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Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of Oenothera biennis cell cultures (ObHEx) and we showed its skin anti-aging properties. The aim of this work is to assess its biological effect specifically on cell senescence. ObHEx action has been evaluated on normal human dermal fibroblasts subjected to stress-induced premature senescence (SIPS) through an ultra-deep proteomic analysis, leading to the most global senescence-associated proteome so far. Mass spectrometry data show that the treatment with ObHEx re-establishes levels of crucial mitotic proteins, strongly downregulated in senescent cells. To validate our proteomics findings, we proved that ObHEx can, in part, restore the activity of ‘senescence-associated-ß-galactosidase’, the most common hallmark of senescent cells. Furthermore, to assess if the upregulation of mitotic protein levels translates into a cell cycle re-entry, FACS experiments have been carried out, demonstrating a small but significative reactivation of senescent cell proliferation by ObHEx. In conclusion, the deep senescence-associated global proteome profiling published here provides a panel of hundreds of proteins deregulated by SIPS that can be used by the community to further understand senescence and the effect of new potential modulators. Moreover, proteomics analysis pointed to a specific promitotic effect of ObHEx on senescent cells. Thus, we suggest ObHEx as a powerful adjuvant against senescence associated with skin aging.

Accumulation of senescent dermal fibroblasts drives skin aging. The reactivation of proliferation is one strategy to modulate cell senescence. Recently, we reported the exact chemical composition of the hydrophilic extract of

Human type 1 diabetes results from a destructive auto-reactive immune response in which CD8(+) T lymphocytes play a critical role. Given the intense ongoing efforts to develop immune intervention to prevent and/or cure the disease, biomarkers suitable for prediction of disease risk and progress, as well as for monitoring of immunotherapy are required. We undertook separate multi-parameter analyses of single naïve and activated/memory CD8(+) T lymphocytes from pediatric and adult patients, with the objective of identifying cellular profiles associated with onset of type 1 diabetes. We observe global perturbations in gene and protein expression and in the abundance of T cell populations characterizing pediatric but not adult patients, relative to age-matched healthy individuals. Pediatric diabetes is associated with a unique population of CD8(+) T lymphocytes co-expressing effector (perforin, granzyme B) and regulatory (transforming growth factor β, interleukin-10 receptor) molecules. This population persists after metabolic normalization and is especially abundant in children with high titers of auto-antibodies to glutamic acid decarboxylase and with elevated HbA1c values. These findings highlight striking differences between pediatric and adult type 1 diabetes, indicate prolonged large-scale perturbations in the CD8(+) T cell compartment in the former, and suggest that CD8(+)CD45RA(-) T cells co-expressing effector and regulatory factors are of interest as biomarkers in pediatric type 1 diabetes.

International audience; Hepatitis B virus X protein (HBx) contributes to Hepatitis B virus (HBV)-related liver cancer. However, its impact on hepatocyte proliferation and genomic stability remains elusive. We studied the role of HBx expression on the progression of cell cycle and liver polyploidization during proliferation and liver carcinogenesis. Full-length HBx transgenic mice (FL-HBx) were developed to investigate liver ploidy as well as hepatocyte proliferation, along normal liver maturation and during cancer initiation (chemical carcinogen treatment). Investigation of postnatal liver development in FL-HBx showed an aberrant G1/S and G2/M transitions, triggered (1) a delay of the formation of hepatocytes binucleation, (2) the early synthesis of polyploidy nuclei (≥4n) and (3) DNA damage appearance. Moreover, HBV infection during hepatocytes proliferation in a humanized liver mouse model led, to modifications in polyploidy of hepatocytes. In initiation of hepatocellular carcinoma, FL-HBx protein decreased ChK1 phosphorylation, Mre11 and Rad51 expression, upregulated IL-6 expression and impaired apoptosis. This was related to DNA damage accumulation in FL-HBx mice. At day 75 after initiation of hepatocellular carcinoma, FL-HBx mice revealed significant cell cycle changes related to the increased amount of 4n nuclei and of markers of cancer progenitor cells. Finally, PLK1 upregulation and p38/ERK activation in FL-HBx mice were implicated in aberrant polyploidization favoring DNA damage propagation and hepatocyte transformation. In conclusion, our data indicate that FL-HBx protein increases DNA damage through the hijack of hepatocyte polyploidization. That leads to enhancement of hepatocellular carcinoma initiation in an inflammatory context.

International audience; Maturation of functional neuronal circuits during central nervous system development relies on sophisticated mechanisms. First, axonal and dendritic growth should reach appropriate targets for correct synapse elaboration. Second, pruning and neuronal death are required to eliminate redundant or inappropriate neuronal connections. Serotonin, in addition to its role as a neurotransmitter, actively participates in postnatal establishment and refinement of brain wiring in mammals. Brain resident macrophages, that is, microglia, also play an important role in developmentally regulated neuronal death as well as in synaptic maturation and elimination. Here, we tested the hypothesis of cross-regulation between microglia and serotonin during postnatal brain development in a mouse model of synaptic refinement. We found expression of the serotonin 5-HT2B receptor on postnatal microglia, suggesting that serotonin could participate in temporal and spatial synchronization of microglial functions. Using two-photon microscopy, acute brain slices, and local delivery of serotonin, we observed that microglial processes moved rapidly toward the source of serotonin in Htr2B(+/+) mice, but not in Htr2B(-/-) mice lacking the 5-HT2B receptor. We then investigated whether some developmental steps known to be controlled by serotonin could potentially result from microglia sensitivity to serotonin. Using an in vivo model of synaptic refinement during early brain development, we investigated the maturation of the retinal projections to the thalamus and observed that Htr2B(-/-) mice present anatomical alterations of the ipsilateral projecting area of retinal axons into the thalamus. In addition, activation markers were upregulated in microglia from Htr2B(-/-) compared to control neonates, in the absence of apparent morphological modifications. These results support the hypothesis that serotonin interacts with microglial cells and these interactions participate in brain maturation.

The evolutionary success of bacteria depends greatly on their capacity to continually generate phenotypic diversity. Structured environments are particularly favorable for diversification because of attenuated clonal interference, which renders selective sweeps nearly impossible and enhances opportunities for adaptive radiation. We examined at the microscale level the emergence and the spatial and temporal dynamics of phenotypic diversity and their underlying causes in Escherichia coli colonies. An important dynamic heterogeneity in the growth, metabolic activity, morphology, gene expression patterns, stress response induction, and death patterns among cells within colonies was observed. Genetic analysis indicated that the phenotypic variation resulted mostly from mutations and that indole production, oxidative stress, and the RpoS-regulated general stress response played an important role in the generation of diversity. We observed the emergence and persistence of phenotypic variants within single colonies that exhibited variable fitness compared to the parental strain. Some variants showed improved capacity to produce biofilms, whereas others were able to use different nutrients or to tolerate antibiotics or oxidative stress. Taken together, our data show that bacterial colonies provide an ecological opportunity for the generation and maintenance of vast phenotypic diversity, which may increase the probability of population survival in unpredictable environments.

International audience; T cell-dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell-independent responses. We used this dissociation to analyze the repertoire diversification of IgM(+)IgD(+)CD27(+) B cells (also known as "IgM memory" B cells), comparing these cells with switched B cells in children

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34(+)CD10(+) progenitor population and which is distinct from B cell-committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin(-)CD34(+)CD10(+)CD24(-) progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor alpha, and CD3epsilon. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.

Natural regulatory T cells (Tregs) are present in high frequencies among tumor-infiltrating lymphocytes and in draining lymph nodes, supposedly facilitating tumor development. To investigate their role in controlling local immune responses, we analyzed intratumoral T cell accumulation and function in the presence or absence of Tregs. Tumors that grew in normal BALB/c mice injected with the 4T1 tumor cell line were highly infiltrated by Tregs, CD4 and CD8 cells, all having unique characteristics. Most infiltrating Tregs expressed low levels of CD25Rs and Foxp3. They did not proliferate even in the presence of IL-2 but maintained a strong suppressor activity. CD4 T cells were profoundly anergic and CD8 T cell proliferation and cytotoxicity were severely impaired. Depletion of Tregs modified the characteristics of tumor infiltrates. Tumors were initially invaded by activated CD4+CD25− T cells, which produced IL-2 and IFN-γ. This was followed by the recruitment of highly cytotoxic CD8+ T cells at tumor sites leading to tumor rejection. The beneficial effect of Treg depletion in tumor regression was abrogated when CD4 helper cells were also depleted. These findings indicate that the massive infiltration of tumors by Tregs prevents the development of a successful helper response. The Tregs in our model prevent Th cell activation and subsequent development of efficient CD8 T cell activity required for the control of tumor growth.

The role of the thymus in T cell commitment of hemopoietic precursor is yet controversial. We previously identified a major T cell progenitor activity in precursor cells isolated from bone marrow-derived spleen colonies. In this study, we characterize the properties of these pre-T cells. We demonstrate that they have unique phenotype and can be generated in a total absence of any thymic influence. Indeed, even when studied at the single-cell level, extrathymic T cell-committed precursors express T cell-specific genes. Moreover, these cells are not committed to a particular T cell differentiation pathway because they can generate both extrathymic CD8αα+ intraepithelial lymphocytes and thymus-derived conventional thymocytes. We also compared these pre-T cells with fully T cell-committed thymic progenitors. When tested in vitro or by direct intrathymic transfer, these cells have a low clonogenic activity. However, after i.v. transfer, thymus repopulation is efficient and these precursors generate very high numbers of peripheral T cells. These results suggest the existence of extra steps of pre-T cell maturation that improve thymus reconstitution capacity and that can be delivered even after full T cell commitment. Consequently, our studies identify a source of extrathymic progenitors that will be helpful in defining the role of the thymus in the earliest steps of T cell differentiation.

CD4+CD25+ regulatory T cells have been extensively studied during the last decade, but how these cells exert their regulatory function on pathogenic effector T cells remains to be elucidated. Naive CD4+ T cells transferred into T cell-deficient mice strongly expand and rapidly induce inflammatory bowel disease (IBD). Onset of this inflammatory disorder depends on IFN-γ production by expanding CD4+ T cells. Coinjection of CD4+CD25+ regulatory T cells protects recipient mice from IBD. In this study, we show that CD4+CD25+ regulatory T cells do not affect the initial activation/proliferation of injected naive T cells as well as their differentiation into Th1 effectors. Moreover, naive T cells injected together with CD4+CD25+ regulatory T cells into lymphopenic hosts are still able to respond to stimuli in vitro when regulatory T cells are removed. In these conditions, they produce as much IFN-γ as before injection or when injected alone. Finally, when purified, they are able to induce IBD upon reinjection into lymphopenic hosts. Thus, prevention of IBD by CD4+CD25+ regulatory T cells is not due to deletion of pathogenic T cells, induction of a non reactive state (anergy) among pathogenic effector T cells, or preferential induction of Th2 effectors rather than Th1 effectors; rather, it results from suppression of T lymphocyte effector functions, leading to regulated responses to self.

CD25(+)CD4(+) regulatory T cells have major roles in controlling immune responses, and use heterogeneous regulatory mechanisms. It is possible that these different activities are mediated by different subsets. Here we show that CD103(+)CD25(+)CD4(+) T cells (that control inflammatory bowel disease) are highly enriched in gut-associated lymphoid tissue and have unique functional properties. In vivo, only this subpopulation is able to control wasting disease and peripheral T cell homeostasis. In vitro, only this subpopulation is able to regulate IL-10 secretion, and it might also mediate infectious suppression. These results demonstrate that regulatory T cells can be divided into discrete subpopulations with defined functional properties and regulatory mechanisms.

Current androgen ablation therapies for prostate cancer are initially successful, but the frequent development of castration resistance urges the generation of alternative therapies and represents an important health concern. Prolactin/signal transducer and activator of transcription 5 (STAT5) signaling is emerging as a putative target for alternative treatment for prostate cancer. However, mechanistic data for its role in development or progression of prostate tumors are scarce. In vivo mouse studies found that local prolactin induced the amplification of prostate epithelial basal/stem cells. Because these cells are proposed cells of origin for prostate cancer and disease recurrence, we looked further into this amplification. Our results indicated that sustained Stat5 activation was associated with the occurrence of abnormal basal/stem cell clusters in prostate epithelium of prostate-specific prolactin-transgenic mice. Analysis of epithelial areas containing these clusters found high proliferation, Stat5 activation, and expression of stem cell antigen 1. Furthermore, enhanced prolactin signaling also led to amplification of a luminal cell population that was positive for stem cell antigen 1. These cells may originate from amplified basal/stem cells and might represent important progenitors for tumor development in prostate epithelium. These data provide a deeper understanding of the initial stages of prostate tumorigenesis induced by prolactin to help determine whether this hormone or its downstream messengers could be useful targets for prostate cancer treatment in the future.

C’est un ensemble de raisons à la fois historiques, politiques et même méthodologiques qui expliquent l’emploi particulier de la dénomination français langue seconde au Canada. Nous verrons que la dénomination français langue étrangère s’inscrit mal dans le contexte canadien et que les didacticiens l’ont abandonnée pour celle de français langue seconde, celle-ci désignant de façon globale une variété de situations linguistiques et de programmes d’enseignement. Historical, political and even methodological factors have led to the specific use of the term français langue seconde in Canada. It will be shown that the term français langue seconde is not appropriate in the Canadian context and that it is no longer used in the language-teaching field, having been superseded by français langue seconde, which encompasses a variety of language-related situations and of teaching programs.

Can spellcheckers or grammar correctors, be they part of a word processor or stand-alone programs, help FSL students become more adept at correcting or editing the written texts they produce ? Does computer-assisted mediation enable learners to correct their work efficiently and how does it compare with human mediation ? What differentiates them ? Texts written by anglophone students were corrected using both methods, i.e., on the one hand, by two teachers with 20 years experience and, on the other hand, using the specialized tools available with MSWord as well as two Canadian text correction programs, Le Correcteur 101 and Antidote. It turns out that the two methods are of very different natures ; computer-based correction can only give some positive results if the users have received appropriate training enabling them to actively and knowledgeably participate in the correcting process, and software programs cannot on their own be relied upon to correct efficiently texts written by intermediate level students., L'apprentissage de la correction / révision de l'écrit par les apprenants de français langue seconde peut-elle tirer profit des correcteurs orthographiques et grammaticaux des traitements de texte ou des correcticiels spécialisés ? La nature de la médiation informatique permet-elle aux apprenants de corriger de façon efficace leurs textes et comment se compare-t-elle avec la correction humaine ? Qu'est-ce qui différencie ces deux médiations ? Des textes rédigés par des anglophones ont été soumis aux deux types de corrections, humaine et informatique, soit deux enseignantes ayant vingt ans d'expérience et la correction informatique effectuée par le correcteur orthographique et grammatical de Word et les deux correcticiels canadiens, Le correcteur 101 et Antidote. Les résultats montrent que la nature de ces médiations n'est pas comparable et que la médiation informatique, pour être efficace, nécessite la participation active, intelligente et instruite de l'utilisateur et que les correcticiels ne peuvent pas corriger efficacement des textes d'étudiants de niveau intermédiaire.

International audience; Regulatory T cells (Treg) are commonly identified by CD25 (IL-2R alpha) surface expression and/or intracellular expression of the FOXP3 transcription factor. In addition, Treg are also characterized by low CD127 (IL-7R alpha) expression when compared to conventional T cells and their biology in the periphery is considered essentially independent of IL-7. We further investigated CD127 expression on Treg and we demonstrated differential CD127 expression depending on Treg subsets considered. Notably, we observed high CD127 expression on inducible costimulatory molecule (ICOS)- and CD103-expressing Treg subsets. Since these two markers reflect activation status, we addressed whether Treg activation modulated CD127 expression. We demonstrated that in contrast to conventional T cells, Treg significantly upregulated CD127 expression during in vitro and in vivo activation using adoptive transfer and contact dermatitis models. High CD127 expression on Treg was also predominantly detected ex vivo in some specific sites, notably bone marrow and skin. Importantly, higher CD127 expression on Treg correlated with higher phosphorylation of STAT5 upon IL-7 exposure. High CD127 expression on Treg also provided survival advantage upon in vitro incubation with IL-7. We thus demonstrated that low CD127 expression is not an intrinsic characteristic of Treg and we identified activated Treg as a potential target of endogenous or therapeutic IL-7.

We have developed a new method for accurate quantification of dead microbial cells. This technique employs the simultaneous use of fluorescent hydrazides and nucleic acid dyes. Fluorescent hydrazides allow detection of cells that cannot be detected with currently used high-affinity nucleic acid dyes. This is particularly important for nongrowing bacterial populations and for multicellular communities containing physiologically heterogeneous cell populations, such as colonies and biofilms.

We characterized CD8+ T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4+CD25+ T cells, CD8+CD25+ T cells appear late in the periphery during ontogeny. Peripheral CD8+CD25+ T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4+CD25+ T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25− T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4+CD25+ T cells. However, unlike CD4+CD25+ T cells CD8+CD25+ T cells from MHC class II-deficient mice strongly proliferate and produce IFN-γ in vitro in response to stimulation in the absence of exogenous IL-2.

International audience; The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive, immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) "memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis, complementarity-determining region 3 (CDR3) spectratyping during a T-independent response, and gene-expression profiling of the different blood and splenic B-cell subsets, we show here that blood IgM(+)IgD(+)CD27(+) cells correspond to circulating splenic marginal zone B cells. Furthermore, analysis of this peripheral subset in healthy children younger than 2 years shows that these B cells develop and mutate their immunoglobulin receptor during ontogeny, prior to their differentiation into T-independent antigen-responsive cells. It is therefore proposed that these IgM(+)IgD(+)CD27(+) B cells provide the splenic marginal zone with a diversified and protective preimmune repertoire in charge of the responses against encapsulated bacteria.

L’apprentissage de la correction / révision de l’écrit par les apprenants de français langue seconde peut-elle tirer profit des correcteurs orthographiques et grammaticaux des traitements de texte ou des correcticiels spécialisés ? La nature de la médiation informatique permet-elle aux apprenants de corriger de façon efficace leurs textes et comment se compare-t-elle avec la correction humaine ? Qu’est-ce qui différencie ces deux médiations ? Des textes rédigés par des anglophones ont été soumis aux deux types de corrections, humaine et informatique, soit deux enseignantes ayant vingt ans d’expérience et la correction informatique effectuée par le correcteur orthographique et grammatical de Word et les deux correcticiels canadiens, Le correcteur 101 et Antidote. Les résultats montrent que la nature de ces médiations n’est pas comparable et que la médiation informatique, pour être efficace, nécessite la participation active, intelligente et instruite de l’utilisateur et que les correcticiels ne peuvent pas corriger efficacement des textes d’étudiants de niveau intermédiaire.Can spellcheckers or grammar correctors, be they part of a word processor or stand-alone programs, help FSL students become more adept at correcting or editing the written texts they produce? Does computer-assisted mediation enable learners to correct their work efficiently and how does it compare with human mediation? What differentiates them? Texts written by anglophone students were corrected using both methods, i.e., on the one hand, by two teachers with 20 years experience and, on the other hand, using the specialized tools available with MSWord as well as two Canadian text correction programs, Le Correcteur 101 and Antidote. It turns out that the two methods are of very different natures ; computer-based correction can only give some positive results if the users have received appropriate training enabling them to actively and knowledgeably participate in the correcting process, and software programs cannot on their own be relied upon to correct efficiently texts written by intermediate level students.

Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation.

L’espace scriptural du manuel scolaire est faconne par de multiples ruptures typographiques qui donnent a voir une succession en apparence chaotique de textes et d’images tout a fait disparates. Or, sous l’heterogeneite de surface du tissu textuel, il est possible de degager un nombre limite d’elements recurrents possedant une fonction didactique specifique. L’analyse d’un vaste corpus de manuels de francais langue seconde et etrangere a permis de construire une typologie des formes textuelles du manuel de langue, qui acquierent, au plan du discours, leur coherence dans le « contrat didactique » que propose le manuel.