Cancer bronchique primitif, voies de signalisation intra-cellulaires et modèles précliniques

Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l ajout d un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l association d un inhibiteur de mTOR et d un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l association de l inhibiteur de mTOR everolimus et de l inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l injection orthotopique d une lignée bioluminescente de CBP-NPC chez des souris nude a permis d établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L association d un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l association d un inhibiteur de mTOR et d un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d envisager un transfert en clinique.Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Clinical review: Intrapericardial fibrinolysis in management of purulent pericarditis

Purulent pericarditis (PP) is a potentially life-threatening disease. Reported mortality rates are between 20 and 30%. Constrictive pericarditis occurs over the course of PP in at least 3.5% of cases. The frequency of persistent PP (chronic or recurrent purulent pericardial effusion occurring despite drainage and adequate antibiotherapy) is unknown because this entity was not previously classified as a complication of PP. No consensus exists on the optimal management of PP. Nevertheless, the cornerstone of PP management is complete eradication of the focus of infection. In retrospective studies, compared to simple drainage, systematic pericardiectomy provided a prevention of constrictive pericarditis with better clinical outcome. Because of potential morbidity associated with pericardiectomy, intrapericardial fibrinolysis has been proposed as a less invasive method for prevention of persistent PP and constrictive pericarditis. Experimental data demonstrate that fibrin formation, which occurs during the first week of the disease, is an essential step in the evolution to constrictive pericarditis and persistent PP. We reviewed the literature using the MEDLINE database. We evaluated the clinical efficacy, outcome, and complications of pericardial fibrinolysis. Seventy-four cases of fibrinolysis in PP were analysed. Pericarditis of tuberculous origin were excluded. Among the 40 included cases, only two treated by late fibrinolysis encountered failure requiring pericardiectomy. No patient encountered clinical or echocardiographic features of constriction during follow-up. Only one serious complication was described. Despite the lack of definitive evidence, potential benefits of fibrinolysis as a less invasive alternative to surgery in the management of PP seem promising. Early consideration should be given to fibrinolysis in order to prevent both constrictive and persistent PP. Nevertheless, in case of failure of fibrinolysis, pericardiectomy remains the primary option for complete eradication of infection

From internal waves to turbulence in a stably stratified fluid

We report on the statistical analysis of stratified turbulence forced by large-scale waves. The setup mimics some features of the tidal forcing of turbulence in the ocean interior at submesoscales. Our experiments are performed in the large-scale Coriolis facility in Grenoble which is 13 m in diameter and 1 m deep. Four wavemakers excite large scale waves of moderate amplitude. In addition to weak internal wave turbulence at large scales, we observe strongly nonlinear waves, the breaking of which triggers intermittently strong turbulence at small scales. A transition to strongly nonlinear turbulence is observed at smaller scales. Our measurements are reminiscent of oceanic observations. Despite similarities with the empirical Garrett & Munk spectrum that assumes weak wave turbulence, our observed energy spectra are rather be attributed to strongly nonlinear internal waves.Comment: accepted for publication in Physical Review Letter

Generation of weakly nonlinear turbulence of internal gravity waves in the Coriolis facility

We investigate experimentally stratified turbulence forced by waves. Stratified turbulence is present in oceans and it is expected to be dominated by nonlinear interaction of internal gravity waves as described by the Garrett & Munk spectrum. In order to reach turbulent regimes dominated by stratification we use the Coriolis facility in Grenoble (France) which large size enables us to reach regimes with both low Froude number and large Reynolds number. Stratification is obtained by using vertically linearly varying salt concentration and we force large scale waves in a $6\times6\times 1$ m$^3$ domain. We perform time-resolved PIV to probe the space-time structure of the velocity field. We observe a wide band spectrum which is made of waves. Discrete modes are observed due to the square shape of the flow container as well as a continuum part which appears consistent with an axisymmetric superposition of random weakly nonlinear waves. Our observations support the interpretation of turbulence of a strongly stratified fluid as wave turbulence of internal waves although our spectrum is quite different from the Garrett & Munk spectrum. Weak turbulence proceeds down to a small cutoff length scale (the buoyancy wavelength) at which a transition to more strongly nonlinear turbulence is expected.Comment: accepted for publication in Physical Review Fluid

Counting function fluctuations and extreme value threshold in multifractal patterns: the case study of an ideal 1/f1/f noise

To understand the sample-to-sample fluctuations in disorder-generated multifractal patterns we investigate analytically as well as numerically the statistics of high values of the simplest model - the ideal periodic $1/f$ Gaussian noise. By employing the thermodynamic formalism we predict the characteristic scale and the precise scaling form of the distribution of number of points above a given level. We demonstrate that the powerlaw forward tail of the probability density, with exponent controlled by the level, results in an important difference between the mean and the typical values of the counting function. This can be further used to determine the typical threshold $x_m$ of extreme values in the pattern which turns out to be given by $x_m^{(typ)}=2-c\ln{\ln{M}}/\ln{M}$ with $c=3/2$. Such observation provides a rather compelling explanation of the mechanism behind universality of $c$. Revealed mechanisms are conjectured to retain their qualitative validity for a broad class of disorder-generated multifractal fields. In particular, we predict that the typical value of the maximum $p_{max}$ of intensity is to be given by $-\ln{p_{max}} = \alpha_{-}\ln{M} + \frac{3}{2f'(\alpha_{-})}\ln{\ln{M}} + O(1)$, where $f(\alpha)$ is the corresponding singularity spectrum vanishing at $\alpha=\alpha_{-}>0$. For the $1/f$ noise we also derive exact as well as well-controlled approximate formulas for the mean and the variance of the counting function without recourse to the thermodynamic formalism.Comment: 28 pages; 7 figures, published version with a few misprints corrected, editing done and references adde

Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells

Metabotropic glutamate receptor 4 (mGluR4) possesses immune modulatory properties in vivo, such that a positive allosteric modulator (PAM) of the receptor confers protection on mice with relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). ADX88178 is a newly-developed, one such mGluR4 modulator with high selectivity, potency, and optimized pharmacokinetics. Here we found that application of ADX88178 in the RR-EAE model system converted disease into a form of mild-yet chronic-neuroinflammation that remained stable for over two months after discontinuing drug treatment. In vitro, ADX88178 modulated the cytokine secretion profile of dendritic cells (DCs), increasing production of tolerogenic IL-10 and TGF-β. The in vitro effects required activation of a Gi-independent, alternative signaling pathway that involved phosphatidylinositol-3-kinase (PI3K), Src kinase, and the signaling activity of indoleamine 2,3-dioxygenase 1 (IDO1). A PI3K inhibitor as well as small interfering RNA targeting Ido1-but not pertussis toxin, which affects Gi protein-dependent responses-abrogated the tolerogenic effects of ADX88178-conditioned DCs in vivo. Thus our data indicate that, in DCs, highly selective and potent mGluR4 PAMs such as ADX88178 may activate a Gi-independent, long-lived regulatory pathway that could be therapeutically exploited in chronic autoimmune diseases such as multiple sclerosis

Nouvelle approche d'optimisation pour le problème d'affectation des unités ferroviaires

RÉSUMÉ: Nous nous intéressons au problème d’affectation des unités ferroviaires. Il s’agit d’un problème de couverture, où des voyages prévus au préalable doivent être associés à des unités ferroviaires. Nous avons travaillé sur ce problème sur un horizon d’une journée, en cherchant à obtenir une solution périodique qui puisse être appliquée sur un intervalle de temps plus long. L’assignation doit respecter un certain nombre de contraintes, notamment la disponibilité des différents types d’unités, la périodicité quotidienne des stationnements dans les dépôts, et des contraintes de couplage et de découplage des unités selon des règles prédéfinies. Ce problème peut être résolu efficacement en découpant en deux phases distinctes : calcul des compositions de trains optimales en ignorant certaines contraintes puis affectation des trains à ces compositions. Cette approche a notamment été utilisée par Giro avec lesquels nous avons travaillé au cours de cette maîtrise. Cette approche heuristique ne permet cependant pas de connaître l’erreur par rapport à la valeur optimale du problème. Nous avons donc cherché à modéliser ce problème en une seule phase, par un problème de plus court chemin dans un graphe avec contraintes supplémentaires, où chaque chemin décrit un enchaînement potentiel de voyages couverts par une unique unité. Nous résolvons ce modèle linéaire par génération de colonnes, avant d’utiliser un solveur en nombres entiers sur les colonnes retenues. Nous avons travaillé sur des instances construites aléatoirement et sur des instances fournies par la SNCF. La plus grosse de nos instances inclut 367 voyages à couvrir, répartis entre 26 gares. Les temps de résolution sur cette instance et celles de taille comparable nous ont poussé à utiliser des techniques heuristiques pour accélérer la résolution du problème en nombres entiers. Nous présentons nos résultats, comparés avec ceux de Giro, et avec les meilleures bornes que nous connaissons pour ce problème. Nous présentons enfin les limites de notre modèle, et les pistes d’amélioration de notre méthode. ABSTRACT: We interested ourselves in the problem of allocating rail units. This is a hedging problem, where pre-scheduled trips have to be associated with railway units. We have worked on this problem over a one-day horizon, seeking to obtain a periodic solution that can be applied over a longer time interval. The assignment must respect a number of constraints, including the availability of different types of units, the daily periodicity of parking in depots, and constraints on coupling and uncoupling units according to predefined rules. This problem can be solved efficiently by dividing it into two distinct phases: calculating optimal train compositions while ignoring certain constraints, and then assigning trains to these compositions. This approach was used in particular by Giro, with whom we worked during the course of this master’s degree. However, this heuristic approach does not allow us to know the error with respect to the optimal value of the problem. We therefore sought to model this problem in a single phase, by means of a shortest path problem in a graph with additional constraints, where each path describes a potential sequence of journeys covered by a single unit. We solve this linear model by column generation, before using an integer solver on the selected columns. We worked on randomly constructed instances and on instances provided by SNCF. The largest of our instances included 367 trips to cover, spread over 26 stations. Solving times on this instance and others of comparable size prompted us to use heuristic techniques to speed up solving the integer problem. We present our results, compared with those of Giro, and with the best bounds we know for this problem. Finally, we present the limitations of our model, and suggest ways of improving our method

Cancer bronchique primitif, voies de signalisation intra-cellulaires et modèles précliniques

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l’espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l’oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l’ajout d’un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l’inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l’association de l’inhibiteur de mTOR everolimus et de l’inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l’injection orthotopique d’une lignée bioluminescente de CBP-NPC chez des souris nude a permis d’établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L’association d’un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d’envisager un transfert en clinique