A highly sensitive in vitro infection assay to explore early stages of mouse mammary tumor virus infection.

Mouse mammary tumor virus (MMTV) infection of adult mice induces a strong response to superantigen (Sag) in their draining lymph nodes, which results from the presentation of Sag by MMTV-infected B cells to Sag-reactive T cells. To date, infection with physiologically relevant doses of MMTV can be detected in vivo only after several days of Sag-mediated T-cell-dependent amplification of infected B cells. Furthermore, no efficient in vitro system of detecting MMTV infection is available. Such a model would allow the dissection of the early phase of infection, the assessment of the contributions of different cell types, and the screening of large panels of molecules for their potential roles in infection and Sag response. For these reasons, we have established an in vitro model for detecting infection which is as sensitive and reproducible as the in vivo model. We found that the viral envelope (Env) protein is crucial for target cell infection but not for presentation of Sag. Furthermore, we show that infection of purified B cells with MMTV induces entry of Sag-responsive T cells into the cell cycle, while other professional antigen-presenting cells, such as dendritic cells, are much less efficient in inducing a response.

CELLULAR AND MOLECULAR STUDY OF SUBCORTICAL BAND HETEROTOPIA IN THE MOUSE MUTANT HECO

The HeCo mouse model is characterized by a subcortical heterotopia formed by misplaced neurons normally migrating into the superficial cortical layers. The mutant mouse has a tendency to epileptic seizures. In my thesis project we discovered the mutated Eml1 gene, a member of the echinoderm microtubule-associated protein (EMAP) family, in HeCo as well as in a family of three children showing complex malformation of cortical development. This discovery formed an important step in exploring the pathogenic mechanisms underlying the HeCo phenotype. In vitro results showed that during cell division the EML1 protein is associated with the midbody and a mutated version of Eml1 highlighted an important role of the protein in the astral MT array during cell cycle. In vivo, we found that already at an early age of cortical development (E13), ectopic progenitors such as RGs (PAX6) and IPCs (TBR2) accumulate in the IZ along the entire neocortex. We demonstrated that in the VZ of the HeCo mouse, spindle orientation and cell cycle exit are perturbed. In later stages (E17), RG fibers are strongly disorganized with deep layer (TBR1) and upper layer (CUX1) neurons trapped within an ectopic mass. At P3, columns of upper layer neurons were present between the heterotopia and the developing cortex; these columns were also present at P7 but at lesser extent. Time lapse video recording (E15.5) revealed that the parameters characterizing the migration of individual neurons are not disturbed in HeCo; however, this analysis showed that the density of migrating neuron was smaller in HeCo. In conclusion, truncated EML1 is likely to play a prominent role during cell cycle but also acts on the cytoskeletal architecture altering the shape of RG fibers thus influencing the pattern of neuronal migration. The signal transduction between external cues and intracellular effector pathways through MTs may be secondary but sustains the heterotopia development and further studies are needed to clarify the impact of EML1 in progenitors versus post-mitotic cells.

Intravenous fish oil blunts the physiological response to endotoxin in healthy subjects

Résumé L'administration par voie orale d'acides gras polyinsaturés de type ω-3 contenus dans l'huile de poisson exerce des effets bénéfiques sur la réponse métabolique et inflammatoire chez des sujets sains soumis à une injection d'endotoxine. Ce modèle expérimental a été validé pour l'investigation clinique. Il simule un sepsis et induit une réponse comparable à un état grippal, accompagné de modifications métaboliques et inflammatoires. L'objectif de cette étude est de déterminer les effets de l'huile de poisson administré par voie intraveineuse sur la réponse à l'endotoxine chez le sujet sain. L'hypothèse est qu'il sera possible de réduire le temps de latence en comparaison avec la voie orale. Pour ce faire, nous avons inclut dans une étude prospective randomisée 16 volontaires sains âgés de 16 à 35 ans et les avons répartis en 2 groupes : l'un recevant une émulsion lipidique contenant les acides gras polyinsaturés EPA et DHA et l'autre, sans traitement, constituant le groupe contrôle. Huit sujets reçoivent une perfusion continue de 0.5g/kg d'huile de poisson durant 6h, 48h et 24h avant la journée test. Lors de cette journée test, tous les volontaires ont reçu une dose d'endotoxine (2mg/kg) au temps t0. Les paramètres vitaux sont monitorés et enregistrés : fréquence cardiaque, respiratoire, pression artérielle, saturation artérielle en oxygène, ainsi que température. Des prises de sang sont effectuées à intervalles réguliers pour déterminer 1) l'incorporation membranaire des thrombocytes en EPA et DHA ; 2) le taux plasmatique d'hormones (insulin, glucagon, cortisol, ACTH et catécholamines), de marqueurs inflammatoires (TNF-α, IL-6, hsCRP), ainsi que de substrats énergétiques (glucose, lactate, acides gras libres et triglycérides). La dépense énergétique est déterminée par calorimétrie indirecte. L'analyse statistique est effectuée par analyse de variance (ANOVA). Les résultats montrent une incorporation significative de EPA et DHA au niveau membranaire des thrombocytes. L'huile de poisson induit une atténuation significative de la réponse neuro-endocrinienne et inflammatoire en réponse à l'injection d'endotoxine avec diminution de la fièvre (-0.7°C), ainsi que du taux plasmatique ,d'ACTH (-68%), TNF-α (-63%) et de noradrénaline (-%) dans le groupe huile de poisson. En conclusion, cette étude montre que la supplémentation de 2 doses d'huile de poisson par voie intraveineuse modifie la composition phospholipidique des membranes des thrombocytes et diminue la réaction inflammatoire et neuroendocrinienne en réponse à l'endotoxine. Ces résultats positifs ouvrent la perspective d'une supplémentation parentérale préopératoire en acides gras polyinsaturés ω-3 pour diminuer le stress lié à la chirurgie majeure.

Connexins protect mouse pancreatic β cells against apoptosis.

Type 1 diabetes develops when most insulin-producing β cells of the pancreas are killed by an autoimmune attack. The in vivo conditions modulating the sensitivity and resistance of β cells to this attack remain largely obscure. Here, we show that connexin 36 (Cx36), a trans-membrane protein that forms gap junctions between β cells in the pancreatic islets, protects mouse β cells against both cytotoxic drugs and cytokines that prevail in the islet environment at the onset of type 1 diabetes. We documented that this protection was at least partially dependent on intercellular communication, which Cx36 and other types of connexin channels establish within pancreatic islets. We further found that proinflammatory cytokines decreased expression of Cx36 and that experimental reduction or augmentation of Cx36 levels increased or decreased β cell apoptosis, respectively. Thus, we conclude that Cx36 is central to β cell protection from toxic insults.

Peroxisome proliferator-activated receptor gamma is required in mature white and brown adipocytes for their survival in the mouse.

The peroxisome proliferator-activated receptor gamma (PPARgamma) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPARgamma functions in mature adipocytes is precluded by lethality of PPARgamma(-/-) fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPARgamma in adipocytes of adult mice by using the tamoxifen-dependent Cre-ER(T2) recombination system. We show that mature PPARgamma-null white and brown adipocytes die within a few days and are replaced by newly formed PPARgamma-positive adipocytes, demonstrating that PPARgamma is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPARgamma antagonists could be used to acutely reduce obesity.

Circadian clock-dependent and -independent rhythmic proteomes implement distinct diurnal functions in mouse liver.

Diurnal oscillations of gene expression controlled by the circadian clock underlie rhythmic physiology across most living organisms. Although such rhythms have been extensively studied at the level of transcription and mRNA accumulation, little is known about the accumulation patterns of proteins. Here, we quantified temporal profiles in the murine hepatic proteome under physiological light-dark conditions using stable isotope labeling by amino acids quantitative MS. Our analysis identified over 5,000 proteins, of which several hundred showed robust diurnal oscillations with peak phases enriched in the morning and during the night and related to core hepatic physiological functions. Combined mathematical modeling of temporal protein and mRNA profiles indicated that proteins accumulate with reduced amplitudes and significant delays, consistent with protein half-life data. Moreover, a group comprising about one-half of the rhythmic proteins showed no corresponding rhythmic mRNAs, indicating significant translational or posttranslational diurnal control. Such rhythms were highly enriched in secreted proteins accumulating tightly during the night. Also, these rhythms persisted in clock-deficient animals subjected to rhythmic feeding, suggesting that food-related entrainment signals influence rhythms in circulating plasma factors.

Evolution of the neurochemical profile after transient focal cerebral ischemia in the mouse brain.

Evolution of the neurochemical profile consisting of 19 metabolites after 30 mins of middle cerebral artery occlusion was longitudinally assessed at 3, 8 and 24 h in 6 to 8 microL volumes in the striatum using localized 1H-magnetic resonance spectroscopy at 14.1 T. Profound changes were detected as early as 3 h after ischemia, which include elevated lactate levels in the presence of significant glucose concentrations, decreases in glutamate and a transient twofold glutamine increase, likely to be linked to the excitotoxic release of glutamate and conversion into glial glutamine. Interestingly, decreases in N-acetyl-aspartate (NAA), as well as in taurine, exceeded those in neuronal glutamate, suggesting that the putative neuronal marker NAA is rather a sensitive marker of neuronal viability. With further ischemia evolution, additional, more profound concentration decreases were detected, reflecting a disruption of cellular functions. We conclude that early changes in markers of energy metabolism, glutamate excitotoxicity and neuronal viability can be detected with high precision non-invasively in mice after stroke. Such investigations should lead to a better understanding and insight into the sequential early changes in the brain parenchyma after ischemia, which could be used for identifying new targets for neuroprotection.

IL-12-mediated STAT4 signaling and TCR signal strength cooperate in the induction of CD40L in human and mouse CD8(+) T cells.

CD40L is one of the key molecules bridging the activation of specific T cells and the maturation of professional and nonprofessional antigen-presenting cells including B cells. CD4(+) T cells have been regarded as the major T-cell subset that expresses CD40L upon cognate activation; however, we demonstrate here that a putative CD8(+) helper T-cell subset expressing CD40L is induced in human and murine CD8(+) T cells in vitro and in mice immunized with antigen-pulsed dendritic cells. IL-12 and STAT4-mediated signaling was the major instructive cytokine signal boosting the ability of CD8(+) T cells to express CD40L both in vitro and in vivo. Additionally, TCR signaling strength modulated CD40L expression in CD8(+) T cells after primary differentiation in vitro as well as in vivo. The induction of CD40L in CD8(+) T cells regulated by IL-12 and TCR signaling may enable CD8(+) T cells to respond autonomously of CD4(+) T cells. Thus, we propose that under proinflammatory conditions, a self-sustaining positive feedback loop could facilitate the efficient priming of T cells stimulated by high affinity peptide displaying APCs.

Intravenous pamidronate as treatment for osteoporosis after heart transplantation: a prospective study.

Fractures due to osteoporosis are one of the major complications after heart transplantation, occurring mostly during the first 6 months after the graft, with an incidence ranging from 18% to 50% for vertebral fractures. Bone mineral density (BMD) decreases dramatically following the graft, at trabecular sites as well as cortical sites. This is explained by the relatively high doses of glucocorticoids used during the months following the graft, and by a long-term increase of bone turnover which is probably due to cyclosporine. There is some evidence for a beneficial effect on BMD of antiresorptive treatments after heart transplantation. The aim of this study was to assess prospectively the effect on BMD of a 3-year treatment of quarterly infusions of 60 mg of pamidronate, combined with 1 g calcium and 1000 U vitamin D per day, in osteoporotic heart transplant recipients, and that of a treatment with calcium and vitamin D in heart transplant recipients with no osteoporosis. BMD of the lumbar spine and the femoral neck was measured by dual-energy X-ray absorptiometry in all patients every 6 months for 2 years and after 3 years. Seventeen patients, (1 woman, 16 men) aged 46+/-4 years (mean +/- SEM) received only calcium and vitamin D. A significant decrease in BMD was observed after 6 months following the graft, at the lumbar spine (- 6.6%) as well as at the femoral neck (-7.8%). After 2 years, BMD tended to recover at the lumbar spine, whereas the loss persisted after 3 years at the femoral neck. Eleven patients (1 woman and 10 men) aged 46+/-4 years (mean +/- SEM) started treatment with pamidronate on average 6 months after the graft, because they had osteoporosis of the lumbar spine and/or femoral neck (BMD T-score below -2.5 SD). Over the whole treatment period, a continuous increase in BMD at the lumbar spine was noticed, reaching 18.3% after 3 years (14.3% compared with the BMD at the time of the graft). BMD at the femoral neck was lowered in the first year by -3.4%, but recovered totally after 3 years of treatment. In conclusion, a 3-year study of treatment with pamidronate given every 3 months to patients with existing osteoporosis led to a significant increase in lumbar spine BMD and prevented loss at the femoral neck. However, since some of these patients were treated up to 14 months after the transplant, they may already have passed through the phase of most rapid bone loss. In patients who were not osteoporotic at baseline, treatment with calcium and vitamin D alone was not able to prevent the rapid bone loss that occurs immediately after transplantation.

T cell receptor V beta repertoire in mice lacking endogenous mouse mammary tumor provirus.

When endogenous mouse mammary tumor virus (MMTV) superantigens (SAg) are expressed in the first weeks of life an efficient thymic deletion of T cells expressing MMTV SAg-reactive T cell receptor (TcR) V beta segments is observed. As most inbred mouse strains and wild mice contain integrated MMTV DNA, knowing the precise extent of MMTV influence on T cell development is required in order to study T cell immunobiology in the mouse. In this report, backcross breeding between BALB.D2 (Mtv-6, -7, -8 and -9) and 38CH (Mtv-) mice was carried out to obtain animals either lacking endogenous MMTV or containing a single MMTV locus, i.e. Mtv-6, -7, -8 or -9. The TcR V beta chain (TcR V beta) usage in these mice was analyzed using monoclonal antibodies specific for TcR V beta 2, V beta 3, V beta 4, V beta 5, V beta 6, V beta 7, V beta 8, V beta 11, V beta 12 and V beta 14 segments. Both Mtv-8+ mice and Mtv-9+ mice deleted TcR V beta 5+ and V beta 11+ T cells. Moreover, we also observed the deletion of TcR V beta 12+ cells by Mtv-8 and Mtv-9 products. Mtv-6+ and Mtv-7+ animals deleted TcR V beta 3+ and V beta 5+ cells, and TcR V beta 6+, V beta 7+ and V beta 8.1+ cells, respectively. Unexpectedly, TcR V beta 8.2+ cells were also deleted in some backcross mice expressing Mtv-7. TcR V beta 8.2 reactivity to Mtv-7 was shown to be brought by the 38CH strain and to result from an amino acid substitution (Asn-->Asp) in position 19 on the TcR V beta 8.2 fragment. Reactivities of BALB.D2 TcR V beta 8.2 and 38CH TcR V beta 8.2 to the exogenous infectious viruses, MMTV(SW) and MMTV(SHN), were compared. Finally, the observation of increased frequencies of TcR V beta 2+, V beta 4+ and V beta 8+ CD4+ T cell subsets in Mtv-8+ and Mtv-9+ mice, and TcR V beta 4+ CD4+ T cells in Mtv-6+ and Mtv-7+ mice, when compared with the T cell repertoire of Mtv- mice, is consistent with the possibility that MMTV products contribute to positive selection of T cells.

Blunting the response to endotoxin in healthy sujects : effects of various dose of intravenous fish oil

Enjeux et contexte La recherche de cette dernière décennie sur les acides gras n-3 PUFA contenus dans l'huile de poisson a montré que ceux-ci, et particulièrement l'ΕΡΑ et le DHA, avaient des propriétés anti¬inflammatoires et anti arythmiques puissantes, potentiellement utiles chez les septiques et « cardiaques ». Les mécanismes sous-jacents sont nombreux, incluant l'incorporation des acides gras dans les membranes de phopholipides, la réduction de la production de médiateurs pro-inflammatoires (prostaglandines, leukotrienes, thromboxane), l'augmentation de la production de résolvines et protectines dérivées du DHA, et la régulation de voies de signalisation cellulaire. Cependant, les doses de n-3 PUFA utilisées dans les études cliniques et chez le sujet sain avant le travail de Yann-Karim Pittet étaient nettement supérieures aux doses nutritionnelles de l'ordre de 5-8 g/j par voie orale ou 1 g/kg par voie intraveineuse. De plus, la voie entérale avait la réputation de nécessiter plusieurs jours à semaines de traitement avant d'aboutir à une incorporation d'acides gras membranaire suffisante pour avoir un impact clinique; quant au temps minimal requis pour obtenir cet effet par voie IV, il était inconnu. Depuis, le développement d'émulsions lipidiques intraveineuses destinées à la nutrition parentérale a permis d'imaginer l'administration de prétraitements IV rapides. Pour les étudier, notre laboratoire a développé un modèle d'endotoxine (LPS d'E.Coli) qui mime les réponses physiologique, endocrinienne et biologique du sepsis chez le sujet sain, utilisant des doses de 2 ng/kg IV. Les réponses sont totalement réversibles en 8 heures. Dans le but de réduire à la fois la dose de lipides et le temps de perfusion, ce travail a étudié l'influence de 3 doses dégressives de n-3 PUFA sur les réponses à l'endotoxine, et sur l'incorporation membranaire de ces acides gras. Méthodes Etude prospective chez 3 groupes consécutifs de sujets sains soumis à un challenge d'endotoxine. Intervention : perfusions d'huile de poisson (0.5 et 0.2 g/kg de n-3 PUFA, Omegaven® 10%) ou placebo, administrées en 3 heures ou en 1 heure, soit le jour avant ou le jour-même du test d'endotoxine. Mesures : variables physiologiques (T°, fc, tension artérielle, calorimétrie indirecte) Laboratoire - prises de sang à T0, 60, 120 et 360 min après l'injection de LPS: TNF-α, hs-CRP, hormones de stress, composition en acides gras des membranes plaquettaires. Statistiques Les résultats ont été rapportés en moyennes et écarts types. Des aires sous la courbe (AUC) ont été calculées avec la méthode des parallélépipèdes pour toutes les variables déterminées de manière répétée. L'effet du temps a été exploré par des two-way ANOVA pour mesures répétées. Les comparaisons post-hoc ont été réalisées avec des tests de Dunnett's ou de Scheffe. Les modifications de composition membranaires ainsi que les AUC ont été analysées par des tests non-paramétriques (Kruskal-Wallis). Résultats Après LPS, la température, les concentrations d'ACTH et TNF-α ont augmenté dans les 3 groupes. Ces réponse ont été significativement atténuées (p<0.0001) par l'huile de poisson comparé à ce que nous avions observé dans le groupe contrôle de Pluess et al (ICM 2007). Les concentrations les plus faibles d'ACTH, de TNF-α, et les AUC les plus basses des températures, ont été observées après une dose unique de 0.2 g/kg de n-3 PUFA administrée 1 heure avant le LPS. Par contre, l'incorporation membranaire d'EPA est dose-dépendante. Conclusions Sachant que la réponse à l'endotoxine est reproductible, cette étude montre que 3 doses différentes d'huile de poisson atténuent de manière différente cette réponse. La perfusion de 0.2 g/kg administrée juste avant l'endotoxine s'est avérée la plus efficace à atténuer la réponse fébrile, les cytokines et les hormones de stress, suggérant une capture de l'endotoxine par l'émulsion lipidique qui se surajoute aux effets systémiques et membranaires.

Longitudinal neurochemical modifications in the aging mouse brain measured in vivo by (1)H magnetic resonance spectroscopy.

Alterations to brain homeostasis during development are reflected in the neurochemical profile determined noninvasively by (1)H magnetic resonance spectroscopy. We determined longitudinal biochemical modifications in the cortex, hippocampus, and striatum of C57BL/6 mice aged between 3 and 24 months . The regional neurochemical profile evolution indicated that aging induces general modifications of neurotransmission processes (reduced GABA and glutamate), primary energy metabolism (altered glucose, alanine, and lactate) and turnover of lipid membranes (modification of choline-containing compounds and phosphorylethanolamine), which are all probably involved in the frequently observed age-related cognitive decline. Interestingly, the neurochemical profile was different in male and female mice, particularly in the levels of taurine that may be under the control of estrogen receptors. These neurochemical profiles constitute the basal concentrations in cortex, hippocampus, and striatum of healthy aging male and female mice.

The role of macrophage migration inhibitory factor in mouse islet transplantation.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by many tissues including pancreatic beta-cells. METHODS: This study investigates the impact of MIF on islet transplantation using MIF knock-out (MIFko) mice. RESULTS: Early islet function, assessed with a syngeneic marginal islet mass transplant model, was enhanced when using MIFko islets (P<0.05 compared with wild-type [WT] controls). This result was supported by increased in vitro resistance of MIFko islets to apoptosis (terminal deoxynucleotide tranferase-mediated dUTP nick-end labeling assay), and by improved glucose metabolism (lower blood glucose levels, reduced glucose areas under curve and higher insulin release during intraperitoneal glucose challenges, and in vitro in the absence of MIF, P<0.01). The beneficial impact of MIFko islets was insufficient to delay allogeneic islet rejection. However, the rejection of WT islet allografts was marginally delayed in MIFko recipients by 6 days when compared with WT recipient (P<0.05). This effect is supported by the lower activity of MIF-deficient macrophages, assessed in vitro and in vivo by cotransplantation of islet/macrophages. Leukocyte infiltration of the graft and donor-specific lymphocyte activity (mixed lymphocyte reaction, interferon gamma ELISPOT) were similar in both groups. CONCLUSION: These data indicate that targeting MIF has the potential to improve early function after syngeneic islet transplantation, but has only a marginal impact on allogeneic rejection.