Subcellular localization of an intracellular serine protease of 68 kDa in Leishmania (Leishmania) amazonensis promastigotes

Here we report the subcellular localization of an intracellular serine protease of 68 kDa in axenic promastigotes of Leishmania (Leishmania) amazonensis, using subcellular fractionation, enzymatic assays, immunoblotting, and immunocytochemistry. All fractions were evaluated by transmission electron microscopy and the serine protease activity was measured during the cell fractionation procedure using a-N-r-tosyl-L-arginine methyl ester (L-TAME) as substrate, phenylmethylsulphone fluoride (PMSF) and L-1-tosylamino-2-phenylethylchloromethylketone (TPCK) as specific inhibitors. The enzymatic activity was detected mainly in a membranous vesicular fraction (6.5-fold enrichment relative to the whole homogenate), but also in a crude plasma membrane fraction (2.0-fold). Analysis by SDS-PAGE gelatin under reducing conditions demonstrated that the major proteolytic activity was found in a 68 kDa protein in all fractions studied. A protein with identical molecular weight was also recognized in immunoblots by a polyclonal antibody against serine protease (anti-SP), with higher immunoreactivity in the vesicular fraction. Electron microscopic immunolocalization using the same polyclonal antibody showed the enzyme present at the cell surface, as well as in cytoplasmic membranous compartments of the parasite. Our findings indicate that the internal location of this serine protease in L. amazonensis is mainly restricted to the membranes of intracellular compartments resembling endocytic/exocytic elements.

Electron cryo-microscopy reveals mechanism of action of propranolol on artificial membranes.

The pharmacological activity of several amphiphilic drugs is often related to their ability to interact with biological membranes. Propranolol is an efficient multidrug resistance (MDR) modulator; it is a nonselective beta-blocker and is thought to reduce hypertension by decreasing the cardiac frequency and thus blood pressure. It is used in drug delivery studies in order to treat systemic hypertension. We are interested in the interaction of propranolol with artificial membranes, as liposomes of controllable size are used as biocompatible and protective structures to encapsulate labile molecules, such as proteins, nucleic acids or drugs, for pharmaceutical, cosmetic or chemical applications. We present here a study of the interaction of propranolol, a cationic surfactant, with pure egg phosphatidylcholine (EPC) vesicles. The gradual transition from liposome to micelle of EPC vesicles in the presence of propranolol was monitored by time-resolved electron cryo-microscopy (cryo-EM) under different experimental conditions. The liposome-drug interaction was studied with varying drug/lipid (D/L) ratios and different stages were captured by direct thin-film vitrification. The time-series cryo-EM data clearly illustrate the mechanism of action of propranolol on the liposome structure: the drug disrupts the lipid bilayer by perturbing the local organization of the phospholipids. This is followed by the formation of thread-like micelles, also called worm-like micelles (WLM), and ends with the formation of spherical (globular) micelles. The overall reaction is slow, with the process taking almost two hours to be completed. The effect of a monovalent salt was also investigated by repeating the lipid-surfactant interaction experiments in the presence of KCl as an additive to the lipid/drug suspension. When KCl was added in the presence of propranolol the overall reaction was the same but with slower kinetics, suggesting that this monovalent salt affects the general lipid-to-micelle transition by stabilizing the membrane, presumably by binding to the carbonyl chains of the phosphatidylcholine.

Molecular characterization of human Trypanosoma cruzi isolates from endemic areas in Panama

The present work provides information on Trypanosoma cruzi genotype circulating in endemic areas of Chagas disease in Panama. A total of 26 crude stocks of T. cruzi, isolated from the blood of persons with different clinical profiles of Chagas disease were collected and crio-conserved until used. Most of the stocks had been characterized by means of isoenzyme electrophoresis on cellulose acetate membranes. The clinical profiles of infected persons included 9 (34.6%) asymptomatic and 17 acute (65.4%) including 5 (19.2%) fatal cases, 2 under 5 years old and 3 adults. A multiplex-PCR assay based on the amplification of the non-transcribed spacer of the mini-exon gene was performed. All stocks of T. cruzi included in the study were found to correspond to Tc I group. This result supports the predominance of T. cruzi-I in the transmission cycles affecting the human population in the Republic of Panama.

PPAR beta a player in astrocyte metabolism and morphology

AbstractPPARP is a nuclear receptor responding in vivo to several free fatty acids, and implicated in cell metabolism, differentiation and survival. PPARp is ubiquitously expressed but shows high expression in the developing and adult brain. PPARp is expressed in different cell types such as neurons and astrocytes, where it might play a role in metabolism. To study this nuclear receptor the laboratory engineered a PPARP -/- mouse model. The aim of my PhD was to dissect the role of PPARP in astrocytes.Experiments in primary culture revealed that cortical astrocytes from PPARP -/- mouse have an impaired energetic metabolism. Unstimulated PPARP -/- astrocytes exhibit a 30% diminution in glucose uptake, correlating to a 30% decrease in lactate release and intracellular glucose. After acute stimulation by D- aspartate mimicking glutamate exposure, both WT and -/- astrocytes up-regulate their metabolism to respond to the increasing energy needed (ATP) for glutamate uptake. According to the Astrocyte Neuron Lactate Shuttle Hypothesis (ANLSH), the ratio between glucose uptake/ lactate release is 1. However, stimulated PPARp -/- astrocytes display a higher increase in lactate release than glucose uptake which remains lower than in WT. The extra glucose equivalents could come from the degradation of intra cellular glycogen stores, which indeed decrease in PPARP -/- cells upon stimulation. Lower glucose metabolism correlates with a decreased acute glutamate uptake in PPARP -/- astrocytes. Reciprocally, we also observed an increase of glutamate uptake and ATP production after treatment of WT astrocytes with a PPARp agonist. Glutamate transporter protein expression is not affected. However, their trafficking and localization might be altered as PPARp -/- astrocytes have higher cholesterol levels, which may also affect proper transporter structure in the membrane.Metabolism, transporter localization and cholesterol levels are respectively linked to cell mobility, cell cytoskeleton and cellular membrane composition. All three functions are important in astrocytes to in vivo acquire star shaped morphology, in a process known as stellation. PPARP -/- astrocytes showed an impaired acquired stellation in presence of neurons or chemical stimuli, as well as more actin stress fibers and cell adhesion structures. While non stellation of astrocytes is mainly an in vitro phenomenon, it reveals PPARp -/- primary astrocytes inability to respond to different exterior stimuli. These morphological phenotypes correlate with a slower migration in cell culture wound healing assays.This thesis work demonstrates that PPARp is implicated in cortical astrocyte glucose metabolism. PPARp absence leads to an unusual intracellular glycogen use. Added to the effect on acute glutamate uptake and astrocyte migration, PPARp could be an interesting target for neuroprotection therapies.RésuméPPARP est un récepteur nucléaire qui a pour ligands naturels certains acides gras libres. Il est impliqué dans le métabolisme, la différentiation et la survie des cellules. PPARP est ubiquitaire, et a une expression élevée dans le cerveau en développement ainsi qu'adulte. PPARp est exprimé dans différents types cellulaires tels que les neurones et les astrocytes, où il régule potentiellement leurs métabolismes. Pour étudier ce récepteur nucléaire, le laboratoire a créé un modèle de souris PPARp -/-. L'objectif de ma thèse est de comprendre le rôle de PPARp dans les astrocytes.Les expériences montrent un défaut du métabolisme énergétique dans les astrocytes corticaux primaires tirés de souris PPARp -/-. Sans stimulation, l'entrée du glucose dans les astrocytes PPARP -/- est diminuée de 30% ce qui correspond à une diminution de 30% du relargage du lactate. Après stimulation par du D-Aspartate qui mime une exposition au glutamate, les astrocytes WT et -/- augmentent leur métabolisme en réponse à la demande accrue en énergie (ATP) due à l'entrée du glutamate. D'après l'Astrocyte Neuron Lactate Shuttle Hypothesis (ANLSH), le ratio entre le glucose entrant et le lactate sortant est de 1. Cependant le relargage du lactate dans les astrocytes PPARP-/- est plus élevé que l'entrée du glucose. L'apport supplémentaire de glucose transformé en lactate pourrait provenir de la dégradation des stocks de glycogène intracellulaire, qui sont partiellement diminués après stimulation dans les cellules PPARP -/-. Un métabolisme plus faible du glucose corrèle avec une réduction de l'import du glutamate dans les astrocytes PPARp -/-. Réciproquement, nous observons une augmentation de l'import du glutamate et de la production d'ATP après traitement avec l'agoniste pour PPARp. Bien que l'expression des transporteurs de glutamate ne soit pas affectée, nous ne pouvons pas exclure que leur localisation et leur structure soient altérées du fait du niveau élevé de cholestérol dans les astrocytes PPARp -/-.Le métabolisme, la localisation des transporteurs et le niveau de cholestérol sont tous liés au cytosquelette, à la mobilité, et à la composition des membranes cellulaires. Toutes ces fonctions sont importantes pour les astrocytes pour acquérir leur morphologie in vivo. Les astrocytes PPARP -/- présentent un défaut de stellation, aussi bien en présence de neurones que de stimuli chimiques, ainsi qu'un plus grand nombre de fibres de stress (actine) et de structures d'adhésion cellulaire. Bien que les astrocytes non stellaires soient principalement observés in vitro, le défaut de stellation des astrocytes primaires PPARp -/- indique une incapacité à répondre aux différents stimuli extérieurs. Ces phénotypes morphologiques corrèlent avec une migration plus lente en cas de lésion de la culture.Ce travail de thèse a permis de démontrer l'implication de PPARP dans le métabolisme du glucose des astrocytes corticaux. L'absence de ce récepteur nucléaire amène à l'utilisation du glucose intracellulaire, auquel s'ajoutent les effets sur l'import du glutamate et la migration des astrocytes. PPARp aurait des effets neuroprotecteurs, et de ce fait pourrait être utilisé à des fins thérapeutiques.

Proteomic analysis of the shistosome tegument and its surface membranes

The tegument surface of the adult schistosome, bounded by a normal plasma membrane overlain by a secreted membranocalyx, holds the key to understanding how schistosomes evade host immune responses. Recent advances in mass spectrometry (MS), and the sequencing of the Schistosoma mansoni transcriptome/genome, have facilitated schistosome proteomics. We detached the tegument from the worm body and enriched its surface membranes by differential extraction, before subjecting the preparation to liquid chromatography-based proteomics to identify its constituents. The most exposed proteins on live worms were labelled with impearmeant biotinylation reagents, and we also developed methods to isolate the membranocalyx for analysis. We identified transporters for sugars, amino acids, inorganic ions and water, which confirm the importance of the tegument plasma membrane in nutrient acquisition and solute balance. Enzymes, including phosphohydrolases, esterases and carbonic anhydrase were located with their catalytic domains external to the plasma membrane, while five tetraspanins, annexin and dysferlin were implicated in membrane architecture. In contrast, few parasite proteins could be assigned to the membranocalyx but mouse immune response proteins, including three immunoglobulins and two complement factors, were detected, plus host membrane proteins such as CD44, integrin and a complement regulatory protein, testifying to the acquisitive properties of the secreted bilayer.

Anatomic and functional outcome after 23-gauge sutureless transconjunctival vitrectomy, peeling and intravitreal triamcinolone for idiopathic macular epiretinal membranes

Purpose:To report the functional, anatomic outcome and safety profile of 23-gauge pars plana vitrectomy (PPV) combined with peeling and intravitreal injection of triamcinolone acetonide (TA) in eyes with idiopathic epiretinal membranes (ERM). Methods:Retrospective, nonrandomized study of consecutive patients who underwent 23-gauge transconjunctival sutureless PPV with subsequent membrane peeling and intravitreal TA injection for an idiopathic ERM. All patients were operated between February 2007 and February 2008 at the Jules Gonin University Eye Hospital. The minimum follow-up was 6-months. Results:Thirty-nine eyes of 39 patients were included. The mean follow-up was 7 months (SD: 2.2, range: 6-15 months). Twenty-two (56%) eyes were pseudophakic and 17 (44%) were phakic at the time of surgery. Mean preoperative intraocular pressure (IOP) was 14 mmHg (SD: 3.5). At the final follow-up mean IOP was 14.5 (SD: 2.7) that did not differ significantly from the IOP at baseline (P: 0.14- 2-tailed t test). Five patients (13%) needed temporary topical anti-glaucoma treatment.Mean preoperative BCVA was 0.28 decimal equivalent (logMAR 0.54, SD: 0.2, range: 1.0 - 0.2). and improved significantly (P <0.0001, 2-tailed t test) to a mean of 0.6 decimal equivalent (logMAR 0.22, SD: 0.16, range: 0.6 - 0) at the final follow-up. The visual acuity improved by a mean of 3.2 lines (SD 2.1, range 0- 8). Twenty-nine patients (74%) demonstrated a gain of 3 or more lines.Mean central macular thickness (CMT) was 456 µm (SD: 77) at the baseline that reduced significantly (P <0.0001, 2-tailed t test) at the final follow-up to 327µm (SD: 79). Average CMT reduction was 131µm (SD: 77, range: 36- 380 µm). A subgroup analysis of 15 selected cases that had CMT measurement 1 week after surgery demonstrated that 84% of the total final reduction in CMT occurred during the first week. Conclusions:23-gauge sutureless transconjunctival vitrectomy with the concomitant administration of intravitreal TA is a safe and effective technique for the treatment of idiopathic ERM and may speed up anatomical recovery.

Molecular detection of human astrovirus in an urban sewage treatment plant in Rio de Janeiro, Brazil

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7% of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2% and 4.3% was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

Use of the pyrophosphatase activity as a reliable tonoplast marker in maize roots.

Membranes of maize (Zea mays L., cv LG 11) roots were fractionated by sucrose (in presence or absence of Mg2+) or dextran density gradient centrifugations and the locations of organelles were determined using marker enzymes. Latent UDPase was used as a Golgi marker, catalase for the peroxysomes, cytochrome c oxidase for the mitochondria, UDP-Gal-galactosyltransferase for the amyloplast membranes and NADH-cytochrome c reductase for the ER. Two markers were selected for the plasmalemma, the vanadate-sensitive ATPase and UDP-Glc-sterolglucosyltransferase. The distributions of the PPase and vacuolar ATPase were found to be similar after density gradient centrifugation. The PPase and vacuolar ATPase activities were clearly separated from almost all the other markers tested, however, a partial association of both activities with the ER cannot be completely ruled out. The PPase of maize roots is more active and easier to measure than the vacuolar ATPase and is therefore an excellent candidate for use as a tonoplast marker.

Subcellular localization, distribution and expression of calmodulin in Zea mays roots.

The subcellular localization, distribution and the steady state level of calmodulin from maize roots (Zea mays L., cv. LG 11) were studied. To analyze the subcellular localization, 2-day old root membranes were fractionated by sucrose density gradient centrifugation and immunoblotting was done with an antibody raised against a vertebrate calmodulin (SWant) which recognized the plant calmodulin. Calmodulin was principally associated with high density fractions and particularly plasmalemma. For studying the distribution of calmodulin in various zones of Zea mays roots, a micro method of membrane preparation was developed. Most of the calmodulin was present in microsomes isolated from the root apex corresponding to the first 4 mm of a 15 +/- 2 mm root. An identical distribution was found by studying the steady state level of the protein by Northern blotting using a cDNA clone of Zea mays calmodulin.

Trypanosoma cruzi: parasite antigens sequestered in heart interstitial dendritic cells are related to persisting myocarditis in benznidazole-treated mice

We investigated whether sequestered Trypanosoma cruzi antigens found in heart interstitial dendritic cells (IDCs) contribute to the residual myocarditis found in mice following treatment with benznidazole, a specific chemotherapeutic drug. IDCs are antigen-presenting cells that are MHC-II-receptor dependent. Swiss mice were divided into two experimental groups: the 1st group was infected with the Colombian strain of T. cruzi, which is resistant to treatment with benznidazole, and the 2nd group was infected with clone 21SF-C 3, which has a medium susceptibility to the drug. Treatment of the Colombian strain group started on the 120th day post-infection and for the 21SF-C3 strain group treatment was started on the 90th day. In both groups, treatment lasted for 90 days. The animals were sacrificed either 150 or 200 days post-treatment. The myocardium was analysed by immunohistochemistry using anti-MAC3, 33D1, CD11b and CD11c monoclonal antibodies for IDCs or anti-T. cruzi purified antibodies. Parasite antigens were expressed on the IDC membranes in both treated and untreated mice. Myocarditis subsided following treatment, evidenced by both histological and morphometrical evaluation. A reduction in the number of IDCs carrying T. cruzi antigens in the treated group indicates that the elimination of parasites influences antigen presentation with concomitant decreases in inflammation. There is a correlation between the presence of T. cruzi antigens in these cells and the chronic focal, residual myocarditis seen in treated mice.

8-weeks training program attenuates mitochondrial oxidative stress in the liver of emotionally stressed rats

In recent years it has been shown that emotional stress induced by immobilization may change the balance between pro-oxidant and antioxidant factors inducing oxidative damage. On the other hand, contradictory views exist concerning the role of physical activity on redox metabolism. Consequently, the present work was designed to assess the influence of an 8-week moderate swimming training program in emotionally stressed rats. Sixty 1-month-old male albino Wistar rats weighing 125-135 g were used in this experimental study. They were divided into three groups, as Control (lot A; n=20), Stressed (lot B; n=20) and Stressed & Exercised (lot C; n=20). Rats were stressed by placing the animals in a 25 x 7 cm plastic bottle 1 h/day, 5 days a week for 8 weeks. Protein carbonyl content values in liver homogenates were significantly increased in stressed animals (0.58+/-0.02 vs 0.86+/-0.03; p=0.018) which clearly indicated that emotional stress was associated with oxidative stress. Ultrastructural alterations, predominantly mitochondrial swelling and the decrease of cristae number observed by electron microscopy represented direct evidence of membrane injury. The most striking feature of our study was that we also found differences between stressed rats and stressed rats that performed our 8 week training program. Consequently our results highlight the potential benefit of a moderate training program to reduce oxidative damage induced by emotional stress since it attenuated protein oxidation and mitochondrial alterations.

Clinical predictors for fatal pulmonary embolism in 15520 patients with venous thromboembolism - Findings from the Registro Informatizado de la Enfermedad TromboEmbolica venosa (RIETE) registry

BACKGROUND Clinical predictors for fatal pulmonary embolism (PE) in patients with venous thromboembolism have never been studied. METHODS AND RESULTS Using data from the international prospective Registro Informatizado de la Enfermedad TromboEmbolica venosa (RIETE) registry about patients with objectively confirmed symptomatic acute venous thromboembolism, we determined independent predictive factors for fatal PE. Between March 2001 and July 2006, 15520 consecutive patients (mean age+/-SD, 66.3+/-16.9 years; 49.7% men) with acute venous thromboembolism were included. Symptomatic deep-vein thrombosis without symptomatic PE was observed in 58.0% (n=9008) of patients, symptomatic nonmassive PE in 40.4% (n=6264), and symptomatic massive PE in 1.6% (n=248). At 3 months, the cumulative rates of overall mortality and fatal PE were 8.65% and 1.68%, respectively. On multivariable analysis, patients with symptomatic nonmassive PE at presentation exhibited a 5.42-fold higher risk of fatal PE compared with patients with deep-vein thrombosis without symptomatic PE (P<0.001). The risk of fatal PE was multiplied by 17.5 in patients presenting with a symptomatic massive PE. Other clinical factors independently associated with an increased risk of fatal PE were immobilization for neurological disease, age >75 years, and cancer. CONCLUSIONS PE remains a potentially fatal disease. The clinical predictors identified in the present study should be included in any clinical risk stratification scheme to optimally adapt the treatment of PE to the risk of the fatal outcome.

Identification of biologic markers of the premature rupture of fetal membranes: proteomic approach.

In obstetrics, premature rupture of the membranes (PROM) is a frequent observation which is responsible for many premature deliveries. PROM is also associated with an increased risk of fetal and maternal infections. Early diagnosis is mandatory in order to decrease such complications. Despite that current biological tests allowing the diagnosis of PROM are both sensitive and specific, contamination of the samples by maternal blood can induce false positive results. Therefore, in order to identify new potential markers of PROM (present only in amniotic blood, and absent in maternal blood), proteomic studies were undertaken on samples collected from six women at terms (pairs of maternal plasma and amniotic fluid) as well as on four samples of amniotic fluid collected from other women at the 17(th) week of gestation. All samples (N = 16) were analyzed by two-dimensional (2-D) high-resolution electrophoresis, followed by sensitive silver staining. The gel images were studied using bioinformatic tools. Analyses were focused on regions corresponding to pI between 4.5 and 7 and to molecular masses between 20 and 50 kDa. In this area, 646 +/- 113 spots were detected, and 27 spots appeared to be present on the gels of amniotic fluid, but were absent on those of maternal plasma. Nine out of these 27 spots were also observed on the gels of the four samples of amniotic fluids collected at the 17(th) week of pregnancy. Five of these 9 spots were unambiguously detected on preparative 2-D gels stained by Coomassie blue, and were identified by mass spectrometry analyses. Three spots corresponded to fragments of plasma proteins, and 2 appeared to be fragments of proteins not known to be present in plasma. These 2 proteins were agrin (SWISS-PROT: O00468) and perlecan (SWISS-PROT: P98160). Our results show that proteomics is a valuable approach to identify new potential biological markers for future PROM diagnosis.

Metabolic and physiologic effects of an endotoxin challenge in healthy obese subjects

Rapport de synthèseEnjeux et contexteL'épidémie d'obésité est un enjeu majeur de santé publique, et l'augmentation parallèle du nombre de patients obèses admis aux soins intensifs appelle à une meilleure connaissance des spécificités de la physiopathologie de cette population. De nombreuses anomalies physiologiques associées à l'obésité sont connues, notamment une inflammation sub-clinique chronique. Cependant, les connaissances concernant la réponse inflammatoire lors d'une agression des sujets obèses sont pour l'heure assez limitées. Bien que les réponses inflammatoires in vitro du tissu adipeux soient augmentées, les données in vivo sont pour l'instant non-conclusives.L'injection intraveineuse d'endotoxine est un test hautement reproductible provoquant une inflammation de durée limitée. Il s'agit d'un test validé pour l'étude in vivo lors des réponses inflammatoires. L'endotoxine est un lipopolysaccharide contenu dans les membranes externes des bactéries gram- négatives, notamment de E.Coli. Notre équipe possède une expérience de ces tests avec endotoxine acquise lors d'une série de recherches sur les propriétés modulatrices de l'inflammation des acides gras polyinsaturés oméga-3.Lors de l'élaboration de ce projet, la réponse du sujet obèse à l'endotoxine restait méconnue. L'objectif de l'essai est d'étudier les spécificités des réponses à l'endotoxine, notre hypothèse étant que les réponses physiologiques, métaboliques et endocrines sont amplifiées chez cette catégorie de sujets.Présentation de l'étudeAfin de tester notre hypothèse, nous avons conçu une étude prospective randomisée, avec 2 procédures (injection d'endotoxine vs de placebo) en cross-over: le protocole d'investigation durait chaque fois 8h. Huit volontaires obèses grade I (BMI médian de 33.8 kg/m2) sans morbidité ont été enrôlés. Les variables étudiées étaient: les fréquences cardiaque et respiratoire, la température, la tension artérielle, le débit cardiaque et la saturation veineuse en oxygène, ainsi qu'une calorimétrie indirecte en continu. Les symptômes tels que myalgie, céphalée et nausée ont également été consignés. Des marqueurs hormonaux et inflammatoires (Cortisol, ACTH, catécholamines, insuline, glucose, glucagon, leptine, TNF-alpha, IL-6 et CRP) ont été dosés de manière répétée.Statistiques : Pour limiter les effets de la variabilité inter-individuelle et permettre une comparaison des réponses, le calcul des aires sous la courbe (AUC) selon la méthode trapézoïdale a été utilisé. Le groupe étudié étant de « petite taille », bien ceci soit habituel pour les études de physiologie, et les réponses n'étant pas normalement distribuées, des tests non-paramétriques ont été appliqués : nous savons que la puissance statistique de notre étude est limitée. Considérant les désagréments majeurs (bien que rapidement réversibles) vécus par les volontaires soumis à des infections d'endotoxine, leurs réponses ont été comparées de manières qualitative à celles des non-obèse mesurés lors de précédentes études pour éviter de répliquer ces expériences désagréables et parfaitement prévisibles.Les résultats de cette étude sont parfaitement superposables à celles trouvées chez les sujets de BMI normal, invalidant notre hypothèse de départ d'une éventuelle réponse exacerbée.Conclusions et perspectivesCette étude est la première publication concernant la réponse du patient obèse à un test d'endotoxine. La similitude des résultats chez les patients obèses et non-obèses montre que l'obésité n'est pas en soi un facteur augmentant les réponses inflammatoires.Ces résultats concernent des sujets obèses sains et ne peuvent pas être extrapolés aux sujets obèses avec comorbidités, appelant à de futures investigations chez cette catégorie de patients.

Aggravation of chronic stress effects on hippocampal neurogenesis and spatial memory in LPA₁ receptor knockout mice.

BACKGROUND The lysophosphatidic acid LPA₁ receptor regulates plasticity and neurogenesis in the adult hippocampus. Here, we studied whether absence of the LPA₁ receptor modulated the detrimental effects of chronic stress on hippocampal neurogenesis and spatial memory. METHODOLOGY/PRINCIPAL FINDINGS Male LPA₁-null (NULL) and wild-type (WT) mice were assigned to control or chronic stress conditions (21 days of restraint, 3 h/day). Immunohistochemistry for bromodeoxyuridine and endogenous markers was performed to examine hippocampal cell proliferation, survival, number and maturation of young neurons, hippocampal structure and apoptosis in the hippocampus. Corticosterone levels were measured in another a separate cohort of mice. Finally, the hole-board test assessed spatial reference and working memory. Under control conditions, NULL mice showed reduced cell proliferation, a defective population of young neurons, reduced hippocampal volume and moderate spatial memory deficits. However, the primary result is that chronic stress impaired hippocampal neurogenesis in NULLs more severely than in WT mice in terms of cell proliferation; apoptosis; the number and maturation of young neurons; and both the volume and neuronal density in the granular zone. Only stressed NULLs presented hypocortisolemia. Moreover, a dramatic deficit in spatial reference memory consolidation was observed in chronically stressed NULL mice, which was in contrast to the minor effect observed in stressed WT mice. CONCLUSIONS/SIGNIFICANCE These results reveal that the absence of the LPA₁ receptor aggravates the chronic stress-induced impairment to hippocampal neurogenesis and its dependent functions. Thus, modulation of the LPA₁ receptor pathway may be of interest with respect to the treatment of stress-induced hippocampal pathology.