Biomedical subjects

B Le Varlet

Publications and source records attributed to B Le Varlet.

11 recordsLinked to original sources

PubMed2006

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.

Animals↗

PubMed2004

Determination of sunscreen immune protection factors using human dendritic cell suspensions.

In a previous study, we have used UVB-irradiated human skin explants and the allostimulatory function of Langerhans cells (LC) to determine immune protection factors (IPF) for sunscreens. We sought here to simplify the model by using either human enriched LC suspensions or in vitro generated dendritic cells from human monocytes (MoDC). LC or MoDC suspensions were irradiated with increasing doses of UVB through a piece of translucent strip recovered or not with the sunscreens. The allostimulatory function of the cells was then analysed in a mixed lymphocyte reaction and the UVB dose providing 50% immunosuppression (D50%) was determined graphically. IPF were determined by the ratio of the D50% value in the presence of sunscreen to that of the vehicle alone. In either experimental conditions, the D50% in the presence of sunscreens was significantly higher (p < 0.01) than that obtained with the vehicle, demonstrating the sunscreen immunoprotective effect. IPF values obtained with either DC suspensions were very similar and quite comparable to those previously obtained in the skin explant model. Thus, the present in vitro model provides easy tools to determine a new important biological parameter for sunscreens, i.e. immune protection.

Cell Separation↗

PubMed2003

Decreased expression of keratinocyte beta1 integrins in chronically sun-exposed skin in vivo.

BACKGROUND: Chronic exposure to ultraviolet (UV) radiation induces changes in the skin structure which are mostly found in the superficial dermis and at the dermal-epidermal junction. Keratinocytes and fibroblasts contribute both to the synthesis and to the degradation of the molecules important for the integrity of this skin site. While several studies have reported on alterations of dermal components and of the functions of fibroblasts in vivo and in vitro after UV exposure, recent data suggested that keratinocytes could be the main skin cell type involved in the photoageing process. OBJECTIVES: In this study, we analysed the expression of two keratinocyte molecules namely, beta1 integrin (a proliferation marker) and involucrin (a differentiation marker) in sun-exposed and sun-protected facial skin of 16 healthy patients undergoing facial lifting. METHODS: Methods included histology, immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analysis. RESULTS: Sun-exposed skin displayed the characteristic morphological and molecular features of dermal photoageing, compared with sun-protected skin, including dermal elastosis, diminished fibrillin and type VII collagen expression. Analysis of the epidermis in sun-exposed vs. sun-protected skin showed no histological differences, but dramatic changes in the expression of beta1 integrin and involucrin. In sun-exposed skin, expression of beta1 integrin protein by epidermal basal cells was reduced, paralleling a downregulation of beta1 integrin mRNA, whereas involucrin protein expression was greatly enhanced in the superficial epidermal cell layers. Interestingly, the ratio between involucrin and beta1 integrin protein expression was consistently increased in sun-exposed skin sites. CONCLUSIONS: Collectively these results demonstrate that epidermal homeostasis is impaired by chronic UV exposure, and define beta1 integrin expression as a molecular marker of the epidermal photoageing process.

Aged↗

PubMed2003

Photoageing shows histological features of chronic skin inflammation without clinical and molecular abnormalities.

BACKGROUND: Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis. Mast cells and macrophages, which are found in higher numbers in photoaged skin, have been implicated in this process. OBJECTIVES: To analyse the phenotype of haematopoietic-derived infiltrating cells in photodamaged skin. METHODS: Chronically sun-exposed (preauricular) and control sun-protected (postauricular) skin was recovered from eight healthy subjects undergoing cosmetic surgery (facial lifting). RESULTS: Histological analysis showed that sun-exposed skin harboured more infiltrating mononuclear cells than sun-protected skin. Cellular infiltrates were found at the periphery of areas of elastolysis around hair follicles in sun-exposed sites, whereas they were found in the interfollicular dermis around blood vessels and around hair follicles in sun-protected samples. Immunohistochemical analysis revealed an increased number of mast cells, macrophages and CD4+ CD45RO+ T cells in sun-exposed dermis as well as a higher number of CD1a+ dendritic cells in sun-exposed epidermis, compared with the sun-protected samples. Thus photoageing displays histological features of chronic skin inflammation. However, no molecular sign of inflammation was observed and we even found a decreased expression of interleukin-1beta mRNA in sun-exposed compared with sun-protected sites. Furthermore, the patients' skin looked normal and did not display any clinical inflammation. CONCLUSIONS: Collectively, these data show that chronic ultraviolet irradiation induces alterations of innate immune cells which are recruited in sun-exposed skin without being activated.

Aged↗

PubMed1998

Age-related functional and structural changes in human dermo-epidermal junction components.

Cultured normal human keratinocytes obtained from 14 facial skin biopsies of donors aged 9-79 y were used to study the influence of donor age on the integrin receptors, cell adhesive properties in vitro, and type VII collagen synthesis. Immuno-spectrofluorimetric quantitation of integrins showed a decrease in the beta1- and beta4-subunits in low (0.08 mM) and high (1.8 mM) calcium conditions with aging. Calcium ions decreased the fluorescence intensity by relocating integrins at cell boundaries. Measurements of adhering cells showed that adhesion to bovine serum albumin-, type IV collagen- or laminin 1-coated plastic surfaces initially increased until donor age reached 30 y and then decreased. Specific adhesion to type IV collagen and laminin 1 did not vary with age, but the increase in adhesion to type IV collagen produced by manganese ions increased with age, suggesting an age-dependent feature of beta1 integrin. Synthesis of type VII collagen, increased or not by TGFbeta1 (10 ng per ml), did not vary with the donor age. Global normalized principal component analysis showed that variables related to integrins were strongly correlated, as were those of adhesion. Pre-embedding immunoelectron microscopy of freshly isolated keratinocytes showed that certain hemidesmosomes from aged cells had little or no reaction with anti-beta4-chain antibody. Post-embedding type IV collagen immunostaining and image analysis showed less type IV collagen in adult dermo-epidermal junctions. These findings indicate that there are structural and functional changes in the dermo-epidermal junction components with aging, probably giving a less effective epidermal anchoring system.

Adolescent↗

PubMed1998

Effect of cytokines and nitric oxide on tight junctions in cultured rat retinal pigment epithelium.

PURPOSE: These experiments were designed to study the effect of cytokines and nitric oxide (NO) on rat retinal pigment epithelial (RPE) cell tight junctions in vitro. METHODS: Cultures of confluent RPE cells from retinas of PVG rats (a strain susceptible to development of experimental uveitis) were prepared on filters and incubated with various stimulants. The function of the tight junction was evaluated by measuring the transepithelial electrical resistance (TER) of the cell monolayer and the passive permeation of [3H]inulin across confluent RPE cells. The morphology of the intercellular junctions was visualized by immunolocalization of the tight junction-associated protein zonula occludens-1 (ZO-1) and F-actin. RESULTS: Seventy-two hours after plating, the RPE cell monolayer showed a mean TER level of 67.6+/-18.8 omega/cm2. A decrease in TER was observed after treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). The addition of tumor necrosis factor-alpha (TNF-alpha) accelerated the decrease of TER, whereas NG-monomethyl-L-arginine (L-NMMA) (an NO synthase [NOS] inhibitor) did not further modify the resistance decrease. In contrast, 3-morpholino-sydnonimine (SIN-1), a sydnonimine analog and NO donor, increased the TER. The variations of TER were correlated with the transepithelial fluxes of [3H]inulin and with tight junction morphologic changes of ZO-1 and F-actin immunostaining. CONCLUSIONS: Incubation with LPS associated with IFN-gamma and TNF-alpha induces alterations of RPE tight junctions, whereas NO is involved in the maintenance of their integrity. Cytokines and NO production could play a role in regulation of the blood-ocular barrier function and of the development of ocular inflammation.

Actins↗

PubMed1995

Dibutyryl cyclic adenosine monophosphate and forskolin alter the paracellular pathway in cultured corneal endothelial cells.

PURPOSE: This study describes the effects of the cyclic adenosine monophosphate (cAMP) pathway on the tight junctional barrier of the corneal endothelium, which plays a critical role in maintaining the corneal stroma in an underhydrated, transparent state. METHODS: Subcultured bovine corneal endothelial cells grown on filters were used to study the effects of dibutyryl-cAMP and forskolin on transendothelial electrical resistance and [3H]inulin flux. The tight junction-associated protein ZO-1 (zonula occludens protein-1) and F-actin were visualized by indirect immunofluorescence, and the ultrastructural organization of junctional complexes was studied by freeze-fracture electron microscopy. RESULTS: Cells formed a continuous monolayer of closely apposed hexagonal-type cells separated by a discontinuous belt of tight junctions with a transendothelial electrical resistance of 20.8 +/- 0.6 omega.cm2. Dibutyryl-cAMP (10(-4) M) and forskolin (10(-5) M) increased cell cAMP, significantly decreased the transendothelial resistance by 54% and 43%, respectively, and increased the flux of [3H]inulin from the apical to the basal side of the cells by 56% and 40%, respectively. Both agents also induced condensation of F-actin at the cell borders without any marked changes in the immunostaining of ZO-1 that delineated cell peripheries. However, freeze-fracture studies showed that dibutyryl-cAMP and forskolin induced dispersion of the tight junction network. CONCLUSIONS: These data suggest that activation of the cAMP-dependent pathway, leading to structural changes of the tight junctional network, may modulate the passive fluxes mediated by the paracellular pathway of the corneal endothelial barrier.

Actins↗

PubMed1992

In vitro adhesion of human epidermal Langerhans cells to laminin and fibronectin occurs through beta 1 integrin receptors.

Human epidermal Langerhans cells are dendritic cells that can capture, process, and present antigens to T cells. It was previously shown that these Langerhans cells express the very late activation antigen (VLA) protein family of beta 1 integrins. beta 1 integrins mainly mediate the adhesion of cells to a number of extracellular components, such as laminin, fibronectin, and collagen, which are present in the skin. In this report, we demonstrate that a large percentage of the Langerhans cell population was able in vitro to attach to laminin and fibronectin but not to collagen. An ultrastructural study of adherent Langerhans cells showed that they were spread largely on laminin, with a loss of their round shape, and partially on fibronectin. Langerhans cell binding to laminin or fibronectin induced a decrease of the Birbeck granule number. Specific inhibitions of cell adhesion were performed, and it was demonstrated that VLA-6 was the major receptor involved in Langerhans cell adhesion to laminin. This adhesion was not RGD dependent and was slightly enhanced by Mn2+. VLA-3 and especially VLA-5 mediated Langerhans cell binding to fibronectin through the RGDS sequence of the protein. Mn2+ sharply increased the Langerhans cell adhesion to fibronectin. VLA-6 mediated in vitro Langerhans cell adhesion to laminin, which suggests that in vivo VLA-6 could permit Langerhans cells to attach and migrate through the basement membrane. Moreover, VLA-5 and VLA-3 take part in the in vitro Langerhans cell binding to fibronectin, suggesting that in vivo these fibronectin receptors could facilitate Langerhans cell passage throughout the fibronectin network of the dermis before migration to lymph nodes.

Amino Acid Sequence↗

PubMed1991

Expression and endocytosis of integrin VLA receptors for collagen, fibronectin and laminin by normal human keratinocytes.

The Very Late Activation (VLA) antigen family is involved in cell-extracellular matrix interactions and consists of six heterodimeric cell surface receptors with a common beta 1 and a variable alpha subunit. Using a panel of specific antibodies, we showed that human epidermal basal cells expressed VLA-2, VLA-3 and VLA-6 but failed to express VLA-4. Their functional roles were investigated and VLA-2 appeared as a specific receptor for type IV collagen and also as a laminin receptor. VLA-3 appeared as a receptor for fibronectin and laminin and to a lesser extent as a type I collagen receptor. VLA-6 appeared as a specific receptor for laminin. It also appeared that the VLA-alpha subunit specifically mediates the recognition of ligand but the beta 1 subunit is also involved in adhesion and that both subunits have a synergistic influence. Immunoprecipitation analyses confirmed that VLA-2, VLA-3 and VLA-6 were expressed by basal keratinocytes. Endocytosis of VLA-2 and VLA-3 was observed involving coated vesicles and endosomes that are structures characteristic of a receptor-mediated pathway. These findings provide first evidence that normal human basal keratinocytes are able of endocytosis mediated by receptors. Taken together, these results indicate that multiple VLA receptors function in combination to mediate epidermal basal cell adhesion to extracellular matrix.

Antibodies, Monoclonal↗

PubMed1991

Human epidermal Langerhans cells express integrins of the beta 1 subfamily.

The expression of VLA integrins on human epidermal Langerhans cells (LC) was investigated by indirect immunogold labeling on transmission electron microscopy, followed by a quantitative analysis. Labelings on suspensions enriched in freshly isolated LC were carried out with an antibody recognizing the beta 1 subunit, common to all members of the VLA family, and with antibodies specific for the six different alpha subunits, alpha 1 to alpha 6. Normal human epidermal LC were all beta 1 positive, of which 60% were highly positive. By labelings with different VLA-alpha-chain MoAb, there appeared two subpopulations of LC: one positive and one negative. On average 40% of LC bore small amounts of VLA-1 and VLA-3, 53% and 77% of LC expressed moderate amounts of VLA-2 and VLA-5, respectively, and VLA-4 and VLA-6 were expressed by 67% and 90% of LC, respectively. VLA proteins are mainly extracellular matrix protein receptors. VLA-6 (laminin receptor) and VLA-5 (fibronectin receptor) are expressed mainly by LC, and in this way could subserve LC to leave the epidermal compartment through the basement membrane, to migrate throughout the fibronectin network of the dermis before migrating via the afferent lymphatics to the regional lymph nodes, where they present antigen to T cells. VLA proteins such as VLA-4, VLA-3, or VLA-2, which are found involved in cell-cell contacts, could contribute to the promotion of T-cell activation by facilitating adherence between LC and T cells.

Humans↗

PubMedDate not supplied

Study of housekeeping gene expression in human keratinocytes using OLISA, a long-oligonucleotide microarray and q RT-PCR.

In recent years, applications of microarray platforms have been extended to different areas of research including cosmetic and pharmaceutical. Although microarray technology is still improving its sensitivity and flexibility, researchers often turn toward quantitative RT-PCR for data validation. Assessment of messenger RNA quantity by these methods is based on comparison with internal standard genes, mainly housekeeping genes, so called because their synthesis occurs normally at a constant level. However, numerous studies showed that expression of these genes could vary in given situations. Here, we report results on four housekeeping genes (GAPDH, beta-2 microglobulin, S40 and S26 ribosomal sub-units) with constant expression levels established on OLISA microarray using different keratinocyte cultures. Moreover, qRT-PCR validation demonstrates that S26 ribosomal is a good housekeeping gene on keratinocytes and skin studies. Our data indicate that S26 gene can be routinely used to standardize results to investigate differentially expressed genes in a healthy human skin.

Cells, Cultured↗