Increased male offspring's risk of metabolic-neuroendocrine dysfunction and overweight after fructose-rich diet intake by the lactating mother.

An adverse endogenous environment during early life predisposes the organism to develop metabolic disorders. We evaluated the impact of intake of an iso-caloric fructose rich diet (FRD) by lactating mothers (LM) on several metabolic functions of their male offspring. On postnatal d 1, ad libitum eating, lactating Sprague-Dawley rats received either 10% F (wt/vol; FRD-LM) or tap water (controls, CTR-LM) to drink throughout lactation. Weaned male offspring were fed ad libitum a normal diet, and body weight (BW) and food intake were registered until experimentation (60 d of age). Basal circulating levels of metabolic markers were evaluated. Both iv glucose tolerance and hypothalamic leptin sensitivity tests were performed. The hypothalamus was dissected for isolation of total RNA and Western blot analysis. Retroperitoneal (RP) adipose tissue was dissected and either kept frozen for gene analysis or digested to isolate adipocytes or for histological studies. FRD rats showed increased BW and decreased hypothalamic sensitivity to exogenous leptin, enhanced food intake (between 49-60 d), and decreased hypothalamic expression of several anorexigenic signals. FRD rats developed increased insulin and leptin peripheral levels and decreased adiponectinemia; although FRD rats normally tolerated glucose excess, it was associated with enhanced insulin secretion. FRD RP adipocytes were enlarged and spontaneously released high leptin, although they were less sensitive to insulin-induced leptin release. Accordingly, RP fat leptin gene expression was high in FRD rats. Excessive fructose consumption by lactating mothers resulted in deep neuroendocrine-metabolic disorders of their male offspring, probably enhancing the susceptibility to develop overweight/obesity during adult life.

Demand-based structural change and balanced economic growth

[eng] We analyze the equilibrium of a multi-sector exogenous growth model where the introduction of minimum consumption requirements drives structural change. We show that equilibrium dynamics simultaneously exhibt structural change and balanced growth of aggregate variables as is observed in US when the initial intensity of minimum consumption requirements is sufficiently small. This intensity is measured by the ratio between the aggregate value of the minimum consumption requirements and GDP and, therefore, it is inversely related with the level of economic development. Initially rich economies benefit from an initially low intensity of the minimum consumption requirements and, as a consequence, these economies end up exhibiting balanced growth of aggregate variables, while there is structural change. In contrast, initially poor economies suffer from an initially large intensity of the minimum consumption requirements, which makes the growth of the aggregate variables unbalanced during a very large period. These economies may never exhibit simultaneously balanced growth of aggregate variables and structural change.

Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

ESPEN Guidelines on Enteral Nutrition: Intensive care.

Enteral nutrition (EN) via tube feeding is, today, the preferred way of feeding the critically ill patient and an important means of counteracting for the catabolic state induced by severe diseases. These guidelines are intended to give evidence-based recommendations for the use of EN in patients who have a complicated course during their ICU stay, focusing particularly on those who develop a severe inflammatory response, i.e. patients who have failure of at least one organ during their ICU stay. These guidelines were developed by an interdisciplinary expert group in accordance with officially accepted standards and are based on all relevant publications since 1985. They were discussed and accepted in a consensus conference. EN should be given to all ICU patients who are not expected to be taking a full oral diet within three days. It should have begun during the first 24h using a standard high-protein formula. During the acute and initial phases of critical illness an exogenous energy supply in excess of 20-25 kcal/kg BW/day should be avoided, whereas, during recovery, the aim should be to provide values of 25-30 total kcal/kg BW/day. Supplementary parenteral nutrition remains a reserve tool and should be given only to those patients who do not reach their target nutrient intake on EN alone. There is no general indication for immune-modulating formulae in patients with severe illness or sepsis and an APACHE II Score >15. Glutamine should be supplemented in patients suffering from burns or trauma.

3D culture of postnatal retinal stem cells using self assembling polymers

PURPOSE We have previously shown that retinal stem cells (RSCs) can be isolated from the radial glia population of the newborn mouse retina (Angénieux et al., 2006). These RSCs have a great capacity to renew and to generate a large number of neurons including cells differentiated towards the photoreceptor lineage (Mehri-Soussi et al., 2006). However, recent published results from our lab revealed that such cells have a poor integration and survival rate after grafting. The uncontrolled environment of a retina seems to prevent good integration and survival after grafting in vivo. To bypass this problem, we are evaluating the possibility of generating in vitro a hemi-retinal tissue before transplantation. METHODS RSC were expanded and cells passaged <10 were seeded in a solution containing poly-ethylene-glycol (PEG) polymer based hydrogels crosslinked with peptides that are chosen to be substrates for matrix metalloproteinases. Various doses of cross linkers peptides allowing connections between PEG polymers were tested. Different growth factors were studied to stimulate cell proliferation and differentiation. RESULTS Cells survived only in the presence of EGF and FGF-2 and generated colonies with a sphere shape. No cells migrated within the gel. To improve the migration and the repartition of the cells in the gels, the integrin ligand RGDSP was added into the gel. In the presence of FGF-2 and EGF, newly formed cell clusters appeared by cell proliferation within several days, but again no outspreading of cells was observed. No difference was even seen when the stiffness of the hydrogels or the concentration of the integrin ligand RGDSP were changed. However, our preliminary results show that RSCs still form spheres when laminin is entrapped in the gel, but they started to spread out having a neuronal morphology after around 2 weeks. The neuronal population was assessed by the presence of the neuronal marker b-tubulin-III. This differentiation was achieved after successive steps of stimulations including FGF-2 and EGF, and then only FGF-2. Glial cells were also present. Further characterizations are under process. CONCLUSIONS RSC can be grown in 3D. Preliminary results show that neuronal cell phenotype acquisition can be instructed by exogenous stimulations and factors linked to the gel. Further developments are necessary to form a homogenous tissue containing retinal cells.

Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation.

BACKGROUND: Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent. STUDY DESIGN AND METHODS: This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma. RESULTS: We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation. CONCLUSIONS: Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly.

Compensatory up-regulation of angiotensin II subtype 1 receptors in alpha ENaC knockout heterozygous mice.

BACKGROUND: In mice, a partial loss of function of the epithelial sodium channel (ENaC), which regulates sodium excretion in the distal nephron, causes pseudohypoaldosteronism, a salt-wasting syndrome. The purpose of the present experiments was to examine how alpha ENaC knockout heterozygous (+/-) mice, which have only one allele of the gene encoding for the alpha subunit of ENaC, control their blood pressure (BP) and sodium balance. METHODS: BP, urinary electrolyte excretion, plasma renin activity, and urinary adosterone were measured in wild-type (+/+) and heterozygous (+/-) mice on a low, regular, or high sodium diet. In addition, the BP response to angiotensin II (Ang II) and to Ang II receptor blockade, and the number and affinity of Ang II subtype 1 (AT1) receptors in renal tissue were analyzed in both mouse strains on the three diets. RESULTS: In comparison with wild-type mice (+/+), alpha ENaC heterozygous mutant mice (+/-) showed an intact capacity to maintain BP and sodium balance when studied on different sodium diets. However, no change in plasma renin activity was found in response to changes in sodium intake in alpha ENaC +/- mice. On a normal salt diet, heterozygous mice had an increased vascular responsiveness to exogenous Ang II (P < 0.01). Moreover, on a normal and low sodium intake, these mice exhibited an increase in the number of AT1 receptors in renal tissues; their BP lowered markedly during the Ang II receptor blockade (P < 0.01) and there was a clear tendency for an increase in urinary aldosterone excretion. CONCLUSIONS: alpha ENaC heterozygous mice have developed an unusual mechanism of compensation leading to an activation of the renin-angiotensin system, that is, the up-regulation of AT1 receptors. This up-regulation may be due to an increase in aldosterone production.

Mechanisms underlying functional recovery after in vivo focal cerebral ischemia : from preconditioning to diffusion MRI

Version abregée L'ischémie cérébrale est la troisième cause de mort dans les pays développés, et la maladie responsable des plus sérieux handicaps neurologiques. La compréhension des bases moléculaires et anatomiques de la récupération fonctionnelle après l'ischémie cérébrale est donc extrêmement importante et représente un domaine d'intérêt crucial pour la recherche fondamentale et clinique. Durant les deux dernières décennies, les chercheurs ont tenté de combattre les effets nocifs de l'ischémie cérébrale à l'aide de substances exogènes qui, bien que testées avec succès dans le domaine expérimental, ont montré un effet contradictoire dans l'application clinique. Une approche différente mais complémentaire est de stimuler des mécanismes intrinsèques de neuroprotection en utilisant le «modèle de préconditionnement» : une brève insulte protège contre des épisodes d'ischémie plus sévères à travers la stimulation de voies de signalisation endogènes qui augmentent la résistance à l'ischémie. Cette approche peut offrir des éléments importants pour clarifier les mécanismes endogènes de neuroprotection et fournir de nouvelles stratégies pour rendre les neurones et la glie plus résistants à l'attaque ischémique cérébrale. Dans un premier temps, nous avons donc étudié les mécanismes de neuroprotection intrinsèques stimulés par la thrombine, un neuroprotecteur «préconditionnant» dont on a montré, à l'aide de modèles expérimentaux in vitro et in vivo, qu'il réduit la mort neuronale. En appliquant une technique de microchirurgie pour induire une ischémie cérébrale transitoire chez la souris, nous avons montré que la thrombine peut stimuler les voies de signalisation intracellulaire médiées par MAPK et JNK par une approche moléculaire et l'analyse in vivo d'un inhibiteur spécifique de JNK (L JNK) .Nous avons également étudié l'impact de la thrombine sur la récupération fonctionnelle après une attaque et avons pu démontrer que ces mécanismes moléculaires peuvent améliorer la récupération motrice. La deuxième partie de cette étude des mécanismes de récupération après ischémie cérébrale est basée sur l'investigation des bases anatomiques de la plasticité des connections cérébrales, soit dans le modèle animal d'ischémie transitoire, soit chez l'homme. Selon des résultats précédemment publiés par divers groupes ,nous savons que des mécanismes de plasticité aboutissant à des degrés divers de récupération fonctionnelle sont mis enjeu après une lésion ischémique. Le résultat de cette réorganisation est une nouvelle architecture fonctionnelle et structurelle, qui varie individuellement selon l'anatomie de la lésion, l'âge du sujet et la chronicité de la lésion. Le succès de toute intervention thérapeutique dépendra donc de son interaction avec la nouvelle architecture anatomique. Pour cette raison, nous avons appliqué deux techniques de diffusion en résonance magnétique qui permettent de détecter les changements de microstructure cérébrale et de connexions anatomiques suite à une attaque : IRM par tenseur de diffusion (DT-IR1V) et IRM par spectre de diffusion (DSIRM). Grâce à la DT-IRM hautement sophistiquée, nous avons pu effectuer une étude de follow-up à long terme chez des souris ayant subi une ischémie cérébrale transitoire, qui a mis en évidence que les changements microstructurels dans l'infarctus ainsi que la modification des voies anatomiques sont corrélés à la récupération fonctionnelle. De plus, nous avons observé une réorganisation axonale dans des aires où l'on détecte une augmentation d'expression d'une protéine de plasticité exprimée dans le cône de croissance des axones (GAP-43). En appliquant la même technique, nous avons également effectué deux études, rétrospective et prospective, qui ont montré comment des paramètres obtenus avec DT-IRM peuvent monitorer la rapidité de récupération et mettre en évidence un changement structurel dans les voies impliquées dans les manifestations cliniques. Dans la dernière partie de ce travail, nous avons décrit la manière dont la DS-IRM peut être appliquée dans le domaine expérimental et clinique pour étudier la plasticité cérébrale après ischémie. Abstract Ischemic stroke is the third leading cause of death in developed countries and the disease responsible for the most serious long-term neurological disability. Understanding molecular and anatomical basis of stroke recovery is, therefore, extremely important and represents a major field of interest for basic and clinical research. Over the past 2 decades, much attention has focused on counteracting noxious effect of the ischemic insult with exogenous substances (oxygen radical scavengers, AMPA and NMDA receptor antagonists, MMP inhibitors etc) which were successfully tested in the experimental field -but which turned out to have controversial effects in clinical trials. A different but complementary approach to address ischemia pathophysiology and treatment options is to stimulate and investigate intrinsic mechanisms of neuroprotection using the "preconditioning effect": applying a brief insult protects against subsequent prolonged and detrimental ischemic episodes, by up-regulating powerful endogenous pathways that increase resistance to injury. We believe that this approach might offer an important insight into the molecular mechanisms responsible for endogenous neuroprotection. In addition, results from preconditioning model experiment may provide new strategies for making brain cells "naturally" more resistant to ischemic injury and accelerate their rate of functional recovery. In the first part of this work, we investigated down-stream mechanisms of neuroprotection induced by thrombin, a well known neuroprotectant which has been demonstrated to reduce stroke-induced cell death in vitro and in vivo experimental models. Using microsurgery to induce transient brain ischemia in mice, we showed that thrombin can stimulate both MAPK and JNK intracellular pathways through a molecular biology approach and an in vivo analysis of a specific kinase inhibitor (L JNK1). We also studied thrombin's impact on functional recovery demonstrating that these molecular mechanisms could enhance post-stroke motor outcome. The second part of this study is based on investigating the anatomical basis underlying connectivity remodeling, leading to functional improvement after stroke. To do this, we used both a mouse model of experimental ischemia and human subjects with stroke. It is known from previous data published in literature, that the brain adapts to damage in a way that attempts to preserve motor function. The result of this reorganization is a new functional and structural architecture, which will vary from patient to patient depending on the anatomy of the damage, the biological age of the patient and the chronicity of the lesion. The success of any given therapeutic intervention will depend on how well it interacts with this new architecture. For this reason, we applied diffusion magnetic resonance techniques able to detect micro-structural and connectivity changes following an ischemic lesion: diffusion tensor MRI (DT-MRI) and diffusion spectrum MRI (DS-MRI). Using DT-MRI, we performed along-term follow up study of stroke mice which showed how diffusion changes in the stroke region and fiber tract remodeling is correlating with stroke recovery. In addition, axonal reorganization is shown in areas of increased plasticity related protein expression (GAP 43, growth axonal cone related protein). Applying the same technique, we then performed a retrospective and a prospective study in humans demonstrating how specific DTI parameters could help to monitor the speed of recovery and show longitudinal changes in damaged tracts involved in clinical symptoms. Finally, in the last part of this study we showed how DS-MRI could be applied both to experimental and human stroke and which perspectives it can open to further investigate post stroke plasticity.

The short-rooted phenotype of the brevis radix mutant partly reflects root abscisic acid hypersensitivity.

To gain further insight into abscisic acid (ABA) signaling and its role in growth regulation, we have screened for Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to ABA-mediated root growth inhibition. As a result, we have identified a loss-of-function allele of BREVIS RADIX (BRX) in the Columbia background, named brx-2, which shows enhanced response to ABA-mediated inhibition of root growth. BRX encodes a key regulator of cell proliferation and elongation in the root, which has been implicated in the brassinosteroid (BR) pathway as well as in the regulation of auxin-responsive gene expression. Mutants affected in BR signaling that are not impaired in root growth, such as bes1-D, bzr1-D, and bsu1-D, also showed enhanced sensitivity to ABA-mediated inhibition of root growth. Triple loss-of-function mutants affected in PP2Cs, which act as negative regulators of ABA signaling, showed impaired root growth in the absence of exogenous ABA, indicating that disturbed regulation of ABA sensitivity impairs root growth. In agreement with this result, diminishing ABA sensitivity of brx-2 by crossing it with a 35S:HAB1 ABA-insensitive line allowed significantly higher recovery of root growth after brassinolide treatment. Finally, transcriptomic analysis revealed that ABA treatment negatively affects auxin signaling in wild-type and brx-2 roots and that ABA response is globally altered in brx-2. Taken together, our results reveal an interaction between BRs, auxin, and ABA in the control of root growth and indicate that altered sensitivity to ABA is partly responsible for the brx short-root phenotype.

Molecular epidemiology of Pseudomonas aeruginosa in intensive care units over a 10-year period (1998-2007).

Pseudomonas aeruginosa is one of the leading nosocomial pathogens in intensive care units (ICUs). The source of this microorganism can be either endogenous or exogenous. The proportion of cases as a result of transmission is still debated, and its elucidation is important for implementing appropriate control measures. To understand the relative importance of exogenous vs. endogenous sources of P. aeruginosa, molecular typing was performed on all available P. aeruginosa isolated from ICU clinical and environmental specimens in 1998, 2000, 2003, 2004 and 2007. Patient samples were classified according to their P. aeruginosa genotypes into three categories: (A) identical to isolate from faucet; (B) identical to at least one other patient sample and not found in faucet; and (C) unique genotype. Cases in categories A and B were considered as possibly exogenous, and cases in category C as possibly endogenous. A mean of 34 cases per 1000 admissions per year were found to be colonized or infected by P. aeruginosa. Higher levels of faucet contamination were correlated with a higher number of cases in category A. The number of cases in category B varied from 1.9 to 20 cases per 1000 admissions. This number exceeded 10/1000 admissions on three occasions and was correlated with an outbreak on one occasion. The number of cases considered as endogenous (category C) was stable and independent of the number of cases in categories A and B. The present study shows that repeated molecular typing can help identify variations in the epidemiology of P. aeruginosa in ICU patients and guide infection control measures.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Blood transfusion in sports.

Blood transfusion is an effective and unmediated means of increasing the number of red blood cells in the circulation in order to enhance athletic performance. Blood transfusion became popular in the 1970s among elite endurance athletes and declined at the end of the 1980s with the introduction of recombinant erythropoietin. The successive implementation in 2001 of a direct test to detect exogenous erythropoietin and in 2004 of a test to detect allogeneic blood transfusion forced cheating athletes to reinfuse fully immunologically compatible blood. The implementation of indirect markers of blood doping stored in an Athlete's Biological Passport provides a powerful means to deter any form of blood transfusion.

The JAK2/STAT3 pathway in the embryonic chick heart submitted to anoxia, reoxygenation and oxidant stress

SUMMARYAim: The embryonic/fetal heart is highly sensitive to oxygenation level and a transient uteroplacental hypoperfusion can lead to oxyradicals overproduction. Information about the molecular mechanisms underlying ischemia-reperfusion (I-R) injury in the developing heart is lacking. The Janus Kinase 2 / Signal Transducer and Activator of Transcription 3 (JAK2/STAT3) pathway, required for cardiogenesis and involved in protection of the adult heart against I-R, could also play a key role in the response of the fetal myocardium to transient oxygen deprivation. The aim of the study was to characterize the involvement of JAK2/STAT3 pathway and its interaction with other signalling pathways in the developing heart transiently submitted to anoxia. Furthermore, the response of the embryonic heart to an exogenous oxidant stress (H2O2) in comparison to reoxygenation-induced endogenous oxyradicals has been investigated.Methods: Hearts isolated from 4-day-old chick embryos were submitted to anoxia (30min) and reoxygenation (80min) with or without the antioxidant MPG, the JAK2/STAT3 inhibitor AG490 or exposed to H202 (50|iM-lmM). The time course of phosphorylation of STAT3atyr0Sine7 and Reperfusion Injury Salvage Kinase (RISK) proteins (PI3K, Akt, GSK3B, Glycogen Synthase and ERK2) was determined in homogenate" and in enriched nuclear and cytoplasmic fractions. The STAT3 DNA-binding was determined by EMSA and the expression of STAT3 specific target genes by RT-PCR. The chrono-, dromo- and inotropic disturbances were also investigated by ECG and mechanical recordings.Results: Phosphorylation of STATSaP (P-Tyr STAT3a) was increased by reoxygenation and reduced by MPG or AG490. STAT3 and GSK36 were detected both in nuclear and cytoplasmic fractions while PI3K, Akt, GS and ERK2 were restricted to cytoplasm. Reoxygenation led to nuclear accumulation of STAT3 but unexpectedly without DNA- binding. AG490 decreased the reoxygenation-induced phosphorylation of STABa^, Akt, GS and ERK2 and phosphorylation/inhibition of GSK3B in the nucleus, exclusively. Inhibition of JAK2/STAT3 delayed recovery of atrial rate, worsened RR. variability and prolonged arrhythmias compared to control hearts. Cardiac activity was altered only at concentrations >500μΜ of H2O2. Moreover, ImM of H2O2 suppressed atrial activity in 45% of the hearts, atrioventricular conduction in 66% and augmented P-Tyr STAT3awhich led to an increase in the DNA-binding but no change in the expression of three STAT3 specific target genes (iNOS, MnSOD, Cox-2).Conclusion: In the developing heart, besides its nuclear translocation without transcriptional activity, ROS-activated STAT3a can rapidly interact with RISK proteins present in nucleus and cytoplasm and reduce the anoxia-reoxygenation-induced arrhythmias. Moreover, the embryonic heart is highly resistant to H2O2 and the atrial region is the less affected. The role of JAK2/STAT3 in the response to reoxygenation-induced oxyradicals is different from the response to strong exogenous oxidant stress where STAT3 DNA-binding activity is increased. Such findings provide a first step in understanding the modulation of signalling cascades in the fetal heart submitted to transient intrauterine oxygen deprivation.RESUMEIntroduction: Le coeur embryonnaire et foetal est très sensible au manque d'oxygène et une hypoperfusion utéroplacentaire transitoire peut conduire à une surproduction d'espèces radicalaires (ROS). Dans le coeur en développement les mécanismes moléculaires impliqués en situation d'ischémie-reperfusion (I-R) ne sont pas connus. La voie de signalisation JAK2/STAT3 (Janus Kinase 2 / Signal Transducer and Activator of Transcription 3), impliquée aussi bien dans la cardiogenèse précoce que dans la protection du coeur adulte contre l'I-R, pourrait jouer un rôle clé dans la réponse du myocarde foetal à un déficit en oxygène. Cette étude a permis d'étudier le rôle de la voie JAK2/STAT3 et son interaction avec d'autres voies de signalisation dans un modèle de coeur embryonnaire soumis à un épisode anoxique. En outre, les effets du stress oxydant endogène provoqué par la réoxygénation ont été comparés à ceux du stress oxydatif exogène induit par du peroxyde d'hydrogène (H2O2).Méthodes: Des coeurs isolés d'embryons de poulet âgés de 4 jours ont été soumis à une anoxie (30min) suivie d'une réoxygénation (80min) en présence ou non de l'antioxydant MPG et de l'inhibiteur de JAK2/STAT3 AG490 ou exposés à de 1Ή202 (50μΜ-1πιΜ). L'évolution temporelle de la phosphorylation de 8ΤΑΤ3α*ΓΟδίη6705 (P-Tyr STAT3a) et celle de la phosphorylation des protéines de la voie RISK (Reperfusion Injury Salvage Kinase: PI3K, Akt, GSK3B, glycogène synthase GS et ERK2) ont été déterminés dans l'homogénat et dans les fractions nucléaire et cytopiasmique du myocarde. La liaison de STAT3 à l'ADN a été déterminée par EMSA et l'expression de gènes cibles de STAT3 (iNOS, MnSOD, Cox2) par RT-PCR. Les effets chrono-, dromo- et inotropes ont été déterminés par les enregistrements de l'ECG et de l'activité contractile ventriculaire.Résultats: STAT3 et GSK3B étaient présents dans les fractions nucléaire et cytopiasmique tandis que PI3K, Akt, GS et ERK2 n'étaient détectées que dans la fraction cytopiasmique. L'augmentation de P-Tyr STAT3a provoquée par la réoxygénation était significativement réduite par le MPG ou PAG490. La réoxygénation entraînait l'accumulation nucléaire de STAT3, mais étonnamment sans liaison avec l'ADN. A la réoxygénation TAG490 diminuait la phosphorylation d'Akt, GS et ERK2 ainsi que celle de GSK36 mais exclusivement dans la fraction nucléaire. L'inhibition de JAK2/STAT3 retardait également la récupération du rythme cardiaque et prolongeait la durée des arythmies. L'activité cardiaque n'était perturbée par de ΓΗ2Ο2 qu'à des concentrations >500μΜ. A ImM, ΓΗ2Ο2 supprimait l'activité auriculaire dans 45% des coeurs et la conduction auriculo-ventriculaire dans 66% et augmentait la formation de P-Tyr STAT3a et sa liaison à l'ADN sans modifier l'expression des gènes cibles.Conclusion: Les ROS produits par l'anoxie-réoxygénation activent STAT3a qui subit une translocation dans le noyau sans se lier à l'ADN et interagit rapidement avec des protéines de la voie RISK dans les compartiments nucléaire et cytopiasmique du coeur embryonnaire. Ce dernier, en particulier au niveau des oreillettes, se révèle très résistant au puissant stress oxydatif de l'H202 qui se différencie du stress lié à la réoxygénation en favorisant la liaison de STAT3 à l'ADN. Ces résultats originaux permettent une meilleure compréhension des mécanismes qui peuvent améliorer la récupération du coeur en développement après un épisode hypoxique intra-utérin.

Nosocomial nontyphoidal salmonellosis after antineoplastic chemotherapy: reactivation of asymptomatic colonization?

An increased frequency of nontyphoidal salmonellosis is well established in cancer patients, but it is unclear whether this represents increased susceptibility to exogenous infection or opportunistic, endogenous reactivation of asymptomatic carriage. In a retrospective study, a simple case definition was used to identify the probable presence of reactivation salmonellosis in five cancer patients between 1996 and 2002. Reactivation salmonellosis was defined as the development of nosocomial diarrhea >72 h after admission and following the administration of antineoplastic chemotherapy in an HIV-seronegative cancer patient who was asymptomatic on admission, in the absence of epidemiological evidence of a nosocomial outbreak. Primary salmonellosis associated with unrecognized nosocomial transmission or community acquisition and an unusually prolonged incubation period could not entirely be ruled out. During the same time period, another opportunistic infection, Pneumocystis pneumonia, was diagnosed in six cancer patients. Presumably, asymptomatic intestinal Salmonella colonization was converted to invasive infection by chemotherapy-associated intestinal mucosal damage and altered innate immune mechanisms. According to published guidelines, stool specimens from patients hospitalized for longer than 72 h should be rejected unless the patient is neutropenic or >or=65 years old with significant comorbidity. However, in this study neutropenia was present in only one patient, and four patients were <65 years old. Guidelines should thus be revised in order not to reject stool culture specimens from such patients. In cancer patients, nosocomial salmonellosis can occur as a chemotherapy-triggered opportunistic reactivation infection that may be similar in frequency to Pneumocystis pneumonia.

Effect of physical exercise on glycogen turnover and net substrate utilization according to the nutritional state.

To determine the metabolic effects of a single bout of exercise performed after a meal or in the fasting state, nine healthy subjects were studied over two 8-h periods during which net substrate oxidation was monitored by indirect calorimetry. On one occasion, exercise was performed 90 min after ingestion of a meal labeled with [U-13C]glucose [protocol meal-exercise (M-E)]. On the second occasion, exercise was performed after an overnight fast and was followed 30 min later by ingestion of an identical meal [protocol exercise-meal (E-M)]. Energy balances were similar in both protocols, but carbohydrate balance was positive (42.2 +/- 5.1 g), and lipid balance was negative (-11.1 +/- 2.0) during E-M, whereas they were nearly even during M-E. Total glycogen synthesis was calculated as carbohydrate intake minus oxidation of exogenous 13C-labeled carbohydrate (calculated from 13CO2 production). Total glycogen synthesis was increased by 90% (from 47.6 +/- 3.8 to 90.7 +/- 5.4 g, P < 0.0001) during E-M vs. M-E. Endogenous glycogen breakdown was calculated as net carbohydrate oxidation minus oxidation of exogenous carbohydrate and was increased by 44% (from 35.8 +/- 5.6 to 51.7 +/- 6.6 g, P < 0.004) during E-M. It is concluded that exercise performed in the fasting state stimulates glycogen turnover and fat oxidation.