be demonstrated biochemically. This initiated lesion can be promoted to frank cancer by growth factors released in response to laser surgery. Preliminary results suggest that these lesions can be identified by

  18. Establishment of novel rat models for premalignant breast disease

    Institute of Scientific and Technical Information of China (English)

    Wang Feng; Ma Zhongbing; Wang Fei; Fu Qinye; Fang Yunzhi; Zhang Qiang; Gao Dezong

    2014-01-01

    Background Breast cancer has become one of the most common malignant tumors among females over the past several years.Breast carcinogenesis is a continuous process,which is featured by the normal epithelium progressing to premalignant lesions and then to invasive breast cancer (IBC).Targeting premalignant lesions is an effective strategy to prevent breast cancer.The establishment of animal models is critical to study the mechanisms of breast carcinogenesis,which will facilitate research on breast cancer prevention and drug behaviors.In this study,we established a feasible chemically-induced rat model of premalignant breast cancer.Methods Following the administration of the drugs (carcinogen,estrogen,and progestogen) to Sprague-Dawley (SD) rats,tumors or suspicious tumors were identified by palpation or ultrasound imaging,and were surgically excised for pathological evaluation.A series of four consecutive steps were carried out in order to determine the carcinogen:7,12-dimethylbenzaanthracene (DMBA) or 1-methyl-1-nitrosourea,the route of carcinogen administration,the administration period of estrogen and progestogen,and the DMBA dosage.Results Stable premalignant lesions can be induced in SD rats on administration of DMBA (15 mg/kg,administered three times) followed by administration of female hormones 5-day cycle.Results were confirmed by ultrasound and palpation.Conclusion Under the premise of drug dose and cycle,DMBA combined with estrogen and progestogen can be used as a SD rat model for breast premalignant lesions.

  19. Noninvasive imaging of oral premalignancy and malignancy

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    Wilder-Smith, Petra; Krasieva, T.; Jung, W.; You, J. S.; Chen, Z.; Osann, K.; Tromberg, B.

    2005-04-01

    Objectives: Early detection of cancer and its curable precursors remains the best way to ensure patient survival and quality of life. Despite significant advances in treatment, oral cancer still results in 10,000 U.S. deaths annually, mainly due to the late detection of most oral lesions. Specific aim was to use a combination of non-invasive optical in vivo technologies to test a multi-modality approach to non-invasive diagnostics of oral premalignancy and malignancy. Methods: In the hamster cheek pouch model (120 hamsters), in vivo optical coherence tomography (OCT) and optical Doppler tomography (ODT) mapped epithelial, subepithelial and vascular change throughout carcinogenesis in specific, marked sites. In vivo multi-wavelength multi-photon (MPM) and second harmonic generated (SHG) fluorescence techniques provided parallel data on surface and subsurface tissue structure, specifically collagen presence and structure, cellular presence, and vasculature. Images were diagnosed by 2 blinded, pre-standardized investigators using a standardized scale from 0-6 for all modalities. After sacrifice, histopathological sections were prepared and pathology evaluated on a scale of 0-6. ANOVA techniques compared imaging diagnostics with histopathology. 95% confidence limits of the sensitivity and specificity were established for the diagnostic capability of OCT/ODT+ MPM/SHG using ROC curves and kappa statistics. Results: Imaging data were reproducibly obtained with good accuracy. Carcinogenesis-related structural and vascular changes were clearly visible to tissue depths of 2mm. Sensitivity (OCT/ODT alone: 71-88%; OCT+MPM/SHG: 79-91%) and specificity (OCT alone: 62-83%;OCT+MPM/SHG: 67-90%) compared well with conventional techniques. Conclusions: OCT/ODT and MPM/SHG are promising non-invasive in vivo diagnostic modalities for oral dysplasia and malignancy. Supported by CRFA 30003, CCRP 00-01391V-20235, NIH (LAMMP) RR01192, DOE DE903-91ER 61227, NIH EB-00293 CA91717, NSF BES

  20. Chromosome instability predicts progression of premalignant lesions of the larynx

    NARCIS (Netherlands)

    Bergshoeff, Verona E.; Van der Heijden, Stijn J. A.; Haesevoets, Annick; Litjens, Sophie G. H.; Bot, Fredrik J.; Voogd, Adri C.; Chenault, Michelene N.; Hopman, Anton H. N.; Schuuring, Ed; van der Wal, Jacqueline M.; Manni, Johannes J.; Ramaekers, Frans C. S.; Kremer, Bernd; Speel, Ernst-Jan M.

    2014-01-01

    The histopathology of premalignant laryngeal lesions does not provide reliable information on the risk of malignant transformation, hence we examined new molecular markers which can easily be implemented in clinical practice.Dual-target fluorescence in situ hybridisation (FISH) for chromosome 1 and

  1. Flow Cytometric Ploidy Determination of Oral Premalignant and Malignant Lesions

    Science.gov (United States)

    1990-01-01

    However, subjectivity remains an inherent part of the diagnostic process.9 According to Dabelsteen,10 subjectivity is most apparent in the determination...of Silverman 16 and Pindborg43 and Mincer et al.21 that light microscopio features of premalignancy are often not present in original biopsy specimens...was caused either in whole or in part by the presence of doublets was eliminated by syringina,. filtering and visually examining the population. In

  2. Human keratinocytes are vanilloid resistant.

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    László Pecze

    Full Text Available BACKGROUND: Use of capsaicin or resiniferatoxin (RTX as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1 permanently and selectively removes nociceptive neurons by Ca(2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+-cytotoxicity ([RTX]>15 microM could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar doses of RTX. This additional safety information might be useful for planning future human clinical trials.

  3. Effect of UV-B irradiation on interspecific competition between Ulva pertusa and Grateloupia filicina

    Institute of Scientific and Technical Information of China (English)

    李丽霞; 张培玉; 赵吉强; 周文礼; 唐学玺

    2010-01-01

    We report the effect of UV-B irradiation(9.6 kJ m-2day-1)on interspecific competition between two species of macroalgae,Ulva pertusa(U)and Grateloupia filicina(G),in co-culture.Growth of U.pertusa and G.filicina was inhibited by UV-B irradiation in mono-culture and specific growth rate (μ)declined as a result.Interspecific competition between U.pertusa and G.filicina was closely related to the initial weights when co-cultured.When initial ratios of U.pertusa(U)to G.filicina(G)were U:G=1.2:1 and 1:1,U.pertus...

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

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    Hernandez, Brenda Y.; Zhu, Xuemei; Goodman, Marc T.; Gatewood, Robert; Mendiola, Paul; Quinata, Katrina; Paulino, Yvette C.

    2017-01-01

    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 microbial changes

  5. Immunosuppression, macroencapsulation and ultraviolet-B irradiation as immunoprotection in porcine pancreatic islet xenotransplantation

    Energy Technology Data Exchange (ETDEWEB)

    Sandberg, J.O.; Olsson, N.; Hellerstroem, C.; Andersson, A. [Uppsala Univerity, Dept. of Medical Cell Biology, Uppsala (Sweden); Johnson, R.C. [Baxter Healthcar Corporation, Gene Therapy Unit, Illinois (United States)

    1995-09-01

    Membrane encapsulation or ultraviolet-B irradiation, with or without mild immunosuppressive treatment, was applied in order to prolong the survival of xenogeneic porcine foetal pancreatic grafts. Non-diabetic C57BL/6 mice were transplanted with porcine islet-like cell clusters, either membrane-encapsulated in the epididymal fat pad, or non-encapsulated under the kidney capsule. The animals were treated with daily subcutaneous injections of either cyclosporin A (12.5 mg/kg b.wt.), 15-deoxyspergualin (5.0 mg/kg b.wt.), ethyl (E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-6-isobenzofurany l-4-methyl-4-hexenoate). (RS-61443) (70 mg/kg b.wt.) or with cyclophosphamide (70 mg/kg b.wt.) every second day. A fulminant mononuclear cell infiltration was observed 14 days after transplantation both around the subcapsular graft and outside the membranes in the saline treated control group. The membrane had pores of 0.45 {mu}m and was designed to allow macromolecule transport but prevents cells from crossing. Therefore, xenoantigens can escape from the membrane implants and cause an immune reaction. A significantly weaker mononuclear cell infiltration was, however, seen when the membrane barrier was combined with 15-deoxyspergualin, cyclophosphamide or RS-61443 treatment but the morphology of the encapsulated ICC was not improved. The best subcapsular, non-encapsulated graft survival was obtained in animals treated with 15-deoxyspergualin or cyclophosphamide and the graft insulin content measurements confirmed the morphological data. There was no prolongation of islet-like cell cluster graft survival under the kidney capsule after ultraviolet-B irradiation alone (650 J/m{sup 2} for 90 sec.), and no synergistic effect was observed. It is concluded that neither membrane encapsulation with membrane that allow xenoantigen escape from the implants nor ultraviolet-B irradiation are able to prolong discordant xenograft survival in mice. (Abstract Truncated)

  6. Squamous morules are functionally inert elements of premalignant endometrial neoplasia.

    Science.gov (United States)

    Lin, Ming-Chieh; Lomo, Lesley; Baak, Jan P A; Eng, Charis; Ince, Tan A; Crum, Christopher P; Mutter, George L

    2009-02-01

    Squamous morules are a common component of premalignant glandular lesions that are followed by glandular, rather than squamous, carcinomas. We tested the hypothesis that the appearance of glands associated with morules predicts cancer risk, and undertook molecular testing to determine the clonal and hormonal response properties of admixed squamous and glandular elements. A total of 66 patients with squamous morules in an index endometrial biopsy had follow-up clinical data (average follow-up: interval 31 months, 2.5 biopsies) showing development of carcinoma in 11% (7/66) of cases. The histological appearance of morule-associated glands in the index biopsy was significantly associated with this clinical outcome, with the majority (71%, 5/7) of cancer occurrences following an overtly premalignant lesion (endometrial intraepithelial neoplasia) with squamous morules. Eight endometrial intraepithelial neoplasias with squamous morules were examined by immunohistochemistry for estrogen and progesterone receptors and mitotic activity (Ki-67 antigen percent stained). Glandular components had abundant estrogen and progesterone receptors, and high levels of mitotic activity in all cases. In sharp contrast, all squamous morules were devoid of sex hormone receptors and had undetectable or extremely low-proliferation rates. When mutated, the same specific PTEN mutation was detected in squamous and glandular elements, indicating that both are of common lineage. The clinical and laboratory data are consistent with a model of morule biology in which squamous morules are a hormonally incompetent subpopulation of endometrial glandular lesions. Isolated morules might result from artifactual displacement from their native glandular context, or selective hormonally induced regression of the glandular but not squamous components over time. Subsequent cancer risk, as promoted by estrogens, is greatest when the glandular component has the appearance of endometrial intraepithelial

  7. 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.

  8. Isolation, cultivation and transfection of human keratinocytes.

    Science.gov (United States)

    Zare, Sona; Zarei, Mohammad Ali; Ghadimi, Tayyeb; Fathi, Fardin; Jalili, Ali; Hakhamaneshi, Mohammad Saeed

    2014-04-01

    Human keratinocytes could be used in the repair of damaged skin, in tissue engineering applications, gene therapy and recently, the generation of iPS cells. We isolated human keratinocytes from foreskin and subsequently cultured them on fibronectin, collagen type I, gelatin and laminin-coated dishes that contained three different types of serum-free medium (epilife, KSM or CnT). We developed improved conditions for efficient transfection of these human keratinocytes by testing three common transfection methods and a GFP plasmid vector. The isolated cells showed typical keratinocyte morphology and expressed the epithelial cell specific antigen, cytokeratin 14. Collagen type 1, epilife medium and lipofectamin 2000 gave the best results for isolation and transfection of human keratinocytes. Our protocol can be used as a reproducible, simple and efficient method for isolation, cultivation and genetic manipulation of human keratinocytes, which may be useful in cell and gene therapy applications.

  9. Radiosensitivity of cultured human and mouse keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Parkinson, E.K.; Hume, W.J.; Potten, C.S.

    1986-10-01

    Clonogenic survival assays after ..gamma..-radiation in vitro were performed on freshly isolated and subcultured keratinocytes from mouse skin, mouse tongue and human skin. Survival curves were constructed by fitting the data to a multi-target model of cell survival. When subcultured, keratinocytes from all sites produced survival curves which showed a reduced shoulder region and an increased D/sub 0/ when compared with their freshly isolated counterparts. Freshly isolated human skin keratinocytes were more radiosensitive than mouse keratinocytes from either skin or tongue.

  10. Efficacy of oral brush cytology in the evaluation of the oral premalignant and malignant lesions

    OpenAIRE

    Babshet, M; Nandimath, K; S K Pervatikar; V G Naikmasur

    2011-01-01

    Objective: In the present study, oral brush cytology of premalignant and malignant lesions was performed using tooth brush. The cytopathological diagnosis of brush cytology was compared with that of punch biopsy. The reliability of oral brush cytology using tooth brush was assessed in terms of sensitivity and specificity. Materials and Methods: A total of 67 patients, 32 premalignant lesions and other 35 frank oral carcinomas, were included in the study. All patients underwent oral brush ...

  11. Fate of premalignant clones during the asymptomatic phase preceding lymphoid malignancy.

    Science.gov (United States)

    Moulés, Vincent; Pomier, Carole; Sibon, David; Gabet, Anne-Sophie; Reichert, Michal; Kerkhofs, Pierre; Willems, Luc; Mortreux, Franck; Wattel, Eric

    2005-02-15

    Almost all cancers are preceded by a prolonged period of clinical latency during which a combination of cellular events helps move carcinogen-exposed cells towards a malignant phenotype. Hitherto, investigating the fate of premalignant cells in vivo remained strongly hampered by the fact that these cells are usually indistinguishable from their normal counterparts. Here, for the first time, we have designed a strategy able to reconstitute the replicative history of the bona fide premalignant clone in an animal model, the sheep experimentally infected with the lymphotropic bovine leukemia virus. We have shown that premalignant clones are early and clearly distinguished from other virus-exposed cells on the basis of their degree of clonal expansion and genetic instability. Detectable as early as 0.5 month after the beginning of virus exposure, premalignant cells displayed a two-step pattern of extensive clonal expansion together with a mutation load approximately 6 times higher than that of other virus-exposed cells that remained untransformed during the life span of investigated animals. There was no fixation of somatic mutations over time, suggesting that they regularly lead to cellular death, partly contributing to maintain a normal lymphocyte count during the prolonged premalignant stage. This equilibrium was finally broken after a period of 18.5 to 60 months of clinical latency, when a dramatic decrease in the genetic instability of premalignant cells coincided with a rapid increase in lymphocyte count and lymphoma onset.

  12. Two oncogenes, v-fos and v-ras, cooperate to convert normal keratinocytes to squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Greenhalgh, D.A.; Welty, D.J.; Player, A.; Yuspa, S.H. (National Cancer Institute, Bethesda, MD (USA))

    1990-01-01

    Previous studies have been implicated the ras{sup Ha} oncogene in the initiation of skin carcinogenesis and the fos oncogene in malignant progression of premalignant skin cell lines. To determine if these two oncogenes are sufficient to convert normal keratinocytes to cancer cells, freshly isolated mouse keratinocytes were coinfected with replication-defective ({psi}-2) v-ras{sup Ha} and v-fos viruses in culture. When tested in nude mice within several days of infection, v-fos/v-ras{sup Ha}-coinfected keratinocytes produced squamous cell carcinomas. Introduction of v-fos alone resulted in normal or hyperplastic skin, whereas v-ras{sup Ha} alone produced squamous papillomas. These results indicate that two oncogenes are sufficient to produce the malignant phenotype in epidermal cells. Furthermore, they clearly link the fos oncogene with malignant conversion. Since fos acts as a transcriptional regulator of other genes, malignant conversion may be an indirect consequence of the overexpression of the fos-encoded protein leading to a change in the expression of fos-controlled cellular genes.

  13. Effect of postharvest UV-B irradiation on nutraceutical quality and physical properties of tomato fruits.

    Science.gov (United States)

    Castagna, Antonella; Chiavaro, Emma; Dall'asta, Chiara; Rinaldi, Massimiliano; Galaverna, Gianni; Ranieri, Annamaria

    2013-04-15

    Nutraceutical (ascorbic acid and carotenoids) and physical (colour and firmness) parameters were evaluated in two tomato genotypes (Money maker and high pigment-1) subjected to post harvest UV-B irradiation at different ripening stages (mature green and turning). UV-B treatment increased the concentration of ascorbic acid and carotenoids in Money maker flesh and peel, while high pigment-1 fruits underwent only minor changes, suggesting that hp-1 mutation decreased the fruit ability to respond to UV-B radiation. Colour parameters appeared to be more influenced by harvesting stages than UV-B with the exception of redness (a∗), which in Money maker was found to increase in both flesh and peel of irradiated fruits at turning stage, although not significantly, while control was more red than treated at mature green stage. Firmness was negatively influenced by UV-B, as tomatoes were found to soften after the treatment, although this aspect needs further studies to be clarified.

  14. Standard UV-B irradiation of platelets concentrates to prevent from PT-GVHD for alloimmunization

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Takayoshi [Toranomon Hospital, Tokyo (Japan); Nagahashi, Hisakata; Takenouchi, Kogi; Tayama, Tatsuya; Tadokoro, Kenji; Juji, Takeo

    1994-11-01

    We tried to make an appropriate standard condition for UV-B irradiation of platelets concentrates (PC), which is useful for prophylaxis against post-transfusion graft versus host disease (PT-GVHD) as well as prevention against alloimmunization. Agitation of PC bags during UV-B irradiation is necessary to irradiate evenly cells in the bag, because a lot of UV-B ray should be absorbed by bag membrane and plasma. Amounts of UV-B that each lymphocyte or platelet would actually receive on an average (UVavg) was calculated by the equation as below. UV{sup *}avg=K{sub 1} x (K{sub 2}{sup L} - 1) x UV/(log{sub e}K{sub 2} x L), K{sub 1} and K{sub 2} are permeability index of bag membrane and that in plasma, respectively; while L and UV stands for depth of PC bag and emitting dose of UV-B, respectively. We irradiated PC bags with UV-B in a dose of 541-13,525 J/m{sup 2} of UV{sup *}avg, and examined lymphocytes in the bags about the responder and stimulator activities in mixed lymphocytes culture (MLR), as well as platelet function in the bags. Irradiation more than 5,000 J/m{sup 2} of UV{sup *}avg is needed to suppress responder and stimulator activities, and platelet function is maintained up to 13,525 J/m{sup 2} of UV{sup *}avg. In conclusion, UV-irradiation in a range of 6,000-13,000 J/m{sup 2} of UV{sup *}avg is considered appropriate to prevent from RT-GVHD or alloimmunization. (author).

  15. Keratinocyte Apoptosis is Decreased in Psoriatic Epidermis

    Directory of Open Access Journals (Sweden)

    Fatma Eskioğlu

    2009-12-01

    Full Text Available Background and Design: Abnormal differentiation and hyperproliferation of keratinocytes are the hallmarks of psoriasis vulgaris. Although psoriasis vulgaris is generally accepted as a disease of decreased keratinocyte apoptosis, the results are contradictory. The aim of the current study is to investigate whether decreased keratinocyte apoptosis contributes to the formation of a thickened epidermis as increased keratinocyte proliferation. Material and Method: Forty-three untreated psoriasis vulgaris patients and 20 healthy control subjects were included into the study. Biopsy specimens taken from the enrollee were evaluated by immunohistochemical staining for Ki-67 expressions to show the proliferation of keratinocytes and by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL method to show the apoptotic keratinocytes. Results: Apoptotic index (percentage of the TUNEL positive cells was significantly lower in psoriatic epidermis (0.33±0.64 than in normal epidermis (0.75±0.85; whereas Ki-67 index (percentage of positively staining cells for Ki-67 was significantly higher in psoriatic epidermis (30.86±10.49 than in normal epidermis (11.65±2.98, (p=0.021 and p=0.00; respectively. Conclusion: Decreased keratinocyte apoptosis also contribute to increased epidermal thickness in psoriasis as well as increased keratinocyte proliferation.

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

    Indian Academy of Sciences (India)

    Noé Velázquez-Márquez; Gerardo Santos López; Lucio Jiménez Aranda; Julio Reyes Leyva; Verónica Vallejo Ruiz

    2012-12-01

    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 determined the expression profiles of the glycosidic antigens Tn, sialyl Tn (sTn), Lewis a (Lea), sialyl Lewis a (sLea), Lewis x (Lex) and sialyl Lewis x (sLex) in cervical scrapes with cytological diagnoses of normal, low-grade squamous intraepithelial lesions (LGSIL) and high-grade squamous intraepithelial lesions (HGSIL). Cervical scrapings were collected to detect tumour antigens expressions by flow cytometry using monoclonal antibodies. Cytometry analysis of Tn, sTn, Lea and Lex did not reveal differences at the expression level among groups. The number of positive cells to sLea antigen increased in the HGSIL group with respect to the normal group (=0.0495). The number of positive cells to sLex antigen in the samples increased with respect to the grade of squamous intraepithelial lesion (SIL) ( < 0.001, Mann–Whitney U test). The intensity of expression of this antigen increased in the HGSIL samples with respect to normal samples ( < 0.0068). sLex antigen could be a candidate to be used as biomarker for the early diagnosis of cervical cancer.

  17. Dietary Milk Sphingomyelin Prevents Disruption of Skin Barrier Function in Hairless Mice after UV-B Irradiation.

    Directory of Open Access Journals (Sweden)

    Chisato Oba

    Full Text Available Exposure to ultraviolet-B (UV-B irradiation causes skin barrier defects. Based on earlier findings that milk phospholipids containing high amounts of sphingomyelin (SM improved the water content of the stratum corneum (SC in normal mice, here we investigated the effects of dietary milk SM on skin barrier defects induced by a single dose of UV-B irradiation in hairless mice. Nine week old hairless mice were orally administrated SM (146 mg/kg BW/day for a total of ten days. After seven days of SM administration, the dorsal skin was exposed to a single dose of UV-B (20 mJ/cm2. Administration of SM significantly suppressed an increase in transepidermal water loss and a decrease in SC water content induced by UV-B irradiation. SM supplementation significantly maintained covalently-bound ω-hydroxy ceramide levels and down-regulated mRNA levels of acute inflammation-associated genes, including thymic stromal lymphopoietin, interleukin-1 beta, and interleukin-6. Furthermore, significantly higher levels of loricrin and transglutaminase-3 mRNA were observed in the SM group. Our study shows for the first time that dietary SM modulates epidermal structures, and can help prevent disruption of skin barrier function after UV-B irradiation.

  18. Differentiation of Keratinocytes Modulates Skin HPA Analog.

    Science.gov (United States)

    Wierzbicka, Justyna M; Żmijewski, Michał A; Antoniewicz, Jakub; Sobjanek, Michal; Slominski, Andrzej T

    2017-01-01

    It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

  19. Single cell mechanics of keratinocyte cells.

    Science.gov (United States)

    Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah; Liu, Gang-yu

    2010-11-01

    Keratinocytes represent the major cell type of the uppermost layer of human skin, the epidermis. Using AFM-based single cell compression, the ability of individual keratinocytes to resist external pressure and global rupturing forces is investigated and compared with various cell types. Keratinocytes are found to be 6-70 times stiffer than other cell types, such as white blood, breast epithelial, fibroblast, or neuronal cells, and in contrast to other cell types they retain high mechanic strength even after the cell's death. The absence of membrane rupturing peaks in the force-deformation profiles of keratinocytes and their high stiffness during a second load cycle suggests that their unique mechanical resistance is dictated by the cytoskeleton. A simple analytical model enables the quantification of Young's modulus of keratinocyte cytoskeleton, as high as 120-340 Pa. Selective disruption of the two major cytoskeletal networks, actin filaments and microtubules, does not significantly affect keratinocyte mechanics. F-actin is found to impact cell deformation under pressure. During keratinocyte compression, the plasma membrane stretches to form peripheral blebs. Instead of blebbing, cells with depolymerized F-actin respond to pressure by detaching the plasma membrane from the cytoskeleton underneath. On the other hand, the compression force of keratinocytes expressing a mutated keratin (cell line, KEB-7) is 1.6-2.2 times less than that for the control cell line that has normal keratin networks. Therefore, we infer that the keratin intermediate filament network is responsible for the extremely high keratinocyte stiffness and resilience. This could manifest into the rugged protective nature of the human epidermis.

  20. 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.

  1. Taiwanese Native Plants Inhibit Matrix Metalloproteinase-9 Activity after Ultraviolet B Irradiation

    Directory of Open Access Journals (Sweden)

    Yueh-Lun Lee

    2009-03-01

    Full Text Available Medicinal plants have long been used as a source of therapeutic agents. They are thought to be important anti-aging ingredients in prophylactic medicines. The aim of this study was to screen extracts from Taiwanese plant materials for phenolic contents and measure the corresponding matrix metalloproteinase-9 (MMP-9 activity. We extracted biological ingredients from eight plants native to Taiwan (Alnus formosana, Diospyros discolor, Eriobotrya deflex, Machilus japonica, Pyrrosia polydactylis, Pyrus taiwanensis, Vitis adstricta, Vitis thunbergii. Total phenolic content was measured using the Folin-Ciocalteu method. MMP-9 activities were measured by gelatin zymography. The extracted yields of plants ranged from 3.7 % to 16.9 %. The total phenolic contents ranged from 25.4 to 36.8 mg GAE/g dry material. All of these extracts (except Vitis adstricta Hance were shown to inhibit MMP-9 activity of WS-1 cell after ultraviolet B irradiation. These findings suggest that total phenolic content may influence MMP-9 activity and that some of the plants with higher phenolic content exhibited various biological activities that could serve as potent inhibitors of the ageing process in the skin. This property might be useful in the production of cosmetics.

  2. 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.

  3. Insight into the number of pre-malignancies and malignancies of the skin in a hospital population in the Netherlands

    NARCIS (Netherlands)

    Rijsingen, M.C.J. van; Seubring, I.; Maessen-Visch, B.; Lavrijsen, S.; Bergen, B. van; Groenewoud, J.; Gerritsen, M.J.

    2015-01-01

    BACKGROUND: Skin cancer incidence is rising, placing a burden on healthcare systems worldwide. This problem may even be more extensive than expected, since registration of (pre)malignancies of the skin is poor. OBJECTIVE: To provide insight into the numbers of (pre)malignancies in patients with acti

  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. Influence of epigallocatechin gallate on the immune function of dendritic cells after ultraviolet B irradiation

    Institute of Scientific and Technical Information of China (English)

    DAN LUO; BING RONG ZHOU; XI JI

    2007-01-01

    To investigate the protective effect of epigallocatechin gallate (EGCG) on the immune function of dendritic cells (DCs) after ultraviolet B irradiation (UVB) and its underlying mechanisms, the monocytes were isolated from peripheral blood and cultivated into DCs with cytokines, such as GM-CSF and IL-4. DCs were harvested after cultivation for 7 d and subjected to irradiation with different dosages of UVB. Then, 200 μg/ml of EGCG were added in certain groups immediately after irradiation. DCs simply treated with UVB or treated with both UVB and EGCG were co-cultured with lymphocytes, and MTT assay was used to detect the ability of DCs to stimulate proliferation of lymphocytes. Surface markers CD80, CD86, HLA-DR and CD40 were detected by flow cytometry, and the levels of IL-10and IL-12 secreted from DCs 24 h after cultivation were measured by ELISA. It was demonstrated that UVB irradiation could inhibit the ability of DCs to stimulate the proliferation of lymphocytes and surface expressions of CD80, CD86, HLA-DR and CD40 on DCs in a dose-dependent manner. The inhibition rate of DCs was improved to some extent after treatment with 200 μg/ml of EGCG. When the concentration of EGCG exceeded 100/μg/ml, the enhancing effect of EGCG on the expression of the co-stimulating molecules on DCs could be demonstrated in a dose-dependent manner. UVB showed no significant influence on the secretion of IL-10 and IL-12 from DCs, while EGCG could down-regulate the secretion level of IL-12 and up-regulate that of IL-10. It is concluded that EGCG can antagonize the inhibitory effect on DCs induced by UVB irradiation. This function has some relationship with its protecting effect of the expression of the co-stimulating molecule on the surface of DCs and the secretion level of IL-10and IL-12.

  6. The effect of UV-B irradiation on secondary epidermal infection of mice with herpes simplex virus type 1

    Energy Technology Data Exchange (ETDEWEB)

    El-Ghorr, A.A.; Norval, Mary [Edinburgh Univ. Medical School (United Kingdom). Dept. of Medical Microbiology

    1996-03-01

    Previous studies have indicated that suberythemal ultraviolet B (UV-B) irradiation of C3H mice before primary infection with herpes simplex virus (HSV) type 1 does not result in increased morbidity or mortality, but a suppressed delayed type hypersensitivity (DH) to the virus can be demonstrated. Any effect of UV radiation on pathogenesis during secondary epidermal HSV infection has not been previously examined. Mice were immunized by subcutaneous injection of inactivated HSV and, 5 days later, one group was UV-B-irradiated. The next day all mice were challenged epidermally with HSV. Most of the mice (92%) in the irradiated group developed severe lesions, whilst 59% of the non-irradiated group had mild lesions and 30% no lesions. Infectious virus was not isolated from the adrenal glands after challenge in either group. In addition, the DH to the virus was not affected by the UV exposure. (author).

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  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 neop

  9. Endoscopic laser treatment in pre-malignant and malignant vocal fold epithelial lesions.

    NARCIS (Netherlands)

    Remijn, E.E.; Marres, H.A.M.; Hoogen, F.J.A. van den

    2002-01-01

    Endoscopic laser treatment was performed in 43 patients with pre-malignant or malignant vocal fold epithelial lesions, 10 were treated with endoscopic laser surgery for dysplasia, 12 for carcinoma in situ (CIS), five for verrucous carcinoma and 16 patients for squamous cell carcinoma (SCC). Thirty-t

  10. Premalignant and Malignant Skin Lesions in Two Recipients of Vascularized Composite Tissue Allografts (Face, Hands

    Directory of Open Access Journals (Sweden)

    Jean Kanitakis

    2015-01-01

    Full Text Available Recipients of solid organ transplants (RSOT have a highly increased risk for developing cutaneous premalignant and malignant lesions, favored by the lifelong immunosuppression. Vascularized composite tissue allografts (VCA have been introduced recently, and relevant data are sparse. Two patients with skin cancers (one with basal cell carcinoma and one with squamous cell carcinomas have been so far reported in this patient group. Since 2000 we have been following 9 recipients of VCA (3 face, 6 bilateral hands for the development of rejection and complications of the immunosuppressive treatment. Among the 9 patients, one face-grafted recipient was diagnosed with nodular-pigmented basal cell carcinoma of her own facial skin 6 years after graft, and one patient with double hand allografts developed disseminated superficial actinic porokeratosis, a potentially premalignant dermatosis, on her skin of the arm and legs. Similar to RSOT, recipients of VCA are prone to develop cutaneous premalignant and malignant lesions. Prevention should be applied through sun-protective measures, regular skin examination, and early treatment of premalignant lesions.

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

    NARCIS (Netherlands)

    Wessels, Ronni

    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 OCT-tec

  12. Chitin modulates innate immune responses of keratinocytes.

    Directory of Open Access Journals (Sweden)

    Barbara Koller

    Full Text Available BACKGROUND: Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. CONCLUSIONS/SIGNIFICANCE: We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined.

  13. 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.

  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. Transcriptional profiling of summer wheat, grown under different realistic UV-B irradiation regimes.

    Science.gov (United States)

    Zinser, Christian; Seidlitz, Harald K; Welzl, Gerhard; Sandermann, Heinrich; Heller, Werner; Ernst, Dieter; Rau, Werner

    2007-07-01

    increased UV-B radiation. Flowering and ear development were delayed concomitantly, whereas total grain weight was not influenced at any of the UV-B irradiation regimes.

  16. Karyotypic analysis of gene transformed human keratinocyte line

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ INTRODUCTION In order to solve the difficult problem of long term in vitro culture of human keratinocytes, the technique of gene transfer was utilized to transform human keratinocytes with simian virus 40 (SV40).

  17. Thermolysin in human cultured keratinocyte isolation

    Directory of Open Access Journals (Sweden)

    A. Gragnani

    Full Text Available BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

  18. 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.

  19. Proteomic and Metabolomic Analyses of Leaf from Clematis terniflora DC. Exposed to High-Level Ultraviolet-B Irradiation with Dark Treatment.

    Science.gov (United States)

    Yang, Bingxian; Wang, Xin; Gao, Cuixia; Chen, Meng; Guan, Qijie; Tian, Jingkui; Komatsu, Setsuko

    2016-08-05

    Clematis terniflora DC. has potential pharmaceutical value; on the contrary, high-level UV-B irradiation with dark treatment led to the accumulation of secondary metabolites. Metabolomic and proteomic analyses of leaf of C. terniflora were performed to investigate the systematic response mechanisms to high-level UV-B irradiation with dark treatment. Metabolites related to carbohydrates, fatty acids, and amino acids and/or proteins related to stress, cell wall, and amino acid metabolism were gradually increased in response to high-level UV-B irradiation with dark treatment. On the basis of cluster analysis and mapping of proteins related to amino acid metabolism, the abundances of S-adenosylmethionine synthetase and cysteine synthase as well as 1,1-diphenyl-2-picrylhydrazyl scavenging activity were gradually increased in response to high-level UV-B irradiation with dark treatment. Furthermore, the abundance of dihydrolipoyl dehydrogenase/glutamate dehydrogenase and the content of γ-aminobutyric acid were also increased following high-level UV-B irradiation with dark treatment. Taken together, these results suggest that high-level UV-B irradiation with dark treatment induces the activation of reactive oxygen species scavenging system and γ-aminobutyric acid shunt pathway in leaf of C. terniflora.

  20. [Effect of trypsin on the rat keratinocyte separation and subculture].

    Science.gov (United States)

    Ouyang, An-Li; Zhou, Yan; Hua, Ping; Tan, Wen-Song

    2002-01-01

    The effect of trypsin on the separation an subculture of the keratinocytes was investigated in this work. It was found that when 0.25% trypsin was employed for 5 minutes to separate keratinocytes, the number of active keratinocytes and the cells capable of forming colony were higher than those of other experimental conditions. The maximum attached ratio of primary keratinocytes was obtained when skin tissues were treated at 0.05% concentration of trypsin. With the increase of the trypsin concentrations, the attached ratio, attachment rate constant, and colony forming efficiency were all increased. Thus, 0.25% concentration of trypsin was recommended for separating and subculturing the keratinocytes.

  1. PCNA--a cell proliferation marker in vocal chord cancer. Part I: Premalignant laryngeal lesions.

    Science.gov (United States)

    Pignataro, L D; Broich, G; Lavezzi, A M; Biondo, B; Ottaviani, F

    1995-01-01

    Laryngeal hyperkeratotic lesions can progress to fully developed malignant carcinoma in some cases. These premalignant lesions are proliferative disorders whose potential for further tumour progression is perhaps difficult to assess by mere histology. Immunostaining with PCNA, a protein correlated with cell proliferation, has been used to study tissue behavior in 30 cases of premalignant laryngeal vocal chord lesions treated by epithelial stripping in microlaryngoscopy, 15 of whom had no progression and 15 had recurrence and final development of full malignancy. The results showed a statistically significantly higher PCNA-index in the cases which underwent further tumour progression towards malignancy. PCNA testing may thus be suggested as a marker for tumour progression potential and help in determining clinical treatment choices.

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

    OpenAIRE

    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 neoplasia (CIN) detected by cervical cytological screening are extensively visualised by colposcopy and successively treated by, for instance, large loop electro-surgical excision of the transformation...

  3. Cervical glandular atypia associated with squamous intraepithelial neoplasia: a premalignant lesion?

    OpenAIRE

    Brown, L J; Wells, M

    1986-01-01

    Recent studies have described premalignant changes in the endocervical epithelium, but morphological criteria for the diagnosis of cervical glandular atypia of lesser severity than adenocarcinoma in situ have not been established. Adenocarcinoma in situ is often associated with cervical intraepithelial neoplasia (CIN). The endocervical mucosa in 105 cases of CIN grade III was evaluated and compared with that of 100 controls. Sixteen cases of cervical glandular atypia and one case of adenocarc...

  4. Assessment of the natural course and treatment of premalignant uterine cervical lesions in pregnancy

    Directory of Open Access Journals (Sweden)

    Šljivančanin Dragiša

    2013-01-01

    Full Text Available Introduction. Premalignant changes of the uterine cervix occur with similar frequency during pregnancy and in non­pregnant women. Due to the fact that any surgery on the cervix can jeopardize pregnancy, it is important to define the protocol of procedures for the treatment of these changes during pregnancy. Objective. The aim of the study was to investigate the natural course of premalignant cervical changes during pregnancy and the impact of their treatment on the pregnancy course. Methods. Study involved all patients with colposcopically, cytologically and hystopathologically diagnosed premalignant cervical changes during pregnancy from 2002 to 2008. Patients were divided into two groups according to the applied treatment during pregnancy: surgery or monitoring by regular colposcopic and cytological examinations. The two groups were compared concerning treatment outcome, persistence or regression of changes and pregnancy duration. Results. Study involved 58 patients. Spontaneous remission of lesions occurred after pregnancy in 63.79% of cases. Highgrade squamous intraepithelial lesion (H­SIL demonstrated a higher rate of persistency in comparison with low­grade squamous intraepithelial lesion (L­SIL (χ2=25.115; p<0.05. Only one finding of L­SIL progressed into H­SIL in the monitored group. Patients who underwent conization during pregnancy had a significantly more frequent preterm deliveries (χ2=14.369; p<0.05. Conclusion. The obtained high rate of spontaneous regression of cervical changes after pregnancy as well as the lower incidence of preterm births in patients who were not treated by conization during pregnancy, confirm that patients with premalignant cervical changes should be, if invasion is excluded, under follow­up throughout pregnancy by regular colposcopic and cytological examinations. Therapeutic conization, due to numerous complications, should be performed only when there is a suspected presence of a more severe form of

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

    Directory of Open Access Journals (Sweden)

    Nagaraju Kamarthi

    2010-01-01

    Full Text Available Background and Objectives: 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. Materials and Methods: 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. Results: 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 < 0.001. Interpretation and Conclusion: 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.

  6. Allelic imbalance in oral lichen planus and assessment of its classification as a premalignant condition

    Science.gov (United States)

    Accurso, Brent T.; Warner, Blake M.; Knobloch, Thomas J.; Weghorst, Christopher M.; Shumway, Brian S.; Allen, Carl M.; Kalmar, John R.

    2012-01-01

    OLP is a relatively common immune-mediated mucosal condition with a predilection for middle-aged women. Although classified as a premalignant condition, this classification remains controversial. Using stringent diagnostic criteria, some authors have found that OLP patients are not at increased risk for oral SCC. Credible but limited genetic evidence also indicates that epithelial tissues from OLP patients diagnosed using stringent criteria differs from premalignant or malignant oral lesions but is similar to epithelium from benign oral lesions. To further investigate this genetic line of evidence, biopsy specimens diagnosed as fibroma, OLP, low-grade dysplasia, high-grade dysplasia, and SCC were retrieved from the archives of the Oral Pathology Consultants at the Ohio State University. Using laser capture microdissection, tissue of interest was captured from each case and DNA subsequently extracted. Fluorescently labeled PCR primers were used to amplify DNA at 3 tumor suppressor gene loci (3p14.2, 9p21, and 17p13) and evaluated for LOH or microsatellite instability (MSI). OLP was found to be significantly different from low-grade dysplasia, high-grade dysplasia, and SCC when LOH/MSI was found at more than 1 loci (P = .011, P = .032, P = .003), but not different from benign fibromas (P = .395). In agreement with previous studies, well-documented cases of OLP diagnosed using stringent criteria exhibit a genetic profile more similar to a benign or reactive process than a premalignant/malignant one. These findings do not support the classification of OLP as a premalignant condition. PMID:21764610

  7. Differential Expression of Motility-Related Protein-1 Gene in Gastric Cancer and Its Premalignant Lesions

    Institute of Scientific and Technical Information of China (English)

    YaoXu; JieZheng; WentianLiu; JunXing; YanyunLi; XinGeng; WeimingZhang

    2004-01-01

    OBJECTIVE To identify genes related to gastric cancer and to analyze their expression profiles in different gastric tissues. METHODS The differentially expressed cDNA bands were assayed by fluorescent differential display from gastric cancer specimens, matched with normal gastric mucosa and premalignant lesions. The motility-related protein-1 (MRP-1/CD9) gene expression was studied by Northern blots and reverse transcription polymerase chain reaction (RT-PCR) in different kinds of gastric tissue. RESULTS A differentially expressed cDNA fragment showed lower expression in all gastric cancers compared to the normal gastric mucosa and premalignant lesions; and it was found to be homologous to the MRP-1/CD9 gene. Northern blot analysis confirmed the differential expression. RT-PCR analysis showed that the MRP-1/CD9 gene was expressed at a much lower rate in gastric cancers (0.31 +0.18) compared to the matched normal gastric tissue (0.49+0.24) and premalignant lesions (0.47+0.18)(P<0.05). Furthermore, its expression in intestinal-type of gastric cancer (0.38+0.16) was higher than that expressed in a diffuse-type of gastric cancer (0.22±0.17)(P<0.05). CCONCLUSION The MRP-1/CD9 gene expression was down-regulated in gastric cancer and its expression may be related to the carcinogenic process and histological type of gastric cancer.

  8. MCM2 - a promising marker for premalignant lesions of the lung: a cohort study

    Directory of Open Access Journals (Sweden)

    Beck Amy F

    2001-06-01

    Full Text Available Abstract Background Because cells progressing to cancer must proliferate, marker proteins specific to proliferating cells may permit detection of premalignant lesions. Here we compared the sensitivities of a classic proliferation marker, Ki-67, with a new proliferation marker, MCM2, in 41 bronchial biopsy specimens representing normal mucosa, metaplasia, dysplasia, and carcinoma in situ. Methods Parallel sections were stained with antibodies against MCM2 and Ki-67, and the frequencies of staining were independently measured by two investigators. Differences were evaluated statistically using the two-sided correlated samples t-test and Wilcoxon rank sum test. Results For each of the 41 specimens, the average frequency of staining by anti-MCM2 (39% was significantly (p Conclusions We conclude that MCM2 is detectable in 2-3 times more proliferating premalignant lung cells than is Ki-67. The promise of MCM2 as a sensitive marker for premalignant lung cells is enhanced by the fact that it is present in cells at the surface of metaplastic lung lesions, which are more likely to be exfoliated into sputum. Future studies will determine if use of anti-MCM2 makes possible sufficiently early detection to significantly enhance lung cancer survival rates.

  9. Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study

    Directory of Open Access Journals (Sweden)

    Shweta Jaitley

    2015-01-01

    Full Text Available The present study was undertaken with an aim of determining the cytological features observed in mucosal smears of oral submucous fibrosis (OSF patients and comparing them with that of features of normal mucosal cells. The observed features were than analyzed for their reliability in detecting malignant changes in this premalignant condition. Objective of the study was to conduct an oral exfoliative cytology (OEC study on 30 clinically diagnosed cases of OSF and 30 cases of clinically normal mucosa with no other systemic disease. We observed that all the smears from clinically normal buccal mucosa showed Class I cytology. The exfoliated cells were of normal size and shape with normal staining intensity and normal nuclear characteristics. All the 30 cases of our study group showed features suggestive of benign atypical cytological changes (Class II cytology. In the present study, despite the small number of cases, cytological features consistently observed in all the cases, were indicative of a premalignant change and emphasized a regular follow-up of patients. Early detection of a premalignant oral lesion promises to improve the survival rate of patients suffering from these conditions.

  10. Role of oral exfoliative cytology in predicting premalignant potential of oral submucous fibrosis: A short study.

    Science.gov (United States)

    Jaitley, Shweta; Agarwal, Pankaj; Upadhyay, Ramballabh

    2015-01-01

    The present study was undertaken with an aim of determining the cytological features observed in mucosal smears of oral submucous fibrosis (OSF) patients and comparing them with that of features of normal mucosal cells. The observed features were than analyzed for their reliability in detecting malignant changes in this premalignant condition. Objective of the study was to conduct an oral exfoliative cytology (OEC) study on 30 clinically diagnosed cases of OSF and 30 cases of clinically normal mucosa with no other systemic disease. We observed that all the smears from clinically normal buccal mucosa showed Class I cytology. The exfoliated cells were of normal size and shape with normal staining intensity and normal nuclear characteristics. All the 30 cases of our study group showed features suggestive of benign atypical cytological changes (Class II cytology). In the present study, despite the small number of cases, cytological features consistently observed in all the cases, were indicative of a premalignant change and emphasized a regular follow-up of patients. Early detection of a premalignant oral lesion promises to improve the survival rate of patients suffering from these conditions.

  11. 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

  12. 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.

  13. Anomalous features of EMT during keratinocyte transformation.

    Directory of Open Access Journals (Sweden)

    Tamar Geiger

    Full Text Available During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT. In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.

  14. File list: Unc.Epd.20.AllAg.Keratinocytes [Chip-atlas[Archive

    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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    Lifescience Database Archive (English)

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  5. Zinc(II and copper(II complexes with pheophytin and mesoporphyrin and their stability to UV-B irradiation: Vis spectroscopy studies

    Directory of Open Access Journals (Sweden)

    Zvezdanović Jelena B.

    2012-01-01

    Full Text Available Stability of Zn(II and Cu(II complexes of porphyrin derivatives (pheophytin and mesoporphyrin to UV-B -irradiation has been studied by absorbance spectroscopy in 95% ethanol. The chosen porphyrins as well as their heavy metal complexes undergo photochemical decomposition obeying first-order kinetics. In general, pheophytin is more stable than mesoporphyrin to UV-B irradiation. On the other hand, stability of Zn(II-complex is smaller than Cu(II-complex both for pheophytin and mesoporphyrin; however while Cu(II-complex with pheophytin is more stable than the one with mesoporphyrin, with Zn(II-complex the situation is vice versa.

  6. Prospective study of fruits and vegetables and risk of oral premalignant lesions in men.

    Science.gov (United States)

    Maserejian, Nancy Nairi; Giovannucci, Edward; Rosner, Bernard; Zavras, Athanasios; Joshipura, Kaumudi

    2006-09-15

    The authors prospectively evaluated fruit and vegetable consumption and the incidence of oral premalignant lesions among 42,311 US men in the Health Professionals Follow-up Study. Diet was assessed every 4 years by food frequency questionnaires. The authors confirmed 207 cases of clinically or histopathologically diagnosed oral premalignant lesions occurring between 1986 and 2002. Multivariate-adjusted relative risks were calculated from proportional hazards models. Significant inverse associations were observed with citrus fruits, citrus fruit juice, and vitamin-C-rich fruits and vegetables, indicating 30-40% lower risks with greater intakes (e.g., citrus fruit juice quintile 5 vs. quintile 1 relative risk = 0.65, 95% confidence interval: 0.42, 0.99). Inverse associations with fruits did not vary by smoking status and were stronger in analyses of baseline consumption, with a 10-year lag time to disease follow-up (quintile 5 vs. quintile 1 relative risk = 0.41, 95% confidence interval: 0.20, 0.82; p = 0.01). No associations were observed with total vegetables or with beta-carotene-rich or lycopene-rich fruits and vegetables. For current smokers, green leafy vegetables (ptrend = 0.05) and beta-carotene-rich fruits and vegetables (ptrend = 0.02) showed significant linear trends of increased risk (one additional serving/day relative risk = 1.7). The risk of oral premalignant lesions was significantly reduced with higher consumption of fruits, particularly citrus fruits and juices, while no consistent associations were apparent for vegetables.

  7. Significance of β-tubulin Expression in Breast Premalignant Lesions and Carcinomas

    Institute of Scientific and Technical Information of China (English)

    Yuxia Gao; Yun Niu; Xiumin Ding; Yong Yu

    2008-01-01

    OBJECTIVE To explore the expression of β-tubulin in premalignant lesions and carcinomas of the breast, and to observe the relationship of its expression with breast cancer pathological features.METHODS The expression of β-tubulin was detected immunohistochemically in 50 specimens of premalignant lesions of the breast (ADH and Peri-PM with ADH), 50 specimens of breast in situ ductal carcinomas (DCIS), and 50 specimens of invasive ductal carcinomas (IDC). Thirty specimens of normal breast tissues served as a control group.RESULTS Immunohistochemical analysis showed that: the differences among the 4 groups (normal breast tissues, breast premalignant lesions, DCIS and IDC, P < 0.05) were significant,and there were also statistically significant differences between any 2 groups (P < 0.05) except for the β-tubulin positive expression comparing DCIS versus IDC (P > 0.05). In addition, β-tubulin was expressed at a higher level in Peri-PM with ADH compared to ADH (P < 0.05). Following the degree of breast epithelial hyperplasia involved, and its development into carcinoma, the β-tubulin positive expression displayed an elevating tendency.We also found a significant positive relationship of β-tubulin expression with lymph node metastasis (P < 0.05), but no significant correlation with histological grading and nuclear grade.CONCLUSION Centrosome defects may be an early event in the development of breast cancer and they can also promote tumor progression. Studies of aberrations of centrosomal proteins provide a new way to explore the mechanism of breast tumorigenesis.

  8. Warburg and Crabtree effects in premalignant Barrett's esophagus cell lines with active mitochondria.

    Directory of Open Access Journals (Sweden)

    Martin T Suchorolski

    Full Text Available BACKGROUND: Increased glycolysis is a hallmark of cancer metabolism, yet relatively little is known about this phenotype at premalignant stages of progression. Periodic ischemia occurs in the premalignant condition Barrett's esophagus (BE due to tissue damage from chronic acid-bile reflux and may select for early adaptations to hypoxia, including upregulation of glycolysis. METHODOLOGY/PRINCIPAL FINDINGS: We compared rates of glycolysis and oxidative phosphorylation in four cell lines derived from patients with BE (CP-A, CP-B, CP-C and CP-D in response to metabolic inhibitors and changes in glucose concentration. We report that cell lines derived from patients with more advanced genetically unstable BE have up to two-fold higher glycolysis compared to a cell line derived from a patient with early genetically stable BE; however, all cell lines preserve active mitochondria. In response to the glycolytic inhibitor 2-deoxyglucose, the most glycolytic cell lines (CP-C and CP-D had the greatest suppression of extra-cellular acidification, but were able to compensate with upregulation of oxidative phosphorylation. In addition, these cell lines showed the lowest compensatory increases in glycolysis in response to mitochondrial uncoupling by 2,4-dinitrophenol. Finally, these cell lines also upregulated their oxidative phosphorylation in response to glucose via the Crabtree effect, and demonstrate a greater range of modulation of oxygen consumption. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that cells from premalignant Barrett's esophagus tissue may adapt to an ever-changing selective microenvironment through changes in energy metabolic pathways typically associated with cancer cells.

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

    Directory of Open Access Journals (Sweden)

    Benjamin C Thompson

    Full Text Available Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV radiation and affects DNA damage and repair. Nicotinamide (vitamin B3 reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2 solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer.

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

    Science.gov (United States)

    Thompson, Benjamin C; Halliday, Gary M; Damian, Diona L

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy depletion and enhancement of DNA repair. We investigated whether nicotinamide modifies DNA repair following exposure to UV radiation and sodium arsenite. HaCaT keratinocytes and ex vivo human skin were exposed to 2μM sodium arsenite and low dose (2J/cm2) solar-simulated UV, with and without nicotinamide supplementation. DNA photolesions in the form of 8-oxo-7,8-dihydro-2'-deoxyguanosine and cyclobutane pyrimidine dimers were detected by immunofluorescence. Arsenic exposure significantly increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine in irradiated cells. Nicotinamide reduced both types of photolesions in HaCaT keratinocytes and in ex vivo human skin, likely by enhancing DNA repair. These results demonstrate a reduction of two different photolesions over time in two different models in UV and arsenic exposed cells. Nicotinamide is a nontoxic, inexpensive agent with potential for chemoprevention of arsenic induced skin cancer.

  11. A method for the immortalization of newborn mouse skin keratinocytes

    Directory of Open Access Journals (Sweden)

    Brianna O Hammiller

    2015-07-01

    Full Text Available Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers, while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures, revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation.

  12. Hyaluronan-Phosphatidylethanolamine Polymers Form Pericellular Coats on Keratinocytes and Promote Basal Keratinocyte Proliferation

    Directory of Open Access Journals (Sweden)

    Caitlin J. Symonette

    2014-01-01

    Full Text Available Aged keratinocytes have diminished proliferative capacity and hyaluronan (HA cell coats, which are losses that contribute to atrophic skin characterized by reduced barrier and repair functions. We formulated HA-phospholipid (phosphatidylethanolamine, HA-PE polymers that form pericellular coats around cultured dermal fibroblasts independently of CD44 or RHAMM display. We investigated the ability of these HA-PE polymers to penetrate into aged mouse skin and restore epidermal function in vivo. Topically applied Alexa647-HA-PE penetrated into the epidermis and dermis, where it associated with both keratinocytes and fibroblasts. In contrast, Alexa647-HA was largely retained in the outer cornified layer of the epidermis and quantification of fluorescence confirmed that significantly more Alexa647-HA-PE penetrated into and was retained within the epidermis than Alexa647-HA. Multiple topical applications of HA-PE to shaved mouse skin significantly stimulated basal keratinocyte proliferation and epidermal thickness compared to HA or vehicle cream alone. HA-PE had no detectable effect on keratinocyte differentiation and did not promote local or systemic inflammation. These effects of HA-PE polymers are similar to those reported for endogenous epidermal HA in youthful skin and show that topical application of HA-PE polymers can restore some of the impaired functions of aged epidermis.

  13. Preventive Effects of Pentoxifylline on the Development of Colonic Premalignant Lesions in Obese and Diabetic Mice

    Science.gov (United States)

    Fukuta, Kazufumi; Shirakami, Yohei; Maruta, Akinori; Obara, Koki; Iritani, Soichi; Nakamura, Nobuhiko; Kochi, Takahiro; Kubota, Masaya; Sakai, Hiroyasu; Tanaka, Takuji; Shimizu, Masahito

    2017-01-01

    Obesity and its related metabolic abnormalities, including enhanced oxidative stress and chronic inflammation, are closely related to colorectal tumorigenesis. Pentoxifylline (PTX), a methylxanthine derivative, has been reported to suppress the production of tumor necrosis factor (TNF)-α and possess anti-inflammatory properties. The present study investigated the effects of PTX on the development of carcinogen-induced colorectal premalignant lesions in obese and diabetic mice. Male C57BL/KsJ-db/db mice, which are severely obese and diabetic, were administered weekly subcutaneous injections of the colonic carcinogen azoxymethane (15 mg/kg body weight) for four weeks and then received drinking water containing 125 or 500 ppm PTX for eight weeks. At the time of sacrifice, PTX administration markedly suppressed the development of premalignant lesions in the colorectum. The levels of oxidative stress markers were significantly decreased in the PTX-treated group compared with those in the untreated control group. In PTX-administered mice, the mRNA expression levels of cyclooxygenase (COX)-2, interleukin (IL)-6, and TNF-α, and the number of proliferating cell nuclear antigen (PCNA)-positive cells in the colonic mucosa, were significantly reduced. These observations suggest that PTX attenuated chronic inflammation and oxidative stress, and prevented the development of colonic tumorigenesis in an obesity-related colon cancer model. PMID:28212276

  14. 微小RNA与口腔癌前病变%Micro RNAs and oral premalignant lesions

    Institute of Scientific and Technical Information of China (English)

    朱晓寒; 付纪

    2012-01-01

    微小RNA 是非编码单链小分子RNA,通常在转录后水平通过降解抑制目标信使RNA 参与基因调控,目前研究发现微小RNA 与口腔癌关系密切,在某些口腔癌前疾病的癌变过程中发挥着重要作用,此文就微小RNA与口腔癌前病变的关系作一综述.%Micro RNAs are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading or repressing target messenger RNAs. The current studies showed micro RNAs were related to many oral cancers and played a key role in the tumorigenesis of some oral premalignant lesions. The relationship of micro RNAs and oral premalignant lesions was reviewed in the article.

  15. Primary Sclerosing Cholangitis as a Premalignant Biliary Tract Disease: Surveillance and Management.

    Science.gov (United States)

    Rizvi, Sumera; Eaton, John E; Gores, Gregory J

    2015-11-01

    Primary sclerosing cholangitis (PSC) is a premalignant biliary tract disease that confers a significant risk for the development of cholangiocarcinoma (CCA). The chronic biliary tract inflammation of PSC promotes pro-oncogenic processes such as cellular proliferation, induction of DNA damage, alterations of the extracellular matrix, and cholestasis. The diagnosis of malignancy in PSC can be challenging because inflammation-related changes in PSC may produce dominant biliary tract strictures mimicking CCA. Biomarkers such as detection of methylated genes in biliary specimens represent noninvasive techniques that may discriminate malignant biliary ductal changes from PSC strictures. However, conventional cytology and advanced cytologic techniques such as fluorescence in situ hybridization for polysomy remain the practice standard for diagnosing CCA in PSC. Curative treatment options of malignancy arising in PSC are limited. For a subset of patients selected by using stringent criteria, liver transplantation after neoadjuvant chemoradiation is a potential curative therapy. However, most patients have advanced malignancy at the time of diagnosis. Advances directed at identifying high-risk patients, early cancer detection, and development of chemopreventive strategies will be essential to better manage the cancer risk in this premalignant disease. A better understanding of dysplasia definition and especially its natural history is also needed in this disease. Herein, we review recent developments in our understanding of the risk factors, pathogenic mechanisms of PSC associated with CCA, as well as advances in early detection and therapies.

  16. Lymphocytes in patients with psoriasis promote proliferation of keratinocytes

    Institute of Scientific and Technical Information of China (English)

    DENG An-mei; ZHONG Ren-qian; CHEN Sun-xiao; ZHOU Ye; KONG Xian-tao

    2002-01-01

    Objective: To analyze the effect of lymphocytes on proliferation of keratinocytes in patients with psoriasis. Methods: Lymphocytes in lesion and peripheral blood were isolated and amplified, then cultured together with normal keratinocytes. By MTT method, the living cells were quantified in the mixed culture.Results: Compared with normal controls, lymphocytes from lesion and peripheral blood of psoriasis both promote the proliferation of keratinocytes (P<0. 01 and P<0. 05 respectively). The concentrations of IL-2 and IFN-γ in the mixture of lesion lymphocytes and keratinocytes were significantly higher than that of controls.Tripterygium glycosides inhibited this promotion. Conclusion: Lymphocytes in patients with psoriasis (mainly Thl cell) play an important role in proliferation of keratinocytes. This psoriasis cell model is useful for studies on signal transduction in psoriasis.

  17. 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.

  18. Bilirubin degradation in methanol induced by continuous UV-B irradiation: a UHPLC--ESI-MS study.

    Science.gov (United States)

    Stanojević, J S; Zvezdanović, J B; Marković, D Z

    2015-04-01

    Degradation of bilirubin in aerobic methanol solution by continuous UV-B irradiation has been investigated in this work. The purpose of this study was to shed more light on bilirubin interaction with the UV-B component of natural sunlight, since bilirubin is a very efficient UV-B absorber located in the skin epidermis. The degradation products have been detected and studied by a combined method of Ultra High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry (UHPLC-ESI-MS). Bilirubin, a toxic pigment which itself is a product of (hemoglobin) degradation in organisms, undergoes its own degradation under aerobic conditions of UV-B continuous irradiation (e.g. photooxidation) that can be partly self-sensitized. Two dipyrrolic structures have been identified as a result of the bilirubin degradation, not including the bilirubin derivative biliverdin whose increase in the irradiated system is synchronous with a time dynamics of bilirubin degradation. It appears that one of dipyrrolic products originates directly from bilirubin and biliverdin molecules, while the other one is probably connected to bilirubin self-sensitized degradation. The precursor role of biliverdin in the degradation process--related to the detected dipyrroles--has not been confirmed.

  19. Biomarkers in premalignant conditions of the gastrointestinal tract: Studies on Barrett’s esophagus and primary sclerosing cholangitis

    NARCIS (Netherlands)

    Timmer, M.R.

    2016-01-01

    In this thesis we have described our research on genetic abnormalities in (pre)malignant conditions of the gastrointestinal tract. The wide variation in biological behavior of cancerous and precancerous conditions may be largely explained by differences in genetic abnormalities. They are a source of

  20. Detection, management, and follow-up of pre-malignant cervical lesions and the role for human papillomavirus.

    NARCIS (Netherlands)

    Hamont, D. van; Bekkers, R.L.M.; Massuger, L.F.A.G.; Melchers, W.J.G.

    2008-01-01

    Cervical cytological pathology is common. Prevention of cervical cancer by detecting the disease process at an early and pre-malignant stage is practised globally either through population-based screening programmes (PSP) or through non-organised ones. High-grade cervical intraepithelial neoplasia (

  1. In vivo photo-detection of chemically induced premalignant lesions and squamous cell carcinoma of the rat palatal mucosa

    NARCIS (Netherlands)

    Nauta, JM; Speelman, OC; vanLeengoed, HLLM; Nikkels, PGJ; Roodenburg, JLN; Witjes, MJH; Vermey, A

    1997-01-01

    Photo-detection using in vivo fluorescence was studied for different stages of chemically induced premalignant lesions and squamous cell carcinoma (SCC) of the Wistar rat palatal mucosa. It was found that the epithelial dysplasia (numerically expressed in the epithelial atypia index (EAI)) of the ra

  2. EXPRESSION OF p16, CYCLIN D1 AND RB PROTEIN IN GASTRIC CARCINOMA AND PREMALIGNANT LESIONS

    Institute of Scientific and Technical Information of China (English)

    缪林; 赵志泉; 季国忠; 范志宁; 金宁; 刘政; 张平; 程铁华

    2003-01-01

    Objective: To investigate the expression of p16, cyclin D1 and Rb protein in gastric carcinoma and premalignant lesions including dysplastic gastric mucosa and intestinal metaplasia gastric mucosa. Methods: Using SP immunohistochemical methods, the expression of pl6, cyclin D1 and Rb proteins was detected in 10 specimens of normal gastric mucosa, 15 specimens of dysplastic gastric mucosa, 15 specimens of intestinal metaplasia gastric mucosa, 30 specimens of gastric carcinoma. The clinical characteristics of the 30 patients with gastric carcinoma were analysed to explore the relationship between the parameter detected and biological action of gastric cancer. Results: Expression of p16 protein was detected in 90% of normal gastric mucosa, 86.67% of dysplastic gastric mucosa, 86.67% of intestinal metaplasia gastric mucosa, 36.67% of gastric carcinoma. The positive rate of p16 protein expression in gastric carcinoma is significantly lower than that in normal gastric mucosa and gastric premalignant lesions mucosa (P<0.01). Expression of cyclin D1 protein was detected in 10% of normal gastric mucosa, 20% of dysplastic gastric mucosa, 20% of intestinal metaplasia gastric mucosa, 53.33% of gastric carcinoma. The positive rate of cyclin D1, protein expression in gastric carcinoma is significantly higher than that in normal gastric mucosa and gastric premalignant lesions mucosa (P<0.05). Expression of Rb protein was detected in 90% of normal gastric mucosa, 80% of dysplastic gastric mucosa, 80% of intestinal metaplasia gastric mucosa, 50% of gastric carcinoma. The positive rate of Rb protein expression in gastric carcinoma is significantly lower than that in normal gastric mucosa (P<0.05). The expression of p16, cyclin D1 gene were associated with the degree of differentiation of gastric carcinoma, lymphnodes metastasis and distant metastasis. Conclusion: p16, Cyclin D1 and Rb gene play important role in gastric carcinoma genesis. The expression of p16, cyclin D1 and Rb gene

  3. Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

    Science.gov (United States)

    Schmitt, Sabrina; Safferling, Kai; Westphal, Kathi; Hrabowski, Manuel; Müller, Ute; Angel, Peter; Wiechert, Lars; Ehemann, Volker; Müller, Benedikt; Holland-Cunz, Stefan; Stichel, Damian; Harder, Nathalie; Rohr, Karl; Germann, Günter; Matthäus, Franziska; Schirmacher, Peter; Grabe, Niels; Breuhahn, Kai

    2013-01-01

    Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF) affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.

  4. Stathmin regulates keratinocyte proliferation and migration during cutaneous regeneration.

    Directory of Open Access Journals (Sweden)

    Sabrina Schmitt

    Full Text Available Cutaneous regeneration utilizes paracrine feedback mechanisms to fine-tune the regulation of epidermal keratinocyte proliferation and migration. However, it is unknown how fibroblast-derived hepatocyte growth factor (HGF affects these mutually exclusive processes in distinct cell populations. We here show that HGF stimulates the expression and phosphorylation of the microtubule-destabilizing factor stathmin in primary human keratinocytes. Quantitative single cell- and cell population-based analyses revealed that basal stathmin levels are important for the migratory ability of keratinocytes in vitro; however, its expression is moderately induced in the migration tongue of mouse skin or organotypic multi-layered keratinocyte 3D cultures after full-thickness wounding. In contrast, clearly elevated stathmin expression is detectable in hyperproliferative epidermal areas. In vitro, stathmin silencing significantly reduced keratinocyte proliferation. Automated quantitative and time-resolved analyses in organotypic cocultures demonstrated a high correlation between Stathmin/phospho-Stathmin and Ki67 positivity in epidermal regions with proliferative activity. Thus, activation of stathmin may stimulate keratinocyte proliferation, while basal stathmin levels are sufficient for keratinocyte migration during cutaneous regeneration.

  5. 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

  6. 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

  7. Attenuating properties of Agastache rugosa leaf extract against ultraviolet-B-induced photoaging via up-regulating glutathione and superoxide dismutase in a human keratinocyte cell line.

    Science.gov (United States)

    Oh, Yuri; Lim, Hye-Won; Huang, Yu-Hua; Kwon, Hee-Souk; Jin, Chang Duck; Kim, Kyunghoon; Lim, Chang-Jin

    2016-10-01

    Agastache rugosa Kuntze, known as a Korean mint, is an herbal medicine that has been used for the treatment of diverse kinds of symptoms in traditional medicine. This work was undertaken to assess the protective properties of A. rugosa leaves against UV-B-induced photoaging in HaCaT keratinocytes. They were evaluated via analyzing reactive oxygen species (ROS), promatrix metalloproteinase-2 (proMMP-2) and -9 (proMMP-9), total glutathione (GSH), total superoxide dismutase (SOD), cellular viability, flavonoid content and in vitro radical scavenging activity. Total flavonoid content of ARE, a hot water extract of A. rugosa leaves, was 22.8±7.6mg of naringin equivalent/g ARE. ARE exhibited ABTS(+) radical scavenging activity with an SC50 of 836.9μg/mL. ARE attenuated the UV-B-induced ROS generation. It diminished the UV-B-induced elevation of proMMP-2 and -9 at both activity and protein levels. On the contrary, ARE was able to enhance the UV-B-reduced total GSH and total SOD activity levels. ARE, at the used concentrations, was unable to interfere with the cellular viabilities of HaCaT keratinocytes under UV-B irradiation. Taken together, ARE possesses a protective potential against UV-B-induced photoaging in HaCaT keratinocytes, possibly based upon up-regulating antioxidant components, including total GSH and SOD. These findings reasonably suggest the use of A. rugosa leaves as a photoprotective resource in manufacturing functional cosmetics.

  8. 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

    2006-03-23

    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.

  9. Screening for pre-malignant conditions in the oral cavity of chronic tobacco chewers

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    Priyanka Mahawar, Shweta Anand, Umesh Sinha, Madhav Bansal, Sanjay Dixit

    2011-01-01

    Full Text Available Oral cancer is a major health problem in tobacco users all over the world. It is one of the ten most common cancers in the world. Oral cancer is almost always preceded by some type of precancerous lesion. The precancerous lesions can be detected upto 15years, prior to their change to an invasive carcinoma. It usually affects between the ages of 15 and 40 years. It may be triggered by factors like frequency and duration of tobacco consumption, alcohol, poor oral hygiene etc. This study was conducted primarily to screen chronic tobacco chewers for the presence of oral pre-malignant conditions and secondly to educate them about the hazards of tobacco and motivate them to quit the habit. This was a cross sectional study conducted at Badi gawaltoli area of Indore. Tobacco chewers using tobacco for more than 5yrs were included in the study. Chronic tobacco chewers were screened for oral pre-malignant lesions followed by an educational intervention about the harmful effects of tobacco. Two follow ups were made to motivate them to quit the habit and to get treatment for their lesions. An open ended semi-structured questionnaire was administered to chronic tobacco chewers to assess their habit of tobacco chewing, smoking, their knowledge regarding lesions in their mouth, hazards of tobacco and any cessation efforts. Among the 80 identified chronic tobacco chewers, 60 were males and 20 were females. Lesions such as leukoplakia, erythroplakia and oral sub-mucosal fibrosis were found in 10 females (50% and 24 males (40%.

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

    Science.gov (United States)

    Fuertes, Mercedes B; Rossi, Lucas E; Peralta, Carlos M; Cabrera, Hugo N; Allevato, Miguel A; Zwirner, Norberto W

    2011-01-01

    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+ abTCR and gdTCR T lymphocytes through the NKG2D receptor. Engagement of NKG2D by MICA triggers IFN-g 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.

  11. Endoscopic submucosal dissection for premalignant lesions and noninvasive early gastrointestinal cancers

    Institute of Scientific and Technical Information of China (English)

    Sadettin Hulagu; Ali Erkan Duman; Neslihan Bozkurt; Gokhan Dindar; Tan Attila; Yesim Gurbuz; Orhan Tarcin; Cem Kalayci; Omer Senturk; Cem Aygun; Orhan Kocaman; Altay Celebi; Tolga Konduk; Deniz Koc; Goktug Sirin; Ugur Korkmaz

    2011-01-01

    AIM: To investigate the indication, feasibility, safety,and clinical utility of endoscopic submucosal dissection(ESD) in the management of various gastrointestinalpathologies.METHODS: The medical records of 60 consecutive patients(34 female, 26 male) who underwent ESD at the gastroenterology department of Kocaeli University from2006-2010 were examined. Patients selected for ESDhad premalignant lesions or non-invasive early cancers of the gastrointestinal tract and had endoscopic andhistological diagnoses. Early cancers were considered to be confined to the submucosa, with no lymph node involvement by means of computed tomography andendosonography.RESULTS: Sixty ESD procedures were performed. The indications were epithelial lesions (n = 39) (33/39 adenoma with high grade dysplasia, 6/39 adenoma with low grade dysplasia), neuroendocrine tumor (n = 7),cancer (n = 7) (5/7 early colorectal cancer, 2/7 early gastric cancer), granular cell tumor (n = 3), gastrointestinal stromal tumor (n = 2), and leiomyoma (n = 2). En bloc and piecemeal resection rates were 91.6% (55/60) and 8.3% (5/60), respectively. Complete and incomplete resection rates were 96.6% (58/60) and 3.3%(2/60), respectively. Complications were major bleeding[n = 3 (5%)] and perforations [n = 5 (8.3%)] (4colon, 1 stomach). Two patients with colonic perforations and two patients with submucosal lymphatic and microvasculature invasion (1 gastric carcinoid tumor,1 colonic adenocarcinoma) were referred to surgery.During a mean follow-up of 12 mo, 1 patient with adenoma with high grade dysplasia underwent a second ESD procedure to resect a local recurrence.CONCLUSION: ESD is a feasible and safe method for treatment of premalignant lesions and early malignant gastrointestinal epithelial and subepithelial lesions. Successful en bloc and complete resection of lesions yield high cure rates with low recurrence.

  12. 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.

  13. 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.

  14. Sphingolipid metabolism in organotypic mouse keratinocyte cultures

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    Madison, K.C.; Swartzendruber, D.C.; Wertz, P.W.; Downing, D.T. (Univ. of Iowa College of Medicine, Iowa City (USA))

    1990-12-01

    Ceramides are the dominant component of the stratum corneum intercellular lipid lamellae, which constitute the epidermal permeability barrier. Only pig and human epidermal ceramides have been extensively characterized and the structures of the ceramides of cultured keratinocytes have not been previously investigated. In the present studies, we have characterized the ceramides synthesized by organotypic lifted mouse keratinocyte cultures for the first time and compared them to the ceramides of intact mouse epidermis. Both mouse epidermis and cultures contained five ceramides, ceramide 1 being the least polar and ceramide 5 the most polar. Ceramide 1 was a group of acylceramides, i.e., very-long-chain omega-hydroxyceramides with an ester-linked nonhydroxy fatty acid. Ceramide 2 contained medium-length saturated nonhydroxy fatty acids. (In culture, the ceramide 2 band was split into two parts with the slightly more polar ceramide 2' containing short-chain saturated nonhydroxy fatty acids.) Ceramide 5 contained short-chain alpha-hydroxy fatty acids. The structures of ceramides 1, 2, and 5 were analagous to those of pig and human epidermis. Mouse epidermal ceramide 3 was quite unusual, containing beta-hydroxy fatty acids, a structure not previously identified among mammalian ceramides. In contrast, culture ceramide 3 was composed of omega-hydroxy fatty acids with a chain-length distribution similar to that of ceramide 1. Mouse ceramide 4 was composed of fatty acids with chromatographic mobility similar to hydroxy fatty acids but with different chemical reactivity; it remains only partially characterized. Culture ceramide 4 was present in quantities too small for analysis. All ceramides in mouse epidermis and cultures contained only sphingosine bases, whereas pig and human ceramides also contain phytosphingosine.

  15. Ultraviolet B light-induced apoptosis in human keratinocytes enriched with epidermal stem cells and normal keratinocytes

    Institute of Scientific and Technical Information of China (English)

    MEI Xue-ling; LIAN Shi

    2011-01-01

    Background The stem-cell compartment is the primary target for the accumulation of oncogenic mutations.Overexposure to solar ultraviolet radiation is responsible for the development and progression of >90% of skin cancers.Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.Methods Keratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen Ⅳ and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0-80 mJ/cm2. The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 Western blotting.Results Keratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm2 UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm2 UVB.Conclusions Human epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.

  16. Transcriptomic profiling of soybean in response to high-intensity UV-B irradiation reveals stress defense signaling

    Directory of Open Access Journals (Sweden)

    Min Young Yoon

    2016-12-01

    Full Text Available The depletion of the ozone layer in the stratosphere has led to a dramatic spike in ultraviolet B (UV-B intensity and increased UV-B light levels. The direct absorption of high-intensity UV-B induces complex abiotic stresses in plants, including excessive light exposure, heat, and dehydration. However, UV-B stress signaling mechanisms in plants including soybean (Glycine max [L.] remain poorly understood. Here, we surveyed the overall transcriptional responses of two soybean genotypes, UV-B-sensitive Cheongja 3 and UV-B-resistant Buseok, to continuous UV-B irradiation for 0 (control, 0.5, and 6 h using RNA-seq analysis. Homology analysis using UV-B-related genes from Arabidopsis thaliana revealed differentially expressed genes (DEGs likely involved in UV-B stress responses. Functional classification of the DEGs showed that the categories of immune response, stress defense signaling, and reactive oxygen species (ROS metabolism were over-represented. UV-B-resistant Buseok utilized phosphatidic acid-dependent signaling pathways (based on subsequent reactions of phospholipase C and diacylglycerol kinase rather than phospholipase D in response to UV-B exposure at high fluence rates, and genes involved in its downstream pathways, such as ABA signaling, mitogen-activated protein kinase cascades, and ROS overproduction, were upregulated in this genotype. In addition, the DEGs for TIR-NBS-LRR and heat shock proteins are positively activated. These results suggest that defense mechanisms against UV-B stress at high fluence rates are separate from the photomorphogenic responses utilized by plants to adapt to low-level UV light. Our study provides valuable information for deep understanding of UV-B stress defense mechanisms and for the development of resistant soybean genotypes that survive under high-intensity UV-B stress.

  17. Proteomic Analyses of NF1-Interacting Proteins in Keratinocytes

    Science.gov (United States)

    2015-04-01

    AWARD NUMBER: W81XWH-14-1-0070 TITLE: Proteomic Analyses of NF1 -Interacting Proteins in Keratinocytes PRINCIPAL INVESTIGATOR: Shyni Varghese...TITLE AND SUBTITLE 5a.CONTRACT NUMBER Proteomic Analyses of NF1 -Interacting Proteins in Keratinocytes 5b. GRANT NUMBER W81XWH-14-1-0070 5c. PROGRAM...in the NF1 null epidermis, we analyzed NF1 expression in a mouse model of psoriasis (imiquimod-induced psoriasis-like skin inflammation) and

  18. Transcriptional profiling of epidermal keratinocytes: comparison of genes expressed in skin, cultured keratinocytes, and reconstituted epidermis, using large DNA microarrays.

    Science.gov (United States)

    Gazel, Alix; Ramphal, Patricia; Rosdy, Martin; De Wever, Bart; Tornier, Carine; Hosein, Nadia; Lee, Brian; Tomic-Canic, Marjana; Blumenberg, Miroslav

    2003-12-01

    Epidermal keratinocytes are complex cells that create a unique three-dimensional (3-D) structure, differentiate through a multistage process, and respond to extracellular stimuli from nearby cells. Consequently, keratinocytes express many genes, i.e., have a relatively large "transcriptome." To determine which of the expressed genes are innate to keratinocytes, which are specific for the differentiation and 3-D architecture, and which are induced by other cell types, we compared the transcriptomes of skin from human subjects, differentiating 3-D reconstituted epidermis, cultured keratinocytes, and nonkeratinocyte cell types. Using large oligonucleotide microarrays, we analyzed five or more replicates of each, which yielded statistically consistent data and allowed identification of the differentially expressed genes. Epidermal keratinocytes, unlike other cells, express many proteases and protease inhibitors and genes that protect from UV light. Skin specifically expresses a higher number of receptors, secreted proteins, and transcription factors, perhaps influenced by the presence of nonkeratinocyte cell types. Surprisingly, mitochondrial proteins were significantly suppressed in skin, suggesting a low metabolic rate. Three-dimensional samples, skin and reconstituted epidermis, are similar to each other, expressing epidermal differentiation markers. Cultured keratinocytes express many cell-cycle and DNA replication genes, as well as integrins and extracellular matrix proteins. These results define innate, architecture-specific, and cell-type-regulated genes in epidermis.

  19. Endoscopic early detection of premalignant changes in stomach with mucosal biopsy in patients with symptomatology of acid peptic disease

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    Ajay A. Gujar

    2016-02-01

    Results: Different types of inflammatory lesions of gastric mucosa detected endoscopically and on histopathology in our study. In 7 patients premalignant changes were detected and 7 patients already had gross malignancies in stomach. Out of the total number of biopsies included in the study, we observed lesions of atrophic type in 4 antral biopsies (2.25% and 3 biopsies of the gastric body (1.69%. All were grade 1 as per Sydney classification. Conclusions: Upper endoscopy with histopathology has excellent output to detect early premalignant changes in stomach mucosa, though it is long way to continue this study to correlate both. [Int J Res Med Sci 2016; 4(2.000: 533-539

  20. Treatment to sustain a Th17-type phenotype to prevent skewing toward Treg and to limit premalignant lesion progression to cancer.

    Science.gov (United States)

    Young, M Rita I; Levingston, Corinne A; Johnson, Sara D

    2016-05-15

    While immune suppression is a hallmark of head and neck squamous cell carcinoma (HSNCC), the immunological impact of premalignant oral lesions, which often precedes development of HNSCC, is unknown. The present study assessed the changes in splenic and draining lymph node CD4(+) cell populations and their production of select cytokines that occur in mice with carcinogen-induced premalignant oral lesions and the changes that occur as lesions progress to oral cancer. These studies found skewing toward Th1 and Th17-type phenotypes in the spleen and lymph nodes of mice with premalignant oral lesions and a shift to Treg as lesions progress to cancer. Since the role of Th17 cells in the progression from premalignant lesions to cancer is not clear, studies determined the immunological and clinical effect of treating mice bearing premalignant oral lesions with a TGF-β type 1 receptor inhibitor plus IL-23 as an approach to sustain the Th17 phenotype. These studies showed that the treatment approach not only sustained the Th17 phenotype, but also increased distal spleen cell and regional lymph node cell production of other stimulatory/inflammatory mediators and slowed premalignant lesion progression to cancer.

  1. 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.

  2. The effects of Origanum hypericifolium essential oil application and ultraviolet B irradiation on mouse skin: An ultrastructural study.

    Science.gov (United States)

    Ili, Pinar

    2016-07-01

    Exposure to UV radiation can cause histopathological and ultrastructural changes in the skin. Origanum hypericifolium, an endemic Turkish plant,essential oil is mainly composed of monoterpenes. The effects of undiluted O. hypericifolium oil on the ultrastructural characteristics of the UVB-irradiated dorsal skin of mice were investigated using transmission electron microscopy. The BALB/c mice were shaved of dorsal hair and randomly housed into 4 groups: 1: control; 2: UVB-irradiated; 3: oil applied; and 4: oil applied and UVB-irradiated. The oil was applied topically to the dorsal skins of the mice on alternate days for 1week prior to UVB exposure. The skins were irradiated for a total dose of 3.5J/cm(2). The sections were stained with hematoxylin and eosin, semithin sections were stained with toluidine blue and ultrathin sections were contrasted with uranyl acetate/lead citrate. There were histopathological changes such as parakeratosis and squamous hyperplasia in the epidermal cell layers (Groups 3 and 4). There were also ultrastructural changes including lacunae formations throughout the stratum corneum layer (Groups 2, 3, and 4), enlargement of intercellular spaces (Groups 2 and 3), reduced desmosomes, narrow and elongated interdigitations, shortened, relatively indistinct and electron dense intermediate keratin filament bundles (Group 3). There were various sizes of cytoplasmic and perinucleolar vacuoles (Groups 3 and 4) and apoptotic bodies phagocytized by keratinocytes (Group 4). I conclude that undiluted oil has side-effects and the potential to inflict injury to the skin. The oil does not ameliorate the negative effects of UVB on epidermal skin cells.

  3. High wavenumber Raman spectroscopy in the characterization of urinary metabolites of normal subjects, oral premalignant and malignant patients

    Science.gov (United States)

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

    2017-01-01

    Urine has emerged as one of the diagnostically potential bio fluids, as it has many metabolites. As the concentration and the physiochemical properties of the urinary metabolites may vary under pathological transformation, Raman spectroscopic characterization of urine has been exploited as a significant tool in identifying several diseased conditions, including cancers. In the present study, an attempt was made to study the high wavenumber (HWVN) Raman spectroscopic characterization of urine samples of normal subjects, oral premalignant and malignant patients. It is concluded that the urinary metabolites flavoproteins, tryptophan and phenylalanine are responsible for the observed spectral variations between the normal and abnormal groups. Principal component analysis-based linear discriminant analysis was carried out to verify the diagnostic potentiality of the present technique. The discriminant analysis performed across normal and oral premalignant subjects classifies 95.6% of the original and 94.9% of the cross-validated grouped cases correctly. In the second analysis performed across normal and oral malignant groups, the accuracy of the original and cross-validated grouped cases was 96.4% and 92.1% respectively. Similarly, the third analysis performed across three groups, normal, oral premalignant and malignant groups, classifies 93.3% and 91.2% of the original and cross-validated grouped cases correctly.

  4. A review of Indian literature for association of smokeless tobacco with malignant and premalignant diseases of head and neck region

    Directory of Open Access Journals (Sweden)

    S Datta

    2014-01-01

    Full Text Available In India, about 60% of tobacco users use smokeless tobacco (ST alone. Head and neck squamous cell carcinoma is one of the most common cancers in India. International Agency for Research on Cancer (IARC monograph (Vol 89 found a significant association between ST use and oral cancer. However, only a few articles from India were included in this monograph. To overcome this lacuna, we have reviewed the articles published from India investigating the association between ST use and malignant and premalignant diseases of head and neck region. Data collection has been performed by computer-aided search of the MedLine and PubMed databases using different combinations of the key words. For malignant lesions, only cohort and case control studies were considered for review. For premalignant lesions and dental diseases other than case control studies, some cross-sectional studies have also been reviewed. Studies found a significant association between ST use and cancer of the oral cavity. The association was stronger for the buccal mucosa compared to tongue and for females compared to males. Significant association noted between cancer of the hypopharynx and oropharynx with ST use but no definitive association noted for cancer of the larynx and nasopharynx. Some dental disease and oral premalignant conditions were also associated with ST use. Indian studies suggest ST use is strongly associated with cancer of the oral cavity, oropharynx and hypopharynx.

  5. Effects of folic acid on epithelial apoptosis and expression of Bcl-2 and p53 in premalignant gastric lesions

    Institute of Scientific and Technical Information of China (English)

    Da-Zhong Cao; Wei-Hao Sun; Xi-Long Ou; Qian Yu; Ting Yu; You-Zhen Zhang; Zi-Ying Wu; Qi-Ping Xue; Yun-Lin Cheng

    2005-01-01

    AIM: To evaluate the effects of folic acid on epithelial apoptosis and expression of Bcl-2 and p53 in the tissues of premalignant gastric lesions.METHODS: Thirty-eight patients, with premalignant gastric lesions including 18 colonic-type intestinal metaplasia(IM)and 20 mild or moderate dysplasia, were randomly divided into a treatment group (n = 19) receiving folic acid 10 mg thrice daily and a control group (n = 19) receiving sucralfate 1 000 mg thrice daily for 3 mo. All patients undervvent endoscopies and four biopsies were taken prior to treatment and repeated after concluding therapy.Folate concentrations in gastric mucosa were measured with chemiluminescent enzyme immunoassay. Epithelial apoptosis and the expression of Bcl-2 and p53 protein in gastric mucosa were detected with flow cytometric assay.RESULTS: The mean of folate concentration in gastric mucosa was 9.03±3.37 μg/g wet wt in the folic acid treatment group, which was significantly higher than 6.83±3.02 μg/g wet wt in the control group. Both the epithelial apoptosis rate and the tumor suppressor p53expression in gastric mucosa significantly increased after folic acid treatment. In contrast, the expression of Bcl-2oncogene protein decreased after folic acid therapy.CONCLUSION: These data indicate that folic acid may play an important role in the chemoprevention of gastric carcinogenesis by enhancing gastric epithelial apoptosis in the patients with premalignant lesions.

  6. Effects of UV-B irradiation on growth and physiological activities of cucumber (Cucumis sativus L.) first leaves. Influence of nutrition; Kyuri daiichi honba no seicho to seiri kassei ni oyobosu UV-B shosha no eikyo. Hiryo no eikyo

    Energy Technology Data Exchange (ETDEWEB)

    Murase, K.; Izuta, T.; Totsuka, T. [Tokyo University of Agriculture and Technology, Tokyo (Japan). Faculty of Agriculture; Kondo, N. [The University of Tokyo, Tokyo (Japan); Shimizu, H.; Nakajima, N. [National Inst. for Environmental Studies, Tsukuba (Japan)

    1997-01-10

    The purpose of this study is to clarify the mechanism of growth retardation in UV-B irradiated plants. Effects of UV-B irradiation on the growth of first leaves of fertilized and unfertilized cucumber seedlings were compared. The growth of cucumber seedling first leaves was promoted by fertilization, compared with the unfertilized case. However, it was remarkably suppressed by UV-B irradiation. While, the growth of first leaves of unfertilized plants did not decline significantly by UV-B irradiation. Furthermore, the effects of UV-B irradiation on the growth of first leaves were examined by adding nitrogen, phosphorus, or potassium which are components of fertilizers. All of components promoted the growth, and the degrees of growth suppression were enhanced. Especially, the effects were remarkable in the case of nitrogen addition. It was suggested that the growth suppression of fertilized first leaves by UV-B irradiation would be caused by the decrease in the sensitivity to plant hormones such as cytokinin and the their activities. 23 refs., 6 figs., 1 tab.

  7. 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.

  8. 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.

  9. Detection of STAT2 in early stage of cervical premalignancy and in cervical cancer

    Institute of Scientific and Technical Information of China (English)

    Liang Zeng; Li-Hua Gao; Li-Jun Cao; De-Yun Feng; Ya Cao; Qi-Zhi Luo; Ping Yu; Ming Li

    2012-01-01

    Objective:To measure the expression pattern ofSTAT2 in cervical cancer initiation and progression in tissue sections from patients with cervicitis, dysplasia, and cervical cancer. Methods:Antibody against humanSTAT2 was confirmed by plasmids transient transfection andWestern blot.Immunohistochemistry was used to detectSTAT2 expression in the cervical biopsies by using the confirmed antibody againstSTAT2 as the primary antibody.Results:It was found that the overall rate of positiveSTAT2 expression in the cervicitis, dysplasia and cervical cancer groups were38.5%,69.4% and76.9%, respectively.TheSTAT2 levels are significantly increased in premalignant dysplasia and cervical cancer, as compared to cervicitis(P<0.05). Noticeably,STAT2 signals were mainly found in the cytoplasm, implying thatSTAT2 was not biologically active.Conclusions:These findings reveal an association between cervical cancer progression and augmentedSTAT2 expression.In conclusion,STAT2 increase appears to be an early detectable cellular event in cervical cancer development.

  10. 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

  11. 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.

  12. Akt mediates an angiogenic switch in transformed keratinocytes.

    Science.gov (United States)

    Segrelles, Carmen; Ruiz, Sergio; Santos, Mirentxu; Martínez-Palacio, Jesús; Lara, M Fernanda; Paramio, Jesús M

    2004-07-01

    Akt signaling is involved in tumorigenesis via a number of different mechanisms that result in increased proliferation and decreased apoptosis. Previous data have demonstrated that Akt-mediated signaling is functionally involved in keratinocyte transformation. This work investigates the involvement of angiogenesis as a mediator of tumorigenesis in Akt-transformed keratinocytes. Tumors produced by subcutaneous injection of the latter showed increased angiogenic profiles associated with increased vascular endothelial growth factor (VEGF) protein levels. However, in contrast to v-ras(Ha)-transformed keratinocytes, VEGF mRNA levels were not increased. The induction of VEGF protein by Akt is associated with increased phosphorylation and thus activation of p70S6K and eIF4E-binding protein 1, leading to increased VEGF translation. In addition, we observed increased metaloproteinases 2 and 9 expression, but not thrombospondin 1, in tumors derived from Akt-transformed keratinocytes. Collectively, these results demonstrate that Akt is an important mediator of angiogenesis in malignant keratinocytes through a post-transcriptional mechanism.

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

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    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.

  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. Increased 5-hydroxymethylcytosine and Ten-eleven Translocation Protein Expression in Ultraviolet B-irradiated HaCaT Cells

    Science.gov (United States)

    Wang, Dan; Huang, Jin-Hua; Zeng, Qing-Hai; Gu, Can; Ding, Shu; Lu, Jian-Yun; Chen, Jing; Yang, Sheng-Bo

    2017-01-01

    Background: DNA hydroxymethylation refers to a chemical modification process in which 5-methylcytosine (5mC) is catalyzed to 5- hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family proteins. Recent studies have revealed that aberrant TETs expression or 5hmC level may play important roles in the occurrence and development of various pathological and physiological processes including cancer and aging. This study aimed to explore the relation between aberrant DNA hydroxymethylation with skin photoaging and to investigate the levels of TETs, 5mC, and 5hmC expression 24 h after 40 mJ/cm2 and 80 mJ/cm2 doses of ultraviolet B (UVB) irradiation to HaCaT cells. Methods: To explore whether aberrant DNA hydroxymethylation is also related to skin photoaging, 40 mJ/cm2 and 80 mJ/cm2 doses of UVB were chosen to treat keratinocytes (HaCaT cells). After 24 h of UVB irradiation, 5mC and 5hmC levels were determined by immunohistochemistry (IHC) and immunofluorescence (IF), and at the same time, the expression levels of matrix metalloproteinase 1 (MMP-1) and TETs were assessed by reverse transcription-polymerase chain reaction or Western blot analysis. Results: After 40 mJ/cm2 and 80 mJ/cm2 doses of UVB exposure, both IHC and IF results showed that 5hmC levels increased significantly, while the 5mC levels did not exhibit significant changes in HaCaT cells, compared with HaCat cells without UVB exposure. Moreover, compared with HaCat cells without UVB exposure, the levels of TET1, TET2, and TET3 mRNA and protein expression were significantly upregulated (mRNA: P = 0.0022 and 0.0043 for TET1; all P expression increased dose dependently in 40 mJ/cm2 and 80 mJ/cm2 UVB-irradiated groups. Conclusion: UVB radiation could cause increased 5hmC and TET expression, which might become a novel biomarker in UVB-related skin aging. PMID:28229992

  16. Keratinocyte-derived follistatin regulates epidermal homeostasis and wound repair

    Science.gov (United States)

    Antsiferova, Maria; Klatte, Jennifer E; Bodó, Enikö; Paus, Ralf; Jorcano, José L; Matzuk, Martin M; Werner, Sabine; Kögel, Heidi

    2014-01-01

    Activin is a growth and differentiation factor that controls development and repair of several tissues and organs. Transgenic mice overexpressing activin in the skin were characterized by strongly enhanced wound healing, but also by excessive scarring. In this study, we explored the consequences of targeted activation of activin in the epidermis and hair follicles by generation of mice lacking the activin antagonist follistatin in keratinocytes. We observed enhanced keratinocyte proliferation in the tail epidermis of these animals. After skin injury, an earlier onset of keratinocyte hyperproliferation at the wound edge was observed in the mutant mice, resulting in an enlarged hyperproliferative epithelium. However, granulation tissue formation and scarring were not affected. These results demonstrate that selective activation of activin in the epidermis enhances reepithelialization without affecting the quality of the healed wound. PMID:19079322

  17. Analysis of global sumoylation changes occurring during keratinocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Phillip R Heaton

    Full Text Available Sumoylation is a highly dynamic process that plays a role in a multitude of processes ranging from cell cycle progression to mRNA processing and cancer. A previous study from our lab demonstrated that SUMO plays an important role in keratinocyte differentiation. Here we present a new method of tracking the sumoylation state of proteins by creating a stably transfected HaCaT keratinocyte cell line expressing an inducible SNAP-SUMO3 protein. The SNAP-tag allows covalent fluorescent labeling that is denaturation resistant. When combined with two-dimensional gel electrophoresis, the SNAP-tag technology provides direct visualization of sumoylated targets and can be used to follow temporal changes in the global cohort of sumoylated proteins during dynamic processes such as differentiation. HaCaT keratinocyte cells expressing SNAP-SUMO3 displayed normal morphological and biochemical features that are consistent with typical keratinocyte differentiation. SNAP-SUMO3 also localized normally in these cells with a predominantly nuclear signal and some minor cytoplasmic staining, consistent with previous reports for untagged SUMO2/3. During keratinocyte differentiation the total number of proteins modified by SNAP-SUMO3 was highest in basal cells, decreased abruptly after induction of differentiation, and slowly rebounded beginning between 48 and 72 hours as differentiation progressed. However, within this overall trend the pattern of change for individual sumoylated proteins was highly variable with both increases and decreases in amount over time. From these results we conclude that sumoylation of proteins during keratinocyte differentiation is a complex process which likely reflects and contributes to the biochemical changes that drive differentiation.

  18. File list: DNS.Epd.10.AllAg.Keratinocytes [Chip-atlas[Archive

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  13. File list: ALL.Epd.10.AllAg.Keratinocytes [Chip-atlas[Archive

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  14. 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.

  15. 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.

  16. Astrocytes derived from trisomic human embryonic stem cells express markers of astrocytic cancer cells and premalignant stem-like progenitors

    Directory of Open Access Journals (Sweden)

    Iverson Linda E

    2010-04-01

    Full Text Available Abstract Background Trisomic variants of human embryonic stem cells (hESCs arise spontaneously in culture. Although trisomic hESCs share many properties with diploid hESCs, they also exhibit features of cancer stem cells. Since most hESC-based therapies will utilize differentiated derivatives, it is imperative to investigate the potential of trisomic hESCs to undergo malignant transformation during differentiation prior to their use in the clinical setting. Methods Diploid and trisomic hESCs were differentiated into astrocytic progenitors cells (APCs, RNA extracted and hybridized to human exon-specific microarrays. Global gene expression profiles of diploid and trisomic APCs were compared to that of an astrocytoma cell line and glioblastoma samples, analyzed by others, using the same microarray platform. Results Bioinformatic analysis of microarray data indicates that differentiated trisomic APCs exhibit global expression profiles with similarities to the malignant astrocytoma cell line. An analogous trend is observed in comparison to glioblastoma samples indicating that trisomic APCs express markers of astrocytic cancer cells. The analysis also allowed identification of transcripts predicted to be differentially expressed in brain tumor stem cells. These data indicate that in vitro differentiation of trisomic hESCs along astrocytic pathways give rise to cells exhibiting properties of premalignant astrocytic stem/progenitor cells. Conclusions Given their occult nature, opportunities to study premalignant stem/progenitor cells in human have been few. The ability to propagate and direct the differentiation of aneuploid hESCs provides a powerful in vitro system for investigating biological properties of human cells exhibiting features of premalignant stem cells. This in vitro culture system can be used to elucidate changes in gene expression occurring enroute to malignant transformation and to identify molecular markers of cancer stem

  17. Dental metal-induced innate reactivity in keratinocytes.

    Science.gov (United States)

    Rachmawati, Dessy; Buskermolen, Jeroen K; Scheper, Rik J; Gibbs, Susan; von Blomberg, B Mary E; van Hoogstraten, Ingrid M W

    2015-12-25

    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 mucosa. Fresh foreskin-derived keratinocytes and skin and gingiva KC cell lines were studied for IL-8 release as a most sensitive parameter for NF-kB activation. First, we verified that viral-defense mediating TLR3 is a key innate immune receptor in both skin- and mucosa derived keratinocytes. Second, we found that, in line with our earlier finding that ionized gold can mimic viral dsRNA in triggering TLR3, gold is very effective in KC activation. It would appear that epithelial TLR3 can play a key role in both skin- and mucosa localized irritation reactivities to gold. Subsequently we found that not only gold, but also nickel, copper and mercury salts can activate innate immune reactivity in keratinocytes, although the pathways involved remain unclear. Although current alloys have been optimized for minimal leakage of metal ions, secondary factors such as mechanical friction and acidity may still facilitate such leakage. Subsequently, these metal ions may create local irritation, itching and swelling by triggering innate immune reactions, potentially also facilitating the development of metal specific adaptive immunity.

  18. Efficacy of Allogenic Cultured Keratinocyte Grafts for Burn Wounds

    Science.gov (United States)

    1993-06-01

    antigen expression induced by interferon gamma on C57BL/6 cultured keratinocytes. Notably, this antigen appears in vivo whether or not the animal has been...exposed to interferon gamma . This is extremely important because there is spontaneous generation of this antigen after grafting. Examples of this

  19. FOXO1 expression in keratinocytes promotes connective tissue healing

    Science.gov (United States)

    Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

    2017-01-01

    Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

  20. Effect of silver nanoparticles on human primary keratinocytes.

    Science.gov (United States)

    Szmyd, Radoslaw; Goralczyk, Anna Grazyna; Skalniak, Lukasz; Cierniak, Agnieszka; Lipert, Barbara; Filon, Francesca Larese; Crosera, Matteo; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

  1. Recovery and Cultivation of Keratinocytes From Shipped Mouse Skin.

    Science.gov (United States)

    Yang, Hsin-Ya; La, Thi Dinh; Gurenko, Zhanna; Steenhuis, Pieter; Liu, Wei; Isseroff, R Rivkah

    2015-02-01

    Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations.

  2. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    Science.gov (United States)

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  3. P16 and p53 expression in (pre)malignant epidermal tumors of renal transplant recipients and immunocompetent individuals.

    NARCIS (Netherlands)

    Blokx, W.A.M.; Jong, E.M.G.J. de; Wilde, P.C.M. de; Bulten, J.; Link, M.M.G.M.; Ruiter, D.J.; Kerkhof, P.C.M. van de

    2003-01-01

    Ultraviolet (UV) radiation is a prevailing factor implicated in the etiology of keratinocytic intraepidermal neoplasia (KIN) and squamous cell carcinomas (SCCs), as evidenced by the high frequency of UV-related mutations in the p53 and p16 tumor suppressor genes. In renal transplant recipients (RTRs

  4. Isovitexin-2'-O-beta-[6-O-E-p-coumaroylglucopyranoside] from UV-B irradiated leaves of rice, Oryza sativa L. inhibits fertility of Helicoverpa armigera.

    Science.gov (United States)

    Caasi-Lit, Merdelyn T; Tanner, Gregory J; Nayudu, Murali; Whitecross, Malcolm I

    2007-01-01

    UV-B irradiated rice leaves (Oryza sativa L.) contained four closely related flavonoids, with either an isoorientin or isovitexin aglycone. These flavonoids have previously been purified and characterized, and were added to artificial diets of the African bollworm (Helicoverpa armigera Hübner) at 0.1x concentration found in irradiated rice leaves. Consumption of different diets had relatively small effects on laval, pupal and adult duration, weight and survival, indicating the insects lived near normal life cycles on all diets. However, one of the compounds, flavonoid IIa, isovitexin-2''-O-beta-[6-O-E-p-coumaroylglucopyranoside], dramatically reduced the number of fertile eggs laid to 7% of control insects (Pisoorientin-2''-O-beta-[6-O-E-p-coumaroylglucopyranoside] or isoorientin-2''-O-beta-[6-O-E-p-feruloylglucopyranoside], were not significantly different to control diets.

  5. Tannic acid binding of cell surfaces in normal, premalignant, and malignant squamous epithelium of the human uterine cervix.

    Science.gov (United States)

    Davina, J H; Lamers, G E; van Haelst, U J; Kenemans, P; Stadhouders, A M

    1984-01-01

    Alterations in tannic acid (TA) binding capacity of cell surface carbohydrates in normal, premalignant, and malignant squamous epithelium of the human uterine cervix have been studied using electron microscopic visualization in combination with microdensitometric evaluation. While in normal epithelium there is distinct binding in four to five cell layers of the deep intermediate zone, cells of carcinoma in situ and invasive cancer lesions lack TA binding. In moderate dysplasia an intermediate reacting pattern is found. Deep intermediate cells in areas bordering the carcinoma in situ lesions do not show any binding, although their ultrastructure cannot be distinguished from similar cells in normal tissue. The TA deposition within the deep intermediate zone is probably related to the presence here of glycoprotein-containing membrane-coating granules. The finding that TA binding discriminates between cells in normal squamous epithelium and morphologically normal cells in juxtaposition with lesional areas in premalignant and malignant epithelium opens the possibility for a more reliable cytologic diagnosis of cervical epithelial neoplasia.

  6. 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.

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

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    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.

  8. Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

    Science.gov (United States)

    Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav

    2005-08-21

    Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

  9. Transcriptional network of p63 in human keratinocytes.

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    Silvia Pozzi

    Full Text Available p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin.

  10. Nevus depigmentosus treated by melanocyte-keratinocyte transplantation

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    Sanjeev V Mulekar

    2011-01-01

    Full Text Available Background : Nevus depigmentosus is a congenital, nonprogressive hypopigmented disorder. Various therapeutic methods have been attempted to repigment nevus with variable results. Objective : The objective of this study is to report our experience of treatment of nevus depigmentosus with a combination of noncultured melanocyte-keratinocyte transplantation (MKTP and excimer laser sessions. Materials and Methods : Six patients (male 1, female 5 of nevus depigmentosus were treated with a combination of noncultured melanocyte-keratinocyte transplantation and excimer laser. One patient was lost to follow-up. Remaining five patients were observed for a period ranging from 7 to 30 months. Results : Two patients responded poorly to MKTP. The remaining three patients responded with repigmentation ranging from 80% to 100% but the quality of repigmentation was unsatisfactory in two of them. Conclusion : Though repigmentation of nevus depigmentosus is possible by grafting techniques, the results are inconsistent and recurrence is possible.

  11. Inhibition of JNK promotes differentiation of epidermal keratinocytes.

    Science.gov (United States)

    Gazel, Alix; Banno, Tomohiro; Walsh, Rebecca; Blumenberg, Miroslav

    2006-07-21

    In inflamed tissue, normal signal transduction pathways are altered by extracellular signals. For example, the JNK pathway is activated in psoriatic skin, which makes it an attractive target for treatment. To define comprehensively the JNK-regulated genes in human epidermal keratinocytes, we compared the transcriptional profiles of control and JNK inhibitor-treated keratinocytes, using DNA microarrays. We identified the differentially expressed genes 1, 4, 24, and 48 h after the treatment with SP600125. Surprisingly, the inhibition of JNK in keratinocyte cultures in vitro induces virtually all aspects of epidermal differentiation in vivo: transcription of cornification markers, inhibition of motility, withdrawal from the cell cycle, stratification, and even production of cornified envelopes. The inhibition of JNK also induces the production of enzymes of lipid and steroid metabolism, proteins of the diacylglycerol and inositol phosphate pathways, mitochondrial proteins, histones, and DNA repair enzymes, which have not been associated with differentiation previously. Simultaneously, basal cell markers, including integrins, hemidesmosome and extracellular matrix components, are suppressed. Promoter analysis of regulated genes finds that the binding sites for the forkhead family of transcription factors are over-represented in the SP600125-induced genes and c-Fos sites in the suppressed genes. The JNK-induced proliferation appears to be secondary to inhibition of differentiation. The results indicate that the inhibition of JNK in epidermal keratinocytes is sufficient to initiate their differentiation program and suggest that augmenting JNK activity could be used to delay cornification and enhance wound healing, whereas attenuating it could be a differentiation therapy-based approach for treating psoriasis.

  12. In Vitro Toxicity of Aluminum Nanoparticles in Human Keratinocytes

    Science.gov (United States)

    2008-03-01

    absorption of aluminum has been reported after application of underarm deodorant (Becaria, 2002:311). Two adult volunteers were dermally exposed to one...NPs can be classified as a sensitizer agent. Since sensitization has been identified in persons exposed to aluminum in deodorant , vaccines, and...IN VITRO TOXICITY OF ALUMINUM NANOPARTICLES IN HUMAN KERATINOCYTES THESIS Stephanie McCormack-Brown, Major, USAF, BSC AFIT/GIH/ENV

  13. A Model to Predict the Risk of Keratinocyte Carcinomas.

    Science.gov (United States)

    Whiteman, David C; Thompson, Bridie S; Thrift, Aaron P; Hughes, Maria-Celia; Muranushi, Chiho; Neale, Rachel E; Green, Adele C; Olsen, Catherine M

    2016-06-01

    Basal cell and squamous cell carcinomas of the skin are the commonest cancers in humans, yet no validated tools exist to estimate future risks of developing keratinocyte carcinomas. To develop a prediction tool, we used baseline data from a prospective cohort study (n = 38,726) in Queensland, Australia, and used data linkage to capture all surgically excised keratinocyte carcinomas arising within the cohort. Predictive factors were identified through stepwise logistic regression models. In secondary analyses, we derived separate models within strata of prior skin cancer history, age, and sex. The primary model included terms for 10 items. Factors with the strongest effects were >20 prior skin cancers excised (odds ratio 8.57, 95% confidence interval [95% CI] 6.73-10.91), >50 skin lesions destroyed (odds ratio 3.37, 95% CI 2.85-3.99), age ≥ 70 years (odds ratio 3.47, 95% CI 2.53-4.77), and fair skin color (odds ratio 1.75, 95% CI 1.42-2.15). Discrimination in the validation dataset was high (area under the receiver operator characteristic curve 0.80, 95% CI 0.79-0.81) and the model appeared well calibrated. Among those reporting no prior history of skin cancer, a similar model with 10 factors predicted keratinocyte carcinoma events with reasonable discrimination (area under the receiver operator characteristic curve 0.72, 95% CI 0.70-0.75). Algorithms using self-reported patient data have high accuracy for predicting risks of keratinocyte carcinomas.

  14. 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 influe

  15. Acitretin treatment of premalignant and malignant skin disorders in renal transplant recipients: clinical effects of a randomized trial comparing two doses of acitretin.

    NARCIS (Netherlands)

    Sevaux, R.G.L. de; Smit, J.V.; Jong, E.M.G.J. de; Kerkhof, P.C.M. van de; Hoitsma, A.J.

    2003-01-01

    INTRODUCTION: After renal transplantation, the incidence of premalignant and malignant skin lesions is high. Treatment with acitretin improves the number and aspect of actinic keratoses and appears to reduce the incidence of squamous cell carcinomas, but treatment is hampered by frequent side effect

  16. 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.

  17. C/EBPδ gene targets in human keratinocytes.

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    Serena Borrelli

    Full Text Available C/EBPs are a family of B-Zip transcription factors--TFs--involved in the regulation of differentiation in several tissues. The two most studied members--C/EBPα and C/EBPβ--play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets--MafB and SOX2--affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation.

  18. Salivary trefoil factor 3 enhances migration of oral keratinocytes.

    Science.gov (United States)

    Storesund, Trond; Hayashi, Katsuhiko; Kolltveit, Kristin M; Bryne, Magne; Schenck, Karl

    2008-04-01

    Trefoil factor 3 (TFF3) is a member of the mammalian TFF family. Trefoil factors are secreted onto mucosal surfaces of the entire body and exert different effects according to tissue location. Trefoil factors may enhance mucosal healing by modulating motogenic activity, inhibiting apoptosis, and promoting angiogenesis. Trefoil factor 3 is secreted from the submandibular gland and is present in whole saliva. The aim of this study was to assess the migratory and proliferative effects of TFF3 on primary oral human keratinocytes and oral cancer cell lines. The addition of TFF3 increased the migration of both normal oral keratinocytes and the cancer cell line D12, as evaluated by a two-dimensional scratch assay. By contrast, no increase in proliferation or energy metabolism was observed after stimulation with TFF3. Trefoil factor 3-enhanced migration was found to be driven partly by the extracellular signal-related kinase (Erk1/2) pathway, as shown by addition of the mitogen-activated protein kinase (MAPK) inhibitor PD 98059. Previous functional studies on trefoil peptides have all been based on cells from monolayered epithelium like the intestinal mucosa; this is the first report to show that normal and cancerous keratinocytes from stratified epithelium respond to TFF stimuli. Taken together, salivary TFF3 is likely to contribute to oral wound healing.

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

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    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.

  20. Agent based modelling helps in understanding the rules by which fibroblasts support keratinocyte colony formation.

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    Tao Sun

    Full Text Available BACKGROUND: Autologous keratincoytes are routinely expanded using irradiated mouse fibroblasts and bovine serum for clinical use. With growing concerns about the safety of these xenobiotic materials, it is desirable to culture keratinocytes in media without animal derived products. An improved understanding of epithelial/mesenchymal interactions could assist in this. METHODOLOGY/PRINCIPAL FINDINGS: A keratincyte/fibroblast o-culture model was developed by extending an agent-based keratinocyte colony formation model to include the response of keratinocytes to both fibroblasts and serum. The model was validated by comparison of the in virtuo and in vitro multicellular behaviour of keratinocytes and fibroblasts in single and co-culture in Greens medium. To test the robustness of the model, several properties of the fibroblasts were changed to investigate their influence on the multicellular morphogenesis of keratinocyes and fibroblasts. The model was then used to generate hypotheses to explore the interactions of both proliferative and growth arrested fibroblasts with keratinocytes. The key predictions arising from the model which were confirmed by in vitro experiments were that 1 the ratio of fibroblasts to keratinocytes would critically influence keratinocyte colony expansion, 2 this ratio needed to be optimum at the beginning of the co-culture, 3 proliferative fibroblasts would be more effective than irradiated cells in expanding keratinocytes and 4 in the presence of an adequate number of fibroblasts, keratinocyte expansion would be independent of serum. CONCLUSIONS: A closely associated computational and biological approach is a powerful tool for understanding complex biological systems such as the interactions between keratinocytes and fibroblasts. The key outcome of this study is the finding that the early addition of a critical ratio of proliferative fibroblasts can give rapid keratinocyte expansion without the use of irradiated mouse

  1. 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.

  2. Arsenite suppression of BMP signaling in human keratinocytes

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    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

  3. Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

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    Bonnie H Y Yeung

    Full Text Available Stanniocalcin-1 (STC1, a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam formation, followed by cell migration. In this study, staurosporine (STS treatment induced human keratinocyte (HaCaT e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK, the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

  4. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    Science.gov (United States)

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

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

    NARCIS (Netherlands)

    Karim, R.; Tummers, B.; Meyers, C.; Biryukov, J.L.; Alam, S.; Backendorf, C.; Jha, V.; Offringa, R.; van Ommen, G.J.; Melief, C.J.; Guardavaccaro, D.; Boer, J.M.; van der Burg, S.H.

    2013-01-01

    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

  6. Inhibition of Akt signaling by exclusion from lipid rafts in normal and transformed epidermal keratinocytes

    DEFF Research Database (Denmark)

    Calay, Damien; Vind-Kezunovic, Dina; Frankart, Aurelie;

    2010-01-01

    of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway. Raft disruption was achieved in normal human keratinocytes and precancerous (HaCaT) or transformed (A431) keratinocytes by cholesterol extraction or inactivation with methyl-beta-cyclodextrin, filipin III, or 5-cholestene-5-beta-ol. Lipid raft disruption did not affect...

  7. Distinct roles for ROCK1 and ROCK2 in the regulation of keratinocyte differentiation.

    Directory of Open Access Journals (Sweden)

    Frances E Lock

    Full Text Available BACKGROUND: The human epidermis is comprised of several layers of specialized epithelial cells called keratinocytes. Normal homoeostasis of the epidermis requires that the balance between keratinocyte proliferation and terminal differentiation be tightly regulated. The mammalian serine/threonine kinases (ROCK1 and ROCK2 are well-characterised downstream effectors of the small GTPase RhoA. We have previously demonstrated that the RhoA/ROCK signalling pathway plays an important role in regulation of human keratinocyte proliferation and terminal differentiation. In this paper we addressed the question of which ROCK isoform was involved in regulation of keratinocyte differentiation. METHODOLOGY AND PRINCIPAL FINDINGS: We used RNAi to specifically knockdown ROCK1 or ROCK2 expression in cultured human keratinocytes. ROCK1 depletion results in decreased keratinocyte adhesion to fibronectin and an increase in terminal differentiation. Conversely, ROCK2 depletion results in increased keratinocyte adhesion to fibronectin and inhibits terminal differentiation. CONCLUSION: These data suggest that ROCK1 and ROCK2 play distinct roles in regulating keratinocyte adhesion and terminal differentiation.

  8. Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

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    Dongyuan Lü

    Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

  9. 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

  10. Effects triggered by platinum nanoparticles on primary keratinocytes.

    Science.gov (United States)

    Konieczny, Piotr; Goralczyk, Anna Grazyna; Szmyd, Radoslaw; Skalniak, Lukasz; Koziel, Joanna; Filon, Francesca Larese; Crosera, Matteo; Cierniak, Agnieszka; Zuba-Surma, Ewa K; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Pyza, Elzbieta; Semik, Danuta; Woznicka, Olga; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    The platinum (Pt)-group elements (PGEs) represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]). These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 μg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes, decreasing cell metabolism, but these changes have no effects on cell viability or migration. Moreover, smaller NPs exhibited more deleterious effect on DNA stability than the big ones. Analyzing activation of caspases, we found changes in activity of caspase 9 and caspase 3/7 triggered mainly by smaller NPs. Changes were not so significant in the case of larger nanoparticles. Importantly, we found that PtNPs have antibacterial properties, as is the case with silver NPs (AgNPs). In comparison to our previous study regarding the effects of AgNPs on cell biology, we found that PtNPs do not exhibit such deleterious effects on primary keratinocytes as AgNPs and that they also can be used as potential antibacterial agents

  11. Keratinocyte-derived laminin-332 protein promotes melanin synthesis via regulation of tyrosine uptake.

    Science.gov (United States)

    Chung, Heesung; Jung, Hyejung; Lee, Jung-Hyun; Oh, Hye Yun; Kim, Ok Bin; Han, Inn-Oc; Oh, Eok-Soo

    2014-08-01

    Melanocytes, which produce the pigment melanin, are known to be closely regulated by neighboring keratinocytes. However, how keratinocytes regulate melanin production is unclear. Here we report that melanin production in melanoma cells (B16F10 and MNT-1) was increased markedly on a keratinocyte-derived extracellular matrix compared with a melanoma cell-derived extracellular matrix. siRNA-mediated reduction of keratinocyte-derived laminin-332 expression decreased melanin synthesis in melanoma cells, and laminin-332, but not fibronectin, enhanced melanin content and α-melanocyte-stimulating hormone-regulated melanin production in melanoma cells. Similar effects were observed in human melanocytes. Interestingly, however, laminin-332 did not affect the expression or activity of tyrosinase. Instead, laminin-332 promoted the uptake of extracellular tyrosine and, subsequently, increased intracellular levels of tyrosine in both melanocytes and melanoma cells. Taken together, these data strongly suggest that keratinocyte-derived laminin-332 contributes to melanin production by regulating tyrosine uptake.

  12. Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA.

    Science.gov (United States)

    Zhu, Ping; Yang, Chuan; Chen, Li-Hong; Ren, Meng; Lao, Guo-Juan; Yan, Li

    2011-07-01

    The migration and proliferation of keratinocytes is critical to wound re-epithelialization and defects in this function are associated with the clinical phenomenon of chronic non-healing wounds. Advanced glycation end products (AGEs) occur through non-enzymatic glycation of long-lived proteins in diabetes and play important roles in diabetic complications. However, specific roles for AGEs in keratinocyte migration and proliferation, and the underlying molecular mechanisms, have not been fully established. The aim of the current study was to elucidate the interaction between AGE-modified bovine serum albumin (AGE-BSA) and keratinocytes. As a result, we found that AGE-BSA had no effect on the viability of keratinocytes for up to 48 h of incubation with 50 μg/ml of AGE-BSA. AGE-BSA (but not non-glycated BSA) exerted a concentration-dependent suppression of keratinocyte migration at a range of concentrations. The expression of matrix metalloproteinase-9 (MMP-9) was significantly up-regulated in keratinocytes incubated with increasing AGE-BSA, but tissue inhibitor of metalloproteinases-1 (TIMP-1) expression was down-regulated. AGE-BSA also profoundly depressed phospho-focal adhesion kinase-Tyr397 (p-FAK) and α2β1 integrin expression, while total-FAK expression levels remained constant, in keratinocytes. The proliferative capacity of keratinocytes was diminished after 72 h AGE-BSA incubation. Taken together, these findings suggested that in the presence of AGE-BSA, keratinocytes lose their migratory and proliferation abilities. These data also indicated that, in the context of the chronic hyperglycemia in diabetes, the effects of AGE-BSA on keratinocyte migration might be mediated through MMP-9/TIMP-1, p-FAK and α2β1 integrin.

  13. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents.

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    Melville B Vaughan

    Full Text Available BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent. METHODOLOGY/PRINCIPAL FINDINGS: Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras, were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63, basement-membrane formation (collagen IV, laminin-5, and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes. CONCLUSIONS/SIGNIFICANCE: The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

  14. Selenoproteins are essential for proper keratinocyte function and skin development.

    Directory of Open Access Journals (Sweden)

    Aniruddha Sengupta

    Full Text Available Dietary selenium is known to protect skin against UV-induced damage and cancer and its topical application improves skin surface parameters in humans, while selenium deficiency compromises protective antioxidant enzymes in skin. Furthermore, skin and hair abnormalities in humans and rodents may be caused by selenium deficiency, which are overcome by dietary selenium supplementation. Most important biological functions of selenium are attributed to selenoproteins, proteins containing selenium in the form of the amino acid, selenocysteine (Sec. Sec insertion into proteins depends on Sec tRNA; thus, knocking out the Sec tRNA gene (Trsp ablates selenoprotein expression. We generated mice with targeted removal of selenoproteins in keratin 14 (K14 expressing cells and their differentiated descendents. The knockout progeny had a runt phenotype, developed skin abnormalities and experienced premature death. Lack of selenoproteins in epidermal cells led to the development of hyperplastic epidermis and aberrant hair follicle morphogenesis, accompanied by progressive alopecia after birth. Further analyses revealed that selenoproteins are essential antioxidants in skin and unveiled their role in keratinocyte growth and viability. This study links severe selenoprotein deficiency to abnormalities in skin and hair and provides genetic evidence for the role of these proteins in keratinocyte function and cutaneous development.

  15. Effects triggered by platinum nanoparticles on primary keratinocytes

    Directory of Open Access Journals (Sweden)

    Konieczny P

    2013-10-01

    Full Text Available Piotr Konieczny,1,* Anna Grazyna Goralczyk,1,* Radoslaw Szmyd,1,* Lukasz Skalniak,1,* Joanna Koziel,2 Francesca Larese Filon,3 Matteo Crosera,4 Agnieszka Cierniak,1 Ewa K Zuba-Surma,5 Julia Borowczyk,5 Eliza Laczna,5 Justyna Drukala,5 Elzbieta Pyza,6 Danuta Semik,6 Olga Woznicka,6 Andrzej Klein,1 Jolanta Jura11Department of General Biochemistry, 2Department of Microbiology, Jagiellonian University, Kraków, Poland; 3Department of Public Health Sciences, 4Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy; 5Department of Cell Biology, 6Department of Cell Biology and Imaging, Jagiellonian University, Kraków, Poland*These authors equally contributed to this workAbstract: The platinum (Pt-group elements (PGEs represent a new kind of environmental pollutant and a new hazard for human health. Since their introduction as vehicle-exhaust catalysts, their emissions into the environment have grown considerably compared with their low natural concentration in the earth crust. PGE emissions from vehicle catalysts can be also in the form of nanometer-sized particles (Pt nanoparticles [PtNPs]. These elements, both in their metallic form or as ions solubilized in biological media, are now recognized as potent allergens and sensitizers. Human skin is always exposed to toxic particles; therefore, in the present study we addressed the question of whether polyvinylpyrrolidone-coated PtNPs may have any negative effects on skin cells, including predominantly epidermal keratinocytes. In this study, PtNPs of two sizes were used: 5.8 nm and 57 nm, in concentrations of 6.25, 12.5, and 25 µg/mL. Both types of NPs were protected with polyvinylpyrrolidone. Primary keratinocytes were treated for 24 and 48 hours, then cytotoxicity, genotoxicity, morphology, metabolic activity, and changes in the activation of signaling pathways were investigated in PtNP-treated cells. We found that PtNPs trigger toxic effects on primary keratinocytes

  16. Cervical pre-malignant lesions in HIV infected women attending Care and Treatment Centre in a tertiary hospital, Dar es Salaam, Tanzania.

    Science.gov (United States)

    Balandya, Belinda S; Pembe, Andrea B; Mwakyoma, Henry A

    2011-09-01

    The aims of this study was to determine proportion of HIV infected women with cervical pre-malignant lesions; and compare the use of Visual Inspection of the cervix after application of Acetic acid (VIA) and Papanicolau (Pap) smear in screening for cervical premalignant lesions in HIV positive women attending Care and Treatment Centre (CTC) at Muhimbili National Hospital (MNH), Dar es Salaam, Tanzania. A total of 316 women aged 18-70 years had a Pap smear taken for cytology, followed by spraying onto the cervix with 4% acetic acid and then inspecting it. Cytology was considered negative when there was no Cervical Intraepithelial Neoplasia (CIN) lesion reported from the Pap smear taken, and positive if CIN lesion 1, 2 or 3 was reported. Detection of a well-defined, opaque acetowhite lesion close to the squamocolumnar junction or close to the external cervical os constituted a positive VIA. Out of 316 women, 132 women had acetowhite lesions on VIA, making the proportion of abnormal cervical lesions to be 42.4%. One hundred and one out of 312 women (32.4%) had CIN lesions detected on Pap smear. The proportion of agreement between these two tests was 0.3. The proportion of agreement was moderate in women with advanced WHO HIV clinical stage of the disease and in women not on ART (Anti Retroviral Therapy). Women with CD-4 count less than 200 cells/mm3 had more abnormal cervical lesions. There is considerable proportion of HIV positive women with premalignant lesions of the cervix. Considering the proportion of HIV women with abnormal lesions and the difficulty in logistics of doing Pap smear in low resource settings, these results supports the recommendation to introduce screening of premalignant lesions of the cervix using VIA to all HIV infected women.

  17. 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.

  18. Alterations in vitamin D signaling pathway in gastric cancer progression: a study of vitamin D receptor expression in human normal, premalignant, and malignant gastric tissue.

    Science.gov (United States)

    Wen, Yanghui; Da, Mingxu; Zhang, Yongbin; Peng, Lingzhi; Yao, Jibin; Duan, Yaoxing

    2015-01-01

    Amount of studies in cells and animal models have proved vitamin D has multifarious antitumor effects. However, epidemiological studies showed inconsistent result on gastric cancer. The antitumor role is mainly mediated by the vitamin D receptor (VDR). Our hypothesis is that VDR may be abnormally (poorly) expressed in gastric cancer tissue. Present study is aimed at discovering and analyzing VDR expression in a series of human gastric tissues, including normal, premalignant, and malignant gastric tissue, and correlated VDR to the clinicopathological parameters of gastric cancer patients. VDR expression was detected by immunohistochemistry. The χ(2) test was used to analyze the VDR expression as well as the relationship between VDR and the clinicopathological factors of gastric cancer patients. Compared with normal (82.61%) and premalignant tissues (73.64%), VDR was lower expressed in cancer tissues (57.61%), with a statistically significant difference (P = 0.001). Among cancer tissues, VDR was higher expressed in well and moderate differentiated tissues contrasted with tissues with poor differentiation, and higher expressed in small tumors (gastric tissues. VDR expression has been on the decline from the premalignant stage, finally low expressed in gastric cancer tissues, especial in poorly differentiated tissues. VDR could be a potential prognostic factor for patients with gastric cancer.

  19. 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.

  20. Vitiligo patient-derived keratinocytes exhibit characteristics of normal wound healing via epithelial to mesenchymal transition.

    Science.gov (United States)

    Banerjee, Poulomi; Venkatachalam, Sandhyaa; Mamidi, Murali Krishna; Bhonde, Ramesh; Shankar, Krupa; Pal, Rajarshi

    2015-05-01

    Vitiligo is an autoimmune disorder that leads to depigmentation of skin via melanocyte dysfunction. Keratinocyte-induced toxicity is one among the several etiological factors implicated for vitiligo, and hence, autologous keratinocyte grafting is projected as one of the primary mode of treatment for vitiligo. However, reports indicate that perilesional keratinocytes not only display signatures of apoptosis but also could secrete cytokines and mediators which have antagonistic effect on proliferation or survival. Therefore, we investigated how vitiligo patients' derived keratinocytes respond to surplus amounts of inflammatory cytokines and whether they recapitulate events that take place during conventional wound healing. The primary objective of our study was to determine whether keratinocytes isolated from a vitiligo patient would undergo epithelial-mesenchymal transition similar to their normal counterparts upon induction with inflammatory cytokines such as TGF-b1 and EGF. We found that these keratinocytes undergo EMT during wound repair accompanied with increase in the levels of mesenchymal markers and ECM proteins; decrease in the levels of epithelial markers and enhanced migratory ability. Besides, we also demonstrated that EMT induction leads to activation of SMAD and MAPK pathways via Ras, Raf, PAI 1, Snail, Slug and ZO1. To our knowledge, this is the first report on the characterization of primary keratinocytes isolated from vitiligo patients with respect to their wound healing capacity.

  1. 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.

  2. Characterization of extracellular matrix macromolecules in primary cultures of equine keratinocytes

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    Pollitt Christopher C

    2010-03-01

    Full Text Available Abstract Background Most research to date involving laminins and extracellular matrix protein function in both normal and pathological conditions involves in vitro culture of keratinocytes. Few methods are established to allow for prolonged propagation of keratinocytes from equine tissues, including the hoof lamellae. In this study we modified cell isolation and culture techniques to allow for proliferation and sub-culturing of equine lamellar keratinocytes. Additionally, the production and processing of extracellular matrix molecules by skin and lamellar keratinocytes were studied. Results Physical and proteolytic tissue separation in combination with media containing a calcium concentration of 0.6 mM in combination with additional media supplements proved optimal for proliferation and subculture of equine lamellar keratinocytes on collagen coated substratum. Immunofluorescence and immunoblotting studies confirmed that equine skin and lamellar keratinocytes produce Ln-332 in vitro and processing of this molecule follows that of other species. As well, matrix components including integrin alpha-6 (α6 and the hemidesmsome proteins, bullous pemphigoid antigen 1 (BP180 bullous pemphigoid antigen 2 (BP230 and plectin are also expressed. Conclusions Isolation of equine keratinocytes and study of the matrix and adhesion related molecules produced by them provides a valuable tool for future work in the veterinary field.

  3. Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

    Science.gov (United States)

    Bush, Katie A; Downing, Brett R; Walsh, Sarah E; Pins, George D

    2007-02-01

    The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

  4. Yokukansan, a Traditional Japanese Medicine, Adjusts Glutamate Signaling in Cultured Keratinocytes

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    Maki Wakabayashi

    2014-01-01

    Full Text Available Glutamate plays an important role in skin barrier signaling. In our previous study, Yokukansan (YKS affected glutamate receptors in NC/Nga mice and was ameliorated in atopic dermatitis lesions. The aim of this study was to assess the effect of YKS on skin and cultured human keratinocytes. Glutamate concentrations in skin of YKS-treated and nontreated NC/Nga mice were measured. Then, glutamate release from cultured keratinocytes was measured, and extracellular glutamate concentrations in YKS-stimulated cultured human keratinocytes were determined. The mRNA expression levels of NMDA receptor 2D (NMDAR2D and glutamate aspartate transporter (GLAST were also determined in YKS-stimulated cultured keratinocytes. The glutamate concentrations and dermatitis scores increased in conventional mice, whereas they decreased in YKS-treated mice. Glutamate concentrations in cell supernatants of cultured keratinocytes increased proportionally to the cell density. However, they decreased dose-dependently with YKS. YKS stimulation increased NMDAR2D in a concentration-dependent manner. Conversely, GLAST decreased in response to YKS. Our findings indicate that YKS affects peripheral glutamate signaling in keratinocytes. Glutamine is essential as a transmitter, and dermatitis lesions might produce and release excess glutamate. This study suggests that, in keratinocytes, YKS controls extracellular glutamate concentrations, suppresses N-methyl-D-aspartate (NMDA receptors, and activates glutamate transport.

  5. Differential effects of detergents on keratinocyte gene expression.

    Science.gov (United States)

    van Ruissen, F; Le, M; Carroll, J M; van der Valk, P G; Schalkwijk, J

    1998-04-01

    We have studied the effect of various detergents on keratinocyte gene expression in vitro, using an anionic detergent (sodium dodecyl sulfate), a cationic detergent cetyltrimethylammoniumbromide (CTAB), and two nonionic detergents, Nonidet P-40 and Tween-20. We measured the effect of these detergents on direct cellular toxicity (lactate dehydrogenase release), on the expression of markers for normal differentiation (cytokeratin 1 and involucrin expression), and on disturbed keratinocyte differentiation (SKALP) by northern blot analysis. As reported in other studies, large differences were noted in direct cellular toxicity. In a culture model that mimics normal epidermal differentiation we found that low concentrations of sodium dodecyl sulfate could induce the expression of SKALP, a proteinase inhibitor that is not normally expressed in human epidermis but is found in hyperproliferative skin. Sodium dodecyl sulfate caused upregulation of involucrin and downregulation of cytokeratin 1 expression, which is associated with the hyperproliferative/inflammatory epidermal phenotype found in psoriasis, wound healing, and skin irritation. These changes were not induced after treatment of cultures with CTAB, Triton X-100, and Nonidet-P40. This effect appeared to be specific for the class of anionic detergents because sodium dodecyl benzene sulfonate and sodium laurate also induced SKALP expression. These in vitro findings showed only a partial correlation with the potential of different detergents to induce clinical, biophysical, and cell biologic changes in vivo in human skin. Both sodium dodecyl sulfate and CTAB were found to cause induction and upregulation of SKALP and involucrin at low doses following a 24 h patch test, whereas high concentrations of Triton X-100 did not. Sodium dodecyl sulfate induced higher rates of transepidermal water loss, whereas CTAB treated skin showed more signs of cellular toxicity. We conclude that the action of anionic detergents on

  6. Transdifferentiation of adipose-derived stem cells into keratinocyte-like cells: engineering a stratified epidermis.

    Directory of Open Access Journals (Sweden)

    Claudia Chavez-Munoz

    Full Text Available Skin regeneration is an important area of research in the field of tissue-engineering, especially for cases involving loss of massive areas of skin, where current treatments are not capable of inducing permanent satisfying replacements. Human adipose-derived stem cells (ASC have been shown to differentiate in-vitro into both mesenchymal lineages and non-mesenchymal lineages, confirming their transdifferentiation ability. This versatile differentiation potential, coupled with their ease of harvest, places ASC at the advancing front of stem cell-based therapies. In this study, we hypothesized that ASC also have the capacity to transdifferentiate into keratinocyte-like cells and furthermore are able to engineer a stratified epidermis. ASC were successfully isolated from lipoaspirates and cell sorted (FACS. After sorting, ASC were either co-cultured with human keratinocytes or with keratinocyte conditioned media. After a 14-day incubation period, ASC developed a polygonal cobblestone shape characteristic of human keratinocytes. Western blot and q-PCR analysis showed the presence of specific keratinocyte markers including cytokeratin-5, involucrin, filaggrin and stratifin in these keratinocyte-like cells (KLC; these markers were absent in ASC. To further evaluate if KLC were capable of stratification akin to human keratinocytes, ASC were seeded on top of human decellularized dermis and cultured in the presence or absence of EGF and high Ca(2+ concentrations. Histological analysis demonstrated a stratified structure similar to that observed in normal skin when cultured in the presence of EGF and high Ca(2+. Furthermore, immunohistochemical analysis revealed the presence of keratinocyte markers such as involucrin, cytokeratin-5 and cytokeratin-10. In conclusion this study demonstrates for the first time that ASC have the capacity to transdifferentiate into KLC and engineer a stratified epidermis. This study suggests that adipose tissue is potentially a

  7. Comparison of the Transcriptional Profiles of Melanocytes from Dark and Light Skinned Individuals under Basal Conditions and Following Ultraviolet-B Irradiation.

    Science.gov (United States)

    López, Saioa; Smith-Zubiaga, Isabel; García de Galdeano, Alicia; Boyano, María Dolores; García, Oscar; Gardeazábal, Jesús; Martinez-Cadenas, Conrado; Izagirre, Neskuts; de la Rúa, Concepción; Alonso, Santos

    2015-01-01

    We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype.

  8. Comparison of the Transcriptional Profiles of Melanocytes from Dark and Light Skinned Individuals under Basal Conditions and Following Ultraviolet-B Irradiation

    Science.gov (United States)

    López, Saioa; Smith-Zubiaga, Isabel; García de Galdeano, Alicia; Boyano, María Dolores; García, Oscar; Gardeazábal, Jesús; Martinez-Cadenas, Conrado; Izagirre, Neskuts; de la Rúa, Concepción; Alonso, Santos

    2015-01-01

    We analysed the whole-genome transcriptional profile of 6 cell lines of dark melanocytes (DM) and 6 of light melanocytes (LM) at basal conditions and after ultraviolet-B (UVB) radiation at different time points to investigate the mechanisms by which melanocytes protect human skin from the damaging effects of UVB. Further, we assessed the effect of different keratinocyte-conditioned media (KCM+ and KCM-) on melanocytes. Our results suggest that an interaction between ribosomal proteins and the P53 signaling pathway may occur in response to UVB in both DM and LM. We also observed that DM and LM show differentially expressed genes after irradiation, in particular at the first 6h after UVB. These are mainly associated with inflammatory reactions, cell survival or melanoma. Furthermore, the culture with KCM+ compared with KCM- had a noticeable effect on LM. This effect includes the activation of various signaling pathways such as the mTOR pathway, involved in the regulation of cell metabolism, growth, proliferation and survival. Finally, the comparison of the transcriptional profiles between LM and DM under basal conditions, and the application of natural selection tests in human populations allowed us to support the significant evolutionary role of MIF and ATP6V0B in the pigmentary phenotype. PMID:26244334

  9. Radiosensitivity of keratinocytes from tongue and skin; enhanced radioresistance following serial cultivation

    Energy Technology Data Exchange (ETDEWEB)

    Parkinson, E.K.; Hume, W.J.; Potten, C.S.

    1986-01-01

    A study has been carried out of the radiation response of keratinocytes from human skin, mouse skin and mouse tongue to 0-10 Gy of ..gamma..-radiation, carried out in suspension at 20/sup 0/C. The Dsub(o)values for primary cultures of keratinocytes was similar to those obtained in vivo for mice, suggesting that this in vitro assay could be used to measure the sensitivity of keratinocytes treated with various cytotoxic agents. Sensitivity appears to change on subculturing and hence subcultures may be less appropriate for determining in vivo cell sensitivities.

  10. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation

    Science.gov (United States)

    Bennett, Darin C.; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K. K.; McElwee, Kevin J.; Cheng, Kimberly M.

    2015-01-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51 × faster), ostrich oil (1.46 × faster), and rhea oil (1.64 × faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35 × slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  11. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

    Science.gov (United States)

    Bennett, Darin C; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K K; McElwee, Kevin J; Cheng, Kimberly M

    2015-09-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions.

  12. 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.

  13. 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

    2001-01-01

    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 increas

  14. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    Science.gov (United States)

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  15. 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

  16. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals.

    Science.gov (United States)

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

    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.

  17. Cooperative response of keratinocytes and melanocytes to UV radiation during PUVA therapy

    Science.gov (United States)

    Stolnitz, Mikhail M.; Baskakov, Pavel V.; Peshkova, Anna Y.

    1999-03-01

    The mathematical model of processes in UV-irradiated furocoumarin-sensitized epidermis is presented taking into account the mutual influence of keratinocytes and melanocytes populations. The model describes epidermis as a hierarchical structure on tissue (keratinocytes-melanocytes cooperation, melanin screen formation), cellular (proliferation and differentiation, transitions between subpopulations), subcellular (cell movement on mitotic cycle, generation, maturing and migration of melanosomes), and molecular (melanin synthesis, processes of DNA damage and repair, molecular signal transduction) levels.

  18. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Directory of Open Access Journals (Sweden)

    Thomas Zuliani

    Full Text Available Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  19. Specificity in stress response: epidermal keratinocytes exhibit specialized UV-responsive signal transduction pathways.

    Science.gov (United States)

    Adachi, Makoto; Gazel, Alix; Pintucci, Giuseppe; Shuck, Alyssa; Shifteh, Shiva; Ginsburg, Dov; Rao, Laxmi S; Kaneko, Takehiko; Freedberg, Irwin M; Tamaki, Kunihiko; Blumenberg, Miroslav

    2003-10-01

    UV light, a paradigmatic initiator of cell stress, invokes responses that include signal transduction, activation of transcription factors, and changes in gene expression. Consequently, in epidermal keratinocytes, its principal and frequent natural target, UV regulates transcription of a distinctive set of genes. Hypothesizing that UV activates distinctive epidermal signal transduction pathways, we compared the UV-responsive activation of the JNK and NFkappaB pathways in keratinocytes, with the activation of the same pathways by other agents and in other cell types. Using of inhibitors and antisense oligonucleotides, we found that in keratinocytes only UVB/UVC activate JNK, while in other cell types UVA, heat shock, and oxidative stress do as well. Keratinocytes express JNK-1 and JNK-3, which is unexpected because JNK-3 expression is considered brain-specific. In keratinocytes, ERK1, ERK2, and p38 are activated by growth factors, but not by UV. UVB/UVC in keratinocytes activates Elk1 and AP1 exclusively through the JNK pathway. JNKK1 is essential for UVB/UVC activation of JNK in keratinocytes in vitro and in human skin in vivo. In contrast, in HeLa cells, used as a control, crosstalk among signal transduction pathways allows considerable laxity. In parallel, UVB/UVC and TNFalpha activate the NFkappaB pathway via distinct mechanisms, as shown using antisense oligonucleotides targeted against IKKbeta, the active subunit of IKK. This implies a specific UVB/UVC responsive signal transduction pathway independent from other pathways. Our results suggest that in epidermal keratinocytes specific signal transduction pathways respond to UV light. Based on these findings, we propose that the UV light is not a genetic stress response inducer in these cells, but a specific agent to which epidermis developed highly specialized responses.

  20. Functional genomic and radiosensitivity of human keratinocytes: from differentiated to stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamartine, J.; Rachidi, W.; Franco, N.; Lemaitre, G.; Vaigot, P.; Le Minter, P.; Waksman, G.; Martin, M.T. [Evry Univ. Lab. of Genomic and Radiobiology of Keratinocytes, Service de Genomique Fonctionnelle, CEA, 91 (France)

    2006-07-01

    Despite improvements in radiation techniques, patients can still experience radiation toxicity on the skin. Keratinocytes from the basal layer of the epidermis have been long proposed by ICRP as the main target of ionizing radiation in human skin, both for early and late effects of radiation. But the exact roles of these cells in radiation skin damage are still largely unknown., This is why a new program was started to define the radiosensitivity of human keratinocytes according to their differentiation. In a first study was characterized the response of differentiated keratinocytes to low and high doses of gamma radiation (1). To examine whether the response to low doses was different from that induced by high doses, cultured primary keratinocytes isolated from adult normal skin were irradiated with single doses of 1 c Gray or 2 c Gy. A major finding of this study was the identification of an important number of low dose specific genes (140), most of which were modulated at 48 h. Clustering analysis also revealed low dose specific profiles. These results show for the first time that low dose ionizing irradiation is able to induce specific transcriptional responses in human keratinocytes. Then came the part to characterize the radiosensitivity of human basal keratinocytes. The results show for the first time that keratinocytes stem cells from human epidermis are more resistant than proliferative basal keratinocytes. In summary, using cellular biology and functional genomics, we are improving the knowledge on the effects of ionizing radiation on human epidermis, one of the main target tissue of radiation in the human body. (N.C.)

  1. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    OpenAIRE

    Marcella Mauro; Matteo Crosera; Marco Pelin; Chiara Florio; Francesca Bellomo; Gianpiero Adami; Piero Apostoli; Giuseppe Palma; Massimo Bovenzi; Marco Campanini; Francesca Larese Filon

    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 huma...

  2. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Science.gov (United States)

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  3. H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

    OpenAIRE

    Vaughan, Melville B.; Ruben D Ramirez; Andrews, Capri M.; Woodring E Wright; Jerry W Shay

    2009-01-01

    BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in...

  4. Prospective study of vitamins C, E, and A and carotenoids and risk of oral premalignant lesions in men.

    Science.gov (United States)

    Maserejian, Nancy Nairi; Giovannucci, Edward; Rosner, Bernard; Joshipura, Kaumudi

    2007-03-01

    Case-control studies indicate that vitamins C, E, A and carotenoids decrease risk of oral premalignant lesions (OPLs) and oral cancer, but clinical trials have failed to find protective effects of beta-carotene and suggest that vitamin E may increase risk. The authors prospectively evaluated the association between intake of vitamins C, E, A and carotenoids and incidence of OPL. Participants were 42,340 men in the Health Professionals Follow-up Study who provided information on supplement use and diet every 2-4 years by food frequency questionnaire. The authors confirmed 207 clinically or histopathologically diagnosed OPL events occurring between 1986 and 2002 by medical record review. Multivariate-adjusted relative risks (RR) of OPL were calculated with proportional hazards models. Total intake of vitamin C, vitamin A or carotenoids was not significantly associated with OPL risk. Dietary vitamin C was significantly associated with reduced risk (quintile 5 vs. 1, RR = 0.52, 95% CI 0.31-0.85, p(trend) = 0.04), but no association with supplemental vitamin C was observed. Inverse associations were apparent for beta-cryptoxanthin and alpha-carotene intake. No clear relationship emerged with beta-carotene, lycopene or lutein/zeaxanthin. Vitamin E was associated with increased risk (quintile 5 vs. 1, RR = 1.86, 95% CI 1.06-3.19), particularly among current smokers and with supplemental intake (current-smokers, supplement dose tertile 3 vs. 1, RR = 3.07, 95% CI 1.28-7.34, p(trend) = 0.01). For current smokers, beta-carotene also increased risk. Vitamin C from dietary sources, but not supplements, was associated with a reduced risk of OPL. The observed increased risk for current smokers with high vitamin E or beta-carotene intake should be explored further.

  5. Inactivation of SLIT2-ROBO1/2 pathway in premalignant lesions of uterine cervix: clinical and prognostic significances.

    Directory of Open Access Journals (Sweden)

    Sraboni Mitra

    Full Text Available The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30% and ROBO1 (22% followed by ROBO2 (9%. In CACX, alteration of the genes were in the following order: Deletion:ROBO1 (48% > SLIT2 (35% > ROBO2 (33%, Methylation:SLIT2 (34% > ROBO1 (29% > ROBO2 (26%. Overall alterations of SLIT2 and/or ROBO1 (44% and SLIT2 and/or ROBO2 (39% were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥ 5 women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV and early sexual debut (<19 years had worst prognosis. Our data suggests the importance of abrogation of SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease.

  6. Inactivation of SLIT2-ROBO1/2 pathway in premalignant lesions of uterine cervix: clinical and prognostic significances.

    Science.gov (United States)

    Mitra, Sraboni; Mazumder-Indra, Dipanjana; Mondal, Ranajit K; Basu, Partha S; Roy, Anup; Roychoudhury, Susanta; Panda, Chinmay K

    2012-01-01

    The SLIT2-ROBO1/2 pathways control diverse biological processes, including growth regulation. To understand the role of SLIT2 and ROBO1/2 in cervical carcinogenesis, firstly their RNA expression profiles were screened in 21 primary uterine cervical carcinoma (CACX) samples and two CACX cell lines. Highly reduced expressions of these genes were evident. Concomitant alterations [deletion/methylation] of the genes were then analyzed in 23 cervical intraepithelial neoplasia (CIN) and 110 CACX samples. In CIN, SLIT2 was deleted in 22% samples compared to 9% for ROBO1 and none for ROBO2, whereas comparable methylation was observed for both SLIT2 (30%) and ROBO1 (22%) followed by ROBO2 (9%). In CACX, alteration of the genes were in the following order: Deletion:ROBO1 (48%) > SLIT2 (35%) > ROBO2 (33%), Methylation:SLIT2 (34%) > ROBO1 (29%) > ROBO2 (26%). Overall alterations of SLIT2 and/or ROBO1 (44%) and SLIT2 and/or ROBO2 (39%) were high in CIN followed by significant increase in stage I/II tumors, suggesting deregulation of these interactions in premalignant lesions and early invasive tumors. Immunohistochemical analysis of SLIT2 and ROBO1/2 in CACX also showed reduced expression concordant with molecular alterations. Alteration of all these genes predicted poor patient outcome. Multiparous (≥ 5) women with altered SLIT2 and ROBO1 along with advanced tumor stage (III/IV) and early sexual debut (SLIT2-ROBO1 and SLIT2-ROBO2 interactions in the initiation and progression of CACX and also for early diagnosis and prognosis of the disease.

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

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    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

  8. The expression of P63 protein in some keratinocyte original tissues and cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective:To examine the expression patterns of p63 in tissues of particular keratinocyte original hyperproliferate diseases and variety cell types for determining if P63 is the marker of proliferative potential keratinocytes.Methods:P63 protein Was detected and analyzed by immunoreacdvity method and Western blot in biopsy specimens of keratinocyte original disorders including squamous cell carcinomas SCC,basal cell carcinomas BCC,Bowen's disease and other tissues or cells,such as psoriasis vulgaris,normal skin tissues,primary cultured keratinocytes,immortal HaCaT cells,and epidermoid carcinoma cells A431.Results:P63 protein was expressed in the nuclei of basal and suprabasal layer of the epidermis,germinative cells of sebaceous glands in normal epidermal.P63 was strongly and diffusely detected in the majority of tumor cells in BCC and poorly-differentiated SCC.In Bowen's disease,p63expresses are remarkable in all cell layers.In the psoriasis plaque epidermal,p63 expressed mainly in basal cells and part of spinous cells.P63 expressed more strongly in primary cultured keratinocytes than in A431 cells or HaCaT cells.Conclusion:P63 is a nuclei marker of undifferentiated keratinocytes with the proliferative potential and may disrupt the terminal differentiation.The overexpression of p63 reflects immaturity of the tumor cells.The immunohistochemical staining of p63 may be useful for investigating the origin and differentiation of tumor cells.

  9. Vitamin D signaling regulates oral keratinocyte proliferation in vitro and in vivo.

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    Yuan, Feng-Ning F; Valiyaparambil, Jayasanker; Woods, Michael C; Tran, Huy; Pant, Rima; Adams, John S; Mallya, Sanjay M

    2014-05-01

    The secosteroidal hormone 1,25-dihyroxyvitamin D [1,25(OH)(2)D(3)] and its receptor, the vitamin D receptor (VDR), are crucial regulators of epidermal proliferation and differentiation. However, the effects of 1,25(OH)(2)D(3)-directed signaling on oral keratinocyte pathophysiology have not been well studied. We examined the role of 1,25(OH)(2)D(3) in regulating proliferation and differentiation in cultured oral keratinocytes and on the oral epithelium in vivo. Using lentiviral-mediated shRNA to silence VDR, we generated an oral keratinocyte cell line with stable knockdown of VDR expression. VDR knockdown significantly enhanced proliferation and disrupted calcium- and 1,25(OH)(2)D(3)-induced oral keratinocyte differentiation, emphasizing the anti-proliferative and pro-differentiation effects of 1,25(OH)(2)D(3) in oral keratinocytes. Using vitamin D(3)-deficient diets, we induced chronic vitamin D deficiency in mice as evidenced by decreased serum 25-hydroxyvitamin D (25OHD) concentrations. The vitamin D-deficient mice manifested increased proliferation of the tongue epithelium, but did not develop any morphological or histological abnormalities in the oral epithelium, suggesting that vitamin D deficiency alone is insufficient to alter oral epithelial homeostasis and provoke carcinogenesis. Immunohistochemical analyses of human and murine oral squamous cell carcinomas showed increased VDR expression. Overall, our results provide strong support for a crucial role for vitamin D signaling in oral keratinocyte pathophysiology.

  10. Nerve allograft regeneration and immunological tolerance of rats with sciatic nerve transplantation after intragastric pretreatment with ultraviolet B-irradiated donor splenocytes

    Institute of Scientific and Technical Information of China (English)

    Rui Mi; Qiang Liu; Dou Wu

    2008-01-01

    BACKGROUND: Nerve allograft rejection is an unavoidable problem for nerve allografts. Traumatic peripheral nerve injuries are commonly reconstructed using autogenous nerve grafts. However, this form of reconstruction is limited by insufficient autologous nerves for large gap repairs and by morbidity at the nerve donor site.OBJECTIVE: To examine sciatic nerve regeneration and immune tolerance reaction after intragastric administration of ultraviolet B-irradiated (UVB) donor splenocytes.DESIGN, TIME AND SETTING: A complete randomized grouping design and controlled experiment. The experiments were conducted in the Department of Orthopedics, the First Affiliated Hospital to Shanxi Medical University, China, between March and October 2007.MATERIALS: Fourteen adult male SD rats and fourteen male Wistar rats, weighing 250-300g, were randomly matched as donors and acceptors. A further seven male SD rats (weight 250-300g, age 12-16 weeks) were used for nerve isografts. Immune preparations and the Epics XL flow cytometer were purchased from B-D Company, USA. A computer-assisted electromyograph machine was provided by Keypoint P, Dantel Company, Denmark.METHODS: Splenocytes from Wistar rats were isolated, purified, and cultured, and then irradiated with ultraviolet B. In the first control group (Group 1), the SD rats received a syngeneic SD nerve isograft. In the second control group (Group 2), the SD rats received a nerve allograft from Wistar rats without pretreatment. In the oral-tolerance treated group (Group 3), the SD recipient rats were inoculated with 2.5×107 Lewis UVB-irradiated donor splenocyte cells by intestinal tract administration at seven days before transplantation.MAIN OUTCOME MEASURES: (1) The recent end and the middle and distal end of the transplanted nerve were cut at 8 and 12 weeks after operation. Recovery of nerve regeneration was measured with HE staining using the total number, density, and diameter of the nerve fibers. (2) The level of CD25+T

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

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    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.

  12. Prevalence of human papillomavirus in archival samples obtained from patients with cervical pre-malignant and malignant lesions from Northeast Brazil

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    Prado José CM

    2010-04-01

    Full Text Available Abstract Background Human Papillomavirus (HPV is considered as a necessary, but not sufficient, cause of cervical cancer. In this study, we aimed to assess the prevalence of HPV in a series of pre-malignant and malignant cervical lesion cases, to identify the virus genotypes, and to assess their distribution pattern according to lesion type, age range, and other considered variables. The samples were submitted to histopathological revision examination and analysed by polymerase chain reaction (PCR for the presence of HPV DNA, followed by HPV typing by dot blot hybridisation. Findings Of the analysed samples, 53.7% showed pre-malignant cervical lesions, and 46.3% presented with cervical cancer. Most cancer samples (84.1% were classified as invasive carcinoma. The mean age of these cancer patients was 47.3 years. The overall HPV prevalence was 82.4% in patients with pre-malignant lesions and 92.0% in the cancer patients. HPV 16 was the most prevalent type, followed by HPV 18 and 58, including both single and double infections. Double infection was detected in 11.6% of the samples, and the most common combination was HPV 16+18. Conclusions Cervical cancer appears to occur in women in a lower age range in the studied area, compared to the situation in other Brazilian regions. Furthermore, among the patients with CIN 3 and those with cancer, we observed a higher proportion of married women, women with more than one sexual partner, smokers, and individuals with less than an elementary education, relative to their counterparts. Findings The overall HPV prevalence was 82.4% in patients with pre-malignant lesions and 92.0% in the cervical cancer patients from Northeast Brazil. HPV 16 was the most prevalent type, followed by HPV 18 and 58. The most common double infection was HPV 16+18. Cervical cancer appears to occur in women in a lower age range in the Northeast Brazil. Among the patients with CIN 3 and those with cancer, we observed a higher

  13. Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

    Science.gov (United States)

    Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

    2014-04-01

    The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

  14. Keratinocyte growth factor (KGF) is required for postnatal thymic regeneration.

    Science.gov (United States)

    Alpdogan, Onder; Hubbard, Vanessa M; Smith, Odette M; Patel, Neel; Lu, Sydney; Goldberg, Gabrielle L; Gray, Daniel H; Feinman, Jared; Kochman, Adam A; Eng, Jeffrey M; Suh, David; Muriglan, Stephanie J; Boyd, Richard L; van den Brink, Marcel R M

    2006-03-15

    Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that mediates epithelial cell proliferation and differentiation in a variety of tissues, including the thymus. We studied the role of KGF in T-cell development with KGF-/- mice and demonstrated that thymic cellularity and the distribution of thymocyte subsets among KGF-/-, wildtype (WT), and KGF+/- mice were similar. However, KGF-/- mice are more vulnerable to sublethal irradiation (450 cGy), and a significant decrease was found in thymic cellularity after irradiation. Defective thymopoiesis and peripheral T-cell reconstitution were found in KGF-/- recipients of syngeneic or allogeneic bone marrow transplant, but using KGF-/- mice as a donor did not affect T-cell development after transplantation. Despite causing an early developmental block in the thymus, administration of KGF to young and old mice enhanced thymopoiesis. Exogenous KGF also accelerated thymic recovery after irradiation, cyclophosphamide, and dexamethasone treatment. Finally, we found that administering KGF before bone marrow transplantation (BMT) resulted in enhanced thymopoiesis and peripheral T-cell numbers in middle-aged recipients of an allogeneic BM transplant. We conclude that KGF plays a critical role in postnatal thymic regeneration and may be useful in treating immune deficiency conditions.

  15. Keratinocyte gene therapy for systemic diseases. Circulating interleukin 10 released from gene-transferred keratinocytes inhibits contact hypersensitivity at distant areas of the skin.

    Science.gov (United States)

    Meng, X; Sawamura, D; Tamai, K; Hanada, K; Ishida, H; Hashimoto, I

    1998-01-01

    This study has examined the systemic effects of a circulating gene product, human interleukin 10 (IL-10), released from transduced keratinocytes. IL-10 is an anti-inflammatory cytokine which has an inhibitory effect on contact hypersensitivity (CHS). An expression vector (phIL-10) was constructed for human IL-10 and was injected into the dorsal skin of hairless rats. Local expression of IL-10 mRNA and protein was detected by reverse-transcriptase polymerase chain reaction and immunohistochemical staining, respectively. Enzyme-linked immunosorbent assay showed that the amount of IL-10 in the local keratinocytes and in the circulation increased with the dose of phIL-10 transferred. To determine whether circulating IL-10 could inhibit the effector phase of CHS at a distant area of the skin, various doses of phIL-10 were injected into the dorsal skin of sensitized rats before challenge on the ears. Our results showed that the degree of swelling of the ears of phIL-10- treated rats was significantly lower than that in the negative control animals. These results suggest that IL-10 released from transduced keratinocytes can enter the bloodstream and cause biological effects at distant areas of the skin. This study demonstrates that it may be possible to treat systemic disease using keratinocyte gene therapy. PMID:9502789

  16. 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.

  17. Remifentanil protects human keratinocytes against hypoxia-reoxygenation injury through activation of autophagy.

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    Jae-Young Kwon

    Full Text Available The proliferation, differentiation, and migration of keratinocytes are essential in the early stages of wound healing. Hypoxia-Reoxygenation (H/R injury to keratinocytes can occur in various stressful environments such as surgery, trauma, and various forms of ulcers. The effects of remifentanil on human keratinocytes under hypoxia-reoxygenation have not been fully studied. Therefore, we investigated the effects of remifentanil on the proliferation, apoptosis, and autophagic activation of human keratinocytes during hypoxic-reoxygenation. Human keratinocytes were cultured under 1% oxygen tension for 24 h. The cells were then treated with various concentrations of remifentanil (0.01, 0.1, 0.5, and 1 ng/mL for 2 h. Thereafter, the cells were reoxygenated for 12 h at 37°C. We measured cell viability via MTT assay. Using quantitative real-time PCR and western blot analysis, we measured the expression levels of proteins associated with apoptosis and autophagy. Quantification of apoptotic cells was performed using flow cytometer analysis and autophagic vacuoles were observed under a fluorescence microscope. Remifentanil treatment brought about an increase in the proliferation of human keratinocytes damaged by hypoxia-reoxygenation and decreased the apoptotic cell death, enhancing autophagic activity. However, the autophagy pathway inhibitor 3-MA inhibited the protective effect of remifentanil in hypoxia-reoxygenation injury. In conclusion, the current study demonstrated that remifentanil treatment stimulated autophagy and reduced apoptotic cell death in a hypoxia-reoxygenation model of human keratinocytes. Our results provide additional insights into the relationship between apoptosis and autophagy.

  18. Cytokine secretion profiles of human keratinocytes during Trichophyton tonsurans and Arthroderma benhamiae infections.

    Science.gov (United States)

    Shiraki, Yumi; Ishibashi, Yoshio; Hiruma, Masataro; Nishikawa, Akemi; Ikeda, Shigaku

    2006-09-01

    Dermatophytes cause intractable superficial infections in humans. Arthroderma benhamiae, a zoophilic dermatophyte, triggers severe inflammatory responses in humans, while Trichophyton tonsurans, an anthropophilic dermatophyte, triggers minimal ones. Cytokines and other factors derived from keratinocytes play important roles in inflammatory and immune responses in the skin. The authors performed an in vitro investigation to determine the human keratinocyte cytokine profiles during dermatophyte infection. The human keratinocyte cell line PHK16-0b was infected with A. benhamiae or T. tonsurans for 24 h, and the cytokines secreted were analysed using a human cytokine antibody array. Marked differences were observed in the cytokine profiles of the cells infected with the two dermatophytes. A. benhamiae infection resulted in the secretion of a broad spectrum of cytokines, including proinflammatory cytokines, chemokines, and immunomodulatory cytokines. In contrast, T. tonsurans-infected keratinocytes secreted only limited cytokines, including eotaxin-2, interleukin (IL)-8 and IL-16. cDNA microarray analysis confirmed that A. benhamiae infection upregulated genes encoding IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-13, IL-15, IL-16, IL-17 and interferon (IFN)-gamma, while T. tonsurans infection upregulated only a few genes, such as those encoding IL-1beta and IL-16. RT-PCR demonstrated that infection by both dermatophytes enhanced IL-8 mRNA expression in keratinocytes. These results suggest that A. benhamiae-induced secretion of several cytokines from keratinocytes may be involved in a severe inflammatory response, and that the limited cytokine secretion from keratinocytes in response to T. tonsurans infection may result in a minimal inflammatory response in the skin. These cytokine profiles may aid in proving the clinical features of dermatophytosis.

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

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    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

  20. Co-overexpression of bcl-2 and c-myc in uterine cervix carcinomas and premalignant lesions

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    Z. Protrka

    2011-03-01

    Full Text Available To establish the role of co-overexpression of bcl-2 and c-myc protooncogenes in uterine cervix carcinogenesis, we examined 138 tissue samples of low grade cervical squamous intraepithelial lesions (SIL, high grade SIL, portio vaginalis uteri (PVU carcinoma in situ and PVU carcinoma invasive, stage IA-IIA (study group and 36 samples without SIL or malignancy (control group. The expression of bcl-2 and c-myc was detected immunohistochemically using a monoclonal antibody. Fisher’s exact test (P<0.05 was used to assess statistical significance. Overexpression of bcl-2 was found to increase in direct relation to the grade of the cervical lesions. High sensitivity was of great diagnostic significance for the detection of these types of changes in the uterine cervix. On the basis of high predictive values it can be said that in patients with bcl-2 overexpression there is a great possibility that they have premalignant or malignant changes in the uterine cervix. Co-overexpression of bcl-2 and c-myc oncogenes was found only in patients with PVU invasive carcinoma (6/26-23.0%. Statistically significant difference was not found in the frequency of co-overexpression in patients with PVU invasive carcinoma in relation to the control group (Fisher’s test; P=0.064. The method's sensitivity of determining these oncogenes with the aim of detecting PVU invasive carcinoma was 23%, while specificity was 72.2%. On the basis of high predictive values (100%, speaking in statistical terms, it can be concluded that all patients with co-overexpression of bcl-2 and c-myc oncogenes will have PVU invasive carcinoma. We confirmed in our research that co-overexpression of bcl-2 and c-myc oncogenes was increased only in PVU invasive carcinoma. However, a more extensive series of samples and additional tests are required to establish the prognostic significance of bcl-2 and c-myc co-overexpression in cervical carcinogenesis.

  1. Dose-response relationships of oral habits associated with the risk of oral pre-malignant lesions among men who chew betel quid.

    Science.gov (United States)

    Yen, Amy Ming-Fang; Chen, Shao-Ching; Chen, Tony Hsiu-Hsi

    2007-08-01

    Betel quid, cigarettes and alcohol are well-recognized risk factors for oral cancer. However, the combined effect of the frequency and duration of these oral habits on the risk for developing oral pre-malignancies among betel quid users has not been fully addressed. In this study, an oral screening programme for men chewing betel quid was carried out by well-trained dentists for early detection of oral pre-malignancy lesions. Using generalized logit model and proportional odds model, we found that, compared with the occasional user, the adjusted odds ratios of developing leukoplakia for men chewing one to 10 pieces of betel quid, 11-20 pieces, and more than 20 pieces per day were estimated as 2.14 (95% confidence interval [CI] 1.62-2.81), 2.99 (95% CI 2.06-4.27), and 5.37 (95% CI 3.76-7.47), respectively. The corresponding figures for erythroleukoplakia were 3.69 (95% CI 1.55-8.79), 13.78 (95% CI 5.76-32.98), and 36.64 (95% CI 15.94-84.16), respectively. Similar results were found while the duration was considered. The dose-response relationships were not as noteworthy for cigarette and alcohol drinking.

  2. Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

    Science.gov (United States)

    Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N

    2016-05-01

    Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM.

  3. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    Science.gov (United States)

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization.

  4. Improvement of human keratinocyte migration by a redox active bioelectric dressing.

    Directory of Open Access Journals (Sweden)

    Jaideep Banerjee

    Full Text Available Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.

  5. Improvement of human keratinocyte migration by a redox active bioelectric dressing.

    Science.gov (United States)

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K; Bellman, Karen; Dickinson, Bryan C; Suri, Prerna; Subramaniam, Vish V; Chang, Christopher J; Sen, Chandan K

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.

  6. 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.

  7. A Glutathione-Nrf2-Thioredoxin Cross-Talk Ensures Keratinocyte Survival and Efficient Wound Repair.

    Directory of Open Access Journals (Sweden)

    Michèle Telorack

    2016-01-01

    Full Text Available The tripeptide glutathione is the most abundant cellular antioxidant with high medical relevance, and it is also required as a co-factor for various enzymes involved in the detoxification of reactive oxygen species and toxic compounds. However, its cell-type specific functions and its interaction with other cytoprotective molecules are largely unknown. Using a combination of mouse genetics, functional cell biology and pharmacology, we unraveled the function of glutathione in keratinocytes and its cross-talk with other antioxidant defense systems. Mice with keratinocyte-specific deficiency in glutamate cysteine ligase, which catalyzes the rate-limiting step in glutathione biosynthesis, showed a strong reduction in keratinocyte viability in vitro and in the skin in vivo. The cells died predominantly by apoptosis, but also showed features of ferroptosis and necroptosis. The increased cell death was associated with increased levels of reactive oxygen and nitrogen species, which caused DNA and mitochondrial damage. However, epidermal architecture, and even healing of excisional skin wounds were only mildly affected in the mutant mice. The cytoprotective transcription factor Nrf2 was strongly activated in glutathione-deficient keratinocytes, but additional loss of Nrf2 did not aggravate the phenotype, demonstrating that the cytoprotective effect of Nrf2 is glutathione dependent. However, we show that deficiency in glutathione biosynthesis is efficiently compensated in keratinocytes by the cysteine/cystine and thioredoxin systems. Therefore, our study highlights a remarkable antioxidant capacity of the epidermis that ensures skin integrity and efficient wound healing.

  8. 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.

  9. Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes.

    Science.gov (United States)

    Hung, Sandy S C; Pébay, Alice; Wong, Raymond C B

    2015-08-20

    Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

  10. Human Keratinocyte Growth and Differentiation on Acellular Porcine Dermal Matrix in relation to Wound Healing Potential

    Directory of Open Access Journals (Sweden)

    Robert Zajicek

    2012-01-01

    Full Text Available A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7–10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs, CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.

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

    Science.gov (United States)

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-01

    The effects and the underlying mechanisms of hydrogen sulfide (H2S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H2S 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 H2S) 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 H2S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation.

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

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    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.

  13. 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.

  14. 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.

  15. Study of HLA-DR synthesis in cultured human keratinocytes.

    Science.gov (United States)

    Wikner, N E; Huff, J C; Norris, D A; Boyce, S T; Cary, M; Kissinger, M; Weston, W L

    1986-11-01

    Within the normal human epidermis only Langerhans and indeterminate cells express HLA-DR. Human keratinocytes (HK), however, may also express HLA-DR in certain disease states characterized by mononuclear cell infiltrates. Previous studies have shown that HK synthesize HLA-DR in response to stimulation by interferon gamma (INF-gamma). The purposes of this study were to define conditions under which cultured HK might express HLA-DR and to compare the HLA-DR synthesis of HK with that of monocytes. HLA-DR expression by HK as determined by indirect immunofluorescence of HK cultures was absent under standard low calcium conditions and remained absent with the addition of calcium, serum, mitogens, and supernatants from Pam-212 cells containing epidermal thymocyte-activating factor. HLA-DR expression in HK was induced by cocultivation with concanavalin A-stimulated peripheral blood mononuclear cells (PBMC), but not unstimulated PBMC. This effect was time-dependent and directly related to the number of PBMC. HLA-DR expression was also induced in a time- and dose-dependent manner by addition of supernatant from stimulated PBMC (SS) or by addition of recombinant INF-gamma but not by addition of interleukin (IL)-1 or IL-2. Induction by either SS or INF-gamma was blocked by an antiserum to INF-gamma. As determined by a semiquantitative immunoprecipitation technique, HLA-DR synthesis by HK was directly related to INF-gamma concentration. The pattern of HLA-DR peptides produced by HK was similar to that of monocytes, but the relative quantity synthesized was far less than that of monocytes.

  16. 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 (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 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 TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) 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 TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-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 H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2.

  17. 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

    2013-06-07

    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.

  18. Enhancing effect of tazarotene on the HLA-DR expression of cultured human keratinocytes induced by interferon-gamma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-gan; TAN Sheng-shun

    2005-01-01

    Objective: To investigate the effect of tazarotene on the expression of HLA-DR induced by IFN-γ. Methods: (1) Keratinocytes from normal human skin were cultured in vitro;(2) Tazarotene, IFN-γ and the combination of the two compounds were incubated with the keratinocytes in medium, respectively. The expression of HLA-DR in keratinocytes was determined using immunocytochemistry techniques at 24h after incubation. Results: (1) There was rare expression of HLA-DR in normal human keratinocytes; (2) 10-6mol/L tazarotene failed to induce the expression of HLA-DR in keratinocytes at 24h after incubation; (3) 500 U/ml IFN-γ obviously induced the HLA-DR expression in keratinocytes at 24h after treatment; (4) After 24h, 10-7-10-5 mol/L tazarotene had a significantly enhancing effect on the expression of HLA-DR induced by IFN-γ (P<0.005). Conclusion: Tazarotene up-regulates the expression of HLA-DR in keratinocytes cultured in vitro when combined with IFN-γ . Therefore, the reduction of HLA-DR positive keratinocytes in psoriatic lesions may be attributed to not direct interaction of tazarotene in combination with IFN-γ but other pathways.

  19. The transcriptional coactivator DRIP/mediator complex is involved in vitamin D receptor function and regulates keratinocyte proliferation and differentiation.

    Science.gov (United States)

    Oda, Yuko; Chalkley, Robert J; Burlingame, Alma L; Bikle, Daniel D

    2010-10-01

    Mediator is a multisubunit coactivator complex that facilitates transcription of nuclear receptors. We investigated the role of the mediator complex as a coactivator for vitamin D receptor (VDR) in keratinocytes. Using VDR affinity beads, the vitamin D receptor interacting protein (DRIP)/mediator complex was purified from primary keratinocytes, and its subunit composition was determined by mass spectrometry. The complex included core subunits, such as DRIP205/MED1 (MED1), that directly binds to VDR. Additional subunits were identified that are components of the RNA polymerase II complex. The functions of different mediator components were investigated by silencing its subunits. The core subunit MED1 facilitates VDR activity and regulating keratinocyte proliferation and differentiation. A newly described subunit MED21 also has a role in promoting keratinocyte proliferation and differentiation, whereas MED10 has an inhibitory role. Blocking MED1/MED21 expression caused hyperproliferation of keratinocytes, accompanied by increases in mRNA expression of the cell cycle regulator cyclin D1 and/or glioma-associated oncogene homolog. Blocking MED1 or MED21 expression also resulted in defects in calcium-induced keratinocyte differentiation, as indicated by decreased expression of differentiation markers and decreased translocation of E-cadherin to the membrane. These results show that keratinocytes use the transcriptional coactivator mediator to regulate VDR functions and control keratinocyte proliferation and differentiation.

  20. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    Science.gov (United States)

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  1. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    Science.gov (United States)

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death.

  2. A modeling approach to study the effect of cell polarization on keratinocyte migration.

    Directory of Open Access Journals (Sweden)

    Matthias Jörg Fuhr

    Full Text Available The skin forms an efficient barrier against the environment, and rapid cutaneous wound healing after injury is therefore essential. Healing of the uppermost layer of the skin, the epidermis, involves collective migration of keratinocytes, which requires coordinated polarization of the cells. To study this process, we developed a model that allows analysis of live-cell images of migrating keratinocytes in culture based on a small number of parameters, including the radius of the cells, their mass and their polarization. This computational approach allowed the analysis of cell migration at the front of the wound and a reliable identification and quantification of the impaired polarization and migration of keratinocytes from mice lacking fibroblast growth factors 1 and 2--an established model of impaired healing. Therefore, our modeling approach is suitable for large-scale analysis of migration phenotypes of cells with specific genetic defects or upon treatment with different pharmacological agents.

  3. Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK).

    Science.gov (United States)

    Manni, Vanessa; Lisi, Antonella; Rieti, Sabrina; Serafino, Annalucia; Ledda, Mario; Giuliani, Livio; Sacco, Donatella; D'Emilia, Enrico; Grimaldi, Settimio

    2004-02-01

    This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.

  4. Vaccinia virus induces rapid necrosis in keratinocytes by a STAT3-dependent mechanism.

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    Yong He

    Full Text Available Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.Mice treated topically with a STAT3 inhibitor (Stattic developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.

  5. 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.

  6. 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

  7. Establishment of a novel method for primary culture of normal human cervical keratinocytes

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-zhen; L(U) Xiu-ping; PAN Zi-xuan; ZHANG Wei; CHEN Zhao-ri; WANG Hui; LIU Hua

    2013-01-01

    Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS).However,it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor.Therefore,the culture of these cells is extremely difficult.In this study,we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.Methods Normal cervical epithelial tissue pieces were obtained and digested with type Ⅰ collagenase to dissociate the cells and a single cell suspension produced.The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type Ⅰ from rat tail,with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS.After attachment,the medium were replaced with K-SFM without FBS.The expression of basal keratins of the ectocervical epithelium,K5,K14 and K19 were assayed by immunofiuorescence with monoclonal antibodies to identify the cell purity.Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5%FBS than in K-SFM alone,as well as to tissue culture plastic coated with collagen type Ⅰ than plastic alone.The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type Ⅰ from rat tail was the best method for culture of normal cervical epithelial cells.K5,K14 and K19 were assayed and keratinocyte purity was nearly 100%.Conclusion A novel,simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

  8. Investigation on etretin effects on expression of Fas/FasL ligand and apoptosis in cultured human keratinocytes

    Institute of Scientific and Technical Information of China (English)

    Ping Liu; Shunsheng Tan; Yanping Xi; Zhenping Cao

    2005-01-01

    Objective: To further illuminate a possibme mechanism of Fas/FasL in the treatment of psoriasis, the expression of it and apoptosis of KC were investigated. Methods: With cell culture,immunocytochemistry, the expression of Fas/FasL protein after the treatment with etretin was observed in cultured human normal keratinocytes. Then, the state of apoptosis in cultured keratinocyte after stimuwasn't involved in apoptosis in cultured normol human keratinocytes. But during limited period, the apoptosis of KC could be induced by etretin, thus it can antagonize benign proliferate of keratinocytes. Our data showed up-regulation of the expression of Fas/FasL and apoptosis in cultured human keratinocytes stimulated by etretin, and its function may be involved in the therapeutic machanism of psoriasis.

  9. KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Objective: To establish two stably KGF-transfected, immortalized cell lines. Methods: HaCaT-keratinocytes and KMST-6-fibroblasts were transfected by liposome mediated gene transfer. Transfection effectivity, gene integration and configuration of the transgenic protein were investigated by ELISA, DANN-PCR and β-Gal-staining. Results: Most effective GF producing clones were tested by a colorimetric XTT-test. Conclusion: This is a significant acceleration of cell proliferation and mitosis of human keratinocytes in an Air Liquid Interface (ALI) test system.

  10. [Cultured epidermis in the treatment of leg ulcer: "edge effect" and correlation with keratinocyte proliferation index].

    Science.gov (United States)

    Giannotti, V; Pimpinelli, N; Mariotti, V; Borgognoni, L; Reali, U M

    1990-04-01

    Cultured keratinocyte grafting (KG) of chronic leg ulcers produces an impressive stimulation of host epithelization by an effect on the edge of the ulcer, which starts to grow in rapidly ("edge effect"). In 5 patients with chronic leg ulcers treated by KG, we have studied the proliferation index of the epidermis of the edge and of the graft area and that of secondary culture keratinocytes. The aim of the study was to correlate this parameter with the clinical evolution of the treated lesions. We found a significant correlation between the proliferation index of the edge epidermis and the clinical evidence of "edge effect".

  11. High glucose inhibits ClC-2 chloride channels and attenuates cell migration of rat keratinocytes

    Directory of Open Access Journals (Sweden)

    Pan F

    2015-08-01

    Full Text Available Fuqiang Pan, Rui Guo, Wenguang Cheng, Linlin Chai, Wenping Wang, Chuan Cao, Shirong LiDepartment of Plastic and Reconstructive Surgery, Southwestern Hospital, Third Military Medical University, Chongqing, People’s Republic of China Background: Accumulating evidence has demonstrated that migration of keratinocytes is critical to wound epithelialization, and defects of this function result in chronic delayed-healing wounds in diabetes mellitus patients, and the migration has been proved to be associated with volume-activated chloride channels. The aim of the study is to investigate the effects of high glucose (HG, 25 mM on ClC-2 chloride channels and cell migration of keratinocytes.Methods: Newborn Sprague Dawley rats were used to isolate and culture the keratinocyte in this study. Immunofluorescence assay, real-time polymerase chain reaction, and Western blot assay were used to examine the expression of ClC-2 protein or mRNA. Scratch wound assay was used to measure the migratory ability of keratinocytes. Transwell cell migration assay was used to measure the invasion and migration of keratinocytes. Recombinant lentivirus vectors were established and transducted to keratinocytes. Whole-cell patch clamp was used to perform the electrophysiological studies.Results: We found that the expression of ClC-2 was significantly inhibited when keratinocytes were exposed to a HG (25 mM medium, accompanied by the decline of volume-activated Cl- current (ICl,vol, migration potential, and phosphorylated PI3K as compared to control group. When knockdown of ClC-2 by RNAi or pretreatment with wortmannin, similar results were observed, including ICl,vol and migration keratinocytes were inhibited.Conclusion: Our study proved that HG inhibited ClC-2 chloride channels and attenuated cell migration of rat keratinocytes via inhibiting PI3K signaling.Keywords: high glucose, keratinocytes, ClC-2, cell migration, PI3K

  12. Analysis of Sensitivity, Specificity, and Positive and Negative Predictive Values of Smear and Colposcopy in Diagnosis of Premalignant and Malignant Cervical Lesions.

    Science.gov (United States)

    Barut, Mert Ulaş; Kale, Ahmet; Kuyumcuoğlu, Umur; Bozkurt, Murat; Ağaçayak, Elif; Özekinci, Server; Gül, Talip

    2015-12-10

    BACKGROUND This study aimed to examine the positive and negative predictive value in the diagnosis of premalignant and malignant lesions of cervical colposcopy, the sensitivity and specificity of smear, and to evaluate the correlation with histopathology of abnormal cytology and colposcopy. MATERIAL AND METHODS The criteria for inclusion of patients with unhealthy cervix in the study were: Erosion, Chronic cervicitis, and Healed lacerations, Hypertrophied cervix, bleeding on touch, suspicious growth/ulcer/polyp on the cervix, and abnormal discharges from the cervix. Women with frank carcinoma cervix, pregnant females, patients with bleeding per vaginum at the time of examination, and those who had used vaginal medications, vaginal contraceptives or douches in the last 48 h of examination were excluded from the study. Demographic analysis was performed for 450 patients who were admitted to the clinic. Sensitivity, specificity, positive predictive value and negative predictive values of patients to identify cervical pathologies of smear and colposcopy were histopathologically calculated. The statistical software package SPSS 15.0 (SPSS Inc., Chicago, IL, USA) and Spearman's and Chi-Square tests were used for statistical analysis. RESULTS Sensitivity, specificity, PPD and NDP of smear were 0.57%, 0.76%, 0.26%, 0.92% respectively. Sensitivity, specificity, PPD and NDP of colposcopy were 0.92%, 0.67%, 0.52%, 0.96% respectively. A statistically significant correlation was found between abnormal cytology and histopathology, and abnormal colposcopy finding and histopathology. CONCLUSIONS Women with clinical diagnosis of unhealthy cervix should be evaluated by cytology to detect any premalignant or malignant lesions. It was concluded that Pap smear, colposcopy and histopathology should be collectively evaluated to evaluate cervical findings in low socio-economic regions.

  13. Melanocytes and keratinocytes have distinct and shared responses to ultraviolet radiation and arsenic.

    Science.gov (United States)

    Cooper, K L; Yager, J W; Hudson, L G

    2014-01-30

    The rise of melanoma incidence in the United States is a growing public health concern. A limited number of epidemiology studies suggest an association between arsenic levels and melanoma risk. Arsenic acts as a co-carcinogen with ultraviolet radiation (UVR) for the development of squamous cell carcinoma and proposed mechanisms include generation of oxidative stress by arsenic and UVR and inhibition of UVR-induced DNA repair by arsenic. In this study, we investigate similarities and differences in response to arsenic and UVR in keratinocytes and melanocytes. Normal melanocytes are markedly more resistant to UVR-induced cytotoxicity than normal keratinocytes, but both cell types are equally sensitive to arsenite. Melanocytes were more resistant to arsenite and UVR stimulation of superoxide production than keratinocytes, but the concentration of arsenite necessary to inhibit the activity of the DNA repair protein poly(ADP-ribose)polymerase and enhance retention of UVR-induced DNA damage was essentially equivalent in both cell types. These findings suggest that although melanocytes are less sensitive than keratinocytes to initial UVR-mediated DNA damage, both of these important target cells in the skin share a mechanism related to arsenic inhibition of DNA repair. These findings suggest that concurrent chronic arsenic exposure could promote retention of unrepaired DNA damage in melanocytes and act as a co-carcinogen in melanoma.

  14. Activated protein C: A regulator of human skin epidermal keratinocyte function

    Institute of Scientific and Technical Information of China (English)

    Kelly; McKelvey; Christopher; John; Jackson; Meilang; Xue

    2014-01-01

    Activated protein C(APC) is a physiological anticoagulant, derived from its precursor protein C(PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor(EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC’s function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

  15. Influence of different buffers (HEPES/MOPS) on keratinocyte cell viability and microbial growth.

    Science.gov (United States)

    Dias, Kássia de Carvalho; Barbugli, Paula Aboud; Vergani, Carlos Eduardo

    2016-06-01

    This study assessed the effect of the buffers 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 3-(N-morpholino) propanesulfonic acid (MOPS) on keratinocyte cell viability and microbial growth. It was observed that RPMI buffered with HEPES, supplemented with l-glutamine and sodium bicarbonate, can be used as a more suitable medium to promote co-culture.

  16. 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.

  17. Cdc42 expression in keratinocytes is required for the maintenance of the basement membrane in skin

    DEFF Research Database (Denmark)

    Wu, Xunwei; Quondamatteo, Fabio; Brakebusch, Cord

    2006-01-01

    , structure and number of hemidesomosomes were not significantly changed in the Cdc42 mutant skin compared with the control mice and no blister formation was observed in mutant skin. These data indicate that Cdc42 in keratinocytes is important for maintenance of the basement membrane of skin....

  18. Development of a full-thickness human gingiva equivalent constructed from immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    J.K. Buskermolen; C.M.A. Reijnders; S.W. Spiekstra; T. Steinberg; C.J. Kleverlaan; A.J. Feilzer; A.D. Bakker; S. Gibbs

    2016-01-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines

  19. Activated protein C: A regulator of human skin epidermal keratinocyte function.

    Science.gov (United States)

    McKelvey, Kelly; Jackson, Christopher John; Xue, Meilang

    2014-05-26

    Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

  20. 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...

  1. The Parkinsonism-associated protein DJ-1/Park7 prevents glycation damage in human keratinocyte.

    Science.gov (United States)

    Advedissian, Tamara; Deshayes, Frédérique; Poirier, Françoise; Viguier, Mireille; Richarme, Gilbert

    2016-04-22

    Reducing sugars and dicarbonyls form covalent adducts with proteins through a nonenzymatic process known as glycation, which inactivates proteins, is increased in diabetic patients and is associated with diabetic complications, including retinopathy, cataracts, nephropathy, neuropathy, cardiomyopathy and skin defects. We recently characterized DJ-1/Park7 as a protein deglycase that repairs proteins from glycation by glyoxal and methylglyoxal, two major glycating agents which are responsible for up to 65% of glycation events. In this study, we investigated the ability of DJ-1 to prevent protein glycation in keratinocytes. Glycation of collagen and keratinocyte proteins was tested by measuring ultraviolet absorption and fluorescence emission. Protein glycation in HaCaT keratinocytes was investigated by immunodetection with anti-advanced glycation endproduct antibodies, after DJ-1 depletion or overexpression. In vitro, DJ-1 prevented glycation of collagen and keratinocyte protein extracts. In cell culture, DJ-1 depletion by small interfering RNAs resulted in a 3-fold increase in protein glycation levels. Moreover, protein glycation levels were decreased several-fold in cells overexpressing DJ-1 after addition of the Nrf2 inducer sulforaphane or after transfection with a DJ-1 plasmid. Thus, the DJ-1 deglycase plays a major role in preventing protein glycation in eukaryotic cells and might be important for preventing skin glycation.

  2. Kinetin Improves Barrier Function of the Skin by Modulating Keratinocyte Differentiation Markers

    Science.gov (United States)

    An, Sungkwan; Cha, Hwa Jun; Ko, Jung-Min; Han, Hyunjoo; Kim, Su Young; Kim, Kyung-Suk; Lee, Song Jeong; An, In-Sook; Kim, Sangwon; Youn, Hae Jeong

    2017-01-01

    Background Kinetin is a plant hormone that regulates growth and differentiation. Keratinocytes, the basic building blocks of the epidermis, function in maintaining the skin barrier. Objective We examined whether kinetin induces skin barrier functions in vitro and in vivo. Methods To evaluate the efficacy of kinetin at the cellular level, expression of keratinocyte differentiation markers was assessed. Moreover, we examined the clinical efficacy of kinetin by evaluating skin moisture, transepidermal water loss (TEWL), and skin surface roughness in patients who used kinetin-containing cream. We performed quantitative real-time polymerase chain reaction to measure the expression of keratinocyte differentiation markers in HaCaT cells following treatment. A clinical trial was performed to assess skin moisture, TEWL, and evenness of skin texture in subjects who used kinetin-containing cream for 4 weeks. Results Kinetin increased involucrin, and keratin 1 mRNA in HaCaT cells. Moreover, use of a kinetin-containing cream improved skin moisture and TEWL while decreasing roughness of skin texture. Conclusion Kinetin induced the expression of keratinocyte differentiation markers, suggesting that it may affect differentiation to improve skin moisture content, TEWL, and other signs of skin aging. Therefore, kinetin is a potential new component for use in cosmetics as an anti-aging agent that improves the barrier function of skin. PMID:28223740

  3. Candida albicans phospholipomannan triggers inflammatory responses of human keratinocytes through Toll-like receptor 2.

    Science.gov (United States)

    Li, Min; Chen, Qing; Shen, Yongnian; Liu, Weida

    2009-07-01

    The Toll-like receptors (TLRs) play an important role in the recognition of Candida albicans components and activation of innate immunity. Phospholipomannan (PLM), a glycolipid, is expressed at the surface of C. albicans cell wall, which acts as a member of the pathogen-associated molecular patterns family. In this study, we sought to clarify whether C. albicans-native PLM could induce an inflammation response in human keratinocytes and to determine the underlying mechanisms. Exposure of cultured human primary keratinocytes to PLM led to the increased gene expression and secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8). PLM hydrolysed with beta-d-mannoside mannohydrolase failed to induce gene expression and secretion of IL-6 and IL-8. PLM up-regulated the mRNA and protein levels of TLR2, whereas the mRNA level of TLR4 was not altered. Keratinocytes challenged with PLM resulted in the activation of NF-kappaB and mitogen-activated protein kinase (MAPKs) including p38. Anti-TLR2 neutralizing antibody, NFkappaB and p38MAPK inhibitors blocked the PLM-induced secretion of IL-6, IL-8 in keratinocytes, but no such effect was observed in pretreatment with anti-TLR4-neutralizing antibody and lipopolysaccharide inhibitor (polymyxin B). These data suggest C. albicans-native PLM may contribute to the inflammatory responses of cutaneous candidiasis in the TLR2-NF-kappaB and p38MAPK signalling pathway dependent manner.

  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.......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 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...

  5. 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

  6. In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos.

    Science.gov (United States)

    Murai, Hidetaka; Tadokoro, Ryosuke; Sakai, Ken-Ichiro; Takahashi, Yoshiko

    2015-04-01

    During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin-containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross-talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus-mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication-Competent Avian sarcoma-leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back-transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)(+) Neo(r+) -electroporated melanocytes from Hypeco nera were selectively grown in G418-supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS-mOrange, mOrange(+) keratinocytes and transplanted EGFP(+) melanocytes were frequently juxtaposed each other. High-resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.

  7. Polymerized laminin-332 matrix supports rapid and tight adhesion of keratinocytes, suppressing cell migration.

    Directory of Open Access Journals (Sweden)

    Yoshinobu Kariya

    Full Text Available Laminin-332 (α3ß3γ2 (Lm332 supports the stable anchoring of basal keratinocytes to the epidermal basement membrane, while it functions as a motility factor for wound healing and cancer invasion. To understand these contrasting activities of Lm332, we investigated Lm332 matrices deposited by normal human keratinocytes and other Lm332-expressing cell lines. All types of the cells efficiently deposited Lm332 on the culture plates in specific patterns. On the contrary, laminins containing laminin ß1 and/or γ1 chains, such as Lm511 and Lm311, were not deposited on the culture plates even if secreted into culture medium. The Lm332 deposition was not inhibited by function-blocking antibodies to the α3 and α6 integrins but was inhibited by sodium selenate, suggesting that sulfated glycosaminoglycans on cell surface, e.g. heparan sulfate proteoglycans, might be involved in the process. HEK293 cells overexpressing exogenous Lm332 (Lm332-HEK almost exclusively deposited Lm332 on the plates. The deposited Lm332 matrix showed a mesh-like network structure as analyzed by electron microscopy, suggesting that Lm332 was highly polymerized. When biological activity was analyzed, the Lm332 matrix rather suppressed the migration of keratinocytes as compared with purified Lm332, which highly promoted the cell migration. The Lm332 matrix supported adhesion of keratinocytes much more strongly and stably than purified Lm332. Integrin α3ß1 bound to the Lm332 matrix at a three times higher level than purified Lm332. Normal keratinocytes prominently showed integrin α6ß4-containing, hemidesmosome-like structures on the Lm332 matrix but not on the purified one. These results indicate that the polymerized Lm332 matrix supports stable cell adhesion by interacting with both integrin α6ß4 and α3ß1, whereas unassembled soluble Lm332 supports cell migration.

  8. Distinct epidermal keratinocytes respond to extremely low-frequency electromagnetic fields differently.

    Directory of Open Access Journals (Sweden)

    Chao-Ying Huang

    Full Text Available Following an increase in the use of electric appliances that can generate 50 or 60 Hz electromagnetic fields, concerns have intensified regarding the biological effects of extremely low-frequency electromagnetic fields (ELF-EMFs on human health. Previous epidemiological studies have suggested the carcinogenic potential of environmental exposure to ELF-EMFs, specifically at 50 or 60 Hz. However, the biological mechanism facilitating the effects of ELF-EMFs remains unclear. Cellular studies have yielded inconsistent results regarding the biological effects of ELF-EMFs. The inconsistent results might have been due to diverse cell types. In our previous study, we indicated that 1.5 mT, 60 Hz ELF-EMFs will cause G1 arrest through the activation of the ATM-Chk2-p21 pathway in human keratinocyte HaCaT cells. The aim of the current study was to investigate whether ELF-EMFs cause similar effects in a distinct epidermal keratinocyte, primary normal human epidermal keratinocytes (NHEK, by using the same ELF-EMF exposure system and experimental design. We observed that ELF-EMFs exerted no effects on cell growth, cell proliferation, cell cycle distribution, and the activation of ATM signaling pathway in NHEK cells. We demonstrated that the 2 epidermal keratinocytes responded to ELF-EMFs differently. To further validate this finding, we simultaneously exposed the NHEK and HaCaT cells to ELF-EMFs in the same incubator for 168 h and observed the cell growths. The simultaneous exposure of the two cell types results showed that the NHEK and HaCaT cells exhibited distinct responses to ELF-EMFs. Thus, we confirmed that the biological effects of ELF-EMFs in epidermal keratinocytes are cell type specific. Our findings may partially explain the inconsistent results of previous studies when comparing results across various experimental models.

  9. Oral keratinocytes support non-replicative infection and transfer of harbored HIV-1 to permissive cells

    Directory of Open Access Journals (Sweden)

    Giacaman Rodrigo A

    2008-07-01

    Full Text Available Abstract Background Oral keratinocytes on the mucosal surface are frequently exposed to HIV-1 through contact with infected sexual partners or nursing mothers. To determine the plausibility that oral keratinocytes are primary targets of HIV-1, we tested the hypothesis that HIV-1 infects oral keratinocytes in a restricted manner. Results To study the fate of HIV-1, immortalized oral keratinocytes (OKF6/TERT-2; TERT-2 cells were characterized for the fate of HIV-specific RNA and DNA. At 6 h post inoculation with X4 or R5-tropic HIV-1, HIV-1gag RNA was detected maximally within TERT-2 cells. Reverse transcriptase activity in TERT-2 cells was confirmed by VSV-G-mediated infection with HIV-NL4-3Δenv-EGFP. AZT inhibited EGFP expression in a dose-dependent manner, suggesting that viral replication can be supported if receptors are bypassed. Within 3 h post inoculation, integrated HIV-1 DNA was detected in TERT-2 cell nuclei and persisted after subculture. Multiply spliced and unspliced HIV-1 mRNAs were not detectable up to 72 h post inoculation, suggesting that HIV replication may abort and that infection is non-productive. Within 48 h post inoculation, however, virus harbored by CD4 negative TERT-2 cells trans infected co-cultured peripheral blood mononuclear cells (PBMCs or MOLT4 cells (CD4+ CCR5+ by direct cell-to-cell transfer or by releasing low levels of infectious virions. Primary tonsil epithelial cells also trans infected HIV-1 to permissive cells in a donor-specific manner. Conclusion Oral keratinocytes appear, therefore, to support stable non-replicative integration, while harboring and transmitting infectious X4- or R5-tropic HIV-1 to permissive cells for up to 48 h.

  10. 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.

  11. 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.

  12. Distinct epidermal keratinocytes respond to extremely low-frequency electromagnetic fields differently.

    Science.gov (United States)

    Huang, Chao-Ying; Chuang, Chun-Yu; Shu, Wun-Yi; Chang, Cheng-Wei; Chen, Chaang-Ray; Fan, Tai-Ching; Hsu, Ian C

    2014-01-01

    Following an increase in the use of electric appliances that can generate 50 or 60 Hz electromagnetic fields, concerns have intensified regarding the biological effects of extremely low-frequency electromagnetic fields (ELF-EMFs) on human health. Previous epidemiological studies have suggested the carcinogenic potential of environmental exposure to ELF-EMFs, specifically at 50 or 60 Hz. However, the biological mechanism facilitating the effects of ELF-EMFs remains unclear. Cellular studies have yielded inconsistent results regarding the biological effects of ELF-EMFs. The inconsistent results might have been due to diverse cell types. In our previous study, we indicated that 1.5 mT, 60 Hz ELF-EMFs will cause G1 arrest through the activation of the ATM-Chk2-p21 pathway in human keratinocyte HaCaT cells. The aim of the current study was to investigate whether ELF-EMFs cause similar effects in a distinct epidermal keratinocyte, primary normal human epidermal keratinocytes (NHEK), by using the same ELF-EMF exposure system and experimental design. We observed that ELF-EMFs exerted no effects on cell growth, cell proliferation, cell cycle distribution, and the activation of ATM signaling pathway in NHEK cells. We demonstrated that the 2 epidermal keratinocytes responded to ELF-EMFs differently. To further validate this finding, we simultaneously exposed the NHEK and HaCaT cells to ELF-EMFs in the same incubator for 168 h and observed the cell growths. The simultaneous exposure of the two cell types results showed that the NHEK and HaCaT cells exhibited distinct responses to ELF-EMFs. Thus, we confirmed that the biological effects of ELF-EMFs in epidermal keratinocytes are cell type specific. Our findings may partially explain the inconsistent results of previous studies when comparing results across various experimental models.

  13. 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.

  14. NOVEL NON-CALCEMIC SECOSTEROIDS THAT ARE PRODUCED BY HUMAN EPIDERMAL KERATINOCYTES PROTECT AGAINST SOLAR RADIATION

    Science.gov (United States)

    Slominski, Andrzej T.; Janjetovic, Zorica; Kim, Tae-Kang; Wasilewski, Piotr; Rosas, Sofia; Hanna, Sherie; Sayre, Robert M.; Dowdy, John C.; Li, Wei; Tuckey, Robert C.

    2015-01-01

    CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→ 17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defence against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. PMID:25617667

  15. Keratinocyte growth factor gene therapy ameliorates ulcerative colitis in rats

    Institute of Scientific and Technical Information of China (English)

    Chun-Jie Liu; Ji-De Jin; Tong-De Lv; Zu-Ze Wu; Xiao-Qin Ha

    2011-01-01

    AIM: To investigate the effect of keratinocyte growth factor (KGF) gene therapy in acetic acid-induced ulcerative colitis in rat model. METHODS: The colitis of Sprague-Dawley rats was induced by intrarectal infusion of 1 mL 5% (v/v) acetic acid. Twenty-four hours after exposed to acetic acid, rats were divided into three experimental groups: control group, attenuated Salmonella typhimurium Ty21a strain (SP) group and SP strain carrying human KGF gene (SPK) group, and they were separately administered orally with 10% NaHCO3, SP or SPK. Animals were sacrificed and colonic tissues were harvested respectively on day 3, 5, 7 and 10 after administration. Weights of rats, colonic weight/ length ratio and stool score were evaluated. Histological changes of colonic tissues were examined by hematoxylin and eosin (HE) staining method. The expression of KGF, KGF receptor (KGFR) and TNF-α were measured either by enzyme-linked immunosorbent assay or Western blotting. Immunohistochemistry was used to detect the cellular localization of KGFR and Ki67. In addition, superoxide dismutase (SOD) activity and malondialdehyde (MDA) contents in the homogenate were measured. RESULTS: Body weight and colonic weight/length ratio were declined in SPK group compared with SP and control groups (body weight: 272.78 ± 17.92 g vs 243.72 ± 14.02 g and 240.68 ± 12.63 g, P < 0.01; colonic weight/length ratio: 115.76 ± 7.47 vs 150.32 ± 5.99 and 153.67 ± 5.50 mg/cm, P < 0.01). Moreover, pathological changes of damaged colon were improved in SPK group as well. After administration of SPK strain, KGF expression increased markedly from the 3rd d, and remained at a high level till the 10th d. Furthermore, KGFR expression and Ki67 expression elevated, whereas TNF-α expression was inhibited in SPK group. In the group administered with SPK, SOD activity increased significantly (d 5: 26.18 ± 5.84 vs 18.12 ± 3.30 and 18.79 ± 4.74 U/mg, P < 0.01; d 7: 35.48 ± 3.35 vs 22.57 ± 3.44 and 21.69 ± 3.94 U

  16. Biological properties of differently-aged human keratinocytes:population doubling time growth curve and cell cycle analysis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To explore the biological properties of keratinocytes from differently-aged healthy human beings. Methods Keratinocytes from fetus,teenager and middle-aged groups were separated and cultured. The population doubling time (PDT) and cell growth curve in different cells were compared,and the cell cycles were analyzed by flow cytometry. Results ① In primary culture of keratinocytes,the adherence time in middle-aged group was longer than that in fetus and teenager groups. However,all cell morphology sh...

  17. 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...

  18. 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.

  19. 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

  20. The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

    Science.gov (United States)

    Bebes, Attila; Kis, Kornélia; Nagy, Tünde; Kurunczi, Anita; Polyánka, Hilda; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-01-01

    Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

  1. 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

    keratinocytes from incisional mouse skin wounds and adjacent normal skin keratinocytes. Changes in gene expression were determined by comparative cDNA array analyses, and the approach was validated by in situ hybridization. The analyses identified 48 candidate genes not previously associated with 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....

  2. 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.

  3. Alcohol metabolism by oral streptococci and interaction with human papillomavirus leads to malignant transformation of oral keratinocytes.

    Science.gov (United States)

    Tao, Lin; Pavlova, Sylvia I; Gasparovich, Stephen R; Jin, Ling; Schwartz, Joel

    2015-01-01

    Poor oral hygiene, ethanol consumption, and human papillomavirus (HPV) are associated with oral and esophageal cancers. However, the mechanism is not fully known. This study examines alcohol metabolism in Streptococcus and its interaction with HPV-16 in the malignant transformation of oral keratinocytes. The acetaldehyde-producing strain Streptococcus gordonii V2016 was analyzed for adh genes and activities of alcohol and aldehyde dehydrogenases. Streptococcus attachment to immortalized HPV-16 infected human oral keratinocytes, HOK (HPV/HOK-16B), human oral buccal keratinocytes, and foreskin keratinocytes was studied. Acetaldehyde, malondialdehyde, DNA damage, and abnormal proliferation among keratinocytes were also quantified. We found that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB, and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol, and ethanol, respectively. S. gordonii V2016 did not show a detectable aldehyde dehydrogenase. AdhE is the major alcohol dehydrogenase in S. gordonii. Acetaldehyde and malondialdehyde production from permissible Streptococcus species significantly increased the bacterial attachment to keratinocytes, which was associated with an enhanced expression of furin to facilitate HPV infection and several malignant phenotypes including acetaldehyde adduct formation, abnormal proliferation, and enhanced migration through integrin-coated basement membrane by HPV-infected oral keratinocytes. Therefore, expression of multiple alcohol dehydrogenases with no functional aldehyde dehydrogenase contributes to excessive production of acetaldehyde from ethanol by oral streptococci. Oral Streptococcus species and HPV may cooperate to transform oral keratinocytes after ethanol exposure. These results suggest a significant clinical interaction, but further validation is warranted.

  4. Biological properties of differently-aged human keratinocytes:population doubling time growth curve and cell cycle analysis

    Institute of Scientific and Technical Information of China (English)

    Hui-qun Ma; Jie Feng; Lech Chyczewski; Jacek Niklinski

    2009-01-01

    Objective To explore the biological properties of keratinocytes from differently-aged healthy human beings. Methods Keratinocytes from fetus, teenager and middle-aged groups were separated and cultured. The population doubling time (PDT) and cell growth curve in different cells were compared, and the cell cycles were analyzed by flow cytometry. Results ① In primary culture of keratinocytes, the adherence time in middle-aged group was longer than that in fetus and teenager groups. However, all cell morphology showed no obvioas differences. In subculture of kecatinocytes, with donator's age increasing, time of cell adherence prolonged, passage number decreused and differences in cell morphology were obrioas. ② The average PDT of keratinocytes was shorter in fetus group than in teenager and middle-aged groups. Bat difference in cell growth curve between different passages was not observed. ③ Keratinocytes showed G2/M period in fetus group but G0/G1 period in teenager and middle-aged groups mainly. Conclusion As age increases, the biological properties of keratinocytes change obviously.

  5. Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.

    Directory of Open Access Journals (Sweden)

    Elena Rahn

    Full Text Available Herpes simplex virus type 1 (HSV-1 can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s. Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.

  6. Keratinocyte differentiation and upregulation of ceramide synthesis induced by an oat lipid extract via the activation of PPAR pathways.

    Science.gov (United States)

    Chon, Su-Hyoun; Tannahill, Ruth; Yao, Xiang; Southall, Michael D; Pappas, Apostolos

    2015-04-01

    Activation of peroxisome proliferator-activated receptors (PPARs) has been shown to have an important role in skin barrier function by regulating differentiation and lipid synthesis in keratinocytes. Oat (Avena sativa) has long been used as a soothing agent to relieve skin irritations, and the clinical benefits of topical oat formulations have been proven; however, the mechanistic understanding of oat's mode of action remains unknown. We investigated whether an oat lipid extract could activate PPARs and subsequently increase epidermal lipid synthesis and differentiation markers. Primary human epidermal keratinocytes and transformed cell lines were treated with PPAR agonists and oat lipid extracts to investigate the PPAR agonism. PPAR target genes and epidermal differentiation markers were analysed using quantitative real-time PCR and HPTLC analysis. Oat lipid extract demonstrated robust dual agonism for PPARα and PPARβ/δ, and increased direct PPAR target gene induction in primary human keratinocytes. In addition, oat oil treatment increased both receptor expression and, consistent with the literature on PPARs, oat oil treatment resulted in a significant upregulation of differentiation genes (involucrin, SPRRs and transglutaminase 1) and ceramide processing genes (β-glucocerebrosidase, sphingomyelinases 3 and ABCA12). Further, oat oil treatment in keratinocytes significantly increased ceramide levels (70%), suggesting a functional translation of PPAR activation by oat oil in keratinocytes. Taken together, these results demonstrate that oat lipids possess robust dual agonistic activities for PPARα and PPARβ/δ, increase their gene expression and induce differentiation and ceramide synthesis in keratinocytes, which can collectively improve skin barrier function.

  7. Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes.

    Science.gov (United States)

    Liu, Szu-Hsiu; Chu, I-Ming; Pan, I-Horng

    2008-08-01

    In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte-keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.

  8. Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin.

    Science.gov (United States)

    Bigliardi-Qi, M; Sumanovski, L T; Büchner, S; Rufli, T; Bigliardi, P L

    2004-01-01

    We have previously shown that human epidermal keratinocytes express a functionally active micro-opiate receptor, which adds a new dimension to the recently developed research in neuroimmunodermatology and neurogenic inflammation in skin diseases. Human keratinocytes specifically bind and also produce beta-endorphin, the endogenous micro-opiate receptor ligand. Using confocal imaging microscopy, we could now demonstrate that micro-opiate receptors are not only expressed in keratinocytes, but also on unmyelinated peripheral nerve fibers in the dermis and epidermis. Some of the peripheral nerve fibers also express the ligand beta-endorphin. The keratinocytes positive for beta-endorphin staining are clustered around the terminal ends of the unmyelinated nerve fibers. Therefore the opiate receptor system seems to be crucial in the direct communication between nerves and skin. The keratinocytes can influence the unmyelinated nerve fibers in the epidermis directly via secreting beta-endorphin. On the other hand, nerve fibers can also secrete beta-endorphin and influence the migration, differentiation and probably also the cytokine production pattern of keratinocytes.

  9. Relationship Between Apoptosis and PCNA Expression of Keratinocytes in Condylomata Acuminata

    Institute of Scientific and Technical Information of China (English)

    樊翌明; 马泽粦; 冯进云; 吴志华; 李顺凡

    2002-01-01

    Objective: To investigate the relationship betweenapoptosis and proliferating cell nuclear antigen (PCNA)expression of keratinocytes in Condylomata acuminata (CA). Methods: PCNA expression was observed byimmunohistochemistry technique (ABC method) in 51 CAspecimens and 18 normal specimens of foreskin or vaginalmucosae. 55 specimens (40 in the CA group and 15 in thecontrol group) were randomly sampled for in situ labelingof apoptotic cells using the TUNEL method. Results: Positive expression of PCNA in CA and controlgroups were 90.2% and 77.8%, respectively, and theproliferation index in CA group was significantly higherthan that in the control group (P0.05). The proliferation indexshowed a significant negative correlation with theapoptosis-proliferation ratio (r=-0.62, P=0.01) in the CAgroup. Conclusion: It is suggested that the proliferativeappearance of CA could be due to the imbalance betweencell growth and cell death which is caused by moreproliferation and less apoptosis in keratinocytes.

  10. Long-term subculture of human keratinocytes under an anoxic condition.

    Science.gov (United States)

    Kino-oka, Masahiro; Agatahama, Yuka; Haga, Yuki; Inoie, Masukazu; Taya, Masahito

    2005-07-01

    The serial subculturing of human keratinocyte cells under the anoxic and normoxic conditions was examined. The cumulative number of population doublings in the subcultures under the former condition increased 2.1-fold while maintaining an appreciable growth rate of cells, as compared with that under the latter condition. Moreover, the migration ability, which was estimated by the rotation rate of paired cells, was maintained accompanied by fully developed filopodia of F-actin filaments under the anoxic condition, despite of the poor development of stress fibers at the center of the cellular body. The cells passaged under the anoxic condition possessed the sufficient clonogenic potential to form epithelial sheets, supporting the view that the long-term subculture of keratinocytes under the anoxic condition can be applied for cell expansion in the practical production of epithelial sheets.

  11. CARD14-Mediated Activation of Paracaspase MALT1 in Keratinocytes: Implications for Psoriasis.

    Science.gov (United States)

    Van Nuffel, Elien; Schmitt, Anja; Afonina, Inna S; Schulze-Osthoff, Klaus; Beyaert, Rudi; Hailfinger, Stephan

    2017-03-01

    Mutations in caspase recruitment domain-containing protein 14(CARD14) have been linked to susceptibility to psoriasis. CARD14 is an intracellular scaffold protein that regulates proinflammatory gene expression. Recent studies have offered novel insights into the mechanisms of CARD14-mediated signaling in keratinocytes and the molecular impact of psoriasis-associated CARD14 mutations. CARD14 forms a signaling complex with BCL10 and the paracaspase MALT1, and this process is enhanced upon pathogenic CARD14 mutation, culminating in the activation of MALT1 protease activity and psoriasis-associated gene expression. This review summarizes the current knowledge of CARD14/MALT1-mediated signaling in keratinocytes and its therapeutic implications in psoriasis.

  12. Amygdalin analogues inhibit IFN-γ signalling and reduce the inflammatory response in human epidermal keratinocytes.

    Science.gov (United States)

    Paoletti, Iole; De Gregorio, Vincenza; Baroni, Adone; Tufano, Maria Antonietta; Donnarumma, Giovanna; Perez, Juan Jesus

    2013-12-01

    Peptide T (PT), an octapeptide fragment located in the V2 region of the HIV-1 gp120-coating protein, appears to be beneficial in the treatment of psoriasis. Our previous investigations suggest that keratinocytes play a key role in conditioning the therapeutic effects of PT in psoriasis. The aim of this study was to explore the effects of PT and the peptidomimetic natural products, Dhurrin and Prunasin, on the expression of the IL-6, IL-8, IL-23, HSP70 and ICAM-1 on IFN-γ and TNF-α-NHEK activated cells. Moreover, we analysed the interference of PT and its analogues through STAT-3 activation. Our results show that the analogues tested exhibit the beneficial biological effects of PT, suggesting the primary role of keratinocytes upon which PT and the peptidomimetics act directly, by reducing proinflammatory responses. Its reduction appears to be important for therapeutic approach in psoriasis pathogenesis.

  13. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    Science.gov (United States)

    Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

    2015-01-01

    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.

  14. COP1 contributes to UVB-induced signaling in human keratinocytes.

    Science.gov (United States)

    Kinyó, Agnes; Kiss-László, Zsuzsanna; Hambalkó, Szabolcs; Bebes, Attila; Kiss, Mária; Széll, Márta; Bata-Csörgo, Zsuzsanna; Nagy, Ferenc; Kemény, Lajos

    2010-02-01

    UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.

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

    DEFF Research Database (Denmark)

    Stanley, Alanna; Pedersen, Esben; Brakebusch, Cord

    2016-01-01

    Keratinocytes, in response to irritants, secrete pro-inflammatory mediators which recruit and activate immune and mesenchymal cells, including fibroblasts, to repair the skin. Fibroblasts respond by synthesising collagen and promoting the crosslinking extracellular matrix (ECM). We recently showed...... 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...... and ICD affects the architectural organisation of the dermal collagen, and that a higher inflammatory state in the tissue (i.e. when Rac1 is deleted in the keratinocytes or ICD is induced in the skin, or a combination of both) influences the diameter of the collagen fibrils. It is proposed...

  16. Chrysin protects epidermal keratinocytes from UVA- and UVB-induced damage.

    Science.gov (United States)

    Wu, Nan-Lin; Fang, Jia-You; Chen, Marcelo; Wu, Chia-Jung; Huang, Chieh-Chen; Hung, Chi-Feng

    2011-08-10

    Chrysin (5,7-dihydroxyflavone), a natural flavonoid occurring in various plants and foods such as propolis and honey, reportedly opposes inflammation and carcinogenesis, but has rarely been applied in skin care. This study, therefore, aimed to explore the roles of chrysin in protection against UV-induced damage in HaCaT keratinocytes. Results showed that chrysin can attenuate apoptosis, reactive oxygen species (ROS) production, and cyclooxygenase 2 (COX-2) expression induced by UVB and UVA. Chrysin predominantly reversed the down-regulation of aquaporin 3 (AQP-3) by UVB. It predominantly reversed JNK activation and also mildly inhibited p38 activation triggered by UVA and UVB. Animal studies revealed that chrysin's topical application demonstrated efficient percutaneous absorption and no skin irritation. Overall, results demonstrated significant benefits of chrysin on the protection of keratinocytes against UVA- and UVB-induced injuries and suggested its potential use in skin photoprotection.

  17. Combined treatment with sodium butyrate and PD153035 enhances keratinocyte differentiation

    Science.gov (United States)

    Carrion, Sandra Leon; Sutter, Carrie Hayes; Sutter, Thomas R.

    2014-01-01

    Epidermal growth factor (EGF) receptor (EGFR) signaling is a critical determinant of keratinocyte proliferation and differentiation in both normal and diseased skin. Here we explore the effects of combined treatment with the differentiation-promoting agent sodium butyrate (SB) and the EGFR inhibitor (EGFRI) PD153035 on terminal differentiation of normal human epidermal keratinocytes (NHEKs). Cells treated with SB showed increased expression of the levels of mRNA and protein of the differentiation markers filaggrin and transglutaminase 1. Co-treatment with EGF significantly blunted these effects of SB. Combined treatment with SB and PD153035 alleviated these inhibitory actions of EGF, resulting in improved effects of decreased cell growth and increased terminal differentiation, relative to the individual treatments. These results indicate that the combined use of a differentiation-promoting agent and an EGFR inhibitor may offer an additional approach to the management of hyperproliferative skin diseases. PMID:24451036

  18. The effect of the plasma needle on the human keratinocytes related to the wound healing process

    Science.gov (United States)

    Korolov, Ihor; Fazekas, Barbara; Széll, Márta; Kemény, Lajos; Kutasi, Kinga

    2016-01-01

    In the present study we aim to verify the influence of a non-thermal atmospheric pressure plasma on the wound healing process. In this process the major contributors are the keratinocytes, which migrate to fill in the gap created by the wound. Therefore, we performed the direct treatment of HPV-immortalized human keratinocytes, protected by a layer of phosphate buffered saline (PBS) solution, with the glow discharge generated in flowing helium by a plasma needle. To mimick a wound, a 4 mm scratch was performed on the cell culture (scratch assay). We conducted two types of experiments: (i) cell proliferation and (ii) wound-healing model experiments. The plasma needle configuration, the plasma treatment conditions and the thickness of the protecting PBS layer were set based on viability experiments. The proliferation studies showed that short, 5-10 s, and low power treatments, such as 18 W and 20 W input power, could positively influence the cell proliferation when keratinocytes were protected by PBS. On the other hand, the plasma treatment of cell medium covered keratinocytes resulted in the decrease of proliferation. The wound-healing model (scratch assay) studies showed, that there was a maximum in the wound reduction as a function of the input power and treatment time, namely, at 18 W and 5 s. Furthermore, the wound reduction strongly depended on the treated cell—PBS interaction time. To mimic an infected wound, the scratch assay was covered with a 1× {{10}9} cfu ml-1 Propionibacterium acnes suspension. The plasma treatment of this infected assay resulted in closing of the scratch, while in the non-treated assay the wound did not close at all.

  19. Image enhancement of optical images for binary system of melanocytes and keratinocytes

    Science.gov (United States)

    Takanezawa, S.; Baba, A.; Sako, Y.; Ozaki, Y.; Date, A.; Toyama, K.; Morita, S.

    2013-05-01

    Automatic determination of the cell shapes of large numbers of melanocytes based on optical images of human skin models have been largely unsuccessful (the complexities introduced by dendrites and the melanin pigmentation over the keratinocytes to give unclear outlines). Here, we present an image enhancement procedure for enhancing the contrast of images with removing the non-uniformity of background. The brightness is normalized also for the non-uniform population density of melanocytes.

  20. Low dose chronic treatment of human keratinocytes with inorganic arsenic causes hyperproliferation and altered protein phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Steinberg, M.L. [City College of New York, NY (United States); Su, L.; Snow, E.T. [New York Univ. Medical Center, Tuxedo, NY (United States)]|[City College of New York, NY (United States)

    1997-10-01

    Chronic exposure to arsenate [As(V)] or arsenite [As(III)] causes hyperproliferation of normal and SV40-transformed human epidermal keratinocytes. Line 327 SV40-infected human keratinocytes were grown in the presence of either As(III) or As(V) (0.01 to 10 {mu}M) in complete medium for seven days prior to harvesting and counting. Both As(III) and As(V) were cytotoxic at micromolar concentrations, however submicromolar arsenic caused a significant increase in cell growth. Cell numbers in cultures exposed to As(V) were increased more than 186% relative to controls, and an even larger stimulation in cell growth was observed after treatment with 50 nM As(III). Normal non-SV40 T-antigen. Preliminary cell cycle analysis using unselected, log-phase cultures of arsenic-treated keratinocytes shows an increased proportion of cells in S- and G2/M-phase. Isoelectric focusing of phosphotyrosine-containing proteins from cells labeled with {sup 32}P-inorganic phosphate showed that the hyperproliferation of keratinocytes grown in low concentrations of arsenic is accompanied by altered tyrosine-specific protein phosphorylation. A number of phosphorylated proteins were observed in As-treated cells that were not observed in the controls; and minor bands at IEPs of 3.0, 4.2, 7.2, 7.5 and 8.2. These results, together with the lack of direct enzyme inhibition by arsenic shown by Su et al., this volume, suggest that arsenic-induced skin lesions and carcinogenesis may be the result of altered cell cycle control rather than DNA damage or reduced DNA repair.

  1. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    OpenAIRE

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Jennifer H. Shin; PARK, YEUNSOO; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repr...

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

    OpenAIRE

    Abhay Kyadarkunte; Milind Patole; Varsha Pokharkar

    2014-01-01

    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 u...

  4. 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.

  5. Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

    Science.gov (United States)

    Wikner, N E; Persichitte, K A; Baskin, J B; Nielsen, L D; Clark, R A

    1988-09-01

    Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

  6. Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

    Science.gov (United States)

    Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

    2016-07-07

    The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing.

  7. A sensitive sensor cell line for the detection of oxidative stress responses in cultured human keratinocytes.

    Science.gov (United States)

    Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

    2014-06-25

    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.

  8. Upregulation of ANGPTL6 in mouse keratinocytes enhances susceptibility to psoriasis

    Science.gov (United States)

    Tanigawa, Hiroki; Miyata, Keishi; Tian, Zhe; Aoi, Jun; Kadomatsu, Tsuyoshi; Fukushima, Satoshi; Ogata, Aki; Takeda, Naoki; Zhao, Jiabin; Zhu, Shunshun; Terada, Kazutoyo; Endo, Motoyoshi; Morinaga, Jun; Sugizaki, Taichi; Sato, Michio; Morioka, Masaki Suimye; Manabe, Ichiro; Mashimo, Youichi; Hata, Akira; Taketomi, Yoshitaka; Yamamoto, Kei; Murakami, Makoto; Araki, Kimi; Jinnin, Masatoshi; Ihn, Hironobu; Oike, Yuichi

    2016-01-01

    Psoriasis is a chronic inflammatory skin disease marked by aberrant tissue repair. Mutant mice modeling psoriasis skin characteristics have provided useful information relevant to molecular mechanisms and could serve to evaluate therapeutic strategies. Here, we found that epidermal ANGPTL6 expression was markedly induced during tissue repair in mice. Analysis of mice overexpressing ANGPTL6 in keratinocytes (K14-Angptl6 Tg mice) revealed that epidermal ANGPTL6 activity promotes aberrant epidermal barrier function due to hyperproliferation of prematurely differentiated keratinocytes. Moreover, skin tissues of K14-Angptl6 Tg mice showed aberrantly activated skin tissue inflammation seen in psoriasis. Levels of the proteins S100A9, recently proposed as therapeutic targets for psoriasis, also increased in skin tissue of K14-Angptl6 Tg mice, but psoriasis-like inflammatory phenotypes in those mice were not rescued by S100A9 deletion. This finding suggests that decreasing S100A9 levels may not ameliorate all cases of psoriasis and that diverse mechanisms underlie the condition. Finally, we observed enhanced levels of epidermal ANGPTL6 in tissue specimens from some psoriasis patients. We conclude that the K14-Angptl6 Tg mouse is useful to investigate psoriasis pathogenesis and for preclinical testing of new therapeutics. Our study also suggests that ANGPTL6 activation in keratinocytes enhances psoriasis susceptibility. PMID:27698489

  9. Spatial Distribution of Stem Cell-Like Keratinocytes in Dissected Compound Hair Follicles of the Dog.

    Directory of Open Access Journals (Sweden)

    Dominique J Wiener

    Full Text Available Hair cycle disturbances are common in dogs and comparable to some alopecic disorders in humans. A normal hair cycle is maintained by follicular stem cells which are predominately found in an area known as the bulge. Due to similar morphological characteristics of the bulge area in humans and dogs, the shared particularity of compound hair follicles as well as similarities in follicular biomarker expression, the dog is a promising model to study human hair cycle and stem cell disorders. To gain insight into the spatial distribution of follicular keratinocytes with stem cell potential in canine compound follicles, we microdissected hair follicles in anagen and telogen from skin samples of freshly euthanized dogs. The keratinocytes isolated from different locations were investigated for their colony forming efficiency, growth and differentiation potential as well as clonal growth. Our results indicate that i compound and single hair follicles exhibit a comparable spatial distribution pattern with respect to cells with high growth potential and stem cell-like characteristics, ii the lower isthmus (comprising the bulge harbors most cells with high growth potential in both, the anagen and the telogen hair cycle stage, iii unlike in other species, colonies with highest growth potential are rather small with an irregular perimeter and iv the keratinocytes derived from the bulbar region exhibit characteristics of actively dividing transit amplifying cells. Our results now provide the basis to conduct comparative studies of normal dogs and those with hair cycle disorders with the possibility to extend relevant findings to human patients.

  10. Effects of Arsenic on Cell Proliferation and Its Related Gene Expression in Human Epidermal Keratinocyte

    Institute of Scientific and Technical Information of China (English)

    顾军; 毕新岭; 米庆胜; 文军慧

    2002-01-01

    Objective:To study the effects of low concentration of arsenic (As2O3) on DNA synthesisand related transcription factor gene E2F1 expression in keratinocyte. Methods: Human epidermal kerati-nocyte (cell line HaCaT) cultured in vitro was used. After treatment with various concentrations of arse-nic, DNA synthesis and E2F1 expression in HaCaT cells were detected by using 3 H-TdR method and RT-PCR. Results: Arsenic caused a modest increase of keratinocyte DNA synthesis when the concentrationreached the range within 0.5-16 nmol/L, but the amount of incorporated 3 H-TdR decreased and returnedto baseline level when the concentration of arsenic increased to over 16 nmol/L. RT-PCR analysis showedthe level of E2F1 mRNA was elevated in HaCaT cells with the increase of DNA synthesis. Conclusion:Ar-senic of a certain concentration could increase DNA synthesis and enhance E2F1 expression in HaCaT cellline, which might be one of the pathological mechanisms of skin disease related to arsenic.

  11. The impact of skin viability on drug metabolism and permeation -- BSA toxicity on primary keratinocytes.

    Science.gov (United States)

    Haberland, A; Schreiber, S; Maia, C Santos; Rübbelke, M K; Schaller, M; Korting, H C; Kleuser, B; Schimke, I; Schäfer-Korting, M

    2006-04-01

    For testing cutaneous absorption of drugs, ingredients of cosmetics and also for risk assessment of industrial compounds predictable in vitro test protocols are under investigation using excised skin or reconstructed human epidermis. Since the metabolizing enzymes expressed by viable skin can influence the absorption behaviour of substances by changing their structure and thereby their physicochemical characteristics, the metabolic capacity should be considered in the design of the test protocols of compounds susceptible to metabolism. Then data, generated using viable reconstructed epidermis may reflect the in vivo situation. Interestingly, bovine serum albumin (BSA) commonly used in receptor media in permeation studies to facilitate solubility of highly lipophilic substances strongly inhibited the metabolism of topically applied prednicarbate in reconstructed epidermis. Here, we show that 5% BSA is toxic to reconstructed epidermis and keratinocytes which was consistent with the earlier findings. While media toxicity (deficiency media) was at least partly the cause of both apoptotic and necrotic processes in keratinocytes, BSA only slightly increased the rate of necrotic cells. Moreover, caspase inhibitors did not reduce BSA toxicity. Yet, the results show that BSA toxicity on keratinocytes has to be carefully considered if this protein is used in permeation studies with reconstructed epidermis.

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

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    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.

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

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    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.

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

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    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.

  15. Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

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    Sollberger, Gabriel; Strittmatter, Gerhard E; Grossi, Serena; Garstkiewicz, Martha; Auf dem Keller, Ulrich; French, Lars E; Beer, Hans-Dietmar

    2015-05-01

    Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1β. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

  16. Hydrogen-enriched water restoration of impaired calcium propagation by arsenic in primary keratinocytes

    Science.gov (United States)

    Yu, Wei-Tai; Chiu, Yi-Ching; Lee, Chih-Hung; Yoshioka, Tohru; Yu, Hsin-Su

    2013-11-01

    Endemic contamination of artesian water for drinking by arsenic is known to cause several human cancers, including cancers of the skin, bladder, and lungs. In skin, multiple arsenic-induced Bowen's disease (As-BD) can develop into invasive cancers after decades of arsenic exposure. The characteristic histological features of As-BD include full-layer epidermal dysplasia, apoptosis, and abnormal proliferation. Calcium propagation is an essential cellular event contributing to keratinocyte differentiation, proliferation, and apoptosis, all of which occur in As-BD. This study investigated how arsenic interferes calcium propagation of skin keratinocytes through ROS production and whether hydrogen-enriched water would restore arsenic-impaired calcium propagation. Arsenic was found to induce oxidative stress and inhibit ATP- and thapsigaragin-induced calcium propagation. Pretreatment of arsenic-treated keratinocytes by hydrogen-enriched water or beta-mercaptoethanol with potent anti-oxidative effects partially restored the propagation of calcium by ATP and by thapsigaragin. It was concluded that arsenic may impair calcium propagation, likely through oxidative stress and interactions with thiol groups in membrane proteins.

  17. Effects of honeybee (Apis mellifera venom on keratinocyte migration in vitro

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    Sang Mi Han

    2013-01-01

    Full Text Available Background: Since the ancient times the skin aging application of honeybee venom (BV is practiced and persisted until nowadays. The present study evaluated the effect of the honeybee venom (BV on keratinocyte migration in wound healing model in vitro. Objective: To access BV further as a cosmetic ingredient and a potential external application for topical uses, we performed studies to investigate the biologic effect of BV treatment on keratinocyte proliferation and migration in vitro. Material and Methods: BV cytotoxicity was assessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT assay over 24 h. To assess BV genotoxicity, damage to human epidermal keratinocyte (HEK was evaluated using the Comet assay. HEK migration was evaluated using a commercial wound healing kit. The skin pro-inflammatory cytokines interleukin (IL-8 and tumor necrosis factor (TNF-α were examined to evaluate the pro-inflammatory response to BV. Results: It was found that BV (<100 μg/ml was not cytotoxic and stimulated more HEK proliferation and migration compared to negative control, and did not induce DNA damage. There were also decreases in IL-8 and TNF-α expression levels in HEK at all time points. Conclusion: These findings highlight the potential of topical application of BV for promoting cell regeneration and wound treatment.

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

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    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.

  19. Oncogenic KRAS sensitizes premalignant, but not malignant cells, to Noxa-dependent apoptosis through the activation of the MEK/ERK pathway.

    Science.gov (United States)

    Conti, Annalisa; Majorini, Maria Teresa; Elliott, Richard; Ashworth, Alan; Lord, Christopher J; Cancelliere, Carlotta; Bardelli, Alberto; Seneci, Pierfausto; Walczak, Henning; Delia, Domenico; Lecis, Daniele

    2015-05-10

    KRAS is mutated in about 20-25% of all human cancers and especially in pancreatic, lung and colorectal tumors. Oncogenic KRAS stimulates several pro-survival pathways, but it also triggers the trans-activation of pro-apoptotic genes. In our work, we show that G13D mutations of KRAS activate the MAPK pathway, and ERK2, but not ERK1, up-regulates Noxa basal levels. Accordingly, premalignant epithelial cells are sensitized to various cytotoxic compounds in a Noxa-dependent manner. In contrast to these findings, colorectal cancer cell sensitivity to treatment is independent of KRAS status and Noxa levels are not up-regulated in the presence of mutated KRAS despite the fact that ERK2 still promotes Noxa expression. We therefore speculated that other survival pathways are counteracting the pro-apoptotic effect of mutated KRAS and found that the inhibition of AKT restores sensitivity to treatment, especially in presence of oncogenic KRAS. In conclusion, our work suggests that the pharmacological inhibition of the pathways triggered by mutated KRAS could also switch off its oncogene-activated pro-apoptotic stimulation. On the contrary, the combination of chemotherapy to inhibitors of specific pro-survival pathways, such as the one controlled by AKT, could enhance treatment efficacy by exploiting the pro-death stimulation derived by oncogene activation.

  20. Synergistic Cytotoxic Effects of Ganoderma lucidum and Bacillus Calmette Guérin on Premalignant Urothelial HUC-PC Cells and Its Regulation on Proinflammatory Cytokine Secretion

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    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.

  1. A decorin-deficient matrix affects skin chondroitin/dermatan sulfate levels and keratinocyte function

    Science.gov (United States)

    Nikolovska, Katerina; Renke, Jana K.; Jungmann, Oliver; Grobe, Kay; Iozzo, Renato V.; Zamfir, Alina D.; Seidler, Daniela G.

    2016-01-01

    Decorin is a small leucine-rich proteoglycan harboring a single glycosaminoglycan chain, which, in skin, is mainly composed of dermatan sulfate (DS). Mutant mice with targeted disruption of the decorin gene (Dcn−/−) exhibit an abnormal collagen architecture in the dermis and reduced tensile strength, collectively leading to a skin fragility phenotype. Notably, Ehlers-Danlos patients with mutations in enzymes involved in the biosynthesis of DS display a similar phenotype, and recent studies indicate that DS is involved in growth factor binding and signaling. To determine the impact of the loss of DS-decorin in the dermis, we analyzed the glycosaminoglycan content of Dcn−/− and wild-type mouse skin. The total amount of chondroitin/dermatan sulfate (CS/DS) was increased in the Dcn−/− skin, but was overall less sulfated with a significant reduction in bisulfated ΔDiS2,X (X=4 or 6) disaccharide units, due to the reduced expression of uronyl 2-O sulfotransferase (Ust). With increasing age, sulfation declined; however, Dcn−/− CS/DS was constantly undersulfated vis-à-vis wild-type. Functionally, we found altered fibroblast growth factor (Fgf)-7 and -2 binding due to changes in the micro-heterogeneity of skin Dcn−/− CS/DS. To better delineate the role of decorin, we used a 3D Dcn−/− fibroblast cell culture model. We found that the CS/DS extracts of wild-type and Dcn−/− fibroblasts were similar to the skin sugars, and this correlated with the lack of uronyl 2-O sulfotransferase in the Dcn−/− fibroblasts. Moreover, Ffg7 binding to total CS/DS was attenuated in the Dcn−/− samples. Surprisingly, wild-type CS/DS significantly reduced the binding of Fgf7 to keratinocytes in concentration dependent manner unlike the Dcn−/− CS/DS that only affected the binding at higher concentrations. Although binding to cell-surfaces was quite similar at higher concentrations, keratinocyte proliferation was differentially affected. Higher concentration of

  2. Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

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    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

  3. A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

    Science.gov (United States)

    Jubin, K; Martin, Y; Lawrence-Watt, D J; Sharpe, J R

    2011-12-01

    Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

  4. Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes

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    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

  5. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    Energy Technology Data Exchange (ETDEWEB)

    Senthilkumar, P.K. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Robertson, L.W. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States); Ludewig, G., E-mail: Gabriele-ludewig@uiowa.edu [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States)

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  6. Mean Annual UV-B Irradiance

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    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...

  7. Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

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    Ravinder Kumar

    2012-01-01

    Full Text Available Background: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. Methods: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. Results: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. Conclusion: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.

  8. Characterization of fetal keratinocytes, showing enhanced stem cell-like properties: a potential source of cells for skin reconstruction.

    Science.gov (United States)

    Tan, Kenneth K B; Salgado, Giorgiana; Connolly, John E; Chan, Jerry K Y; Lane, E Birgitte

    2014-08-12

    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.

  9. MicroRNA Let-7b inhibits keratinocyte migration in cutaneous wound healing by targeting IGF2BP2.

    Science.gov (United States)

    Wu, Yan; Zhong, Julia Li; Hou, Ning; Sun, Yaolan; Ma, Benting; Nisar, Muhammad Farrukh; Teng, Yan; Tan, Zhaoli; Chen, Keping; Wang, Youliang; Yang, Xiao

    2017-02-01

    Wound healing is a complex process which involves proliferation and migration of keratinocyte for closure of epidermal injuries. A member of microRNA family, let-7b, has been expressed in mammalian skin, but its exact role in keratinocyte migration is still not in knowledge. Here, we showed that let-7b regulates keratinocyte migration by targeting the insulin-like growth factor IGF2BP2. Overexpression of let-7b led to reduced HaCaT cell migration, while knockdown of let-7b resulted in enhanced migration. Furthermore, let-7b was decreased during wound healing in wild-type mice, which led us to construct the transgenic mice with overexpression of let-7b in skin. The re-epithelialization of epidermis of let-7b transgenic mice was reduced during wound healing. Using bioinformatics prediction software and a reporter gene assay, we found that IGF2BP2 was a target of let-7b, which contributes to keratinocyte migration. Introduction of an expression vector of IGF2BP2 also rescued let-7b-induced migration deficiency, which confirms that IGF2BP2 is an important target for let-7b regulation. Our findings suggest that let-7b significantly delayed the re-epithelialization possibly due to reduction of keratinocyte migration and restraints IGF2BP2 during skin wound healing.

  10. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

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    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  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.

  12. 口腔黏膜肿瘤的光动力治疗%Photodynamic Therapy for Premalignant and Malignant Lesions of Oral Mucosa

    Institute of Scientific and Technical Information of China (English)

    邱海霞; 顾瑛; 王颖; 朱建国; 曾晶; 黄乃艳; 陈虹霞

    2011-01-01

    Objective To observe the short-term effect of photodynamic therapy on premalignant and malignant lesions of oral mucosa.Methods One patient with histologically confirmed oral lichen planus and one patient with squamous cell carcinomas were included in the study. PSD-007 at 5 mg/kg body weight was intravenously injected 6 h before laser irradiation. A semiconductor laser emitting at 630 nm was used as a light source. The power density of 150 mW/cm2 was used, the exposure time was 10-30 minutes, and the total light dosage was 90-270 J/cm2 at one light spot. Clinical and pathological examination was conducted 4-5 weeks after PDT to evaluate the short-term effect.Results Eighteen foci in the two patients were treated. Of these foci, 15 got complete remission (CR) after once treatment, two foci got partial remission( PR ) after one treatment, and got CR after a repeat treatment, one focus got partial remission(PR) after the first treatment and became stable disease(SD) at second treatment, and got CR after the third treatment. No significantly adverse events occurred during and after PDT.Conclusions PDT has the advantage of good selectivity, excellent tumor targeting and minimal damage to the function of oral cavity,suggesting that PDT is a sage, effective and minimally invasive procedure for premalignant and malignant lesions of oral mucosa.%目的 观察光动力疗法治疗口腔黏膜癌前病变及口腔癌的临床疗效.方法 临床及病理确诊的口腔黏膜扁平苔藓和鳞癌患者各1例,静脉注射光敏剂PSD-007 5 mg/kg后6 h,应用波长630nm的半导体激光以点状光纤照射,激光功率密度150mW/cm2,每个光斑照射10-30min,能量密度90-270 J/cm2.观察患者术中和术后的不良反应.术后4~5周进行近期临床疗效评价.结果 2例患者累计治疗18处病灶,其中15处病灶经1次治疗,2处病灶经2次治疗,1处病灶经3次治疗后痊愈,未见瘢痕形成.2例患者病灶愈合后均无口腔

  13. Characterisation of the kynurenine pathway in skin-derived fibroblasts and keratinocytes.

    Science.gov (United States)

    Sheipouri, Diba; Grant, Ross; Bustamante, Sonia; Lovejoy, David; Guillemin, Gilles J; Braidy, Nady

    2015-06-01

    Acute UVB exposure triggers inflammation leading to the induction of indoleamine 2,3 dioxygenase (IDO1), one of the first enzymes in the kynurenine pathway (KP) for tryptophan degradation. However, limited studies have been undertaken to determine the catabolism of tryptophan within the skin. The aim of this study was two fold: (1) to establish if the administration of the proinflammatory cytokine interferon-gamma (IFN-γ) and/or UVB radiation elicits differential KP expression patterns in human fibroblast and keratinocytes; and (2) to evaluate the effect of KP metabolites on intracellular nicotinamide adenine dinucleotide (NAD(+) ) levels, and cell viability. Primary cultures of human fibroblasts and keratinocytes were used to examine expression of the KP at the mRNA level using qPCR, and at the protein level using immunocytochemistry. Cellular responses to KP metabolites were assessed by examining extracellular lactate dehydrogenase (LDH) activity and intracellular NAD(+) levels. Major downstream KP metabolites were analyzed using GC/MS and HPLC. Our data shows that the KP is fully expressed both in human fibroblasts and keratinocytes. Exposure to UVB radiation and/or IFN-γ causes significant changes in the expression pattern of downstream KP metabolites and enzymes. Exposure to various concentrations of KP metabolites showed marked differences in cell viability and intracellular NAD(+) production, providing support for involvement of the KP in the de novo synthesis of NAD(+) in the skin. This new information will have a significant impact on our understanding of the pathogenesis of UV related skin damage and the diagnosis of KP related disease states.

  14. Knowledge building insights on biomarkers of arsenic toxicity to keratinocytes and melanocytes.

    Science.gov (United States)

    Isokpehi, Raphael D; Udensi, Udensi K; Anyanwu, Matthew N; Mbah, Andreas N; Johnson, Matilda O; Edusei, Kafui; Bauer, Michael A; Hall, Roger A; Awofolu, Omotayo R

    2012-01-01

    Exposure to inorganic arsenic induces skin cancer and abnormal pigmentation in susceptible humans. High-throughput gene transcription assays such as DNA microarrays allow for the identification of biological pathways affected by arsenic that lead to initiation and progression of skin cancer and abnormal pigmentation. The overall purpose of the reported research was to determine knowledge building insights on biomarker genes for arsenic toxicity to human epidermal cells by integrating a collection of gene lists annotated with biological information. The information sets included toxicogenomics gene-chemical interaction; enzymes encoded in the human genome; enriched biological information associated with genes; environmentally relevant gene sequence variation; and effects of non-synonymous single nucleotide polymorphisms (SNPs) on protein function. Molecular network construction for arsenic upregulated genes TNFSF18 (tumor necrosis factor [ligand] superfamily member 18) and IL1R2 (interleukin 1 Receptor, type 2) revealed subnetwork interconnections to E2F4, an oncogenic transcription factor, predominantly expressed at the onset of keratinocyte differentiation. Visual analytics integration of gene information sources helped identify RAC1, a GTP binding protein, and TFRC, an iron uptake protein as prioritized arsenic-perturbed protein targets for biological processes leading to skin hyperpigmentation. RAC1 regulates the formation of dendrites that transfer melanin from melanocytes to neighboring keratinocytes. Increased melanocyte dendricity is correlated with hyperpigmentation. TFRC is a key determinant of the amount and location of iron in the epidermis. Aberrant TFRC expression could impair cutaneous iron metabolism leading to abnormal pigmentation seen in some humans exposed to arsenicals. The reported findings contribute to insights on how arsenic could impair the function of genes and biological pathways in epidermal cells. Finally, we developed visual analytics

  15. Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome

    Science.gov (United States)

    Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-01-01

    Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells. PMID:27529420

  16. Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

    Science.gov (United States)

    Alvarez-Salas, L M; Velazquez, A; Lopez-Bayghen, E; Woodworth, C D; Garrido, E; Gariglio, P; DiPaolo, J A

    1995-05-01

    Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

  17. Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

    Science.gov (United States)

    Chernyavsky, Alex; Chen, Yumay; Wang, Ping H; Grando, Sergei A

    2015-11-01

    The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, β2 and β4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity.

  18. Bullous pemphigoid and linear IgA dermatosis sera recognize a similar 120-kDa keratinocyte collagenous glycoprotein with antigenic cross-reactivity to BP180

    NARCIS (Netherlands)

    Pas, HH; Kloosterhuis, GJ; Heeres, K; vanderMeer, JB; Jonkman, MF

    1997-01-01

    Circulating IgG from a large subset of bullous pein; phigoid (SP) patients reacted on immunoblot with a 120-kDa protein in conditioned keratinocyte culture medium and in keratinocyte cell extracts, A protein with a similar molecular weight was recognized by circulating IgA from a subset of patients

  19. Protein engineering,expression,and activity of a novel fusion protein possessing keratinocyte growth factor 2 and fibronectin

    Institute of Scientific and Technical Information of China (English)

    Wonmo Kang; Junhyeog Jang

    2009-01-01

    Growth factor-induced proliferation and differentiation often require adhesion of cells to the extracellular matrix proteins such as fibronectin(FN).In this study,we aimed to investigate the effect of protein engineering of the keratinocyte growth factor 2(KGF2)fused to the FN on the mitogenic activity of KGF2.The fusion protein(KGF2-FN10),which was expressed in Escherichia coli,showed significantly enhanced mitogenic activity of KGF2 on human keratinocytes.Moreover,KGF2-FN10 fusion protein showed significantly increased activity to differentiate keratinocytes from native KGF2.In conclusion,these results suggest that KGF2-FN10 fusion protein has certain advantages over native KGF2 and may offer a novel strategy to potentiate the therapeutic effect of KGF2.

  20. [Mucocutaneous diseases and murine models with death of keratinocytes induced by lichenoid tissue reaction/interface dermatitis].

    Science.gov (United States)

    Okiyama, Naoko

    2015-01-01

    A set of histopathological elements with death of epidermal basal cell layer keratinocytes along with inflammatory cell infiltration distinguishes lichenoid tissue reaction (LTR)/interface dermatitis (IFD) from other inflammatory mucocutaneous diseases. The LTR/IFD can be seen in skin disorders like as lichen planus, acute graft-versus-host disease, lupus erythematosus, dermatomyositis, and toxic epidermal necrolysis/Stevesn-Johnson syndrome. Clinical and basic researches suggested that cytotoxic CD8 T cells producing interferon-γ and FasL are final effector cells to cause apoptosis of keratinocyte. Some murine models of LTR/IFD have been established, for example, LTR/IFD reactions of keratinocyte-specific ovalbumin (OVA)-transgenic mice after OVA-specific T-cell-receptor(+)CD8 T cells. By analysis of the murine model, a new class of immunosuppressant, a JAK inhibitor, has been suggested as a new candidate for treatment of LTR/IFD.

  1. Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

    Directory of Open Access Journals (Sweden)

    Sharon Wong

    2013-12-01

    Full Text Available Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays.Methods: Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA. Haematoxylin and eosin (H&E immunostaining was performed to study the morphological changes in the skin cells. Results: Radiation is mostly absorbed by the epidermal layers and observed to damage the epidermal keratinocytes leading to the activation of apoptotic proteins. Apoptotic proteins (p53, p21 and PCNA were confirmed to be up-regulated in radiation exposed skin cells as compared to normal skin cells with no radiation. There is strong correlation of apoptotic protein expressions with increased radiation dosage and dose fractionation. Statistical analysis with ANOVA revealed a significant increase of PCNA and p21 expression with increased radiation dosage and dose fractionation (p < 0.05. Immunohistochemically, 14 % (range 10.71% to 17.29% of the keratinocytes were positive for PCNA and 22.5% (range 18.28% to 27.2% for p21 after 2Gy of irradiation.  The most widespread, intense and uniform staining for PCNA

  2. Bach1-dependent and -independent Regulation of Heme Oxygenase-1 in Keratinocytes*

    OpenAIRE

    2010-01-01

    Bach1 is a member of the basic leucine zipper transcription factor family, and the Bach1/small Maf heterodimer specifically represses transcriptional activity directed by the Maf recognition element (MARE). Because Bach1 is a repressor of the oxidative stress response, we examined the function(s) of Bach1 in keratinocytes subjected to oxidative stress. Oxidative stress induced by H2O2 led to an increase in MARE activity and expression of heme oxygenase-1 (HO-1), an inducible antioxidant defen...

  3. Reciprocal role of vitamin D receptor on β-catenin regulated keratinocyte proliferation and differentiation.

    Science.gov (United States)

    Hu, Lizhi; Bikle, Daniel D; Oda, Yuko

    2014-10-01

    The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), suppresses the proliferation while promoting the differentiation of keratinocytes through the vitamin D receptor (VDR). β-Catenin, on the other hand, promotes proliferation and blocks epidermal differentiation, although it stimulates hair follicle differentiation. In intestinal epithelia VDR binds β-catenin and blocks its proliferative effects. In this study we investigated the role of 1,25(OH)2D3/VDR on β-catenin regulated gene transcription during keratinocyte proliferation and differentiation. 1,25(OH)2D3 suppressed promoter reporter activity driven by synthetic and natural TCF/β-catenin response elements. Over-expression of VDR further suppressed these TCF/β-catenin promoter activities. 1,25(OH)2D3 also suppressed the mRNA expression of the β-catenin regulated gene Gli1 through VDR. These data were consistent with our previous observations that VDR silencing resulted in keratinocyte hyperproliferation with increased expression of Gli1 in vitro, whereas VDR null skin showed hyperproliferation in vivo. In contrast, 1,25(OH)2D3 induced expression of another β-catenin regulated gene, PADI1, important for both epidermal and hair follicle differentiation. Deletion of VDR resulted in defects in hair differentiation in vivo, with decreased expression of β-catenin regulated hair differentiation genes such as PADI1, hair keratin KRT31 and calcium binding protein S100a3. These genes possess vitamin D response elements (VDRE) adjacent to TCF/β-catenin response elements and are regulated by both VDR and β-catenin signaling. Therefore, we propose that VDR and β-catenin interact reciprocally to promote VDR stimulation of genes involved with differentiation that contain both VDR and β-catenin response elements while inhibiting β-catenin stimulation of genes involved with proliferation. Thus the major finding of this study is that while 1,25(OH)2D3/VDR inhibits the actions of β-catenin to

  4. 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.

    OpenAIRE

    Bikle, D D; Nemanic, M K; Gee, E; Elias, P

    1986-01-01

    Human foreskin keratinocytes in vitro metabolize 25-hydroxyvitamin D3 to a number of metabolites, including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This metabolite remains mostly within the cell and does not accumulate in the medium under the conditions of these experiments. With time, 1,25(OH)2D3 is catabolized, and more polar metabolites appear in both the cells and the medium. The production of 1,25(OH)2D3 has an apparent Michaelis constant (Km) for 25-hydroxyvitamin D3 of 5.4 X 10(-8) M. ...

  5. Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bachmann, M.; Chang, S.; Slor, H.; Kukulies, J.; Mueller, W.E. (Universitaet, Mainz (Germany, F.R.))

    1990-12-01

    During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with epidermal growth factor receptors.

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

    Science.gov (United States)

    De Abrew, K Nadira; Thomas-Virnig, Christina L; Rasmussen, Cathy A; Bolterstein, Elyse A; Schlosser, Sandy J; Allen-Hoffmann, B Lynn

    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.

  7. Activation of TLR3 in keratinocytes increases expression of genes involved in formation of the epidermis, lipid accumulation and epidermal organelles

    Science.gov (United States)

    Borkowski, Andrew W.; Park, Kyungho; Uchida, Yoshikazu; Gallo, Richard L.

    2013-01-01

    Injury to the skin, and the subsequent release of non-coding double-stranded RNA from necrotic keratinocytes, has been identified as an endogenous activator of Toll-like receptor 3 (TLR3). Since changes in keratinocyte growth and differentiation follow injury, we hypothesized that TLR3 might trigger some elements of the barrier repair program in keratinocytes. Double-stranded RNA was observed to induce TLR3-dependent increases in human keratinocyte mRNA abundance for ABCA12 (ATP-binding cassette, sub-family A, member 12), glucocerebrosidase, acid sphingomyelinase, and transglutaminase 1. Additionally, treatment with double-stranded RNA resulted in increases in sphingomyelin and morphologic changes including increased epidermal lipid staining by oil-red O and TLR3-dependent increases in lamellar bodies and keratohyalin granules. These observations show that double-stranded RNA can stimulate some events in keratinocytes that are important for skin barrier repair and maintenance. PMID:23353987

  8. Triterpenoid α-amyrin stimulates proliferation of human keratinocytes but does not protect them against UVB damage

    DEFF Research Database (Denmark)

    Biskup, Edyta; Gołębiowski, Marek; Gniadecki, Robert;

    2012-01-01

    Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (HaCaT) by a......Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (Ha...

  9. Increased Skin Tumor Incidence and Keratinocyte Hyper-Proliferation in a Mouse Model of Down Syndrome.

    Science.gov (United States)

    Yang, Annan; Currier, Duane; Poitras, Jennifer L; Reeves, Roger H

    2016-01-01

    Down syndrome (DS) is a genetic disorder caused by the presence of an extra copy of human chromosome 21 (Hsa21). People with DS display multiple clinical traits as a result of the dosage imbalance of several hundred genes. While many outcomes of trisomy are deleterious, epidemiological studies have shown a significant risk reduction for most solid tumors in DS. Reduced tumor incidence has also been demonstrated in functional studies using trisomic DS mouse models. Therefore, it was interesting to find that Ts1Rhr trisomic mice developed more papillomas than did their euploid littermates in a DMBA-TPA chemical carcinogenesis paradigm. Papillomas in Ts1Rhr mice also proliferated faster. The increased proliferation was likely caused by a stronger response of trisomy to TPA induction. Treatment with TPA caused hyperkeratosis to a greater degree in Ts1Rhr mice than in euploid, reminiscent of hyperkeratosis seen in people with DS. Cultured trisomic keratinocytes also showed increased TPA-induced proliferation compared to euploid controls. These outcomes suggest that altered gene expression in trisomy could elevate a proliferation signalling pathway. Gene expression analysis of cultured keratinocytes revealed upregulation of several trisomic and disomic genes may contribute to this hyperproliferation. The contributions of these genes to hyper-proliferation were further validated in a siRNA knockdown experiment. The unexpected findings reported here add a new aspect to our understanding of tumorigenesis with clinical implications for DS and demonstrates the complexity of the tumor repression phenotype in this frequent condition.

  10. Gene Expression Response of Trichophyton rubrum during Coculture on Keratinocytes Exposed to Antifungal Agents

    Science.gov (United States)

    Komoto, Tatiana Takahasi; Bitencourt, Tamires Aparecida; Silva, Gabriel; Beleboni, Rene Oliveira; Marins, Mozart; Fachin, Ana Lúcia

    2015-01-01

    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. PMID:26257814

  11. 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.

  12. Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

    Science.gov (United States)

    Miniaci, Maria Concetta; Irace, Carlo; Capuozzo, Antonella; Piccolo, Marialuisa; Di Pascale, Antonio; Russo, Annapina; Lippiello, Pellegrino; Lepre, Fabio; Russo, Giulia; Santamaria, Rita

    2016-02-01

    L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

  13. 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.

  14. Analyses of the Secondary Particle Radiation and the DNA Damage it Causes to Human Keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lebel E. A.; Tafrov S.; Rusek, A.; Sivertz, M. B.; Yip, K.; Thompson, K. H.

    2011-11-01

    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.

  15. Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

    Directory of Open Access Journals (Sweden)

    Xiaoru Zhang

    Full Text Available 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.

  16. Interferon-γ Protects from Staphylococcal Alpha Toxin-Induced Keratinocyte Death through Apolipoprotein L1.

    Science.gov (United States)

    Brauweiler, Anne M; Goleva, Elena; Leung, Donald Y M

    2016-03-01

    Staphylococcus aureus is a bacterial pathogen that frequently infects the skin, causing lesions and cell destruction through its primary virulence factor, alpha toxin. Here we show that interferon gamma (IFN-?) protects human keratinocytes from cell death induced by staphylococcal alpha toxin. We find that IFN-? prevents alpha toxin binding and reduces expression of the alpha toxin receptor, a disintegrin and metalloproteinase 10 (ADAM10). We determine that the mechanism for IFN-?-mediated resistance to alpha toxin involves the induction of autophagy, a process of cellular adaptation to sublethal damage. We find that IFN-? potently stimulates activation of the primary autophagy effector, light chain 3 (LC3). This process is dependent on upregulation of apolipoprotein L1. Depletion of apolipoprotein L1 by small interfering RNA significantly increases alpha toxin-induced lethality and inhibits activation of light chain 3. We conclude that IFN-? plays a significant role in protecting human keratinocytes from the lethal effects of staphylococcal alpha toxin through apolipoprotein L1-induced autophagy.

  17. MALT1 Protease Activity Controls the Expression of Inflammatory Genes in Keratinocytes upon Zymosan Stimulation.

    Science.gov (United States)

    Schmitt, Anja; Grondona, Paula; Maier, Tabea; Brändle, Marc; Schönfeld, Caroline; Jäger, Günter; Kosnopfel, Corinna; Eberle, Franziska C; Schittek, Birgit; Schulze-Osthoff, Klaus; Yazdi, Amir S; Hailfinger, Stephan

    2016-04-01

    The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.

  18. Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

    Science.gov (United States)

    Secor, Patrick R; Jennings, Laura K; James, Garth A; Kirker, Kelly R; Pulcini, Elinor Delancey; McInnerney, Kate; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K; Bothner, Brian; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    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.

  19. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

    Science.gov (United States)

    Konger, R L; Malaviya, R; Pentland, A P

    1998-02-04

    We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

  20. Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation.

    Science.gov (United States)

    Sommer, Anselm; Fries, Anja; Cornelsen, Isabell; Speck, Nancy; Koch-Nolte, Friedrich; Gimpl, Gerald; Andrä, Jörg; Bhakdi, Sucharit; Reiss, Karina

    2012-07-06

    Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.

  1. Hyperthermia-induced micronucleus formation in a human keratinocyte cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hintzsche, Henning; Riese, Thorsten [Universitaet Wuerzburg, Institut fuer Pharmakologie und Toxikologie, Versbacher Str. 9, 97078 Wuerzburg (Germany); Stopper, Helga, E-mail: Stopper@toxi.uni-wuerzburg.de [Universitaet Wuerzburg, Institut fuer Pharmakologie und Toxikologie, Versbacher Str. 9, 97078 Wuerzburg (Germany)

    2012-10-15

    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 Degree-Sign C to 42 Degree-Sign 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 Degree-Sign C or higher. Cell proliferation was unaffected up to 40 Degree-Sign C and decreased at 41 Degree-Sign C and 42 Degree-Sign C. Expression of the heat shock protein Hsp70 was elevated, particularly at temperatures of 40 Degree-Sign 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.

  2. Asian dust storm particles induce a broad toxicological transcriptional program in human epidermal keratinocytes.

    Science.gov (United States)

    Choi, Hyun; Shin, Dong Wook; Kim, Wonnyon; Doh, Seong-Jae; Lee, Soo Hwan; Noh, Minsoo

    2011-01-15

    Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongolian deserts results in increased incidence of a range of diseases including asthma, contact dermatitis and conjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast of North America. In order to study toxicological mechanisms in human skin, we evaluated the effects of dust particles collected during Asian dust storms (Asian dust particles) on gene expression in human epidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased gene expressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of aryl hydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcription of the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggesting that Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that protein extract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes to the increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggest that Asian dust particles can exert toxicological effects on human skin through the activation of the cellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines, and changes in the expression of proteins essential in normal epidermal differentiation.

  3. Enrichment of breast cancer stem cells using a keratinocyte serum-free medium

    Institute of Scientific and Technical Information of China (English)

    LIU Zhen-zhen; CHEN Ping; LU Zhen-duo; CUI Shu-de; DONG Zi-ming

    2011-01-01

    Background Keratinocyte serum-free medium (K-SFM) is a defined medium used to support the growth of primary keratinocytes and embryonic stem cell. The aim of this research was to optimize enrichment of breast cancer stem cells (CSCs) using K-SFM.Methods A K-SFM was used to enrich CSCs from two breast cancer cell lines and a primary culture of breast cancer.RPMI-1640 supplemented with 10% fetal calf serum (FCS) was used as a control. CSCs were identified with flow cytometry using CD44+/CD24-as molecular markers. The expression of a variety of CSC markers (Oct-4, ABCG2, Nanog,N-cadherin, and E-cadherin) was analyzed with real-time PCR.Results Much higher percentage of CSCs was achieved with K-SFM: 17.3% for MCF-7 cells, 17.4% for SKBR-3, and 20.0% for primary breast cancer culture. Less than 1% CSC was achieved using RPMI-1640 supplemented with 10% FCS. In comparison to the CSCs obtained with RPMI-1640, CSCs in the K-SFM expressed higher levels of Oct-4,ABCG2, Nanog and N-cadherin, and lower level of E-cadherin.Conclusion K-SFM is an optimal culture medium to maintain and to enrich breast CSCs.

  4. 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.

  5. Keratinocyte growth factor improves alterations of lung permeability and bronchial epithelium in allergic rats.

    Science.gov (United States)

    Tillie-Leblond, I; Gosset, P; Le Berre, R; Janin, A; Prangère, T; Tonnel, A B; Guery, B P H

    2007-07-01

    Chronic allergic asthma is associated with marked inflammatory reaction, microvascular leakage and epithelium injury. As previously shown in a rat model of chronic asthma, these alterations increase lung permeability and distal airway fluid clearance. Keratinocyte growth factor (KGF) has been shown to induce epithelial cell proliferation and to protect from acute lung injuries. Therefore, the current authors evaluated the potential role of KGF treatment on lung permeability and airway inflammation in rats with chronic asthma. KGF (1 mg x kg(-1)) was administered intravenously before the last ovalbumin (OVA) challenge in sensitised rats. Permeability was assessed by the leak of radiolabelled albumin from the alveolar and systemic compartments. Histopathological analysis was also performed. Treatment with KGF decreased the leak of both markers and decreased the level of extravascular lung water in sensitised rats challenged with OVA. KGF treatment also reduced the inflammatory cell number in bronchoalveolar lavage fluid but not in bronchial mucosa. KGF markedly limited the allergen-induced alterations in epithelium integrity and the expression of the intercellular junction proteins beta-catenin and zonula occludens protein-1. In conclusion, keratinocyte growth factor administration markedly limits lung permeability and airway inflammation, an effect associated with a decrease in epithelium alterations during chronic allergic asthma. These data open new prospects in the therapeutic strategy of asthma.

  6. Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

    Science.gov (United States)

    Witzmann, F A; Monteiro-Riviere, N A; Inman, A O; Kimpel, M A; Pedrick, N M; Ringham, H N; Riviere, J E

    2005-12-30

    Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

  7. 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.

  8. 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.

  9. Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    C.M.A. Reijnders; A. van Lier; S. Roffel; D. Kramer; R.J. Scheper; S. Gibbs

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

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

    NARCIS (Netherlands)

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

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  11. Gene Editing for the Efficient Correction of a Recurrent COL7A1 Mutation in Recessive Dystrophic Epidermolysis Bullosa Keratinocytes.

    Science.gov (United States)

    Chamorro, Cristina; Mencía, Angeles; Almarza, David; Duarte, Blanca; Büning, Hildegard; Sallach, Jessica; Hausser, Ingrid; Del Río, Marcela; Larcher, Fernando; Murillas, Rodolfo

    2016-04-05

    Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.

  12. Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes

    NARCIS (Netherlands)

    El Ghalbzouri, A; Jonkman, MF; Dijkman, R; Ponec, M

    2005-01-01

    This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence

  13. Triterpenoid α-amyrin stimulates proliferation of human keratinocytes but does not protect them against UVB damage

    DEFF Research Database (Denmark)

    Biskup, Edyta; Gołębiowski, Marek; Gniadecki, Robert;

    2012-01-01

    Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (HaCaT) by a...

  14. CCN1 promotes IL-1β production in keratinocytes by activating p38 MAPK signaling in psoriasis

    Science.gov (United States)

    Sun, Yue; Zhang, Jie; Zhai, Tianhang; Li, Huidan; Li, Haichuan; Huo, Rongfen; Shen, Baihua; Wang, Beiqing; Chen, Xiangdong; Li, Ningli; Teng, Jialin

    2017-01-01

    CCN1, an extracellular protein also known as cysteine-rich protein 61 (Cyr61), is a novel pro-inflammatory factor involved in the pathogenesis of rheumatoid arthritis. As an inflammatory disease, psoriasis is characterized by keratinocyte activation-induced epidermal hyperplasia and cytokine-mediated inflammation. We demonstrated in our previous study that CCN1 promoted keratinocyte activation in psoriasis. However, the role of CCN1 in regulating inflammation in psoriasis is still unknown. Here, we showed that CCN1 increased inflammatory cytokine IL-1β production in keratinocytes. Furthermore, endogenous ATP and caspase-1 were required for mature IL-1β production stimulated by CCN1 in keratinocytes. After binding to the receptor of integrin α6β1, CCN1 activated the downstream p38 MAPK signaling pathway, thus inducing the expression of IL-1β. In addition, we inhibited CCN1 function in mouse models of psoriasis, and decreased IL-1β production was observed in vivo. Overall, we showed that CCN1 increased IL-1β production via p38 MAPK signaling, indicating a role for CCN1 protein in regulating inflammation in psoriasis. PMID:28266627

  15. Inhibition of mTOR by apigenin in UVB-irradiated keratinocytes: A new implication of skin cancer prevention.

    Science.gov (United States)

    Bridgeman, Bryan B; Wang, Pu; Ye, Boping; Pelling, Jill C; Volpert, Olga V; Tong, Xin

    2016-05-01

    Ultraviolet B (UVB) radiation is the major environmental risk factor for developing skin cancer, the most common cancer worldwide, which is characterized by aberrant activation of Akt/mTOR (mammalian target of rapamycin). Importantly, the link between UV irradiation and mTOR signaling has not been fully established. Apigenin is a naturally occurring flavonoid that has been shown to inhibit UV-induced skin cancer. Previously, we have demonstrated that apigenin activates AMP-activated protein kinase (AMPK), which leads to suppression of basal mTOR activity in cultured keratinocytes. Here, we demonstrated that apigenin inhibited UVB-induced mTOR activation, cell proliferation and cell cycle progression in mouse skin and in mouse epidermal keratinocytes. Interestingly, UVB induced mTOR signaling via PI3K/Akt pathway, however, the inhibition of UVB-induced mTOR signaling by apigenin was not Akt-dependent. Instead, it was driven by AMPK activation. In addition, mTOR inhibition by apigenin in keratinocytes enhanced autophagy, which was responsible, at least in part, for the decreased proliferation in keratinocytes. In contrast, apigenin did not alter UVB-induced apoptosis. Taken together, our results indicate the important role of mTOR inhibition in UVB protection by apigenin, and provide a new target and strategy for better prevention of UV-induced skin cancer.

  16. Expression of TNF-related apoptosis-inducing ligand (TRAIL in keratinocytes mediates apoptotic cell death in allogenic T cells

    Directory of Open Access Journals (Sweden)

    Kiefer Paul

    2009-11-01

    Full Text Available Abstract The objective of the present study was to evaluate the aptitude of TRAIL gene expression for inducing apoptosis in co-cultivated T-cells. This should allow preparing a strategy for the development of a durable, allogenic skin substitute based on the induction of an immune-privileged transplant. In order to counteract the significant potential of rejection in transplanted allogenic keratinocytes, we created a murine keratinocyte cell line which expressed TRAIL through stable gene transfer. The exogenic protein was localized on the cellular surface and was not found in soluble condition as sTRAIL. Contact to TRAIL expressing cells in co-culture induced cell death in sensitive Jurkat-cells, which was further intensified by lymphocyte activation. This cytotoxic effect is due to the induction of apoptosis. We therefore assume that the de-novo expression of TRAIL in keratinocytes can trigger apoptosis in activated lymphocytes and thus prevent the rejection of keratinocytes in allogenic, immune-privileged transplants.

  17. 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.

  18. 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...

  19. 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)

  20. New Insights into the Molecular Distinction of Dysplastic Nevi and Common Melanocytic Nevi-Highlighting the Keratinocyte-Melanocyte Relationship.

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    Eliades, Philip; Tsao, Hensin

    2016-10-01

    Mitsui et al. approach the problem of differentiating dysplastic nevi from common melanocytic nevi through a molecular lens. Whereas most of the literature on this topic shines the spotlight toward melanocytes, the focus of this paper is shifted to the tumor microenvironment. Using microarrays, reverse transcriptase-PCR, and immunohistochemistry, their results emphasize the role of keratinocyte dysplasia within dysplastic nevi.

  1. Dysregulation of suppressor of cytokine signaling 3 in keratinocytes causes skin inflammation mediated by interleukin-20 receptor-related cytokines.

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    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.

  2. The Staphylococcus aureus extracellular adherence protein promotes bacterial internalization by keratinocytes independent of fibronectin-binding proteins.

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    Bur, Stephanie; Preissner, Klaus T; Herrmann, Mathias; Bischoff, Markus

    2013-08-01

    Staphylococcus aureus, the leading causal pathogen of skin infections, is strongly associated with skin atopy, and a number of bacterial adhesins allow the microbe to adhere to and invade eukaryotic cells. One of these adhesive molecules is the multifunctional extracellular adherence protein (Eap), which is overexpressed in situ in authentic human wounds and was shown to delay wound healing in experimental models. Yet, its role during invasion of keratinocytes is not clearly defined. By using a gentamicin/lysostaphin protection assay we demonstrate here that preincubation of HaCaT cells or primary keratinocytes with Eap results in a concentration-dependent significant increase in staphylococcal adhesion, followed by an even more pronounced internalization of bacteria by eukaryotic cells. Flow cytometric analysis revealed that Eap increased both the number of infected eukaryotic cells and the bacterial load per infected cell. Moreover, treatment of keratinocytes with Eap strongly enhanced the internalization of coagulase-negative staphylococci, as well as of E. coli, and markedly promoted staphylococcal invasion into extended-culture keratinocytes, displaying expression of keratin 10 and involucrin as differentiation markers. Thus, wound-related staphylococcal Eap may provide a major cellular invasin function, thereby enhancing the pathogen's ability to hide from the host immune system during acute and chronic skin infection.

  3. 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

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    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.

  4. 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

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    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.

  5. 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.

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    Bhatia, Vikram; Goel, Madhu Mati; Makker, Annu; Tewari, Shikha; Yadu, Alka; Shilpi, Priyanka; Kumar, Sandeep; Agarwal, S P; Goel, Sudhir K

    2014-01-01

    Promoter methylation and relative gene expression of O(6)-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.

  6. Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

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    Lyrio, Eloah C D; Campos-Souza, Ivy C; Corrêa, Luiz C D; Lechuga, Guilherme C; Verícimo, Maurício; Castro, Helena C; Bourguignon, Saulo C; Côrte-Real, Suzana; Ratcliffe, Norman; Declercq, Wim; Santos, Dilvani O

    2015-07-01

    Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

  7. 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. PMID:25025687

  8. 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.

  9. Induction of Interleukin-22 (IL-22) production in CD4+ T Cells by IL-17A Secreted from CpG-Stimulated Keratinocytes

    Science.gov (United States)

    Li, Zheng Jun; Choi, Dae-Kyoung; Sohn, Kyung-Cheol; Lim, Seul Ki; Im, Myung; Lee, Young; Seo, Young-Joon; Kim, Chang Deok

    2016-01-01

    Background Interleukin-17A (IL-17A) is mainly secreted from Th17 cells that are activated by various stimuli including CpG oligodeoxynucleotide, a Toll-like receptor 9 (TLR9) ligand. Recently, it has been demonstrated that keratinocytes play an important role in the pathogenesis of psoriasis. Objective To investigate the potential role of keratinocytes, we examined whether TLR9 ligand CpG induces IL-17A expression in keratinocytes. Methods We used HaCaT keratinocytes as a model system, and determined CpG-induced IL-17A using enzyme-linked immunosorbent assay and Western blot. Results When HaCaT keratinocytes were treated with CpG, the expression of several cytokines including IL-17A, tumor necrosis factor-α and CCL20 was markedly increased. Treatment with nuclear factor (NF)-κB inhibitor significantly blocked the CpG-induced IL-17A production, indicating that CpG induced IL-17A expression through the NF-κB signaling pathway. In addition, IL-17A secreted from keratinocytes stimulated the CD4+ T cells, resulting in strong induction of IL-22 production. Conclusion Since IL-22 is an important mediator for psoriatic inflammation, our data suggest that keratinocytes can participate in the pathogenesis of psoriasis via the TLR9-dependent IL-17A production. PMID:27746637

  10. Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

    Science.gov (United States)

    Xu, Song; Li, Li; Li, Min; Zhang, Mengli

    2017-01-01

    The mechanistic target of Rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors Rapamycin, everolimus, Torin 1, and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with Rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by Rapamycin, everolimus, or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the downregulation of BiP and PERK, activation of Histone H2A and JNK, and cleavage of caspase-3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB triggered cellular response.

  11. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

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    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.

  12. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

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    Herbert, Katharine J. [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia); Holloway, Adele [Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000 (Australia); Cook, Anthony L. [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia); Chin, Suyin P. [Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000 (Australia); Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia)

    2014-11-15

    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. Keratinocyte-targeted overexpression of the glucocorticoid receptor delays cutaneous wound healing.

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    Ana Sanchis

    Full Text Available Delayed wound healing is one of the most common secondary adverse effects associated to the therapeutic use of glucocorticoid (GC analogs, which act through the ligand-dependent transcription factor GC-receptor (GR. GR function is exerted through DNA-binding-dependent and -independent mechanisms, classically referred to as transactivation (TA and transrepression (TR. Currently both TA and TR are thought to contribute to the therapeutical effects mediated by GR; however their relative contribution to unwanted side effects such as delayed wound healing is unknown. We evaluated skin wound healing in transgenic mice with keratinocyte-restricted expression of either wild type GR or a mutant GR that is TA-defective but efficient in TR (K5-GR and K5-GR-TR mice, respectively. Our data show that at days (d 4 and 8 following wounding, healing in K5-GR mice was delayed relative to WT, with reduced recruitment of granulocytes and macrophages and diminished TNF-α and IL-1β expression. TGF-β1 and Kgf expression was repressed in K5-GR skin whereas TGF-β3 was up-regulated. The re-epithelialization rate was reduced in K5-GR relative to WT, as was formation of granulation tissue. In contrast, K5-GR-TR mice showed delays in healing at d4 but re-established the skin breach at d8 concomitant with decreased repression of pro-inflammatory cytokines and growth factors relative to K5-GR mice. Keratinocytes from both transgenic mice closed in vitro wounds slower relative to WT, consistent with the in vivo defects in cell migration. Overall, the delay in the early stages of wound healing in both transgenic models is similar to that elicited by systemic treatment with dexamethasone. Wound responses in the transgenic keratinocytes correlated with reduced ERK activity both in vivo and in vitro. We conclude that the TR function of GR is sufficient for negatively regulating early stages of wound closure, while TA by GR is required for delaying later stages of healing.

  14. Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

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    Sharon Wong

    2013-12-01

    Full Text Available Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays. Methods: Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA. Haematoxylin and eosin (H&E immunostaining was performed to study the morphological changes in the skin cells. Results: Radiation is mostly absorbed by the epidermal layers and observed to damage the epidermal keratinocytes leading to the activation of apoptotic proteins. Apoptotic proteins (p53, p21 and PCNA were confirmed to be up-regulated in radiation exposed skin cells as compared to normal skin cells with no radiation. There is strong correlation of apoptotic protein expressions with increased radiation dosage and dose fractionation. Statistical analysis with ANOVA revealed a significant increase of PCNA and p21 expression with increased radiation dosage and dose fractionation (p < 0.05. Immunohistochemically, 14 % (range 10.71% to 17.29% of the keratinocytes were positive for PCNA and 22.5% (range 18.28% to 27.2% for p21 after 2Gy of irradiation. The most widespread, intense and uniform staining for PCNA and

  15. Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

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    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

  16. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Buskermolen, Jeroen K; Reijnders, Christianne M A; Spiekstra, Sander W; Steinberg, Thorsten; Kleverlaan, Cornelis J; Feilzer, Albert J; Bakker, Astrid D; Gibbs, Susan

    2016-08-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  17. 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; Filon, Francesca Larese

    2015-07-17

    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.

  18. Modulation of the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated human keratinocytes and melanocytes.

    Science.gov (United States)

    Decean, H; Perde-Schrepler, M; Tatomir, C; Fischer-Fodor, E; Brie, I; Virag, P

    2013-10-01

    The human epidermis exerts immunoregulatory functions through the variety of cytokines and other molecules elaborated by keratinocytes and melanocytes. Their constitutive production is very low; however, considerably increased upon stimulation. In vivo, keratinocytes and melanocytes have a typical exposure in the skin, referred as melanocyte epidermal unit. In the present study we co-cultivated these cells in vitro proposing to elucidate some communication links in close cell-to-cell association. We assessed the amounts of IL-6, IL-8, and matrix metalloproteinases (MMP-2 and MMP-9) in individually and co-cultured cells, exposed or not to UVB radiation. Normal human epidermal keratinocytes and melanocytes were grown in specific media and supplements. Cells were exposed to UVB radiation (100 mJ/cm(2)) to create comparable stress to the environmental one. Cytokines were determined with ELISA and confirmed with Western blot and metalloproteinases with gel zimography. Pure cultures of keratinocytes and melanocytes released low amounts of cytokines and metalloproteinases, these secretions being enhanced by UVB irradiation. In co-cultures, the cell-to-cell proximity triggered signals which markedly augmented the cytokines' secretions, whereas metalloproteinases were down-regulated. UVB irradiation did not influence either of these secretions in co-cultures. Concurrently with the highest levels of the pro-inflammatory cytokines, MMP-9 was up-regulated creating pro-inflammatory conditions and premises for changes in cellular survival, differentiation and phenotype. A complex network of interactions occurred between keratinocytes and melanocytes in co-cultures, resulting in modulated pro-inflammatory cytokines and metalloproteinases productions. Therefore, any disturbances in the microenvironmental signaling system and its molecular constituents may result in inflammation or even tumorigenesis in the epidermis.

  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. 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.

  1. Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis

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    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.

  2. MiR-26a inhibits proliferation and migration of HaCaT keratinocytes through regulating PTEN expression.

    Science.gov (United States)

    Yu, Nanze; Yang, Yang; Li, Xiongwei; Zhang, Mingzi; Huang, Jiuzuo; Wang, Xiaojun; Long, Xiao

    2016-12-05

    MicroRNAs (miRNAs) have been shown to be associated with differentiation, migration and apoptosis in keratinocyte. Although it has been reported that microRNA-26a (miR-26a) plays important roles in tumor cells, its biological functions in keratinocytes are still not well elucidated. In this study, we confirmed expression of miR-26a in human keratinocytes using RT-PCR and further studied the role of miR-26a in cell proliferation and cell migration. Ectopic expression of MiR-26a mimic or inhibitor increased or decreased miR-26a expression respectively in HaCaT cells. Proliferation of HaCaT keratinocyte can be suppressed or promoted by overexpression or down-expression of miR-26a. In scratch wound-healing assay and Boyden chamber cell migration assay, upregulating miR-26a expression blocked cell migration, while downregulating miR-26a expression enhanced the migration. Using quantitative RT-PCR (qRT-PCR) and western blot, we further discovered that both mRNA and protein level of phosphatase and tensin homolog deleted from chromosome 10(PTEN) were regulated by miR-26a in HaCaT cells. Meanwhile the level of active form of AKT was also regulated by the miR-26a. In rescue experiment, knockdown of PTEN in the miR-26a mimic transduced cells recovered the migration ability of HaCaT cells. Together these results suggest that miR-26a modulates the proliferation and migration of keratinocytes via regulating PTEN/AKT signaling pathway.

  3. 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

  4. 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

    using a combination of reverse transcriptase polymerase chain reaction, Northern and Western blotting, and immunohistochemistry. PPARdelta was the predominant PPAR subtype in human keratinocytes and highly expressed in basal cells and suprabasal cells. Induction of PPARalpha and PPARgamma expression...... nuclear receptor corepressor and silence mediator for retinoid and thyroid hormone receptors. We critically evaluated the effects of selective PPAR ligands and a synthetic fatty acid analog, tetradecylthioacetic acid. Tetradecylthioacetic acid activated all human PPAR subtypes in the ranking order...... a dose-dependent induction was observed with L165041. Simultaneous addition of L165041 and BRL49653 synergistically induced strong involucrin expression. Additionally, L165041 potently induced CD36 mRNA expression. Administration of tetradecylthioacetic acid resulted in a dramatic decrease...

  5. 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.

  6. Low-level laser irradiation promotes the proliferation and maturation of keratinocytes during epithelial wound repair

    Science.gov (United States)

    Sperandio, Felipe F.; Simões, Alyne; Corrêa, Luciana; Aranha, Ana Cecília C.; Giudice, Fernanda S.; Hamblin, Michael R.; Sousa, Suzana C.O.M.

    2015-01-01

    Low-level laser therapy (LLLT) has been extensively employed to improve epithelial wound healing, though the exact response of epithelium maturation and stratification after LLLT is unknown. Thus, this study aimed to assess the in vitro growth and differentiation of keratinocytes (KCs) and in vivo wound healing response when treated with LLLT. Human KCs (HaCaT cells) showed an enhanced proliferation with all the employed laser energy densities (3, 6 and 12 J/cm2, 660nm, 100mW), together with an increased expression of Cyclin D1. Moreover, the immunoexpression of proteins related to epithelial proliferation and maturation (p63, CK10, CK14) all indicated a faster maturation of the migrating KCs in the LLLT-treated wounds. In that way, an improved epithelial healing was promoted by LLLT with the employed parameters; this improvement was confirmed by changes in the expression of several proteins related to epithelial proliferation and maturation. PMID:25411997

  7. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    Science.gov (United States)

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses.

  8. Arabinogalactan Proteins from Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes in vitro.

    Science.gov (United States)

    Zahid, Abderrakib; Despres, Julie; Benard, Magalie; Nguema-Ona, Eric; Leprince, Jerome; Vaudry, David; Rihouey, Christophe; Vicré-Gibouin, Maité; Driouich, Azeddine; Follet-Gueye, Marie-Laure

    2016-10-13

    Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However little is currently known regarding their potential activity towards skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. This article is protected by copyright. All rights reserved.

  9. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity Using Human Keratinocyte Cells

    Science.gov (United States)

    Shvedova, Anna A.; Castranova, Vincent; Kisin, Elena R.; Schwegler-Berry, Diane; Murray, Ashley R.; Gandelsman, Vadim Z.; Maynard, Andrew; Baron, Paul

    2003-01-01

    Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

  10. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    Science.gov (United States)

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage.

  11. Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

    Science.gov (United States)

    Baroni, A; Paoletti, I; Greco, R; Satriano, R A; Ruocco, E; Tufano, M A; Perez, J J

    2005-11-01

    Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

  12. Transcriptional profiling defines the effects of nickel in human epidermal keratinocytes.

    Science.gov (United States)

    Gazel, Alix; Rosdy, Martin; Tornier, Carine; De Fraissinette, Anne De Brugerolle; Blumenberg, Miroslav

    2008-12-01

    Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air-liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni-regulated genes were determined at two time points, immediate-early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL-18, galectin-3, and LITAF. These secreted proteins may be important in Ni-caused allergic reactions. Ni induced inhibitors of the NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB binding sites were found to be overrepresented in the Ni-suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni-induced genes. Significant parallels were found between the Ni-regulated genes and the genes regulated by TGFbeta, EGF, glucocorticoids, or Oncostatin-M. The comprehensive identification of Ni-regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin.

  13. Melittin Modulates Keratinocyte Function through P2 Receptor-dependent ADAM Activation*

    Science.gov (United States)

    Sommer, Anselm; Fries, Anja; Cornelsen, Isabell; Speck, Nancy; Koch-Nolte, Friedrich; Gimpl, Gerald; Andrä, Jörg; Bhakdi, Sucharit; Reiss, Karina

    2012-01-01

    Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent. PMID:22613720

  14. 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.

  15. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis.

    Science.gov (United States)

    Hussain, H; Shornick, L P; Shannon, V R; Wilson, J D; Funk, C D; Pentland, A P; Holtzman, M J

    1994-01-01

    Human epidermal cells exhibited none of the cytosolic lipoxygenase activity that is prominent in mucosal epithelial cells, but instead contained a microsomal activity that converted arachidonic acid to 12-hydroxyeicosatetraenoic acid (12-HETE). Identification of the extractable 12-HETE-forming activity as a 12-lipoxygenase (distinct from cytochrome P-450) included (S)-12-stereospecificity of product formation, trapping of 12-hydroperoxyeicosatetraenoic acid as an intermediate reaction product, and lack of NADPH dependence for activity. Epidermal cell poly(A)+ RNA contained high levels of a 2.3-kb mRNA that selectively hybridized with human platelet 12-lipoxygenase cDNA, and partial cDNA sequence of this mRNA indicated identity to platelet 12-lipoxygenase. The epidermal 12-lipoxygenase was not recognized by antibodies against the leukocyte-type 12- and 15-lipoxygenases (found in leukocytes, reticulocytes, and mucosal epithelial cells) but was detected by an antiplatelet 12-lipoxygenase antibody. The epidermal 12-lipoxygenase antigen was selectively expressed in germinal layer keratinocytes in healthy and psoriatic skin, and these layers exhibited hyperplasia and increased immunostaining in inflamed psoriatic skin. Together with previous results, these observations indicate that 1) epidermis generates 12-HETE by either cytochrome P-450 or lipoxygenase-based mechanisms depending on reaction conditions, and 2) 12-lipoxygenases (originally described in hematopoietic cell types) may be expressed in at least two distinct isoforms in epithelial barriers in humans, and in the case of the skin, a microsomal (platelet-type) 12-lipoxygenase is selectively overexpressed in germinal layer keratinocytes during psoriatic inflammation.

  16. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  17. Keratinocytes function as accessory cells for presentation of endogenous antigen expressed in the epidermis.

    Science.gov (United States)

    Kim, Brian S; Miyagawa, Fumi; Cho, Young-Hun; Bennett, Clare L; Clausen, Björn E; Katz, Stephen I

    2009-12-01

    The precise contribution(s) of skin dendritic cells (DCs) to immune responses in the skin has not been well delineated. We developed an intradermal (i.d.) injection model in which CD8+ T (OT-I) cells that express ovalbumin (OVA) peptide-specific TCRs (Valpha2/Vbeta5) are delivered directly to the dermis of transgenic (Tg) mice expressing OVA in the epidermis. After i.d. injection, these mice reliably develop skin graft-versus-host disease (GVHD) by day 7. To determine the relative contribution of Langerhans cells (LCs) to the ensuing GVHD-like reaction, we generated K14-OVA x Langerin-diphtheria-toxin-receptor (Langerin-DTR) Tg mice to allow conditional ablation of LCs in the epidermis. To delineate the role of dermal DCs (dDCs) in the reaction, we also generated K14-OVA Tg chimeras using beta(2)-microglobulin-deficient (beta(2)m) congenic donor bone marrow cells. Dermal DCs in these mice cannot present OVA to autoreactive T cells (OT-I cells), whereas the LCs are antigen presentation-competent. Unexpectedly, OT-I cell injection into diphtheria toxin (DT)-treated beta(2)m --> K14-OVA x Langerin-DTR Tg mice resulted in skin GVHD. Thus, in vivo, both LC and dDC appear to be dispensable for the induction of keratinocyte-directed, CD8-mediated effector immune responses. Furthermore and surprisingly, OVA-expressing epidermal cells depleted of LCs that could not initiate allogeneic epidermal lymphocyte reactions activated naive OT-I cells in vitro. These results indicate that keratinocytes may function as accessory cells competent to prime naive skin-reactive T cells.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://network.nature.com/group/jidclub.

  18. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1989-07-01

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

  20. Flavonoid myricetin inhibits TNF-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-κB pathways in human keratinocytes.

    Science.gov (United States)

    Lee, Da Hee; Lee, Chung Soo

    2016-08-05

    Flavonoid myricetin has been shown to exhibit anti-inflammatory and anti-oxidant effects. Nevertheless, the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we examined the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in relation to the Akt, mTOR and NF-κB pathways, which regulate the transcription genes involved in immune and inflammatory responses. TNF-α stimulated production of the inflammatory mediators and reactive oxygen species in keratinocytes, and activation of the Akt, mTOR and NF-κB pathways in HaCaT cells and primary keratinocytes. Myricetin, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-κB activation), rapamycin (mTOR inhibitor) and N-acetylcysteine attenuated TNF-α-induced activation of Akt, mTOR and NF-κB. Myricetin and N-acetylcysteine attenuated the TNF-α-stimulated production of cytokines and chemokines, and production of reactive oxygen species in keratinocytes. The results show that myricetin may reduce TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR and NF-κB pathways. The effect of myricetin appears to be associated with inhibition of the production of reactive oxygen species. Further, myricetin appears to attenuate the proinflammatory mediator-induced inflammatory skin diseases.

  1. Gracilaria bursa-pastoris (Gmelin) Silva extract attenuates ultraviolet B radiation-induced oxidative stress in human keratinocytes.

    Science.gov (United States)

    Piao, M J; Kim, K C; Zheng, J; Yao, C W; Cha, J W; Kang, H K; Yoo, E S; Koh, Y S; Ko, M H; Lee, N H; Hyun, Jin Won

    2014-01-01

    The purpose of this study was to assess the protective effects of an ethanol extract derived from the red alga Gracilaria bursa-pastoris (Gmelin) Silva (GBE) on ultraviolet B (UVB)-irradiated human HaCaT keratinocytes. GBE exhibited scavenging activity against intracellular reactive oxygen species that were induced by either hydrogen peroxide or UVB radiation. In addition, both the superoxide anion and the hydroxyl radical were scavenged by GBE in cell-free systems. GBE absorbed light in the UVB range (280-320 nm) of the electromagnetic spectrum and lessened the extent of UVB-induced oxidative damage to cellular lipids, proteins, and DNA. Finally, GBE-treated keratinocytes showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies. These results suggest that GBE exerts cytoprotective actions against UVB-stimulated oxidative stress by scavenging ROS and absorbing UVB rays, thereby attenuating injury to cellular constituents and preventing cell death.

  2. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Woodworth, C.D.; Doniger, J.; DiPaolo, J.A.

    1989-01-01

    Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

  3. Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes

    OpenAIRE

    2012-01-01

    Background: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. Aim: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. Methods: The skin grafts wer...

  4. Protein-kinase-Cmu expression correlates with enhanced keratinocyte proliferation in normal and neoplastic mouse epidermis and in cell culture.

    Science.gov (United States)

    Rennecke, J; Rehberger, P A; Fürstenberger, G; Johannes, F J; Stöhr, M; Marks, F; Richter, K H

    1999-01-05

    In order to gain insight into the biological function of a PKC iso-enzyme, the protein kinase Cmu, we analyzed the expression pattern of this protein in mouse epidermis and keratinocytes in culture. Daily analysis of neonatal mouse epidermis immediately after birth showed a time-dependent reduction in the PKCmu content. Expression of the proliferating-cell nuclear antigen (PCNA), indicative of the proliferative state of cells, was reduced synchronously with PKCmu as the hyperplastic state of the neonatal tissue declined. In epidermal mouse keratinocytes, fractionated according to their maturation state, PKCmu expression was restricted to PCNA-positive basal-cell fractions. In primary cultures of those cells, growth arrest and induction of terminal differentiation by Ca2+ resulted in strongly reduced PKCmu expression, concomitantly with the loss of PCNA expression. Treatment of PMK-R1 keratinocytes with 100 nM of the mitogen 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in activation of PKCmu, reflected by translocation from the cytosolic to the particulate fraction and by shifts in electrophoretic mobility. DNA synthesis was significantly inhibited by the PKCmu inhibitor Goedecke 6976, while Goedecke 6983 did not inhibit PKCmu. Carcinomas generated according to the 2-stage carcinogenesis protocol in mouse skin consistently exhibited high levels of PKCmu. These data correlate PKCmu expression with the proliferative state of murine keratinocytes and point to a role of PKCmu in growth stimulation. A correlation between PKCmu expression and enhanced cell proliferation was also observed for NIH3T3 fibroblasts transfected with and overexpressing human PKCmu.

  5. RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes.

    Science.gov (United States)

    Jackson, Ben; Peyrollier, Karine; Pedersen, Esben; Basse, Astrid; Karlsson, Richard; Wang, Zhipeng; Lefever, Tine; Ochsenbein, Alexandra M; Schmidt, Gudula; Aktories, Klaus; Stanley, Alanna; Quondamatteo, Fabio; Ladwein, Markus; Rottner, Klemens; van Hengel, Jolanda; Brakebusch, Cord

    2011-03-01

    RhoA is a small guanosine-5'-triphosphatase (GTPase) suggested to be essential for cytokinesis, stress fiber formation, and epithelial cell-cell contacts. In skin, loss of RhoA was suggested to underlie pemphigus skin blistering. To analyze RhoA function in vivo, we generated mice with a keratinocyte-restricted deletion of the RhoA gene. Despite a severe reduction of cofilin and myosin light chain (MLC) phosphorylation, these mice showed normal skin development. Primary RhoA-null keratinocytes, however, displayed an increased percentage of multinucleated cells, defective maturation of cell-cell contacts. Furthermore we observed increased cell spreading due to impaired RhoA-ROCK (Rho-associated protein kinase)-MLC phosphatase-MLC-mediated cell contraction, independent of Rac1. Rho-inhibiting toxins further increased multinucleation of RhoA-null cells but had no significant effect on spreading, suggesting that RhoB and RhoC have partially overlapping functions with RhoA. Loss of RhoA decreased directed cell migration in vitro caused by reduced migration speed and directional persistence. These defects were not related to the decreased cell contraction and were independent of ROCK, as ROCK inhibition by Y27632 increased directed migration of both control and RhoA-null keratinocytes. Our data indicate a crucial role for RhoA and contraction in regulating cell spreading and a contraction-independent function of RhoA in keratinocyte migration. In addition, our data show that RhoA is dispensable for skin development.

  6. Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection.

    Directory of Open Access Journals (Sweden)

    Giuseppe Valacchi

    Full Text Available One of the main functions of cutaneous tissues is to protect our body from the outdoor insults. Ozone (O3 is among the most toxic stressors to which we are continuously exposed and because of its critical location, the skin is one of the most susceptible tissues to the oxidative damaging effect of O3. O3 is not able to penetrate the skin, and although it is not a radical per se, the damage is mainly a consequence of its ability to induce oxidative stress via the formation of lipid peroxidation products.In this study we investigated the protective effect of defined "antioxidant" mixtures against O3 induced oxidative stress damage in human keratinocytes and understand their underlying mechanism of action.Results showed that the mixtures tested were able to protect human keratinocytes from O3-induced cytotoxicity, inhibition of cellular proliferation, decrease the formation of HNE protein adducts, ROS, and carbonyls levels. Furthermore, we have observed the decreased activation of the redox sensitive transcription factor NF-kB, which is involved in transcribing pro-inflammatory cytokines and therefore constitutes one of the main players associated with O3 induced skin inflammation. Cells exposed to O3 demonstrated a dose dependent increase in p65 subunit nuclear expression as a marker of NF-kB activation, while pre-treatment with the mixtures abolished NF-kB nuclear translocation. In addition, a significant activation of Nrf2 in keratinocytes treated with the mixtures was also observed.Overall this study was able to demonstrate a protective effect of the tested compounds versus O3-induced cell damage in human keratinocytes. Pre-treatment with the tested compounds significantly reduced the oxidative damage induced by O3 exposure and this protective effect was correlated to the abolishment of NF-kB nuclear translocation, as well as activation of Nrf2 nuclear translocation activating the downstream defence enzymes involved in cellular detoxification

  7. Cytotoxicity and chromosome aberrations in normal human oral keratinocytes induced by chemical carcinogens: Comparison of inter-individual variations.

    Science.gov (United States)

    Tsutsui, T; Kawamoto, Y; Suzuki, N; Gladen, B C; Barrett, J C

    1991-01-01

    Normal human keratinocytes from the oral cavity were cultured in vitro in serum-free medium. Cultures from different individuals were established, and the responses of the cells to different chemicals were compared. The cells, grown at clonal densities, were treated separately with an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine; MNNG), two arsenical salts (sodium arsenate or sodium arsenite), sodium fluoride or two polyaromatic hydrocarbons (benzo[a]pyrene or 7,12-dimethylbenz[a]-anthracene). There were no significant differences in the colony-forming efficiencies (22.8 +/- 4.2%) of control (untreated) cells from five different individuals. At selected doses, each of the chemicals reduced the colony-forming efficiencies of the treated cells. The cytotoxicity of most of the chemicals did not differ significantly among cells derived from different individuals, with the exception of sodium arsenate at two doses and sodium fluoride at the highest dose tested. Induction of chromosome aberrations by MNNG, sodium arsenite, sodium arsenate and sodium flouride was analysed with cells derived from up to nine individuals. There was little difference in the inducibilities of chromosome aberrations among cultured keratinocytes from different donors. Treatment of cells from nine donors with one dose of sodium fluoride revealed a statistically significant inter-individual variation. These findings provide a model system to study the effects of carcinogens on the target cells for oral cancers. The results can be compared with findings for cells from other epithelial tissues, since the culture conditions support the growth of keratinocytes regardless of origin. Little inter-individual variation was observed in the response of oral keratinocytes to the chemicals examined.

  8. Fat in the skin: Triacylglycerol metabolism in keratinocytes and its role in the development of neutral lipid storage disease

    OpenAIRE

    2014-01-01

    Keratinocyte differentiation is essential for skin development and the formation of the skin permeability barrier. This process involves an orchestrated remodeling of lipids. The cleavage of precursor lipids from lamellar bodies by β-glucocerebrosidase, sphingomyelinase, phospholipases and sterol sulfatase generates ceramides, non-esterified fatty acids and cholesterol for the lipid-containing extracellular matrix, the lamellar membranes in the stratum corneum. The importance of triacylglycer...

  9. Role of VEGF receptors in normal and psoriatic human keratinocytes: evidence from irradiation with different UV sources.

    Directory of Open Access Journals (Sweden)

    Jian-Wei Zhu

    Full Text Available Vascular endothelial growth factor (VEGF promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.

  10. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    Science.gov (United States)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  11. Selective peroxidation and externalization of phosphatidylserine in normal human epidermal keratinocytes during oxidative stress induced by cumene hydroperoxide.

    Science.gov (United States)

    Shvedova, Anna A; Tyurina, Julia Y; Kawai, Kazuaki; Tyurin, Vladimir A; Kommineni, Choudari; Castranova, Vincent; Fabisiak, James P; Kagan, Valerian E

    2002-06-01

    Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar

  12. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    Science.gov (United States)

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  13. Urethral reconstruction with a 3D porous bacterial cellulose scaffold seeded with lingual keratinocytes in a rabbit model.

    Science.gov (United States)

    Huang, Jian-Wen; Lv, Xiang-Guo; Li, Zhe; Song, Lu-Jie; Feng, Chao; Xie, Min-Kai; Li, Chao; Li, Hong-Bin; Wang, Ji-Hong; Zhu, Wei-Dong; Chen, Shi-Yan; Wang, Hua-Ping; Xu, Yue-Min

    2015-09-11

    The goal of this study was to evaluate the effects of urethral reconstruction with a three-dimensional (3D) porous bacterial cellulose (BC) scaffold seeded with lingual keratinocytes in a rabbit model. A novel 3D porous BC scaffold was prepared by gelatin sponge interfering in the BC fermentation process. Rabbit lingual keratinocytes were isolated, expanded, and seeded onto 3D porous BC. BC alone (group 1, N  =  10), 3D porous BC alone (group 2, N  =  10), and 3D porous BC seeded with lingual keratinocytes (group 3, N  =  10) were used to repair rabbit ventral urethral defects (2.0   ×   0.8 cm). Scanning electron microscopy revealed that BC consisted of a compact laminate while 3D porous BC was composed of a porous sheet buttressed by a dense outer layer. The average pore diameter and porosity of the 3D porous BC were 4.23   ±   1.14 μm and 67.00   ±   6.80%, respectively. At 3 months postoperatively, macroscopic examinations and retrograde urethrograms of urethras revealed that all urethras maintained wide calibers in group 3. Strictures were found in all rabbits in groups 1 and 2. Histologically, at 1 month postoperatively, intact epithelium occurred in group 3, and discontinued epithelium was found in groups 1 and 2. However, groups 2 and 3 exhibited similar epithelial regeneration, which was superior to that of group 1 at 3 months (p  3D porous BC seeded with lingual keratinocytes enhanced urethral tissue regeneration. 3D porous BC could potentially be used as an optimized scaffold for urethral reconstruction.

  14. Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

    Directory of Open Access Journals (Sweden)

    Ortt Kori

    2007-05-01

    Full Text Available Abstract Background p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63 and those lacking this domain (ΔNp63. Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes. Results To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. Conclusion This unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.

  15. 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.

  16. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

    OpenAIRE

    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.

    2009-01-01

    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  17. Influence of cyclodextrins on the proliferation of HaCaT keratinocytes in vitro.

    Science.gov (United States)

    Hipler, U C; Schönfelder, U; Hipler, C; Elsner, P

    2007-10-01

    Safety and efficacy of pharmaceutical agents can be greatly improved by encapsulation within, or covalent attachment to, a biomaterial carrier. Drug delivery systems must deliver the necessary amount of drug to the targeted site for a necessary period of time, both efficiently and precisely. Various kinds of high-performance biomaterials are being constantly developed for this purpose. Cyclodextrins are potential candidates for such a role, because of their ability to alter physical, chemical, and biological properties of guest molecules through the formation of inclusion complexes. The alpha-, beta-, and gamma-cyclodextrins are widely used natural cyclodextrins, consisting of six, seven, and eight D-glucopyranose residues, respectively, linked by -1,4 glycosidic bonds into a macro cycle. Each cyclodextrin has its own ability to form inclusion complexes with specific guests, an ability, which depends on a proper fit of the guest molecule into the hydrophobic cyclodextrin cavity. The most common pharmaceutical application of cyclodextrins is to enhance the solubility, stability, and bioavailability of drug molecules. Such kinds of ligand-receptor complexes can be used for different applications, e.g., for a transdermal therapeutic system (TTS) or in biofunctional textiles. The aim of this study was the investigation of the influence of the different cyclodextrins on the cell proliferation using HaCaT keratinocytes as an in vitro test system. Moreover, the study was performed to find harmless and nontoxic cyclodextrin concentrations for dermal applications. By means of different independent in vitro tests could be confirmed that alpha-, beta-, and gamma-cyclodextrins in concentrations up to 0.1% (w/v) do not show any antiproliferative influence on HaCaT keratinocytes. Sometimes even proliferative effects could be found. However, all used cyclodextrins (besides gamma-cyclodextrin and its derivatives) in concentrations of 0.5 and 1% (w/v), respectively, exert a

  18. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hanwen [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States); Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, University of South Carolina, Columbia, SC 29208 (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States)

    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.

  19. Kaempferol-3-O-rutinoside from Afgekia mahidoliae promotes keratinocyte migration through FAK and Rac1 activation.

    Science.gov (United States)

    Petpiroon, Nareerat; Suktap, Chalermlat; Pongsamart, Sunanta; Chanvorachote, Pithi; Sukrong, Suchada

    2015-07-01

    The restoration of the epidermal epithelium through re-epithelialization is a critical process in wound healing. Directed keratinocyte migration to the wound is required, and the retardation of this process may result in a chronic, non-healing wound. The present study contributes to research aiming to identify promising compounds that promote wound healing using a human keratinocyte model. The effects of three kaempferol glycosides from an Afgekia mahidoliae leaf extract, kaempferol-3-O-arabinoside, kaempferol-3-O-glucoside, and kaempferol-3-O-rutinoside, on keratinocyte migration were determined. Interestingly, kaempferol-3-O-rutinoside exhibited a pronounced effect on wound closure in comparison to the parental kaempferol and other glycosides. The mechanism by which kaempferol-3-O-rutinoside enhances cell migration involves the induction of filopodia and lamellipodia formation, increased cellular levels of phosphorylated FAK (Tyr 397) and phosphorylated Akt (Ser 473), and up-regulation of active Rac1-GTP. The data obtained in this study may support the development of this compound for use in wound healing therapies.

  20. Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract

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    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.

  1. Tanshinone IIA Inhibits Growth of Keratinocytes through Cell Cycle Arrest and Apoptosis: Underlying Treatment Mechanism of Psoriasis

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    Fu-Lun Li

    2012-01-01

    Full Text Available The aim of the present investigation was to elucidate the cellular mechanisms whereby Tanshinone IIA (Tan IIA leads to cell cycle arrest and apoptosis in vitro in keratinocytes, the target cells in psoriasis. Tan IIA inhibited proliferation of mouse keratinocytes in a dose- and time-dependent manner and induced apoptosis, resulting in S phase arrest accompanied by down-regulation of pCdk2 and cyclin A protein expression. Furthermore, Tan IIA-induced apoptosis and mitochondrial membrane potential changes were also further demonstrated by DNA fragmentation, single-cell gel electrophoresis assay (SCGE, and flow cytometry methods. Apoptosis was partially blocked by the caspase-3 inhibitor Ac-DEVD-CHO. Mitochondrial regulation of apoptosis further downstream was investigated, showing changes in the mitochondrial membrane potential, cytochrome c release into the cytoplasm, and enhanced activation of cleaved caspase-3 and Poly ADP-ribose polymerase (PARP. There was also no translocation of apoptosis-inducing factor (AIF from mitochondria to the nucleus in apoptotic keratinocytes, indicating Tan IIA-induced apoptosis occurs mainly through the caspase pathway. Our findings provide the molecular mechanisms by which Tan IIA can be used to treat psoriasis and support the traditional use of Salvia miltiorrhiza Bungee (Labiatae for psoriasis and related skin diseases.

  2. Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair

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    Tao Wang

    2015-12-01

    Full Text Available Programmed cell death 4 (PDCD4 is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes’ biology and indicate that PDCD4 plays a role in epidermal homeostasis.

  3. PI3-kinase-dependent activation of apoptotic machinery oc-curs on commitment of epidermal keratinocytes to terminal differentiation

    Institute of Scientific and Technical Information of China (English)

    Sam M Janes; Tyler A Ofstad; Douglas H Campbell; Ayad Eddaoudi; Gary Warnes; Derek Davies; Fiona M Watt

    2009-01-01

    We have investigated the earliest events in commitment of human epidermal keratinocytes to terminal differen-tiation. Phosphorylated Akt and caspase activation were detected in cells exiting the basal layer of the epidermis. Activation of Akt by retroviral transduction of primary cultures of human keratinocytes resulted in an increase in abortive clones founded by transit amplifying cells, while inhibition of the upstream kinase, Pl3-kinase, inhibited suspension-induced terminal differentiation. Caspase inhibition also blocked differentiation, the primary mediator being caspase 8. Caspase activation was initiated by 2 h in suspension, preceding the onset of expression of the termi-nal differentiation marker involucrin by several hours. Incubation of suspended cells with fibronectin or inhibition of PI3-kinase prevented caspase induction. At 2 h in suspension, keratinocytes that had become committed to terminal differentiation had increased side scatter, were 7-aminoactinomycin D (7-AAD) positive and annexin V negative; they exhibited loss of mitochondrial membrane potential and increased cardiolipin oxidation, but with no increase in reac-tive oxygen species. These properties indicate that the onset of terminal differentiation, while regulated by Pl3-kinase and caspases, is not a classical apoptotic process.

  4. RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation.

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    Meleri Jones

    2014-01-01

    Full Text Available Varicella zoster virus (VZV is the etiological agent of chickenpox and shingles, diseases characterized by epidermal skin blistering. Using a calcium-induced keratinocyte differentiation model we investigated the interaction between epidermal differentiation and VZV infection. RNA-seq analysis showed that VZV infection has a profound effect on differentiating keratinocytes, altering the normal process of epidermal gene expression to generate a signature that resembles patterns of gene expression seen in both heritable and acquired skin-blistering disorders. Further investigation by real-time PCR, protein analysis and electron microscopy revealed that VZV specifically reduced expression of specific suprabasal cytokeratins and desmosomal proteins, leading to disruption of epidermal structure and function. These changes were accompanied by an upregulation of kallikreins and serine proteases. Taken together VZV infection promotes blistering and desquamation of the epidermis, both of which are necessary to the viral spread and pathogenesis. At the same time, analysis of the viral transcriptome provided evidence that VZV gene expression was significantly increased following calcium treatment of keratinocytes. Using reporter viruses and immunohistochemistry we confirmed that VZV gene and protein expression in skin is linked with cellular differentiation. These studies highlight the intimate host-pathogen interaction following VZV infection of skin and provide insight into the mechanisms by which VZV remodels the epidermal environment to promote its own replication and spread.

  5. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yi; Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC (United States)

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  6. 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.

  7. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

    Directory of Open Access Journals (Sweden)

    Leslie A. Mehalick

    2015-12-01

    Full Text Available Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine for human oral gingival epithelial (GE keratinocytes, oral gingival fibroblasts (GF, dendritic cells (DC, and squamous cell carcinoma (SCC cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2–10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0–80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

  8. 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.

  9. Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia.

    Science.gov (United States)

    Dai, D; Li, L; Huebner, A; Zeng, H; Guevara, E; Claypool, D J; Liu, A; Chen, J

    2013-01-01

    Genes involved in the planar cell polarity (PCP) signaling pathway are essential for a number of developmental processes in mammals, such as convergent extension and ciliogenesis. Tissue-specific PCP effector genes of the PCP signaling pathway are believed to mediate PCP signals in a tissue- and cell type-specific manner. However, how PCP signaling controls the morphogenesis of mammalian tissues remains unclear. In this study, we investigated the role of inturned (Intu), a tissue-specific PCP effector gene, during hair follicle formation in mice. Tissue-specific disruption of Intu in embryonic epidermis resulted in hair follicle morphogenesis arrest because of the failure of follicular keratinocyte to differentiate. Targeting Intu in the epidermis resulted in almost complete loss of primary cilia in epidermal and follicular keratinocytes, and a suppressed hedgehog signaling pathway. Surprisingly, the epidermal stratification and differentiation programs and barrier function were not affected. These results demonstrate that tissue-specific PCP effector genes of the PCP signaling pathway control the differentiation of keratinocytes through the primary cilia in a cell fate- and context-dependent manner, which may be critical in orchestrating the propagation and interpretation of polarity signals established by the core PCP components.

  10. Nicotinamide Enhances Repair of Arsenic and Ultraviolet Radiation-Induced DNA Damage in HaCaT Keratinocytes and Ex Vivo Human Skin

    OpenAIRE

    Thompson, Benjamin C.; Halliday, Gary M.; Damian, Diona L

    2015-01-01

    Arsenic-induced skin cancer is a significant global health burden. In areas with arsenic contamination of water sources, such as China, Pakistan, Myanmar, Cambodia and especially Bangladesh and West Bengal, large populations are at risk of arsenic-induced skin cancer. Arsenic acts as a co-carcinogen with ultraviolet (UV) radiation and affects DNA damage and repair. Nicotinamide (vitamin B3) reduces premalignant keratoses in sun-damaged skin, likely by prevention of UV-induced cellular energy ...

  11. PAI-1 transcriptional regulation during the G0 --> G1 transition in human epidermal keratinocytes.

    Science.gov (United States)

    Qi, Li; Allen, Rosalie R; Lu, Qi; Higgins, Craig E; Garone, Rosemarie; Staiano-Coico, Lisa; Higgins, Paul J

    2006-10-01

    Plasminogen activator inhibitor type-1 (PAI-1) is the major negative regulator of the plasmin-dependent pericellular proteolytic cascade. PAI-1 gene expression is normally growth state regulated but frequently elevated in chronic fibroproliferative and neoplastic diseases affecting both stromal restructuring and cellular migratory activities. Kinetic modeling of cell cycle transit in synchronized human keratinocytes (HaCaT cells) indicated that PAI-1 transcription occurred early after serum stimulation of quiescent (G0) cells and prior to entry into a cycling G1 condition. PAI-1 repression (in G0) was associated with upstream stimulatory factor-1 (USF-1) occupancy of two consensus E box motifs (5'-CACGTG-3') at the PE1 and PE2 domains in the PF1 region (nucleotides -794 to -532) of the PAI-1 promoter. Chromatin immunoprecipitation (ChIP) analysis established that the PE1 and PE2 site E boxes were occupied by USF-1 in quiescent cells and by USF-2 in serum-activated, PAI-1-expressing keratinocytes. This reciprocal and growth state-dependent residence of USF family members (USF-1 vs. USF-2) at PE1/PE2 region chromatin characterized the G0 --> G1 transition period and the transcriptional status of the PAI-1 gene. A consensus E box motif was required for USF/E box interactions, as a CG --> AT substitution at the two central nucleotides inhibited formation of USF/probe complexes. The 5' flanking sites (AAT or AGAC) in the PE2 segment were not necessary for USF binding. USF recognition of the PE1/PE2 region E box sites required phosphorylation with several potential involved residues, including T153, maping to the USF-specific region (USR). A T153A substitution in USF-1 did not repress serum-induced PAI-1 expression whereas the T153D mutant was an effective suppressor. As anticipated from the ChIP results, transfection of wild-type USF-2 failed to inhibit PAI-1 induction. Collectively, these data suggest that USF family members are important regulators of PAI-1 gene

  12. A distal region of the human TGM1 promoter is required for expression in transgenic mice and cultured keratinocytes

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    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

  13. A Novel Controllable Hydrogen Sulfide-Releasing Molecule Protects Human Skin Keratinocytes Against Methylglyoxal-Induced Injury and Dysfunction

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    Chun-tao Yang

    2014-09-01

    Full Text Available Background/Aim: Delayed wound healing is a common skin complication of diabetes, which is associated with keratinocyte injury and dysfunction. Levels of methylglyoxal (MGO, an α-dicarbonyl compound, are elevated in diabetic skin tissue and plasma, while levels of hydrogen sulfide (H2S, a critical gaseous signaling molecule, are reduced. Interestingly, the gas has shown dermal protection in our previous study. To date, there is no evidence demonstrating whether MGO affects keratinocyte viability and function or H2S donation abolishes these effects and improves MGO-related impairment of wound healing. The current study was conducted to examine the effects of MGO on the injury and function in human skin keratinocytes and then to evaluate the protective action of a novel H2S-releasing molecule. Methods: An N-mercapto-based H2S donor (NSHD-1 was synthesized and its ability to release H2S was observed in cell medium and cells, respectively. HaCaT cells, a cell line of human skin keratinocyte, were exposed to MGO to establish an in vitro diabetic wound healing model. NSHD-1 was added to the cells before MGO exposure and the improvement of cell function was observed in respect of cellular viability, apoptosis, oxidative stress, mitochondrial membrane potential (MMP and behavioral function. Results: Treatment with MGO decreased cell viability, induced cellular apoptosis, increased intracellular reactive oxygen species (ROS content and depressed MMP in HaCaT cells. The treatment also damaged cell behavioral function, characterized by decreased cellular adhesion and migration. The synthesized H2S-releasing molecule, NSHD-1, was able to increase H2S levels in both cell medium and cells. Importantly, pretreatment with NSHD-1 inhibited MGO-induced decreases in cell viability and MMP, increases in apoptosis and ROS accumulation in HaCaT cells. The pretreatment was also able to improve adhesion and migration function. Conclusion: These results demonstrate that

  14. Noncovalent PEGylation by polyanion complexation as a means to stabilize keratinocyte growth factor-2 (KGF-2).

    Science.gov (United States)

    Khondee, Supang; Olsen, Christopher M; Zeng, Yuhong; Middaugh, C Russell; Berkland, Cory

    2011-11-14

    Repifermin, a truncated form of fibroblast growth factor-10 (FGF-10) also known as keratinocyte growth factor-2 (KGF-2), is a heparin-binding protein with potent regenerative properties. The protein unfolds and aggregates at relatively low temperature (~37 °C). Electrostatic interactions between polyanions and several FGFs have been reported to enhance the thermal stability of these proteins. Polyethylene glycol (PEG) was grafted to the polyanions pentosan polysulfate (PPS) and dextran sulfate (DS) as an alternative means to stabilize and noncovalently PEGylate KGF-2. Physical characteristics of KGF-2:polyanion-PEG complexes were examined using a variety of methods including circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning calorimetry, and dynamic light scattering. When compared to KGF-2 alone, subtle changes in CD spectra and fluorescence emission maxima were found when KGF-2 was formulated with the synthetic PEG-polyanions. Highly PEGylated polyanions (DS-PEG5) did not bind KGF-2 as well as conjugates with fewer PEG chains. The molecular weight of PEG did not have a noticeable effect on KGF-2 binding to the various PEG-polyanion conjugates. At optimal molar ratios, PPS-PEG and DS-PEG conjugates were able to stabilize KGF-2 by increasing the melting temperature by approximately 9-17 °C. Thus, polyanion-PEG conjugates improved the stability of KGF-2 and also offered a new electrostatic PEGylation scheme that may be extrapolated to other heparin-binding proteins.

  15. Differential cytotoxic effects of graphene and graphene oxide on skin keratinocytes

    Science.gov (United States)

    Pelin, Marco; Fusco, Laura; León, Verónica; Martín, Cristina; Criado, Alejandro; Sosa, Silvio; Vázquez, Ester; Tubaro, Aurelia; Prato, Maurizio

    2017-01-01

    Impressive properties make graphene-based materials (GBMs) promising tools for nanoelectronics and biomedicine. However, safety concerns need to be cleared before mass production of GBMs starts. As skin, together with lungs, displays the highest exposure to GBMs, it is of fundamental importance to understand what happens when GBMs get in contact with skin cells. The present study was carried out on HaCaT keratinocytes, an in vitro model of skin toxicity, on which the effects of four GBMs were evaluated: a few layer graphene, prepared by ball-milling treatment (FLG), and three samples of graphene oxide (GOs, a researc