Postmenopausal circulating levels of 2- and 16α-hydroxyestrone and risk of endometrial cancer.

Background:It has been suggested that the relative importance of oestrogen-metabolising pathways may affect the risk of oestrogen-dependent tumours including endometrial cancer. One hypothesis is that the 2-hydroxy pathway is protective, whereas the 16α-hydroxy pathway is harmful.Methods:We conducted a case-control study nested within three prospective cohorts to assess whether the circulating 2-hydroxyestrone : 16α-hydroxyestrone (2-OHE1 : 16α-OHE1) ratio is inversely associated with endometrial cancer risk in postmenopausal women. A total of 179 cases and 336 controls, matching cases on cohort, age and date of blood donation, were included. Levels of 2-OHE1 and 16α-OHE1 were measured using a monoclonal antibody-based enzyme assay.Results:Endometrial cancer risk increased with increasing levels of both metabolites, with odds ratios in the top tertiles of 2.4 (95% CI=1.3, 4.6; P(trend)=0.007) for 2-OHE1 and 1.9 (95% CI=1.1, 3.5; P(trend)=0.03) for 16α-OHE1 in analyses adjusting for endometrial cancer risk factors. These associations were attenuated and no longer statistically significant after further adjustment for oestrone or oestradiol levels. No significant association was observed for the 2-OHE1 : 16α-OHE1 ratio.Conclusion:Our results do not support the hypothesis that greater metabolism of oestrogen via the 2-OH pathway, relative to the 16α-OH pathway, protects against endometrial cancer.

Le cancer du sein sans envahissement ganglionnaire. Au-delà des consensus internationaux: une approche pragmatique [Node negative breast cancer. Beyond international consensus: a pragmatic approach].

Apart from therapeutic advances related to new treatments, our practices in the management of early breast cancer have been modified by to key organizational settings (1) mass screening, substantially altering the presentation and epidemiology of breast cancer and (2) the development of guidelines to ensure that any patient management is in agreement with the demonstrated impact in the adjuvant treatment. In daily practice, the impact of screening and guidelines recommendations has put us now in a paradoxical situation: while the majority of non-metastatic breast cancers treated in the hexagon are node negative, most of the results of clinical studies on chemotherapy and targeted therapies today arise from populations predominantly node positive. Therefore, it seemed legitimate to convene a working group around a reflection on the directions of adjuvant chemotherapy in a growing node negative population in order to better respond to the questions of the field oncologists, trying to address the discrepancies between different existing guidelines.

Targeted nasal vaccination provides antibody-independent protection against Staphylococcus aureus.

Despite showing promise in preclinical models, anti-Staphylococcus aureus vaccines have failed in clinical trials. To date, approaches have focused on neutralizing/opsonizing antibodies; however, vaccines exclusively inducing cellular immunity have not been studied to formally test whether a cellular-only response can protect against infection. We demonstrate that nasal vaccination with targeted nanoparticles loaded with Staphylococcus aureus antigen protects against acute systemic S. aureus infection in the absence of any antigen-specific antibodies. These findings can help inform future developments in staphylococcal vaccine development and studies into the requirements for protective immunity against S. aureus.

Deficiency of glucose-dependent insulinotropic polypeptide receptor prevents ovariectomy-induced obesity in mice.

Menopause and premature gonadal steroid deficiency are associated with increases in fat mass and body weight. Ovariectomized (OVX) mice also show reduced locomotor activity. Glucose-dependent-insulinotropic-polypeptide (GIP) is known to play an important role both in fat metabolism and locomotor activity. Therefore, we hypothesized that the effects of estrogen on the regulation of body weight, fat mass, and spontaneous physical activity could be mediated in part by GIP signaling. To test this hypothesis, C57BL/6 mice and GIP-receptor knockout mice (Gipr(-/-)) were exposed to OVX or sham operation (n = 10 per group). The effects on body composition, markers of insulin resistance, energy expenditure, locomotor activity, and expression of hypothalamic anorexigenic and orexigenic factors were investigated over 26 wk in all four groups of mice. OVX wild-type mice developed obesity, increased fat mass, and elevated markers of insulin resistance as expected. This was completely prevented in OVX Gipr(-/-) animals, even though their energy expenditure and spontaneous locomotor activity levels did not significantly differ from those of OVX wild-type mice. Cumulative food intake in OVX Gipr(-/-) animals was significantly reduced and associated with significantly lower hypothalamic mRNA expression of the orexigenic neuropeptide Y (NPY) but not of cocaine-amphetamine-related transcript (CART), melanocortin receptors (MCR-3 and MCR-4), or thyrotropin-releasing hormone (TRH). GIP receptors thus interact with estrogens in the hypothalamic regulation of food intake in mice, and their blockade may carry promising potential for the prevention of obesity in gonadal steroid deficiency.

The role of the transcription factor Tcf-1 for the development and the function of NK cells

Natural Killer (NK) cells are innate immune cells that can eliminate malignant and foreign cells and that play an important role for the early control of viral and fungal infections. Further, they are important regulators of the adaptive and innate immune responses. During their development in the bone marrow (BM) NK cells undergo several maturation steps that directly establish an effector program. The transcriptional network that controls NK cell development and maturation is still incompletely understood. Based on earlier findings that NK cell numbers are reduced in the absence of the transcription factor T cell factor-1 (Tcf-1), my thesis has addressed the precise role of this transcription factor for NK cell development, maturation and function and whether Tcf-1 acts as a nuclear effector of the canonical Wnt signaling pathway to mediate its effects. It is shown that Tcf-1 is selectively required for the emergence of mature BM NK cells. Surprisingly, the emergence of BM NK cells depends on the repressor function of Tcf-1 and is independent of the Wnt pathway. In BM and peripheral NK cells Tcf-1 is found to suppress Granzyme B (GzmB) expression, a key cytotoxic effector molecule required to kill target cells. We provide evidence that GzmB over-expression in the absence of Tcf-1 results in accelerated spontaneous death of bone marrow NK cells and of cytokine stimulated peripheral NK cells. Moreover, Tcf-1 deficient NK cells show reduced target cell killing, which is due to enhanced GzmB-dependent NK cell death induced by the recognition of tumour target cells. Collectively, these data provide significant new insights into the transcriptional regulation of NK cell development and function and suggest a novel mechanism that protects NK cells from the deleterious effects of highly cytotoxic effector molecules. - Les cellules NK (de l'anglais Natural Killer) font partie du système immunitaire inné et sont capables d'éliminer à elles seules les cellules cancéreuses ou infectées. Ces cellules participent dans la régulation et la coordination des réponses innée et adaptative. Lors de leur développement dans la moelle osseuse, les cellules NK vont acquérir leurs fonctions effectrices, un processus contrôlé par des facteurs de transcription mais encore peu connu. Des précédentes travaux ont montré qu'une diminution du nombre de cellules NK corrélait avec l'absence du facteur de transcription Tcf-1 (T cell factor-1), suggérant un rôle important de Tcf-1 dans le développement de cellules NK. Cette thèse a pour but de mieux comprendre le rôle du facteur de transcription Tcf-1 lors du développement et la maturation des cellules NK, ainsi que son interaction avec la voie de signalisation Wnt. Nous avons montré que Tcf-1 est essentiel pour la transition des cellules immatures NK (iNK) à des cellules matures NK (mNK) dans la moelle osseuse, et cela de manière indépendamment de la voie de signalisation Wnt. De manière intéressante, nous avons observé qu'en absence du facteur de transcription Tcf-1, les cellules NK augmentaient l'expression de la protéine Granzyme B (GzmB), une protéine essentielle pour l'élimination des cellules cancéreuses ou infectées. Ceci a pour conséquence, une augmentation de la mort des cellules mNK dans la moelle osseuse ainsi qu'une diminution de leur fonction «tueuses». Ces résultats montrent pour la première fois, le rôle répresseur du facteur de transcription Tcf-1 dans l'expression de la protéine GzmB. L'ensemble de ces résultats apporte de nouveaux éléments concernant le rôle de Tcf-1 dans la régulation du développement et de la fonction des cellules NK et suggèrent un nouveau mécanisme cellulaire de protection contre les effets délétères d'une dérégulation de l'expression des molécules cytotoxique.

A common ancestor DNA motif for invertebrate and vertebrate hormone response elements.

The ecdysone-responsive DNA sequence of the Drosophila hsp27 gene promoter contains four direct and inverted repeats reminiscent of those that compose the vertebrate palindromic estrogen response element (ERE) and the thyroid hormone/retinoic acid response element (TRE/RRE). Interestingly, a 3 bp substitution in the wild-type Hsp27 ecdysone response element (EcdRE) increases both its similarity with the vertebrate ERE and TRE/RRE and its capacity to confer ecdysone responsiveness to a heterologous promoter. Remarkably, increasing the spacing between the inverted repeats of this strong EcdRE by two nucleotides converts it into an ERE. Inversely, decreasing the spacing between the two inverted repeats of the vertebrate consensus palindromic ERE, from three to one nucleotide, converts it into a functional EcdRE. Thus, the only difference between an invertebrate EcdRE and a vertebrate palindromic ERE or TRE/RRE is in the spacing between the conserved inverted repeated motifs forming these palindromic HREs. The finding that the sequence motif 5'-GGTCA-3' present in the vertebrate ERE and TRE/RRE is also a functionally important characteristic of an invertebrate HRE, suggests that a common ancestor regulatory DNA sequence gave rise to all HREs known so far. We discuss the possibility that this progenitor motif is the GGTCA sequence.

Cardiovascular influences of alpha1b-adrenergic receptor defect in mice.

BACKGROUND: The alpha1-adrenergic receptors (alpha1-ARs) play a key role in cardiovascular homeostasis. However, the functional role of alpha1-AR subtypes in vivo is still unclear. The aim of this study was to evaluate the cardiovascular influences of alpha1b-AR. METHODS AND RESULTS: In transgenic mice lacking alpha1-AR (KO) and their wild-type controls (WT), we evaluated blood pressure profile and cardiovascular remodeling induced by the chronic administration (18 days via osmotic pumps) of norepinephrine, angiotensin II, and subpressor doses of phenylephrine. Our results indicate that norepinephrine induced an increase in blood pressure levels only in WT mice. In contrast, the hypertensive state induced by angiotensin II was comparable between WT and KO mice. Phenylephrine did not modify blood pressure levels in either WT or KO mice. The cardiac hypertrophy and eutrophic vascular remodeling evoked by norepinephrine was observed only in WT mice, and this effect was independent of the hypertensive state because it was similar to that observed during subpressor phenylephrine infusion. Finally, the cardiac hypertrophy induced by thoracic aortic constriction was comparable between WT and KO mice. CONCLUSIONS: Our data demonstrate that the lack of alpha1b-AR protects from the chronic increase of arterial blood pressure induced by norepinephrine and concomitantly prevents cardiovascular remodeling evoked by adrenergic activation independently of blood pressure levels.

Role of the inflammasome in murine experimental models of arthritis

Summary : The purpose of this study was to investigate the role of the inflammasome in human and experimental murine models (such as ΑΙΑ and K/BxN) of rheumatoid arthritis (RA)RA, affecting 1% of the population is the most frequent inflammatory disease characterized by synovial hyperplasia and cartilage and bone erosion, leading to joint destruction. In general, women are 3 times more affected by RA suggesting a role of estrogen in this disease. The inflammasome is a multiproteic complex triggering the activation of caspase-1 leading to the activation of IL-1 β, an important pro-inflammatory cytokine implicated in arthritis. The inflammasome has been implicated in several inflammatory diseases and particularly in gout. To highlight a possible role of the inflammasome in murine arthritis, we obtained ASC, caspase-1 and NALP3 +/+ and -/- littermate mice to perform ΑΙΑ and K/BxN arthritis. NALP3 -/- and caspase-1 -/- mice were as arthritic as wild type littermate mice in both ΑΙΑ and K/BxN models implicating that the NALP3 inflammasome is not involved in experimental arthritis. By contrast, ΑΙΑ severity was significantly diminished in ASC- deficient male and female mice, and in the K/BxN model, in ASC-deficient female mice. These results were supported by histological scoring and acute phase protein serum amyloid A (SAA) levels that were equivalent between NALP+/+ and NALP3-/- mice and diminished in ASC -/- mice. In ΑΙΑ and K/BxN murine experimental models, we observed a sexdependent phenotype. We studied the role of estradiol in both the ALA and the K/BxN models. Castrated female or male ASC -/- mice that received estradiol had a decreased arthritis severity. This implies a protective role of estrogen in the absence of ASC. In the ΑΙΑ model, proliferation assay were performed using splenocytes from mBSA- immunized ASC +/+ and -/- mice. The mBSA-induced proliferation was significantly lower in ASC-/- splenocytes. Moreover the CD3-specific proliferation of purified splenic Τ cells was significantly lower in ASC-/- cells. Finally, Τ cells from ASC-/- mice produced significantly decreased levels of IFN-gamma associated with increased levels of IL-10. These results imply a possible role of ASC in the TCR-signaling pathway and Τ cell cytokine production. In parallel the expression of the different inflammasome components were analyzed in biopsies from rheumatoid arthritis (RA) and osteoarthritis (OA) patiens. The expression of the 14 different NALPs, their effector protein ASC, and caspase-1 and -5 was readily measurable by RT-PCR in a similar proportion in RA and OA synovial samples, with the exception of NALP-5 and NALP-13, which weren't found in samples from either disease. The corresponding NALP1, -3, -12 and ASC proteins were expressed at similar levels in both OA and RA biopsies, as determined by immunohistochemistry and Western-blot analysis. By contrast, caspase-1 levels were significantly enhanced in RA synovial tissues compared to those from OA patients. NALP-1, -2, -3, -10, -12 and -14, as well as ASC, caspase-1, and -5 were detected in RNA from unstimulated and stimulated RA synoviocytes. In FLS, only ASC and caspase-1 were expressed at the protein level. NALP1, 3 and 12 were not detected. However, upon stimulation, no secreted IL-Ιβ was detectable in either RA or in OA synoviocytes culture medium. Résumé : Le but de ce projet était d'étudier le rôle de l'inflammasome dans des modèles expérimentaux d'arthrite tels que les modèles ΑΙΑ et K/BxN ainsi que dans la polyarthrite humaine (RA). La polyarthrite est une maladie inflammatoire très fréquente avec 1 % de la population affectée et touche 3 fois plus les femmes que les hommes, suggérant un rôle des hormones sexuelles dans cette pathologie. L'inflammasome est un complexe multiprotéique qui permet l'activation de la caspase-1, une cystéine protéase qui va ensuite cliver et activer rinterleukine-ΐβ (IL-Ιβ). L'inflammasome a été impliqué ces dernières années dans de nombreuses maladies inflammatoires notamment dans la goutte. Pour mettre en évidence un éventuel rôle de l'inflammasome dans l'arthrite expérimentale nous avons obtenu des souris déficientes pour certains des composants de l'inflammasome tels que ASC, NALP3 et caspase-1. Les souris NALP3 déficientes et caspase-1 déficientes sont aussi arthritiques que les souris wild type correspondantes que ce soit dans le modèle ΑΙΑ ou K/BxN. Par contre les souris mâles et femelles ASC-déficientes sont moins arthritiques que les souris +/+ correspondantes dans le modèle ΑΙΑ. Dans le modèle KRN, le même phénotype (diminution de la sévérité de l'arthrite) est observé uniquement chez les femelles ASC-/- Ce phénotype est corrélé avec l'histologie ainsi qu'avec le dosage du serum amyloid A (SAA) qui reflète l'inflammation systémique et qui est diminué chez les souris ASC-déficientes. Nous avons ensuite étudié le rôle de Γ estradiol (une des formes active des estrogènes) dans les modèles K/BxN et ΑΙΑ. Les souris castrées maies ou femelles déficientes pour ASC ayant reçu de l'estradiol ont une arthrite moins sévère ce qui implique que les estradiol ont un effet protecteur en l'absence de ASC. Dans le modèle ΑΙΑ, nous nous sommes aussi intéressés à la réponse immune. Des tests de prolifération ont été effectués sur des splénocytes en présence de mBSA (qui est l'antigène utilisé dans le modèle ΑΙΑ). Les splénocytes ASC -/- ont une proliferation qui est diminuée en présence de l'antigène. De plus la proliferation de cellules Τ spléniques purifiées en présence d'anti-CD3 est diminuée chez les cellules Τ ASC-/-. Ces résultats nous indiquent une éventuelle implication de ASC dans la signalisation par le récépteur des cellules T. En parallèle l'expression des différents composants de l'inflammasome a été analysée dans des biopsies de patients atteints de polyarthrite rhumatoide (RA) et d'arthrose (OA). L'expression des 14 différents NALPs, de l'adaptateur ASC, ainsi que des caspase-1 et -5 était similaires dans les échantillons RA et OA, à l'exception de NALP5 et 13 qui n'étaient pas détéctables. L'expression protéique de NALP1, 3, 12 et ASC effectuée par Western blot et immunohistochimie était similaire dans les biopsies RA et OA. Par contre la quantité de la caspase-1 mesurée par ELISA était augmentée de façon significative dans les extraits protéiques de biopsies RA. NALP-1, -2. -3, -10, -12, and -14 ainsi que ASC, caspase-1 et -5 étaient exprimés de façon similaire par les synoviocytes RA non stimulés et stimulés. Dans les synoviocytes seuls ASC et caspase-1 étaient détéctable au niveau protéique. NALP-1, -3 et -12 n'était pas détéctables. Cependant après stimulation il n'y avait d'IL-Ιβ sécrété que ce soit dans les surnageants de cultures de synoviocytes RA ou OA.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up.

BACKGROUND: Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up. METHODS: BIG 1-98 is a randomised, phase 3, double-blind trial of postmenopausal women with hormone receptor-positive early breast cancer that compares 5 years of tamoxifen or letrozole monotherapy, or sequential treatment with 2 years of one of these drugs followed by 3 years of the other. Randomisation was done with permuted blocks, and stratified according to the two-arm or four-arm randomisation option, participating institution, and chemotherapy use. Patients, investigators, data managers, and medical reviewers were masked. The primary efficacy endpoint was disease-free survival (events were invasive breast cancer relapse, second primaries [contralateral breast and non-breast], or death without previous cancer event). Secondary endpoints were overall survival, distant recurrence-free interval (DRFI), and breast cancer-free interval (BCFI). The monotherapy comparison included patients randomly assigned to tamoxifen or letrozole for 5 years. In 2005, after a significant disease-free survival benefit was reported for letrozole as compared with tamoxifen, a protocol amendment facilitated the crossover to letrozole of patients who were still receiving tamoxifen alone; Cox models and Kaplan-Meier estimates with inverse probability of censoring weighting (IPCW) are used to account for selective crossover to letrozole of patients (n=619) in the tamoxifen arm. Comparison of sequential treatments to letrozole monotherapy included patients enrolled and randomly assigned to letrozole for 5 years, letrozole for 2 years followed by tamoxifen for 3 years, or tamoxifen for 2 years followed by letrozole for 3 years. Treatment has ended for all patients and detailed safety results for adverse events that occurred during the 5 years of treatment have been reported elsewhere. Follow-up is continuing for those enrolled in the four-arm option. BIG 1-98 is registered at clinicaltrials.govNCT00004205. FINDINGS: 8010 patients were included in the trial, with a median follow-up of 8·1 years (range 0-12·4). 2459 were randomly assigned to monotherapy with tamoxifen for 5 years and 2463 to monotherapy with letrozole for 5 years. In the four-arm option of the trial, 1546 were randomly assigned to letrozole for 5 years, 1548 to tamoxifen for 5 years, 1540 to letrozole for 2 years followed by tamoxifen for 3 years, and 1548 to tamoxifen for 2 years followed by letrozole for 3 years. At a median follow-up of 8·7 years from randomisation (range 0-12·4), letrozole monotherapy was significantly better than tamoxifen, whether by IPCW or intention-to-treat analysis (IPCW disease-free survival HR 0·82 [95% CI 0·74-0·92], overall survival HR 0·79 [0·69-0·90], DRFI HR 0·79 [0·68-0·92], BCFI HR 0·80 [0·70-0·92]; intention-to-treat disease-free survival HR 0·86 [0·78-0·96], overall survival HR 0·87 [0·77-0·999], DRFI HR 0·86 [0·74-0·998], BCFI HR 0·86 [0·76-0·98]). At a median follow-up of 8·0 years from randomisation (range 0-11·2) for the comparison of the sequential groups with letrozole monotherapy, there were no statistically significant differences in any of the four endpoints for either sequence. 8-year intention-to-treat estimates (each with SE ≤1·1%) for letrozole monotherapy, letrozole followed by tamoxifen, and tamoxifen followed by letrozole were 78·6%, 77·8%, 77·3% for disease-free survival; 87·5%, 87·7%, 85·9% for overall survival; 89·9%, 88·7%, 88·1% for DRFI; and 86·1%, 85·3%, 84·3% for BCFI. INTERPRETATION: For postmenopausal women with endocrine-responsive early breast cancer, a reduction in breast cancer recurrence and mortality is obtained by letrozole monotherapy when compared with tamoxifen montherapy. Sequential treatments involving tamoxifen and letrozole do not improve outcome compared with letrozole monotherapy, but might be useful strategies when considering an individual patient's risk of recurrence and treatment tolerability. FUNDING: Novartis, United States National Cancer Institute, International Breast Cancer Study Group.

Functional analysis of altered channel-activating protease-1 (CAP1) expression in the skin

Summary : The skin is a complex organ that protects the body against entry of pathogens and supplies a relatively dry and impermeable barrier to water loss. This barrier function is mainly provided by the epidermis, which is the outermost layer of the skin. Serine proteases are involved in skin physiology and it is known that mutations or alterations in their expression can lead to skin diseases. In order to investigate the importance of the regulated expression of CAPI/Prss8, a membrane bound serine protease expressed in the epidermis, we developed transgenic mice ectopically expressing CAPI/Prss8 in the skin. These animals exhibited a phenotype characterized by scaly skin, epidermal hypertrophy, inflammation and scratching behavior. This phenotype could be completely abolished in mice lacking the proteinase activated receptor 2 (PAR2) revealing PAR2 as a potential in vivo downstream target of CAP 1 /Prss8. We could also provide evidence of a CAP1 /Prss8 function independent of its catalytic activity. Additionally, mice ectopically expressing PAR2 in the skin developed a skin phenotype very similar to CAPI/Prss8 transgenic animals, supporting the hypothesis of PAR2 activation by CAPI/Prss8. We could furthermore demonstrate an inhibitory effect of the serine protease inhibitor nexin-I on CAPI/Prss8, since nexin-1 transgenic expression negated the skin phenotype observed in CAPI/Prss8 transgenic mice. CAP1/Prss8 and PAR2 transgenic animals, and the understanding of the interaction between CAPl/Prss8 and PAR2, can be helpful in developing potential CAPI/Prss8 and PAR2 inhibitory molecules that may be used as drugs to treat ichthyoses-like skin diseases. Résumé : La peau est un organe complexe qui protège le corps contre l'entrée des pathogènes et forme une barrière imperméable qui empêche la déshydratation. Cette fonction de barrière est surtout fournie par l'épiderme, la couche la plus superficielle de la peau. Le bon fonctionnement de cet organe est permis, entre autres, par les protéases à sérine qui sont des enzymes dont l'altération peut causer des maladies de la peau. Pour étudier l'importance de la régulation de CAP1/Prss8, une protéase à sérine exprimée au niveau de l'épiderme, des souris génétiquement modifiées, dans lesquelles CAP1/Prss8 est exprimé d'une manière ectopique dans la peau, ont été générées. Les animaux transgéniques pour CAP1/Prss8 présentent une peau squameuse, un épiderme hypertrophique, des processus inflammatoires et se grattent. Ce phénotype a pu être complètement guéri lorsque le gène de PAR2, un récepteur qui règle l'activité des cellules de l'épiderme, est inactivé chez la souris. Ceci montre que PAR2 est une cible de CAP1/Prss8 dans le système étudié. Des études expérimentales suggèrent de plus que l'effet de CAP1/Prss8 dans ce modèle ne dépend pas de son activité enzymatique. En dernière analyse, il a été démontré que l'expression transgénique de nexin-1, un inhibiteur des protéases à sérine exprimé dans la peau, a la capacité d'améliorer la peau squameuse et l'épiderme hypertrophique causés par CAP1/Prss8 transgénique. Les animaux transgéniques pour CAP1/Prss8 et PAR2, et la compréhension du mécanisme d'interaction entre eux, pourraient aider à développer et à tester des molécules inhibitrices de CAP1 /Prss8 et PARI qui pourraient alors être utilisées comme médicaments pour traiter des maladies de la peau comme les ichthyoses.

Autocrine secretion of IGF2 regulates adult ß cell functional plasticity

In the pathogenesis of type 2 diabetes, hyperglycemia appears when ß cell mass and insulin secretory capacity are no longer sufficient to compensate for insulin resistance. The reduction in ß cell mass results from increased apoptosis. Therefore, finding strategies to preserve ß cell mass and function may be useful for the treatment or prevention of diabetes. Glucagon-like peptide-1 (GLP-1) protects ß cells against apoptosis, increases their glucose competence, and induces their proliferation. Previous studies in the lab of Prof. Bernard Thorens showed that the GLP-1 anti- apoptotic effect was mediated by robust up-regulation of IGF-1R expression, and this was paralleled with an increase in Akt phosphorylation. This effect was dependent not only on increased IGF-1R expression but also on the autocrine secretion of insulin-like growth factor 2 (IGF2). They also demonstrated that GLP-1 up-regulated IGF-1R expression by a protein a kinase A-dependent translational control mechanism. The main aim of this PhD work has been to further investigate the role of the IGF2/IGF-1 Receptor autocrine loop in ß cell function and to determine the physiological role of IGF2 in ß cell plasticity and its regulation by nutrients. This PhD thesis is divided in 3 chapters. The first chapter describes the role of IGF2/IGF-1R autocrine loop in ß cell glucose competence and proliferation. Here using MIN6 cells and primary mouse islets as an experimental model we demonstrated that the glucose competence of these cells was dependent on the level of IGF-1R expression and on IGF2 secretion. Furthermore, we showed that GLP-1-induced primary ß cell proliferation was significantly reduced by Igf-lr gene inactivation and by IGF2 immunoneutralization or knockdown. In the second chapter we examined the role of this IGF2/IGF-1R autocrine loop on the ß cell functional plasticity during ageing, pregnancy, and in response to acute induction of insulin resistance using mice with ß cell-specific inactivation of ig/2. Here we showed a gender-dependent role of ß cell IGF2 in ageing and high fat diet-induced metabolic stress; we demonstrated that the autocrine secretion of IGF2 is essential for ß cell mass adaptation during pregnancy. Further we also showed that this autocrine loop plays an important role in ß cell expansion in response to acute induction of insulin resistance. The aim of the third chapter was to investigate whether we can modulate the expression and secretion of IGF2 by nutrients in order to increase the activity of autocrine loop. Here we showed that glutamine induces IGF2 biosynthesis and its fast secretion through the regulated pathway, a mechanism enhanced in the presence of glucose. Furthermore, we demonstrated that glutamine-mediated Akt phosphorylation is dependent on IGF2 secretion, indicating that glutamine controls the activity of the IGF2/IGF1R autocrine loop through IGF2 up-regulation. In summary, this PhD work highlights that autocrine secretion of IGF2 is required for compensatory ß cell adaptation to ageing, pregnancy, and insulin resistance. Moreover IGF2/IGF1R autocrine loop is regulated by two feeding-related cues, GLP-1 to increase IGF-1R expression and glutamine to control IGF2 biosynthesis and secretion. -- Dans le diabète de type 2, lorsque la sécrétion d'insuline des cellules Beta du pancréas n'est plus suffisante pour compenser la résistance à l'insuline, une hyperglycémie est observée. Cette baisse de sécrétion d'insuline est Causée par la diminution de la masse de cellules Beta suite à l'augmentation du phénomène de mort cellulaire ou « apoptose ». En diabétologie, une des stratégies médicales concerne la préservation des cellules Beta du pancréas. Une des protéines intervenant dans cette fonction est GLP-1 (Glucagon-like peptide-1). GLP-1 est capable de protéger les cellules Beta contre la mort cellulaire et d'induire leur prolifération. Des études précédemment menées dans le laboratoire du Professeur Bernard Thorens ont montrées que l'activité « anti-apoptotique » de GLP-1 est le résultat l'une augmentation de l'expression du gène IGF-1R sous la dépendance de la sécrétion autocrine d'IGF2 (Insulin-Like Growth Factor). Le but de mon travail de thèse aura été d'étudier le mécanisme de la régulation de GLP-1 par IGF2 et plus précisément de déterminer le rôle physiologique d'IGF2 dans la plasticité des cellules ß ainsi que sa régulation par les nutriments. Ce manuscrit est ainsi divisé en trois chapitres : Le premier chapitre décrit la fonction d'IGF2/IGF- R1 dans la réponse des cellules Beta au glucose ainsi que dans leur capacité à proliférer. Dans ce chapitre nous avons montré l'importance du niveau d'expression d'IGFR-1 et de la sécrétion d'IGF2 dans la régulation du métabolisme du glucose. Dans un deuxième chapitre, nous étudions la boucle de régulation IGF2/IGF-R1 sur la plasticité des cellules Beta lors du vieillissement, de la grossesse ainsi que dans un modèle de souris résistantes à l'insuline. Cette étude met en évidence un dimorphisme sexuel dans le rôle d'IGF2 lors du vieillissement et lors d'un stress métabolique. Nous montrons également l'importance d'IGF2 pour l'adaptation des cellules Beta tout au long de la grossesse ou lors du phénomène de résistance à l'insuline. Dans un troisième chapitre, nous mettons en évidence la possibilité de moduler l'expression et la sécrétion d'IGF2 par les nutriments. En conclusion, ce travail de thèse aura permis de mettre en évidence l'importance d'IGF2 dans la plasticité des cellules ß, une plasticité indispensable lors du vieillissement, de la grossesse ou encore dans le cas d'une résistance à l'insuline.

Role of gluconic acid production in the regulation of biocontrol traits of Pseudomonas fluorescens CHA0.

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Deltagcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0.

In vivo over-expression of interleukin-10 increases resistance to focal brain ischemia in mice.

Early studies showed that the administration of the anti-inflammatory cytokine interleukin-10 (IL10) protects against permanent middle cerebral artery occlusion (MCAO) in mice. In this study, transgenic mice expressing murine IL10 (IL10T) directed by the major histocompatibility complex Ea promoter were produced and used to explore the effect of chronically increased IL10 levels on MCAO-related molecular mechanisms. IL10 was over-expressed in astrocytes, microglia, and endothelial brain cells in IL10T compared with wild type mice. Four days following MCAO, IL10T mice showed a 40% reduction in infarct size which was associated to significantly reduced levels of active caspase 3 compared with wild type mice. Under basal conditions, anti-inflammatory factors such as nerve growth factor and GSH were up-regulated and the pro-inflammatory cytokine IL1beta was down-regulated in the brain of IL10T animals. In addition, these mice displayed increased basal GSH levels in microglial and endothelial cells as well as a marked increase in manganese superoxide dismutase in endothelial lining blood vessels. Following ischemia, IL10T mice showed a marked reduction in pro-inflammatory cytokines, including tumor necrosis factor-alpha, interferon-gamma, and IL1beta. Our data indicate that constitutive IL10 over-expression is associated with a striking resistance to cerebral ischemia that may be attributed to changes in the basal redox properties of glial/endothelial cells.

Granulocyte-macrophage colony-stimulating factor elicits bone marrow-derived cells that promote efficient colonic mucosal healing.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) therapy is effective in treating some Crohn's disease (CD) patients and protects mice from colitis induced by dextran sulfate sodium (DSS) administration. However, its mechanisms of action remain elusive. We hypothesized that GM-CSF affects intestinal mucosal repair. METHODS: DSS colitic mice were treated with daily pegylated GM-CSF or saline and clinical, histological, and inflammatory parameters were kinetically evaluated. Further, the role of bone marrow-derived cells in the impact of GM-CSF therapy on DSS colitis was addressed using cell transfers. RESULTS: GM-CSF therapy reduced clinical signs of colitis and the release of inflammatory mediators. GM-CSF therapy improved mucosal repair, with faster ulcer reepithelialization, accelerated hyperproliferative response of epithelial cells in ulcer-adjacent crypts, and lower colonoscopic ulceration scores in GM-CSF-administered mice relative to untreated mice. We observed that GM-CSF-induced promotion of mucosal repair is timely associated with a reduction in neutrophil numbers and increased accumulation of CD11b(+) monocytic cells in colon tissues. Importantly, transfer of splenic GM-CSF-induced CD11b(+) myeloid cells into DSS-exposed mice improved colitis, and lethally irradiated GM-CSF receptor-deficient mice reconstituted with wildtype bone marrow cells were protected from DSS-induced colitis upon GM-CSF therapy. Lastly, GM-CSF-induced CD11b(+) myeloid cells were shown to promote in vitro wound repair. CONCLUSIONS: Our study shows that GM-CSF-dependent stimulation of bone marrow-derived cells during DSS-induced colitis accelerates colonic tissue repair. These data provide a putative mechanism for the observed beneficial effects of GM-CSF therapy in Crohn's disease.

Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer.

BACKGROUND: A 70-gene signature was previously shown to have prognostic value in patients with node-negative breast cancer. Our goal was to validate the signature in an independent group of patients. METHODS: Patients (n = 307, with 137 events after a median follow-up of 13.6 years) from five European centers were divided into high- and low-risk groups based on the gene signature classification and on clinical risk classifications. Patients were assigned to the gene signature low-risk group if their 5-year distant metastasis-free survival probability as estimated by the gene signature was greater than 90%. Patients were assigned to the clinicopathologic low-risk group if their 10-year survival probability, as estimated by Adjuvant! software, was greater than 88% (for estrogen receptor [ER]-positive patients) or 92% (for ER-negative patients). Hazard ratios (HRs) were estimated to compare time to distant metastases, disease-free survival, and overall survival in high- versus low-risk groups. RESULTS: The 70-gene signature outperformed the clinicopathologic risk assessment in predicting all endpoints. For time to distant metastases, the gene signature yielded HR = 2.32 (95% confidence interval [CI] = 1.35 to 4.00) without adjustment for clinical risk and hazard ratios ranging from 2.13 to 2.15 after adjustment for various estimates of clinical risk; clinicopathologic risk using Adjuvant! software yielded an unadjusted HR = 1.68 (95% CI = 0.92 to 3.07). For overall survival, the gene signature yielded an unadjusted HR = 2.79 (95% CI = 1.60 to 4.87) and adjusted hazard ratios ranging from 2.63 to 2.89; clinicopathologic risk yielded an unadjusted HR = 1.67 (95% CI = 0.93 to 2.98). For patients in the gene signature high-risk group, 10-year overall survival was 0.69 for patients in both the low- and high-clinical risk groups; for patients in the gene signature low-risk group, the 10-year survival rates were 0.88 and 0.89, respectively. CONCLUSIONS: The 70-gene signature adds independent prognostic information to clinicopathologic risk assessment for patients with early breast cancer.