Anti-vascular endothelial growth factor acts on retinal microglia/macrophage activation in a rat model of ocular inflammation.

PURPOSE: To evaluate whether anti-vascular endothelial growth factor (VEGF) neutralizing antibodies injected in the vitreous of rat eyes influence retinal microglia and macrophage activation. To dissociate the effect of anti-VEGF on microglia and macrophages subsequent to its antiangiogenic effect, we chose a model of acute intraocular inflammation. METHODS: Lewis rats were challenged with systemic lipopolysaccharide (LPS) injection and concomitantly received 5 µl of rat anti-VEGF-neutralizing antibody (1.5 mg/ml) in the vitreous. Rat immunoglobulin G (IgG) isotype was used as the control. The effect of anti-VEGF was evaluated at 24 and 48 h clinically (uveitis scores), biologically (cytokine multiplex analysis in ocular media), and histologically (inflammatory cell counts on eye sections). Microglia and macrophages were immunodetected with ionized calcium-binding adaptor molecule 1 (IBA1) staining and counted based on their differential shapes (round amoeboid or ramified dendritiform) on sections and flatmounted retinas using confocal imaging and automatic quantification. Activation of microglia was also evaluated with inducible nitric oxide synthase (iNOS) and IBA1 coimmunostaining. Coimmunolocalization of VEGF receptor 1 and 2 (VEGF-R1 and R2) with IBA1 was performed on eye sections with or without anti-VEGF treatment. RESULTS: Neutralizing rat anti-VEGF antibodies significantly decreased ocular VEGF levels but did not decrease the endotoxin-induced uveitis (EIU) clinical score or the number of infiltrating cells and cytokines in ocular media (interleukin [IL]-1β, IL-6, tumor necrosis factor [TNF]-α, and monocyte chemoattractant protein [MCP]-1). Eyes treated with anti-VEGF showed a significantly decreased number of activated microglia and macrophages in the retina and the choroid and decreased iNOS-positive microglia. IBA1-positive cells expressed VEGF-R1 and R2 in the inflamed retina. CONCLUSIONS: Microglia and macrophages expressed VEGF receptors, and intravitreous anti-VEGF influenced the microglia and macrophage activation state. Taking into account that anti-VEGF drugs are repeatedly injected in the vitreous of patients with retinal diseases, part of their effects could result from unsuspected modulation of the microglia activation state. This should be further studied in other ocular pathogenic conditions and human pathology.

Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

Retinoic acid signaling affects cortical synchrony during sleep.

Delta oscillations, characteristic of the electroencephalogram (EEG) of slow wave sleep, estimate sleep depth and need and are thought to be closely linked to the recovery function of sleep. The cellular mechanisms underlying the generation of delta waves at the cortical and thalamic levels are well documented, but the molecular regulatory mechanisms remain elusive. Here we demonstrate in the mouse that the gene encoding the retinoic acid receptor beta determines the contribution of delta oscillations to the sleep EEG. Thus, retinoic acid signaling, which is involved in the patterning of the brain and dopaminergic pathways, regulates cortical synchrony in the adult.

Sustained activation and tumor targeting of NKT cells using a CD1d-anti-HER2-scFv fusion protein induce antitumor effects in mice

Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated. A single injection of the high-affinity CD1d ligand alpha-galactosylceramide (alphaGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when alphaGalCer was loaded on a recombinant soluble CD1d molecule (alphaGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-gamma secretion as well as DC maturation in mice. Most importantly, when alphaGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free alphaGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy

The neurobiological basis of prefrontal cortex impairment in the phencyclidine model of schizophrenia : convergent reduction of synaptic glutamate release, energy metabolism and cognitive performance

RÉSUMÉ : Le traitement répété à la phencyclidine (PCP), un bloqueur du récepteur NMDA (NMDAR), reproduit chez les rongeurs une partie de la symptomatologie typique de la schizophrénie. Le blocage prolongé du NMDAR par la PCP mime une hypofunction du NMDAR, une des principales altérations supposées exister dans les cerveaux des patients schizophréniques. Le but de notre étude était d'examiner les conséquences neurochimiques, métaboliques et fonctionnelles du traitement répété à la phencyclidine in vivo, au niveau du cortex préfrontal (cpf), une région cérébrale qui joue un rôle dans les déficits cognitifs observés chez les patients schizophréniques. Pour répondre à cette question, les rats ou les souris ont reçu chaque jour une injection soit de PCP (5 mg/kg), soit de solution saline, pendant 7 ou 14 jours. Les animaux ont ensuite été sacrifiés au moins 24 heures après le dernier traitement. Des tranches aiguës du cpf ont été préparées rapidement, puis stimulées avec une concentration élevée de KCI, de manière à induire une libération de glutamate à partir des terminaisons synaptiques excitatrices. Les résultats montrent que les tranches du cpf des animaux traités à la PCP ont libéré une quantité de glutamate significativement inférieure par rapport à celles des animaux contrôle. Ce déficit de libération a persisté 72 heures après la fin du traitement, tandis qu'il n'était pas observé dans le cortex visuel primaire, une autre région corticale. En outre, le traitement avec des antipsychotiques, l'halopéridol ou l'olanzapine, a supprimé le déficit induit par la PCP. Le même déficit de libération a été remarqué sur des synaptosomes obtenus à partir du cpf des animaux traités à la phenryclidine. Cette observation indique que la PCP induit une modification plastique adaptative du mécanisme qui contrôle la libération du glutamate dans les terminaisons synaptiques. Nous avons découvert que cette modification implique la sous-régulation d'un NMDAR présynaptique, qui serait doué d'un rôle d'autorécepteur stimulateur de la libération du glutamate. Grâce à des tests comportementaux conduits en parallèle et réalisés pour évaluer la fonctionnalité du cpf, nous avons observé chez les souris traitées à la PCP une flexibilité comportementale réduite lors d'un test de discrimination de stimuli visuels/tactiles. Le déficit cognitif était encore présent 4 jours après la dernière administration de PCP. La technique de l'autoradiographie quantitative du [14C]2-deoxyglucose a permis d'associer ce déficit à une réduction de l'activité métabolique cérébrale pendant le déroulement du test, particulièrement au niveau du cpf. Dans l'ensemble, nos résultats suggèrent que le blocage prolongé du NMDAR lors de l'administration répétée de PCP produit un déficit de libération du glutamate au niveau des terminaisons synaptiques excitatrices du cpf. Un tel déficit pourrait être provoqué par la sousrégulation d'un NMDAR présynaptique, qui aurait une fonction de stimulateur de libération; la transmission excitatrice du cpf s'en trouverait dans ce cas réduite. Ce résultat est en ligne avec l'activité métabolique et fonctionnelle réduite du cpf et l'observation de déficits cognitifs induits lors de l'administration de la PCP. ABSTRACT : Sub-chronic treatment with phencyclidine (PCP), an NMDA receptor (NMDAR) channel blocker, reproduces in rodents part of the symptomatology associated to schizophrenia in humans. Prolonged pharmacological blockade of NMDAR with PCP mimics NMDAR hypofunction, one of the main alterations thought to take place in the brains of schizophrenics. Our study was aimed at investigating the neurochemical, metabolic and behavioral consequences of repeated PCP administration in vivo, focusing on the functioning of the prefrontal cortex (pfc), a brain region highly relevant for the cognitive deficits observed in schizophrenic patients. Rats or mice received a daily administration of either PCP (5 mg/kg) or saline for 7 or 14 days. At least 24 hours after the last treatment the animals were sacrificed. Acute slices of the pfc were quickly prepared and challenged with high KCl to induce synaptic glutamate release. Pfc slices from PCP-treated animals released significantly less glutamate than slices from salinetreated animals. The deficit persisted 72 hours after the end of the treatment, while it was not observed in another cortical region: the primary visual cortex. Interestingly, treatment with antipsychotic drugs, either haloperidol or olanzapine, reverted the glutamate release defect induced by PCP treatment. The same release defect was observed in synaptosomes prepared from the pfc of PCP-treated animals, indicating that PCP induces a plastic adaptive change in the mechanism controlling glutamate release in the glutamatergic terminals. We discovered that such change most likely involves the down-regulation of a newly identified, pre-synaptic NMDAR with stimulatory auto-receptor function on glutamate release. In parallel sets of behavioral experiments challenging pfc function, mice sub-chronically treated with PCP displayed reduced behavioral flexibility (reversal learning) in a visual/tactile-cued discrimination task. The cognitive deficit was still evident 4 days after the last PCP administration and was associated to reduced brain metabolic activity during the performance of the behavioral task, notably in the pfc, as determined by [14C]2-deoxyglucose quantitative autoradiography. Clverall, our findings suggest that prolonged NMDAR blockade by repeated PCP administration results in a defect of glutamate release from excitatory afferents in the pfc, possibly ascribed to down-regulation of apre-synaptic stimulatory NMDAR. Deficient excitatory neurotransmission in the pfc is consistent with the reduced metabolic and functional activation of this area and the observed PCP-induced cognitive deficits.

Modulatory effects of 5Hz rTMS over the primary somatosensory cortex in focal dystonia--an fMRI-TMS study.

Dystonia is associated with impaired somatosensory ability. The electrophysiological method of repetitive transcranial magnetic stimulation (rTMS) can be used for noninvasive stimulation of the human cortex and can alter cortical excitability and associated behavior. Among others, rTMS can alter/improve somatosensory discrimation abilities, as shown in healthy controls. We applied 5Hz-rTMS over the left primary somatosensory cortex (S1) in 5 patients with right-sided writer's dystonia and 5 controls. We studied rTMS effects on tactile discrimination accuracy and concomitant rTMS-induced changes in hemodynamic activity measured by functional magnetic resonance imaging (fMRI). Before rTMS, patients performed worse on the discrimination task than controls even though fMRI showed greater task-related activation bilaterally in the basal ganglia (BG). In controls, rTMS led to improved discrimination; fMRI revealed this was associated with increased activity of the stimulated S1, bilateral premotor cortex and BG. In dystonia patients, rTMS had no effect on discrimination; fMRI showed similar cortical effects to controls except for no effects in BG. Improved discrimination after rTMS in controls is linked to enhanced activation of S1 and BG. Failure of rTMS to increase BG activation in dystonia may be associated with the lack of effect on sensory discrimination in this group and may reflect impaired processing in BG-S1 connections. Alternatively, the increased BG activation seen in the baseline state without rTMS may reflect a compensatory strategy that saturates a BG contribution to this task.

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase.

OBJECTIVE Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes. METHODS ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR. RESULTS ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG. CONCLUSIONS ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.

New insights into pathophysiology of vestibular migraine.

Vestibular migraine (VM) is a common disorder in which genetic, epigenetic, and environmental factors probably contribute to its development. The pathophysiology of VM is unknown; nevertheless in the last few years, several studies are contributing to understand the neurophysiological pathways involved in VM. The current hypotheses are mostly based on the knowledge of migraine itself. The evidence of trigeminal innervation of the labyrinth vessels and the localization of vasoactive neuropeptides in the perivascular afferent terminals of these trigeminal fibers support the involvement of the trigemino-vascular system. The neurogenic inflammation triggered by activation of the trigeminal-vestibulocochlear reflex, with the subsequent inner ear plasma protein extravasation and the release of inflammatory mediators, can contribute to a sustained activation and sensitization of the trigeminal primary afferent neurons explaining VM symptoms. The reciprocal connections between brainstem vestibular nuclei and the structures that modulate trigeminal nociceptive inputs (rostral ventromedial medulla, ventrolateral periaqueductal gray, locus coeruleus, and nucleus raphe magnus) are critical to understand the pathophysiology of VM. Although cortical spreading depression can affect cortical areas involved in processing vestibular information, functional neuroimaging techniques suggest a dysmodulation in the multimodal sensory integration and processing of vestibular and nociceptive information, resulting from a vestibulo-thalamo-cortical dysfunction, as the pathogenic mechanism underlying VM. The elevated prevalence of VM suggests that multiple functional variants may confer a genetic susceptibility leading to a dysregulation of excitatory-inhibitory balance in brain structures involved in the processing of sensory information, vestibular inputs, and pain. The interactions among several functional and structural neural networks could explain the pathogenic mechanisms of VM.

TRAIL receptor-mediated JNK activation and Bim phosphorylation critically regulate Fas-mediated liver damage and lethality.

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family with potent apoptosis-inducing properties in tumor cells. In particular, TRAIL strongly synergizes with conventional chemotherapeutic drugs to induce tumor cell death. Thus, TRAIL has been proposed as a promising future cancer therapy. Little, however, is known regarding what the role of TRAIL is in normal untransformed cells and whether therapeutic administration of TRAIL, alone or in combination with other apoptotic triggers, may cause tissue damage. In this study, we investigated the role of TRAIL in Fas-induced (CD95/Apo-1-induced) hepatocyte apoptosis and liver damage. While TRAIL alone failed to induce apoptosis in isolated murine hepatocytes, it strongly amplified Fas-induced cell death. Importantly, endogenous TRAIL was found to critically regulate anti-Fas antibody-induced hepatocyte apoptosis, liver damage, and associated lethality in vivo. TRAIL enhanced anti-Fas-induced hepatocyte apoptosis through the activation of JNK and its downstream substrate, the proapoptotic Bcl-2 homolog Bim. Consistently, TRAIL- and Bim-deficient mice and wild-type mice treated with a JNK inhibitor were protected against anti-Fas-induced liver damage. We conclude that TRAIL and Bim are important response modifiers of hepatocyte apoptosis and identify liver damage and lethality as a possible risk of TRAIL-based tumor therapy.

Malt1-dependent RelB cleavage promotes canonical NF-kappaB activation in lymphocytes and lymphoma cell lines.

The protease activity of the paracaspase Malt1 contributes to antigen receptor-mediated lymphocyte activation and lymphomagenesis. Malt1 activity is required for optimal NF-κB activation, but little is known about the responsible substrate(s). Here we report that Malt1 cleaved the NF-κB family member RelB after Arg-85. RelB cleavage induced its proteasomal degradation and specifically controlled DNA binding of RelA- or c-Rel-containing NF-κB complexes. Overexpression of RelB inhibited expression of canonical NF-κB target genes and led to impaired survival of diffuse large B-cell lymphoma cell lines characterized by constitutive Malt1 activity. These findings identify a central role for Malt1-dependent RelB cleavage in canonical NF-κB activation and thereby provide a rationale for the targeting of Malt1 in immunomodulation and cancer treatment.

NLRP3 inflammasome activation: The convergence of multiple signalling pathways on ROS production?

The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is a multiprotein complex that activates caspase 1, leading to the processing and secretion of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and IL-18. The NLRP3 inflammasome is activated by a wide range of danger signals that derive not only from microorganisms but also from metabolic dysregulation. It is unclear how these highly varied stress signals can be detected by a single inflammasome. In this Opinion article, we review the different signalling pathways that have been proposed to engage the NLRP3 inflammasome and suggest a model in which one of the crucial elements for NLRP3 activation is the generation of reactive oxygen species (ROS).

Omega-3 fatty acids prevent inflammation and metabolic disorder through inhibition of NLRP3 inflammasome activation.

Omega-3 fatty acids (ω-3 FAs) have potential anti-inflammatory activity in a variety of inflammatory human diseases, but the mechanisms remain poorly understood. Here we show that stimulation of macrophages with ω-3 FAs, including eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and other family members, abolished NLRP3 inflammasome activation and inhibited subsequent caspase-1 activation and IL-1β secretion. In addition, G protein-coupled receptor 120 (GPR120) and GPR40 and their downstream scaffold protein β-arrestin-2 were shown to be involved in inflammasome inhibition induced by ω-3 FAs. Importantly, ω-3 FAs also prevented NLRP3 inflammasome-dependent inflammation and metabolic disorder in a high-fat-diet-induced type 2 diabetes model. Our results reveal a mechanism through which ω-3 FAs repress inflammation and prevent inflammation-driven diseases and suggest the potential clinical use of ω-3 FAs in gout, autoinflammatory syndromes, or other NLRP3 inflammasome-driven inflammatory diseases.

Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid rafts.

Antigen-specific T-cell activation implicates a redistribution of plasma membrane-bound molecules in lipid rafts, such as the coreceptors CD8 and CD4, the Src kinases Lek and Fyn, and the linker for activation of T cells (LAT), that results in the formation of signaling complexes. These molecules partition in lipid rafts because of palmitoylation of cytoplasmic, membrane proximal cysteines, which is essential for their functional integrity in T-cell activation. Here, we show that exogenous dipalmitoyl-phosphatidylethanolamine (DPPE), but not the related unsaturated dioleoyl-phosphatidylethanolamine (DOPE), partitions in lipid rafts. DPPE inhibits activation of CD8(+) T lymphocytes by sensitized syngeneic antigen-presenting cells or specific major histocompatibility complex (MHC) peptide tetramers, as indicated by esterase release and intracellular calcium mobilization. Cytotoxic, T lymphocyte (CTL)-target cell conjugate formation is not affected by DPPE, indicating that engagement of the T-cell receptor by its cognate ligand is intact in lipid-treated cells. In contrast to other agents known to block raft-dependent signaling, DPPE efficiently inhibits the MHC peptide-induced recruitment of palmitoylated signaling molecules to lipid rafts and CTL activation without affecting cell viability or lipid raft integrity.