58 research outputs found

    The Application of PCR and STR DNA Profiling for the Identification of Haematoxylin Eosin Histological Slides in a Case of Sample Mix-Up Involving Synonymous Patients

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    A good laboratory practice ensures that biopsy material is correctly identified and associated with a given patient. Nevertheless, there are cases where the proof of origin of a tissue sample may be questioned. In this case study we have identified the source of cervical cancer glass slide sections stained with H/E, (hematoxylin eosin), after the request of a patient of Northern Greek origin who suspected sample mix-up when she coincidentally found out that a synonymous patient was examined for cervical cancer at the same time period in the same hospital in Greece. The patient was prepared to legally challenge the administrators of the downstream chemotherapeutic regimen. A combination of organic gradient clean up and silica membrane method was used for DNA isolation. Powerplex-16® system (Promega U.S.A) was used to generate complete DNA profiles from histological slides and the reference blood sample collected from the patient. Histochemical slides often yield inadequate STR profiles for successful DNA typing. Complete profiling in this case could be attributed to the adequate removal of stain and fixatives inhibitors and the isolation of good quality DNA for PCR or STR, protocols. Matching of histochemical slide DNA with patient blood DNA prevented legal action

    Hypoxia-inducible factor 1α is a new target of microphthalmia-associated transcription factor (MITF) in melanoma cells

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    In melanocytes and melanoma cells α-melanocyte stimulating hormone (α-MSH), via the cAMP pathway, elicits a large array of biological responses that control melanocyte differentiation and influence melanoma development or susceptibility. In this work, we show that cAMP transcriptionally activates Hif1a gene in a melanocyte cell–specific manner and increases the expression of a functional hypoxia-inducible factor 1α (HIF1α) protein resulting in a stimulation of Vegf expression. Interestingly, we report that the melanocyte-specific transcription factor, microphthalmia-associated transcription factor (MITF), binds to the Hif1a promoter and strongly stimulates its transcriptional activity. Further, MITF “silencing” abrogates the cAMP effect on Hif1a expression, and overexpression of MITF in human melanoma cells is sufficient to stimulate HIF1A mRNA. Our data demonstrate that Hif1a is a new MITF target gene and that MITF mediates the cAMP stimulation of Hif1a in melanocytes and melanoma cells. Importantly, we provide results demonstrating that HIF1 plays a pro-survival role in this cell system. We therefore conclude that the α-MSH/cAMP pathway, using MITF as a signal transducer and HIF1α as a target, might contribute to melanoma progression

    Identification of a transcriptional signature for the wound healing continuum

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    There is a spectrum/continuum of adult human wound healing outcomes ranging from the enhanced (nearly scarless) healing observed in oral mucosa to scarring within skin and the nonhealing of chronic skin wounds. Central to these outcomes is the role of the fibroblast. Global gene expression profiling utilizing microarrays is starting to give insight into the role of such cells during the healing process, but no studies to date have produced a gene signature for this wound healing continuum. Microarray analysis of adult oral mucosal fibroblast (OMF), normal skin fibroblast (NF), and chronic wound fibroblast (CWF) at 0 and 6 hours post-serum stimulation was performed. Genes whose expression increases following serum exposure in the order OMF < NF < CWF are candidates for a negative/impaired healing phenotype (the dysfunctional healing group), whereas genes with the converse pattern are potentially associated with a positive/preferential healing phenotype (the enhanced healing group). Sixty-six genes in the enhanced healing group and 38 genes in the dysfunctional healing group were identified. Overrepresentation analysis revealed pathways directly and indirectly associated with wound healing and aging and additional categories associated with differentiation, development, and morphogenesis. Knowledge of this wound healing continuum gene signature may in turn assist in the therapeutic assessment/treatment of a patient's wounds

    Exploring hypotheses of the actions of TGF-beta 1 in epidermal wound healing using a 3D computational multiscale model of the human epidermis

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    In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-beta 1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-beta 1 literature-derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units ( keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-beta 1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged ( by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-beta 1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-beta 1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing

    Exon Array Analysis of Head and Neck Cancers Identifies a Hypoxia Related Splice Variant of LAMA3 Associated with a Poor Prognosis

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    The identification of alternatively spliced transcript variants specific to particular biological processes in tumours should increase our understanding of cancer. Hypoxia is an important factor in cancer biology, and associated splice variants may present new markers to help with planning treatment. A method was developed to analyse alternative splicing in exon array data, using probeset multiplicity to identify genes with changes in expression across their loci, and a combination of the splicing index and a new metric based on the variation of reliability weighted fold changes to detect changes in the splicing patterns. The approach was validated on a cancer/normal sample dataset in which alternative splicing events had been confirmed using RT-PCR. We then analysed ten head and neck squamous cell carcinomas using exon arrays and identified differentially expressed splice variants in five samples with high versus five with low levels of hypoxia-associated genes. The analysis identified a splice variant of LAMA3 (Laminin α 3), LAMA3-A, known to be involved in tumour cell invasion and progression. The full-length transcript of the gene (LAMA3-B) did not appear to be hypoxia-associated. The results were confirmed using qualitative RT-PCR. In a series of 59 prospectively collected head and neck tumours, expression of LAMA3-A had prognostic significance whereas LAMA3-B did not. This work illustrates the potential for alternatively spliced transcripts to act as biomarkers of disease prognosis with improved specificity for particular tissues or conditions over assays which do not discriminate between splice variants

    Delayed Cutaneous Wound Healing and Aberrant Expression of Hair Follicle Stem Cell Markers in Mice Selectively Lacking Ctip2 in Epidermis

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    This is the publisher’s final pdf. The published article is copyrighted by PLoS and can be found at: http://www.plosone.org/home.action.Background: COUP-TF interacting protein 2 [(Ctip2), also known as Bcl11b] is an important regulator of skin homeostasis, and is overexpressed in head and neck cancer. Ctip2(ep-/-) mice, selectively ablated for Ctip2 in epidermal keratinocytes, exhibited impaired terminal differentiation and delayed epidermal permeability barrier (EPB) establishment during development, similar to what was observed in Ctip2 null (Ctip2(-/-)) mice. Considering that as an important role of Ctip2, and the fact that molecular networks which underlie cancer progression partially overlap with those responsible for tissue remodeling, we sought to determine the role of Ctip2 during cutaneous wound healing. \ud \ud Methodology/Principal Findings: Full thickness excisional wound healing experiments were performed on Ctip2(L2/L2) and Ctip2(ep-/-) animals per time point and used for harvesting samples for histology, immunohistochemistry (IHC) and immunoblotting. Results demonstrated inherent defects in proliferation and migration of Ctip2 lacking keratinocytes during re-epithelialization. Mutant mice exhibited reduced epidermal proliferation, delayed keratinocyte activation, altered cell-cell adhesion and impaired ECM development. Post wounding, Ctip2(ep-/-) mice wounds displayed lack of E-Cadherin suppression in the migratory tongue, insufficient expression of alpha smooth muscle actin (alpha SMA) in the dermis, and robust induction of K8. Importantly, dysregulated expression of several hair follicle (HF) stem cell markers such as K15, NFATc1, CD133, CD34 and Lrig1 was observed in mutant skin during wound repair. \ud \ud Conclusions/Significance: Results confirm a cell autonomous role of keratinocytic Ctip2 to modulate cell migration, proliferation and/or differentiation, and to maintain HF stem cells during cutaneous wounding. Furthermore, Ctip2 in a non-cell autonomous manner regulated granulation tissue formation and tissue contraction during wound closure

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

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    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 ameliorates the disease. Recently, dehydrocostuslactone (DCE) and custonolide (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-\u3b3, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-\u3b3-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. Taken together, our findings encourage the employment of DCE and CS in psoriasis, as they could efficiently counteract the pro-inflammatory effects of IFN-\u3b3 and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit hyperproliferation in the psoriatic epidermis

    Signature transcriptomale des kératinocytes humains dans un modèle de réépithélisation (validation fonctionnelle des molécules impliquées)

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    Le travail qui fait l objet de cette thèse contribue à une meilleure compréhension des événements moléculaires qui régissent la cicatrisation cutanée. En étudiant la réponse biologique spécifique des kératinocytes, nous avons utilisé la technologie des puces à ADN et un système original de blessure développé dans notre laboratoire 1) pour identifier les gènes qui sont induits ou réprimés dans les kératinocytes humains en culture en réponse à une blessure mécanique 2) pour caractériser les voies de signalisation qui contrôlent l expression de ces gènes. Nous avons montré que la blessure d un tapis de kératinocytes provoque la stimulation et l inhibition de l expression de 161 nouveaux marqueurs de la réparation épidermique. Une analyse pharmacologique réalisée en présence des inhibiteurs spécifiques de voies de signalisation ERK1/2,p38[MAPK] et PI3K, montre que ces voies exercent une contrôle sélectif sur le processus de réparation en contrôlant l expression des gènes spécifiques. Une étude fonctionnelle réalisée à la suite de notre analyse transcriptomale nous a permis de montrer que la stimulation d HB-EGF, d ETS1 et de NDRG1 est une condition sine qua non pour la migration et la prolifération des kératinocytes en réponse à une blessure, la surexpression ou l inhibition de l expression de chacun d eux entraînant des altérations cinétiques du processus de fermeture de blessure. Par ailleurs, nous démontrons pour la première fois que HIF1a joue un rôle fondamental dans la migration cellulaire en contrôlant le niveau d expression de la sous-unité ax3 de la laminine 5, une protéine qui joue un rôle essentiel dans la migration, la prolifération, et l adhésion des kératinocytes.NICE-BU Sciences (060882101) / SudocSudocFranceF
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