-
[show abstract]
[hide abstract]
ABSTRACT: Severe burns in children are conventionally treated with split-thickness skin autografts or epidermal sheets. However, neither early complete healing nor quality of epithelialization is satisfactory. An alternative approach is to graft isolated keratinocytes. We evaluated paediatric foreskin and auricular skin as donor sources, autologous keratinocyte transplantation, and compared the graft efficiency to the in vitro capacities of isolated keratinocytes to divide and reconstitute epidermal tissue. Keratinocytes were isolated from surgical samples by enzymatic digestion. Living cell recovery, in vitro proliferation and epidermal reconstruction capacities were evaluated. Differentiation status was analysed, using qRT-PCR and immunolabelling. Eleven children were grafted with foreskin-derived (boys) or auricular (girls) keratinocyte suspensions dripped onto deep severe burns. The aesthetic and functional quality of epithelialization was monitored in a standardized way. Foreskin keratinocyte graft in male children provides for the re-epithelialization of partial deep severe burns and accelerates wound healing, thus allowing successful wound closure, and improves the quality of scars. In accordance, in vitro studies have revealed a high yield of living keratinocyte recovery from foreskin and their potential in terms of regeneration and differentiation. We report a successful method for grafting paediatric males presenting large severe burns through direct spreading of autologous foreskin keratinocytes. This alternative method is easy to implement, improves the quality of skin and minimizes associated donor site morbidity. In vitro studies have highlighted the potential of foreskin tissue for graft applications and could help in tissue selection with the prospect of grafting burns for girls. Copyright © 2013 John Wiley & Sons, Ltd.
Journal of Tissue Engineering and Regenerative Medicine 03/2013; · 3.28 Impact Factor
-
Jean-Philippe Giot,
Isabelle Paris,
Pierre Levillain,
Vincent Huguier,
Sandrine Charreau,
Adrianna Delwail,
Martine Garcia,
Julien Garnier, François-Xavier Bernard,
Guy Dagregorio,
Gérard Guillet,
Franck Morel,
Jean-Claude Lecron,
Laure Favot
[show abstract]
[hide abstract]
ABSTRACT: Hypertensive leg ulcer (HLU) is an inflammatory disease characterized by intense pain, alteration of vascularization, and skin necrosis. The optimal treatment relies on surgical removal of necrotic tissues covered by a split-skin graft. We studied the histomorphology of the lesions and investigated the involvement of inflammatory cells and cytokines to further define the physiopathology of HLU. We report epidermis acanthosis and a preferential occlusion of the precapillary arterioles with infiltration of neutrophils, macrophages, and T lymphocytes in the dermis. OSM, IL-1β, and IL-6 were overexpressed in the ulcer, whereas the Th17-derived cytokines were not. In vitro, the addition of IL-1β and OSM promoted acanthosis and destructuring of reconstructed epidermis. Exogenous IL-1β and OSM synergistically induced epidermal acanthosis in mice. These data show that OSM and IL-1β are not only a biological characteristic signature of HLU, but these cytokines reflect a specific inflammatory state, directly involved in the pathogenesis. We suggest that anti-cytokine biotherapies could be an alternative strategy to surgery to treat HLU.
American Journal Of Pathology 01/2013; · 4.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Sebocytes originate from the same lineage as keratinocytes, and both cell types may have similarities in terms of growth and differentiation. We were interested in studying the behaviour of human sebocytes when cultured in conditions validated for epidermal reconstruction. For this purpose, we established a HPV16-E6/7-immortalized human sebocyte cell line (SEBO662) growing in keratinocyte defined media. Postconfluent SEBO662 cells in monolayers express the early sebocyte marker, cytokeratin 7 (K7), do not express Epithelia Membrane Antigen (EMA) and do not exhibit strong lipogenic activity. However, when placed at the air-liquid interface, SEBO662 multilayers spontaneously differentiate into a sebaceous-like structure as shown by the strong polarized expression of the late sebaceous marker EMA, the overexpression of some lipogenic markers and lipid production on the upper side of the epithelium. This work highlights the value of simple 3D models for exhibiting spontaneous differentiation and polarization.
Experimental Dermatology 04/2012; 21(4):314-6. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cutaneous homeostasis and defenses are maintained by permanent cross-talk among particular epidermal keratinocytes and immune cells residing or recruited in the skin, through the production of cytokines. If required, a coordinated inflammatory response is triggered, relayed by specific cytokines. Due to numerous reasons, troubles in the resolution of this phenomenon could generate a cytokine-mediated vicious circle, promoting skin chronic inflammation, the most common being atopic dermatitis and psoriasis. In this paper, we discuss the biological effects of cytokine on keratinocytes, more particularly on specific or shared cytokines involved in atopic dermatitis or psoriasis. We report and discuss monolayer or 3D in vitro models of keratinocytes stimulated by specific sets of cytokines to mimic atopic dermatitis or psoriasis. IL-22, TNFa, IL-4, and IL-13 combination is able to mimic an "atopic dermatitis like" state. In psoriasis lesions, over expression of IL-17 is observed whereas IL-4 and IL-13 were not detected; the replacement of IL-4 and IL-13 by IL-17 from this mix is able to mimic in vitro a "psoriasis like" status on keratinocytes. We conclude that specific cytokine environment deregulation plays a central role on skin morphology and innate immunity, moving towards specific pathologies and opening the way to new therapeutic strategies.
Journal of Allergy 01/2012; 2012:718725.
-
Coralie Lamiche,
Jonathan Clarhaut,
Pierre-Olivier Strale,
Sophie Crespin,
Nathalie Pedretti, François-Xavier Bernard,
Christian C Naus,
Vincent C Chen,
Leonard J Foster,
Norah Defamie,
Marc Mesnil,
Françoise Debiais,
Laurent Cronier
[show abstract]
[hide abstract]
ABSTRACT: For decades, cancer was associated with gap-junction defects. However, more recently it appeared that the gap junction proteins (connexins) could be re-expressed and participate to cancer cell dissemination during the late stages of tumor progression. Since primary tumors of prostate cancer (PCa) are known to be connexin deficient, it was interesting to verify whether their bone-targeted metastatic behaviour could be influenced by the re-expression of the connexin type (connexin43) which is originally present in prostate tissue and highly expressed in bone where it participates to the differentiation of osteoblastic cells. Thus, we investigated the effect of the increased Cx43 expression, by retroviral infection, on the metastatic behaviour of two well-characterized cell lines (PC-3 and LNCaP) representing different stages of PCa progression. It appeared that Cx43 differently behaved in those cell lines and induced different phenotypes. In LNCaP, Cx43 was functional, localized at the plasma membrane and its high expression was correlated with a more aggressive phenotype both in vitro and in vivo. In particular, those Cx43-expressing LNCaP cells exhibited a high incidence of osteolytic metastases generated by bone xenografts in mice. Interestingly, LNCaP cells were also able to decrease the proliferation of cocultured osteoblastic cells. In contrast, the increased expression of Cx43 in PC-3 cells led to an unfunctional, cytoplasmic localization of the protein and was correlated with a reduction of proliferation, adhesion and invasion of the cells. In conclusion, the localization and the functionality of Cx43 may govern the ability of PCa cells to metastasize in bones.
Clinical and Experimental Metastasis 11/2011; 29(2):111-22. · 3.52 Impact Factor
-
Soufiane Ghannam,
Cécile Dejou,
Nathalie Pedretti,
Jean-Philipe Giot,
Karim Dorgham,
Hassan Boukhaddaoui,
Virginie Deleuze, François-Xavier Bernard,
Christian Jorgensen,
Hans Yssel,
Jérôme Pène
[show abstract]
[hide abstract]
ABSTRACT: CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human β-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.
The Journal of Immunology 02/2011; 186(3):1411-20. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Keratinocytes play a crucial role in the regulation of skin inflammation, responding to environmental and immune cells stimuli. They produce soluble factors that can act in an autocrine or paracrine manner on immune cells or directly on aggressors. A screening of the activities of 36 cytokines on keratinocyte gene expression identified IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha as potent cytokines in inducing cutaneous inflammation. These five proinflammatory cytokines synergistically increased production of CXCL8 and beta-defensin 2 (BD2). In addition, ex vivo studies on human skin explants demonstrated upregulation of BD2, S100A7, and CXCL8 expression in response to the same combination of cytokines. In vivo intradermal injection of these five cytokines in mouse increased CXCL1, CXCL2, CXCL3, S100A9, and BD3 expression, associated with neutrophil infiltration. We confirmed and extended this synergistic effect using quantitative real-time PCR analysis and observed increased expression of nine chemokines and 12 antimicrobial peptides. Production of CXCL, CXCL5, and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of BD2, BD3, and S100A7 was associated with an increased antimicrobial activity. Finally, the transcriptional profile observed in this in vitro model of inflammatory keratinocytes correlated with the one of lesional psoriatic skin. Our results demonstrate the important potentiating activities of IL-17A, IL-22, oncostatin M, TNF-alpha, and IL-1alpha on keratinocytes. This is particularly interesting in the context of psoriasis where these cytokines are overexpressed and could synergize to play an important role in upregulation of chemokines and antimicrobial peptides production.
The Journal of Immunology 03/2010; · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The present study investigated the lipolytic (break of fat stored) effect of a citrus-based polyphenolic dietary supplement (SINETROL) at human adipocytes (ex vivo), body fat (clinical) and biochemical levels (inhibition of phosphodiesterase). Free fatty acids (FFA) release was used as indicator of human adipocyte lipolysis and SINETROL activity has been compared with known lipolytic products (isoproterenol, theopylline and caffeine). SINETROL stimulated significantly the lipolytic activity in a range of 6 fold greater than the control. Moreover, SINETROL has 2.1 greater activity than guarana 12% caffeine while its content in caffeine is 3 times lower. Clinically, two groups of 10 volunteers with BMI relevant of overweight were compared during 4 and 12 weeks with 1.4 g/day SINETROL and placebo supplementation. In the SINETROL Group the body fat (%) decreased with a significant difference of 5.53% and 15.6% after 4 and 12 weeks, respectively, while the body weight (kg) decreased with a significant difference of 2.2 and 5.2 kg after 4 and 12 weeks, respectively. These observed effects are linked to SINETROL polyphenolic composition and its resulting synergistic activity. SINETROL is a potent inhibitor of cAMP-phosphodiesterase (PDE) (97%) compared to other purified compounds (cyanidin-3 glycoside, narangin, caffeine). These results suggest that SINETROL has a strong lipolytic effect mediated by cAMP-PDE inhibition. SINETROL may serve to prevent obesity by decreasing BMI.
Phytomedicine: international journal of phytotherapy and phytopharmacology 08/2008; 15(10):783-92. · 2.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: As revealed by immunohistochemistry and RT-QPCR, the focal adhesion protein tensin1 is expressed in cultured human dermal fibroblasts and reduced by 60% after transfection with tensin1 siRNA. Tensin1 silenced fibroblast exhibited a strongly reduced capacity to contract collagen gels. Aged fibroblasts, generated with the Hayflick replicative senescence model, exhibit as siRNA silences fibroblasts, a reduced tensin1 expression and an impaired gel contraction capacity. Based on these results, we speculate that in human dermal fibroblasts, tensin1 plays an important role in cell-matrix interaction and that a reduced expression might contribute to the dermal alterations observed during skin ageing.
Experimental Dermatology 07/2008; 17(9):788-9. · 3.54 Impact Factor
-
Katia Boniface,
Caroline Diveu,
Franck Morel,
Nathalie Pedretti,
Josy Froger,
Elisa Ravon,
Martine Garcia,
Emilie Venereau,
Laurence Preisser,
Emmanuel Guignouard,
Gérard Guillet,
Guy Dagregorio,
Jérôme Pène,
Jean-Pierre Moles,
Hans Yssel,
Sylvie Chevalier, François-Xavier Bernard,
Hugues Gascan,
Jean-Claude Lecron
[show abstract]
[hide abstract]
ABSTRACT: Cutaneous inflammatory diseases such as psoriasis vulgaris and atopic dermatitis are associated with altered keratinocyte function, as well as with a particular cytokine production profile of skin-infiltrating T lymphocytes. In this study we show that normal human epidermal keratinocytes express a functional type II oncostatin-M (OSM) receptor (OSMR) consisting of the gp130 and OSMRbeta components, but not the type I OSMR. The type II OSMR is expressed in skin lesions from both psoriatic patients and those with atopic dermatitis. Its ligand, OSM, induces via the recruitment of the STAT3 and MAP kinase pathways a gene expression profile in primary keratinocytes and in a reconstituted epidermis that is characteristic of proinflammatory and innate immune responses. Moreover, OSM is a potent stimulator of keratinocyte migration in vitro and increases the thickness of a reconstituted epidermis. OSM transcripts are enhanced in both psoriatic and atopic dermatitic skin as compared with healthy skin and mirror the enhanced production of OSM by T cells isolated from diseased lesions. Results from a microarray analysis comparing the gene-modulating effects of OSM with those of 33 different cytokines indicate that OSM is a potent keratinocyte activator similar to TNF-alpha, IL-1, IL-17, and IL-22 and that it acts in synergy with the latter cytokines in the induction of S100A7 and beta-defensin 2 expression, characteristic of psoriatic skin. Taken together, these results demonstrate that OSM and its receptor play an important role in cutaneous inflammatory responses in general and that the specific effects of OSM are associated with distinct inflammatory diseases depending on the cytokine environment.
The Journal of Immunology 05/2007; 178(7):4615-22. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cytokines are key factors in the cross talk between the immune system and other systems including hepatic, nervous, cardiac and cutaneous systems, leading to an adaptive and integrated response of the organism to stress. They are also involved in the regulation of many processes, including hematopoiesis, the immune response and inflammation. IL-10 is one of the most important anti-inflammatory cytokines. Five cytokines structurally related to IL-10 have been described and presently form this family of cytokines: IL-19, IL-20, IL-22, IL-24 and IL-26. In contrast to IL-10, these cytokines display pro-inflammatory activities in different tissues, including skin. Indeed, some of them induce an inflammatory keratinocyte gene expression profile and an epidermis histology resembling psoriatic lesions. In this review, we discuss recent knowledge about the effects of cytokines of the IL-10 family on keratinocytes and their potential role in psoriasis, a cutaneous inflammatory disease.
European cytokine network 01/2006; 16(4):309-19. · 1.73 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: IL-22 belongs to a family of cytokines structurally related to IL-10, including IL-19, IL-20, IL-24, and IL-26. In contrast to IL-10, IL-22 has proinflammatory activities. IL-22 signals through a class II cytokine receptor composed of an IL-22-binding chain, IL-22RA1, and the IL-10RB subunit, which is shared with the IL-10R. In the present study, we show that short-term cultured human epidermal keratinocytes express a functional IL-22R but no IL-10R. Accordingly, IL-22 but not IL-10 induces STAT3 activation in keratinocytes. Using a cDNA array screening approach, real-time RT-PCR, and Western blot analysis, we demonstrate that IL-22 up-regulates, in a dose-dependent manner, the expression of S100A7, S100A8, S100A9, a group of proinflammatory molecules belonging to the S100 family of calcium-binding proteins, as well as the matrix metalloproteinase 3, the platelet-derived growth factor A, and the CXCL5 chemokine. In addition, IL-22 induces keratinocyte migration in an in vitro injury model and down-regulates the expression of at least seven genes associated with keratinocyte differentiation. Finally, we show that IL-22 strongly induces hyperplasia of reconstituted human epidermis. Taken together, these results suggest that IL-22 plays an important role in skin inflammatory processes and wound healing.
The Journal of Immunology 04/2005; 174(6):3695-702. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In order to validate a model for predictive screening of dermatological drugs, we used a customized cDNA macro-array system containing 475 skin-related genes to analyze the gene expression patterns in human keratinocytes from different origins: (1) normal human epidermal keratinocyte mono-layer cultures, (2) the commercially available SkinEthic reconstituted human epidermis model, and (3) biopsies of normal human epidermis. Few markers of those that were detected significantly in keratinocyte mono-layers or in reconstituted epidermis were undetected or detected at very low level in the normal epidermis biopsies. A comparative expression of more than 100 markers could be evidenced in both normal epidermis and reconstituted epidermis samples; however, only 90% of these were detected in keratinocyte mono-layers: expression of several terminal differentiation markers, such as filaggrin, loricrin, and corneodesmosin were strongly detected in normal epidermis and reconstituted epidermis, but were not significantly expressed in keratinocyte mono-layers. Under the experimental conditions described herein, the reconstituted human epidermis model was found to significantly reproduce the gene expression profile of normal human epidermis. Using the same methodology, we then investigated the effects of all-trans retinoic acid, 9-cis retinoic acid, all-trans retinol and a commercialized tretinoin-containing cream (Retacnyl) on the gene expression profiles of reconstituted human epidermis. According to the nature and the length of the treatments, more than 40 genes were found significantly modified. Among the genes whose expression was decreased, we found cytokeratins 1, 10, 2E, and 6B, several cornified envelope precursors, integrins alpha 3, alpha 6, beta 1, beta 4, some components of desmosomes, of hemi-desmosomes and of the epidermal basement membrane. Transcriptional upregulation was observed for keratins 18 and 19, autocrine and paracrine growth factors such as HB-EGF, IGF 1, PDGF-A, calgranulins A and B, interleukin-1 alpha and the other IL-1-related markers, type II IL-1 receptor and type I IL-1-receptor antagonist. Our results confirm most of the known effects of retinoids on human epidermis, but also give new insights into their complex pharmacological activity on skin. The reconstituted human epidermis used proves to be a highly predictive model for efficacy evaluation of skin-targeted compounds, such as retinoids.
Experimental Dermatology 03/2002; 11(1):59-74. · 3.54 Impact Factor
