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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. PMID:24184596

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

    Science.gov (United States)

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

    2014-01-01

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

  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

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

    DEFF Research Database (Denmark)

    Faurschou, A.; Gniadecki, R.

    2008-01-01

    , ERK1/2 and p38. The specific peptide blocking the activity of the atypical protein kinase C (aPKC) species zeta and iota/lambda abrogated the effects of TNF-alpha on Akt and ERK1/2 but increased the activation of p38. The TNF-alpha-dependent phosphorylation of Akt-ERK1/2 was slightly decreased by NF......B- and OH-dependent pathway resulting in the activation of survival and mitogenic pathways mediated by Akt and ERK1/2, and a signalling pathway conveyed by p38 that contributes to Akt activation but is suppressed by aPKC. Our data may be utilized in the development of more selective anti...... 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...

  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. 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.

  7. 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. PMID:27573915

  8. 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.

  9. 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.

  10. Anogenital malignancies and premalignancies: facts and controversies.

    Science.gov (United States)

    Kutlubay, Zekayi; Engin, Burhan; Zara, Tuba; Tüzün, Yalçın

    2013-01-01

    Anogenital malignancies and premalignancies are an important personal/public health problem due to their effects on individuals' physical, mental, and sexual health. Also, due to their etiological association with human papillomavirus (HPV) infection, anogenital malignancies and premalignancies constitute an immense public health burden. In addition to HPV infection, immunosuppression, HIV infection, chronic dermatoses, such as lichen sclerosis, previous radiotherapy and chemotherapy treatments, and smoking, are the other important etiopathologic factors in the development of anogenital malignancies and premalignancies. The incidence of anal squamous cell carcinoma (SCC) has increased considerably in the past decade, mainly due to the growing number of cases in high-risk groups, such as men who have sex with men, immunosuppressed individuals, and patients with HIV infection. Also, an increase in vulvar intraepithelial neoplasia (VIN) and VIN-related invasive vulvar cancer has been noted in women younger than age 50 years due to its association with HPV infections over the past decade. SCC of the scrotum seems to be the first cancer linked to occupational exposure. Bowen's disease, Bowenoid papulosis, and erythroplasia of Queyrat are the most widely seen premalignancies of anogenital region and are all forms of squamous intraepithelial neoplasia. Histopathologically, these conditions share identical histologic features of SCC in situ, but their clinical features differ. Early diagnosis is vital to improve prognosis, especially in anogenital malignancies. Also, if a delay occurs in diagnosis, treatment options used will be associated with significant negative effects on the patient's psychological well-being and quality of life; hence, management of anogenital malignancies and premalignancies should be organized in a multidisciplinary fashion. PMID:23806153

  11. Premalignant cystic neoplasms of the pancreas.

    Science.gov (United States)

    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.

  12. Histochemical identification of malignant and premalignant lesions

    Science.gov (United States)

    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

  13. 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.

  14. Breast Reconstruction for Premalignant and Malignant Disease—An Update

    OpenAIRE

    Apfelberg, David B.; Maldowney, Bart; Laub, Donald R; Maser, Morton R.; Lash, Harvey

    1981-01-01

    New concepts in the control of breast cancer and improvements in plastic surgery techniques have facilitated subsequent breast reconstruction. In a six-year period 72 breast reconstructions were carried out in 57 women after surgical treatment for premalignant or malignant breast disease and, in some cases, radiation therapy. The average age of the patients was 48 years and the average interval between the primary cancer operation and breast reconstruction was 42 months. Our experience in the...

  15. Expression of CD133 in various premalignant and proliferative lesions

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    Rahmi Amtha

    2015-06-01

    Full Text Available Background: In Jakarta, oral squamous cell carcinoma (OSCC usually detected in late stage with very low survival rate ofabout 1.1 years. OSCC may be preceded by premalignant lesion, so that early detection of the lesion may decrease the mortality rate due to oral malignancy. CD133 is a hematopoietic stem cell that play role in tissue regeneration, inflammation and tumor. Upregulated of CD133 was reported on tumor progression. Purpose: The aim of study is to determine circulating CD133 expression on premalignant (PML and proliferative (PL lesion. Method: Observational research was carried out on patients who seek treatment of PML and PL at Oral Medicine clinic. CD133 was taken from peripheral blood serum, examined using PCR. Data was analyzed by Chi square test. Result: 15 subjects (each of five subjects for PML, PL and control consist of 40% male and 60% female. Age group of above 41 years old was most affected PML and PL (66.7%. Tongue is common site for oral lesion (40%. There is a significant different of circulating CD133 rate among all groups lesion (p=0.039. Conclusion: CD133 express differently in premalignant and proliferative lesions.

  16. Antarctic marine bacteria versus UV-B irradiation

    International Nuclear Information System (INIS)

    strains only six were not able to survive 10 hours UV-B irradiation. The most sensitive strains were isolated from habitats protected from UV radiation (krill stomach, sea ice). The additional experiment, showing the lethal effect of UV-B for bacteria, throws light on the importance of shadowed environmental niches for bacterial survival in regions of the highest UV irradiance. Total cell number did not change significantly when exposed to UV-B radiation for 10h while without UV bacterial number increased by 27% during this period. The biovolume of strains used in this study which ranged from 0.14 to 13.7 (micro)m3 cell-1 also did not change significantly after 10 hours irradiation. Preliminary results show that the API 20NE test can be used to show changes in enzymatic abilities. Even 10 hours irradiation were not able to inhibit all enzymatic processes demonstrated by API 20NE system. The high variability in UV-B sensitivity of Antarctic bacteria may led to shifts from normal bacterial strains to more resistant. and thus to establish bacterial community adapted to life at risen UV-B level in the upper layer of Antarctic marine ecosystems. (author)

  17. Primary structure of keratinocyte transglutaminase

    International Nuclear Information System (INIS)

    The nucleotide and deduced amino acid sequences of the coding regions of human and rat keratinocyte transglutaminases (protein-glutamine: amine γ-glutamyltransferase; EC 2.3.2.13) have been determined. These yield proteins of ∼90 kDa that are 92% identical, indicative of the conservation of important structural features. Alignments of amino acid sequences show substantial similarity among the keratinocyte transglutaminase, human clotting factor XIII catalytic subunit, guinea pig liver tissue transglutaminase, and the human erythrocyte band-4.2 protein. The keratinocyte enzyme is most similar to factor XIII, whereas the band-4.2 protein is most similar to the tissue transglutaminase. A salient feature of the keratinocyte transglutaminase is its 105-residue extension beyond the N terminus of the tissue transglutaminase. This extension and the unreltaed activation peptide of factor XIII (a 37-residue extension) appear to be added for specialized functions after divergence of the tissue transglutaminase from their common lineage

  18. Administration of a vaccine composed of dendritic cells pulsed with premalignant oral lesion lysate to mice bearing carcinogen-induced premalignant oral lesions stimulates a protective immune response

    OpenAIRE

    De Costa, Anna-Maria A.; Justis, Danielle N.; Schuyler, Corinne A.; M. Rita I. Young

    2012-01-01

    The use of dendritic cell (DC) vaccines as treatment for malignancy is complicated by immune evasion tactics often employed by carcinomas such as head and neck squamous cell carcinoma (HNSCC). The present study aims to determine if an immune response can be elicited by administering a DC vaccine during the premalignant stages of HNSCC, prior to development of immune escape. Mice treated with the carcinogen 4-nitroquinoline 1-oxide (4NQO) in drinking water develop premalignant oral lesions tha...

  19. 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.

  20. Keratinocyte dysfunction in vitiligo epidermis: cytokine microenvironment and correlation to keratinocyte apoptosis

    OpenAIRE

    Moretti, Silvia; Fabbri, Paolo; Baroni, Gianna; Berti, Samantha; Ban, Daniele; Berti, Emilio; Nassini, Romina; Lotti, Torello; Massi, Daniela

    2009-01-01

    Vitiligo is a skin disorder characterized by loss of functional melanocytes. Keratinocytes contribute to melanocyte homeostasis, and keratinocyte alteration may play a role in melanocyte dysfunction in vitiligo. In particular, the release of melanogenic mediators and the level of functioning keratinocytes may affect melanocyte dysfunction in vitiligo epidermis. Keratinocyte-derived mediators involved in pigmentation, analysed by in situ hybridization, and epidermal apo...

  1. 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

  2. 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...

  3. Identification of genes expressed in premalignant breast disease by microscopy-directed cloning.

    OpenAIRE

    Jensen, R. A.; Page, D. L.; J. T. Holt

    1994-01-01

    Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RN...

  4. THE CLINICAL AND MORPHOLOGICAL STUDY OF 75 CASES OF ORAL PREMALIGNANT LESIONS

    Directory of Open Access Journals (Sweden)

    Syed Salman

    2015-02-01

    Full Text Available BACKGROUND: Oral cancer is of significant public health importance to India. Oral cancer will remain a major health problem and efforts towards early detection , and prevention will reduce this burden. A premalignant lesion is a disease , syndrome , or finding that , if left untreated , may lead to cancer. OBJECTIVE: To understand the prevalence , clinical and morphological profile of oral premalignant lesions. MATERIALS AND METHODS : Clinical material for present study comp rises 75 cases with oral premalignant lesions. The parame ters studied were types of oral premalignant lesions , age distribution , sex distribution , the local habits of addiction and the sites of involvement. RESULTS : Maximum number of cases was between 21 - 3 0 years of age. The sex incidence was more in males with the ratio of 9:1. Out of total 75 cases , 64% cases were of oral submucous fibrosis , 17.3% cases were of leukoplakia , 8% cases of erythroplakia , 6.6% cases of traumatic dental ulcer and 4% cases of li chen planus. In cases of oral submucous fibrosis , betel nut chewing was the commonest habit (89.5%. In cases of Lichen planus no addictions were found. Buccal mucosa was the commonest site involved in premalignant lesions. CONCLUSION: Premalignant lesions are seen mainly in early adulthood. The number of cases is increased due to adverse oral habits. Its prevalence can be reduced if awareness is created among such patients. This study mandates close cooperation between dentist and ENT surgeons.

  5. 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.

  6. Clinical significance of gelsolin-like actin-capping protein expression in oral carcinogenesis: an immunohistochemical study of premalignant and malignant lesions of the oral cavity

    International Nuclear Information System (INIS)

    Gelsolin-like actin-capping protein (CapG) is a ubiquitous gelsolin-family actin-modulating protein involved in cell signalling, receptor-mediated membrane ruffling, phagocytosis, and motility. CapG has generated great interest due to its oncogenic function in the control of cell migration or invasion in a variety of cancer cells. We previously applied proteomic methods to characterize differentially expressed proteins in oral squamous-cell carcinoma (OSCC) cells and detected significantly high expression levels of CapG in OSCC-derived cell lines compared to human normal oral keratinocytes. In the current study, to further determine the potential involvement of CapG in OSCC, we evaluated the status of CapG protein and mRNA expression in human oral premalignant lesions (OPLs) and primary OSCCs and correlated the results with clinicopathologic variables. Matched normal and tumour tissue sections of 79 human primary OSCCs and 28 OPLs were analyzed for CapG expression by immunohistochemistry (IHC). Correlations between CapG-immunohistochemical staining scores of OSCCs and clinicopathologic features were evaluated by Fisher's exact test. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was used to estimate CapG expression at the mRNA level. In IHC, substantial up-regulation of CapG protein was observed in primary OSCCs (52%) and OPLs (64%), whereas corresponding normal tissues showed consistently weak or absent immunoreactivity of CapG. qRT-PCR data were consistent with the protein expression status. Moreover, CapG expression was correlated with the TNM stage grading of OSCCs. Our finding of frequent dysregulated expression of CapG in premalignant and malignant lesions together with an association with an advanced clinical disease stage suggests that CapG could contribute to cancer development and progression and that CapG may have potential as a biomarker and a therapeutic target for OSCC

  7. 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)

  8. Argyrophilic nucleolar organizer regions in inflammatory, premalignant, and malignant oral lesions: A quantitative and qualitative assessment

    Directory of Open Access Journals (Sweden)

    Elangovan T

    2008-01-01

    Full Text Available Background and Objective: Argyrophilic nucleolar organizer regions (AgNORs have found widespread application in the past, especially in tumor histopathology. This study was undertaken to evaluate the significance of various AgNOR parameters and to assess their role in differentiating hyperplastic, premalignant, and malignant lesions. Materials and Methods: The study sample consisted of archival biopsy specimens of ten squamous cell carcinomas, ten premalignant lesions, and five inflammatory lesions. Two biopsies from normal mucosa acted as control. AgNORs were assessed both quantitatively and qualitatively. The data were analyzed using Student′s independent t-test, one-way analysis of variance (ANOVA, and multiple range test (Tukey-HSD. Results: Quantitatively significant difference existed in the number of AgNORs between the normal mucosa, inflammatory lesions, and carcinomas, but the premalignant lesions failed to differ significantly from the normal mucosa. The number of AgNORs was found to be related to epithelial proliferation. Qualitatively, in terms of size, shape, and pattern of distribution, the normal mucosa and inflammatory lesion were alike, but the premalignant and malignant lesions differed significantly from the normal, with a marked degree of AgNOR pleomorphism being observed in carcinomas. Conclusions: AgNOR quantity is strictly proportional to the proliferative activity of the cell and does not necessarily indicate malignancy. It is the qualitative characteristics of AgNOR that help to differentiate hyperplastic, premalignant, and malignant lesions.

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

    Science.gov (United States)

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

    2012-10-01

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

  10. Role of taurine accumulation in keratinocyte hydration.

    Science.gov (United States)

    Janeke, Guido; Siefken, Wilfried; Carstensen, Stefanie; Springmann, Gunja; Bleck, Oliver; Steinhart, Hans; Höger, Peter; Wittern, Klaus-Peter; Wenck, Horst; Stäb, Franz; Sauermann, Gerhard; Schreiner, Volker; Doering, Thomas

    2003-08-01

    Epidermal keratinocytes are exposed to a low water concentration at the stratum corneum-stratum granulosum interface. When epithelial tissues are osmotically perturbed, cellular protection and cell volume regulation is mediated by accumulation of organic osmolytes such as taurine. Previous studies reported the presence of taurine in the epidermis of several animal species. Therefore, we analyzed human skin for the presence of the taurine transporter (TAUT) and studied the accumulation of taurine as one potential mechanism protecting epidermal keratinocytes from dehydration. According to our results, TAUT is expressed as a 69 kDa protein in human epidermis but not in the dermis. For the epidermis a gradient was evident with maximal levels of TAUT in the outermost granular keratinocyte layer and lower levels in the stratum spinosum. No TAUT was found in the basal layer or in the stratum corneum. Keratinocyte accumulation of taurine was induced by experimental induction of skin dryness via application of silica gel to human skin. Cultured human keratinocytes accumulated taurine in a concentration- and osmolarity-dependent manner. TAUT mRNA levels were increased after exposure of human keratinocytes to hyperosmotic culture medium, indicating osmosensitive TAUT mRNA expression as part of the adaptation of keratinocytes to hyperosmotic stress. Keratinocyte uptake of taurine was inhibited by beta-alanine but not by other osmolytes such as betaine, inositol, or sorbitol. Accumulation of taurine protected cultured human keratinocytes from both osmotically induced and ultraviolet-induced apoptosis. Our data indicate that taurine is an important epidermal osmolyte required to maintain keratinocyte hydration in a dry environment. PMID:12880428

  11. Effects of UV-B irradiation on growth, survival, pigmentation and nitrogen metabolism enzymes in Cyanobacteria

    International Nuclear Information System (INIS)

    The effects of artificial UV-B irradiation on growth, survival, pigmentation, nitrate reductase (NR), glutamine synthetase (GS) and total protein profile have been studied in a number of N2-fixing cyanobacterial strains isolated from rice (paddy) fields in India. Different organisms show different effects in terms of growth and survival. Complete killing of Anabaena sp. and Nostoc carmium occurs after 120 min of UV-B exposure, whereas the same occurs only after 150 min of exposure in the case of Nostoc commune and Scytonema sp. Growth patterns of the cells treated with UV-B revealed that Nostoc commune and Scytonema sp. are comparatively more tolerant than Anabaena sp. and Nostoc carmium. Pigment content, particularly phycocyanin, was severely decreased following UV-B irradiation in all strains tested so far. In vivo NR activity was found to increase, while in vivo GS activity was decreased following exposure to UV-B for different durations in all test organisms; although complete inhibition of GS activity did not occur even after 120 min of UV-B exposure. (author)

  12. Effects of UV-B irradiation on growth, survival, pigmentation and nitrogen metabolism enzymes in Cyanobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Sinha, R.P.; Hader, D.P. [Institut fuer Botanik und Pharmazeutische Biologie, Friedrich-Alexander Universitaet, Erlangen (Germany); Kumar, H.D.; Kumar, A. [Banaras Hindu University, Varanasi (India)

    1995-12-31

    The effects of artificial UV-B irradiation on growth, survival, pigmentation, nitrate reductase (NR), glutamine synthetase (GS) and total protein profile have been studied in a number of N{sub 2}-fixing cyanobacterial strains isolated from rice (paddy) fields in India. Different organisms show different effects in terms of growth and survival. Complete killing of Anabaena sp. and Nostoc carmium occurs after 120 min of UV-B exposure, whereas the same occurs only after 150 min of exposure in the case of Nostoc commune and Scytonema sp. Growth patterns of the cells treated with UV-B revealed that Nostoc commune and Scytonema sp. are comparatively more tolerant than Anabaena sp. and Nostoc carmium. Pigment content, particularly phycocyanin, was severely decreased following UV-B irradiation in all strains tested so far. In vivo NR activity was found to increase, while in vivo GS activity was decreased following exposure to UV-B for different durations in all test organisms; although complete inhibition of GS activity did not occur even after 120 min of UV-B exposure. (author). 37 refs, 6 figs.

  13. 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.

  14. 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.

  15. 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.

  16. Intrinsic differences between oral and skin keratinocytes.

    Directory of Open Access Journals (Sweden)

    Anna Turabelidze

    Full Text Available Keratinocytes cover both the skin and some oral mucosa, but the morphology of each tissue and the behavior of the keratinocytes from these two sites are different. One significant dissimilarity between the two sites is the response to injury. Oral mucosal wounds heal faster and with less inflammation than equivalent cutaneous wounds. We hypothesized that oral and skin keratinocytes might have intrinsic differences at baseline as well as in the response to injury, and that such differences would be reflected in gene expression profiles.

  17. 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).

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  19. 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

  20. Unexpected premalignant gynecological lesions in women undergoing vaginal hysterectomy for utero-vaginal prolapse

    Directory of Open Access Journals (Sweden)

    Assem A.M. Elbiaa

    2015-09-01

    Full Text Available Aim of the study was to estimate the incidence of unexpected premalignant gynecological lesions in women undergoing vaginal hysterectomy for utero-vaginal prolapse. Material and methods : Eighty women with asymptomatic utero-vaginal prolapse were included in this prospective study for vaginal hysterectomy after preoperative preparation and after written informed consent. Women included in this study were screened preoperatively by high vaginal swab, Pap smear, endometrial biopsy and trans-vaginal ultrasound. Surgically removed uteri and ovaries were sent for histopathological examination. Results of histopathological examination as gold standard were compared with conventional gynecological screening methods. Results : Histopathological examination of surgically removed uteri and ovaries after vaginal hysterectomy for uterovaginal prolapse showed abnormal findings in 61.25% (49/80 of studied cases (10 chronic cervicitis; 20 cervical intra-epithelial neoplasia-1 [CIN-1]; 5 CIN-2; 2 CIN-3; 10 simple endometrial hyperplasia without atypia and 2 simple serous ovarian cyst. Also, histopathological examination showed premalignant changes in 33.75% (27/80 of studied cases (20 CIN-1; 5 CIN-2 and 2 CIN-3, which mean 50% sensitivity of pre-operative Pap smear to detect premalignant cervical changes. Conclusions : Asymptomatic women with utero-vaginal prolapse may have associated premalignant lesions which may not be detected by conventional screening methods, and this should be explained preoperatively for women undergoing surgery, especially if conservative management was considered.

  1. PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

    Science.gov (United States)

    Ho, Yeung; Li, Xiting; Jamison, Stephanie; Harding, Heather P; McKinnon, Peter J; Ron, David; Lin, Wensheng

    2016-07-01

    Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation. PMID:27181404

  2. 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

  3. 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

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

    Science.gov (United States)

    Kanitakis, Jean; Petruzzo, Palmina; Gazarian, Aram; Testelin, Sylvie; Devauchelle, Bernard; Badet, Lionel; Dubernard, Jean-Michel; Morelon, Emmanuel

    2015-01-01

    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. PMID:26550517

  5. 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.

  6. 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.

  7. Cultured keratinocyte grafting on various biologic matrices

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Objective: To make attempts to use cell constructs from subconfluent keratinocyte cultures, which contain a much glue (TissucollR) and directly applied onto full thickness wounds in athymic mice or combined with allogenic split thickness overgrafts and compared with cultured sheet grafts. This keratinocyte fibrin glue suspension (KFGS) has also been used in burns up to 88% burned TBSA as well as in chronic wounds. Keratinocytes were also seeded onto various biomaterials (BiobraneR, HYAFF LaserskinR, IntegraTM, TissuFascieR) as carriers. Results: Human suspended keratinocytes were effective to reorganize to skin in vivo both in nude mice and in patients and superior if compared to sheet grafts. 3~ 5 d after seeding onto various biomaterials, cell reached subconfluence and were ready for grafting. These cell-membrane constructs were always tured on microspheres in spinner cultures could increase the cell yield, and the subconfluently covered microspheres were directly grafted onto" the wound. Conclusion: These experiments demonstrated that keratinocytes can grow on a variety of carrier materials in vitro and these cell constructs were able to spontaneously reform a multilayer neoepithelium in vivo. The current screening looks for the ideal carrier for keratinocytes that also would serve as a temporary wound cover and induce dermis formation by tissue conduction which further may be enhanced by gene therapy.

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

    International Nuclear Information System (INIS)

    The role of fullerene as a pro-oxidant or anti-oxidant in Ultraviolet B ray (UV-B)-induced disorders in mouse skin was investigated. Fullerene gave no photo-toxic effect to UV-B-irradiated mouse skin. Since erythema was concentrated at the pore circumference in a UV-B irradiation experiment in mouse skin, the sebaceous gland pairs was strongly implicated as a site for the generation of reactive oxygen species (ROS). In a histological evaluation of the skin stained with CH3MDFDA (ROS index) and YO-Pro-1 (apoptosis index), the fluorescence intensity of a sebaceous gland significantly increased with UV-B irradiation. With the application of fullerene to UV-irradiated mouse skin, no toxicity was recognized in comparison with the control, and erythema, the ROS index, and the apoptosis index decrease with the application of fullerene. Ascorbyl radical (AA·) increased with the application of ascorbate (AA) to UV-B-irradiated mouse skin, and AA· decreased with the application of fullerene. The co-application of AA and fullerene, which suppressed AA· in vitro, significantly suppressed erythema, and also suppressed both the ROS index and apoptosis index in mouse skin after UV-B irradiation. In both mouse skin at 48 h after UV-B irradiation and in an attempt to reproduce this phenomenon artificially in vitro, a similar high AA· peak (AA·/H· > 4) was observed in electron spin resonance (ESR) charts. The binding of fullerene with AA impairs the Fenton reaction between AA and Fe-protein based on the observation of ascorbate-specific UV absorption and a linear equation for the calibration curve. Therefore, fullerene may impair the intercalation of AA to a heme pocket by binding with AA. These results suggest that the co-application of AA and fullerene is effective against oxidative skin damage caused by UV-B irradiation, and the development of an AA· inhibitor such as fullerene should be useful for reducing organ damage associated with Fe-protein oxidation.

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

    NARCIS (Netherlands)

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

    2008-01-01

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

  10. 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.

  11. Effect of moderate UV-B irradiation on Synechocystis PCC 6803 biliproteins

    International Nuclear Information System (INIS)

    In the present study, we investigated the mechanism of UV-B radiation induced damage to the light harvesting apparatus of the cyanobacterium Synechocystis 6803. Liquid chromatography analysis and spectroscopy investigations performed on phycobilisomes or isolated biliproteins irradiated with moderate UV-B intensity (1.3 W/m2) revealed rapid destruction of β-phycocyanin and a slower damage of the other biliproteins, α-phycocyanin and both α and β-allophycocyanin. EPR spin trapping measurements revealed that carbon centered adducts of the spin trap DMPO were formed. This evidence indicates that free radicals produced from bilins probably attack the polypeptide chain of protein inducing its degradation. Our results show that the bilin chromophore is the main target of UV-B irradiation, causing structural changes, which in turn induce reaction of the chromophore with atmospheric oxygen and lead to production of reactive radicals. Our results also demonstrate that β-phycocyanin is the most affected biliprotein, probably due to the presence of two bilins as chromophore

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

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    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.

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

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    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.

  14. Chitin modulates innate immune responses of keratinocytes.

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    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.

  15. 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).

  16. Surgical management of premalignant lesions of the oral cavity with the CO2 laser.

    Science.gov (United States)

    Pinheiro, A L; Frame, J W

    1996-01-01

    The management of patients with premalignant and malignant lesions of the oral cavity can present problems. The potentially invasive nature of premalignant lesions together with their large extent influences the treatment. The common modalities of treatment of these lesions are surgical excision, cryotherapy, electrosurgery and radiotherapy. Recently, CO2 laser surgery has become available. Less pain, little bleeding, minimal post-operative edema, reduced risk of infection, and low recurrence rates were advantages observed following CO2 laser surgery in the mouth when compared to other modalities of treatment. Healing following CO2 laser surgery progressed well with little postoperative scarring and re-epithelialization was complete after 4-6 weeks. The newly formed epithelium appeared normal and was soft on palpation. PMID:9206362

  17. Functional Characterization of Cultured Keratinocytes after Acute Cutaneous Burn Injury

    OpenAIRE

    Gauglitz, Gerd G; Siegfried Zedler; Felix von Spiegel; Jasmin Fuhr; Guido Henkel von Donnersmarck; Eugen Faist

    2012-01-01

    BACKGROUND: In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable--keratinocyte-derived--cytokine and growth factor milieu. METHODS: In this study the morphology and cytokine expression profile of keratinocytes fro...

  18. 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...

  19. Unexpected premalignant gynecological lesions in women undergoing vaginal hysterectomy for utero-vaginal prolapse

    OpenAIRE

    Assem A.M. Elbiaa; Abdelazim, Ibrahim A.; Farghali, Mohamed M.; Hussain, M.; Omu, A.E.

    2015-01-01

    Aim of the study Aim of the study was to estimate the incidence of unexpected premalignant gynecological lesions in women undergoing vaginal hysterectomy for utero-vaginal prolapse. Material and methods Eighty women with asymptomatic utero-vaginal prolapse were included in this prospective study for vaginal hysterectomy after preoperative preparation and after written informed consent. Women included in this study were screened preoperatively by high vaginal swab, Pap smear, endometrial biops...

  20. Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study

    OpenAIRE

    Jain Anshu; Maheshwari Veena; Alam Kiran; Mehdi Ghazala; Sharma S

    2009-01-01

    Background: Oral cancers are a major health problem in India. Recently, parameters of cell proliferation and cell death have emerged as important diagnostic and prognostic tools. Aims: The aim was to study apoptosis in premalignant and malignant squamous cell lesions of the oral cavity and to evaluate its prognostic role in oral cancers. Materials and Methods: The study included 175 patients presenting with oral lesions. Evaluation of apoptotic index (AI) (using light microscopy) was per...

  1. 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.

  2. Premalignant lesions of prostate and their association with nodular hyperplasia and carcinoma prostate

    Directory of Open Access Journals (Sweden)

    Rekhi Bharat

    2004-01-01

    Full Text Available BACKGROUND : A relatively new development in the arena of prostatic histopathological study is the premalignant proliferative changes in the glandular epithelium, possibly relating to carcinoma. Two major categories have come up, namely prostatic intraepithelial neoplasia (PIN and atypical adenomatous hyperplasia (AAH. AIMS : The aims of present study were to identify foci of the two putative premalignant conditions viz. PIN and AAH in ducto-acinar lining epithelia of 200 prostatectomy specimens and their association with nodular hyperplasia and adenocarcinoma prostate. MATERIAL AND METHODS : Micro sections from 200 prostatectomy specimens, received in the Department of Pathology, PGIMS, Rohtak, were extensively studied for the presence and association of premalignant conditions. Significant values were obtained by employing Chi-square (x2 test, with P value < 0.05 as significant. RESULTS : Out of 177 cases of nodular hyperplasia, 53 (29.9% showed PIN and 38 (20.3% showed presence of AAH. All 6 cases (100% of pure carcinoma revealed foci of PIN. Out of the remaining 23 cases of carcinoma with nodular hyperplasia, foci of PIN were observed in 16 cases (94.1% and AAH in 2 cases (11.7%. High-grade PIN was observed in 20 cases (86.9% of the total 23 cases of carcinoma, with/without nodular hyperplasia and 20 cases (11.2% of nodular hyperplasia. Low-grade PIN was observed in 33 cases (18.6% of nodular hyperplasia and in only 1 case (5.8% of carcinoma prostate with nodular hyperplasia. CONCLUSION : PIN, especially high-grade type was the most commonly observed premalignant lesion, in cases of adenocarcinoma, thereby suggesting it to be the likely precursor of carcinoma prostate. AAH showed a weaker association with carcinoma.

  3. Genomics and premalignant breast lesions: clues to the development and progression of lobular breast cancer

    OpenAIRE

    Mastracci, Teresa L; Boulos, Fouad I; Andrulis, Irene L.; Lam, Wan L.

    2007-01-01

    Advances in genomic technology have improved our understanding of the genetic events that parallel breast cancer development. Because almost all mammary carcinomas develop in the terminal duct lobular units of the breast, understanding the events involved in mammary gland development make it possible to recognize those events that, when altered, contribute to breast neoplasia. In this review we focus on lobular carcinomas, discussing the pathology, development, and progression of premalignant...

  4. Ornithine decarboxylase activity is a marker of premalignancy in longstanding Helicobacter pylori infection.

    OpenAIRE

    Patchett, S E; Katelaris, P H; Zhang, Z. W.; Alstead, E M; Domizio, P; Farthing, M J

    1996-01-01

    BACKGROUND: Longstanding Helicobacter pylori infection may increase the risk of developing gastric adenocarcinoma. The sequence of chronic active gastritis leading to gastritis with atrophy and subsequent intestinal metaplasia is thought to be a key step in gastric carcinogenesis. Ornithine decarboxylase (ODC) activity is increased in some pre-malignant gastrointestinal conditions and is essential for malignant transformation in vitro. AIMS: To measure ODC activity in the antrum of H pylori i...

  5. 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

  6. 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.

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

    International Nuclear Information System (INIS)

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

  10. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-06-01

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

  12. [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.

  13. 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.

  14. 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.

  15. 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.

  16. 11β-Hydroxysteroid dehydrogenase 1 contributes to the pro-inflammatory response of keratinocytes

    International Nuclear Information System (INIS)

    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 × 10−13 M cortisol, whereas 1 × 10−5 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 concentrations

  17. 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

    2016-08-01

    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. PMID:27323210

  18. Human keratinocyte sensitivity towards inflammatory cytokines varies with culture time

    Directory of Open Access Journals (Sweden)

    G. Elliott

    1992-01-01

    Full Text Available Proliferating keratinocyte cultures have been reported to synthesize higher concentrations of prostaglandin (PG E than confluent ones. As interleukin-1 (IL-1 stimulates keratinocyte PGE synthesis we investigated whether the degree of confluency of the keratinocyte culture modified the response of the cells to IL-1. It was found that IL-1α (100 U/ml stimulated PGE2 synthesis by proliferating (7 days in culture but not differentiating (14 days in culture keratinocytes. Similar effects were observed using tumour necrosis factor-α. Both arachidonic acid (AA and the calcium ionophore A23187 stimulated PGE2 synthesis by 7 and 14 day cultures although the increase was greatest when 7 day cultures were used. Our data indicate that there is a specific down-regulation of the mechanism(s by which some inflammatory cytokines stimulate keratinocyte eicosanoid synthesis as cultured keratinocytes begin to differentiate.

  19. Anomalous features of EMT during keratinocyte transformation.

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    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.

  20. Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor

    OpenAIRE

    Chapman, Sandra; Liu, Xuefeng; Meyers, Craig; Schlegel, Richard; Alison A McBride

    2010-01-01

    Primary human keratinocytes are useful for studying the pathogenesis of many different diseases of the cutaneous and mucosal epithelia. In addition, they can form organotypic tissue equivalents in culture that can be used as epidermal autografts for wound repair as well as for the delivery of gene therapy. However, primary keratinocytes have a finite lifespan in culture that limits their proliferative capacity and clinical use. Here, we report that treatment of primary keratinocytes (originat...

  1. Nitric oxide produced by ultraviolet-irradiated keratinocytes stimulates melanogenesis.

    OpenAIRE

    Roméro-Graillet, C; Aberdam, E; Clément, M.; Ortonne, J P; Ballotti, R

    1997-01-01

    Ultraviolet (UV) radiation is the main physiological stimulus for human skin pigmentation. Within the epidermal-melanin unit, melanocytes synthesize and transfer melanin to the surrounding keratinocytes. Keratinocytes produce paracrine factors that affect melanocyte proliferation, dendricity, and melanin synthesis. In this report, we show that normal human keratinocytes secrete nitric oxide (NO) in response to UVA and UVB radiation, and we demonstrate that the constitutive isoform of keratino...

  2. HER2 Status in Premalignant, Early, and Advanced Neoplastic Lesions of the Stomach

    Directory of Open Access Journals (Sweden)

    A. Ieni

    2015-01-01

    Full Text Available Objectives. HER2 expression in gastric cancer (GC has received attention as a potential target for therapy with Trastuzumab. We reviewed the current knowledge on HER2 status in premalignant gastric lesions and in early (EGC and advanced (AGC GC to discuss the possible pathogenetic and prognostic roles of HER2 overexpression in GC. Results. HER2 overexpression was documented in gastric low-grade (LG and high-grade intraepithelial neoplasia (HG-IEN, with higher frequency in gastric type dysplasia. HER2 overexpression was significantly associated with disease recurrence and poor prognosis in EGC representing an independent risk factor for lymph node metastases. HER2 overexpression was more frequent in AGC characterized by high grade, advanced stage, and high Ki-67 labeling index. The discordance in HER2 status was evidenced between primitive GC and synchronous or metachronous metastases. Conclusions. HER2 overexpression in premalignant gastric lesions suggests its potential involvement in the early steps of gastric carcinogenesis. The assessment of HER2 status in EGC may be helpful for the identification of patients who are at low risk for developing nodal metastases. Finally, the possible discordance in HER2 status between primary GC and its synchronous metastases support routine assessment of HER2 both in the primary GC and in its metastatic lesions.

  3. 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.

  4. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

    Science.gov (United States)

    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression. PMID:23667900

  5. 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.

  6. 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.

  7. 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. PMID:25658450

  8. The effect of pantothenic acid deficiency on keratinocyte proliferation and the synthesis of keratinocyte growth factor and collagen in fibroblasts.

    Science.gov (United States)

    Kobayashi, Daisaku; Kusama, Miho; Onda, Masaaki; Nakahata, Norimichi

    2011-01-01

    It has been reported that pantothenic acid (vitamin B5) and panthenol, an alcohol derivative of pantothenic acid, have beneficial moisturizing effects on the skin. However, few studies have investigated the mechanism of action of pantothenic acid on skin tissues. We tried to clarify the role of pantothenic acid on skin function by using keratinocytes and fibroblasts. The depletion of pantothenic acid from the culture medium suppressed keratinocyte proliferation and promoted differentiation. Moreover, pantothenic acid depletion decreased the synthesis of keratinocyte growth factor and procollagen 4a2 in fibroblasts. These results suggest that pantothenic acid is essential for maintaining keratinocyte proliferation and differentiation. PMID:21258175

  9. 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 (

  10. High prevalence of premalignant lesions in prophylactically removed breasts from women at hereditary risk for breast cancer.

    NARCIS (Netherlands)

    Hoogerbrugge-van der Linden, N.; Bult, P.; Widt-Levert, L.M. de; Beex, L.V.A.M.; Kiemeney, L.A.L.M.; Ligtenberg, M.J.L.; Massuger, L.F.A.G.; Boetes, C.; Manders, P.; Brunner, H.G.

    2003-01-01

    PURPOSE: Women with a hereditary predisposition for breast cancer have an extremely high risk of developing invasive breast carcinoma, and many women consider prophylactic mastectomy to avoid this risk. The use of prophylactic mastectomy is still debated. Identification of frequent premalignant lesi

  11. 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

  12. N-acetyltransferase in human skin and keratinocytes

    NARCIS (Netherlands)

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

    2014-01-01

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

  13. 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.

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    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%.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Effects of UVB irradiation on keratinocyte growth factor (KGF) and receptor (KGFR) expression in cultured human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Y.; Lee, H.S.T.; Kooshesh, F.; Fujisawa, H.; Sauder, D.N.; Kondo, S. [Univ. of Toronto, Sunnybrook Health Science Centre, Div. of Dermatology, Toronto (Canada)

    1996-06-01

    Keratinocyte growth factor (KGF) and its receptor (KGFR) are thought to play important roles in normal keratinocyte growth and differentiation. Since UVB radiation is known to influence keratinocyte growth, we sought to determine whether UVB would alter the expression of KGF and KGFR. Using a reverse-transcription coupled polymerase chain reaction (RT-PCR), the present study examined the expression of KGF and KGFR mRNA in cultured normal human keratinocytes exposed to UVB irradiation. Total cellular RNA was extracted from cultured keratinocytes at various time points after irradiation, reverse transcribed and used for PCR amplification using primers specific for KGF and KGFR. Constitutive expression of KGFR mRNA, but not KGF mRNA, was detected in normal cultured human keratinocytes. After UVB irradiation at 300 J/m{sup 2}, the KGF mRNA remained undetectable while the KGFR mRNA level was significantly decreased. The down-regulation of KGFR mRNA expression was also confirmed by Northern blot analysis. Immunohistochemical studies demonstrated a decreased positive signal of KGFR in human keratinocytes after UVB irradiation. Our results suggest a possible role for the KGF-KGFR signalling pathway in the skin after exposure to UVB, and that UVB-induced growth inhibition of keratinocytes in hyperproliferative skin disorders may be related to downregulation of KGFR. (au) 39 refs.

  1. Effect of ultraviolet B irradiation on delayed-type hypersensitivity, cytotoxic T lymphocyte activity, and skin graft rejection

    International Nuclear Information System (INIS)

    The influence of ultraviolet B irradiation on the induction of delayed-type hypersensitivity reactions to alloantigens by epidermal cells (EC), on the generation of cytotoxic T lymphocyte activity to alloantigens, and on skin graft rejection was studied. After the skin was irradiated with UVB in vitro, EC were obtained. The EC were injected subcutaneously, and the DTH reaction was compared with that induced by non-UVB-irradiated EC. A reduction in the DTH reaction was observed (from 62% to 99.1%). CTL activity in these mice was assessed after in vitro stimulation. CTL activity in mice sensitized with UVB-irradiated EC was significantly reduced. Furthermore, mice sensitized with UVB-irradiated EC did not reject a subsequent skin allograft in an accelerated fashion, whereas mice sensitized with non-UVB-irradiated EC did. The mechanism(s) of these reactions and the clinical application of the UVB irradiation prior to grafting are discussed

  2. 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.

  3. 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.

  4. Scanning Ion Conductance Microscopy of Live Keratinocytes

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

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

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

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    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.

  6. 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.

  7. Establishment of primary keratinocyte culture from horse tissue biopsates

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    Jernej OGOREVC

    2015-12-01

    Full Text Available Primary cell lines established from skin tissue can be used in immunological, proteomic and genomic studies as in vitro skin models. The goal of our study was to establish a primary keratinocyte cell culture from tissue biopsates of two horses. The primary keratinocyte cell culture was obtained by mechanical and enzymatic dissociation and with explant culture method. The result was a heterogeneous primary culture comprised of keratinocytes and fibroblasts. To distinguish epithelial and mesenchymal cells immunofluorescent characterisation was performed, using antibodies against cytokeratin 14 and vimentin. We successfully at attained a primary cell line of keratinocytes, which could potentially be used to study equine skin diseases, as an animal model for human diseases, and for cosmetic and therapeutic product testing.

  8. Apoptosis in premalignant and malignant squamous cell lesions of the oral cavity: A light microscopic study

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    Jain Anshu

    2009-04-01

    Full Text Available Background: Oral cancers are a major health problem in India. Recently, parameters of cell proliferation and cell death have emerged as important diagnostic and prognostic tools. Aims: The aim was to study apoptosis in premalignant and malignant squamous cell lesions of the oral cavity and to evaluate its prognostic role in oral cancers. Materials and Methods: The study included 175 patients presenting with oral lesions. Evaluation of apoptotic index (AI (using light microscopy was performed on hematoxylin and eosin-stained sections. Statistical Analysis Used: Student′s t test was performed. Results: The mean AI increased progressively with increasing dysplasia, with the maximum AI in well-differentiated (WD squamous cell carcinoma, and a fall was noted with progression toward higher grades. The difference between WD SCC and poorly-differentiated SCC was significant (P < 0.05. Cases with lymph node metastasis had significantly (P < 0.05 lower mean AI values. Conclusion: Apoptosis can be fairly accurately assessed using light microscopy. Tumors that exhibit less apoptosis tend to show aggressive behavior and have a greater potential for metastasis.

  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. Functional analysis of ZFP36 proteins in keratinocytes.

    Science.gov (United States)

    Prenzler, Frauke; Fragasso, Annunziata; Schmitt, Angelika; Munz, Barbara

    2016-08-01

    The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner. PMID:27182009

  11. Distinctive molecular responses to ultraviolet radiation between keratinocytes and melanocytes.

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    Sun, Xiaoyun; Kim, Arianna; Nakatani, Masashi; Shen, Yao; Liu, Liang

    2016-09-01

    Solar ultraviolet radiation (UVR) is the major risk factor for skin carcinogenesis. To gain new insights into the molecular pathways mediating UVR effects in the skin, we performed comprehensive transcriptomic analyses to identify shared and distinctive molecular responses to UVR between human keratinocytes and melanocytes. Keratinocytes and melanocytes were irradiated with varying doses of UVB (10, 20 and 30 mJ/cm(2) ) then analysed by RNA-Seq at different time points post-UVB radiation (4, 24 and 72 h). Under basal conditions, keratinocytes and melanocytes expressed similar number of genes, although they each expressed a distinctive subset of genes pertaining to their specific cellular identity. Upon UVB radiation, keratinocytes displayed a clear pattern of time- and dose-dependent changes in gene expression that was different from melanocytes. The early UVB-responsive gene set (4 h post-UVR) differed significantly from delayed UVB-responsive gene sets (24 and 72 h). We also identified multiple novel UVB signature genes including PRSS23, SERPINH1, LCE3D and CNFN, which were conserved between melanocyte and keratinocyte lines from different individuals. Taken together, our findings elucidated both common and distinctive molecular features between melanocytes and keratinocytes and uncovered novel UVB signature genes that might be utilized to predict UVB photobiological effects on the skin. PMID:27119462

  12. 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

    2016-01-01

    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

  13. Functional characterization of cultured keratinocytes after acute cutaneous burn injury.

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    Gerd G Gauglitz

    Full Text Available BACKGROUND: In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable--keratinocyte-derived--cytokine and growth factor milieu. METHODS: In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters. RESULTS: Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11 in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß. CONCLUSION: Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor

  14. 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.

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

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

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    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.

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

    International Nuclear Information System (INIS)

    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). 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. 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(P)H oxidase. 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

  18. Polymeric membranes modulate human keratinocyte differentiation in specific epidermal layers.

    Science.gov (United States)

    Salerno, Simona; Morelli, Sabrina; Giordano, Francesca; Gordano, Amalia; Bartolo, Loredana De

    2016-10-01

    In vitro models of human bioengineered skin substitutes are an alternative to animal experimentation for testing the effects and toxicity of drugs, cosmetics and pollutants. For the first time specific and distinct human epidermal strata were engineered by using membranes and keratinocytes. To this purpose, biodegradable membranes of chitosan (CHT), polycaprolactone (PCL) and a polymeric blend of CHT-PCL were prepared by phase-inversion technique and characterized in order to evaluate their morphological, physico-chemical and mechanical properties. The capability of membranes to modulate keratinocyte differentiation inducing specific interactions in epidermal membrane systems was investigated. The overall results demonstrated that the membrane properties strongly influence the cell morpho-functional behaviour of human keratinocytes, modulating their terminal differentiation, with the creation of specific epidermal strata or a fully proliferative epidermal multilayer system. In particular, human keratinocytes adhered on CHT and CHT-PCL membranes, forming the structure of the epidermal top layers, such as the corneum and granulosum strata, characterized by withdrawal or reduction from the cell cycle and cell proliferation. On the PCL membrane, keratinocytes developed an epidermal basal lamina, with high proliferating cells that stratified and migrated over time to form a complete differentiating epidermal multilayer system. PMID:27371895

  19. 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.

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

    Science.gov (United States)

    Arul, Santhosh; Dayalan, Haripriya; Jegadeesan, Muhilan; Damodharan, Prabhavathy

    2016-12-01

    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. PMID:27453822

  1. Quantitative analysis of laminin 5 gene expression in human keratinocytes.

    Science.gov (United States)

    Akutsu, Nobuko; Amano, Satoshi; Nishiyama, Toshio

    2005-05-01

    To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains alpha3, beta3, and gamma2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein-labeled probes with the ABI PRISM 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde-3-phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 x 10(2) to 1 x 10(8) gene copies. Basal gene expression level of LAMB3 was about one-tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of alpha3, beta3, and gamma2 synthesized in radio-labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme-linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms. PMID:15854126

  2. 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

  3. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    Science.gov (United States)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  4. 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.

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

    Directory of Open Access Journals (Sweden)

    Sornam Ganesan

    2015-01-01

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

  6. 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.

  7. Immunoglobulin-free light chain monomer-dimer patterns help to distinguish malignant from premalignant monoclonal gammopathies: a pilot study.

    Science.gov (United States)

    Kaplan, Batia; Golderman, Sizilia; Aizenbud, Boris; Esev, Konstantin; Kukuy, Olga; Leiba, Merav; Livneh, Avi; Ben-Zvi, Ilan

    2014-09-01

    Multiple myeloma (MM) and AL amyloidosis (AL) are two malignant forms of monoclonal gammopathies. For the purposes of prognosis and treatment, it is important to distinguish these diseases from the premalignant forms of monoclonal gammopathies, such as monoclonal gammopathy of unknown significance (MGUS) and smoldering myeloma (SMM). Routine serum/urine tests for monoclonal protein are insufficient for differential diagnosis. Thus, invasive procedures, such as tissue aspiration or biopsy, are applied. In this study, we aimed at characterization of serum-free light chain (FLC) monomer-dimer patterns to distinguish the malignant from the premalignant forms of monoclonal gammopathies. A quantitative Western blotting was applied to estimate the FLC monomer and dimer levels in AL, MM, MGUS, and SMM patients, and in control subjects (healthy individuals and patients with AA amyloidosis). AL and MM patients displayed an abnormally increased dimerization of monoclonal FLC, accompanied by higher clonality values of FLC dimers, as compared to that of monomers. These abnormalities of FLC patterns were not observed in patients with MGUS, SMM, AA amyloidosis, and healthy individuals. Analysis of FLC patterns helped to differentiate AL and MM from MGUS and SMM, a goal difficult to achieve using routine serum tests. Also, our technique might serve as a complimentary diagnostic tool in the cases with suspected AL amyloidosis, where the diagnosis of MM is excluded, while the results of amyloid typing by routine immunohistochemical techniques are inconclusive. PMID:24866208

  8. 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.

  9. Dental metal-induced innate reactivity in keratinocytes

    NARCIS (Netherlands)

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

    2015-01-01

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

  10. 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.

  11. Decrease in class pi glutathione transferase mRNA levels by ultraviolet irradiation of cultured rat keratinocytes

    International Nuclear Information System (INIS)

    The effect of ultraviolet (UV) B irradiation on pi class glutathione transferase (GST-P) gene expression was examined in cultured rat keratinocytes. Immunoblotting demonstrated GST-P to be the major GST form in the cells, and it was significantly decreased following irradiation. Northern blot analysis revealed that the mRNA decreased to 10-25% of the initial value 24 h after irradiation at a dose of 40 mJ/cm2. No remarkable changes were observed at earlier time points. Hydrogen peroxide treatment enhanced GST-P mRNA expression, with a 70% increase at 250 μM concentration. Alterations in possible trans-acting factors were examined to clarify the mechanism of repression by UV irradiation. c-Jun mRNA was induced 3.5-fold at 4 h after irradiation, but by 24 h fell to a lower level than that observed initially. c-Fos mRNA was increased 10-fold at 1 h but was completely suppressed at 12 and 24 h. Thus, the changes of c-Jun and c-Fos mRNA differed from that of GST-P mRNA. The level of mRNA for silencer factor-B was decreased to less than 10% at 12 h. UV irradiation of cells transfected with the chloramphenicol acetyltransferase (CAT) reporter gene containing enhancer (GPE I) or silencer regions of the GST-P gene did not suppress CAT activity. Although basal expression of the GST-P gene was mainly dependent on GPE I, altered expression of c-jun, c-fos and other genes coding for factors possibly trans-acting on GPE I did not appear to be responsible for the decreased GST-P mRNA levels. (author)

  12. Premalignant PTEN-deficient thymocytes activate microRNAs miR-146a and miR-146b as a cellular defense against malignant transformation

    OpenAIRE

    Burger, Megan L.; Xue, Ling; Sun, Yuefang; Kang, Chulho; Winoto, Astar

    2014-01-01

    miR-146a and miR-146b are upregulated during premalignancy in the thymus of T cell–specific PTEN-deficient mice.Transgenic expression of mir-146a/b delays PTEN-deficient lymphomagenesis through repression of TCR signals critical for c-myc activation.

  13. Curcumin down regulates smokeless tobacco-induced NF-κB activation and COX-2 expression in human oral premalignant and cancer cells

    International Nuclear Information System (INIS)

    Smokeless tobacco (ST) consumption is a major cause of oral cancer in South East Asia including India. Recently, we showed that exposure to smokeless tobacco extract (STE) (khaini) results in increased expression and activation of nuclear factor-κB (NF-κB) and its downstream target cyclooxygenase-2 (COX-2) in human oral cell systems in vitro. The present study was designed to test the hypothesis that curcumin may inhibit the activation of NF-κB in ST exposed oral premalignant and cancer cells. Exposure of oral premalignant and cancer cells to curcumin resulted in significant decrease in cell viability and induced apoptosis. STE-induced nuclear translocation and DNA-binding activity of NF-κB were inhibited in curcumin pretreated oral premalignant and cancer cells in vitro. Curcumin treatment led to decreased expression of NF-κB and COX-2. The tobacco specific nitrosamine, 4-(methylnitrosamino-)-1-(3-pyridyl)-1-butanone (NNK), is one of the carcinogenic components of STE (khaini). We demonstrate that curcumin pretreatment abrogated NNK-induced activation of NF-κB and COX-2 expression, suggesting that NNK is one of the factors in STE (khaini) modulated by curcumin. In conclusion, our findings demonstrate for the first time that curcumin downregulates STE (khaini) or NNK-induced NF-κB and COX-2 in oral premalignant and cancer cells in vitro

  14. Toxicity of silver nanoparticles in monocytes and keratinocytes

    DEFF Research Database (Denmark)

    Orłowski, Piotr; Krzyzowska, Malgorzata; Winnicka, Anna;

    2012-01-01

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

  15. Transcriptional network of p63 in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    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.

  16. Neoplastic transformation of immortalized human epidermal keratinocytes by ionizing radiation.

    OpenAIRE

    Thraves, P; Salehi, Z; Dritschilo, A; Rhim, J S

    1990-01-01

    Efforts to investigate the progression of events that cause human cells to become neoplastic in response to ionizing radiation have been aided by the development of tissue culture systems of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus type 12 and simian virus 40 have been transformed by exposure to x-ray irradiation. Such transformants showed morphological alterations, formed colonies in soft agar, and induced carcinomas when...

  17. 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.

  18. Antioxidants Protect Keratinocytes against M. ulcerans Mycolactone Cytotoxicity

    OpenAIRE

    Alvar Grönberg; Louise Zettergren; Kerstin Bergh; Mona Ståhle; Johan Heilborn; Kristian Angeby; Small, Pamela L.; Hannah Akuffo; Sven Britton

    2010-01-01

    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 p...

  19. Antioxidants Protect Keratinocytes against M. ulcerans Mycolactone Cytotoxicity

    OpenAIRE

    Grönberg, Alvar; Zettergren, Louise; Bergh, Kerstin; Ståhle, Mona; Heilborn, Johan; Ängeby, Kristian; Small, Pamela L.; Akuffo, Hannah; Britton, Sven

    2010-01-01

    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 pr...

  20. Response of human epidermal keratinocytes to UV light

    International Nuclear Information System (INIS)

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

  1. 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.

  2. High-Glucose Environment Enhanced Oxidative Stress and Increased Interleukin-8 Secretion From Keratinocytes

    OpenAIRE

    Lan, Cheng-Che E.; Wu, Ching-Shuang; Huang, Shu-Mei; Wu, I-Hui; Chen, Gwo-Shing

    2013-01-01

    Impaired wound healing frequently occurs in patients with diabetes. Interleukin (IL)-8 production by keratinocyte is responsible for recruiting neutrophils during healing. Intense inflammation is associated with diabetic wounds, while reduction of neutrophil infiltration is associated with enhanced healing. We hypothesized that increased neutrophil recruitment by keratinocytes may contribute to the delayed healing of diabetic wounds. Using cultured human keratinocytes and a diabetic rat model...

  3. Micronucleus formation in human keratinocytes is dependent on radiation quality and tissue architecture.

    Science.gov (United States)

    Snijders, Antoine M; Mannion, Brandon J; Leung, Stanley G; Moon, Sol C; Kronenberg, Amy; Wiese, Claudia

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. PMID:25041929

  4. Keratinocyte Migration, Proliferation, and Differentiation in Chronic Ulcers From Patients With Diabetes and Normal Wounds

    OpenAIRE

    Usui, Marcia L.; Mansbridge, Jonathan N.; Carter, William G.; Fujita, Mayumi; Olerud, John E

    2008-01-01

    Epithelialization of normal acute wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore barrier function. The keratinocytes in the epidermis of chronic ulcers fail to execute this series of events. To better understand the epithelial dynamics of chronic ulcers, we used immunohistochemistry to evaluate proliferation, differentiation, adhesion, and migration in keratinocytes along the margin of chronic ulcers from patients with dia...

  5. Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion.

    Science.gov (United States)

    Prince, Tessa; McBain, Andrew J; O'Neill, Catherine A

    2012-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Marchal Joëlle

    2005-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Ju-Suk Nam

    2014-03-01

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

  8. Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice.

    OpenAIRE

    Guo, L.; Yu, Q C; E. Fuchs

    1993-01-01

    Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. Synthesized by cells of the dermal component of skin, KGF's potent mitogenic activity is on the epidermal component, which harbors the receptors for this factor. To explore the possible role of KGF in mesenchymal-epithelial interactions in skin, we used a human keratin 14 promoter to target expression of human KGF cDNA to the stratified squamous epithelia of transgenic mice. Mice expressing KGF in their...

  9. 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.

  10. 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. PMID:25356006

  11. Efficient keratinocyte differentiation strictly depends on JNK-induced soluble factors in fibroblasts.

    Science.gov (United States)

    Schumacher, Marion; Schuster, Christian; Rogon, Zbigniew M; Bauer, Tobias; Caushaj, Nevisa; Baars, Sebastian; Szabowski, Sibylle; Bauer, Christine; Schorpp-Kistner, Marina; Hess, Jochen; Holland-Cunz, Stefan; Wagner, Erwin F; Eils, Roland; Angel, Peter; Hartenstein, Bettina

    2014-05-01

    Previous studies demonstrated that fibroblast-derived and JUN-dependent soluble factors have a crucial role on keratinocyte proliferation and differentiation during cutaneous wound healing. Furthermore, mice with a deficiency in Jun N-terminal kinases (JNKs) , JNK1 or JNK2, showed impaired skin development and delayed wound closure. To decipher the role of dermal JNK in keratinocyte behavior during these processes, we used a heterologous coculture model combining primary human keratinocytes and murine fibroblasts. Although cocultured JNK1/JNK2-deficient fibroblasts did not affect keratinocyte proliferation, temporal monitoring of the transcriptome of differentiating keratinocytes revealed that efficient keratinocyte differentiation not only requires the support by fibroblast-derived soluble factors, but is also critically dependent on JNK1 and JNK2 signaling in these cells. Moreover, we showed that the repertoire of fibroblast transcripts encoding secreted proteins is severely disarranged upon loss of JNK under the coculture conditions applied. Finally, our data demonstrate that efficient keratinocyte terminal differentiation requires constant presence of JNK-dependent and fibroblast-derived soluble factors. Taken together, our results imply that mesenchymal JNK has a pivotal role in the paracrine cross talk between dermal fibroblasts and epidermal keratinocytes during wound healing. PMID:24335928

  12. Arsenite suppression of BMP signaling in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-06-15

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Ling Yang

    2011-01-01

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

  15. Understanding Intratumoral Heterogeneity: Lessons from the Analysis of At-Risk Tissue and Premalignant Lesions in the Colon.

    Science.gov (United States)

    Sievers, Chelsie K; Leystra, Alyssa A; Clipson, Linda; Dove, William F; Halberg, Richard B

    2016-08-01

    Advances in DNA sequencing have created new opportunities to better understand the biology of cancers. Attention is currently focused on precision medicine: does a cancer carry a mutation that is targetable with already available drugs? But, the timing at which multiple, targetable mutations arise during the adenoma to carcinoma sequence remains unresolved. Borras and colleagues identified mutations and allelic imbalance in at-risk mucosa and early polyps in the human colon. Their analyses indicate that mutations in key genes can arise quite early during tumorigenesis and that polyps are often multiclonal with at least two clones. These results are consistent with the "Big Bang" model of tumorigenesis, which postulates that intratumoral heterogeneity is a consequence of a mutational burst in the first few cell divisions following initiation that drives divergence from a single founder with unique but related clones coevolving. Emerging questions center around the ancestry of the tumor and impact of early intratumoral heterogeneity on tumor establishment, growth, progression, and most importantly, response to therapeutic intervention. Additional sequencing studies in which samples, especially at-risk tissue and premalignant neoplasms, are analyzed from animal models and humans will further our understanding of tumorigenesis and lead to more effective strategies for prevention and treatment. Cancer Prev Res; 9(8); 638-41. ©2016 AACRSee related article by Borras, et al., Cancer Prev Res 2016;9(6):417-427. PMID:27199343

  16. 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...

  17. Sodium fluoride influences the expression of keratins in cultured keratinocytes

    OpenAIRE

    Prado, Euridice; Wurtz, Tilmann; Ferbus, Didier; Shabana, El-Hassan; FOREST, Nadine; Berdal, Ariane

    2010-01-01

    Epithelia in lung, skin, and kidney are often exposed to fluoride, and tissue damage in lung and kidney due to fluoride is well documented. Nevertheless, the biological effects of fluoride on epithelia are poorly investigated. In the present study, we report effects of sodium fluoride (NaF) on the differentiation of a human epithelial cell line, HaCaT. These cells may serve as a keratinocyte model, because they express a wide spectrum of keratins (Ks), and they associate into stratified tissu...

  18. 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

  19. 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

  20. Characterisation of human fibroblasts as keratinocyte feeder layer using p63 isoforms status.

    Science.gov (United States)

    Auxenfans, Céline; Thépot, Amélie; Justin, Virginie; Hautefeuille, Agnès; Shahabeddin, Lili; Damour, Odile; Hainaut, Pierre

    2009-01-01

    Large-scale culture of primary keratinocytes allows the production of large epidermal sheet surfaces for the treatment of extensive skin burns. This method is dependent upon the capacity to establish cultures of proliferating keratinocytes in conditions compatible with their clonal expansion while maintaining their capacity to differentiate into the typical squamous pattern of human epidermis. Feeder layers are critical in this process because the fibroblasts that compose this layer serve as a source of adhesion, growth and differentiation factors. In this report, we have characterise the expression patterns of p63 isoforms in primary keratinocytes cultured on two different feeder layer systems, murine 3T3 and human fibroblasts. We show that with the latter, keratinocytes express a higher ratio of Delta N to TAp63 isoform, in relation with higher clonogenic potential. These results indicate that human fibroblasts represent an adequate feeder layer system to support the culture of primary human keratinocytes. PMID:20042803

  1. Stimulatory effect of Brazilian propolis on hair growth through proliferation of keratinocytes in mice.

    Science.gov (United States)

    Miyata, Shota; Oda, Yozo; Matsuo, Chika; Kumura, Haruto; Kobayashi, Ken

    2014-12-10

    Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.

  2. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Keratinocyte migration in the developing eyelid requires LIMK2.

    Directory of Open Access Journals (Sweden)

    Dennis S Rice

    Full Text Available In vitro studies have identified LIMK2 as a key downstream effector of Rho GTPase-induced changes in cytoskeletal organization. LIMK2 is phosphorylated and activated by Rho associated coiled-coil kinases (ROCKs in response to a variety of growth factors. The biochemical targets of LIMK2 belong to a family of actin binding proteins that are potent modulators of actin assembly and disassembly. Although numerous studies have suggested that LIMK2 regulates cell morphology and motility, evidence supportive of these functions in vivo has remained elusive. In this study, a knockout mouse was created that abolished LIMK2 biochemical activity resulting in a profound inhibition of epithelial sheet migration during eyelid development. In the absence of LIMK2, nascent eyelid keratinocytes differentiate and acquire a pre-migratory phenotype but the leading cells fail to nucleate filamentous actin and remain immobile causing an eyes open at birth (EOB phenotype. The failed nucleation of actin was associated with significant reductions in phosphorylated cofilin, a major LIMK2 biochemical substrate and potent modulator of actin dynamics. These results demonstrate that LIMK2 activity is required for keratinocyte migration in the developing eyelid.

  5. 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

  6. UV-B Radiation Induces Macrophage Migration Inhibitory Factor–Mediated Melanogenesis through Activation of Protease-Activated Receptor-2 and Stem Cell Factor in Keratinocytes

    OpenAIRE

    Enomoto, Akiko; Yoshihisa, Yoko; Yamakoshi, Takako; Ur Rehman, Mati; Norisugi, Osamu; HARA Hiroshi; Matsunaga, Kenji; Makino, Teruhiko; Nishihira, Jun; Shimizu, Tadamichi

    2011-01-01

    UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF ha...

  7. Nitric Oxide Donors Suppress Chemokine Production by Keratinocytes in Vitro and in Vivo

    Science.gov (United States)

    Giustizieri, Maria Laura; Albanesi, Cristina; Scarponi, Claudia; De Pità, Ornella; Girolomoni, Giampiero

    2002-01-01

    Nitric oxide (NO) is involved in the modulation of inflammatory responses. In psoriatic skin, NO is highly produced by epidermal keratinocytes in response to interferon-γ and tumor necrosis factor-α. In this study, we investigated whether the NO donors, S-nitrosoglutathione (GS-NO) and NOR-1, could regulate chemokine production by human keratinocytes activated with interferon-γ and tumor necrosis factor-α. In addition, we studied the effects of the topical application of a GS-NO ointment on chemokine expression in lesional psoriatic skin. NO donors diminished in a dose-dependent manner and at both mRNA and protein levels the IP-10, RANTES, and MCP-1 expression in keratinocytes cultured from healthy patients and psoriatic patients. In contrast, constitutive and induced interleukin-8 production was unchanged. GS-NO-treated psoriatic skin showed reduction of IP-10, RANTES, and MCP-1, but not interleukin-8 expression by keratinocytes. Moreover, the number of CD14+ and CD3+ cells infiltrating the epidermis and papillary dermis diminished significantly. NO donors also down-regulated ICAM-1 protein expression without affecting mRNA accumulation in vitro, and suppressed keratinocyte ICAM-1 in vivo. Finally, NO donors inhibited nuclear factor-κB and STAT-1, but not AP-1 activities in transiently transfected keratinocytes. These results define NO donors as negative regulators of chemokine production by keratinocytes. PMID:12368213

  8. 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.

  9. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Systemic suppression of delayed-type hypersensitivity by supernatants from UV-irradiated keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Rivas, J.M.; Ullrich, S.E. (Univ. of Texas, Houston (United States))

    1992-12-15

    Exposing murine keratinocyte cultures to UV radiation causes the release of a suppressive cytokine that mimics the immunosuppressive effects of total-body UV exposure. Injecting supernatants from UV-irradiated keratinocyte cultures into mice inhibits their ability to generate a delayed-type hypersensitivity reaction against allogeneic histocompatibility Ag, and spleen cells from mice injected with supernatant do not respond to alloantigen in the in vitro MLR. A unique feature of the immunosuppression induced by either total-body UV-exposure or injecting the suppressive cytokine from UV-irradiated keratinocytes is the selectivity of suppression. Although cellular immune reactions such as delayed-type hypersensitivity are suppressed antibody production is unaffected. Because the selective nature to the UV-induced immunosuppression is similar to the biologic activity of IL-10, the authors examined the hypothesis that UV exposure of keratinocytes causes the release of IL-10. Keratinocyte monolayers were exposed to UV radiation and at specific times after exposure mRNA was isolated or the culture supernatant from the cells was collected. These data indicate that activated keratinocytes are capable of secreting IL-10 and suggest that the release of IL-10 by UV-irradiated keratinocytes plays an essential role in the induction of systemic immunosuppression after total-body UV exposure. 44 refs., 3 figs., 2 tabs.

  12. Protective effects of myricetin against ultraviolet-B-induced damage in human keratinocytes.

    Science.gov (United States)

    Huang, Jheng-Hua; Huang, Chieh-Chen; Fang, Jia-You; Yang, Cheng; Chan, Chi-Ming; Wu, Nan-Lin; Kang, Shung-Wen; Hung, Chi-Feng

    2010-02-01

    Myricetin is a flavonoid similar to quercetin, which is commonly found in natural foods such as berries, vegetables, teas, wine, and herbs. It is considered to be an antioxidant which is capable of quenching photoaging-causing free radicals within the skin. In this study, we investigated the mechanisms underlying protective effect of myricetin on ultraviolet-B (UVB)-induced damage to keratinocytes. We found that myricetin concentration-dependently attenuated UVB-induced keratinocyte death as determined by a cell viability assay. Pretreatment with myricetin also reduced the UVB-induced malondialdehyde level. Moreover, UVB-induced H(2)O(2) generation in keratinocytes was inhibited by myricetin according to flow cytometry, suggesting that myricetin can act as a free radical scavenger when keratinocytes experience photodamage. Furthermore, UVB-induced activation of c-jun-NH(2) terminal kinase (JNK) in keratinocytes was inhibited by myricetin. UVB-induced pre-G(1) phase arrest leading to apoptotic changes in keratinocytes was blocked by myricetin. Taken together, the protective mechanisms of keratinocyte by myricetin against UVB-induced photodamage occur by the inhibition of UVB-induced intracellular hydrogen peroxide production, lipid peroxidation and JNK activation. Therefore, myricetin is suitable for further development as an anti-aging agent for skin care. PMID:19778600

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

    Science.gov (United States)

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

    2015-01-01

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

  14. 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.

  15. 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.

  16. 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.

  17. 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

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

    Directory of Open Access Journals (Sweden)

    Raghav K Chhetri

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

  19. 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.

  20. 3D co-cultures of keratinocytes and melanocytes and cytoprotective effects on keratinocytes against reactive oxygen species by insect virus-derived protein microcrystals

    International Nuclear Information System (INIS)

    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

  1. 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

  2. Reorganization of the interchromosomal network during keratinocyte differentiation.

    Science.gov (United States)

    Sehgal, Nitasha; Seifert, Brandon; Ding, Hu; Chen, Zihe; Stojkovic, Branislav; Bhattacharya, Sambit; Xu, Jinhui; Berezney, Ronald

    2016-06-01

    The well-established human epidermal keratinocyte (HEK) differentiation model was investigated to determine possible alterations in chromosome territory (CT) association during differentiation. The seven human chromosomes (1, 4, 11, 12, 16, 17, and 18) selected for this analysis are representative of the chromosome size and gene density range of the overall human genome as well as including a majority of genes involved in epidermal development and differentiation (CT1, 12, and 17). Induction with calcium chloride (Ca(2+)) resulted in morphological changes characteristic of keratinocyte differentiation. Combined multi-fluorescence in situ hybridization (FISH) and computational image analysis on the undifferentiated (0 h) and differentiated (24 h after Ca(2+) treatment) HEK revealed that (a) increases in CT volumes correspond to overall nuclear volume increases, (b) radial positioning is gene density-dependent at 0 h but neither gene density- nor size-dependent at 24 h, (c) the average number of interchromosomal associations for each CT is gene density-dependent and similar at both time points, and (d) there are striking differences in the single and multiple pairwise interchromosomal association profiles. Probabilistic network models of the overall interchromosomal associations demonstrate major reorganization of the network during differentiation. Only ~40 % of the CT pairwise connections in the networks are common to both 0 and 24 h HEK. We propose that there is a probabilistic chromosome positional code which can be significantly altered during cell differentiation in coordination with reprogramming of gene expression. PMID:26490167

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-01

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. PMID:27216463

  5. Modulation of keratinocyte gene expression and differentiation by PPAR-selective ligands and tetradecylthioacetic acid

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  6. Calcium--a central regulator of keratinocyte differentiation in health and disease.

    Science.gov (United States)

    Elsholz, Floriana; Harteneck, Christian; Muller, Walter; Friedland, Kristina

    2014-01-01

    Regular keratinocyte differentiation is crucial for the formation of an intact epidermal barrier and is triggered by extracellular calcium. Disturbances of epidermal barrier formation and aberrant keratinocyte differentiation are involved in the pathophysiology of several skin diseases, such as psoriasis, atopic dermatitis, basal and squamous skin cancer, and genetic skin diseases such as Darier's disease and Olmstedt syndrome. In this review, we summarize current knowledge about the underlying molecular mechanisms of calcium-induced differentiation in keratinocytes. We provide an overview of calcium's genomic and non-genomic mechanisms to induce differentiation and discuss the calcium gradient in the epidermis, giving rise to cornified skin and lipid envelope formation. We focus on the calcium-sensing receptor, transient receptor potential channels, and STIM/Orai as the major constituents of calcium sensing and calcium entry in the keratinocytes. Finally, skin diseases linked to impaired differentiation will be discussed, paying special attention to disturbed TRP channel expression and TRP channel mutations.

  7. 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

  8. Failure of extracts from Malassezia pachydermatis to stimulate canine keratinocyte proliferation in vitro.

    Science.gov (United States)

    Chen, Tai-An; Halliwell, Richard E W; Hill, Peter B

    2002-12-01

    Epidermal hyperplasia is one of the major histopathological features seen in dogs with Malassezia dermatitis. The aim of this study was to investigate the effects of extracts and culture supernatants from Malassezia pachydermatis on the proliferation of canine keratinocytes. Keratinocyte cultures were established from normal dog skin, and cell monolayers were co-cultured with Malassezia extracts (prepared either with or without protease inhibitors) and supernatants derived from organisms grown in liquid culture. The proliferation of keratinocytes was measured using a colourimetric assay. Neither the culture supernatants nor the Malassezia extracts had significant effects on the proliferation rate of canine keratinocytes, regardless of whether protease inhibitors were present or not. The results indicate that the epidermal hyperplasia seen in Malassezia dermatitis is unlikely to be caused directly by secretion of products from the organism. PMID:12464065

  9. 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.

  10. 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

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

    International Nuclear Information System (INIS)

    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

  12. Pimecrolimus Enhances TLR2/6-Induced Expression of Antimicrobial Peptides in Keratinocytes

    OpenAIRE

    Büchau, Amanda S.; Schauber, Jürgen; Hultsch, Thomas; Stuetz, Anton; Richard L Gallo

    2008-01-01

    Calcineurin inhibitors are potent inhibitors of T-cell-receptor mediated activation of the adaptive immune system. The effects of this class of drug on the innate immune response system are not known. Keratinocytes are essential to innate immunity in skin and rely on toll-like receptors (TLRs) and antimicrobial peptides to appropriately recognize and respond to injury or microbes. In this study we examined the response of cultured human keratinocytes to pimecrolimus. We observed that pimecrol...

  13. Microtubule Disruption in Keratinocytes Induces Cell-Cell Adhesion through Activation of Endogenous E-Cadherin

    OpenAIRE

    Kee, Sun-Ho; Steinert, Peter M.

    2001-01-01

    The association of the cytoskeleton with the cadherin–catenin complex is essential for strong cell-cell adhesion in epithelial cells. In this study, we have investigated the effect of microtubule organization on cell-cell adhesion in differentiating keratinocytes. When microtubules of normal human epidermal keratinocytes (NHEKs) grown in low calcium media (0.05 mM) were disrupted with nocodazole or colcemid, cell-cell adhesion was induced through relocalization of the ...

  14. Tonic Inhibition of TRPV3 by Mg2+ in Mouse Epidermal Keratinocytes

    OpenAIRE

    Luo, Jialie; Stewart, Randi; Berdeaux, Rebecca; Hu, Hongzhen

    2012-01-01

    The transient receptor potential vanilloid 3 channel (TRPV3) is abundantly expressed in epidermal keratinocytes and plays important roles in sensory biology and skin health. Mg2+ deficiency causes skin disorders under certain pathological conditions such as type 2 diabetes mellitus. In this study, we investigated the effect of Mg2+ on TRPV3 in primary epidermal keratinocytes. Extracellular Mg2+ ([Mg2+]o) inhibited TRPV3-mediated membrane current and calcium influx. TRPV3 activation induced a ...

  15. Involucrin and envelope competence in human keratinocytes: Modulation by hydrocortisone, retinyl acetate and growth arrest

    OpenAIRE

    Rice, Rh; Cline, PR

    1983-01-01

    Involucrin accumulation and ionophore-assisted envelope for mation, markers of keratinocyte differentiation, were found to be highly dependent on culture conditions in the malignant epidermal keratinocyte line, SCC-13, derived from a human squamous cell carcinoma. In confluent cultures, approximately one-half of the cells were competent to form envelopes when grown in medium without hydrocortisone or retinyl acetate supplementation. Ad dition of hydrocortisone to the medi...

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

    OpenAIRE

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

    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...

  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. Combined treatment with sodium butyrate and PD153035 enhances keratinocyte differentiation

    OpenAIRE

    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 differentia...

  19. Biologic mechanisms for the regulation of normal human keratinocyte proliferation and differentiation.

    OpenAIRE

    Wilke, M. S.; Hsu, B. M.; Wille, J J; Pittelkow, M R; Scott, R. E.

    1988-01-01

    Normal human keratinocytes can be grown in serum-free medium, and the integrated control of their proliferation and differentiation can be modulated experimentally. The growth of cultured human keratinocytes can also be specifically arrested at either reversible or irreversible growth arrest states. Reversible growth arrest is induced by culture in medium containing TGF-beta or ethionine or in medium deficient of isoleucine. Irreversible growth arrest is induced by culture in razoxane-contain...

  20. 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...

  1. Tualang Honey protects keratinocytes from ultraviolet radiation induced inflammation and DNA damage†

    OpenAIRE

    Ahmad, Israr; Jimenez, Hugo; Yaacob, Nik Soriani; Yusuf, Nabiha

    2012-01-01

    Malaysian tualang honey possesses strong antioxidant and anti-inflammatory properties. Here, we evaluated the effect of tualang honey on early biomarkers of photocarcinogenesis employing PAM212 mouse keratinocyte cell line. Keratinocytes were treated with tualang honey (1.0%, v/v) before a single UVB (150 mJ/cm2) irradiation. We found that treatment of tualang honey inhibited UVB-induced DNA damage, and enhanced repair of UVB-mediated formation of cyclobutane pyrimidine dimers (CPDs) and 8-ox...

  2. 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.

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. 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.

  6. 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.

  7. Gene expression studies on human keratinocytes transduced with human growth hormone gene for a possible utilization in gene therapy

    International Nuclear Information System (INIS)

    Taking advantage of the recent progress in the DNA-recombinant techniques and of the potentiality of normal human keratinocytes primary culture to reconstitute the epidermis, it was decided to genetically transform these keratinocytes to produce human growth hormone under controllable conditions that would be used in gene therapy at this hormone deficient patients. The first step to achieve this goal was to standardize infection of keratinocytes with retrovirus producer cells containing a construct which included the gene of bacterial b-galactosidase. The best result was obtained cultivating the keratinocytes for 3 days in a 2:1 mixture of retrovirus producer cells and 3T3-J2 fibroblasts irradiated with 60 Gy, and splitting these infected keratinocytes on 3T3-J2 fibroblasts feeder layer. Another preliminary experiment was to infect normal human keratinocytes with interleukin-6 gene (hIL-6) that, in pathologic conditions, could be reproduced by keratinocytes and secreted to the blood stream. Thus, we verify that infected keratinocytes secrete an average amount of 500 ng/106 cell/day of cytokin during the in vitro life time, that certify the stable character of the injection. These keratinocytes, when grafted in mice, secrete hIL-6 to the blood stream reaching levels of 40 pg/ml of serum. After these preliminary experiments, we construct a retroviral vector with the human growth hormone gene (h GH) driven by human metallothionein promoter (h PMT), designated DChPMTGH. Normal human keratinocytes were infected with DChPMTGH producer cells, following previously standardized protocol, obtaining infected keratinocytes secreting to the culture media 340 ng h GH/106 cell/day without promoter activation. This is the highest level of h GH secreted in human keratinocytes primary culture described in literature. The h GH value increases approximately 10 times after activation with 100 μM Zn+2 for 8-12 hours. (author). 158 refs., 42 figs., 6 tabs

  8. In vitro culture conditions to study keratinocyte differentiation using the HaCaT cell line

    OpenAIRE

    Deyrieux, Adeline F.; Wilson, V G

    2007-01-01

    In vitro models to study the process of keratinocyte differentiation have been hindered by the stringent culture requirements and limitations imposed by the inherent properties of the cells. Primary keratinocytes only have a finite life span, while transformed cell lines exhibit many phenotypic features not found in normal cells. The spontaneously immortalized HaCaT cell line has been a widely employed keratinocyte model due to its ease of propagation and near normal phenotype, but protocols ...

  9. Basal and stress-inducible expression of HSPA6 in human keratinocytes is regulated by negative and positive promoter regions

    OpenAIRE

    Ramirez, Vincent P.; Stamatis, Michael; Shmukler, Anastasia; Aneskievich, Brian J.

    2014-01-01

    Epidermal keratinocytes serve as the primary barrier between the body and environmental stressors. They are subjected to numerous stress events and are likely to respond with a repertoire of heat shock proteins (HSPs). HSPA6 (HSP70B′) is described in other cell types with characteristically low to undetectable basal expression, but is highly stress induced. Despite this response in other cells, little is known about its control in keratinocytes. We examined endogenous human keratinocyte HSPA6...

  10. A review on quality of life in keratinocyte carcinoma patients.

    Science.gov (United States)

    Waalboer-Spuij, R; Nijsten, T E C

    2013-06-01

    Health-related quality of life issues in patients with cutaneous malignancies is being re-explored. This is motivated by the heavy burden they put on dermatological care, it is more and more considered a chronic disease and new non-invasive therapies are being introduced. The purpose of this review is to identify the relevant quality of life (QOL) issues and to summarize the instruments used for investigating QOL in keratinocyte carcinoma patients. With a systematic literature search in Embase, MEDLINE OvidSP, PubMed publisher and Cochrane Central, 10 questionnaires and 4 studies reporting on quality of life issues were identified. Generic (UK Sickness Impact profile [UKSIP], Short Form 36-item Health Survey [SF-36], Functional Assessment of Cancer Therapy-General [FACT-G]) and dermatology specific (Dermatology Life Quality Index [DLQI] and Skindex-29, -16, -17) instruments demonstrated little to no QOL impairment. This may be explained by failing to capture the relevant domains such as "emotions", "appearance" and "anxiety". Skin cancer specific questionnaires (Skin Cancer Index [SCI], Skin Cancer Quality of Life Impact Tool [SCQOLIT] and Actinic Keratosis Quality of Life [AKQoL]) demonstrated good validity and responsiveness and represent the effect on QOL properly. However, there are some points of critique to these questionnaires. Optimal management of patients with actinic neoplasia syndrome and the selection and evaluation of therapies may benefit from the use of PROs in this ever increasing population. PMID:23670061

  11. 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.

  12. 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.

  13. 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.

  14. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-15

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

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

    International Nuclear Information System (INIS)

    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

  17. 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.

  18. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway.

    Science.gov (United States)

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin Md; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-03-15

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

  19. Melatonin protects skin keratinocyte from hydrogen peroxide-mediated cell death via the SIRT1 pathway

    Science.gov (United States)

    Lee, Ju-Hee; Moon, Ji-Hong; Nazim, Uddin MD.; Lee, You-Jin; Seol, Jae-Won; Eo, Seong-Kug; Lee, John-Hwa; Park, Sang-Youel

    2016-01-01

    Melatonin (N-acetyl-5-methoxytryptamine), which is primarily synthesized in and secreted from the pineal gland, plays a pivotal role in cell proliferation as well as in the regulation of cell metastasis and cell survival in a diverse range of cells. The aim of this study is to investigate protection effect of melatonin on H2O2-induced cell damage and the mechanisms of melatonin in human keratinocytes. Hydrogen peroxide dose-dependently induced cell damages in human keratinocytes and co-treatment of melatonin protected the keratinocytes against H2O2-induced cell damage. Melatonin treatment activated the autophagy flux signals, which were identified by the decreased levels of p62 protein. Inhibition of autophagy flux via an autophagy inhibitor and ATG5 siRNA technique blocked the protective effects of melatonin against H2O2-induced cell death in human keratinocytes. And we found the inhibition of sirt1 using sirtinol and sirt1 siRNA reversed the protective effects of melatonin and induces the autophagy process in H2O2-treated cells. This is the first report demonstrating that autophagy flux activated by melatonin protects human keratinocytes through sirt1 pathway against hydrogen peroxide-induced damages. And this study also suggest that melatonin could potentially be utilized as a therapeutic agent in skin disease. PMID:26918354

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

    Directory of Open Access Journals (Sweden)

    Stefanie Löffek

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

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

    Science.gov (United States)

    Telorack, Michèle; Meyer, Michael; Ingold, Irina; Conrad, Marcus; Bloch, Wilhelm; Werner, Sabine

    2016-01-01

    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. PMID:26808544

  2. On the interaction of alginate-based core-shell nanocarriers with keratinocytes in vitro.

    Science.gov (United States)

    Nguyen, Hoang Truc Phuong; Allard-Vannier, Emilie; Gaillard, Cédric; Eddaoudi, Imane; Miloudi, Lynda; Soucé, Martin; Chourpa, Igor; Munnier, Emilie

    2016-06-01

    Calcium alginate nanocarriers (CaANCs) were developed as a potential tool for delivery of hydrophobic active molecules such as pharmaceutical and cosmetic active ingredients. In this study, we focused on interactions between CaANCs and keratinocytes in culture and examined toxicity, internalization and drug release. Prior to cellular interactions, cryogenic transmission electron microscopy images showed that CaANCs appear as regular, spherical and dense particles, giving evidence of the surface gelation of CaANCs. Their size, around 200nm, was stable under tested conditions (temperature, culture media, presence of serum and presence of encapsulated dye), and their toxicity on keratinocytes was very low. Flow cytometry assays showed that CaANCs are internalized into keratinocytes by endocytosis with a predominant implication of the caveolae-mediated route. Förster resonance energy transfer (FRET) demonstrated that after a 2h contact, the release of CaANC contents in the cytoplasm of keratinocytes was almost complete. The endocytosis of CaANCs by a lysosome-free pathway, and the rapid release of their contents inside keratinocytes, will allow vectorized molecules to fully exhibit their pharmacological or cosmetic activity. PMID:26962764

  3. Rapid adhesion and proliferation of keratinocytes on the gold colloid/chitosan film scaffold

    International Nuclear Information System (INIS)

    The gold colloid/chitosan film scaffold, which could enhance the attached ratio and accelerate proliferation of newborn mice keratinocytes, was fabricated by nanotechnology and self-assembly technology. This nanometer scaffold was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). The keratinocytes were cultured and observed on three different extracellular matrices (ECM): gold colloid/chitosan film scaffold, chitosan film and cell culture plastic (control groups). 6 h, 12 h, 24 h after inoculation, the cell attached ratios were calculated respectively. In comparison to control groups, this scaffold could significantly (P < 0.01) increase the attached ratio of keratinocytes and promote their growth. Meanwhile, there were not any fusiform fibroblasts growing on this scaffold. The rapidly proliferating keratinocytes were indentified and characterized by immunohistochemistry and transmissive electron microscope (TEM), which showed the cells maintain their biological activity well. The results indicated that gold colloid/chitosan film scaffold was nontoxic to keratinocytes, and was a good candidate for wound dressing in skin tissue engineering.

  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. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

  9. 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.

  10. 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.

  11. Growth of human keratinocytes on hydrophilic film support and application to bioreactor culture

    Energy Technology Data Exchange (ETDEWEB)

    Knooka, M. (Osaka Univ., Suita (Japan)) Prenosil, J. (Swizerland Univ., Zurich (Switzerland))

    1998-10-01

    Human keratinocytes were cultured on an inert polymeric film support in serum-free medium to produce subconfluent autologous skin grafts for healing of burn wounds and chronic leg ulcers. The growth of keratinocytes in Petriperm with such a hydrophilic film bottom as a culture support was superior to that in T-flask owing to better cell adhesion. Based on this principle, an autonomous modular bioreactor, KERATOR for large scale production of skin grafts up to 5280 cm[sup 2] in size was constructed. It is computer controlled, and operations such as cell seeding and medium change are automated. The cell growth rate profiles in the bioreactor and Petriperm are similar, evincing the significance of KERATOR. The harvested skin grafts consist of the polymeric film covered by subconfluent sheets of non-differentiated keratinocyte cells. Such grafts may be applied to wounds in 'upside-down' fashion. 16 refs., 3 figs.

  12. 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

  13. 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.

  14. Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

    International Nuclear Information System (INIS)

    Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium. In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders

  15. Peptides from Tetraspanin CD9 Are Potent Inhibitors of Staphylococcus Aureus Adherence to Keratinocytes.

    Science.gov (United States)

    Ventress, Jennifer K; Partridge, Lynda J; Read, Robert C; Cozens, Daniel; MacNeil, Sheila; Monk, Peter N

    2016-01-01

    Staphylococcus aureus is one of the primary causative agents of skin and wound infections. As bacterial adherence is essential for infection, blocking this step can reduce invasion of host tissues by pathogens. An anti-adhesion therapy, based on a host membrane protein family, the tetraspanins, has been developed that can inhibit the adhesion of S. aureus to human cells. Synthetic peptides derived from a keratinocyte-expressed tetraspanin, CD9, were tested for anti-adhesive properties and at low nanomolar concentrations were shown to inhibit bacterial adhesion to cultured keratinocytes and to be effective in a tissue engineered model of human skin infection. These potential therapeutics had no effect on keratinocyte viability, migration or proliferation, indicating that they could be a valuable addition to current treatments for skin infection. PMID:27467693

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

    Energy Technology Data Exchange (ETDEWEB)

    Adams, Stephanie; Valchanova, Ralitsa S. [Charite-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany); Munz, Barbara, E-mail: barbara.munz@charite.de [Charite-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin (Germany)

    2010-03-10

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

  17. 磷脂酶Dδ缺失加剧UV-B诱导的膜伤害%Suppression of Phospholipase Dδ Enhances the Membrane Damage Induced by UV-B Irradiation

    Institute of Scientific and Technical Information of China (English)

    李艳; 田波; 李唯奇

    2011-01-01

    检测了拟南芥野生型(WS)及磷脂酶Dδ缺失突变体在UV-B辐射下的膜脂分子变化,并比较了二者在紫外辐射下的膜脂含量、双键指数及碳链长度的差异.结果发现,紫外辐射导致植株膜脂发生了降解,其中叶绿体膜脂MGDG和DGDG是膜伤害的主要作用靶点,而且突变体中的膜脂降解比野生型剧烈.上述结果说明磷脂酶Dδ的缺失会加剧紫外辐射诱导的膜伤害,导致植株对紫外辐射更加敏感.%The changes of molecular species in membrane lipids under UV-B irradiation in WS and PLD(ζ)-knockout plants were profiled with ESI-MS/MS based lipidomics.The content of membrane lipids, double bond index ( DBI) and carbon number of the fatty acid were examined in both of them.The results showed that UV-B irradiation induced the degradation of membrane lipids, in which chloroplast membrane lipids such as MCDC and DCDC were the main targets of membrane injury.In addition, the deUadation of membrane lipid in PLD8-def was more severe than that in WS plants.The results suggested that suppression of PLD(ζ) enhanced membrane damage induced by UV-B irradiation.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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

  20. 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.

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

    Science.gov (United States)

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

    2014-02-01

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

  2. 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.

  3. 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.

  4. Human keratinocyte ATP2C1 localizes to the Golgi and controls Golgi Ca2+ stores

    OpenAIRE

    Behne, M J; Tu, Chia-Ling L; Aronchik, I; Epstein, E; Bench, G.; Bikle, D D; Pozzan, T; Mauro, T.M.

    2003-01-01

    Hailey-Hailey disease (MIM16960) is a blistering skin disease caused by mutations in the Ca2+ ATPase ATP2C1. We found that the abnormal Ca2+ signaling seen in Hailey-Hailey disease keratinocytes correlates with decreased protein levels of ATP2C1. Human ATP2C1 protein approximated 115 kDa in size. The ATP2C1 is localized to the Golgi apparatus in human keratinocytes, similar to its localization in yeast and Caenorhabditis elegans. To test whether the ATP2C1 controls Golgi Ca2+ stores, we measu...

  5. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    Science.gov (United States)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  6. Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

    OpenAIRE

    Li, G.; Ho, V. C.; D. L. Mitchell; Trotter, M. J.; Tron, V A

    1997-01-01

    The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compare...

  7. Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

    DEFF Research Database (Denmark)

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

    1997-01-01

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

  8. 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

  9. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes.

    Science.gov (United States)

    Varma, Sandeep R; Sivaprakasam, Thiyagarajan O; Mishra, Abheepsa; Kumar, L M Sharath; Prakash, N S; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body's vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  10. Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocytes in vitro.

    Science.gov (United States)

    Deters, Alexandra M; Lengsfeld, Christian; Hensel, Andreas

    2005-12-01

    In traditional medicine, a variety of plants with high carbohydrate contents were used for dermatological therapies. Contemporary investigations confirmed exogenous carbohydrates as biologically active. The recent study describes the characterization of oligo- and polysaccharides from medicinal herbs and evaluation of composite-dependent physiological activity of carbohydrates on human keratinocytes in vitro. Polysaccharide isolation was followed by size- and charge fractionation. Identification of monosaccharide components was performed by GLC/MS. Primary human keratinocytes (NHK) and cells of the cell line HaCaT were used for investigation of carbohydrate action on cellular proliferation (BrdU-uptake), differentiation specific enzymes (involucrin), cell viability (MTT-reduction) and cytotoxicity. Incubation of keratinocytes with a purified beta-glucan from Reed mace seeds resulted in an improved proliferation followed by an increased differentiation after contact inhibition. Fucosylated oligo- and polysaccharides of human milk and Sea weed induced involucrin expression as maker for early differentiation without an increase in proliferation. Cell viability and proliferation of keratinocytes were enhanced by an arabinogalactan of Kaki fruits. Okra fruit rhamnogalacturonans increased cell proliferation. Heart sease pectin-like polysaccharides reduced the proliferation significantly but improved the cell viability. These results led assume that the carbohydrates of traditional used herbs play a part in their efficacy. PMID:16111846

  11. Regulation of chloride transport in cultured normal and cystic fibrobis keratinocytes

    NARCIS (Netherlands)

    S.M. Kansen (Maarten); J.L.M. Keulemans (J. L M); A.T. Hoogeveen (Andre); B.J. Scholte (Bob); A.B. Vaandrager (Arie); A.W.M. van der Kamp (Arthur); M. Sinaasappel (Maarten); A.G. Bot (Alice); H.R. de Jonge (Hugo); J. Bijman (Jan)

    1992-01-01

    markdownabstractAbstract Cultured normal (N) and cystic fibrosis (CF) keratinocytes were evaluated for their Cl−-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl− channel which has been reported to

  12. 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.

  13. 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.

  14. 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

  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. PMID:27060444

  16. p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

    Directory of Open Access Journals (Sweden)

    Boris Fischer

    2014-01-01

    Full Text Available p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium.

  17. p53 and TAp63 Promote Keratinocyte Proliferation and Differentiation in Breeding Tubercles of the Zebrafish

    Science.gov (United States)

    Fischer, Boris; Metzger, Manuel; Richardson, Rebecca; Knyphausen, Philipp; Ramezani, Thomas; Franzen, Rainer; Schmelzer, Elmon; Bloch, Wilhelm; Carney, Thomas J.; Hammerschmidt, Matthias

    2014-01-01

    p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal appendages which according to our analyses display more advanced stratification and keratinization than regular epidermis, including continuous desquamation and renewal of superficial cells by derivatives of basal keratinocytes. Defects are further enhanced in TAp63/p53 double mutants, pointing to partially redundant roles of the two related factors. Molecular analyses, treatments with chemical inhibitors and epistasis studies further reveal the existence of a linear TAp63/p53->Notch->caspase 3 pathway required both for enhanced proliferation of keratinocytes at the base of the tubercles and their subsequent differentiation in upper layers. Together, these studies identify the zebrafish breeding tubercles as specific epidermal structures sharing crucial features with the cornified mammalian epidermis. In addition, they unravel essential roles of TAp63 and p53 to promote both keratinocyte proliferation and their terminal differentiation by promoting Notch signalling and caspase 3 activity, ensuring formation and proper homeostasis of this self-renewing stratified epithelium. PMID:24415949

  18. Stress protein synthesis in human keratinocytes treated with sodium arsenite, phenyldichloroarsine, and nitrogen mustard

    International Nuclear Information System (INIS)

    Cells from bacteria to man respond to sublethal thermal and certain chemical stresses by synthesis of heat shock, or stress, proteins. The human epidermal keratinocyte is a target for a variety of cytotoxic substances. One response of cells exposed to such agents may be the synthesis of stress proteins. Human epidermal keratinocytes were treated thermally (43 degrees C) or chemically with sodium arsenite and the skin irritants phenyldichloroarsine and mechlorethamine. Proteins synthesized by keratinocytes were radiolabeled with [35S]methionine, separated on polyacrylamide gels under denaturing conditions, and visualized by fluorography. Quantitation by computer-assisted densitometry of fluorograms revealed different patterns of synthesis of two heat shock proteins (hsp's) with apparent molecular weights of 70 and 90 kDa after treatment with heat, sodium arsenite, phenyl-dichloroarsine, or mechlorethamine. Sodium arsenite induced the highest levels of synthesis of these two proteins, approximately 10-fold and 3-fold increases in hsp-70 and hsp-90, respectively. Phenyldichloroarsine at 0.5 microM produced a 2-fold increase in hsp-70 but no significant increase in hsp-90. Mechlorethamine, in contrast, had an apparent inhibitory effect on hsp-70 synthesis. These results suggest that some but not all skin irritants induce the synthesis of heat shock proteins in human keratinocytes

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

    Science.gov (United States)

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

    2014-10-01

    Malassezia spp. are saprophyte yeasts involved in skin diseases with different degrees of severity. The aim of our study was to analyze the response of human epidermal keratinocytes to Malassezia globosa and restricta strains evaluating the host defence mechanisms induced by Malassezia spp. colonization. Our results showed a different modulation of the inflammatory and immunomodulatory cytokine pathways obtained with the different strains of Malassezia tested. In addition, this expression is altered by blocking the TLR2 receptor. In comparison with M. furfur, M. globosa and restricta displayed an unexpected and striking cytotoxicity on keratinocytes. The differences observed could be related to the different modalities of interaction between keratinocytes and Malassezia strains, but also to their growth condition. Taken together, these results indicate that M. globosa or M. restricta colonization exert a different control on the cytokine inflammatory response activated in the human keratinocyte in which TLR2 might be involved. M. globosa and M. restricta may play a synergistic role in the exacerbation of skin diseases in which both are found. PMID:25038621

  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. Keratinocytes-associated chemokines and enzymatically quiescent heparanase induce the binding of resting CD4+ T cells.

    Science.gov (United States)

    Hershkoviz, R; Marikovsky, M; Gilat, D; Lider, O

    1996-02-01

    Whether the chemokines macrophage inflammatory protein-1 beta (MIP-1 beta) and regulated on activation normal T expressed and secreted (RANTES), which interact specifically with glycosaminoglycans and thus mediate the recruitment, attachment, and migration of leukocytes to vascular endothelia and extracellular matrix, are also involved in interactions between CD4+ murine T lymphocytes and keratinocytes was examined. We have previously observed that depending on the local pH, a mammalian extracellular matrix-degrading enzyme, endo-beta-D glucuronidase (heparanase), which cleaves heparin sulfate proteoglycans, can function wither as an enzyme or as an adhesion molecule for CD4+ T lymphocytes. Herein, the involvement of heparanase in T cell-keratinocyte interactions was also probed. At 37 degree C and pH 7.2, radioactively labeled MIP-1 beta, RANTES, and heparanase bound to confluent layers of resting keratinocytes in a saturable and an heparan sulfate- or heparin-dependent manner, and thereby induced the adhesion of resting CD4+ T cells to keratinocytes. At a relatively acidic pH characteristic of inflammatory milieu, enzymatically active heparanase did not bind to the keratinocytes but, rather, inhibited the binding of MIP-1beta, RANTES, and the enzymatically quiescent heparanase to keratinocytes. These results suggest that certain chemokines and heparanase may function to restrict passing leukocytes, notable T lymphocytes, in the cutaneous micro-environment, a site which is continuously challenged with antigens. These keratinocyte-bound lymphocytes can serve as a reservoir of immediate responders to immunological stimuli. PMID:8601723

  2. Effects of growth factors on the proliferation of human keratinocytes and fibroblasts in vitro.

    Science.gov (United States)

    Kim, D S; Korting, H C; Schäfer-Korting, M

    1998-01-01

    Growth/differentiation factor-5 (GDF-5) is a new member of the transforming growth factor-beta (TGF-beta) superfamily of multifunctional peptide growth factors that appear to mediate many key events in cell growth and development. The effects of GDF-5 and other growth factors (epidermal growth factor, EGF; TGF-beta 1) on the proliferation of human keratinocytes and fibroblasts compared with desoximetasone and calcipotriol have been investigated. The proliferation rate was determined by a hemocytometer, MTT assay and the incorporation of [3H]-thymidine. Moreover, cell cycle analyses were performed and the influence on interleukin-1 alpha (IL-1 alpha) production in keratinocytes was measured by enzyme-linked immunosorbent assay (ELISA) because of its pronounced proinflammatory effect. In keratinocytes, GDF-5 stimulated cell proliferation to a minor extent. The drug already proved to be effective at very low concentrations (0.1 ng/ml). Growth stimulatory effects with EGF have been observed only in keratinocyte basal medium (KBM), but not in keratinocyte growth medium (KGM). TGF-beta 1 markedly inhibited the proliferation of keratinocytes at concentrations > 1 ng/ml. Calcipotriol and desoximetasone also showed a dose-dependent cell growth inhibition in epidermal cell cultures. IL-1 alpha synthesis was greatly suppressed by calcipotriol 10(-8)-10(-6) M. EGF at 10 ng/ml, in contrast, strongly stimulated IL-1 alpha production. Neither GDF-5 nor TGF-beta 1 had a significant effect on IL-1 alpha production in keratinocyte monolayer cultures. In fibroblasts, GDF-5 induced very weak antiproliferative effects. Calcipotriol and desoximetasone also inhibited cell growth in fibroblast cultures whereas proliferation and DNA synthesis were strongly stimulated by 1 ng/ml EGF. There was, however, a contradiction between TGF-beta 1 results on fibroblasts. Whereas TGF-beta 1 increased proliferation in cell number determination and in the thymidine incorporation assay, MTT assays showed

  3. Adipose derived mesenchymal stem cells express keratinocyte lineage markers in a co-culture model.

    Science.gov (United States)

    Irfan-Maqsood, M; Matin, M M; Heirani-Tabasi, A; Bahrami, M; Naderi-Meshkin, H; Mirahmadi, M; Hassanzadeh, H; Sanjar Moussavi, N; Raza-Shah, H; Raeesolmohaddeseen, M; Bidkhori, H; Bahrami, A R

    2016-01-01

    Cutaneous wound healing is a complex type of biological event involving proliferation, differentiation, reprograming, trans/de-differentiation, recruitment, migration, and apoptosis of a number of cells (keratinocytes, fibroblasts, endothelial cells, nerve cells and stem cells) to regenerate a multi-layered tissue that is damaged by either internal or external factors. The exact regeneration mechanism of damaged skin is still unknown but the epithelial and other kinds of stem cells located in skin play crucial roles in the healing process. In this work, a co-culture model composed of adipose derived mesenchymal stem cells and keratinocytes was developed to understand the cellular differentiation behaviour in wound healing. Human mesenchymal stem cells were isolated from waste lipoaspirates. Keratinocytes were isolated from neonatal rats skin as well from human adult skin. Both types of cells were cultured and their culturing behaviour was observed microscopically under regular intervals of time. The identity of both cells was confirmed by flow cytometry and qRT-PCR. Cells were co-cultured under the proposed co-culturing model and the model was observed for 7, 14 and 21 days. The cellular behaviour was studied based on change in morphology, colonization, stratification, migration and expression of molecular markers. Expression of molecular markers was studied at transcriptional level and change in cellular morphology and migration capabilities was observed under the invert microscope regularly. Successfully isolated and characterized mesenchymal stem cells were found to express keratinocyte lineage markers i.e. K5, K10, K14, K18, K19 and Involucrin when co-cultured with keratinocytes after 14 and 21 days. Their expression was found to increase by increasing the time span of cell culturing. The keratinocyte colonies started to disappear after 10 days of culturing which might be due to stratification process initiated by possibly transdifferentiated stem cells. It can

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    Xiuju Dai

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

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

    Science.gov (United States)

    Dai, Xiuju; Okazaki, Hidenori; Hanakawa, Yasushi; Murakami, Masamoto; Tohyama, Mikiko; Shirakata, Yuji; Sayama, Koji

    2013-01-01

    Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were quantified by ELISA. We detected the presence of IL-1α, IL-1β, and high levels of IL-31 in sweat samples. To investigate whether sweat activates keratinocytes, normal human keratinocytes were stimulated with concentrated sweat. Western blot analysis demonstrated the activation of NF-κB, ERK, and JNK signaling in sweat-stimulated keratinocytes. Real-time PCR using total RNA and ELISA analysis of supernatants showed the upregulation of IL-8 and IL-1β by sweat. Furthermore, pretreatment with IL-1R antagonist blocked sweat-stimulated cytokine production and signal activation, indicating that bioactive IL-1 is a major factor in the activation of keratinocytes by sweat. Moreover, IL-31 seems to be another sweat stimulator that activates keratinocytes to produce inflammatory cytokine, CCL2. Sweat is secreted onto the skin's surface and does not come into contact with keratinocytes in normal skin. However, in skin with a defective cutaneous barrier, such as atopic dermatitis-affected skin, sweat cytokines can directly act on epidermal keratinocytes, resulting in their activation. In conclusion, eccrine sweat contains proinflammatory cytokines, IL-1 and IL-31, and activates epidermal keratinocytes as a danger signal. PMID:23874436

  7. 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

  8. Analysis of aquaporin 9 expression in human epidermis and cultured keratinocytes

    Directory of Open Access Journals (Sweden)

    Yoshinori Sugiyama

    2014-01-01

    Full Text Available Aquaporin 9 (AQP9 is a member of the aquaglyceroporin family that transports glycerol, urea and other small solutes as well as water. Compared to the expression and function in epidermal keratinocytes of AQP3, another aquaglyceroporin, our knowledge of epidermal AQP9 remains elusive. In this study, we investigated the expression of AQP9 in the human epidermis and cultured keratinocytes. Immunofluorescence studies revealed that AQP9 expression is highly restricted to the stratum granulosum of the human epidermis, where occludin is also expressed at the tight junctions. Interestingly, the AQP3 staining decreased sharply below the cell layers in which AQP9 is expressed. In cultured normal human epidermal keratinocytes (NHEK, knock-down of AQP9 expression in the differentiated cells induced by RNA interference reduced glycerol uptake, which was not as pronounced as was the case with AQP3 knock-down cells. In contrast, similar reduction of urea uptake was detected in AQP9 and AQP3 knock-down cells. These findings suggested that AQP9 expression in NHEK facilitates at least the transport of glycerol and urea. Finally, we analyzed the effect of retinoic acid (RA, a potent stimulator of keratinocyte proliferation, on AQP3 and AQP9 mRNA expression in differentiated NHEK. Stimulation with RA at 1 μM for 24 h augmented AQP3 expression and down-regulated AQP9 expression. Collectively, these results indicate that AQP9 expression in epidermal keratinocytes is regulated in a different manner from that of AQP3.

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

    Directory of Open Access Journals (Sweden)

    Rezaul Karim

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

  10. 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.

  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. 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.

  14. 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...

  15. SV40 Tag transformation of the normal invasive trophoblast results in a premalignant phenotype. I. Mechanisms responsible for hyperinvasiveness and resistance to anti-invasive action of TGFbeta.

    Science.gov (United States)

    Khoo, N K; Bechberger, J F; Shepherd, T; Bond, S L; McCrae, K R; Hamilton, G S; Lala, P K

    1998-07-29

    Invasion of the uterus by first trimester human placental extravillous trophoblast (EVT) cells depends on mechanisms shared by malignant cells. However, unlike tumor invasion, trophoblast invasion of the uterus is stringently controlled in situ by local molecules such as transforming growth factor (TGF)beta. Since EVT cells possess active invasion-associated genes but are nontumorigenic, our objective was to induce premalignant and then malignant phenotype into a normal EVT cell line in order to identify the molecular basis of tumor progression. Simian virus 40 large T antigen (SV40 Tag) was introduced into a normal human first trimester invasive EVT cell line, HTR8, established in our laboratory. Since the HTR8 line has a limited in vitro lifespan of 12-15 passages, SV40 Tag-transformed cells were selected on the basis of extended lifespan. A long-lived line, RSVT-2, was produced and an immortalized subclone, RSVT2/C, was further derived under a forced crisis regimen. We examined transformation-induced alterations in proliferative and invasive abilities, responses to the invasion and proliferation-regulating growth factor TGFbeta and changes in gene expression for invasion-associated enzymes or enzyme inhibitors. RSVT-2 and RSVT2/C cell lines were hyperproliferative and hyperinvasive when compared with the parental HTR8 cell line. They were also variably resistant to the anti-proliferative and anti-invasive signals from TGFbeta. Since both cell lines remained non-tumorigenic in nude mice, these properties indicate that they attained a premalignant phenotype. Both cell lines showed reduced expression of tissue inhibitor of metalloproteases (TIMP)-1, while TIMP-2 and plasminogen activator inhibitor (PAI)-I expression was was also reduced in RSVT2/C cells, thus contributing to their hyperinvasiveness. Their resistance to the anti-invasive action of TGFbeta was explained by the failure of TGFbeta to upregulate TIMPs and PAI-I, in contrast to the TGFbeta

  16. 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.

  17. Autologous keratinocyte suspension in platelet concentrate accelerates and enhances wound healing – a prospective randomized clinical trial on skin graft donor sites: platelet concentrate and keratinocytes on donor sites

    OpenAIRE

    Guerid S.; Darwiche S.E.; Berger M.M.; Applegate L.A.; Benathan M.; Raffoul W.

    2013-01-01

    BACKGROUND: Wound healing involves complex mechanisms, which, if properly chaperoned, can enhance patient recovery. The abilities of platelets and keratinocytes may be harnessed in order to stimulate wound healing through the formation of platelet clots, the release of several growth factors and cytokines, and cell proliferation. The aim of the study was to test whether autologous keratinocyte suspensions in platelet concentrate would improve wound healing. The study was conducted at the Laus...

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

    Directory of Open Access Journals (Sweden)

    John Wai-man Yuen

    2012-01-01

    Full Text Available Bacillus Calmette-Guérin (BCG is conventionally used as an adjuvant immunotherapy to reduce the recurrence of bladder cancer. To address the issues of efficacy and safety, an ethanol extract of Ganoderma lucidum (GLe was evaluated for its interaction with BCG. In a model of premalignant human uroepithelial cells (HUC-PC, GLe exerted immediate cytotoxic effects while BCG showed a delayed response, given that both were immunological active in inducing the secretion of interleukin (IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1. Synergistic cytotoxic effects were observed when cells were either coincubated with both drugs or firstly preincubated with GLe. Synergism between GLe and BCG was demonstrated to achieve a complete cytostasis in 24 hours, and such effects were progressed in the subsequent 5 days. However, the pretreatment of GLe resulted in suppression of IL-6, IL-8, and MCP-1 secretions without affecting the cytotoxicity. Given that numerous proinflammatory cytokines are associated with the high side effects toll of BCG, results herein suggested the potential implications of GL to supplement the BCG immunotherapy in bladder cancer, for better efficacy and reducing side effects.

  19. 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.

  20. 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

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

    International Nuclear Information System (INIS)

    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

  2. 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.

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

    International Nuclear Information System (INIS)

    Highlights: → Influence of cyclic stretch on melanogenetic paracrine cytokines was investigated. → Keratinocyte-derived endothelin-1 was upregulated with cyclic stretch. → Degree of upregulation increases dose-dependently. → 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-1α, 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.

  4. Sodium Dodecyl Sulfate and C31G as Microbicidal Alternatives to Nonoxynol 9: Comparative Sensitivity of Primary Human Vaginal Keratinocytes

    OpenAIRE

    Krebs, Fred C.; Miller, Shendra R.; Catalone, Bradley J.; Welsh, Patricia A.; Malamud, Daniel; Howett, Mary K; Wigdahl, Brian

    2000-01-01

    A broad-spectrum vaginal microbicide must be effective against a variety of sexually transmitted disease pathogens and be minimally toxic to the cell types found within the vaginal epithelium, including vaginal keratinocytes. We assessed the sensitivity of primary human vaginal keratinocytes to potential topical vaginal microbicides nonoxynol-9 (N-9), C31G, and sodium dodecyl sulfate (SDS). Direct immunofluorescence and fluorescence-activated cell sorting analyses demonstrated that primary va...

  5. X-ray microanalysis of cultured keratinocytes: methodological aspects and effects of the irritant sodium lauryl sulphate on elemental composition.

    Science.gov (United States)

    Grängsjö, A; Pihl-Lundin, I; Lindberg, M; Roomans, G M

    2000-09-01

    Irritant substances have been shown to induce elemental changes in human and animal epidermal cells in situ. However, skin biopsies are a complicated experimental system and artefacts can be introduced by the anaesthesia necessary to take the biopsy. We therefore attempted to set up an experimental system for X-ray microanalysis (XRMA) consisting of cultured human keratinocytes. A number of methodological aspects were studied: different cell types, washing methods and different culture periods for the keratinocytes. It was also investigated whether the keratinocytes responded to exposure to sodium lauryl sulphate (SLS) with changes in their elemental composition. The concentrations of biologically important elements such as Na, Mg, P and K were different in HaCaT cells (a spontaneously immortalized non-tumorigenic cell line derived from adult human keratinocytes) compared to natural human epidermal keratinocytes. The washing procedure and time of culture influenced the intracellular elemental content, and rinsing with distilled water was preferred for further experiments. Changes in the elemental content in the HaCaT cells compatible with a pattern of cell injury followed by repair by cell proliferation were seen after treatment with 3.33 microM and 33 microM SLS. We conclude that XRMA is a useful tool for the study of functional changes in cultured keratinocytes, even though the preparation methods have to be strictly controlled. The method can conceivably be used for predicting effects of different chemicals on human skin. PMID:10971801

  6. 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.

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Yasuaki Kobayashi

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

  9. 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

    Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation...... 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...... in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid...

  10. 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.

  11. Establishment of an Immortalized Skin Keratinocyte Cell Line Derived from the Animal Model Mastomys coucha

    Science.gov (United States)

    Hasche, Daniel; Stephan, Sonja; Savelyeva, Larissa; Westermann, Frank; Rösl, Frank

    2016-01-01

    In the present report we describe the establishment of a spontaneous immortalized skin keratinocyte cell line derived from the skin of the multimammate rodent Mastomys coucha. These animals are used in preclinical studies for a variety of human diseases such as infections with nematodes, bacteria and papillomaviruses, especially regarding cutaneous manifestations such as non-melanoma skin cancer. Here we characterize the cells in terms of their origin and cytogenetic features. Searching for genomic signatures, a spontaneous mutation in the splicing donor sequence of Trp53 (G to A transition at the first position of intron 7) could be detected. This point mutation leads to alternative splicing and to a premature stop codon, resulting in a truncated and, in turn, undetectable form of p53, probably contributing to the process of immortalization. Mastomys coucha-derived skin keratinocytes can be used as an in vitro system to investigate molecular and immunological aspects of infectious agent interactions with their host cells. PMID:27533138

  12. 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

    Lipid rafts are cholesterol-rich plasma membrane domains that regulate signal transduction. Because our earlier work indicated that raft disruption inhibited proliferation and caused cell death, we investigated here the role of membrane cholesterol, the crucial raft constituent, in the regulation...... in deactivation of mammalian target of rapamycin, activation of FoxO3a, and increased sensitivity to apoptosis stimuli. Lipid raft disruption abrogated the binding of Akt and the major Akt kinase, phosphatidylinositol-dependent kinase 1, to the membrane by pleckstrin-homology domains. Thus, the integrity of lipid...... 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...

  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. 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.

  16. 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.

  17. 14-3-3σ regulates keratinocyte proliferation and differentiation by modulating Yap1 cellular localization

    Science.gov (United States)

    Sambandam, Sumitha A.T.; Kasetti, Ramesh Babu; Xue, Lei; Dean, Douglas C.; Lu, Qingxian; Li, Qiutang

    2015-01-01

    The homozygous repeated epilation (Er/Er) mouse mutant of the gene encoding 14-3-3σ displays an epidermal phenotype characterized by hyperproliferative keratinocytes and undifferentiated epidermis. Heterozygous Er/+ mice develop spontaneous skin tumors and are highly sensitive to tumor-promoting DMBA/TPA induction. The molecular mechanisms underlying 14-3-3σ regulation of epidermal proliferation, differentiation, and tumor formation have not been well elucidated. In the present study, we found that Er/Er keratinocytes failed to sequester Yap1 in the cytoplasm, leading to its nuclear localization during epidermal development in vivo and under differentiation-inducing culture conditions in vitro. In addition, enhanced Yap1 nuclear localization was also evident in DMBA/TPA-induced tumors from Er/+ skin. Furthermore, shRNA knockdown of Yap1 expression in Er/Er keratinocytes inhibited their proliferation, suggesting that YAP1 functions as a downstream effector of 14-3-3σ controlling epidermal proliferation. We then demonstrated that keratinocytes express all seven 14-3-3 protein isoforms, some of which form heterodimers with 14-3-3σ, either full-length WT or the mutant form found in Er/Er mice. However Er 14-3-3σ does not interact with Yap1, as demonstrated by co-immunoprecipitation. We conclude that Er 14-3-3σ disrupts the interaction between 14-3-3 and Yap1, thus fails to block Yap1 nuclear transcriptional function, causing continued progenitor expansion and inhibition of differentiation in Er/Er epidermis. PMID:25668240

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

    OpenAIRE

    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-solan...

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

    OpenAIRE

    Prince, Tessa; McBain, Andrew J.; O'Neill, Catherine A.

    2012-01-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. ...

  20. Ceramide Stimulates ABCA12 Expression via Peroxisome Proliferator-activated Receptor δ in Human Keratinocytes*

    OpenAIRE

    Jiang, Yan J.; Uchida, Yoshikazu; Lu, Biao; Kim, Peggy; Mao, Cungui; Akiyama, Masashi; Elias, Peter M.; Holleran, Walter M.; Grunfeld, Carl; Feingold, Kenneth R.

    2009-01-01

    ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise ∼50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorde...

  1. 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

    2016-07-01

    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. PMID:27105673

  2. 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.

  3. Keratinocyte detachment-differentiation connection revisited, or anoikis-pityriasi nexus redux.

    Directory of Open Access Journals (Sweden)

    Tomohiro Banno

    Full Text Available Epidermis, a continuously self-renewing and differentiating organ, produces a protective stratum corneum that shields us from external chemical, physical and microbial threats. Epidermal differentiation is a multi-step process regulated by influences, some unknown, others insufficiently explored. Detachment of keratinocytes from the basement membrane is one such pro-differentiation stimulus. Here, we define the transcriptional changes during differentiation, especially those caused by detachment from the substratum. Using comprehensive transcriptional profiling, we revisited the effects of detachment as a differentiation signal to keratinocytes. We identified the genes regulated by detachment, the corresponding ontological categories and, using metaanalysis, compared the genes and categories to those regulated by other pro-differentiating stimuli. We identified 762 genes overexpressed in suspended keratinocyte, including known and novel differentiation markers, and 1427 in attached cells, including basal layer markers. Detachment induced epidermis development, cornification and desmosomal genes, but also innate immunity, proliferation inhibitors, transcription regulators and MAPKs; conversely the attached cells overexpressed cell cycle, anchoring, motility, splicing and mitochondrial genes, and both positive and negative regulators of apoptosis. Metaanalysis identified which detachment-regulated categories overlap with those induced by suprabasal location in vivo, by reaching confluency in vitro, and by inhibition of JUN kinases. Attached and in vivo basal cells shared overexpression of mitochondrial components. Interestingly, melanosome trafficking components were also overexpressed in the attached and in vivo basal keratinocytes. These results suggest that specific pro-differentiation signals induce specific features of the keratinization process, which are in vivo orchestrated into harmonious epidermal homeostasis.

  4. Primary cell culture from human oral tissue: gingival keratinocytes,gingival fibroblasts and periodontal ligament fibroblasts

    Directory of Open Access Journals (Sweden)

    Supreya Wanichpakorn

    2010-08-01

    Full Text Available Primary cell culture of human oral tissue has many applications for oral biology research. There are two techniques in primary culture, which includes the enzymatic and direct explant technique. The objectives of this study were (1 to isolate and investigate the difference in percentage the success in culturing three cell types from human oral tissue: gingival keratinocytes, gingival fibroblasts and periodontal ligament fibroblasts by using the direct explant technique; (2 to compare the effect of sex and age on the success of tissue culturing. Twenty seven tissue samples were obtained from healthy human gingival tissue, 19 female and 8 male patients aged 14-67 years (37.7±17.5. The tissue was cut into 1x1 mm pieces and placed on plastic culture plates containing Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 1% amphotericin B. For the keratinocytes culture, after the epithelial cells started to multiply around the gingival origin and the diameter was 2-5 mm., the fibroblasts were liminated by mechanical removal under inverted microscope to prevent fibroblast overgrowth and the medium was changed to keratinocyte-SFM (Gibco, BRL supplemented with 5 µg/ml gentamycin. The results revealed that gingival fibroblast gave the highest success rate in culture (96.3%, followed by gingival keratinocytes (88.9% and periodontal ligament fibroblasts (81.5%. There was no significant difference in the success rate of cultivation between younger and older individuals, as between sex of the subjects (p>0.05. The risk of failure in culture techniques is mainly caused by microbiological contamination from the tissue samples.

  5. The epithelial sodium channel mediates the directionality of galvanotaxis in human keratinocytes

    OpenAIRE

    Yang, Hsin-ya; Charles, Roch-Philippe; Hummler, Edith; Baines, Deborah L.; Isseroff, R. Rivkah

    2013-01-01

    Cellular directional migration in an electric field (galvanotaxis) is one of the mechanisms guiding cell movement in embryogenesis and in skin epidermal repair. The epithelial sodium channel (ENaC), in addition to its function of regulating sodium transport in kidney, has recently been found to modulate cell locomotory speed. Here we tested whether ENaC has an additional function of mediating the directional migration of galvanotaxis in keratinocytes. Genetic depletion of ENaC completely bloc...

  6. Transcriptional Profiling of Ectoderm Specification to Keratinocyte Fate in Human Embryonic Stem Cells

    OpenAIRE

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

    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 severa...

  7. 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

    2016-07-01

    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.

  8. p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish.

    OpenAIRE

    Boris Fischer; Manuel Metzger; Rebecca Richardson; Philipp Knyphausen; Thomas Ramezani; Rainer Franzen; Elmon Schmelzer; Wilhelm Bloch; Carney, Thomas J.; Matthias Hammerschmidt

    2014-01-01

    p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal a...

  9. p53 and TAp63 promote keratinocyte proliferation and differentiation in breeding tubercles of the zebrafish

    OpenAIRE

    Fischer, Boris; Metzger, Manuel; Richardson, Rebecca; Knyphausen, Philipp; Ramezani, Thomas; Franzen, Rainer; Schmelzer, Elmon; Bloch, Wilhelm; Carney, Thomas J.; Hammerschmidt, Matthias

    2014-01-01

    p63 is a multi-isoform member of the p53 family of transcription factors. There is compelling genetic evidence that ΔNp63 isoforms are needed for keratinocyte proliferation and stemness in the developing vertebrate epidermis. However, the role of TAp63 isoforms is not fully understood, and TAp63 knockout mice display normal epidermal development. Here, we show that zebrafish mutants specifically lacking TAp63 isoforms, or p53, display compromised development of breeding tubercles, epidermal a...

  10. 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; 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 repr...

  11. Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes

    OpenAIRE

    Sunthamala, Nuchsupha; Pang, Chai Ling; Thierry, Francoise; Teissier, Sebastien; Pientong, Chamsai; Ekalaksananan, Tipaya

    2014-01-01

    The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA...

  12. Keratinocyte galvanotaxis in combined DC and AC electric fields supports an electromechanical transduction sensing mechanism.

    Science.gov (United States)

    Hart, Francis X; Laird, Mhairi; Riding, Aimie; Pullar, Christine E

    2013-02-01

    Sedentary keratinocytes at the edge of a skin wound migrate into the wound, guided by the generation of an endogenous electric field (EF) generated by the collapse of the transepithelial potential. The center of the wound quickly becomes more negative than the surrounding tissue and remains the cathode of the endogenous EF until the wound is completely re-epithelialized. This endogenous guidance cue can be studied in vitro. When placed in a direct current (DC) EF of physiological strength, 100 V/m, keratinocytes migrate directionally toward the cathode in a process known as galvanotaxis. Although a number of membrane-bound (e.g., epidermal growth factor receptor (EGFR), integrins) and cytosolic proteins (cAMP, ERK, PI3K) are known to play a role in the downstream signaling mechanisms underpinning galvanotaxis, the initial sensing mechanism for this response is not understood. To investigate the EF sensor, we studied the migration of keratinocytes in a DC EF of 100 V/m, alternating current (AC) EFs of 40 V/m at either 1.6 or 160 Hz, and combinations of DC and AC EFs. In the AC EFs alone, keratinocytes migrated randomly. The 1.6 Hz AC EF combined with the DC EF suppressed the direction of migration but had no effect on speed. In contrast, the 160 Hz AC EF combined with the DC EF did not affect the direction of migration but increased the migration speed compared to the DC EF alone. These results can be understood in terms of an electromechanical transduction model, but not an electrodiffusion/osmosis or a voltage-gated channel model. PMID:22907479

  13. Repair of ultraviolet light damage to the DNA of cultured human epidermal keratinocytes and fibroblasts

    International Nuclear Information System (INIS)

    Pure cultures of dermal fibroblasts and epidermal keroatinocytes have been obtained from a single biopsy of newborn foreskin. The cells were labeled, exposed to several doses of uv light, and allowed to repair in the dark for 16 h. The number of pyrimidine dimers before and after repair was assessed by measuring the numbers of sites in the DNA sensitive to a specific uv endonuclease. At all doses used, the extent of repair was similar in the cultured keratinocytes and cultured fibroblasts

  14. Effect of Immunosuppressants Tacrolimus and Mycophenolate Mofetil on the Keratinocyte UVB Response†

    OpenAIRE

    Ming, Mei; Zhao, Baozhong; Qiang, Lei; He, Yu-Ying

    2014-01-01

    Non-melanoma skin cancer, derived from epidermal keratinocytes, is the most common malignancy in organ transplant recipients, causes serious morbidity and mortality, and is strongly associated with solar ultraviolet (UV) exposure. Preventing and treating skin cancer in these individuals has been extraordinarily challenging. Following organ transplantation, the immunosuppressants are used to prevent graft rejection. Until now, immunosuppression has been assumed to be the major factor leading t...

  15. Metabolism of the Antibacterial Triclocarban by Human Epidermal Keratinocytes to Yield Protein Adducts

    OpenAIRE

    Schebb, Nils Helge; Buchholz, Bruce A.; Hammock, Bruce D.; Rice, Robert H.

    2012-01-01

    Previous studies of triclocarban suggest that its biotransformation could yield reactive metabolites that form protein adducts. Since the skin is the major route of triclocarban exposure, present work examined this possibility in cultured human keratinocytes. The results provide evidence for considerable biotransformation and protein adduct formation when cytochrome P450 activity is induced in the cells by TCDD, a model Ah receptor ligand. Since detecting low adduct levels in cells and tissue...

  16. Effects of artemether on the proliferation, apoptosis, and differentiation of keratinocytes: potential application for psoriasis treatment

    OpenAIRE

    Wu, Jie; LI Hong; Li, Ming

    2015-01-01

    Artemether exhibits diverse pharmacological effects and has multiple applications. This study aimed to investigate its antiproliferative and apoptogenic effects on HaCaT cells and keratinocyte differentiation-inducing activity in vivo. WST-8 analysis demonstrated that Artemether can inhibit the proliferation of cultured HaCaT cells in a time- and dose-dependent manner. Annexin V/PI dual staining and JC-1 staining further revealed that Artemether can dose-dependently augment HaCaT apoptosis. T...

  17. 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.

  18. 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.

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    Rodrigo A Silva

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

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

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    Emeric Deruy

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-15

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Directory of Open Access Journals (Sweden)

    Soung-Hoon Lee

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

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Alfredo Gragnani

    2003-01-01

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

  7. 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

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

    International Nuclear Information System (INIS)

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

  9. Adiponectin corrects premature cellular senescence and normalizes antimicrobial peptide levels in senescent keratinocytes.

    Science.gov (United States)

    Jin, Taewon; Kim, Min Jeong; Heo, Won Il; Park, Kui Young; Choi, Sun Young; Lee, Mi-Kyung; Hong, Seung-Phil; Kim, Seong-Jin; Im, Myung; Moon, Nam Ju; Seo, Seong Jun

    2016-09-01

    Stress-induced premature senescence or aging causes dysfunction in the human somatic system. Adiponectin (Acrp30) plays a role in functional recovery, especially with adenosine 3',5'-monophosphate (AMP)-activated protein kinase (AMPK) and silent mating type information regulation 2 homolog 1 (SIRT1). Acrp30 stimulation reduced the premature senescence positive ratio induced by hydrogen peroxide (H2O2) and restituted human β-defensin 2 (hBD-2) levels in senescent keratinocytes. Acrp30 recovered AMPK activity in senescent keratinocytes and increased SIRT1 deacetylation activity. As a result, FoxO1 and FoxO3 transcription activity was recovered. Additionally, Acrp30 stimulation suppresses NFκB p65, which induces abnormal expression of hBD-2 induced by H2O2. In the present study, we have shown that Acrp30 reduces premature senescence and recovers cellular function in keratinocytes. These results suggest a role for Acrp30 as an anti-aging agent to improve impaired skin immune barriers. PMID:27349869

  10. Biphasic effects of minoxidil on the proliferation and differentiation of normal human keratinocytes.

    Science.gov (United States)

    Boyera, N; Galey, I; Bernard, B A

    1997-01-01

    Minoxidil is the most used drug with proved effects in the treatment of androgenetic alopecia (AGA), but little is known about its pharmacological activity and target cells in hair follicles. As AGA is characterized by follicle atrophy, accelerated hair cycles and hair fiber thinning, we postulated that keratinocyte proliferation/differentiation is affected and we tested Minoxidil's effects on those parameters. Normal human keratinocytes (NHK) of follicular or epidermal origin were cultured in the presence of Minoxidil (0, 0.1, 1, 10, 100, 1,000 microM) during 5-8 days in various media (high-/low-calcium content, with or without serum). Proliferation was assessed by mitochondrial dehydrogenase activity (XTT), BrdU incorporation, lysosome numeration (neutral red incorporation) and total protein dosage. Drug-induced cytotoxicity was measured by lactate dehydrogenase release in culture supernatant, and pro-differentiating effects were evaluated by relative involucrin expression (ELISA dosage). On this basis, we showed that Minoxidil had biphasic effects on the proliferation and differentiation of NHK: Minoxidil stimulated NHK proliferation at micromolar doses, while antiproliferative, pro-differentiative and partially cytotoxic effects were observed with millimolar concentrations. We can hypothesize that Minoxidil hypertrichotic activity in vivo is possibly mediated by the maintenance of proliferative potential in follicular keratinocytes precociously committed to differentiation. PMID:9413895

  11. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication.

    Science.gov (United States)

    Lee, Wonhye; Debasitis, Jason Cushing; Lee, Vivian Kim; Lee, Jong-Hwan; Fischer, Krisztina; Edminster, Karl; Park, Je-Kyun; Yoo, Seung-Schik

    2009-03-01

    We present a method to create multi-layered engineered tissue composites consisting of human skin fibroblasts and keratinocytes which mimic skin layers. Three-dimensional (3D) freeform fabrication (FF) technique, based on direct cell dispensing, was implemented using a robotic platform that prints collagen hydrogel precursor, fibroblasts and keratinocytes. A printed layer of cell-containing collagen was crosslinked by coating the layer with nebulized aqueous sodium bicarbonate. The process was repeated in layer-by-layer fashion on a planar tissue culture dish, resulting in two distinct cell layers of inner fibroblasts and outer keratinocytes. In order to demonstrate the ability to print and culture multi-layered cell-hydrogel composites on a non-planar surface for potential applications including skin wound repair, the technique was tested on a poly(dimethylsiloxane) (PDMS) mold with 3D surface contours as a target substrate. Highly viable proliferation of each cell layer was observed on both planar and non-planar surfaces. Our results suggest that organotypic skin tissue culture is feasible using on-demand cell printing technique with future potential application in creating skin grafts tailored for wound shape or artificial tissue assay for disease modeling and drug testing. PMID:19108884

  12. A synthetic isoflavone, DCMF, promotes human keratinocyte migration by activating Src/FAK signaling pathway.

    Science.gov (United States)

    Sophors, Phorl; Kim, Young Mee; Seo, Ga Young; Huh, Jung-Sik; Lim, Yoongho; Koh, Dong Soo; Cho, Moonjae

    2016-04-01

    Flavonoids are plant secondary compounds with various pharmacological properties. We previously showed that one flavonoid, trimethoxyisoflavone (TMF), could promote wound healing by inducing keratinocyte migration. Here, we screened TMF derivatives for enhanced activity and identified one compound, 2',6 Dichloro-7-methoxyisoflavone (DCMF), as most effective at promoting migration in a scratch wound assay. Using the HaCaT keratinocyte cell line, we found DCMF treatment induced phosphorylation of both FAK and Src, and increased keratinocyte migration. DCMF-induced Src kinase could promote activation of ERK, AKT, and p38 signaling pathways, and DCMF-induced secretion of matrix metalloproteinase (MMP)-2 and MMP-9 and partial epithelial-mesenchymal transition (EMT), whereas Src inhibition abolished DCMF-induced EMT. Using an in vivo excisional wound model, we observed improved wound closure and re-epithelialization in DCMF-treated mice, as compared to controls. Collectively, our data demonstrate that DCMF induces cell migration and promotes wound healing through activation of Src/FAK, ERK, AKT, and p38 MAPK signaling. PMID:26923073

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

    International Nuclear Information System (INIS)

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

  14. Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

    International Nuclear Information System (INIS)

    Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21WAF1/Cip1 resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3σ, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3σ (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds

  15. 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.

  16. Candidate EDA targets revealed by expression profiling of primary keratinocytes from Tabby mutant mice

    Science.gov (United States)

    Esibizione, Diana; Cui, Chang-Yi; Schlessinger, David

    2009-01-01

    EDA, the gene mutated in anhidrotic ectodermal dysplasia, encodes ectodysplasin, a TNF superfamily member that activates NF-kB mediated transcription. To identify EDA target genes, we have earlier used expression profiling to infer genes differentially expressed at various developmental time points in Tabby (Eda-deficient) compared to wild-type mouse skin. To increase the resolution to find genes whose expression may be restricted to epidermal cells, we have now extended studies to primary keratinocyte cultures established from E19 wild-type and Tabby skin. Using microarrays bearing 44,000 gene probes, we found 385 preliminary candidate genes whose expression was significantly affected by Eda loss. By comparing expression profiles to those from Eda-A1 transgenic skin, we restricted the list to 38 “candidate EDA targets”, 14 of which were already known to be expressed in hair follicles or epidermis. We confirmed expression changes for 3 selected genes, Tbx1, Bmp7, and Jag1, both in keratinocytes and in whole skin, by Q-PCR and Western blotting analyses. Thus, by the analysis of keratinocytes, novel candidate pathways downstream of EDA were detected. PMID:18848976

  17. 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

  18. 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.

  19. Nuclear TK1 expression is an independent prognostic factor for survival in pre-malignant and malignant lesions of the cervix

    International Nuclear Information System (INIS)

    Thymidine kinase 1 (TK1) is a proliferation biomarker that has been found useful for prognostication in cancer patients. Here we investigate for the first time the use of TK1 expression as a prognostic factor for patients with premalignant and malignant lesions of the uterine cervix. TK1 expression was determined by immunohistochemistry in cervical lesions (cervical intraepithelial neoplasia (CIN), n = 216; invasive cervical carcinoma, n = 84). TK1 and Ki-67 expressions and pathological/FIGO stages and age were correlated with 5-year survival by Kaplan-Meier, log rank and COX hazard uni- and multivariate analyses. TK1 labeling index (LI) was significantly correlated with CIN grades and invasive cervical carcinoma stages, while TK1 labeling intensity was only correlated to CIN grades. TK1 LI was significantly higher compared with Ki-67 LI. TK1 LI correlated significantly to 5-year survival in patients with invasive cervical carcinoma, particularly nuclear TK1 LI. In a multivariate analysis, nuclear TK1 expression was independent prognostic factor in patients with in situ/invasive cervical carcinoma or in invasive cervical carcinoma alone. Interestingly, in invasive cervical carcinoma patients with advanced tumors, nuclear TK1 expression could identify patients with significantly better survival rates (80%), while Ki-67 could not. Nuclear TK1 expression in early grade CIN predicts risk for progression to malignancy. Nuclear TK1 expression is also a prognostic factor for treatment outcome, particularly in patients with advanced cervical carcinomas. Nuclear TK1 expression is more useful than Ki-67 and pathological/FIGO stages

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

    Directory of Open Access Journals (Sweden)

    Ami Oizumi

    Full Text Available Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD. A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes", which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii S1P induces the production

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

    Directory of Open Access Journals (Sweden)

    Olerud John E

    2011-06-01

    Full Text Available Abstract Background Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. Results The impact of S. aureus soluble products in biofilm-conditioned medium (BCM or in planktonic-conditioned medium (PCM on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. Conclusions Collectively the results indicate that S

  2. 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

  3. Xenobiotics in vitro: the influence of L-cystine, pantothenat, and miliacin on metabolic and proliferative capacity of keratinocytes.

    Science.gov (United States)

    Obrigkeit, D Hoeller; Oepen, T; Jugert, F K; Merk, H F; Kubicki, J

    2006-01-01

    To investigate the effect of cell growth-stimulating agents on human epidermal keratinocytes, we exposed monolayers of normal human keratinocytes derived from foreskin to different concentrations of the amino acid L-cystine, the member of the vitamin B family D-pantothenat, the phytosterol miliacin, and a combination thereof in keratinocyte growth medium. As a test system for the metabolic capacity, we used the activity of mitochondrial deyhdrogenases as measured by XTT, and for the cell proliferation, we determined the BrdU-uptake. The additives, active ingredients of the hair growth drug PRIORIN, were added in the presence of fully supplemented keratinocyte growth medium or a deficient medium without L-cystine, L-methionine, L-histidin, D-pantothenat, epidermal growth factor, and bovine pituary gland extract. Deficient medium itself reduced the metabolic capacity of keratinocytes to 35% compared with keratinocytes in fully supplemented growth medium. In deficient medium cell, proliferation was not measurable. Increasing doses of L-cystine restored the reduced metabolic capacity from 46% (0.009 mg/L) to 54% (0.09 mg/L) and 92% (0.45 mg/L) in deficient medium. Addition of D-pantothenat (0.43 mg/L) enhanced the metabolic capacity to 150% only in fully supplemented growth medium, compared with untreated controls with growth medium. Miliacin (6 mg/mL) increased not only the metabolic capacity (162%) but also stimulated cell proliferation (215%) as measured by BrdU-uptake in growth medium. The combination of all three additives increased the metabolic capacity (245%) synergistically in growth medium. We were able to show effects of D-panthenol, L-lysine, and miliacin on proliferation and metabolic capacity of keratinocyte monocell culture, which was further increased by combination of the three substances. These basic results suggest a beneficial effect on keratinocyte growth and stimulation by products combining these substances (e.g., Priorin). Furthermore, this work

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

    Directory of Open Access Journals (Sweden)

    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.

  5. HPV prevalence and type-distribution in cervical cancer and premalignant lesions of the cervix: A population-based study from Northern Ireland.

    Science.gov (United States)

    Anderson, Lesley A; O'Rorke, Michael A; Wilson, Robbie; Jamison, Jackie; Gavin, Anna T

    2016-07-01

    Assessment of Human papillomavirus (HPV) prevalence and genotype distribution is important for monitoring the impact of prophylactic HPV vaccination. This study aimed to demonstrate the HPV genotypes predominating in pre-malignant and cervical cancers in Northern Ireland (NI) before the vaccination campaign has effect. Formalin fixed paraffin embedded tissue blocks from 2,303 women aged 16-93 years throughout NI were collated between April 2011 and February 2013. HPV DNA was amplified by PCR and HPV genotyping undertaken using the Roche(®) linear array detection kit. In total, 1,241 out of 1,830 eligible samples (68.0%) tested positive for HPV, with the majority of these [1,181/1,830 (64.5%)] having high-risk (HR) HPV infection; 37.4% were positive for HPV-16 (n = 684) and 5.1% for HPV-18 (n = 93). HPV type-specific prevalence was 48.1%, 65.9%, 81.3%, 92.2%, and 64.3% among cervical intraepithelial neoplasias (CIN) Grades I-III, squamous cell carcinomas (SCC) and adenocarcinoma (AC) cases, respectively. Most SCC cases (81.3%) had only one HPV genotype detected and almost a third (32.0%) of all cervical pathologies were HPV negative including 51.9% of CIN I (n = 283), 34.1% CIN II (n = 145), 18.7% of CIN III (n = 146), 7.8% of SCC (n = 5), and 35.7% of AC (n = 5) cases. This study provides important baseline data for monitoring the effect of HPV vaccination in NI and for comparison with other UK regions. The coverage of other HR-HPV genotypes apart from 16 and 18, including HPV-45, 31, 39, and 52, and the potential for cross protection, should be considered when considering future polyvalent vaccines. J. Med. Virol. 88:1262-1270, 2016. © 2015 Wiley Periodicals, Inc. PMID:26680281

  6. 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.

  7. Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

    Science.gov (United States)

    Sun, Baihui; Ding, Ruoting; Yu, Wenlin; Wu, Yanhong; Wang, Bulin; Li, Qin

    2016-07-01

    Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1. PMID:27155970

  8. 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

  9. 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.

  10. 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. PMID:25639585

  11. Increased oxidative stress and antioxidant expression in mouse keratinocytes following exposure to paraquat

    International Nuclear Information System (INIS)

    Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is a widely used herbicide known to induce skin toxicity. This is thought to be due to oxidative stress resulting from the generation of cytotoxic reactive oxygen intermediates (ROI) during paraquat redox cycling. The skin contains a diverse array of antioxidant enzymes which protect against oxidative stress including superoxide dismutase (SOD), catalase, glutathione peroxidase-1 (GPx-1), heme oxygenase-1 (HO-1), metallothionein-2 (MT-2), and glutathione-S-transferases (GST). In the present studies we compared paraquat redox cycling in primary cultures of undifferentiated and differentiated mouse keratinocytes and determined if this was associated with oxidative stress and altered expression of antioxidant enzymes. We found that paraquat readily undergoes redox cycling in both undifferentiated and differentiated keratinocytes, generating superoxide anion and hydrogen peroxide as well as increased protein oxidation which was greater in differentiated cells. Paraquat treatment also resulted in increased expression of HO-1, Cu,Zn-SOD, catalase, GSTP1, GSTA3 and GSTA4. However, no major differences in expression of these enzymes were evident between undifferentiated and differentiated cells. In contrast, expression of GSTA1-2 was significantly greater in differentiated relative to undifferentiated cells after paraquat treatment. No changes in expression of MT-2, Mn-SOD, GPx-1, GSTM1 or the microsomal GST's mGST1, mGST2 and mGST3, were observed in response to paraquat. These data demonstrate that paraquat induces oxidative stress in keratinocytes leading to increased expression of antioxidant genes. These intracellular proteins may be important in protecting the skin from paraquat-mediated cytotoxicity

  12. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1.

    Science.gov (United States)

    Kim, Chloe S; Mitchell, Isaiah P; Desotell, Anthony W; Kreeger, Pamela K; Masters, Kristyn S

    2016-07-01

    Epidermal growth factor (EGF) is a critical element in dermal repair, but EGF-containing wound dressings have not been successful clinically. However, these dressings have delivered only soluble EGF, and the native environment provides both soluble and matrix-bound EGF. To address our hypothesis that tethered EGF can stimulate cell behaviors not achievable with soluble EGF, we examined single-cell movement and signaling in human immortalized HaCaT keratinocytes treated with soluble or immobilized EGF. Although both EGF treatments increased collective sheet displacement and individual cell speed, only cells treated with immobilized EGF exhibited directed migration, as well as 2-fold greater persistence compared with soluble EGF. Immunofluorescence showed altered EGF receptor (EGFR) trafficking, where EGFR remained membrane-localized in the immobilized EGF condition. Cells treated with soluble EGF demonstrated higher phosphorylated ERK1/2, and cells on immobilized EGF exhibited higher pPLCγ1, which was localized at the leading edge. Treatment with U0126 inhibited migration in both conditions, demonstrating that ERK1/2 activity was necessary but not responsible for the observed differences. In contrast, PLCγ1 inhibition with U73122 significantly decreased persistence on immobilized EGF. Combined, these results suggest that immobilized EGF increases collective keratinocyte displacement via an increase in single-cell migration persistence resulting from altered EGFR trafficking and PLCγ1 activation.-Kim, C. S., Mitchell, I. P., Desotell, A. W., Kreeger, P. K., Masters, K. S. Immobilized epidermal growth factor stimulates persistent, directed keratinocyte migration via activation of PLCγ1. PMID:27025961

  13. UVA and UVB irradiation differentially regulate microRNA expression in human primary keratinocytes.

    Directory of Open Access Journals (Sweden)

    Anne Kraemer

    Full Text Available MicroRNA (miRNA-mediated regulation of the cellular transcriptome is an important epigenetic mechanism for fine-tuning regulatory pathways. These include processes related to skin cancer development, progression and metastasis. However, little is known about the role of microRNA as an intermediary in the carcinogenic processes following exposure to UV-radiation. We now show that UV irradiation of human primary keratinocytes modulates the expression of several cellular miRNAs. A common set of miRNAs was influenced by exposure to both UVA and UVB. However, each wavelength band also activated a distinct subset of miRNAs. Common sets of UVA- and UVB-regulated miRNAs harbor the regulatory elements GLYCA-nTRE, GATA-1-undefined-site-13 or Hox-2.3-undefined-site-2 in their promoters. In silico analysis indicates that the differentially expressed miRNAs responding to UV have potential functions in the cellular pathways of cell growth and proliferation. Interestingly, the expression of miR-23b, which is a differentiation marker of human keratinocytes, is remarkably up-regulated after UVA irradiation. Studying the interaction between miR-23b and its putative skin-relevant targets using a Luciferase reporter assay revealed that RRAS2 (related RAS viral oncogene homolog 2, which is strongly expressed in highly aggressive malignant skin cancer, to be a direct target of miR-23b. This study demonstrates for the first time a differential miRNA response to UVA and UVB in human primary keratinocytes. This suggests that selective regulation of signaling pathways occurs in response to different UV energies. This may shed new light on miRNA-regulated carcinogenic processes involved in UV-induced skin carcinogenesis.

  14. 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; Habauzit, Denis

    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

  15. 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

    Phenylketonuria (PKU) is caused by deficiency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. PAH requires the cofactor BH(4) to function and the rate-limiting step in the synthesis of BH(4) is GTP cyclohydrolase I (GTP-CH). The skin is a potential target tissue for PKU...... 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...

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

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders;

    2002-01-01

    cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether......The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5...

  17. 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.

  18. Lichen metabolites. 2. Antiproliferative and cytotoxic activity of gyrophoric, usnic, and diffractaic acid on human keratinocyte growth.

    Science.gov (United States)

    Kumar, K C; Müller, K

    1999-06-01

    The sensitivity of the human keratinocyte cell line HaCaT to several lichen metabolites isolated from Parmelia nepalensis and Parmelia tinctorum was evaluated. The tridepside gyrophoric acid (6), the dibenzofuran derivative (+)-usnic acid (1), and the didepside diffractaic acid (5) were potent antiproliferative agents and inhibited cell growth, with IC50 values of 1.7, 2.1, and 2.6 microM, respectively. Methyl beta-orcinolcarboxylate (2), ethyl hematommate (3), the didepside atranorin (4), and (+)-protolichesterinic acid (7) did not influence keratinocyte growth at concentrations of 5 microM. Keratinocytes were further tested for their susceptibility to the action of the potent antiproliferative agents on plasma membrane integrity. The release of lactate dehydrogenase activity into the culture medium was unchanged as compared to controls, documenting that the activity of gyrophoric acid (6), (+)-usnic acid (1), and diffractaic acid (5) was due to cytostatic rather than cytotoxic effects. PMID:10395495

  19. Phototoxicity of Phenylenediamine Hair Dye Chemicals in Salmonella typhimurium TA102 and Human Skin Keratinocytes

    OpenAIRE

    Yu, Hongtao; Mosley-Foreman; Choi, Jaehwa; Wang, Shuguang

    2008-01-01

    Phenylenediamines (PD) are dye precursors used to manufacture hair dyes. The three PDs, 1,2-, 1,3-, and 1,4-PD and three chlorinated PDs, 4-chloro-1,2-PD, 4-chloro-1,3- PD, and 4,5-dichloro-1,2-PD were studied for their mutagenic effect in Salmonella typhimurium TA 102, cytotoxicity in human skin keratinocyte cells, and for DNA cleavage. The results show that all six compounds are not toxic/mutagenic in TA 102 bacteria or skin cells, and do not cause DNA cleavage in ΦX 174 phage DNA. If the s...

  20. Psoriasiform dermatitis is driven by IL-36–mediated DC-keratinocyte crosstalk

    OpenAIRE

    Tortola, Luigi; Rosenwald, Esther; Abel, Brian; Blumberg, Hal; Schäfer, Matthias; Coyle, Anthony J.; Renauld, Jean-Christoph; Werner, Sabine; Kisielow, Jan; Kopf, Manfred

    2012-01-01

    Psoriasis is a chronic inflammatory disorder of the skin affecting approximately 2% of the world’s population. Accumulating evidence has revealed that the IL-23/IL-17/IL-22 pathway is key for development of skin immunopathology. However, the role of keratinocytes and their crosstalk with immune cells at the onset of disease remains poorly understood. Here, we show that IL-36R–deficient (Il36r–/–) mice were protected from imiquimod-induced expansion of dermal IL-17–producing γδ T cells and pso...

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

    DEFF Research Database (Denmark)

    Brakebusch, C; Grose, R; Quondamatteo, F;

    2000-01-01

    developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression......, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance...

  2. Infection of human keratinocytes by Streptococcus dysgalactiae subspecies dysgalactiae isolated from milk of the bovine udder.

    Science.gov (United States)

    Roma-Rodrigues, Catarina; Alves-Barroco, Cynthia; Raposo, Luís R; Costa, Mafalda N; Fortunato, Elvira; Baptista, Pedro Viana; Fernandes, Alexandra R; Santos-Sanches, Ilda

    2016-04-01

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) are considered exclusive animal pathogens; however, a putative zoonotic upper limb cellulitis, a prosthetic joint infection and an infective endocarditis were described in humans. To unravel if bovine SDSD isolates are able to infect human cells, the adherence and internalization to human primary keratinocytes of two bovine SDSD strains isolated from milk collected from udder were analyzed. Bacterial adhesion assays and confocal microscopy indicate a high adherence and internalization of SDSD isolates to human cells, suggesting for the first time the ability of bovine isolates to infect human cells. PMID:26655883

  3. 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

  4. Mammalian target of rapamycin (mTOR) regulates TLR3 induced cytokines in human oral keratinocytes

    OpenAIRE

    Zhao, Jiawei; Manjunatha R Benakanakere; Hosur, Kavita B.; Galicia, Johnah C.; Martin, Michael; Kinane, Denis F.

    2010-01-01

    Recent studies implicate the mammalian target of rapamycin (mTOR) pathway in the control of inflammatory responses following Toll-like receptor (TLR) stimulation in myeloid cells but its role in non-myeloid cells such as human keratinocytes is unknown. Here we show that TLR3 signaling can induce robust cytokine secretion including interleukin 1 beta (IL-1β), tumor necrosis factor alpha (TNFα), IL-12p70 and interferon beta (IFN-β), and our data reveal for the first time that inhibiting mTOR wi...

  5. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations that leads to stable epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells

    Energy Technology Data Exchange (ETDEWEB)

    Lochter, A.; Galosy, S.; Muschler, J.; Freedman, N.; Werb, Z.; Bissell, M.J.

    1997-08-11

    Matrix metalloproteinases (MMPs) regulate ductal morphogenesis, apoptosis, and neoplastic progression in mammary epithelial cells. To elucidate the direct effects of MMPs on mammary epithelium, we generated functionally normal cells expressing an inducible autoactivating stromelysin-1 (SL-1) transgene. Induction of SL-1 expression resulted in cleavage of E-cadherin, and triggered progressive phenotypic conversion characterized by disappearance of E-cadherin and catenins from cell-cell contacts, downregulation of cytokeratins, upregulation of vimentin, induction of keratinocyte growth factor expression and activation, and upregulation of endogenous MMPs. Cells expressing SL-1 were unable to undergo lactogenic differentiation and became invasive. Once initiated, this phenotypic conversion was essentially stable, and progressed even in the absence of continued SL-1 expression. These observations demonstrate that inappropriate expression of SL-1 initiates a cascade of events that may represent a coordinated program leading to loss of the differentiated epithelial phenotype and gain of some characteristics of tumor cells. Our data provide novel insights into how MMPs function in development and neoplastic conversion.

  6. Promoter Region Hypermethylation and mRNA Expression of MGMT and p16 Genes in Tissue and Blood Samples of Human Premalignant Oral Lesions and Oral Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Vikram Bhatia

    2014-01-01

    Full Text Available Promoter methylation and relative gene expression of O6-methyguanine-DNA-methyltransferase (MGMT and p16 genes were examined in tissue and blood samples of patients with premalignant oral lesions (PMOLs and oral squamous cell carcinoma (OSCC. Methylation-specific PCR and reverse transcriptase PCR were performed in 146 tissue and blood samples from controls and patients with PMOLs and OSCC. In PMOL group, significant promoter methylation of MGMT and p16 genes was observed in 59% (P=0.0010 and 57% (P=0.0016 of tissue samples, respectively, and 39% (P=0.0135 and 33% (P=0.0074 of blood samples, respectively. Promoter methylation of both genes was more frequent in patients with OSCC, that is, 76% (P=0.0001 and 82% (P=0.0001 in tissue and 57% (P=0.0002 and 70% (P=0.0001 in blood, respectively. Significant downregulation of MGMT and p16 mRNA expression was observed in both tissue and blood samples from patients with PMOLs and OSCC. Hypermethylation-induced transcriptional silencing of MGMT and p16 genes in both precancer and cancer suggests important role of these changes in progression of premalignant state to malignancy. Results support use of blood as potential surrogate to tissue samples for screening or diagnosing PMOLs and early OSCC.

  7. 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

    Science.gov (United States)

    Bhatia, Vikram; 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 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. PMID:24991542

  8. Laser capture microdissection-based in vivo genomic profiling of wound keratinocytes identifies similarities and differences to squamous cell carcinoma

    DEFF Research Database (Denmark)

    Pedersen, Tanja Xenia; Leethanakul, Chidchanop; Patel, Vyomesh;

    2003-01-01

    we present the first analysis of global changes in keratinocyte gene expression during skin wound healing in vivo, and compare these changes to changes in gene expression during malignant conversion of keratinized epithelium. Laser capture microdissection was used to isolate RNA from wound...... reepithelialization. Furthermore, the analyses revealed that the phenotypic resemblance of wound keratinocytes to squamous cell carcinoma is mimicked at the level of gene expression, but notable differences between the two tissue-remodeling processes were also observed. The combination of laser capture...

  9. A crucial role of beta 1 integrins for keratinocyte migration in vitro and during cutaneous wound repair

    DEFF Research Database (Denmark)

    Grose, Richard; Hutter, Caroline; Bloch, Wilhelm;

    2002-01-01

    of beta 1 integrins in keratinocytes caused a severe defect in wound healing. beta 1-null keratinocytes showed impaired migration and were more densely packed in the hyperproliferative epithelium. Surprisingly, their proliferation rate was not reduced in early wounds and even increased in late wounds....... The failure in re-epithelialisation resulted in a prolonged inflammatory response, leading to dramatic alterations in the expression of important wound-regulated genes. Ultimately, beta 1-deficient epidermis did cover the wound bed, but the epithelial architecture was abnormal. These findings demonstrate...

  10. 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...

  11. RhoA is dispensable for skin development, but crucial for contraction and directed migration of keratinocytes

    DEFF Research Database (Denmark)

    Jackson, Ben; Peyrollier, Karine; Pedersen, Esben;

    2011-01-01

    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......, 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...

  12. Mean Annual UV-B Irradiance

    Data.gov (United States)

    U.S. Environmental Protection Agency — Ultraviolet-B (UV-B) radiation is the most energetic part of sunlight reaching the Earth's surface (wavelength region is 280 to 315 nm), and it has been shown to...

  13. 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

    2012-12-01

    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. PMID:23142932

  14. 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.

  15. Chrysanthemum boreale Makino essential oil induces keratinocyte proliferation and skin regeneration.

    Science.gov (United States)

    Kim, Do Yoon; Won, Kyung-Jong; Yoon, Mi-So; Hwang, Dae Il; Yoon, Seok Won; Park, Joo-Hoon; Kim, Bokyung; Lee, Hwan Myung

    2015-01-01

    We investigated the effect of essential oil from the flower of Chrysanthemum boreale Makino (CBMEO) on growth of human keratinocytes (HaCaTs) and explored a possible mechanism for this response. CBMEO was extracted using the steam distillation method. CBMEO contained a total of 33 compounds. CBMEO stimulated HaCaT proliferation (EC50, 0.028 μg/mL) and also induced phosphorylation of Akt and ERK1/2 in HaCaTs (EC50, 0.007 and 0.005 μg/mL, for phosphorylated Akt and ERK1/2, respectively). Moreover, CBMEO promoted wound closure in the dorsal side skin of rat tail. This study demonstrated that CBMEO can stimulate growth of human skin keratinocytes, probably through the Akt and ERK1/2 pathways. Therefore, CBMEO may be helpful in skin regeneration and wound healing in human skin, and may also be a possible cosmetic material for skin beauty. PMID:25167931

  16. 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

  17. 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.

  18. Proteomics unveil corticoid-induced S100A11 shuttling in keratinocyte differentiation

    International Nuclear Information System (INIS)

    Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the 'organelles and membranes' compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis

  19. 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.

  20. 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.

  1. Alkannin, HSP70 inducer, protects against UVB-induced apoptosis in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Yoko Yoshihisa

    Full Text Available Alkannin is an active constituent from the root extract of Alkanna tinctoria of the Boraginaceae family and it may have utility as a heat shock protein 70 (HSP70 inducer in living organisms. Here, the effects of alkannin-induced HSP70 on ultraviolet (UV B (40 mJ/cm(2-induced apoptosis were investigated in human keratinocyte HaCaT cells. Pretreatment of cells with alkannin (1 µM caused significant inhibition of UVB-induced apoptosis and caspase-3 cleavage. On the other hand, the addition of KNK437 (HSP70 inhibitor reversed the action of alkannin increasing UVB-induced apoptosis in a dose-dependent manner. In addition, differences in gene expression associated with the suppression of UVB-induced apoptosis in the presence of alkannin were investigated using Gene Chip assay. Our results indicate that alkannin suppresses UVB-induced apoptosis through the induction of HSP70 in human keratinocytes, and therefore, we suggest the usefulness of using alkannin as an antiaging agent.

  2. Intrinsic Defect in Keratinocyte Function Leads to Inflammation in Hidradenitis Suppurativa.

    Science.gov (United States)

    Hotz, Claire; Boniotto, Michele; Guguin, Aurélie; Surenaud, Mathieu; Jean-Louis, Francette; Tisserand, Pascaline; Ortonne, Nicolas; Hersant, Barbara; Bosc, Romain; Poli, Florence; Bonnabau, Henri; Thiébaut, Rodolphe; Godot, Véronique; Wolkenstein, Pierre; Hocini, Hakim; Lévy, Yves; Hüe, Sophie

    2016-09-01

    Hidradenitis suppurativa (HS) is a chronic, inflammatory, debilitating, follicular disease of the skin. Despite a high prevalence in the general population, the physiopathology of HS remains poorly understood. The use of antibiotics and immunosuppressive agents for therapy suggests a deregulated immune response to microflora. Using cellular and gene expression analyses, we found an increased number of infiltrating CD4(+) T cells secreting IL-17 and IFN-γ in perilesional and lesional skin of patients with HS. By contrast, IL-22-secreting CD4(+) T cells are not enriched in HS lesions contrasting with increased number of those cells in the blood of patients with HS. We showed that keratinocytes isolated from hair follicles of patients with HS secreted significantly more IL-1β, IP-10, and chemokine (C-C motif) ligand 5 (RANTES) either constitutively or on pattern recognition receptor stimulations. In addition, they displayed a distinct pattern of antimicrobial peptide production. These findings point out a functional defect of keratinocytes in HS leading to a balance prone to inflammatory responses. This is likely to favor a permissive environment for bacterial infections and chronic inflammation characterizing clinical outcomes in patients with HS. PMID:27206704

  3. Desmocollin 3-mediated Binding Is Crucial for Keratinocyte Cohesion and Is Impaired in Pemphigus*

    Science.gov (United States)

    Spindler, Volker; Heupel, Wolfgang-Moritz; Efthymiadis, Athina; Schmidt, Enno; Eming, Rüdiger; Rankl, Christian; Hinterdorfer, Peter; Müller, Thomas; Drenckhahn, Detlev; Waschke, Jens

    2009-01-01

    Desmocollin (Dsc) 1–3 and desmoglein (Dsg) 1–4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo- and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering. PMID:19717567

  4. 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. PMID:26581186

  5. 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.

  6. cdk4 Deficiency Inhibits Skin Tumor Development but Does Not Affect Normal Keratinocyte Proliferation

    Science.gov (United States)

    Rodriguez-Puebla, Marcelo L.; Miliani de Marval, Paula L.; LaCava, Margaret; Moons, David S.; Kiyokawa, Hiroaki; Conti, Claudio J.

    2002-01-01

    Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue. PMID:12163365

  7. Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion.

    Science.gov (United States)

    Strittmatter, Gerhard E; Garstkiewicz, Martha; Sand, Jennifer; Grossi, Serena; Beer, Hans-Dietmar

    2016-01-01

    Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1β and -18, and induces unconventional protein secretion (UPS) of mature IL-1β, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1. PMID:27665556

  8. 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. PMID:26767426

  9. 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.

  10. 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.

  11. Cdc42 is crucial for the maturation of primordial cell junctions in keratinocytes independent of Rac1

    DEFF Research Database (Denmark)

    Du, Dan; Pedersen, Esben; Wang, Zhipeng;

    2008-01-01

    -deficient immortalized and primary keratinocytes form only punctate primordial cell contacts in vitro, which cannot mature into belt-like junctions. This defect was independent of enhanced degradation of beta-catenin, but correlated to an impaired activation and localization of aPKCzeta in the Cdc42-null...

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

    Directory of Open Access Journals (Sweden)

    Ayako Uto-Konomi

    Full Text Available Homeostatic regulation of epidermal keratinocytes is controlled by the local cytokine milieu. However, a role for suppressor of cytokine signaling (SOCS, a negative feedback regulator of cytokine networks, in skin homeostasis remains unclear. Keratinocyte specific deletion of Socs3 (Socs3 cKO caused severe skin inflammation with hyper-production of IgE, epidermal hyperplasia, and S100A8/9 expression, although Socs1 deletion caused no inflammation. The inflamed skin showed constitutive STAT3 activation and up-regulation of IL-6 and IL-20 receptor (IL-20R related cytokines, IL-19, IL-20 and IL-24. Disease development was rescued by deletion of the Il6 gene, but not by the deletion of Il23, Il4r, or Rag1 genes. The expression of IL-6 in Socs3 cKO keratinocytes increased expression of IL-20R-related cytokines that further facilitated STAT3 hyperactivation, epidermal hyperplasia and neutrophilia. These results demonstrate that skin homeostasis is strictly regulated by the IL-6-STAT3-SOCS3 axis. Moreover, the SOCS3-mediated negative feedback loop in keratinocytes has a critical mechanistic role in the prevention of skin inflammation caused by hyperactivation of STAT3.

  13. Human atopic dermatitis skin-derived T cells can induce a reaction in mouse keratinocytes in vivo

    DEFF Research Database (Denmark)

    Martel, Britta C; Blom, Lars; Dyring-Andersen, Beatrice;

    2015-01-01

    through keratinocyte activation and consequently cause development of eczematous lesions. Punch biopsies of lesional skin from AD patients were used to establish skin-derived T cell cultures and which were transferred into NOD.Cg-Prkd(scid) Il2rg(tm1Sug) /JicTac (NOG) mice. We found that subcutaneous...

  14. Mesenchymal stem cell-conditioned medium improves the proliferation and migration of keratinocytes in a diabetes-like microenvironment.

    Science.gov (United States)

    Li, Meirong; Zhao, Yali; Hao, Haojie; Dai, Hanren; Han, Qingwang; Tong, Chuan; Liu, Jiejie; Han, Weidong; Fu, Xiaobing

    2015-03-01

    The impairment of wound healing in diabetic patients is an important clinical problem. Proper keratinocyte migration and proliferation are the crucial steps during reepithelialization, and these steps may be impaired in diabetes mellitus (DM) due to hyperglycemia and chronic inflammation in wound site. In this study, we explored the effects of diabetes-like microenvironment with high glucose (HG) and intense inflammation on the migration and proliferation of keratinocytes in vitro. We found that the migration and proliferation of rat keratinocytes were reduced with HG and lipopolysaccharide (LPS) stimulation via Erk signaling pathway in a reactive oxygen species (ROS)-dependent manner. Nevertheless, mesenchymal stem cell-conditioned medium (MSC-CM) counteracts the effects of HG and LPS. Treatment of rat keratinocyte with MSC-CM decreased HG- and/or LPS-induced ROS overproduction. Furthermore, MSC-CM reversed the downregulation of phosphorylation of MEK1/2 and Erk 1/2, which was induced by HG and/or LPS without affecting total levels. Our results may provide a possible mechanism for delayed wound healing in DM and provide a foundation to develop MSC-CM as an alternative therapeutic strategy to ameliorate the poor wound-healing conditions.

  15. The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Gromov, P;

    1995-01-01

    )vaccinia virus expression of full length cDNAs, and (vi) in vitro transcription/translation of full-length cDNAs. This year, special emphasis has been given to the identification of signal transduction components by using 2-D gel immunoblotting of crude keratinocyte lysates in combination with enhanced...

  16. Self-Antigen Presentation by Keratinocytes in the Inflamed Adult Skin Modulates T-Cell Auto-Reactivity.

    Science.gov (United States)

    Meister, Michael; Tounsi, Amel; Gaffal, Evelyn; Bald, Tobias; Papatriantafyllou, Maria; Ludwig, Julia; Pougialis, Georg; Bestvater, Felix; Klotz, Luisa; Moldenhauer, Gerhard; Tüting, Thomas; Hämmerling, Günter J; Arnold, Bernd; Oelert, Thilo

    2015-08-01

    Keratinocytes have a pivotal role in the regulation of immune responses, but the impact of antigen presentation by these cells is still poorly understood, particularly in a situation where the antigen will be presented only in adult life. Here, we generated a transgenic mouse model in which keratinocytes exclusively present a myelin basic protein (MBP) peptide covalently linked to the major histocompatibility complex class II β-chain, solely under inflammatory conditions. In these mice, inflammation caused by epicutaneous contact sensitizer treatment resulted in keratinocyte-mediated expansion of MBP-specific CD4(+) T cells in the skin. Moreover, repeated contact sensitizer application preceding a systemic MBP immunization reduced the reactivity of the respective CD4(+) T cells and lowered the symptoms of the resulting experimental autoimmune encephalomyelitis. This downregulation was CD4(+) T-cell-mediated and dependent on the presence of the immune modulator Dickkopf-3. Thus, presentation of a neo self-antigen by keratinocytes in the inflamed, adult skin can modulate CD4(+) T-cell auto-aggression at a distal organ. PMID:25835957

  17. Immunopathology of american cutaneous leishmaniasis. Modulation of MHC class II gene products by Keratinocytes before and after glucantime therapy

    Directory of Open Access Journals (Sweden)

    Claude Pirmez

    1990-06-01

    Full Text Available Epidermal changes from 32 cutaneous and 3 mucosal American leishmaniasis (ACL active lesions were studied for HLA-DR, -DP expression, Lanerhans cells and lymphocyte infiltration. In addition to a DR and DQ positivity at the surface of the cells of the inflammatory infiltrate, a strong reaction for DR antigens was detected on keratinocytes. Hyperplasia of Langerhans cells was present in al cutaneous lesions and epidermis was infiltrated by T lymphocytes. When healed lesions of 14 of these subjects were re-biopsied 1 to 12 months after the end of pentavalent antimonial therapy, MHC class antigens could no longer be seen on keratinocytes. Our data represrn evidence for hhe reversibility of the abnormal HLA-DR expression by keratinocytes in ACL after Glucantime therapy or spontaneous scar formation, demonstrating that this expresion is restricted to the period of active lesions. The present findings can be regarded as an indirect evidence that keratinocytes may be involved in the immunopathology of ACL.

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

    International Nuclear Information System (INIS)

    We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

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

    Energy Technology Data Exchange (ETDEWEB)

    Walter, M.N.M. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom); Wright, K.T.; Fuller, H.R. [Institute for Science and Technology in Medicine, Keele University RJAH Orthopaedic Hospital, Oswestry, SY10 7AG (United Kingdom); MacNeil, S. [Kroto Research Institute and Centre for Nanoscience and Technology, Sheffield University, Sheffield, S1 2UE (United Kingdom); Johnson, W.E.B., E-mail: w.e.johnson@aston.ac.uk [School of Life and Health Science, Aston University, Aston Triangle, Birmingham, B4 7EJ (United Kingdom)

    2010-04-15

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

  1. 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

    2016-01-01

    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. PMID:27045209

  2. Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

    International Nuclear Information System (INIS)

    Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (α3β3γ2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the α3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials

  3. 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

  4. 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

  5. Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration.

    Science.gov (United States)

    Pfalzgraff, Anja; Heinbockel, Lena; Su, Qi; Gutsmann, Thomas; Brandenburg, Klaus; Weindl, Günther

    2016-01-01

    The stagnation in the development of new antibiotics and the concomitant high increase of resistant bacteria emphasize the urgent need for new therapeutic options. Antimicrobial peptides are promising agents for the treatment of bacterial infections and recent studies indicate that Pep19-2.5, a synthetic anti-lipopolysaccharide (LPS) peptide (SALP), efficiently neutralises pathogenicity factors of Gram-negative (LPS) and Gram-positive (lipoprotein/-peptide, LP) bacteria and protects against sepsis. Here, we investigated the potential of Pep19-2.5 and the structurally related compound Pep19-4LF for their therapeutic application in bacterial skin infections. SALPs inhibited LP-induced phosphorylation of NF-κB p65 and p38 MAPK and reduced cytokine release and gene expression in primary human keratinocytes and dermal fibroblasts. In LPS-stimulated human monocyte-derived dendritic cells and Langerhans-like cells, the peptides blocked IL-6 secretion, downregulated expression of maturation markers and inhibited dendritic cell migration. Both SALPs showed a low cytotoxicity in all investigated cell types. Furthermore, SALPs markedly promoted cell migration via EGFR transactivation and ERK1/2 phosphorylation and accelerated artificial wound closure in keratinocytes. Peptide-induced keratinocyte migration was mediated by purinergic receptors and metalloproteases. In contrast, SALPs did not affect proliferation of keratinocytes. Conclusively, our data suggest a novel therapeutic target for the treatment of patients with acute and chronic skin infections. PMID:27509895

  6. Effect of in vitro and in vivo UV irradiation on the production of ETAF activity by human and murine keratinocytes

    International Nuclear Information System (INIS)

    Cultured epidermal cells and keratinocytes produce a potent hormone-like factor called epidermal cell-derived thymocyte-activating factor (ETAF). ETAF appears to be similar if not identical to a monocyte-derived lymphokine, known as interleukin 1 (IL-1). These two cytokines are able to amplify a diverse number of proliferative and inflammatory processes. Several recent investigations have suggested that UV-induced immunosuppression may be due in part to the inhibition of IL-1/ETAF production by monocytes and keratinocytes, respectively. We therefore decided to directly study the effects of various doses of in vitro and in vivo UV radiation (UVR) on the production of ETAF by normal murine epidermal cells and a murine (Pam 212) and a human (SCC) keratinocyte cell line. Our results surprisingly demonstrated an increase in both the extracellular and the intracellular ETAF activity of the murine epidermal, Pam 212, and SCC after sublethal amounts of in vitro UVR. Likewise, increased ETAF activity of murine epidermal cells was detected after sublethal doses of in vivo UVR. The UV-induced ETAF activity was cycloheximide-sensitive, suggesting that de novo synthesis of ETAF rather than cell membrane leakage was responsible for the increased ETAF activity. The fact that UV irradiation can increase ETAF activity by keratinocytes could have important local and systemic consequences for the host and may provide an efficient, contaminant-free method for generating ETAF activity for further biochemical and immunologic studies

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

    Science.gov (United States)

    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.

  8. APR-246/PRIMA-1(MET) rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations.

    Science.gov (United States)

    Shen, Jinfeng; van den Bogaard, Ellen H; Kouwenhoven, Evelyn N; Bykov, Vladimir J N; Rinne, Tuula; Zhang, Qiang; Tjabringa, Geuranne S; Gilissen, Christian; van Heeringen, Simon J; Schalkwijk, Joost; van Bokhoven, Hans; Wiman, Klas G; Zhou, Huiqing

    2013-02-01

    p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patient keratinocytes cultured either in submerged 2D cultures or in 3D skin equivalents have impaired epidermal differentiation and stratification. Treatment of these patient keratinocytes with the mutant p53-targeting compound APR-246/PRIMA-1(MET) (p53 reactivation and induction of massive apoptosis) that has been successfully tested in a phase I/II clinical trial in cancer patients partially but consistently rescued morphological features and gene expression during epidermal stratification in both 2D and 3D models. This rescue coincides with restoration of p63 target-gene expression. Our data show that EEC patient keratinocytes with p63 mutations can be used for characterization of the abnormal molecular circuitry in patient skin and may open possibilities for the design of novel pharmacological treatment strategies for patients with mutant p63-associated developmental abnormalities. PMID:23355676

  9. 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...

  10. The cytotoxic effect of neonatal lupus erythematosus and maternal sera on keratinocyte cultures is complement-dependent and can be augmented by ultraviolet irradiation

    International Nuclear Information System (INIS)

    To elucidate the role of autoantibodies and ultraviolet (UV) exposure in the pathogenesis of the skin lesions in neonatal lupus erythematosus (NLE), keratinocytes were cultured, as the target cells, from a patient with NLE and from a normal neonate. We demonstrated that the expression of nuclear/cytoplasma Ro/SSA and La/SSB molecules on to the surface of NLE keratinocytes occurred to a much greater extent than that on normal keratinocytes. A dose of 200 mJ/cm2 UVB irradiation on NLE keratinocytes induced a 2.5-3-fold increase in Ro/SSA and La/SSB expression compared to non-irradiated cells. Sera derived from both the NLE patient and from his mother exhibited a cytotoxic effect on NLE keratinocytes, but not on control cells, in the presence of complement. Furthermore, the cytotoxicity of the sera was enhanced in UVB-irradiated NLE keratinocytes, whereas it had no cytotoxic effects on UVB-irradiated control cells. This suggests that the abnormal expression of both Ro/SSA and La/SSB on the surface membrane of NLE keratinocytes induces the autoantibodies and complements to injure the cells. This complement-mediated cytotoxic effect can be augmented by UV irradiation, a concept not incompatible with the exacerbation of the skin eruption in sun-exposed skin sites. (author)

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

    Directory of Open Access Journals (Sweden)

    Sunita Nayak

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

  12. The inflammatory response of keratinocytes and its modulation by vitamin D: the role of MAPK signaling pathways.

    Science.gov (United States)

    Miodovnik, Mor; Koren, Ruth; Ziv, Esther; Ravid, Amiram

    2012-05-01

    The hormonal form of vitamin D, calcitriol, and its analogs are known for their beneficial effect in the treatment of inflammatory skin disorders. Keratinocytes play a role in epidermal inflammatory responses invoked by breeching of the epidermal barrier, by infectious agents and by infiltrating immune cells. We studied the role of calcitriol in the initiation of keratinocyte inflammatory response by the viral and injury mimic polyinosinic-polycytidylic acid (poly(I:C)) and in its maintenance by tumor-necrosis-factor α (TNFα) and investigated the role of the mitogen-activated protein kinase cascades in these processes and their regulation by calcitriol. The inflammatory response of human HaCaT keratinocytes to poly(I:C) or TNFα was assessed by measuring mRNA levels of 13 inflammation-related molecules by real-time PCR microarray and by in-depth investigation of the regulation of interleukin 8, intercellular-adhesion-molecule 1, and TNFα expression. We found that while calcitriol had only a minor effect on the keratinocyte response to poly(I:C) and a modest effect on the early response (2 h) to TNFα, it markedly attenuated the later response (16-24 h) to TNFα. The expression of CYP27B1, the enzyme responsible for calcitriol production, was marginally increased by poly(I:C) and markedly by TNFα treatment. This pattern suggests that while allowing the initial keratinocyte inflammatory response to proceed, calcitriol contributes to its timely resolution. Using pharmacological inhibitors we found that while the p38 MAPK and the extracellular signal-regulated kinase have only a minor role, c-Jun N-terminal kinase plays a pivotal role in the induction of the pro-inflammatory genes and its modulation by calcitriol. PMID:21792935

  13. 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.

  14. Porphyromonas gingivalis induces CCR5-dependent transfer of infectious HIV-1 from oral keratinocytes to permissive cells

    Directory of Open Access Journals (Sweden)

    Dietrich Elizabeth A

    2008-03-01

    Full Text Available Abstract Background Systemic infection with HIV occurs infrequently through the oral route. The frequency of occurrence may be increased by concomitant bacterial infection of the oral tissues, since co-infection and inflammation of some cell types increases HIV-1 replication. A putative periodontal pathogen, Porphyromonas gingivalis selectively up-regulates expression of the HIV-1 coreceptor CCR5 on oral keratinocytes. We, therefore, hypothesized that P. gingivalis modulates the outcome of HIV infection in oral epithelial cells. Results Oral and tonsil epithelial cells were pre-incubated with P. gingivalis, and inoculated with either an X4- or R5-type HIV-1. Between 6 and 48 hours post-inoculation, P. gingivalis selectively increased the infectivity of R5-tropic HIV-1 from oral and tonsil keratinocytes; infectivity of X4-tropic HIV-1 remained unchanged. Oral keratinocytes appeared to harbor infectious HIV-1, with no evidence of productive infection. HIV-1 was harbored at highest levels during the first 6 hours after HIV exposure and decreased to barely detectable levels at 48 hours. HIV did not appear to co-localize with P. gingivalis, which increased selective R5-tropic HIV-1 trans infection from keratinocytes to permissive cells. When CCR5 was selectively blocked, HIV-1 trans infection was reduced. Conclusion P. gingivalis up-regulation of CCR5 increases trans infection of harbored R5-tropic HIV-1 from oral keratinocytes to permissive cells. Oral infections such as periodontitis may, therefore, increase risk for oral infection and dissemination of R5-tropic HIV-1.

  15. Interleukin 6 is Expressed in High Levels in Psoriatic Skin and Stimulates Proliferation of Cultured Human Keratinocytes

    Science.gov (United States)

    Grossman, Rachel M.; Krueger, James; Yourish, Debra; Granelli-Piperno, Angela; Murphy, Daniel P.; May, Lester T.; Kupper, Thomas S.; Sehgal, Pravinkumar B.; Gottlieb, Alice B.

    1989-08-01

    Psoriasis is a common papulosquamous skin disease. The histopathology is characterized by epidermal hyperplasia and inflammation. Recent studies suggest that keratinocyte proliferation and inflammation in psoriasis are manifestations of the same underlying pathological process. Interleukin 6 (IL-6), a cytokine that is a major mediator of the host response to tissue injury and infection, is produced by both keratinocytes and leukocytes in culture. IL-6 expression was studied in psoriatic plaques by immunoperoxidase staining with two different polyclonal anti-recombinant IL-6 antisera and by in situ nucleic acid hybridization with IL-6 cRNA probes. Epidermal and dermal cells in active psoriatic plaques from 35 psoriasis patients stained heavily for IL-6 as compared with nonlesional skin and with plaques after treatment with antimetabolic and antiinflammatory agents. Absorption of the anti-recombinant IL-6 antisera with purified fibroblast-derived IL-6 or with recombinant IL-6, but not bovine serum albumin, removed the immunostaining. Increased levels of IL-6 were detected in the plasma of patients with active psoriasis (mean 3 ng/ml) by using two different bioassays. IL-6 production by proliferating keratinocytes was suggested by IL-6-specific immunostaining in cultured normal and psoriatic keratinocytes and by the detection of mRNA specific for IL-6 in psoriatic epidermis by in situ hybridization. IL-6 stimulated the proliferation of cultured, normal human keratinocytes as assessed by two different assays. Thus, IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affect the function of dermal inflammatory cells.

  16. Inhibition of inflammatory and proliferative responses of human keratinocytes exposed to the sesquiterpene lactones dehydrocostuslactone and costunolide.

    Directory of Open Access Journals (Sweden)

    Claudia Scarponi

    Full Text Available The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE and costunolide (CS, two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit

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

    Directory of Open Access Journals (Sweden)

    Marcella Mauro

    2015-07-01

    Full Text Available Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1 and needle-abraded human skin (experiment 2. Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm−2, while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm−2 and those with intact skin (1.08 ± 0.20 ng·cm−2. To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue® and propidium iodide (PI uptake assays. The results indicate that a long exposure time (i.e., seven days was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10−4 M, 95% CL = 0.8–1.9 × 10−4 M, MTT essay; 3.7 × 10−5 M, 95% CI = 2.2–6.1 × 10−5 M, AlamarBlue® assay that seems to be associated to necrotic events (EC50 value: 1.3 × 10−4 M, 95% CL = 0.9–1.9 × 10−4 M, PI assay. This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

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

    Directory of Open Access Journals (Sweden)

    Philpott Michael P

    2010-02-01

    Full Text Available Abstract Background The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. Results The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH and copy number variations (CNV. FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3 and segmental LOH (6q25.1-6q25.3. Conclusion We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant

  19. 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

  20. 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 and

  1. 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

    2015-07-01

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled-Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm⁻²), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm⁻²) and those with intact skin (1.08 ± 0.20 ng·cm⁻²). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10-4 M, 95% CL = 0.8-1.9 × 10⁻⁴ M, MTT essay; 3.7 × 10⁻⁵ M, 95% CI = 2.2-6.1 × 10⁻⁵ M, AlamarBlue assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10⁻⁴ M, 95% CL = 0.9-1.9 × 10⁻⁴ M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  3. Influence of interleukin-8 (IL-8) and IL-8 receptors on the migration of human keratinocytes, the role of PLC-γ and potential clinical implications

    OpenAIRE

    Jiang, Wen G; Sanders, Andrew J.; Ruge, Fiona; HARDING, KEITH G.

    2011-01-01

    Interleukin (IL)-8 is a pro-inflammatory cytokine that has a direct effect on immune cells, including polymorphonuclear cells. Keratinocytes are a rich source of IL-8. However, there is little knowledge on the role of IL-8 in clinical wound healing and the direct biological effect of IL-8 on keratinocytes. In this study, the effect of recombinant human IL-8 (rhIL-8) on migration and adhesion was tested using HaCaT keratinocytes as a cell model. The cell functions were evaluated using impedanc...

  4. Impact of mTORC1 Inhibition on Keratinocyte Proliferation During Skin Tumor Promotion in Wild-Type and BK5.AktWT Mice

    OpenAIRE

    Rho, Okkyung; Kiguchi, Kaoru; Jiang, Guiyu; DiGiovanni, John

    2013-01-01

    In this study, we examined the impact of rapamycin on mTORC1 signaling during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced keratinocyte proliferation and skin tumor promotion in both wild-type (FVB/N) and BK5.AktWT mice. TPA activated mTORC1 signaling in a time-dependent manner in cultured primary mouse keratinocytes and a mouse keratinocyte cell line. Early activation (15–30 min) of mTORC1 signaling induced by TPA was mediated in part by PKC activation, whereas later activation (2–4 h)...

  5. 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.

  6. Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin

    DEFF Research Database (Denmark)

    Pappinen, Sari; Hermansson, Martin; Kuntsche, Judith;

    2008-01-01

    The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present...... study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning...... calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non...

  7. 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

  8. Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes.

    Science.gov (United States)

    Zhang, Leshuai W; Yu, William W; Colvin, Vicki L; Monteiro-Riviere, Nancy A

    2008-04-15

    Quantum dots nanoparticles have novel optical properties for biomedical applications and electronics, but little is known about their skin permeability and interaction with cells. QD621 are nail-shaped nanoparticles that contain a cadmium/selenide core with a cadmium sulfide shell coated with polyethylene glycol (PEG) and are soluble in water. QD were topically applied to porcine skin flow-through diffusion cells to assess penetration at 1 microM, 2 microM and 10 microM for 24 h. QD were also studied in human epidermal keratinocytes (HEK) to determine cellular uptake, cytotoxicity and inflammatory potential. Confocal microscopy depicted the penetration of QD621 through the uppermost stratum corneum (SC) layers of the epidermis and fluorescence was found primarily in the SC and near hair follicles. QD were found in the intercellular lipid bilayers of the SC by transmission electron microscopy (TEM). Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd) and fluorescence for QD both did not detect Cd nor fluorescence signal in the perfusate at any time point or concentration. In HEK, viability decreased significantly (p<0.05) from 1.25 nM to 10 nM after 24 h and 48 h. There was a significant increase in IL-6 at 1.25 nM to 10 nM, while IL-8 increased from 2.5 nM to 10 nM after 24 h and 48 h. TEM of HEK treated with 10 nM of QD621 at 24 h depicted QD in cytoplasmic vacuoles and at the periphery of the cell membranes. These results indicate that porcine skin penetration of QD621 is minimal and limited primarily to the outer SC layers, yet if the skin were damaged allowing direct QD exposure to skin or keratinocytes, an inflammatory response could be initiated. PMID:18261754

  9. Role of SP-1 in SDS-Induced Adipose Differentiation Related Protein Synthesis in Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Emanuela Corsini

    2007-01-01

    Full Text Available Skin irritation is a complex phenomenon, and keratinocytes play an important role in it. We have recently characterized the expression and protective role of adipose differentiation related protein (ADRP in skin irritation. In particular, ADRP expression is induced to recover functional cell membrane following the cell damage caused by skin irritants. The purpose of this study was to characterize in a human keratinocyte cells line (NCTC 2544 the biochemical events that lead to ADRP expression following SDS treatment, and in particular, to investigate the role of transcription factor SP-1. Analysis of ADRP promoter region revealed the presence of a potential binding site for the transcription factor SP-1 close to the start site. Evaluated by measuring the DNA binding activity, we found that SDS induced a dose and time related SP-1 activation, which was correlated with SDS-induced ADRP mRNA expression. Furthermore, SDS-induced SP-1 activation, ADRP mRNA expression and lipid droplets accumulation could be modulated by mithramycin A, an antibiotic that selectively binds to the GC box preventing SP-1 binding and gene expression. This demonstrated that SDS-induced ADRP expression was mediated in part through the transcription factor SP-1. In addition, SDS-induced SP-1 activation and ADRP expression could be modulated by the calcium chelator BAPTA, indicating a role of calcium in ADRP-induction. Thus, every time an irritant perturbs the membrane barrier, it renders the membrane leaky and allows extracellular calcium to enter the cells, an event that provides the upstream mechanisms initiating the signaling cascade that triggers the activation of SP-1 and culminates in the enhancement of ADRP expression, which helps to restore the normal homeostasis and ultimately repairs the to membrane.

  10. Negative pressure induces p120-catenin-dependent adherens junction disassembly in keratinocytes during wound healing.

    Science.gov (United States)

    Huang, Ching-Hui; Hsu, Chih-Chin; Chen, Carl Pai-Chu; Chow, Shu-Er; Wang, Jong-Shyan; Shyu, Yu-Chiau; Lu, Mu-Jie

    2016-09-01

    A negative-pressure of 125mmHg (NP) has been widely used to treat chronic wounds in modern medicine. Keratinocytes under NP treatment have shown accelerated cell movement and decreased E-cadherin expression. However, the molecular mechanism of E-cadherin regulation under NP remains incompletely understood. Therefore, we investigated the E-cadherin regulation in keratinocytes (HaCaT cells) under NP. HaCaT cells were treated at ambient pressure (AP) and NP for 12h. Cell movement was measured by traditional and electric wound healing assays at the 2 different pressures. Mutants with overexpression of p120-catenin (p120(ctn)) were used to observe the effect of NP on p120(ctn) and E-cadherin expression during wound healing. Cell fractionation and immunoblotting data showed that NP increased Y228-phosphorylated p120(ctn) level and resulted in the translocation of p120(ctn) from the plasma membrane to cytoplasm. Immunofluorescence images revealed that NP decreased the co-localization of p120(ctn) and E-cadherin on the plasma membrane. Knockdown of p120(ctn) reduced E-cadherin expression and accelerated cell movement under AP. Overexpression of the Y228-phosphorylation-mimic p120(ctn) decreased E-cadherin membrane expression under both AP and NP. Phosphorylation-deficient mutants conferred restored adherens junctions (AJs) under NP. The Src inhibitor blocked the phosphorylation of p120(ctn) and impeded cell migration under NP. In conclusion, Src-dependent phosphorylation of p120(ctn) can respond rapidly to NP and contribute to E-cadherin downregulation. The NP-induced disassembly of the AJ further accelerates wound healing. PMID:27220534

  11. Assessment of radiation induced cytogenetic damage in human keratinocytes by comet assay

    International Nuclear Information System (INIS)

    In the present study the effect of gamma radiation on normal human keratinocytes (HaCaT) cells has been analyzed using alkaline comet assay and a comparative study over the sensitivity of different comet parameters such as tail length (TL), olive tail moment (OTM) and percentage tail DNA (TDNA) has also been made. Human keratinocytes (HaCaT) cells were grown in Dulbecco's modified essential medium (DMEM) (10% FCS) at 37 °C in a humidified atmosphere containing 5% CO2. Cultured cells were harvested with 0.025 % trypsin EDTA. The sample (2 X 10 cells/ml) was exposed to gamma radiation of different dose using a 60Co gamma source at dose rate of 2 Gy min-1 and the dosimetry has been carried out using Fricke and FBX dosimeters. After irradiation, to quantify the DNA damage the comet assay (single cell gel electrophoresis) was carried out under alkaline conditions, by the methods outlined by Singh et al. The quantification of the DNA strand breaks in each cells were performed using CASP software. The DNA damage quantification can be accomplished by measuring those comet parameters which exhibit a linear dependence on the amount of DNA damage. In the present study, comet parameters such as OTM, TL and TDNA were recorded and the variation of these parameters and their correlation coefficients for different doses of gamma radiation is plotted. The OTM value is normalized with control value and control for TL and TDNA is adjusted to zero to avoid initial variations in different experiments

  12. Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes.

    Science.gov (United States)

    Jiang, Yan J; Uchida, Yoshikazu; Lu, Biao; Kim, Peggy; Mao, Cungui; Akiyama, Masashi; Elias, Peter M; Holleran, Walter M; Grunfeld, Carl; Feingold, Kenneth R

    2009-07-10

    ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise approximately 50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C(2)-Cer and C(6)-Cer), but not C(8)-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C(6)-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARdelta-mediated signaling pathway, providing a substrate-driven, feed

  13. Biological interactions of quantum dot nanoparticles in skin and in human epidermal keratinocytes

    International Nuclear Information System (INIS)

    Quantum dots nanoparticles have novel optical properties for biomedical applications and electronics, but little is known about their skin permeability and interaction with cells. QD621 are nail-shaped nanoparticles that contain a cadmium/selenide core with a cadmium sulfide shell coated with polyethylene glycol (PEG) and are soluble in water. QD were topically applied to porcine skin flow-through diffusion cells to assess penetration at 1 μM, 2 μM and 10 μM for 24 h. QD were also studied in human epidermal keratinocytes (HEK) to determine cellular uptake, cytotoxicity and inflammatory potential. Confocal microscopy depicted the penetration of QD621 through the uppermost stratum corneum (SC) layers of the epidermis and fluorescence was found primarily in the SC and near hair follicles. QD were found in the intercellular lipid bilayers of the SC by transmission electron microscopy (TEM). Inductively coupled plasma-optical emission spectroscopy (ICP-OES) analysis for cadmium (Cd) and fluorescence for QD both did not detect Cd nor fluorescence signal in the perfusate at any time point or concentration. In HEK, viability decreased significantly (p < 0.05) from 1.25 nM to 10nM after 24 h and 48 h. There was a significant increase in IL-6 at 1.25 nM to 10 nM, while IL-8 increased from 2.5nM to 10nM after 24 h and 48 h. TEM of HEK treated with 10 nM of QD621 at 24 h depicted QD in cytoplasmic vacuoles and at the periphery of the cell membranes. These results indicate that porcine skin penetration of QD621 is minimal and limited primarily to the outer SC layers, yet if the skin were damaged allowing direct QD exposure to skin or keratinocytes, an inflammatory response could be initiated

  14. Keratinocyte growth factor phage model peptides can promote epidermal cell proliferation without tumorigenic effect

    Institute of Scientific and Technical Information of China (English)

    ZONG Xian-lei; JIANG Du-yin; WANG Ji-chang; LIU Jun-li; LIU Zhen-zhong; CAI Jing-long

    2010-01-01

    Background Keratinocyte growth factor (KGF) significantly influences epithelial wound healing. The aim of this study was to isolate KGF phage model peptides from a phage display 7-mer peptide library to evaluate their effect on promoting epidermal cell proliferation. Methods A phage display 7-mer peptide library was screened using monoclonal anti-human KGF antibody as the target. Enzyme linked immunosorbent assay (ELISA) was performed to select monoclonal phages with good binding activity. DNA sequencing was done to find the similarities of model peptides. Three-(4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) assay, immunofluorescence assay and quantitative real-time PCR analysis were employed to evaluate the effect of the phage model peptides on epidermal cells. Results Thirty-three out of fifty-eight (56.9%) of the isolated monoclonal phages exhibited high binding activity by ELISA. Ten of fifteen obtained phage model peptides were similar to KGF or epidermal growth factor (EGF). MTT assay data showed that four (No. 1-4) of the ten phage model peptides could promote epidermal cell proliferation. The expression of keratinocyte growth factor receptor (KGFR) mRNA in the KGF control group and the two phage model peptide groups (No. 1 and No. 2) increased. Expression of c-Fos mRNA and c-Jun mRNA in the KGF control group increased, but did not increase in the four phage model peptide groups (No.1-4). Conclusion Four phage model peptides isolated from the phage display 7-mer peptide library can safely promote epidermal cell proliferation without tumorigenic effect.

  15. 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.

  16. CDK2 Activation in Mouse Epidermis Induces Keratinocyte Proliferation but Does Not Affect Skin Tumor Development

    Science.gov (United States)

    Macias, Everardo; Miliani de Marval, Paula L.; De Siervi, Adriana; Conti, Claudio J.; Senderowicz, Adrian M.; Rodriguez-Puebla, Marcelo L.

    2008-01-01

    It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21Cip1 and p27Kip1. Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4D158N mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4D158N, but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21Cip1 in K5Cdk2, but not in K5Cdk4D158N, epidermis, suggesting that CDK2 overexpression elicits a p21Cip1 response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis. PMID:18599613

  17. 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.

  18. 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. PMID:21401652

  19. Dynamic changes in nicotinamide pyridine dinucleotide content in normal human epidermal keratinocytes and their effect on retinoic acid biosynthesis

    International Nuclear Information System (INIS)

    The function of many enzymes that regulate metabolism and transcription depends critically on the nicotinamide pyridine dinucleotides. To understand the role of NAD(P)(H) in physiology and pathophysiology, it is imperative to estimate both their amount and ratios in a given cell type. In human epidermis and in cultured epidermal keratinocytes, we found that the total dinucleotide content is in the low millimolar range. The dinucleotide pattern changes during proliferation and maturation of keratinocytes in culture. Differences in the concentrations of NAD(P)(H) of 1.5- to 12-fold were observed. This resulted in alteration of the NAD(P)H/NAD(P) ratio, which could impact the differential regulation of both transcriptional and metabolic processes. In support of this notion, we provide evidence that the two-step oxidation of retinol to retinoic acid, a nuclear hormone critical for epidermal homeostasis, can be regulated by the relative physiological amounts of the pyridine dinucleotides

  20. 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.

  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. Eckol inhibits ultraviolet B-induced cell damage in human keratinocytes via a decrease in oxidative stress

    International Nuclear Information System (INIS)

    In previous reports, the antioxidant effects of eckol were shown to protect cells against hydrogen peroxide- and gamma ray-induced oxidative stress. In this study, the role of eckol in protecting human skin keratinocytes (HaCaT) against UVB-induced oxidative cell damage was investigated. Also, triphlorethol-A, one of the chemical components in Ecklonia cava, and quercetin a well known antioxidant, were compared with eckol in terms of antioxidant activity based on chemical structure. Eckol decreased UVB-induced intracellular reactive oxygen species (ROS), decreased injury to cellular components resulting from UVB-induced oxidative stress, and restored cell viability. In addition, eckol reduced UVB-induced apoptosis by inhibiting the disruption of mitochondrial membranes. These results suggest that eckol protects human keratinocytes against UVB-induced oxidative stress by scavenging ROS, thereby lessening injury to cellular components. (author)

  3. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

    International Nuclear Information System (INIS)

    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

  4. 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

  5. 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

  6. Eccrine Sweat Contains IL-1α, IL-1β and IL-31 and Activates Epidermal Keratinocytes as a Danger Signal

    OpenAIRE

    Xiuju Dai; Hidenori Okazaki; Yasushi Hanakawa; Masamoto Murakami; Mikiko Tohyama; Yuji Shirakata; Koji Sayama

    2013-01-01

    Eccrine sweat is secreted onto the skin's surface and is not harmful to normal skin, but can exacerbate eczematous lesions in atopic dermatitis. Although eccrine sweat contains a number of minerals, proteins, and proteolytic enzymes, how it causes skin inflammation is not clear. We hypothesized that it stimulates keratinocytes directly, as a danger signal. Eccrine sweat was collected from the arms of healthy volunteers after exercise, and levels of proinflammatory cytokines in the sweat were ...

  7. Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection.

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    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

  8. 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.

  9. Paeoniflorin attenuates ultraviolet B-induced apoptosis in human keratinocytes by inhibiting the ROS-p38-p53 pathway.

    Science.gov (United States)

    Kong, Lingwen; Wang, Shangshang; Wu, Xiao; Zuo, Fuguo; Qin, Haihong; Wu, Jinfeng

    2016-04-01

    Ultraviolet (UV) light is one of the most harmful environmental factors that contribute to skin damage. Exposure to UV induces extensive generation of reactive oxygen species (ROS), and results in photoaging and skin cancer development. One approach to protecting human skin against UV radiation is the use of antioxidants. In recent years, naturally occurring herbal compounds have gained considerable attention as protective agents for UV exposure. Paeoniflorin (PF) is a novel natural antioxidant, which is isolated from peony root (Radix Paeoniae Alba). The present study evaluated the protective effects of PF on UV‑induced skin damage in vitro, and demonstrated that the effects were mediated via the ROS‑p38‑p53 pathway. The results of the present study demonstrated that treatment with PF (25, 50, and 100 µM) significantly increased the percentage of viable keratinocytes after UV‑B exposure. In addition, cell death analysis indicated that PF treatment markedly reduced UV‑B‑radiation‑induced apoptosis in keratinocytes, which was accompanied by increased procaspase 3 expression and decreased cleaved caspase 3 expression. Treatment with PF markedly reduced the production of ROS, and inhibited the activation of p38 and p53 in human keratinocytes, thus suggesting that the ROS‑p38‑p53 pathway has a role in UV‑B‑induced skin damage. In conclusion, the present study reported that PF was able to attenuate UV‑B‑induced cell damage in human keratinocytes. Notably, these effects were shown to be mediated, at least in part, via inhibition of the ROS-p38-p53 pathway. PMID:26936104

  10. CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure

    OpenAIRE

    Huang, S.; Guo, S.; Guo, F; Yang, Q.; XIAO, X.; Murata, M.; Ohnishi, S.; Kawanishi, S; Ma, N

    2013-01-01

    Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC). To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the human spontaneously immortalized skin keratinocytes (HaCaT) cell line to an environmentally relevant level of arsenic (0.05 ppm) in vitrofor 18 weeks. Following sodium arsenite administration, cell cycle, colo...

  11. Exploiting high-throughput screens to optimize Adeno-Associated Viral Vectors for gene transfer into primary human keratinocytes

    OpenAIRE

    Sallach, Jessica

    2014-01-01

    Chronic non-healing wounds such as diabetic ulcers or burns represent a devastating health problem with significant clinical, physical and social implications. The healing can be frustrating and painful for patients. The difficult healing process requires advanced therapeutic strategies such as the use of primary human keratinocytes (HK) as autologous transplants, which may be considered for clinical use. To improve engraftment or to introduce therapeutic genes into primary HK, efficient and ...

  12. APR-246/PRIMA-1MET rescues epidermal differentiation in skin keratinocytes derived from EEC syndrome patients with p63 mutations

    OpenAIRE

    Shen, Jinfeng; van den Bogaard, Ellen H.; Kouwenhoven, Evelyn N.; Vladimir J.N. Bykov; Rinne, Tuula; Zhang, Qiang; Tjabringa, Geuranne S.; Gilissen, Christian; Van Heeringen, Simon J.; Schalkwijk, Joost; Van Bokhoven, Hans; Wiman, Klas G.; Zhou, Huiqing

    2013-01-01

    p53 and p63 share extensive sequence and structure homology. p53 is frequently mutated in cancer, whereas mutations in p63 cause developmental disorders manifested in ectodermal dysplasia, limb defects, and orofacial clefting. We have established primary adult skin keratinocytes from ectrodactyly, ectodermal dysplasia, and cleft lip/palate (EEC) syndrome patients with p63 mutations as an in vitro human model to study the disease mechanism in the skin of EEC patients. We show that these patien...

  13. 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.

  14. 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 ...

  15. Tanshinone IIA Inhibits Growth of Keratinocytes through Cell Cycle Arrest and Apoptosis: Underlying Treatment Mechanism of Psoriasis

    Directory of Open Access Journals (Sweden)

    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.

  16. Inhibition of Inflammatory Gene Expression in Keratinocytes Using a Composition Containing Carnitine, Thioctic Acid and Saw Palmetto Extract

    Directory of Open Access Journals (Sweden)

    Sridar Chittur

    2011-01-01

    Full Text Available Chronic inflammation of the hair follicle (HF is considered a contributing factor in the pathogenesis of androgenetic alopecia (AGA. Previously, we clinically tested liposterolic extract of Serenoa repens (LSESr and its glycoside, β-sitosterol, in subjects with AGA and showed a highly positive response to treatment. In this study, we sought to determine whether blockade of inflammation using a composition containing LSESr as well as two anti-inflammatory agents (carnitine and thioctic acid could alter the expression of molecular markers of inflammation in a well-established in vitro system. Using a well-validated assay representative of HF keratinocytes, specifically, stimulation of cultured human keratinocyte cells in vitro, we measured changes in gene expression of a spectrum of well-known inflammatory markers. Lipopolysaccharide (LPS provided an inflammatory stimulus. In particular, we found that the composition effectively suppressed LPS-activated gene expression of chemokines, including CCL17, CXCL6 and LTB(4 associated with pathways involved in inflammation and apoptosis. Our data support the hypothesis that the test compound exhibits anti-inflammatory characteristics in a well-established in vitro assay representing HF keratinocyte gene expression. These findings suggest that 5-alpha reductase inhibitors combined with blockade of inflammatory processes could represent a novel two-pronged approach in the treatment of AGA with improved efficacy over current modalities.

  17. 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. PMID:27599115

  18. 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.

  19. Disruption of spectrin-like cytoskeleton in differentiating keratinocytes by PKCδ activation is associated with phosphorylated adducin.

    Directory of Open Access Journals (Sweden)

    Kong-Nan Zhao

    Full Text Available Spectrin is a central component of the cytoskeletal protein network in a variety of erythroid and non-erythroid cells. In keratinocytes, this protein has been shown to be pericytoplasmic and plasma membrane associated, but its characteristics and function have not been established in these cells. Here we demonstrate that spectrin increases dramatically in amount and is assembled into the cytoskeleton during differentiation in mouse and human keratinocytes. The spectrin-like cytoskeleton was predominantly organized in the granular and cornified layers of the epidermis and disrupted by actin filament inhibitors, but not by anti-mitotic drugs. When the cytoskeleton was disrupted PKCδ was activated by phosphorylation on Thr505. Specific inhibition of PKCδ(Thr505 activation with rottlerin prevented disruption of the spectrin-like cytoskeleton and the associated morphological changes that accompany differentiation. Rottlerin also inhibited specific phosphorylation of the PKCδ substrate adducin, a cytoskeletal protein. Furthermore, knock-down of endogenous adducin affected not only expression of adducin, but also spectrin and PKCδ, and severely disrupted organization of the spectrin-like cytoskeleton and cytoskeletal distribution of both adducin and PKCδ. These results demonstrate that organization of a spectrin-like cytoskeleton is associated with keratinocytes differentiation, and disruption of this cytoskeleton is mediated by either PKCδ(Thr505 phosphorylation associated with phosphorylated adducin or due to reduction of endogenous adducin, which normally connects and stabilizes the spectrin-actin complex.

  20. Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair

    Directory of Open Access Journals (Sweden)

    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.

  1. 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. PMID:22935613

  2. Effect of various metals on intercellular adhesion molecule-1 expression and tumour necrosis factor alpha production by normal human keratinocytes.

    Science.gov (United States)

    Guéniche, A; Viac, J; Lizard, G; Charveron, M; Schmitt, D

    1994-01-01

    Nickel, cobalt and chromium are metals very often implicated in allergic contact dermatitis. In vivo, keratinocytes, which are the first target cells, can be directly activated to participate in the local reaction, especially through the expression of the membrane antigen ICAM-1, a ligand of the leucocyte antigen LFA-1, and the production of cytokines. Our aim was to assess the effects of sensitizing metal haptens (nickel, cobalt and chromium) compared with the toxic metal cadmium on the induction of ICAM-1 and the production of TNF alpha by epidermal cells. For this purpose, normal human keratinocytes obtained during plastic skin surgery were cultured in low-calcium defined medium (MCDB153) and the metals were used in non-toxic concentrations. Using FACS analysis, ICAM-1 expression was found to be induced only by nickel. This stimulation appeared as early as 24 h after stimulation. All the metals induced a low expression of TNF alpha detectable by immunocytochemistry correlating with the induction of the nuclear stress protein Hsp72 which is closely linked genetically with the TNF alpha locus. However, only Ni2+, Co2+ and Cr2+ induced a significant release of TNF alpha detectable by ELISA after 48 h stimulation. This secretion was lower than that observed with known stimulants such as lipopolysaccharide. These results indicate that the metals studied are able to induce an aggressive cellular effect, and that nickel, by its ICAM-1 induction, may play a major role in the keratinocyte activation state during allergic contact dermatitis. PMID:7864660

  3. RNA-seq analysis of host and viral gene expression highlights interaction between varicella zoster virus and keratinocyte differentiation.

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. 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.

  6. 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.

  7. Photoprotective Effects of Hydroxychloroqine and TCMs on Human Keratinocytes Damaged from Ultraviolet Irradiation

    Institute of Scientific and Technical Information of China (English)

    LUODan; MINWei; LINXiang-fei; WUDi; MIAOXu

    2004-01-01

    Objective: To investigate damage effects of ultraviolet irradiation on eternal keratinocyte-HaCaT cells and to evaluate photo-protective efficiency of hydroxychloroqine and Traditional Chinese Medicines ( epigallocatechingallate[EGCG], baikal skullcap root and szechwan lovge rhizome) on HaCaT ceils damaged by middle wave ultraviolet(UVB) irradiation. Methods: Subconfluent HaCaT cells were sham or UVB irradiated and treated with above TCM agents. The damage degree of HaCaT cells was observed by a light microscop. Cell growth was recorded by cellcount and cellular activity was detected by MTT method. The secretion amount of IL-6 and TNF-α was measured by ELISA. Results: The irradiation damage of HaCaT cells was depended on the irradiated dosages and cellular activity was reduced by 36%-80%, with a maximum decrease over 90% after 72 h. The intervention of the above drugs may increase the cellular activity by 10%-72%. The photo-protective efficiency was more apparent in F_A~G (from 1.19+0.07 to 1.28 + 0.06, P < 0.01 ) than that in hydroxychloroqine ( from 0.43 + 0.04 to 0.96 + 0.04, P < 0.05). The other two tested drugs also showed photo-protective effect( from 0.44 + 0.07 to 1.21 -+ 0.02, P < 0.05). As to cytokine secretion, EGCG could decline the secretion amount of IL-6 and TNF.a apparently. Hydroxychloroqine and baikal skullcap root baikal skullcap root could only reduce the secretion of ID6. The secretion of IL-6 and TNF-α could not be inhibitedby szechwan lovge rhizome. Conclusion : The injury effect of UVB irradiation on cultured keratinocytes is dose-dependent and the tested drugs have photo-protective potency. Inhibition of cytokine secretion may be one of the mechanisms related to reducing the damage effect of UVB irradiation.

  8. 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

  9. Cytotoxic effects of sodium dodecyl benzene sulfonate on human keratinocytes are not associated with proinflammatory cytokines expression

    Institute of Scientific and Technical Information of China (English)

    Mu Zhanglei; Liu Xiaojing; Zhao Yan; Zhang Jianzhong

    2014-01-01

    Background Keratinocytes play a crucial role in the biological function of skin barrier.The relationship between sodium lauryl sulfate (SLS) and keratinocytes has been studied.However,the cytotoxicity and effects of sodium dodecyl benzene sulfonate (SDBS),a common detergent similar to SLS,on keratinocytes are still not known.This study aimed to investigate the effects of SDBS on cytotoxicity and expression of proinflammatory cytokines in cultured human keratinocytes.Methods This study was carried out using the keratinocytes cell line,HaCaT cells.The cytotoxicity of SDBS on HaCaT cells was evaluated with cell counting kit-8 (CCK-8) and phase-contrast microscopy.After exposure to different concentrations of SDBS,the total RNA of the HaCaT cells was extracted for evaluating the relative mRNA expression of IL-1α,IL-6,IL-8,and TNF-α by qPCR.The supernatants of cells were collected for measuring the levels of IL-6 and IL-8 by enzyme-linked immunosorbent assay (ELISA).Results SDBS at concentrations of 20 Jg/ml and over showed direct cytotoxicity and induced morphological changes of the HaCaT cells.The mRNA expressions of IL-1a,IL-6,IL-8,and TNF-α in different concentrations of SDBS at different time were comparable with that of controls.SDBS at concentrations of 5,10,and 15 μg/ml had no significant effects on IL-6 and IL-8 excretion from HaCaT cells after 24-hour exposure.Moreover,no significant effects on the IL-6 and IL-8 excretion were found after 10 and 15 μg/ml S DBS stimulations for 6,12,and 24 hours,respectively.Conclusion SDBS at higher concentrations had cytotoxicity on HaCaT cells but had no effects on the mRNA expression of IL-1α,IL-6,IL-8,and TNF-α,that was different from SLS.

  10. 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

  11. The Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone enhances keratinocyte migration and induces Mmp13 gene expression in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Paes, Camila, E-mail: camilaquinetti@gmail.com [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nakagami, Gojiro, E-mail: gojiron-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Minematsu, Takeo, E-mail: tminematsu-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Nagase, Takashi, E-mail: tnagase@fb3.so-net.ne.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Huang, Lijuan, E-mail: koureikenhlj@gmail.com [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Sari, Yunita, E-mail: yunita-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Sanada, Hiromi, E-mail: hsanada-tky@umin.ac.jp [University of Tokyo, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer An evidence of the positive effect of AHL on epithelialization process is provided. Black-Right-Pointing-Pointer AHL enhances keratinocyte's ability to migrate in an in vitro scratch wound model. Black-Right-Pointing-Pointer AHL induces the expression of Mmp13. Black-Right-Pointing-Pointer Topical application of AHL represents a possible strategy to treat chronic wounds. -- Abstract: Re-epithelialization is an essential step of wound healing involving three overlapping keratinocyte functions: migration, proliferation and differentiation. While quorum sensing (QS) is a cell density-dependent signaling system that enables bacteria to regulate the expression of certain genes, the QS molecule N-(3-oxododecanoyl) homoserine lactone (AHL) exerts effects also on mammalian cells in a process called inter-kingdom signaling. Recent studies have shown that AHL improves epithelialization in in vivo wound healing models but detailed understanding of the molecular and cellular mechanisms are needed. The present study focused on the AHL as a candidate reagent to improve wound healing through direct modulation of keratinocyte's activity in the re-epithelialization process. Results indicated that AHL enhances the keratinocyte's ability to migrate in an in vitro scratch wound healing model probably due to the high Mmp13 gene expression analysis after AHL treatment that was revealed by real-time RT-PCR. Inhibition of activator protein 1 (AP-1) signaling pathway completely prevented the migration of keratinocytes, and also resulted in a diminished Mmp13 gene expression, suggesting that AP-1 might be essential in the AHL-induced migration. Taken together, these results imply that AHL is a promising candidate molecule to improve re-epithelialization through the induction of migration of keratinocytes. Further investigation is needed to clarify the mechanism of action and molecular pathway of AHL on the keratinocyte migration

  12. Increased expression of 70 kD heat shock protein in cultured primary human keratinocytes induced by human papillomavirus 16 E6/E7 gene

    Institute of Scientific and Technical Information of China (English)

    LIAO Wen-jun; FAN Ping-shen; FU Meng; FAN Xue-li; LIU Yu-feng

    2005-01-01

    Background Heat shock protein 70 (HSP70) is expressed highly in epithelial tumours associated closely with human papillomavirus 16 (HPV16) infections. However, evidence about the direct relationship between HSP70 expression and HPVs infections are still lacking. In the present study, we examined the expression of HSP70 in keratinocytes introduced with HPV16 E6/E7 oncogenes. Methods Stable transfected cells were established by transfection of the plasmids pLXSN16E6/E7 into cultured primary keratinocytes and subsequently selected by plasmid specific selection antibiotic (G418) at the required concentration. The expression of HSP70 in pLXSN16E6/E7 transfected keratinocytes was determined by Western blot. The correlation of HSP70 expression and E6/E7 transfection was further confirmed by doubly labelled immunofluorescent staining.Results Compared to non-transfected keratinocytes, there was a significant trend for higher levels of HSP70 in pLXSN16E6/E7 transfected keratinocytes. Doubly labelled immunofluorescent staining experiment showed that the co-localization of HPV16 E6/E7 and HSP70 in transfected keratinocytes was observed and increased expression of HSP70 was strongly associated with the transfection of HPV16 E6/E7.Conclusions Our studies demonstrated increased levels of HSP70 proteins in keratinocytes stably transfected by HPV16 E6/E7 oncogenes. It suggests that the expression of HSP70 is modulated by HPV16 E6/E7 proteins, which may be involved in HPV16 E6/E7 induced immortalization.

  13. Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

    Science.gov (United States)

    Fleischmajer, R; MacDonald, E D; Contard, P; Perlish, J S

    1993-09-01

    Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

  14. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress

    Science.gov (United States)

    KIM, KI CHEON; PIAO, MEI JING; HEWAGE, SUSARA RUWAN KUMARA MADDUMA; HAN, XIA; KANG, KYOUNG AH; JO, JIN OH; MOK, YOUNG SUN; SHIN, JENNIFER H.; PARK, YEUNSOO; YOO, SUK JAE; HYUN, JIN WON

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2′,7′-dichlorodihydro-fluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components. PMID:26573561

  15. Toxicity of tannic acid-modified silver nanoparticles in keratinocytes: potential for immunomodulatory applications.

    Science.gov (United States)

    Orlowski, Piotr; Soliwoda, Katarzyna; Tomaszewska, Emilia; Bien, Karolina; Fruba, Aleksandra; Gniadek, Marianna; Labedz, Olga; Nowak, Zuzanna; Celichowski, Grzegorz; Grobelny, Jarosław; Krzyzowska, Malgorzata

    2016-09-01

    Hydrolyzable tannins are known to exhibit anti-inflammatory activity, which can be used in combination with silver nanoparticles (AgNPs) for dermal uses. In this study, we investigated the effects of tannic acid-modified 13, 33, 46nm and unmodified 10-65nm AgNPs using the human-derived keratinocyte HaCaT and VK2-E6/E7 cell lines in the form of stationary and spheroids cultures. After exposition to tannic acid-modified AgNPs, VK2-E6/E7 cells showed higher toxicity, increased production of reactive oxygen species (ROS) and activity of JNK stress kinase, while HaCaT cell line demonstrated less ROS production and activation of ERK kinase. AgNPs internalization was detected both in the superficial and internal layers of spheroids prepared from both cell lines. Tannic acid modified AgNPs sized above 30nm did not induce DNA breaks in comet assay performed in both cell lines. Tannic acid-modified but not unmodified AgNPs down-regulated TNF-α and LPS-triggered production of IL-8 in VK2-E6/E7 but not in HaCaT cells. In summary, tannic acid-modified AgNPs sized above 30nm show good toxicological profile both in vitro and possess immunomodulatory properties useful for potential dermal applications in humans. PMID:27216470

  16. Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro.

    Science.gov (United States)

    Mahabal, Shyam; Konala, Vijay Bhaskar Reddy; Mamidi, Murali Krishna; Kanafi, Mohammad Mahboob; Mishra, Suniti; Shankar, Krupa; Pal, Rajarshi; Bhonde, Ramesh

    2016-08-01

    Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT-PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin-EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing. PMID:25698160

  17. Gene Expression Profiling of Human Epidermal Keratinocytes in Simulated Microgravity and Recovery Cultures

    Institute of Scientific and Technical Information of China (English)

    Jade Q. Clement; Shareen M. Lacy; Bobby L. Wilson

    2008-01-01

    Simulated microgravity (SMG) bioreactors and DNA microarray technology are powerful tools to identify "space genes" that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3,4,9, and 10d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15,50, and 60d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were "center genes" that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately down regulated. Another seven of the center genes, which are all metallothionein MT-Ⅰ and MT-Ⅱ isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly upregulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures (≤4d) recovered in 15d; for longer (≥9d) exposures, more than 50d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.

  18. Electrospun tilapia collagen nanofibers accelerating wound healing via inducing keratinocytes proliferation and differentiation.

    Science.gov (United States)

    Zhou, Tian; Wang, Nanping; Xue, Yang; Ding, Tingting; Liu, Xin; Mo, Xiumei; Sun, Jiao

    2016-07-01

    The development of biomaterials with the ability to induce skin wound healing is a great challenge in biomedicine. In this study, tilapia skin collagen sponge and electrospun nanofibers were developed for wound dressing. The collagen sponge was composed of at least two α-peptides. It did not change the number of spleen-derived lymphocytes in BALB/c mice, the ratio of CD4(+)/CD8(+) lymphocytes, and the level of IgG or IgM in Sprague-Dawley rats. The tensile strength and contact angle of collagen nanofibers were 6.72±0.44MPa and 26.71±4.88°, respectively. They also had good thermal stability and swelling property. Furthermore, the nanofibers could significantly promote the proliferation of human keratinocytes (HaCaTs) and stimulate epidermal differentiation through the up-regulated gene expression of involucrin, filaggrin, and type I transglutaminase in HaCaTs. The collagen nanofibers could also facilitate rat skin regeneration. In the present study, electrospun biomimetic tilapia skin collagen nanofibers were succesfully prepared, were proved to have good bioactivity and could accelerate rat wound healing rapidly and effectively. These biological effects might be attributed to the biomimic extracellular matrix structure and the multiple amino acids of the collagen nanofibers. Therefore, the cost-efficient tilapia collagen nanofibers could be used as novel wound dressing, meanwhile effectively avoiding the risk of transmitting animal disease in the future clinical apllication. PMID:27037778

  19. 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.

  20. Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes

    International Nuclear Information System (INIS)

    The mechanism by which radiation-induced genomic instability is initiated, propagated and effected is currently under intense scrutiny. We have investigated the potential role of altered genomic methylation patterns in the cellular response to irradiation and have found evidence for widespread dysregulation of CpG methylation persisting up to 20 population doublings post-irradiation. Similar effects are seen with cells treated with medium from irradiated cells (the 'bystander effect') rather than subjected to direct irradiation. Using an arbitrarily primed methylation sensitive PCR screening method we have demonstrated that irradiation causes reproducible alterations in the methylation profile of a human keratinocyte cell line, HPV-G, and have further characterised one of these sequences as being a member of a retrotransposon element derived sequence family on chromosome 7; MLT1A. Multiple changes were also detected in the screen, which indicate that although the response of cells is predominantly hypermethylation, specific hypomethylation occurs as well. Sequence specific changes are also reported in the methylation of the pericentromeric SAT2 satellite sequence. This is the first demonstration that irradiation results in the induction of heritable methylation changes in mammalian cells, and provides a link between the various non-radiological instigators of genomic instability, the perpetuation of the unstable state and several of its manifestations

  1. Stages of Cell Cannibalism--Entosis--in Normal Human Keratinocyte Culture.

    Science.gov (United States)

    Garanina, A S; Khashba, L A; Onishchenko, G E

    2015-11-01

    Entosis is a type of cell cannibalism during which one cell penetrates into another cell and usually dies inside it. Researchers mainly pay attention to initial and final stages of entosis. Besides, tumor cells in suspension are the primary object of studies. In the present study, we investigated morphological changes of both cells-participants of entosis during this process. The substrate-dependent culture of human normal keratinocytes HaCaT was chosen for the work. A combination of light microscopy and scanning electron microscopy was used to prove that one cell was completely surrounded by the plasma membrane of another cell. We investigated such "cell-in-cell" structures and described the structural and functional changes of both cells during entosis. The outer cell nucleus localization and shape were changed. Gradual degradation of the inner cell nucleus and of the junctions between the inner and the outer cells was revealed. Moreover, repeated redistribution of the outer cell membrane organelles (Golgi apparatus, lysosomes, mitochondria, and autophagosomes), rearrangement of its cytoskeleton, and change in the lysosomal, autophagosomal, and mitochondrial state in both entotic cells were observed during entosis. On the basis of these data, we divided entosis into five stages that make it possible to systematize description of this type of cell death. PMID:26615438

  2. Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes.

    Science.gov (United States)

    Sunthamala, Nuchsupha; Pang, Chai Ling; Thierry, Francoise; Teissier, Sebastien; Pientong, Chamsai; Ekalaksananan, Tipaya

    2014-12-01

    The E2 protein is expressed in the early stage of human papillomavirus (HPV) infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK) harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA) revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively. PMID:26484085

  3. Genome-wide analysis of high risk human papillomavirus E2 proteins in human primary keratinocytes

    Directory of Open Access Journals (Sweden)

    Nuchsupha Sunthamala

    2014-12-01

    Full Text Available The E2 protein is expressed in the early stage of human papillomavirus (HPV infection that is associated with cervical lesions. This protein plays important roles in regulation of viral replication and transcription. To characterize the role of E2 protein in modulation of cellular gene expression in HPV infected cells, genome-wide expression profiling of human primary keratinocytes (HPK harboring HPV16 E2 and HPV18 E2 was investigated using microarray. The Principle Components Analysis (PCA revealed that the expression data of HPV16 E2 and HPV18 E2-transduced HPKs were rather closely clustered. The Venn diagram of modulated genes showed an overlap of 10 common genes in HPV16 E2 expressing HPK and HPV18 E2 expressing HPK. These genes were expressed with significant difference by comparison with control cells. In addition, the distinct sets of modulated genes were detected 14 and 34 genes in HPV16 E2 and HPV18 E2 expressing HPKs, respectively.

  4. Photoprotective Activity of Vulpinic and Gyrophoric Acids Toward Ultraviolet B-Induced Damage in Human Keratinocytes.

    Science.gov (United States)

    Varol, Mehmet; Türk, Ayşen; Candan, Mehmet; Tay, Turgay; Koparal, Ayşe Tansu

    2016-01-01

    Vulpinic and gyrophoric acids are known as ultraviolet filters for natural lichen populations because of their chemical structures. However, to the best of our knowledge, there has been no reference to their cosmetic potential for skin protection against ultraviolet B (UVB)-induced damage and, consequently, we propose to highlight their photoprotective profiles in human keratinocytes (HaCaT). Therefore, vulpinic acid and gyrophoric acid were isolated from acetone extracts of Letharia vulpina and Xanthoparmelia pokornyi, respectively. Their photoprotective activities on irradiated HaCaT cells and destructive effects on non-irradiated HaCaT cells were compared through in vitro experimentation: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, 4',6-diamino-2-phenylindole and tetramethylrhodamine B isothiocyanate-phalloidin staining protocols. Both of the lichen substances effectively prevented cytotoxic, apoptotic and cytoskeleton alterative activities of 2.5 J/cm(2) UVB in a dose-dependent manner. Moreover, vulpinic and gyrophoric acids showed no toxic, apoptotic or cytoskeleton alterative effects on non-irradiated HaCaT cells, except at high doses (≥400 μM) of gyrophoric acid. The findings suggest that vulpinic and gyrophoric acids can be promising cosmetic ingredients to photo-protect human skin cells and should therefore be further investigated by in vitro and in vivo multiple bioassays. PMID:26463741

  5. Effects of silver nanoparticles on human dermal fibroblasts and epidermal keratinocytes.

    Science.gov (United States)

    Galandáková, A; Franková, J; Ambrožová, N; Habartová, K; Pivodová, V; Zálešák, B; Šafářová, K; Smékalová, M; Ulrichová, J

    2016-09-01

    Biomedical application of silver nanoparticles (AgNPs) has been rapidly increasing. Owing to their strong antimicrobial activity, AgNPs are used in dermatology in the treatment of wounds and burns. However, recent evidence for their cytotoxicity gives rise to safety concerns. This study was undertaken as a part of an ongoing programme in our laboratory to develop a topical agent for wound healing. Here, we investigated the potential toxicity of AgNPs using normal human dermal fibroblasts (NHDF) and normal human epidermal keratinocytes (NHEK) with the aim of comparing the effects of AgNPs and ionic silver (Ag-I). Besides the effect of AgNPs and Ag-I on cell viability, the inflammatory response and DNA damage in AgNPs and Ag-I-treated cells were examined. The results showed that Ag-I were significantly more toxic than AgNPs both on NHDF and NHEK. Non-cytotoxic concentrations of AgNPs and Ag-I did not induce DNA strand breaks and did not affect inflammatory markers, except for a transient increase in interleukin 6 levels in Ag-I-treated NHDF. The results showed that AgNPs are more suitable for the intended application as a topical agent for wound healing up to the concentration 25 µg/mL. PMID:26500221

  6. The involvement of Gab1 and PI 3-kinase in β1 integrin signaling in keratinocytes

    International Nuclear Information System (INIS)

    The control of the stem cell compartment in epidermis is closely linked to the regulation of keratinocyte proliferation and differentiation. β1 integrins are expressed 2-fold higher by stem cells than transit-amplifying cells. Signaling from these β1 integrins is critical for the regulation of the epidermal stem cell compartment. To clarify the functional relevance of this differential expression of β1 integrins, we established HaCaT cells with high β1integrin expression by repeated flow cytometric sorting of this population from the parental cell line. In these obtained cells expressing β1 integrins by 5-fold, MAPK activation was markedly increased. Regarding the upstream of MAPK, Gab1 phosphorylation was also higher with high β1 integrin expression, while Shc phosphorylation was not altered. In addition, enhanced phosphatidylinositol 3-kinase activation was also observed. These observations suggest that Gab1 and phosphatidylinositol 3-kinase play pivotal roles in the β1 integrin-mediated regulation of the epidermal stem cell compartment

  7. Induction and inhibition of benzo(a)pyrene metabolism in human epidermal keratinocytes and dermal fibroblasts

    International Nuclear Information System (INIS)

    The effects of different growth conditions and various cytochrome P-450 inducers and inhibitors were examined on the benzo(a)pyrene (BP) metabolism of human skin cells in vitro. First, three different populations of neonatal foreskin fibroblasts were treated with 0.98 μM [G-3H]-BP at 9.6 Ci/mmole for 24 hours and the organic-extractable metabolites in the extracellular, the cytoplasmic, and the nuclear fractions were analyzed by high-pressure liquid chromatography (HPLC). Confluent cultures (contact-inhibited cells), which were nontransformable, metabolized BP to a much greater extent than the transformable populations, randomly-proliferating cultures (cells in logarithmic growth) and synchronized cultures (cells treated in S phase). The major extracellular BP metabolites detected were the 9,10-diol, the 7,8-diol, and the phenols, including all four tetrols observed only in the confluent cells. From this data, the state of confluency (i.e. the model for the in vivo state of skin cells) induced enzymes which catalyze primarily the detoxification pathways leading to the formation of metabolites which are readily excreted whereas cells which are actively dividing or synchronized induced the formation of metabolites from activation pathways that led to transformation. Next, the effects of four inducers (3-methylcholanthrene (3-MCA), phenobarbital (PB), isosafrole, and Arochlor 1254) and an inhibitor (butylated hydroxyanisole (BHA)) on the BP metabolism of human epidermal keratinocytes were evaluated

  8. FIH-1 disrupts an LRRK1/EGFR complex to positively regulate keratinocyte migration.

    Science.gov (United States)

    Peng, Han; Kaplan, Nihal; Yang, Wending; Getsios, Spiro; Lavker, Robert M

    2014-12-01

    Factor inhibiting hypoxia-inducible factor 1 (FIH-1; official symbol HIF1AN) is a hydroxylase that negatively regulates hypoxia-inducible factor 1α but also targets other ankyrin repeat domain-containing proteins such as Notch receptor to limit epithelial differentiation. We show that FIH-1 null mutant mice exhibit delayed wound healing. Importantly, in vitro scratch wound assays demonstrate that the positive role of FIH-1 in migration is independent of Notch signaling, suggesting that this hydroxylase targets another ankyrin repeat domain-containing protein to positively regulate motogenic signaling pathways. Accordingly, FIH-1 increases epidermal growth factor receptor (EGFR) signaling, which in turn enhances keratinocyte migration via mitogen-activated protein kinase pathway, leading to extracellular signal-regulated kinase 1/2 activation. Our studies identify leucine-rich repeat kinase 1 (LRRK1), a key regulator of the EGFR endosomal trafficking and signaling, as an FIH-1 binding partner. Such an interaction prevents the formation of an EGFR/LRRK1 complex, necessary for proper EGFR turnover. The identification of LRRK1 as a novel target for FIH-1 provides new insight into how FIH-1 functions as a positive regulator of epithelial migration.

  9. Biocompatibility Evaluation of Dental Luting Cements Using Cytokine Released from Human Oral Fibroblasts and Keratinocytes

    Directory of Open Access Journals (Sweden)

    Jae-Sung Kwon

    2015-10-01

    Full Text Available Dental luting cements are commonly used in dentistry for cementation of prosthetic restoration. Many previous studies focused on the measurement of the cell viability as the method of cytotoxicity evaluation during biocompatibility study for the material. In this study, the biocompatibility of various dental luting cements were evaluated using the new method of cytokine release measurement in order to better simulate inflammatory reactions in animal or clinical model using two different oral cells; immortalized human gingival fibroblast and immortalized human oral keratinocytes. Cells were exposed to extractions of various commercially available dental luting cements for different durations. Cytokines of IL-1α and IL-8 were measured from the supernatants of the cells and the results were then compared to the conventional MTT viability test. The result from the conventional cell viability study showed a relatively simple and straight forward indication that only one of the dental luting cements tested in this study was cytotoxic with increasing duration of exposure for both cells. Meanwhile, the result from the cytokine measurement study was much more complex at the time point they were measured, type of cells used for the study and the type of cytokines measured, all of which influenced the interpretation of the results. Hence, the better understanding of the cytokine release would be required for the application in biocompatibility evaluation.

  10. Effect of lead on IL-8 production and cell proliferation in human oral keratinocytes

    Institute of Scientific and Technical Information of China (English)

    Thaweboon Srosiri; Poomsawat Sopee; Thaweboon Boonyanit

    2010-01-01

    Objective:To investigate the effect of lead on the production of IL-8 and cell proliferation in normal human oral keratinocytes (NHKs). Methods: NHKs were prepared as outgrowths from normal human buccal mucosa. The cells were treated with three concentrations of lead glutamate (4.5í10-5M, 4.5í10-6M and 4.5í10-7M). NHKs grown in glutamic acid were used as control. The amounts of IL-8 secreted in the culture supernatants were evaluated at 12 and 24 h using enzyme-linked immunospecific assay (ELISA). Cell proliferation was determined by the MTT colorimetric assay. Three cultures were used for each experiment, and three independent experiments were performed. Analysis of variance and Duncan’s multiple range tests were used for statistical analysis. Results:An elevation of IL-8 in culture supernatants of NHKs treated with lead at all concentrations at 12 and 24 h after exposure in a dose-dependent manner was revealed. A significant increase in cell numbers was observed only at 24 h exposed to 4.5í10-5M lead glutamate. Conclusions: The capacity of NHKs, to secrete IL-8, enhanced by lead glutamate, is demonstrated here. Induction of cell proliferation is revealed only after exposure to high lead concentration. The elevation of secreted IL-8 is a probable initial sign for the acute inflammatory response and may be involved in the pathogenesis of lead stomatitis.

  11. A mint purified extract protects human keratinocytes from short-term, chemically induced oxidative stress.

    Science.gov (United States)

    Berselli, Patrizia Valeria Rita; Zava, Stefania; Montorfano, Gigliola; Corsetto, Paola Antonia; Krzyzanowska, Justyna; Oleszek, Wieslaw; Berra, Bruno; Rizzo, Angela Maria

    2010-11-10

    Oxidative stress is strictly correlated to the pathogenesis of many diseases, and a diet rich in fruits and vegetables, or adequately integrated, is currently considered to be a protective and preventive factor. This study aimed to analyze the efficacy of a 1 h preincubation with the highest nontoxic dose of a characterized Mentha longifolia extract (80 μg/mL) in protecting human keratinocytes (NCTC2544) from chemically induced oxidative stress (500 μM H2O2 for 2, 16, and 24 h). As reference synthetic pure compounds rosmarinic acid (360.31 μg/mL), a major mint phenolic constituent, and resveratrol (31.95 mg/mL), a well-known antioxidant, were used. Cellular viability was significantly protected by mint, which limited protein and DNA damage, decreased lipid peroxidation, and preserved glutathione and superoxide dismutase activity in the shorter phases of oxidative stress induction, in extents comparable to or better than those of pure compounds. These data suggest that mint use as only a flavoring has to be revised, taking into consideration its enrichment in foodstuff and cosmetics.

  12. Anti-Inflammatory Action of Keratinocyte-Derived Vaspin: Relevance for the Pathogenesis of Psoriasis.

    Science.gov (United States)

    Saalbach, Anja; Tremel, Jenny; Herbert, Diana; Schwede, Katharina; Wandel, Elke; Schirmer, Christine; Anderegg, Ulf; Beck-Sickinger, Annette G; Heiker, John T; Schultz, Stephan; Magin, Thomas; Simon, Jan C

    2016-03-01

    Impaired cross talk between keratinocytes (KCs) and immune cells is believed to contribute to the pathogenesis of chronic inflammatory skin diseases, such as psoriasis. We have previously identified KCs as a rich source of the serpin protease inhibitor vaspin (serpinA12), originally described as an adipokine in adipose tissue. Herein, we studied whether dysregulated vaspin expression in KCs contributes to the pathogenesis of psoriasis. We found vaspin expression to be closely associated to epidermal differentiation, with low levels in proliferating KCs and high levels in differentiated cells. Consistently, in human psoriasis and in a mouse model of a psoriasis-like skin inflammation, epidermal vaspin expression was significantly down-regulated. Down-regulation of vaspin in KCs resulted in decreased expression of differentiation-associated genes and up-regulation of interferon-inducible and inflammation-associated psoriasis signature genes. Vaspin was also shown to modulate the communication between KCs and inflammatory cells under co-culture conditions. A decrease in vaspin expression in KCs stimulated the secretion of tumor necrosis factor-α, IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1 by co-cultured dendritic cells, macrophages, monocytes, and neutrophils. Consequently, the application of vaspin inhibited myeloid cell infiltration in a mouse model of a psoriasis-like skin inflammation.