Search

International audience; The use of dental stem cells has raised many hopes in the development of new treatments for neurodegenerative diseases. According to current statistics, about 1 in 6 people in the world would be affected by a neurological disease. This number continues to increase as the world's population ages, making neurodegenerative diseases probably the one of the major challenges of public health in the 21st century. Neurodegenerative diseases are characterized mainly by a progressive loss of cognitive abilities and patient autonomy related to loss and degeneration of neurons in brain structures. Unfortunately, today, the only treatments available for this type of disease do only relieve the symptoms, they do not treat them, and few clinical trials have been truly convincing to date. Hence, hope lies for these diseases in the development of other therapeutic approaches. As such, dental stem cells could be a promising area of research because of their rapid growth, their great capacity for differentiation into different types of cells (among neuronal ones for some of them) and how easy they can be obtained, without raising ethical issues as for example for embryonic stem cells.; L’utilisation des cellules souches dentaires a fait naître de nombreux espoirs dans le développement de nouveaux traitements destinés aux maladies neurodégénératives. Si l’on se réfère aux statistiques actuelles, environ 1 personne sur 6 dans le monde serait atteinte par une maladie neurologique. Ce nombre ne cesse d’augmenter au fur et à mesure que la population mondiale vieillit, faisant des maladies neurodégénératives probablement l’un des principaux défis de la santé publique du XXIe siècle. Les maladies neurodégénératives se caractérisent principalement par une perte progressive des facultés cognitives et de l’autonomie des patients liée à une perte et une dégénérescence des neurones des structures cérébrales. Malheureusement, force est de constater que les seuls traitements disponibles actuellement pour ce type de pathologies ne font que soulager les symptômes et non les traiter et que peu d’essais cliniques à ce jour ont été véritablement probants. L’espoir réside donc pour ces maladies neurodégénératives dans le développement de nouvelles approches thérapeutiques. Les cellules souches dentaires pourraient constituer une nouvelle voie de recherche en thérapie cellulaire, de par leur croissance rapide, leur grande capacité de différenciation en différents types cellulaires (y compris en cellules neuronales pour certaines) et la facilité avec laquelle elles peuvent être obtenues sans soulever de problèmes d’éthique comme par exemple pour les cellules souches embryonnaires.

The use of dental stem cells has raised many hopes in the development of new treatments for neurodegenerative diseases. According to current statistics, about 1 in 6 people in the world would be affected by a neurological disease. This number continues to increase as the world's population ages, making neurodegenerative diseases probably the one of the major challenges of public health in the 21st century. Neurodegenerative diseases are characterized mainly by a progressive loss of cognitive abilities and patient autonomy related to loss and degeneration of neurons in brain structures. Unfortunately, today, the only treatments available for this type of disease do only relieve the symptoms, they do not treat them, and few clinical trials have been truly convincing to date. Hence, hope lies for these diseases in the development of other therapeutic approaches. As such, dental stem cells could be a promising area of research because of their rapid growth, their great capacity for differentiation into different types of cells (among neuronal ones for some of them) and how easy they can be obtained, without raising ethical issues as for example for embryonic stem cells., L’utilisation des cellules souches dentaires a fait naître de nombreux espoirs dans le développement de nouveaux traitements destinés aux maladies neurodégénératives. Si l’on se réfère aux statistiques actuelles, environ 1 personne sur 6 dans le monde serait atteinte par une maladie neurologique. Ce nombre ne cesse d’augmenter au fur et à mesure que la population mondiale vieillit, faisant des maladies neurodégénératives probablement l’un des principaux défis de la santé publique du XXIe siècle. Les maladies neurodégénératives se caractérisent principalement par une perte progressive des facultés cognitives et de l’autonomie des patients liée à une perte et une dégénérescence des neurones des structures cérébrales. Malheureusement, force est de constater que les seuls traitements disponibles actuellement pour ce type de pathologies ne font que soulager les symptômes et non les traiter et que peu d’essais cliniques à ce jour ont été véritablement probants. L’espoir réside donc pour ces maladies neurodégénératives dans le développement de nouvelles approches thérapeutiques. Les cellules souches dentaires pourraient constituer une nouvelle voie de recherche en thérapie cellulaire, de par leur croissance rapide, leur grande capacité de différenciation en différents types cellulaires (y compris en cellules neuronales pour certaines) et la facilité avec laquelle elles peuvent être obtenues sans soulever de problèmes d’éthique comme par exemple pour les cellules souches embryonnaires.

International audience; Most of the skin barrier function is attributable to the outermost layer of the epidermis, the stratum corneum, which is composed of flattened, anucleated cells called corneocytes surrounded by a lipid-enriched lamellar matrix. The composition of the stratum corneum is directly dependent on the underlying granular keratinocytes, which are the last living cells in the stratified epidermis. Many components present in the intercorneocyte matrix are delivered by the underlying granular keratinocytes through a secretion process dependent on lysosome-related organelles called lamellar bodies. Because of the importance of lamellar bodies in the maintenance of the epidermal barrier, the mechanisms regulating their biogenesis must be better understood. In this study, we show that the Rab11a GTPase is highly expressed in terminally differentiated keratinocytes, where it is partly associated with lamellar bodies. Rab11a silencing in three-dimensional in vitro reconstructed human epidermis induces a barrier defect, a decrease in the amount of lipid found in the stratum corneum, a reduction in lamellar body density and secretion areas in granular keratinocytes, and the mis-sorting of lamellar body cargoes being driven to the lysosomal degradation pathway. Our results highlight the importance of Rab11a-dependent regulation of lamellar body biogenesis in keratinocytes and consequently on epidermal barrier homeostasis.

International audience; Lysophosphatidic acid (LPA) is a pro-fibrotic mediator acting via specific receptors (LPARs) and is synthesized by autotaxin, that increases with obesity. We tested whether LPA could play a role in adipose tissue (AT)-fibrosis associated with obesity. Fibrosis [type I, III, and IV collagens (COL), fibronectin (FN), TGFβ, CTGF and αSMA] and inflammation (MCP1 and F4/80) markers were quantified: (i) in vivo in inguinal (IAT) and perigonadic (PGAT) AT from obese-diabetic db/db mice treated with the LPAR antagonist Ki16425 (5mg/kg/day ip for 7 weeks); and (ii) in vitro in human AT explants in primary culture for 72h in the presence of oleoyl-LPA (10μM) and/or Ki16425 (10μM) and/or the HIF-1α inhibitor YC-1 (100μM). Treatment of db/db mice with Ki16425 reduced Col I and IV mRNAs in IAT and PGAT while Col III mRNAs were only reduced in IAT. This was associated with reduction of COL protein staining in both IAT and PGAT. AT explants showed a spontaneous and time-dependent increase in ATX expression and production of LPA in the culture medium, along with increased levels of Col I and III, TGFβ and αSMA mRNAs and of COL protein staining. In vitro fibrosis was blocked by Ki16425 and was further amplified by oleoyl-LPA. LPA-dependent in vitro fibrosis was blocked by co-treatment with YC1. Our results show that endogenous and exogenous LPA exert a pro-fibrotic activity in AT in vivo and in vitro. This activity could be mediated by an LPA1R-dependent pathway and could involve HIF-1α.

International audience; BACKGROUND: Phthalic acid esters are widely used in the manufacture of plastics. Numerous studies have shown that these phthalates impair testicular testosterone production in the rat. However, the scarce and contradictory data concerning humans have cast doubt over whether these compounds are also anti-androgenic in man. We therefore investigated the direct effects of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on organo-cultured adult human testis and a human cell line. METHODS: Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with DEHP or MEHP. The effects of ketoconazole, used as a reference molecule, were also assessed. RESULTS: In both models, DEHP and MEHP significantly inhibited testosterone production. The effects of both phthalates appeared to be specific for steroidogenesis, as INSL3 production by Leydig cells was not altered. Furthermore, the phthalates of interest had no effect on inhibin B production by Sertoli cells or on germ cell apoptosis. As only a small fraction of the phthalates added was found in the testis explants, and as these compounds were found to be metabolized, we estimate that the anti-androgenic effects observed occurred at concentrations of phthalates that are of the same order of magnitude as exposures reported in the literature for men. CONCLUSIONS: We provide the first evidence that DEHP and MEHP can inhibit testosterone production in the adult human testis. This is consistent with recent epidemiological findings of an inverse correlation between exposure to MEHP and testosterone concentrations.

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.

International audience; Activation of glucocorticoid receptors (GR) by glucocorticoid hormones (GC) enhances contextual fear memories through the activation of the Erk1/2(MAPK) signaling pathway. However, the molecular mechanism mediating this effect of GC remains unknown. Here we used complementary molecular and behavioral approaches in mice and rats and in genetically modified mice in which the GR was conditionally deleted (GR(NesCre)). We identified the tPA-BDNF-TrkB signaling pathway as the upstream molecular effectors of GR-mediated phosphorylation of Erk1/2(MAPK) responsible for the enhancement of contextual fear memory. These findings complete our knowledge of the molecular cascade through which GC enhance contextual fear memory and highlight the role of tPA-BDNF-TrkB-Erk1/2(MAPK) signaling pathways as one of the core effectors of stress-related effects of GC.

International audience; Hypothalamic glucose sensing is involved in the control of feeding behavior and peripheral glucose homeostasis, and glial cells are suggested to play an important role in this process. Diazepam-binding inhibitor (DBI) and its processing product the octadecaneuropeptide (ODN), collectively named endozepines, are secreted by astroglia, and ODN is a potent anorexigenic factor. Therefore, we investigated the involvement of endozepines in brain glucose sensing. First, we showed that intracerebroventricular administration of glucose in rats increases DBI expression in hypothalamic glial-like tanycytes. We then demonstrated that glucose stimulates endozepine secretion from hypothalamic explants. Feeding experiments indicate that the anorexigenic effect of central administration of glucose was blunted by coinjection of an ODN antagonist. Conversely, the hyperphagic response elicited by central glucoprivation was suppressed by an ODN agonist. The anorexigenic effects of centrally injected glucose or ODN agonist were suppressed by blockade of the melanocortin-3/4 receptors, suggesting that glucose sensing involves endozepinergic control of the melanocortin pathway. Finally, we found that brain endozepines modulate blood glucose levels, suggesting their involvement in a feedback loop controlling whole-body glucose homeostasis. Collectively, these data indicate that endozepines are a critical relay in brain glucose sensing and potentially new targets in treatment of metabolic disorders.

International audience; Thermoresponsive tissue culture substrates based on PNIPAM brushes are used to harvest confluent cell sheets for tissue engineering. The prospect of clinical use imposes the utilization of culture medium free of bovine serum, thus suggesting conjugation with adhesion peptides containing the RGD minimal recognition sequence. The optimum position of the RGD along the chain should ensure both cell adhesion at 37 °C and cell detachment at T(L) below the lower critical solution temperature of PNIPAM. Design guidelines are formulated from considerations of brush confinement by the cells: (i) Cell adhesion at 37 °C is controlled by the RGDs accessible without brush compression. (ii) Cell detachment at T(L) is driven by a disjoining force due to confinement of the swollen brush by cells retaining integrin-RGD bonds formed at 37 °C. These suggest placing the RGDs at the grafting surface or its vicinity. Randomly placed RGDs do not enable efficient detachment because a large fraction of the integrin-RGD bonds are not sufficiently tensioned at T(L), in line with experimental observations (Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K.; Okano, T. Immobilization of celladhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest. Tissue Eng. 2004, 10, 1125-1135). The theory framework enables analysis of culture media based on polymer brushes conjugated with adhesion peptides in general.

International audience; Epidemiological and experimental evidence indicates that nicotine is protective for Parkinson disease vulnerable dopamine neurons, but the underlying mechanism of this effect remains only partly characterized. To address this question, we established rat midbrain cultures maintained in experimental conditions that favor the selective and spontaneous loss of dopamine neurons. We report here that nicotine afforded neuroprotection to dopamine neurons (EC(50)=0.32 μM) but only in a situation where cytosolic Ca(2+) (Ca(2+)(cyt)) was slightly and chronically elevated above control levels by concurrent depolarizing treatments. By a pharmacological approach, we demonstrated that the rise in Ca(2+)(cyt) was necessary to sensitize dopamine neurons to the action of nicotine through a mechanism involving α-bungarotoxin-sensitive (presumably α7) nicotinic acetylcholine receptors (nAChRs) and secondarily T-type voltage-gated calcium channels. Confirming the role played by α7 nAChRs in this effect, nicotine had no protective action in midbrain cultures prepared from genetically engineered mice lacking this receptor subtype. Signaling studies revealed that Ca(2+)(cyt) elevations evoked by nicotine and concomitant depolarizing treatments served to activate a survival pathway involving the calcium effector protein calmodulin and phosphatidylinositol 3-kinase. Collectively, our data support the idea that the protective action of nicotine for dopamine neurons is activity-dependent and gated by Ca(2+)(cyt).

International audience; Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

International audience; Different models have been developed to understand the biology of skin or to test pharmaceutical/cosmetic products. These models can be in vitro models that possess advantages such as mono and co-culture models in 2D, which are very reproducible, or organotypic models (skin explant and reconstructed skin) that present a 3D organisation. Animal or human in vivo models allow studies that are closer to reality. In virtuo models developed on computers control all known parameters and do not require animals. The major limitations of these models are the lack of 3D structure for in vitro culture, the variability of results from organotypic models, ethical problems inherent to human and animal tests and the presence of numerous unknown parameters in in virtuo systems. Despite their limitations, skin explants seem to be an interesting model for studies. Skin explants may be kept from a few hours to 10-14 days on supports or directly in culture medium. These explants are generally cultivated at 37 °C, 5% CO(2), preferentially in serum-free conditions. Three basic techniques are used to characterise these models: histological stains, proliferation, apoptosis and cytotoxicity tests. Skin explants could be a very convenient model to study wound-healing, inflammation processes, autoimmune diseases, malignant transformation, stress, ageing, and to serve as screening tests.

Let There Be Light Retinitis pigmentosa, a disease that can result from a wide variety of genetic defects, causes degeneration of photoreceptor cells in the retina and leads to blindness. In the course of the disease, it is generally the rod photoreceptor cells that degenerate first. Cone photoreceptor cells may persist, but in a damaged and nonfunctional state. Busskamp et al. (p. 413 , published online 24 June; see the cover; see the Perspective by Cepko ) have now applied a gene therapy approach to mouse models of retinitis pigmentosa. Inducing expression of a bacterial light-activated ion pump, halorho dopsin, in the damaged cone cells improved visual responses in the diseased mouse retinas. Thus, it may be possible to rescue cone photoreceptors therapeutically, even after they have already been damaged.

International audience; Regulation of microtubule dynamics is crucial for axon growth and guidance as well as for the establishment of synaptic connections. STOPs (Stable Tubule Only Polypeptides) are microtubule-associated proteins that regulate microtubule stabilization but are also able to interact with actin or Golgi membranes. Here, we have investigated the involvement of STOPs during the development of the olfactory system. We first describe the spatio-temporal expression patterns of N- and E-STOP, the two neuronal-specific isoforms of STOP. E- and N-STOP are expressed in the axonal compartment of olfactory sensory neurons, but are differentially regulated during development. Interestingly, each neuronal isoform displays a specific gradient distribution within the olfactory nerve layer. Then, we have examined the development of the olfactory system in the absence of STOPs. Olfactory axons display a normal outgrowth and targeting in STOP-null mice, but maturation of the synapses in the glomerular neuropil is altered.

ObjectiveAdiponectin is a protein abundantly secreted by the adipose tissue (AT). Plasma adiponectin levels are decreased in obese, insulin-resistant, and type 2 diabetic patients. Various multimeric complexes, i.e. high-, middle-, and low-molecular weight isoforms (HMW, MMW and LMW), are present in plasma. Here, we investigated the effect of weight reducing diet on the distribution of adiponectin isoforms in plasma and on their secretion in AT explants from obese subjects.DesignA total of 20 obese subjects (age 37.8±7.3 years, body mass index 33.9±5.0 kg/m2) underwent eight weeks of very low-calorie diet (VLCD). A needle biopsy of subcutaneous abdominal AT and blood samples were taken before and after dietary intervention. AT explants were incubated in culture medium for 4 h. ELISA assay and western blot analyses were used to identify adiponectin complexes in culture media and in plasma.ResultsThe distribution of adiponectin polymers in plasma was different from that secreted in human AT explants. Before VLCD, the relative amount of HMW isoform was 75.5±9.1% of total adiponectin in culture media and 52.2±11.2% in plasma. Despite the diet-induced weight loss and improvement of insulin sensitivity, VLCD neither induced change in total adiponectin level nor in the ratio of HMW to total adiponectin in plasma and in culture media of AT explants.ConclusionsThe profile of adiponectin polymeric isoforms secreted by AT explants into culture media differs from the plasma profile. A dietary intervention leading to weight loss and improvement of insulin sensitivity was not associated with modifications of AT secretion of total or HMW adiponectin.

Marinobacter vinifirmus was shown to degrade toluene as sole carbon and energy source under aerobiosis and at NaCl concentrations in the range 30-150 g/L. Maximum toluene consumption rate, total CO2, and biomass productions were measured in the presence of 60 g/L of NaCl. Under these conditions, 90% of the carbon from toluene was recovered as CO2 and biomass. Maximum specific toluene consumption rate was about 0.12 mgC toluene mgC biomass(-1) h(-1) at NaCl concentrations between 30 and 60 g/L. It decreased to 0.03 mgC toluene mgC biomass(-1) h(-1) at 150 g/L. Besides toluene, M. vinifirmus degraded benzene, ethylbenzene, and p-xylene. Benzene and toluene were utilized to a lesser extent by another Marinobacter sp., Marinobacter hydrocarbonoclasticus.

Current thrombolytic therapy for acute ischemic stroke with tissue-type plasminogen activator (tPA) has clear global benefits. Nevertheless, evidences argue that in addition to its prohemorrhagic effect, tPA might enhance excitotoxic necrosis. In the brain parenchyma, tPA, by binding to and then cleaving the amino-terminal domain (ATD) of the NR1 subunit of N-methyl-D-aspartate (NMDA) glutamate receptors, increases calcium influx to toxic levels. We show here that tPA binds the ATD of the NR1 subunit by a two-sites system (KD=24 nmol/L). Although tenecteplase (TNK) and reteplase also display two-sites binding profiles, the catalytically inactive mutant TNKS478A displays a one-site binding profile and desmoteplase (DSPA), a kringle 2 (K2) domain-free plasminogen activator derived from vampire bat, does not interact with NR1. Moreover, we show that in contrast to tPA, DSPA does not promote excitotoxicity. These findings, together with three-dimensional (3D) modeling, show that a critical step for interaction of tPA with NR1 is the binding of its K2 domain, followed by the binding of its catalytic domain, which in turn cleaves the NR1 subunit at its ATD, leading to a subsequent potentiation of NMDA-induced calcium influx and neurotoxicity. This could help design safer new generation thrombolytic agents for stroke treatment. © 2008 ISCBFM All rights reserved., This work was supported by grants from INSERM, the French Ministry of Research and Technology, the Regional Council of Lower Normandy, the Regional Platform for Cell Imaging of Upper Normandy, the Medical Research Foundation, the University of Caen, Fonds Europeéns de Développement Régional, the Paul Hamel Foundation, the Diagnosis in Molecular Imaging Program (FP6-project DiMI-LSHB-CT-2005-512146-), and PAION Deutschland GmbH.

Our aim was to test cell and trabecular responses to mechanical loading in vitro in a tissue bone explant culture model. We used a new three-dimensional culture model, the ZetOS system, which provides the ability to exert cyclic compression on cancellous bone cylinders (bovine sternum) cultured in forced flow circumfusion chambers, and allows to assess mechanical parameters of the cultivated samples. We evaluated bone cellular parameters through osteocyte viability test, gene and protein expression, and histomorphometric bone formation rate, in nonloaded versus loaded samples. The microarchitecture of bone cores was appraised by in vivo micro-CT imaging. After 3 weeks, the samples receiving daily cyclic compression exhibited increased osteoblast differentiation and activity associated with thicker, more plate-like-shaped trabeculae and higher Young's modulus and ultimate force as compared to unloaded samples. Osteoclast activity was not affected by mechanical strain, although it was responsive to drug treatments (retinoic acid and bisphosphonate) during the first 2 weeks of culture. Thus, in the ZetOS apparatus, we reproduce in vitro the osteogenic effects of mechanical strain known in vivo, making this system a unique and an essential laboratory aid for ex vivo testing of lamellar bone remodeling.

International audience; Analysis of synaptic transmission, synaptic plasticity, axonal processing, synaptic timing or electrical coupling requires the simultaneous recording of both the pre- and postsynaptic compartments. Paired-recording technique of monosynaptically connected neurons is also an appropriate technique to probe the function of small molecules (calcium buffers, peptides or small proteins) at presynaptic terminals that are too small to allow direct whole-cell patch-clamp recording. We describe here a protocol for obtaining, in acute and cultured slices, synaptically connected pairs of cortical and hippocampal neurons, with a reasonably high probability. The protocol includes four main stages (acute/cultured slice preparation, visualization, recording and analysis) and can be completed in approximately 4 h.

International audience; This study was designed to determine whether Coxsackie adenovirus receptor (CAR) and alpha nu beta3/alpha nu beta5 integrin co-receptors are involved in adenovirus gene transfer in the rat cochlea. We find that CAR and integrin co-receptors are expressed in every cell subtype transduced by the adenoviral vector Ad5 DeltaE1-E3/cytomegalovirus/green fluorescent protein (GFP) on cochlear slices in vitro. The spiral ganglion neurons, which do not express CAR, were not transduced by the virus. Blocking these receptors by monoclonal antibodies decreased transgene expression, whereas disrupting tight junctions with ethylenediaminetetraacetic acid led to an increased transgene expression. However, sensory hair cells and strial cells also expressing CAR and alpha nu integrins were not transduced by the vector. GFP expression was also studied in vivo. Perilymphatic perfusion of adenovirus in vivo did not affect hearing and only cells lining the perilymphatic spaces were transduced. Endolymphatic perfusion resulted in low-frequency hearing loss and although some cells of the organ of Corti were efficiently transduced, the sensory and the strial cells were not. Transduced sensory and strial cells were occasionally observed in cochleas after single shot of adenovirus. Pretreatment with anti-CAR and anti-alpha nu antibodies decreases GFP expression in vivo, suggesting that the CAR/alpha nu integrin pathway is involved in adenovirus transduction in the cochlea.

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals.

International audience; Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.

International audience; Initiation of hepatitis C virus (HCV) infection is mediated by docking of the viral envelope to the hepatocyte cell surface membrane followed by entry of the virus into the host cell. Aiming to elucidate the impact of this interaction on host cell biology, we performed a genomic analysis of the host cell response following binding of HCV to cell surface proteins. As ligands for HCV-host cell surface interaction, we used recombinant envelope glycoproteins and HCV-like particles (HCV-LPs) recently shown to bind or enter hepatocytes and human hepatoma cells. Gene expression profiling of HepG2 hepatoma cells following binding of E1/E2, HCV-LPs, and liver tissue samples from HCV-infected individuals was performed using a 7.5-kd human cDNA microarray. Cellular binding of HCV-LPs to hepatoma cells resulted in differential expression of 565 out of 7,419 host cell genes. Examination of transcriptional changes revealed a broad and complex transcriptional program induced by ligand binding to target cells. Expression of several genes important for innate immune responses and lipid metabolism was significantly modulated by ligand-cell surface interaction. To assess the functional relevance and biological significance of these findings for viral infection in vivo, transcriptional changes were compared with gene expression profiles in liver tissue samples from HCV-infected patients or controls. Side-by-side analysis revealed that the expression of 27 genes was similarly altered following HCV-LP binding in hepatoma cells and viral infection in vivo. In conclusion, HCV binding results in a cascade of intracellular signals modulating target gene expression and contributing to host cell responses in vivo. Reprogramming of cellular gene expression induced by HCV-cell surface interaction may be part of the viral strategy to condition viral entry and replication and escape from innate host cell responses.

International audience; Cell replacement therapy using mesencephalic precursor cells is an experimental approach for the treatment of Parkinson's disease (PD). A significant problem associated with this procedure is the poor survival of grafted neurons. Impaired energy metabolism is considered to contribute to neuronal cell death after transplantation. Creatine is a substrate for mitochondrial and cytosolic creatine kinases (CK) and buffers cellular ATP resources. Furthermore, elevated cellular creatine levels facilitate metabolic channeling and show antiapoptotic properties. Exogenous creatine supplementation therefore might offer a tool for improvement of dopaminergic neuron survival. The present study aimed at investigating the effects of creatine on cell survival of rat embryonic day 14 (E14) ventral mesencephalic neurons grown as organotypic free-floating roller tube (FFRT) cultures. We found that the brain-specific isoform of CK (BB-CK) and the ubiquitous mitochondrial isoform (uMt-CK) are expressed at high levels in FFRT cultures and colocalize with tyrosine hydroxylase immunoreactive (TH-ir) cells. Exposure of these cultures to creatine induced an increase in the content of the BB-CK isotype. Creatine (5 mM) administration starting at day in vitro (DIV) 7 resulted in a significant increase (+35%) in TH-ir cell density at DIV21. In addition, we observed that creatine treatment provided neuroprotection against 1-methyl-4-phenyl pyridinium ion (MPP+)-induced TH-ir cell loss in the FFRT culture system, resulting in a significantly higher density (+19%) of TH-ir neurons in creatine-treated cultures compared to corresponding controls. The decrease of TH-ir neurons in the MPP+-treated group corresponded with an increase in immunoreactivity for active caspase-3, an effect that was not seen in the group receiving creatine supplementation. In conclusion, our data imply that creatine administration is beneficial for the survival of TH-ir neurons encountering harmful conditions.

Chorioamniotic infection is a leading cause of preterm premature rupture of fetal membranes (amnion and chorion). Bacterial infection induces an inflammatory response characterized by elevated production of proinflammatory cytokines; the latter activate the production of both PGs that stimulate uterine contractions, and matrix metalloproteinases (MMPs) that degrade the extracellular matrix of the chorioamniotic membranes. The inflammatory response is under the control of cAMP content, which is partly regulated by phosphodiesterases (PDE). In this study, we investigated the role of the PDE4 family in the inflammatory process triggered by LPS in a model of amniochorionic explants. We found that PDE4 family is the major cAMP-PDE expressed in human fetal membranes and that PDE4 activity is increased by LPS treatment. Selective inhibition of PDE4 activity affected LPS signaling, because PDE4 inhibitors (rolipram and/or cilomilast) reduced the release of the proinflammatory cytokine TNF-α and increased the release of the anti-inflammatory cytokine IL-10. PDE4 inhibition reduced cyclooxygenase-2 protein expression and PGE2 production and also modulated MMP-9, a key mediator of the membrane rupture process, by inhibiting pro-MMP-9 mRNA expression and pro-MMP-9 activity. These results demonstrate that the PDE4 family participates in the regulation of the inflammatory response associated with fetal membrane rupture during infection. The PDE4 family may be an appropriate pharmacological target for the management of infection-induced preterm delivery.

International audience; Recent developments in tooth-tissue engineering require that we understand the regulatory processes to be preserved to achieve histomorphogenesis and cell differentiation, especially for enamel tissue engineering. Using mouse first lower molars, our objectives were: (1) to determine whether the cap-stage dental mesenchyme can control dental epithelial histogenesis, (2) to test the role of the primary enamel knot (PEK) in specifying the potentialities of the dental mesenchyme, and (3) to evaluate the importance of positional information in epithelial cells. After tissue dissociation, the dental epithelium was further dissociated into individual cells, re-associated with dental mesenchyme, and cultured. Epithelial cells showed a high plasticity: Despite a complete loss of positional information, they rapidly underwent typical dental epithelial histogenesis. This was stimulated by the mesenchyme. Experiments performed at E13 demonstrated that the initial potentialities of the mesenchyme are not specified by the PEK. Positional information of dental epithelial cells does not require the memorization of their history.