Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa.

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.

Phenotypic switching in Pseudomonas brassicacearum involves GacS- and GacA-dependent Rsm small RNAs.

The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonization under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as noncoding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small RNA (sRNA) genes rsmX, rsmY, and rsmZ. Naturally occurring mutations in the gacS-gacA system accounted for phenotypic switching, which was characterized by downregulation of antifungal secondary metabolites (2,4-diacetylphloroglucinol and cyanide), indoleacetate, exoenzymes (lipase and protease), and three different N-acyl-homoserine lactone molecules. Moreover, in addition to abrogating these biocontrol traits, gacS and gacA mutations resulted in reduced expression of the type VI secretion machinery, alginate biosynthesis, and biofilm formation. In a gacA mutant, the expression of rsmX was completely abolished, unlike that of rsmY and rsmZ. Overexpression of any of the three sRNAs in the gacA mutant overruled the pleiotropic changes and restored the wild-type phenotypes, suggesting functional redundancy of these sRNAs. In conclusion, our data show that phenotypic switching in P. brassicacearum results from mutations in the gacS-gacA system.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

Two GacA-dependent small RNAs modulate the quorum-sensing response in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

Adiposity in children born small for gestational age.

Epidemiological studies indicate that children born small for gestational age (SGA) have an increased risk of metabolic and cardiovascular disorders as adults. This suggests that foetal undernutrition leads to permanent metabolic alterations, which predispose to metabolic abnormalities upon exposure to environmental factors such as low physical activity and/or high-energy intake in later life (thrifty phenotype hypothesis). However, this relationship is not restricted to foetal undernutrition or intrauterine growth retardation, but is also found for children born premature, or for high birth weight children. Furthermore, early post-natal nutrition, and more specifically catch-up growth, appear to modulate cardiovascular risk as well. Intrauterine growth retardation can be induced in animal models by energy/protein restriction, or ligation of uterine arteries. In such models, altered glucose homeostasis, including low beta-cell mass, low insulin secretion and insulin resistance is observed after a few weeks of age. In humans, several studies have confirmed that children born SGA have insulin resistance as adolescents and young adults. Alterations of glucose homeostasis and increased lipid oxidation can indeed be observed already in non-diabetic children born SGA at early pubertal stages. These children also have alterations of stature and changes in body composition (increased fat mass), which may contribute to the pathogenesis of insulin resistance. Permanent metabolic changes induced by foetal/early neonatal nutrition (metabolic inprinting) may involve modulation of gene expression through DNA methylation, or alterations of organ structure. It is also possible that events occurring during foetal/neonatal development lead to long-lasting alterations of the hypothalamo-pituitary-adrenal axis or the hypothalamo-pituitary-insulin-like growth factor-1 axis.

New insights into the role of microRNAs in pancreatic ß-cell maturation and plasticity

Le diabète est une maladie chronique caractérisée par une élévation du taux de sucre dans le sang aussi appelé « glycémie » reflétant un état pathologique. L'élévation de la glycémie au long cours a des répercussions délétères sur nombreux de nos tissus et organes d'où l'apparition de complications sévères chez les sujets diabétiques pouvant atteindre les yeux, les reins, le système nerveux, le système cardiovasculaire et les membres inférieurs. La carence en une hormone essentielle à notre organisme, l'insuline, est au coeur du développement de la maladie. L'insuline induit la captation du glucose circulant dans le sang en excès suite à une prise alimentaire riche en glucides et favorise son utilisation et éventuellement son stockage dans les tissus tels que le foie, le tissu adipeux et les muscles. Ainsi, l'insuline est vitale pour réguler et maintenir stable notre niveau de glycémie. Les cellules bêta du pancréas sont les seules entités de notre corps capables de produire de l'insuline et une perte de fonctionnalité associée à leur destruction ont été mises en cause dans le processus pathologique du diabète de type 2. Cependant la pleine fonctionnalité et la maturation des cellules bêta n'apparaissent qu'après la naissance lorsque le pancréas en développement a atteint sa masse adulte définitive. Enfin, une fois la masse des cellules bêta définitive établie, leur nombre et volume restent relativement constants au cours de la vie adulte chez un sujet sain. Néanmoins, au cours de périodes critiques les besoins en insuline sont augmentés tel qu'observé chez les femmes enceintes et les personnes obèses qui ont une perte de sensibilité à l'insuline qui se traduit par la nécessité de sécréter plus d'insuline afin de maintenir une glycémie normale. Dans l'hypothèse où la compensation n'a pas lieu ou n'est pas aboutie, le diabète se développe. Le processus de maturation postnatale ainsi que les événements compensatoires sont donc des étapes essentielles et de nombreuses questions sont encore non résolues concernant l'identification des mécanismes les régulant. Parmi les acteurs potentiels figurent de petites molécules d'ARN découvertes récemment appelées microARNs et qui ont été rapidement suggérées très prometteuses dans l'identification de nouvelles cibles thérapeutiques dans le cadre du diabète et d'autres pathologies. Les microARNs vont réguler l'expression de notre génome sans en modifier la séquence, phénomène également appelé épigénétique, ce qui résulte en des différences de comportement et de fonction cellulaires. Les microARNs sont donc susceptibles de jouer un rôle clé dans l'ensemble des processus biologiques et notre environnement associé à nos prédispositions génétiques peuvent grandement modifier leur niveau et donc leur action, qui à son tour se répercutera sur notre état physiologique. En effet nous avons identifié des changements de microARNs dans les cellules d'îlots pancréatiques de modèles animaux (rats et souris) associés à un état de résistance à l'insuline (grossesse et obésité). Par le biais d'expériences in vitro sur des cellules bêta extraites de rats et conservées en culture, nous avons pu analyser de plus près l'implication des microARNs dans la capacité des cellules bêta à sécréter de l'insuline mais aussi à se multiplier et à survivre au sein d'un environnement toxique. Ainsi, nous avons identifié des microARNs qui participent positivement à la compensation des cellules bêta, sous la direction d'hormones telles les estrogènes ou d'une hormone libérée par l'intestin au cours de la digestion (l'inerétine GLP1) et qui est largement utilisée comme agent thérapeutique dans la médication contre le diabète. Dans un second temps nous avons utilisé une stratégie similaire afin de déterminer le rôle de microARNs préalablement détectés comme étant changés au cours du développement postnatal des cellules bêta chez le rat. Cette étude a également mené à l'identification de microARNs participant à la maturation et à l'expansion de la masse des cellules bêta sous l'influence de la composition du régime alimentaire et des besoins en insuline adéquats qui en dépendent. Ces études apportent la vision de nouveaux mécanismes moléculaires impliquant les microARNs et démontrant leur importance pour le bon fonctionnement des cellules bêta et leur capacité d'adaptation à l'environnement. -- Les cellules bêta sont une composante des îlots pancréatiques de Langerhans et sont des cellules hautement différenciées qui ont l'unique capacité de sécréter de l'insuline sous l'influence des nutriments suite à une prise alimentaire. L'insuline facilite l'incorporation de glucose dans ses tissus cibles tels le foie, le tissu adipeux et les muscles. Bien que les besoins en insuline soient relativement constants au cours de la vie d'un individu sain, certaines conditions associées à un état de résistance à l'insuline, telles la grossesse ou l'obésité, requièrent une libération d'insuline majorée. En cas de résistance à l'insuline, une dysfonction des cellules bêta plus ou moins associée à leur mort cellulaire, conduisent à une sécrétion d'insuline insuffisante et au développement d'une hyperglycémie chronique, caractéristique du diabète de type 2. Jusqu'à présent, les mécanismes moléculaires sous- jacents à la compensation des cellules bêta ou encore menant à leur dysfonction restent peu connus. Découverts récemment, les petits ARNs non-codant appelés microARNs (miARNs), suscitent un intérêt grandissant de par leur potentiel thérapeutique pour la prise en charge et le traitement du diabète. Les miARNs sont de puissants régulateurs de l'expression génique qui lient directement le 3'UTR de leurs ARN messagers cibles afin d'inhiber leur traduction ou d'induire leur dégradation, ce qui leur permet de contrôler des fonctions biologiques multiples. Ainsi, nous avons pris pour hypothèse que les miARNs pourraient jouer un rôle essentiel en maintenant la fonction des cellules bêta et des processus compensatoires afin de prévenir le développement du diabète. Lors d'une première étude, une analyse transcriptomique a permis l'identification de miARNs différemment exprimés au sein d'îlots pancréatiques de rattes gestantes. Parmi eux, le miR-338-3p a démontré la capacité de promouvoir la prolifération et la survie des cellules bêta exposées à des acides gras saturés et des cytokines pro-inflammatoires, sans altérer leur propriété sécrétrice d'insuline. Nous avons également identifié deux hormones reconnues pour leurs propriétés bénéfiques pour la physiologie de la cellule bêta, l'estradiol et l'incrétine GLP1, qui régulent les niveaux du miR-338-3p. Ce miARN intègre parfaitement les voies de signalisation de ces deux hormones dépendantes de l'AMP cyclique, afin de contrôler l'expression de nombreux gènes conduisant à son action biologique. Dans un projet ultérieur, notre objectif était de déterminer la contribution de miARNs dans l'acquisition de l'identité fonctionnelle des cellules bêta en période postnatale. En effet, directement après la naissance les cellules bêta sont reconnues pour être encore immatures et incapables de sécréter de l'insuline spécifiquement en réponse à l'élévation de la glycémie. Au contraire, la réponse insulinique induite par les acides aminés ainsi que la biosynthèse d'insuline sont déjà fonctionnelles. Nos recherches ont permis de montrer que les changements de miARNs corrélés avec l'apparition du phénotype sécrétoire en réponse au glucose, sont régis par la composition nutritionnelle du régime alimentaire et des besoins en insuline qui en découlent. En parallèle, le taux de prolifération des cellules bêta est considérablement réduit. Les miARNs que nous avons étudiés coordonnent des changements d'expression de gènes clés impliqués dans l'acquisition de propriétés vitales de la cellule bêta et dans la maintenancé de son identité propre. Enfin, ces études ont permis de clairement démontrer l'importance des miARNs dans la régulation de la fonction des cellules bêta pancréatiques. -- Beta-cells are highly differentiated cells localized in the pancreatic islets and are characterized by the unique property of secreting insulin in response to nutrient stimulation after meal intake. Insulin is then in charge of facilitating glucose uptake by insulin target tissues such as liver, adipose tissue and muscles. Despite insulin needs stay more or less constant throughout life of healthy individuals, there are circumstances such as during pregnancy or obesity which are associated to insulin resistance, where insulin needs are increased. In this context, defects in beta-cell function, sometimes associated with beta-cell loss, may result in the release of inappropriate amounts of insulin leading to chronic hyperglycemia, properly defined as type 2 diabetes mellitus. So far, the mechanisms underlying beta- cell compensation as well as beta-cell failure remain to be established. The recently discovered small non-coding RNAs called microRNAs (miRNAs) are emerging as interesting therapeutic targets and are bringing new hope for the treatment of diabetes. miRNAs display a massive potential in regulating gene expression by directly binding to the 3'UTR of messenger RNAs and by inhibiting their translation and/or stability, enabling them to modify a wide range of biological functions. In view of this, we hypothesized that miRNAs may play an essential role in preserving the functional beta-cell mass and permitting to fight against beta-cell exhaustion and decompensation that can lead to diabetes development. In a first study, global profiling in pancreatic islets of pregnant rats, a model of insulin resistance, led to the identification of a set of differentially expressed miRNAs. Among them, miR-338- 3p was found to promote beta-cell proliferation and survival upon exposure of islet cells to pro- apoptotic stimuli such as saturated fatty acids or pro-inflammatory cytokines, without impairment in their capacity to release insulin. We also discovered that miR-338-3p changes are driven by two hormones, the estradiol and the incretin GLP1, both well known for their beneficial impact on beta- cell physiology. Consistently, we found that miR-338-3p integrates the cAMP-dependent signaling pathways regulated by these two hormones in order to control the expression of numerous genes and execute its biological functions. In a second project, we aimed at determining whether miRNAs contribute to the acquisition of beta-cell identity. Indeed, we confirmed that right after birth beta-cells are still immature and are unable to secrete insulin specifically in response to elevated concentrations of glucose. In contrast, amino acid-stimulated insulin release as well as insulin biosynthesis are already fully functional. In parallel, newborn beta-cells are proliferating intensively within the expanding pancreas. Interestingly, we demonstrated that the miRNA changes and the subsequent acquisition of glucose responsiveness is influenced by the diet composition and the resulting insulin needs. At the same time, beta-cell proliferation declines. The miRNAs that we have identified orchestrate expression changes of essential genes involved in the acquisition of specific beta-cell properties and in the maintenance of a mature beta-cell identity. Altogether, these studies clearly demonstrate that miRNAs play important roles in the regulation of beta-cell function.

Proliferation is a prerequisite for bacterial superantigen-induced T cell apoptosis in vivo.

Staphylococcal enterotoxin B (SEB) is a bacterial superantigen that binds to major histocompatibility complex class II molecules and selectively interacts with T cells that bear certain T cell receptor (TCR) V beta domains. Administration of SEB in adult mice results in initial proliferation of V beta 8+ T cells followed by a state of unresponsiveness resulting from a combination of clonal deletion and clonal anergy in the SEB-reactive population. At this time, it is unclear what relationship exists between the T cells that have proliferated and those that have been deleted or have become anergic. Here we show that only a fraction of the potentially reactive V beta 8+ T cells proliferate in response to SEB in vivo, and that all the cells that have proliferated eventually undergo apoptosis. Virtually no apoptosis can be detected in the nonproliferating V beta 8+ T cells. These data demonstrate a causal relationship between proliferation and apoptosis in response to SEB in vivo, and they further indicate that T cells bearing the same TCR V beta segment can respond differently to the same superantigen. The implications of this differential responsiveness in terms of activation and tolerance are discussed.

Targeting the PI3K p110alpha isoform inhibits medulloblastoma proliferation, chemoresistance, and migration.

PURPOSE: The phosphoinositide 3-kinase (PI3K)/Akt pathway is frequently activated in human cancer and plays a crucial role in medulloblastoma biology. We were interested in gaining further insight into the potential of targeting PI3K/Akt signaling as a novel antiproliferative approach in medulloblastoma. EXPERIMENTAL DESIGN: The expression pattern and functions of class I(A) PI3K isoforms were investigated in medulloblastoma tumour samples and cell lines. Effects on cell survival and downstream signaling were analyzed following down-regulation of p110alpha, p110beta, or p110delta by means of RNA interference or inhibition with isoform-specific PI3K inhibitors. RESULTS: Overexpression of the catalytic p110alpha isoform was detected in a panel of primary medulloblastoma samples and cell lines compared with normal brain tissue. Down-regulation of p110alpha expression by RNA interference impaired the growth of medulloblastoma cells, induced apoptosis, and led to decreased migratory capacity of the cells. This effect was selective, because RNA interference targeting of p110beta or p110delta did not result in a comparable impairment of DAOY cell survival. Isoform-specific p110alpha inhibitors also impaired medulloblastoma cell proliferation and sensitized the cells to chemotherapy. Medulloblastoma cells treated with p110alpha inhibitors further displayed reduced activation of Akt and the ribosomal protein S6 kinase in response to stimulation with hepatocyte growth factor and insulin-like growth factor-I. CONCLUSIONS: Together, our data reveal a novel function of p110alpha in medulloblastoma growth and survival.

Small dense lipoproteins, apolipoprotein B, and risk of coronary events in HIV-infected patients on antiretroviral therapy: the Swiss HIV Cohort Study.

OBJECTIVES: HIV infection and exposure to certain antiretroviral drugs is associated with dyslipidemia and increased risk for coronary events. Whether this risk is mediated by highly atherogenic lipoproteins is unclear. We investigated the association of highly atherogenic small dense low-density lipoproteins (LDLs) and apolipoprotein B and coronary events in HIV-infected individuals receiving antiretroviral therapy. METHODS: We conducted a case-control study nested into the Swiss HIV Cohort Study to investigate the association of small dense LDL and apolipoprotein B and coronary events in 98 antiretroviral drug-treated patients with a first coronary event (19 fatal and 79 nonfatal coronary events with 53 definite and 15 possible myocardial infarctions, 11 angioplasties or bypasses) and 393 treated controls matched for age, gender, and smoking status. Lipids were measured by ultracentrifugation. RESULTS: In models including cholesterol, triglycerides, high-density lipoprotein cholesterol, blood pressure, central obesity, diabetes, and family history, there was an independent association between small dense LDL and coronary events [odds ratio (OR) for 1 mg/dL increase: 1.06, 95% confidence interval (CI): 1.00 to 1.11] and apolipoprotein B (OR for 10 mg/dL increase: 1.16, 95% CI: 1.02 to 1.32). When adding HIV and antiretroviral therapy-related variables, ORs were 1.04 (95% CI: 0.99 to 1.10) for small dense LDL and 1.13 (95% CI: 0.99 to 1.30) for apolipoprotein B. In both models, blood pressure and HIV viral load was independently associated with the odds for coronary events. CONCLUSIONS: HIV-infected patients receiving antiretroviral therapy with elevate small dense LDL and apolipoprotein B are at increased risk for coronary events as are patients without sustained HIV suppression.

Factors promoting the exposure to bioaerosols among crop workers

Introduction. Agricultural workers are among the professional groups most at risk of developing acute or chronic respiratory problems. Despite this fact, the etiology of these occupational diseases is poorly known, even in important sectors of agriculture such as the crops sector. Cereals can be colonized by a large number of fungal species throughout the plants' growth, but also during grain storage. Some of these fungi deliver toxins that can have a serious impact on human health when they are ingested via wheat products. Although International and European legislation on contaminants in food, including mycotoxins, include measures to ensure protection of public health by setting down the maximum levels for certain contaminants, the risks associated with the inhalation of such molecules during grain handling remains poorly documented. Goal of study. This project's objective was to characterize worker exposure to pathogenic, irritative or allergenic microorganisms and to identify the abiotic or biotic factors that reduce the growth of these microorganisms in crops. Indeed, the proliferation of microorganisms on wheat is dependent on temperature, rainfall and human disturbance (e.g. usage of tillage, addition of fungicides). A change in the concentration of these microorganisms in the substrate will directly result in a change in the concentration of aerosolized particles of the same microorganisms. Therefore, the exposure of worker to bioaérosols will also change. The Vaud region of Switzerland is a perfect region for conduct such a project as weather conditions vary and agricultural land management programs are divers at a small geographic scale. Methods. Bioaerosols and wheat dust have been sampled during wheat harvesting of summer 2010 at 100 sites uniformly distributed in the Vaud region that are representative of the different agriculture practices. Personal exposure has been evaluated for different wheat related activities: harvesting, grain unload, baling straw, the cleaning of harvesters and silos. Aerosols have been sampled at a rate of 2L/min between 15 min to 4 hours (t) on a 5m PVC filter for estimating the total dust inhaled, on gelatine filter for the identification and quantification of molds, and on a 0.45um polycarbonate filter for endotoxin quantification. Altitude, temperature and annual average rainfall were considered for each site. The physical and chemical characteristics of soils were determined using the methods in effect at Sol Council (Nyon). Total dust has been quantified following NIOSH 0500 method. Reactive endotoxine activity has been determined with Limulus Amebocyte Lysate Assay. All molds have been identified by the pyrosequencing of ITS2 amplicons generated from bioaerosol or wheat dust genomic DNA. Results & Conclusions. Our results confirm the previous quantitative data on the worker exposure to wheat dust. In addition, they show that crop workers are systematically exposed to complex mixtures of allergens, irritants or cytotoxic components. The novelty of our study is the systematic detection of molds such as Fusarium - that is a mycotoxins producer - in the bioaerosols. The results are interpreted by taking in account the agriculture practice, the Phosphorus : Carbon : Nitrogen ratio of the soil, the altitude and the average of rainy days per year.

Meta-analysis of estrogen response in MCF-7 distinguishes early target genes involved in signaling and cell proliferation from later target genes involved in cell cycle and DNA repair.

ABSTRACT: BACKGROUND: Many studies have been published outlining the global effects of 17 beta-estradiol (E2) on gene expression in human epithelial breast cancer derived MCF-7 cells. These studies show large variation in results, reporting between ~100 and ~1500 genes regulated by E2, with poor overlap. RESULTS: We performed a meta-analysis of these expression studies, using the Rank product method to obtain a more accurate and stable list of the differentially expressed genes, and of pathways regulated by E2. We analyzed 9 time-series data sets, concentrating on response at 3-4 hrs (early) and at 24 hrs (late). We found >1000 statistically significant probe sets after correction for multiple testing at 3-4 hrs, and >2000 significant probe sets at 24 hrs. Differentially expressed genes were examined by pathway analysis. This revealed 15 early response pathways, mostly related to cell signaling and proliferation, and 20 late response pathways, mostly related to breast cancer, cell division, DNA repair and recombination. CONCLUSIONS: Our results show that meta-analysis identified more differentially expressed genes than the individual studies, and that these genes act together in networks. These results provide new insight into E2 regulated mechanisms, especially in the context of breast cancer.

Production of Pseudomonas aeruginosa Intercellular Small Signaling Molecules in Human Burn Wounds

Pseudomonas aeruginosa has developed a complex cell-to-cell communication system that relies on low-molecular weight excreted molecules to control the production of its virulence factors. We previously characterized the transcriptional regulator MvfR, that controls a major network of acute virulence functions in P. aeruginosa through the control of its ligands, the 4-hydroxy-2-alkylquinolines (HAQs)-4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS). Though HHQ and PQS are produced in infected animals, their ratios differ from those in bacterial cultures. Because these molecules are critical for the potency of activation of acute virulence functions, here we investigated whether they are also produced during human P. aeruginosa acute wound infection and whether their ratio is similar to that observed in P. aeruginosa-infected mice. We found that a clinically relevant P. aeruginosa isolate produced detectable levels of HAQs with ratios of HHQ and PQS that were similar to those produced in burned and infected animals, and not resembling ratios in bacterial cultures. These molecules could be isolated from wound tissue as well as from drainage liquid. These results demonstrate for the first time that HAQs can be isolated and quantified from acute human wound infection sites and validate the relevance of previous studies conducted in mammalian models of infection.

TCRgamma silencing during alphabeta T cell development depends upon pre-TCR-induced proliferation.

During thymus development, immature T cells become committed to two distinct lineages based upon expression of alphabeta or gammadelta TCR. In the alphabeta lineage, developing thymocytes progressively extinguish transcription of the TCRgamma genes by a poorly understood process known as gamma silencing. We show that alphabeta lineage thymocytes in mice lacking a functional pre-TCR undergo limited proliferation and fail to silence TCRgamma genes during development. Stimulation of pre-TCR-deficient immature thymocytes with anti-CD3 Abs does not directly down-regulate TCRgamma transcription but restores TCRgamma silencing following proliferation. Collectively our data reveal an important role for pre-TCR induced proliferation in activating the TCRgamma silencer in alphabeta lineage thymocytes, a process that may reinforce alphabeta or gammadelta lineage commitment.

Large A-fiber activity is required for microglial proliferation and p38 MAPK activation in the spinal cord: different effects of resiniferatoxin and bupivacaine on spinal microglial changes after spared nerve injury.

BACKGROUND: After peripheral nerve injury, spontaneous ectopic activity arising from the peripheral axons plays an important role in inducing central sensitization and neuropathic pain. Recent evidence indicates that activation of spinal cord microglia also contributes to the development of neuropathic pain. In particular, activation of p38 mitogen-activated protein kinase (MAPK) in spinal microglia is required for the development of mechanical allodynia. However, activity-dependent activation of microglia after nerve injury has not been fully addressed. To determine whether spontaneous activity from C- or A-fibers is required for microglial activation, we used resiniferatoxin (RTX) to block the conduction of transient receptor potential vanilloid subtype 1 (TRPV1) positive fibers (mostly C- and Adelta-fibers) and bupivacaine microspheres to block all fibers of the sciatic nerve in rats before spared nerve injury (SNI), and observed spinal microglial changes 2 days later. RESULTS: SNI induced robust mechanical allodynia and p38 activation in spinal microglia. SNI also induced marked cell proliferation in the spinal cord, and all the proliferating cells (BrdU+) were microglia (Iba1+). Bupivacaine induced a complete sensory and motor blockade and also significantly inhibited p38 activation and microglial proliferation in the spinal cord. In contrast, and although it produced an efficient nociceptive block, RTX failed to inhibit p38 activation and microglial proliferation in the spinal cord. CONCLUSION: (1) Blocking peripheral input in TRPV1-positive fibers (presumably C-fibers) is not enough to prevent nerve injury-induced spinal microglial activation. (2) Peripheral input from large myelinated fibers is important for microglial activation. (3) Microglial activation is associated with mechanical allodynia.

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.