Personne :
Larouche, Danielle

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Département d'épidémiologie, Faculté de Médecine, Université Laval
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Voici les éléments 1 - 10 sur 38
  • Publication
    Production of a bilayered self-assembled skin substitute using a tissue-engineered acellular dermal matrix
    (Mary Ann Liebert, 2015-09-28) Beaudoin-Cloutier, Chanel; Bernard, Geneviève; Germain, Lucie; Larouche, Danielle; Auger, François A.; Gauvin, Robert; Guignard, Rina; Lacroix, Dan.; Moulin, Véronique; Lavoie, Amélie
    Our bilayered self-assembled skin substitutes (SASS) are skin substitutes showing a structure and functionality very similar to native human skin. These constructs are used, in life-threatening burn wounds, as permanent autologous grafts for the treatment of such affected patients even though their production is exacting. We thus intended to shorten their current production time to improve their clinical applicability. A self-assembled decellularized dermal matrix (DM) was used. It allowed the production of an autologous skin substitute from patient's cells. The characterization of SASS reconstructed using a decellularized dermal matrix (SASS-DM) was performed by histology, immunofluorescence, transmission electron microscopy, and uniaxial tensile analysis. Using the SASS-DM, it was possible to reduce the standard production time from about 8 to 4 and a half weeks. The structure, cell differentiation, and mechanical properties of the new skin substitutes were shown to be similar to the SASS. The decellularization process had no influence on the final microstructure and mechanical properties of the DM. This model, by enabling the production of a skin substitute in a shorter time frame without compromising its intrinsic tissue properties, represents a promising addition to the currently available burn and wound treatments.
  • Publication
    Accès libre
    Expression of C4.4A in an in vitro human tissue-engineered skin model
    (Hindawi, 2017-09-07) Germain, Lucie; Larouche, Danielle; Rochette-Drouin, Olivier; Ploug, Michael; Jacobsen, Benedikte
    A multi-LU-domain-containing protein denoted C4.4A exhibits a tightly regulated membrane-associated expression in the suprabasal layers of stratified squamous epithelia such as skin and the esophagus, and the expression of C4.4A is dysregulated in various pathological conditions. However, the biological function of C4.4A remains unknown. To enable further studies, we evaluated the expression of C4.4A in monolayer cultures of normal human keratinocytes and in tissue-engineered skin substitutes (TESs) produced by the self-assembly approach, which allow the formation of a fully differentiated epidermis tissue. Results showed that, in monolayer, C4.4A was highly expressed in the centre of keratinocyte colonies at cell-cell contacts areas, while some cells located at the periphery presented little C4.4A expression. In TES, emergence of C4.4A expression coincided with the formation of the stratum spinosum. After the creation of a wound within the TES, C4.4A expression was observed in the suprabasal keratinocytes of the migrating epithelium, with the exception of the foremost leading keratinocytes, which were negative for C4.4A. Our results are consistent with previous data in mouse embryogenesis and wound healing. Based on these findings, we conclude that this human TES model provides an excellent surrogate for studies of C4.4A and Haldisin expressions in human stratified epithelia.
  • Publication
    Stem cells of the skin and cornea : their clinical applications in regenerative medicine
    (Rapid Science Publishers, 2011-02-01) Germain, Lucie; Larouche, Danielle; Gauvin, Robert; Proulx, Stéphanie; Fradette, Julie
    Purpose of review: The use of stem cells is of great interest for the treatment of various pathologies and ultimately for the restoration of organ function. Progress pointing towards future treatments of skin and corneal epithelial stem cell defects are reviewed, including the transplantation of living tissue-engineered substitutes. Recent findings: This article focuses on substitutes optimized for permanent replacement of skin and cornea. New skin substitutes for burn care are currently under development. More complex tissue-engineered skin substitutes in which stroma, adipose tissue, capillaries, and neurons are combined with the epithelium are being developed. Some dermal/epidermal substitutes have been applied to the treatment of patients. Cultured corneal epithelial cells have been characterized and more complete corneal substitutes are being designed. Long-term clinical results on the transplantation of cultured corneal stem cells for the treatment of limbal stem cell deficiency have been reported. Summary: Advances in tissue engineering for the development of substitutes that will benefit patients suffering from skin or corneal stem cell deficiencies are reviewed. These products are often a combination of cells, scaffolds and other factors. Key considerations in the development of corneal and skin substitutes for clinical applications are discussed.
  • Publication
    Vibrissa hair bulge houses two populations of skin epithelial stem cells distinct by their keratin profile
    (Federation of American Societies for Experimental Biology, 2007-12-27) Germain, Lucie; Tong, Xuemei; Larouche, Danielle; Fradette, Julie; Coulombe, Pierre A.
    Defining the properties of postnatal stem cells is of interest given their relevance for tissue homeostasis and therapeutic applications, such as skin tissue engineering for burn patients. In hair follicles, the bulge region of the outer root sheath houses stem cells. We show that explants from the prominent bulge area, but not the bulb, in rodent vibrissa follicles can produce epidermis in a skin model of tissue engineering. Using morphological criteria and keratin expression, we typified epithelial stem cells of vibrissa bulge. Two types of slow-cycling cells (Bb, Bs1) featuring a high colony-forming capacity occur in the bulge. Bb cells are located in the outermost basal layer, express K5, K15, K17, and K19, and feature a loosely organized keratin network. Bs1 cells localize to the suprabasal layers proximal to Bb cells and express K5/K17, corre lating with a network of densely bundled filaments. These prominent bundles are missing in K17-null mice, which lack vibrissa. Atypically, both the Bb and Bs1 keratinocytes lack K14 expression. These findings show heterogeneity within the hair follicle stem cell reposi tory, establish that a subset of slow-cycling cells are suprabasal in location, and point to a special role for K5/K17 filaments in a newly defined subset of stem cells. Our results are discussed in the context of long-term survival of engineered tissues after grafting that requires the presence of stem cells.
  • Publication
    Considerations in the choice of a skin donor site for harvesting keratinocytes containing a high proportion of stem cells for culture in vitro
    (Butterworth-Heinemann, 2010-12-03) Germain, Lucie; Larouche, Danielle; Paquet, Claudie; Fugère, Claudia.; Genest, Hervé; Auger, François A.; Gauvin, Robert; Têtu, Félix-Andre; Bouchard, Maurice; Roy, Aphonse; Fradette, Julie; Lavoie, Amélie; Beauparlant, Annie.
    The treatment of severely burned patients has benefited from the grafting of skin substitutes obtained by expansion of epithelial cells in culture. The aim of this study was to evaluate whether the anatomic site chosen for harvesting skin had an impact on the quality of the derived cell cultures. Considering that hair follicles contain epithelial stem cells, we compared hairy skin sites featuring different densities and sizes of hair follicles for their capacity to generate high quality keratinocyte cultures. Three anatomic sites from adult subjects were compared: scalp, chest skin and p-auricular (comprising pre-auricular and post-auricular) skin. Keratin (K) 19 was used as a marker to evaluate the proportion of stem cells. Keratinocytes were isolated using the two-step thermolysin and trypsin cell extraction method, and cultured in vitro. The proportion of K19-positive cells harvested from p-auricular skin was about twice that of the scalp. This K19-positive cell content also remained higher during the first subcultures. In contrast to these in vitro results, the number of K19-positive cells estimated in situ on skin sections was about double in scalp as in p-auricular skin. Chest skin had the lowest number of K19-positive cells. These results indicate that in addition to the choice of an adult anatomic site featuring a high number of stem cells in situ, the quality of the cultures greatly depends on the ability to extract stem cells from the skin biopsy
  • Publication
    Accès libre
    Évaluation de la relation entre les apports en antioxydants et le niveau d'expression de marqueurs inflammatoires dans le tissu mammaire normal de femmes atteintes du cancer du sein
    (2017) Larouche, Danielle; Diorio, Caroline
    Le régime alimentaire joue un rôle dans le développement du cancer du sein, mais le mode d’action des facteurs nutritionnels sur le tissu mammaire est mal compris. Un des mécanismes potentiels est la création d’un stress oxydatif qui favoriserait le processus tumoral et l’inflammation. Par conséquent, la consommation d’antioxydants pourrait contribuer à réduire l’inflammation dans les tissus et à prévenir le cancer du sein. Cependant, peu d’études ont exploré la relation entre les apports en antioxydants et l’expression de marqueurs inflammatoires dans le tissu mammaire. Ce projet visait à évaluer la relation entre les apports en antioxydants et l’expression de 11 marqueurs inflammatoires dans le tissu mammaire normal de 160 femmes atteintes d’un cancer du sein. Les données alimentaires ont été obtenues par un questionnaire de fréquence alimentaire auto-administré mesurant les apports alimentaires et la prise de suppléments de l’année précédente. L’expression des marqueurs inflammatoires a été évaluée par immunohistochimie. La corrélation entre les apports en antioxydants et l’expression des marqueurs inflammatoires a été analysée par le coefficient de corrélation partiel de Spearman. Les analyses ont été effectuées pour l’ensemble de l’échantillon et pour les femmes pré-ménopausées et post-ménopausées prises séparément. Après la correction de Bonferroni, les apports élevés en bêta-tocophérol corrélaient avec une diminution de l’expression de l’IL-10 pour l’ensemble de l’échantillon (r=-0,26) et chez les femmes post-ménopausées (r=-0,39). Parmi toutes les femmes, les apports en zinc corrélaient négativement avec l’expression de l’IL-10 (r=-0,26) et parmi les femmes post-ménopausées, les apports en sélénium corrélaient négativement avec l’expression de la lactoferrine (r=-0,39). Aucune association significative n’a été observée chez les femmes pré-ménopausées. Nos résultats suggèrent que le bêta-tocophérol, le zinc et le sélénium pourraient agir sur le tissu mammaire par des mécanismes affectant l’expression de certains marqueurs inflammatoires et que ceci serait influencé par le statut ménopausique.
  • Publication
    Characterization of a 150 kDa accessory receptor for TGF-beta 1 on keratinocytes : direct evidence for a GPI anchor and ligand binding of the released form.
    (Wiley, 2001-09-05) Tam, Betty Y. Y.; Germain, Lucie; Larouche, Danielle; Hooper, N.M; Philip, Anie
    Fibroblasts play a critical role in wound repair and in the development of fibrotic diseases, and transforming growth factor-β (TGF-β) has been shown to profoundly modulate fibroblast function. However, there is limited information on the TGF-β receptor types, isoform specificity, and complex formation in skin fibroblasts. Here, we report that normal adult human skin fibroblasts display two isoform-specific, cell surface glycosyl phosphatidylinositol (GPI)-anchored, TGF-β binding proteins in addition to the type I, II, and III TGF-β receptors. The identities of these proteins are confirmed on the basis of their affinity for TGF-β isoforms, immunoprecipitation with specific antireceptor antibodies, and other biochemical analyses. Immunoprecipitation results also indicated oligomeric complex formation between type I and II and between type II and III TGF-β receptors. Furthermore, by using affinity labeling and two-dimensional electrophoresis, we demonstrate the occurrence of type I and II heterodimers and type I homodimers of TGF-β receptors on these cells. Because the type I receptor does not bind TGF-β in the absence of type II receptor, these results indicate that one molecule of TGF-β induces the formation of a heterooligomeric complex containing more than one molecule each of type I and II TGF-β receptors on these cells. These cells respond to TGF-β by markedly down-regulating all five binding proteins and by potently augmenting DNA synthesis. These results allow the expansion of the proposed heteromeric TGF-β receptor signaling paradigm using mutantcells that are unresponsive to TGF-β and cell lines that have been transfected to overexpress these receptors, to include normal TGF-β-responsive cells. In addition, the definition of TGF-β receptor profiles in human skin fibroblasts provides important information for studying their alterations in these cells in various skin diseases.
  • Publication
    A role for DLK in microtubule reorganization to the cell periphery and in the maintenance of desmosomal and tight junction integrity
    (Elsevier Science, 2016-10-07) Bidoggia, Julie; Guérin, Sylvain; Germain, Lucie; Larouche, Danielle; Blouin, Richard; Hirai, Syu-Ichi; Simard-Bisson, Carolyne
    Dual leucine zipper-bearing kinase (DLK) is an inducer of keratinocyte differentiation, a complex process also involving microtubule reorganization to the cell periphery. However, signaling mechanisms involved in this process remain to be elucidated. Here, we demonstrate that DLK enhances and is required for microtubule reorganization to the cell periphery in human cell culture models and in Dlk knockout mouse embryos. In tissue-engineered skins with reduced DLK expression, cortical distribution of two microtubule regulators, LIS1 and HSP27, is impaired as well as desmosomal and tight junction integrity. Altered cortical distribution of desmosomal and tight junction proteins was also confirmed in Dlk knockout mouse embryos. Finally, desmosomal and tight junction defects were also observed after microtubule disruption in nocodazole-treated tissue-engineered skins, thus confirming a role for microtubules in the maintenance of these types of cell junctions. Globally, this study demonstrates that DLK is a key regulator of microtubule reorganization to the cell periphery during keratinocyte differentiation and that this process is required for the maintenance of desmosomal and tight junction integrity.
  • Publication
    Normal human merkel cells are present in epidermal cell populations isolated and cultured from glabrous and hairy skin sites
    (Elsevier Science, 2015-12-08) Caouette, Louise; Germain, Lucie; Larouche, Danielle; Couture, Véronique; Fugère, Claudia.; Guignard, Rina; Fradette, Julie; Roy, Alphonse; Beauparlant, Annie.
    The Merkel cell is a highly specialized cell that primarily acts as a slowly adapting mechanoreceptor. Merkel cells are scarce in normal skin but can be identified by the expression of distinct keratin filaments. Merkel cells constitute a very unique population and many questions still remain as to their origin, number, proliferative capacity, and functions in cutaneous biology. The dissociation of epidermal cells from skin is a widely used technique to extract and culture keratinocytes. We took advantage of a two-step extraction method to quantify keratin-20-expressing Merkel cells among total cutaneous cells obtained from either hairy or glabrous skin biopsies. Flow cytometry analysis revealed that keratin-20-labeled Merkel cells represent between 3.6% and 5.7% of freshly dissociated basal epidermal cells. No significant differences were seen between samples derived from glabrous palmar and hairy anatomic sites, from children and adult, respectively. We also report on the presence of Merkel cells in primary and first subcultures of epidermal cells indicating their capacity to remain viable after extraction from skin of various anatomic sites. To our knowledge, this is the first demonstration of nontumorigenic human Merkel cells in culture in vitro. The persistence of a small number of Merkel cells in culture suggests that, with the development of appropriate culture conditions, these cells could be amplified and further studied to unravel long-standing questions relative to their paracrine function or epithelial origin.
  • Publication
    A computer-controlled apparatus for the characterization of mechanical and viscoelastic properties of tissue-engineered vascular constructs
    (Springer, 2011-01-25) Germain, Lucie; Larouche, Danielle; Auger, François A.; Gauvin, Robert; Lévesque, Philippe
    Tissue-engineered blood vessels can be partly characterized by analyzing their mechanical properties using burst pressure testing, compliance measurement, creep and cyclic testing. Studying these parameters provides information on the capability of a fabrication method to produce tissue-engineered blood vessels (TEBV) and allow for the optimization of their resistance and viscoelastic properties. This study presents the design and fabrication of an apparatus allowing accurate and reliable measurements of the mechanical properties of tissue-engineered vascular constructs. A computer-controlled system was designed to monitor pressure and diameter variations of vascular constructs submitted to hydrostatic loading. The system was programmed to control the motorized portion of the setup and allow simultaneous data acquisition, analysis and real-time display. Data acquisition cards allow for synchronous monitoring of pressure and diameter of the constructs through a pressure transducer and a CCD camera. Image analysis and pressure data computation resulted in compliance, creep and dynamic characterization of the tested tissues. This experimental setup succeeded in measuring the burst pressure, compliance, creep and cyclic behavior of tissue-engineered vascular media (TEVM), adventitia (TEVA) and a combination of a media and an adventitia (TEVMA) reconstructed by the self-assembly method. Our apparatus has proven to be a precise and reliable tool for the characterization of the mechanical properties of vascular constructs.