Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

Bristle pattern formation in the thorax formation of Drosophila: a systems level approach

A fundamental question in developmental biology is how tissues are patterned to give rise to differentiated body structures with distinct morphologies. The Drosophila wing disc offers an accessible model to understand epithelial spatial patterning. It has been studied extensively using genetic and molecular approaches. Bristle patterns on the thorax, which arise from the medial part of the wing disc, are a classical model of pattern formation, dependent on a pre-pattern of trans-activators and –repressors. Despite of decades of molecular studies, we still only know a subset of the factors that determine the pre-pattern. We are applying a novel and interdisciplinary approach to predict regulatory interactions in this system. It is based on the description of expression patterns by simple logical relations (addition, subtraction, intersection and union) between simple shapes (graphical primitives). Similarities and relations between primitives have been shown to be predictive of regulatory relationships between the corresponding regulatory factors in other Systems, such as the Drosophila egg. Furthermore, they provide the basis for dynamical models of the bristle-patterning network, which enable us to make even more detailed predictions on gene regulation and expression dynamics. We have obtained a data-set of wing disc expression patterns which we are now processing to obtain average expression patterns for each gene. Through triangulation of the images we can transform the expression patterns into vectors which can easily be analysed by Standard clustering methods. These analyses will allow us to identify primitives and regulatory interactions. We expect to identify new regulatory interactions and to understand the basic Dynamics of the regulatory network responsible for thorax patterning. These results will provide us with a better understanding of the rules governing gene regulatory networks in general, and provide the basis for future studies of the evolution of the thorax-patterning network in particular.

Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood

Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.

Melan-A/MART-1-specific CD8 T cells: from thymus to tumor.

The self-antigen Melan-A/MART-1 is frequently involved in T-cell responses against malignant melanoma. The use of fluorescent tetramers incorporating the immunodominant Melan-A/MART-1 peptide has provided new insights into HLA-A2-restricted T-cell responses against this antigen in cancer patients and in healthy individuals. Direct evidence has been provided that a large Melan-A/MART-1-specific CD8 T-cell pool is generated during thymic selection. Although several other examples of naive self-peptide-specific T-cell repertoires are known, this is the only one directly accessible to analysis in healthy individuals

Intravaginal and subcutaneous immunization induced vaccine specific CD8 T cells and tumor regression in the bladder.

PURPOSE: Vaccines targeting tumor associated antigens are in development for bladder cancer. Most of these cancers are nonmuscle invasive at diagnosis and confined in the mucosa and submucosa. However, to our knowledge how vaccination may induce the regression of tumors at such mucosal sites has not been examined previously. We compared different immunization routes for the ability to induce vaccine specific antitumor CD8 T cells in the bladder and bladder tumor regression in mice. MATERIALS AND METHODS: In the absence of a murine bladder tumor model expressing a tumor antigen relevant for human use we established an orthotopic model expressing the HPV-16 tumor antigen E7 as a model. We used an adjuvant E7 polypeptide to induce CD8 T cell mediated tumor regression. RESULTS: Subcutaneous and intravaginal but not intranasal vaccination induced a high number of TetE7(+)CD8(+) T cells in the bladder as well as bladder tumor regression. The entry of vaccine specific T cells in the bladder was not the only key since persistent regression of established bladder tumors by intravaginal or subcutaneous immunization was associated with tumor infiltration of total CD4 and CD8 T cells. This resulted in an increase in TetE7(+)CD8(+) T cells and a decrease in T regulatory cells, leading to an increased number of effector interferon-γ secreting vaccine specific CD8 T cells in the regressing bladder tumor. CONCLUSIONS: These data show that immunization routes should be tailored to each mucosal tumor site. Subcutaneous or intravaginal vaccination may be of additional value to treat patients with bladder cancer.

Cytokine targeting in tumors using a bispecific antibody directed against carcinoembryonic antigen and tumor necrosis factor alpha.

The use of tumor necrosis factor alpha (TNFalpha) in cancer therapy is limited by its short circulatory half-life and its severe systemic side effects. To overcome these limitations, we evaluated the capability of a bispecific antibody (BAb) directed against carcinoembryonic antigen (CEA) and human TNFalpha to target this cytokine in tumors. A BAb was constructed by coupling the Fab' fragments from an anti-CEA monoclonal antibody (MAb) to the Fab' fragments from an anti-TNFalpha MAb via a stable thioether linkage. The double specificity of the BAb for CEA and TNFalpha was demonstrated using a BIAcoreTM two-step analysis. The affinity constants of the BAb for CEA immobilized on a sensor chip and for soluble TNFalpha added to the CEA-BAb complex were as high as those of the parental MAbs (1.7 x 10(9) M-1 and 6.6 x 10(8) M-1, respectively). The radiolabeled 125I-labeled BAb retained high immunoreactivity with both CEA and TNFalpha immobilized on a solid phase. In nude mice xenografted with the human colorectal carcinoma T380, the 125I-labeled BAb showed a tumor localization and biodistribution comparable to that of 131I-labeled anti-CEA parental F(ab')2 with 25-30% of the injected dose (ID)/g tumor at 24 h and 20% ID/g tumor at 48 h. To target TNFalpha to the tumor, a two-step i.v. injection protocol was used first, in which a variable dose of 125I-labeled BAb was injected, followed 24 or 48 h later by a constant dose of 131I-labeled TNFalpha (1 microg). Mice pretreated with 3 microg of BAb and sacrificed 2, 4, 6, or 8 h after the injection of TNFalpha showed a 1.5- to 2-fold increased concentration of 131I-labeled TNFalpha in the tumor as compared to control mice, which received TNFalpha alone. With a higher dose of BAb (25 microg), mice showed a better targeting of TNFalpha with a 3.2-fold increased concentration of 131I-labeled TNFalpha in the tumor: 9.3% versus 2.9% ID/g in control mice 6 h after TNFa injection. In a one-step injection protocol using a premixed BAb-TNFalpha preparation, similar results were obtained 6 h postinjection (3.5-fold increased TNFalpha tumor concentration). A longer retention time of TNFalpha was observed leading to an 8.1-fold increased concentration of TNFalpha in the tumor 14 h postinjection (4.4 versus 0.5% ID/g tumor for BAb-treated and control mice, respectively). These results show that our BAb is able, first, to localize in a human colon carcinoma and, there, to immunoabsorb the i.v.-injected TNFalpha, leading to its increased concentration at the tumor site.

Fibronectin distribution in the chick embryo during formation of the blastula.

The distribution of the fibronectin-rich extracellular matrix (ECM) in the chick embryo during formation of the blastula has been evaluated semiquantitatively using an electron microscopical immunogold staining technique. During the first 10 h of postlaying development, fibronectin was found in both embryonic area pellucida and extra-embryonic area opaca of the blastoderm. In the area pellucida, the fibronectin was (1) associated with the basal lamina of the epiblast, (2) present between epiblastic and hypoblastic cells and (3) occasionally internalized in hypoblastic cells. Along the embryonic axis, a transient and high density of ECM was associated with the front of the anteriorly and rapidly expanding hypoblast. Very high density of fibronectin was observed in the marginal zone of the area pellucida, where the epiblastic and deeper cell layers show contacts and intense re-arrangements. In the area opaca, fibronectin was at first found only sporadically between contacting cells, but its density increased steadily and markedly during the first day of development. These rapid and significant changes in the regional distribution of fibronectin-rich ECM are discussed with respect to the early morphogenesis of the chick embryo.

Influence of physico-chemical material characteristics on staphylococcal biofilm formation--a qualitative and quantitative in vitro analysis of five different calcium phosphate bone grafts.

Various compositions of synthetic calcium phosphates (CaP) have been proposed and their use has considerably increased over the past decades. Besides differences in physico-chemical properties, resorption and osseointegration, artificial CaP bone graft might differ in their resistance against biofilm formation. We investigated standardised cylinders of 5 different CaP bone grafts (cyclOS, chronOS (both β-TCP (tricalcium phosphate)), dicalcium phosphate (DCP), calcium-deficient hydroxyapatite (CDHA) and α-TCP). Various physico-chemical characterisations e.g., geometrical density, porosity, and specific surface area were investigated. Biofilm formation was carried out in tryptic soy broth (TSB) and human serum (SE) using Staphylococcus aureus (ATCC 29213) and S. epidermidis RP62A (ATCC 35984). The amount of biofilm was analysed by an established protocol using sonication and microcalorimetry. Physico-chemical characterisation showed marked differences concerning macro- and micropore size, specific surface area and porosity accessible to bacteria between the 5 scaffolds. Biofilm formation was found on all scaffolds and was comparable for α-TCP, chronOS, CDHA and DCP at corresponding time points when the scaffolds were incubated with the same germ and/or growth media, but much lower for cyclOS. This is peculiar because cyclOS had an intermediate porosity, mean pore size, specific surface area, and porosity accessible to bacteria. Our results suggest that biofilm formation is not influenced by a single physico-chemical parameter alone but is a multi-step process influenced by several factors in parallel. Transfer from in vitro data to clinical situations is difficult; thus, advocating the use of cyclOS scaffolds over the four other CaP bone grafts in clinical situations with a high risk of infection cannot be clearly supported based on our data.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

Itinéraires de formation et lignes biographiques

This paper proposes an analysis of training courses and their connection to other biographical lines, by highlighting their evolution according to social origin and gender. The variable of having received a diploma does not fully explain the longitudinal characteristics of the courses: the social inequalities prove to be also inequalities from the point of view of the followed itinerary. The range of available trajectories as well as the possibility of deviating in order to fulfil a custom trajectory depends on the social characteristics. The training course is connected to other biographical lines, in particular the family and the profession.