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The majority of oncogenic drivers are intracellular proteins, thus constraining their immunotherapeutic targeting to mutated peptides (neoantigens) presented by individual human leukocyte antigen (HLA) allotypes.sup.1. However, most cancers have a modest mutational burden that is insufficient to generate responses using neoantigen-based therapies.sup.2,3. Neuroblastoma is a paediatric cancer that harbours few mutations and is instead driven by epigenetically deregulated transcriptional networks.sup.4. Here we show that the neuroblastoma immunopeptidome is enriched with peptides derived from proteins that are essential for tumourigenesis and focus on targeting the unmutated peptide QYNPIRTTF, discovered on HLA-A*24:02, which is derived from the neuroblastoma dependency gene and master transcriptional regulator PHOX2B. To target QYNPIRTTF, we developed peptide-centric chimeric antigen receptors (CARs) using a counter-panning strategy with predicted potentially cross-reactive peptides. We further hypothesized that peptide-centric CARs could recognize peptides on additional HLA allotypes when presented in a similar manner. Informed by computational modelling, we showed that PHOX2B peptide-centric CARs also recognize QYNPIRTTF presented by HLA-A*23:01 and the highly divergent HLA-B*14:02. Finally, we demonstrated potent and specific killing of neuroblastoma cells expressing these HLAs in vitro and complete tumour regression in mice. These data suggest that peptide-centric CARs have the potential to vastly expand the pool of immunotherapeutic targets to include non-immunogenic intracellular oncoproteins and widen the population of patients who would benefit from such therapy by breaking conventional HLA restriction. Targeting peptides from unmutated cancer drivers that are expressed in tumours but not in normal tissues using peptide-centric chimeric antigen receptors shows potential as treatment for cancer., Author(s): Mark Yarmarkovich [sup.1] , Quinlen F. Marshall [sup.1] , John M. Warrington [sup.1] , Rasika Premaratne [sup.2] , Alvin Farrel [sup.1] [sup.3] , David Groff [sup.1] , Wei Li [...]

The state of somatic energy stores in metazoans is communicated to the brain, which regulates key aspects of behaviour, growth, nutrient partitioning and development.sup.1. The central melanocortin system acts through melanocortin 4 receptor (MC4R) to control appetite, food intake and energy expenditure.sup.2. Here we present evidence that MC3R regulates the timing of sexual maturation, the rate of linear growth and the accrual of lean mass, which are all energy-sensitive processes. We found that humans who carry loss-of-function mutations in MC3R, including a rare homozygote individual, have a later onset of puberty. Consistent with previous findings in mice, they also had reduced linear growth, lean mass and circulating levels of IGF1. Mice lacking Mc3r had delayed sexual maturation and an insensitivity of reproductive cycle length to nutritional perturbation. The expression of Mc3r is enriched in hypothalamic neurons that control reproduction and growth, and expression increases during postnatal development in a manner that is consistent with a role in the regulation of sexual maturation. These findings suggest a bifurcating model of nutrient sensing by the central melanocortin pathway with signalling through MC4R controlling the acquisition and retention of calories, whereas signalling through MC3R primarily regulates the disposition of calories into growth, lean mass and the timing of sexual maturation. MC3R deficiency is associated with a delay in the onset of puberty, and a reduction in growth and lean mass., Author(s): B. Y. H. Lam [sup.1] [sup.2] , A. Williamson [sup.1] [sup.2] [sup.3] , S. Finer [sup.4] , F. R. Day [sup.3] , J. A. Tadross [sup.1] [sup.2] [sup.5] , [...]

Glutamate-gated kainate receptors are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission at the postsynapse and modulate transmitter release at the presynapse.sup.1-7. In the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins.sup.8-11. Here we report cryo-electron microscopy structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-NETO2 complexes, with one or two NETO2 subunits associating with GluK2. We find that NETO2 accesses only two broad faces of kainate receptors, intermolecularly crosslinking the lower lobe of ATD.sup.A/C, the upper lobe of LBD.sup.B/D and the lower lobe of LBD.sup.A/C, illustrating how NETO2 regulates receptor-gating kinetics. The transmembrane helix of NETO2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by NETO2. The authors report the structures of glutamate-gated kainate receptors in complex with NETO2 in both the resting and the desensitized states and reveal how kainate receptors in the brain are regulated by NETO2., Author(s): Lingli He [sup.1] [sup.2] [sup.3] , Jiahui Sun [sup.4] [sup.5] , Yiwei Gao [sup.1] [sup.3] , Bin Li [sup.1] [sup.3] , Yuhang Wang [sup.1] [sup.3] , Yanli Dong [sup.1] [...]

The adenosine A.sub.1 receptor (A.sub.1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain.sup.1,2. However, development of analgesic orthosteric A.sub.1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects.sup.3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A.sub.1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A.sub.1R co-bound to adenosine, MIPS521 and a G.sub.i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts. MIPS521, a positive allosteric modulator of the adenosine A.sub.1 receptor, has analgesic properties in a rat model of neuropathic pain through a mechanism by which MIPS521 stabilizes the complex between adenosine, receptor and G protein., Author(s): Christopher J. Draper-Joyce [sup.1] [sup.10] , Rebecca Bhola [sup.2] , Jinan Wang [sup.3] , Apurba Bhattarai [sup.3] , Anh T. N. Nguyen [sup.1] , India Cowie-Kent [sup.2] , Kelly [...]

[alpha]-Amyrin, a natural pentacyclic triterpene, has an antihyperglycemic effect in mice and dual PPARS/y action in 3T3-L1 adipocytes, and potential in the control of type 2 diabetes (T2D). About 80% of glucose uptake occurs in skeletal muscle cells, playing a significant role in insulin resistance (IR) and T2D. Peroxisome-proliferator activated receptors (PPARs), in particular PPAR[delta] and PPAR[gamma], are involved in the regulation of lipids and carbohydrates and, along with adenosine-monophosphate (AMP) - activated protein kinase (AMPK) and protein kinase B (Akt), are implicated in translocation of glucose transporter 4 (GLUT4); however, it is still unknown whether [alpha]-Amyrin can affect these pathways in skeletal muscle cells. Our objective was to determine the action of [alpha]-Amyrin in PPARS, PPAR[gamma], AMPK, and Akt in C2C12 myoblasts. The expression of PPAR[delta], PPAR[gamma], fatty acid transporter protein (FATP), and GLUT4 was quantified using reverse transcription quantitative PCR and Western blot. [alpha]-Amyrin increased these markers along with phospho- AMPK (p-AMPK) but not p-Akt. Molecular docking showed that [alpha]-Amyrin acts as an AMPK-allosteric activator, and may be related to GLUT4 translocation, as evidenced by confocal microscopy. These data support that [alpha]-Amyrin could have an insulin-mimetic action in C2C12 myoblasts and should be considered as a bioactive molecule for new multitarget drugs with utility in T2D and other metabolic diseases. Key words: [alpha]-Amyrin, p-AMPK activation, dual PPAR[delta]/[gamma], GLUT4 translocation, triterpene. L'[alpha]-Amyrine, un triterpene pentacyclique naturel, a des effets anti-hyperglycemiques chez les souris et une double action PPAR[delta]/[gamma] dans les adipocytes 3T3-L1, avec le potentiel de participer a la maitrise du diabete de type 2 (DT2). Environ 80 % de la captation du glucose survient dans les cellules musculaires squelettiques, ce qui joue un role important dans la resistance a l'insuline et le DT2. Les recepteurs des proliferateurs de peroxysome actives (PPAR), en particulier les PPAR[delta] et les PPAR[gamma], participent a la regulation des lipides et des glucides et, de meme que la proteine kinase activee par l'adenosine monophosphate (AMPK) et que la proteine kinase B (Akt), jouent un role dans la translocation par le GLUT4 (pour << glucose transporter 4 >>). Cependant, on ne sait toujours pas si l'[alpha]-Amyrine peut affecter ces voies de signalisation dans les cellules musculaires squelettiques. L'objectif de ce travail etait d'etablir l'action de l'[alpha]-Amyrine sur les PPARS, les PPAR[gamma], l'AMPKet l'Akt dans des myoblasts C2C12. Nous avons quantifie l'expression des PPAR[delta], des PPAR[gamma], de FATP et de GLUT4 a l'aide de la technique de PCR quantitatif en temps reel et de l'immunobuvardage de Western. L'[alpha]-Amyrine entrainait une hausse de ces marqueurs avec celle de p-AMPK, mais pas de p-Akt. A l'aide de l'arrimage moleculaire, nous avons observe que l' [alpha]-amyrine agit comme un activateur allosterique de l'AMPK, et qu'elle peut etre liee a la translocation par le GLUT4, telle que mise en evidence par la microscopie confocale. Ces donnees viennent appuyer le fait que l'[alpha]-Amyrine pourrait mimer l'action de l'insuline dans les myoblasts C2C12 et que l'on devrait la considerer comme etant une molecule bioactive pour de nouveaux medicaments a cibles multiples dotes d'une utilite dans le DT2 et autres maladies du metabolisme. [Traduit par la Redaction] Mots-cles: [alpha]-Amyrine, activation de p-AMPK, double PPARS/y, translocation par le GLUT4, triterpene., Introduction The [alpha]-Amyrin, a pentacyclic triterpene found in a variety of medicinal plants, has hepatoprotective (Nogueira et al. 2019), anti-inflammatory (Da Silva Junior et al. 2017), and antihyperglycemic properties (Giacoman-Martinez [...]

Alamandine (ALA) and its receptor MrgD were recently identified as components of the renin-angiotensin system, which confer protection against cardio-fibrosis and renal-fibrosis; however, the effects of ALA on pulmonary fibrosis are unknown. This study was designed to serve two goals: (i) to evaluate the ALA/MrgD axis ability in the prevention of angiotensin II (Ang II) - induced pulmonary fibrosis in fibroblasts, and (ii) to determine the effect of ALA in bleomycin (BLM) - treated C57B/6 mice. In vivo experiments revealed that the treatment of C57B/6 mice with ALA prevented BLM-induced fibrosis, and these findings were similar to those reported for pirfenidone. The antifibrosis actions of ALA were mediated via alleviation of oxidative injury and autophagy induction. In addition, in vitro studies revealed that ALA treatment attenuated Ang II-induced [alpha]-collagen I, CTGF, and [alpha]-SMA production in fibroblast which was blocked by D-Pro7-Ang-(1-7), a MrgD antagonist. This led to alleviation of oxidative injury and induction of autophagy similar to that reported for rapamycin. This study demonstrated that ALA via MrgD receptor reduced pulmonary fibrosis through attenuation of oxidative injury and induction of autophagy. Key words: alamandine, pulmonary fibrosis, MrgD receptor, NOX4, autophagy. On a recemment etabli que l'alamandine (ALA) et son recepteur MrgD font partie des composantes du systeme renine-angiotensine (SRA), qui confere une protection contre la fibrose cardiaque et la fibrose renale. Cependant, les effets de l'ALA sur la fibrose pulmonaire sont inconnus. Cette etude a ete concue a deux fins : (i) evaluer la capacite de l'axe ALA/MrgD a prevenir la fibrose pulmonaire engendree par l'angiotensine II (Ang II) dans les fibroblastes, (ii) etablir l'effet de l'ALA chez des souris C57B/6 auxquelles etait administree de la bleomycine (BLM). Les experiences in vivo ont revele que l'administration d'ALA chez les souris C57B/6 permettait de prevenir la fibrose engendree par la BLM, effets similaires a ceux qui ont ete rapportes avec la pirfenidone. Les actions anti-fibrosantes de l'ALA etaient mediees par l'intermediaire de l'attenuation des lesions oxydatives et la production d'autophagie. De plus, les etudes in vitro ont revele que l'administration d'ALA permettait d'attenuer la production d'[alpha]-collagene I, de CTGF et d'[alpha]-SMA engendree par l'Ang II dans les fibroblastes. Ces phenomenes etaient inhibes par un antagoniste des recepteurs MrgD, le D-Pro7-Ang-(1-7), ce qui a mene a une attenuation des lesions oxydatives et a la production d'autophagie, de maniere similaire a ce qui a ete rapporte avec la rapamycine. Cette etude a montre que, par l'intermediaire des recepteurs MrgD, l'ALA permettait d'attenuer la fibrose pulmonaire par l'attenuation des lesions oxydatives et la production d'autophagie. [Traduit par la Redaction] Mots-cles : alamandine, fibrose pulmonaire, recepteurs MrgD, NOX4, autophagie., Introduction The renin-angiotensin system (RAS) is significantly involved in the development and pathogenesis of pulmonary fibrosis disease (Li et al. 2008; Shenoy et al. 2010; Tung et al. 2010). Previously [...]

Background: Abnormalities of inflammation have been implicated in the pathophysiology of depression and suicide, based on observations of increased levels of proinflammatory cytokines in the serum of people who were depressed and died by suicide. More recently, abnormalities in cytokines and innate immunity receptors such as toll-like receptors have also been observed in the postmortem brains of people who were depressed and died by suicide. In addition to toll-like receptors, another subfamily of innate immunity receptors known as NOD-like receptors containing pyrin (NLRPs) are the most widely present NOD-like receptors in the central nervous system. NLRPs also form inflammasomes that play an important role in brain function. We studied the role of NLRPs in depression and suicide. Methods: We determined the protein and mRNA expression of NLRP1, NLRP3 and NLRP6 and the components of their inflammasomes (i.e., adaptor molecule apoptosis-associated speck-like protein [ASC], caspase1, caspase3, interleukin [IL]-1 p and IL-18) postmortem in the prefrontal cortex of people who were depressed and died by suicide, and in healthy controls. We determined mRNA levels using quantitative polymerase chain reaction, and we determined protein expression using Western blot immunolabelling. Results: We found that the protein and mRNA expression levels of NLRP1, NLRP3, NLRP6, caspase3 and ASC were significantly increased in people who were depressed and died by suicide compared to healthy controls. Limitations: Some people who were depressed and died by suicide were taking antidepressant medication at the time of their death. Conclusion: Similar to toll-like receptors, NLRP and its inflammasomes were upregulated in people who were depressed and died by suicide compared to healthy controls. Innate immunity receptors in general--and NLRPs and inflammasomes in particular--may play an important role in the pathophysiology of depression and suicide., Introduction Suicide is a major public health concern. According to the National Center for Health Statistics (US Centers for Disease Control and Prevention), (1) 48344 suicides took place in 2018 [...]

The calcium-sensing receptor (CaSR), a cell-surface sensor for Ca.sup.2+, is the master regulator of calcium homeostasis in humans and is the target of calcimimetic drugs for the treatment of parathyroid disorders.sup.1. CaSR is a family C G-protein-coupled receptor.sup.2 that functions as an obligate homodimer, with each protomer composed of a Ca.sup.2+-binding extracellular domain and a seven-transmembrane-helix domain (7TM) that activates heterotrimeric G proteins. Here we present cryo-electron microscopy structures of near-full-length human CaSR in inactive or active states bound to Ca.sup.2+ and various calcilytic or calcimimetic drug molecules. We show that, upon activation, the CaSR homodimer adopts an asymmetric 7TM configuration that primes one protomer for G-protein coupling. This asymmetry is stabilized by 7TM-targeting calcimimetic drugs adopting distinctly different poses in the two protomers, whereas the binding of a calcilytic drug locks CaSR 7TMs in an inactive symmetric configuration. These results provide a detailed structural framework for CaSR activation and the rational design of therapeutics targeting this receptor. Cryo-EM structures of human calcium-sensing receptor reveal intrinsic asymmetry in the receptor homodimer upon activation that is stabilized by calcimimetic drugs adopting distinct poses in the two protomers, priming one protomer for G-protein coupling., Author(s): Yang Gao [sup.1] [sup.2] , Michael J. Robertson [sup.1] [sup.2] , Sabrina N. Rahman [sup.4] , Alpay B. Seven [sup.1] [sup.2] , Chensong Zhang [sup.1] [sup.2] , Justin G. [...]

Background: Orexin-A (OrxA) administration in the posterior paraventricular nucleus of the thalamus (pPVT) reinstates extinguished cocaine-seeking behaviour following extended access to the drug (a model of dependence). The pPVT receives and integrates information associated with emotionally salient events and sends excitatory inputs to brain regions involved in the expression of emotional states, such as those driving cocaine-seeking behaviour (i.e., the nucleus accumbens, the central nucleus of the amygdala [CeA], the basolateral amygdala, the bed nucleus of the stria terminalis [BNST] and the prefrontal cortex). Methods: We monitored the activation pattern of these regions (measured by Fos) during cocaine-seeking induced by OrxA administered to the pPVT. The BNST and CeA emerged as being selectively activated. To test whether the functionality of these regions was pivotal during OrxA- induced cocaine-seeking behaviour, we transiently inactivated these regions concomitantly with OrxA administration to the pPVT. We then tested the participation of corticotropin-releasing factor receptors ([CRF.sub.1]) in the CeA during OrxA-induced cocaine-seeking using the [CRF.sub.1] antagonist CP154526. Results: We observed selective activation of the CeA and BNST during cocaine-seeking induced by OrxA administered to the pPVT, but only transient inactivation of the CeA prevented cocaine-seeking behaviour. Administration of CP154526 to the CeA prevented OrxA-induced cocaine-seeking behaviour. Limitations: The use of only male rats could have been a limitation. Other limitations could have been the use of an indirect approach to test the hypothesis that administration of OrxA to the pPVT drives cocaine- seeking via [CRF.sub.1] signalling in the CeA, and a lack of analysis of the participation of CeA subregions. Conclusion: Cocaine-seeking behaviour induced by OrxA administered to the pPVT is driven by activation of the CeA via [CRF.sub.1] signalling., Introduction The paraventricular nucleus of the thalamus (PVT) has gained considerable attention in the study of drug-related behaviours because of its prominent projections to pivotal components of the neurocircuitry of [...]

Ghrelin, a peptide hormone released from the gastric endocrine glands, shows analgesic activity apart from its various physiological effects. Nevertheless, the effects of ghrelin receptor (GHS-R) agonists on morphine analgesia and tolerance have not yet been elucidated. The purpose of this study was to evaluate the effects of the ghrelin receptor agonist hexarelin and antagonist [D-Lys3]-GHRP-6 on morphine antinociception and tolerance in rats. A total of 104 Wistar albino male adult rats (weighing approximately 220-240 g) were used in the experiments. To induce morphine tolerance, a three-day cumulative dose regimen was used in the rats. Then, randomly selected rats were evaluated for morphine tolerance on day 4. The analgesic effects of hexarelin (0.2 mg * [kg.sup.-1]), [D-Lys3]-GHRP-6 (10 mg * [kg.sup.-1]), and morphine (5 mg * [kg.sup.-1]) were measured at 30-min intervals (0, 30, 60, 90, and 120 min) by tail-flick and hot-plate analgesia tests. The findings suggest that hexarelin in combination with morphine attenuates analgesic tolerance to morphine. On the other hand, ghrelin receptor antagonist [D-Lys3]-GHRP-6 has no significant analgesic activity on the morphine tolerance in analgesia tests. Furthermore, co-administration of hexarelin and morphine increases the analgesic effect. In conclusion, these data indicate that administration of GHS-R agonist hexarelin with morphine enhances the antinociception and attenuates morphine tolerance. Key words: opioid, ghrelin receptors, hexarelin, GHRP-6, morphine tolerance. La ghreline est une hormone peptidique liberee des glandes endocrines gastriques presentant une activite analgesique entre autres effets physiologiques varies. Neanmoins, les effets des agonistes des recepteurs de la ghreline (GHS-R) sur les effets analgesiques de la morphine et la tolerance a celle-ci n'ont pas encore ete elucides. Cette etude avait pour but d'evaluer les effets de l'hexareline, un agoniste des recepteurs de la ghreline, et du [D-Lys3]-GHRP-6, un antagoniste, sur les effets antinociceptifs de la morphine et de la tolerance a celle-ci chez le rat. Dans le cadre de ces experiences, nous avons utilise au total 104 rats Wistar albinos males adultes pesant environ de 220 a 240 g. Nous avons provoque une tolerance a la morphine chez les rats a l'aide d'une administration cumulative sur trois jours. Puis, nous avons selectionne des rats aleatoirement pour une evaluation de la tolerance a la morphine au jour 4. Nous avons mesure les effets analgesiques de l'hexareline (a 0,2 mg * [kg.sup.-1]), du [D-Lys3]-GHRP-6 (a 10 mg * [kg.sup.-1]) et de la morphine (a 5 mg * [kg.sup.-1]) a 30 min d'intervalle (0, 30, 60, 90 et 120 min) a l'aide des tests du coup de queue (<< tail-flick >>) et de la plaque chauffante. Les resultats laissent entrevoir que l'hexareline en association avec la morphine permet d'attenuer la tolerance a la morphine. Par ailleurs, l'antagoniste des recepteurs de la ghreline (le [D-Lys3]-GHRP-6) ne disposait pas d'une activite analgesique notable sur la tolerance a la morphine dans le cadre des tests d'analgesie. En outre, l'administration concomitante d'hexareline et de morphine permet d'augmenter l'effet analgesique. En conclusion, ces donnees montrent que l'administration d'un agoniste des GHS-R (l'hexareline) avec de la morphine entraine un accroissement de l'antinociception et une attenuation de la tolerance a la morphine. Mots-cles: opioide, recepteurs de la ghreline, hexareline, GHRP-6, tolerance a la morphine., Introduction Ghrelin, a 28 amino acid polypeptide hormone secreted mostly by gastric endocrine X (A) cells, has been shown to stimulate the release of growth hormone (GH), cause weight gain, [...]

Stromal cells in adult bone marrow that express leptin receptor (LEPR) are a critical source of growth factors, including stem cell factor (SCF), for the maintenance of haematopoietic stem cells and early restricted progenitors.sup.1-6. LEPR.sup.+ cells are heterogeneous, including skeletal stem cells and osteogenic and adipogenic progenitors.sup.7-12, although few markers have been available to distinguish these subsets or to compare their functions. Here we show that expression of an osteogenic growth factor, osteolectin.sup.13,14, distinguishes peri-arteriolar LEPR.sup.+ cells poised to undergo osteogenesis from peri-sinusoidal LEPR.sup.+ cells poised to undergo adipogenesis (but retaining osteogenic potential). Peri-arteriolar LEPR.sup.+osteolectin.sup.+ cells are rapidly dividing, short-lived osteogenic progenitors that increase in number after fracture and are depleted during ageing. Deletion of Scf from adult osteolectin.sup.+ cells did not affect the maintenance of haematopoietic stem cells or most restricted progenitors but depleted common lymphoid progenitors, impairing lymphopoiesis, bacterial clearance, and survival after acute bacterial infection. Peri-arteriolar osteolectin.sup.+ cell maintenance required mechanical stimulation. Voluntary running increased, whereas hindlimb unloading decreased, the frequencies of peri-arteriolar osteolectin.sup.+ cells and common lymphoid progenitors. Deletion of the mechanosensitive ion channel PIEZO1 from osteolectin.sup.+ cells depleted osteolectin.sup.+ cells and common lymphoid progenitors. These results show that a peri-arteriolar niche for osteogenesis and lymphopoiesis in bone marrow is maintained by mechanical stimulation and depleted during ageing. A peri-arteriolar niche in the bone marrow for osteogenesis and lymphopoiesis is maintained by mechanical stimulation and is depleted during ageing., Author(s): Bo Shen [sup.1] , Alpaslan Tasdogan [sup.1] , Jessalyn M. Ubellacker [sup.1] , Jingzhu Zhang [sup.1] , Elena D. Nosyreva [sup.2] , Liming Du [sup.1] , Malea M. Murphy [...]

The germinal centre is a dynamic microenvironment in which B cells that express high-affinity antibody variants produced by somatic hypermutation are selected for clonal expansion by limiting the numbers of T follicular helper cells.sup.1,2. Although much is known about the mechanisms that control the selection of B cells in the germinal centre, far less is understood about the clonal behaviour of the T follicular helper cells that help to regulate this process. Here we report on the dynamic behaviour of T follicular helper cell clones during the germinal centre reaction. We find that, similar to germinal centre B cells, T follicular helper cells undergo antigen-dependent selection throughout the germinal centre reaction that results in differential proliferative expansion and contraction. Increasing the amount of antigen presented in the germinal centre leads to increased division of T follicular helper cells. Competition between T follicular helper cell clones is mediated by the affinity of T cell receptors for peptide-major-histocompatibility-complex ligands. T cells that preferentially expand in the germinal centre show increased expression of genes downstream of the T cell receptor, such as those required for metabolic reprogramming, cell division and cytokine production. These dynamic changes lead to marked remodelling of the functional T follicular helper cell repertoire during the germinal centre reaction. T follicular helper cells undergo antigen-dependent selection in germinal centres, with higher-affinity T cell receptors supporting stronger proliferation leading to their clonal dominance., Author(s): Julia Merkenschlager [sup.1] , Shlomo Finkin [sup.1] , Victor Ramos [sup.1] , Julian Kraft [sup.1] , Melissa Cipolla [sup.1] , Carla R. Nowosad [sup.2] , Harald Hartweger [sup.1] , [...]

Milk contains bioactive molecules with important functions as defensive proteins; among them are the whey protein lactoferrin and proteins of the milk fat globule membrane (MFGM) present in buttermilk. The aim of this study has been to investigate the effects of lactoferrin, whey, and buttermilk as modulators of intestinal innate immunity and oxidative stress on intestinal epithelial cells, to evaluate its potential use for the development of functional foods. The mRNA expression levels of innate immune system Toll-like receptors (TLR2, TLR4, and TLR9), lipid peroxidation (malondialdehyde + 4-hydroxyalkenals) and protein expression levels of carbonyl were analyzed in enterocyte-like Caco-2/TC7 cells treated for 24 h with different concentrations of lactoferrin, whey, or buttermilk. None of the substances analyzed caused oxidative damage; however, whey significantly decreased the levels of lipid peroxidation. Furthermore, both lactoferrin and whey reduced the oxidative stress induced by lipopolysaccharide. With respect to TLR receptors, lactoferrin, whey, and buttermilk specifically altered the expression of TLR2, TLR4, and TLR9 receptors, with a strong decrease in the expression levels of TLR4. These results suggest that lactoferrin, whey, and buttermilk are potentially interesting ingredients for functional foods because they seem to modulate oxidative stress and the inflammatory response induced by the activation of TLRs. Key words: intestine, oxidative stress, TLR2, TLR4, TLR9. Le lait contient des molecules actives possedant d'importantes proprietes fonctionnelles comme proteines de defense; parmi elles se trouvent la lactoferrine du lactoserum et les proteines de la membrane du globule gras du lait (MFGM) presentes dans le lactoserum. L'objectif de cette etude consistait a examiner l'effet de la lactoferrine, du lactoserum et du babeurre en tant que modulateurs de l'immunite innee intestinale et du stress oxydant sur les cellules epitheliales intestinales, afin d'evaluer leur utilisation potentielle dans l'elaboration d'aliments fonctionnels. L'expression de l'ARNm des recepteurs de type Toll de l'immunite innee (TLR2, TLR4 et TLR9), la peroxydation lipidique (MDA et 4-HDA) et les niveaux de carbonyles de proteines ont ete analyses dans les cellules de type enterocytaire Caco-2/TC7 traitees pendant 24 heures avec differentes concentrations de lactoferrine, de lactoserum ou de babeurre. Aucune des substances analysees ne provoquait de dommage oxydant, toutefois, le lactoserum diminuait significativement les niveaux de peroxydation lipidique. De plus, la lactoferrine et le lactoserum etaient tous deux capables de reduire le stress oxydant induit par les lipopolysaccharides. En ce qui concerne les recepteurs TLR, la lactoferrine, le lactoserum et le babeurre modifiaient significativement l'expression de TLR2, TLR4 et TLR9, avec une forte diminution de l'expression de TLR4. Ces resultats suggerent que la lactoferrine, le lactoserum et le babeurre pourraient etre des ingredients potentiels interessants des aliments fonctionnels, car ils semblent moduler le stress oxydant et la reponse inflammatoire induite par l'activation des TLR. [Traduit par la Redaction] Mots-cles : intestin, stress oxydant, TLR2, TLR4, TLR9., Introduction Bovine milk is the most consumed type of milk, and several other dairy products have also been produced and consumed worldwide for millennia. Evidence from recent studies, system atic [...]

Pathological degeneration of axons disrupts neural circuits and represents one of the hallmarks of neurodegeneration.sup.1-4. Sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) is a central regulator of this neurodegenerative process.sup.5-8, and its Toll/interleukin-1 receptor (TIR) domain exerts its pro-neurodegenerative action through NADase activity.sup.9,10. However, the mechanisms by which the activation of SARM1 is stringently controlled are unclear. Here we report the cryo-electron microscopy structures of full-length SARM1 proteins. We show that NAD.sup.+ is an unexpected ligand of the armadillo/heat repeat motifs (ARM) domain of SARM1. This binding of NAD.sup.+ to the ARM domain facilitated the inhibition of the TIR-domain NADase through the domain interface. Disruption of the NAD.sup.+-binding site or the ARM-TIR interaction caused constitutive activation of SARM1 and thereby led to axonal degeneration. These findings suggest that NAD.sup.+ mediates self-inhibition of this central pro-neurodegenerative protein. NAD.sup.+ is shown to be a ligand of the armadillo/heat repeat motifs (ARM) domain of SARM1, and it is suggested that this binding of NAD.sup.+ mediates self-inhibition of SARM1., Author(s): Yuefeng Jiang [sup.1] , Tingting Liu [sup.1] , Chia-Hsueh Lee [sup.2] , Qing Chang [sup.3] , Jing Yang [sup.1] , Zhe Zhang [sup.1] Author Affiliations: (1) State Key Laboratory [...]

Venezuelan equine encephalitis virus (VEEV) is a neurotropic alphavirus transmitted by mosquitoes that causes encephalitis and death in humans.sup.1. VEEV is a biodefence concern because of its potential for aerosol spread and the current lack of sufficient countermeasures. The host factors that are required for VEEV entry and infection remain poorly characterized. Here, using a genome-wide CRISPR-Cas9-based screen, we identify low-density lipoprotein receptor class A domain-containing 3 (LDLRAD3)--a highly conserved yet poorly characterized member of the scavenger receptor superfamily--as a receptor for VEEV. Gene editing of mouse Ldlrad3 or human LDLRAD3 results in markedly reduced viral infection of neuronal cells, which is restored upon complementation with LDLRAD3. LDLRAD3 binds directly to VEEV particles and enhances virus attachment and internalization into host cells. Genetic studies indicate that domain 1 of LDLRAD3 (LDLRAD3(D1)) is necessary and sufficient to support infection by VEEV, and both anti-LDLRAD3 antibodies and an LDLRAD3(D1)-Fc fusion protein block VEEV infection in cell culture. The pathogenesis of VEEV infection is abrogated in mice with deletions in Ldlrad3, and administration of LDLRAD3(D1)-Fc abolishes disease caused by several subtypes of VEEV, including highly virulent strains. The development of a decoy-receptor fusion protein suggests a strategy for the prevention of severe VEEV infection and associated disease in humans. LDLRAD3 is a receptor for infection with Venezuelan equine encephalitis virus, and in mouse models deletion of Ldlrad3 or treatment with a soluble LDLRAD3 decoy molecule abrogates infection and disease caused by this virus., Author(s): Hongming Ma [sup.1] , Arthur S. Kim [sup.1] [sup.2] , Natasha M. Kafai [sup.1] [sup.2] , James T. Earnest [sup.1] , Aadit P. Shah [sup.1] , James Brett Case [...]

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors.sup.1-4, followed by fusion of the virus and cell membranes to release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein.sup.5-7. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage.sup.8-10. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide.sup.11,12. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp614.sup.13-15 and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site. Cryo-electron microscopy structures of consecutive binding events of ACE2 in complex with the spike protein of SARS-CoV-2 reveal the mechanisms of receptor binding by the spike protein and activation for membrane fusion by the spike protein of SARS-CoV-2., Author(s): Donald J. Benton [sup.1] , Antoni G. Wrobel [sup.1] , Pengqi Xu [sup.2] [sup.3] , Chloë Roustan [sup.4] , Stephen R. Martin [sup.1] , Peter B. Rosenthal [sup.5] , [...]

Fibroblast-to-myofibroblast (FibroMF) differentiation is crucial for embryogenesis and organ fibrosis. Although transforming growth factor-[beta] (TGF[beta]) is the primary mediator of FibroMF differentiation, the type-I receptor (TGF[beta]RI) responsible for this has not yet been confirmed. In the current study, we investigated the ALK1 and ALK5 expressions in TGF[beta]1-stimulated NIH 3T3 fibroblasts to compare with the data from the Gene Expression Omnibus (GEO) repository. In our results, whereas TGF[beta]1 treatment promoted FibroMF differentiation accompanied by increased ALK5 expression and reduced ALK1 expression, TGF[beta]1induced FibroMF differentiation and increased [alpha]-smooth muscle actin ([alpha]SMA) and ALK5 expression were inhibited by cotreatment with ALK5 inhibitor SB431542. GEO database analysis indicated increased ALK5 expression and reduced ALK1 expression in fibrotic compared to normal mouse or human tissues correlating with organ fibrosis progression. Finally, the inhibitors of Akt, mTOR, and [beta]-catenin suppressed TGF[beta]1-induced ALK5 expression, indicating that the Akt pathway promotes FibroMF differentiation via ALK5 expression and fibrosis. Key words: ALK1, ALK5, TGF[beta]1, myofibroblast, fibrosis. La differentiation du fibroblaste en myofibroblaste (Fibro en MF) est un phenomene crucial pour l'embryogenese et la fibrose des organes. Bien que le facteur de croissance transformant [beta] (TGF[beta]) soit le principal mediateur de la differentiation du Fibro en MF, le recepteur de type 1 (TGF[beta]R1) n'a pas encore ete confirme comme en etant responsable. Dans les presents travaux, nous avons etudie l'expression de l'ALK1 et de l'ALK5 dans les fibroblastes NIH 3T3 stimules avec le TGF[beta]1 aux fins de comparaisonavec les donnees de la base GEO (pour<< Gene Expression Omnibus>>). Selon nos resultats, l'administrationde TGF[beta]1 favorisait la differentiation du Fibro en MF accompagnee d'une augmentation de l'expression de l'ALK5 et d'une diminution de l'expression de l'ALK1. La differentiation du Fibro en MF engendree par le TGF[beta]1 ainsi que la hausse de l'actine du muscle lisse [alpha] (ou [alpha]SMA pour <>) et de l'expression de l'ALK5 etaient inhibees par l'administration concomitante de SB431542, un inhibiteur de l'ALK5. L'etude de la base de donnees GEO a montre une augmentation de l'expression de l'ALK5 avec une diminution de l'expression de l'ALK1 dans le tissu fibreux comparativement aux tissus normaux de souris ou d'humains de maniere proportionnelle a revolution de la fibrose. Enfin, les inhibiteurs de l'Akt, de mTOR et de la [beta]-catenine inhibaient l'augmentation de l'expression de l'ALK5 engendree par le TGF[beta]1, ce qui montrait que la voie de signalisation de l'Akt favorise la differentiation du Fibro en MF par l'intermediaire de l'augmentation de l'expression de l'ALK5 et de la fibrose. [Traduit par la Redaction] Mots-cles: ALK1, ALK5, TGF[beta]1, myofibroblaste, fibrose., Introduction Fibroblast-to-myofibroblast (FibroMF) differentiation is the hallmark of embryogenesis (Roulis and Flavell 2016), wound healing, and organ fibrosis (Weiskirchen et al. 2019). The integral role of the transforming growth factor-[beta] [...]

Bone morphogenetic proteins (BMPs) were once considered only to have a role in bone formation. It is now known that they have pivotal roles in other organ diseases, including heritable pulmonary arterial hypertension (PAH), where genetic mutations in the type II BMP receptor (BMPR2) are the commonest cause of receptor dysfunction. However, it has also recently been demonstrated that aquaporin 1 (Aqp1) dysfunction may contribute to PAH, highlighting that PAH development may involve more than one pathogenic pathway. Whether reduction in BMPR2 affects Aqp1 is unknown. We therefore studied Aqp1 in BMPR2-silenced human pulmonary microvascular endothelial cells (HPMECs). We demonstrated reduced Aqp1 mRNA protein, and function in the BMPR2-silenced cells. Additionally, BMPR2-silenced cells exhibited lower expression of BMP-signaling molecules. In conclusion, decreased BMPR2 appears to affect Aqp1 at the mRNA, protein, and functional levels. This observation may identify a contributory mechanism for PAH. Key words: Aqp1, BMPR2, PAH, HPMEC. On a deja considere que les proteines morphogeniques osseuses (BMP pour << bone morphogenetic proteins >>) jouent uniquement un role dans la formation d'os. On sait maintenant qu'elles jouent un role central dans d'autres maladies, y compris l'hypertension arterielle pulmonaire hereditaire (HAP), ou des mutations genetiques dans les recepteurs aux BMP de type 2 (BMPR2) sont la cause la plus frequente de dysfonctionnement des recepteurs. Toutefois, on a aussi montre recemment qu'un dysfonctionnement de l'aquaporine 1 (Aqp1) pourrait contribuer a l'HAP, ce qui met en relief que l'apparition de l'HAP pourrait mettre enjeu plus d'une voie de signalisation de pathogenie. On ne sait pas si l'abaissement des BMPR2 affecte l'Aqp1. Nous avons donc etudie l'Aqp1 dans des cellules endotheliales de microvaisseaux pulmonaires humains (CEMPH) dont les BMPR2 etaient reprimes. Nous avons montre un abaissement de l'Aqp1 en ARNm, en proteines et quant au fonctionnement dans des cellules dont les BMPR2 etaient reprimes. De plus, les cellules dont les BMPR2 etaient reprimes presentaient une plus faible expression de molecules de signalisation des BMP. En conclusion, l'abaissement des BMPR2 semble affecter l'Aqp1 au niveau de l'ARNm, des proteines et du fonctionnement. Cette observation pourrait permettre de distinguer un mode d'action qui contribuerait a l'HAP. [Traduit par la Redaction] Mots-cles: Aqp1, BMPR2, hypertension arterielle pulmonaire hereditaire, cellules endotheliales de microvaisseaux pulmonaires humains., Introduction Bone morphogenetic proteins (BMPs) are a group of signaling molecules that belong to the transforming growth factor-p (TGF-[beta]) superfamily of proteins. BMPs have multiple roles in the development and [...]

The purpose of this study was to explore the potential contracting effect of leptin on isolated guinea pig tracheal smooth muscle (TSM), the possible mechanism, and the impact of epithelium denudation or allergen sensitization, respectively. An in vitro experiment investigated the effect of leptin at a concentration of 250-1000 nmol/L on isolated guinea pig TSM with an intact or denuded epithelium. Ovalbumin and IgE were used to test the impact of active and passive sensitization. The isolated TSM strips were incubated in Krebs solution and aerated with carbogen (95% [O.sub.2] and 5% C[O.sub.2]) via an automated tissue organ bath system (n = 4 for each group). Isometric contractions were recorded digitally using iox2 data acquisition software. The possible mechanism of leptin-induced TSM contraction was examined by preincubation with leptin receptor (Ob-R) antagonist. Leptin had significant concentration-dependent contraction effects on guinea pig TSM (p < 0.05). Epithelium denuding and active or passive sensitization significantly increased the potency of the leptin. Preincubation with a leptin receptor (Ob-R) antagonist significantly reduced the contraction effects, suggesting an Ob-R-mediated mechanism. Leptin had a contracting effect on airway smooth muscles potentiated by either epithelium denuding or sensitization, and the Ob-R mechanism was a possible effect mediator. Key words: airway smooth muscles, guinea pig, leptin, Ob-R. Cette etude portait sur l'eventuel effet contractile de la leptine sur le muscle lisse tracheal (MLT) de cobaye, le mode d'action possible et l'incidence de la denudation de l'endothelium ou de la sensibilisation aux allergenes. A l'aide d'experiences in vitro, nous avons etudie l'effet de la leptine a certaines concentrations (250-1000 nmol/L) sur le MLT isole de cobaye avec un epithelium intact ou denude. Nous avons evalue l'incidence de la sensibilisation active ou passive a l'aide d'ovalbumine et d'IgE. Nous avons incube les bandelettes de MLT isolees dans une solution de Krebs ventilee a l'aide de carbogene ([O.sub.2] a 95 % et C[O.sub.2] a 5 %) dans un bain de tissu organique automatise (n = 4 pour chaque groupe). Nous avons enregistre les contractions isometriques numerisees a l'aide du logiciel d'acquisition de donnees iox2. Nous avons examine le mode d'action eventuel des contractions du MLT provoquees par la leptine apres une incubation en presence d'un antagoniste des recepteurs de la leptine (Ob-R). Nous avons observe que la leptine avait des effets contractiles marques et proportionnels a la concentration sur le MLT de cobaye (p < 0,05). La denudation de l'epithelium et la sensibilisation active ou passive entrainaient une augmentation notable de la puissance de la leptine. L'incubation prealable en presence d'un antagoniste des recepteurs de la leptine (Ob-R) entrainait clairement une diminution des effets contractiles, ce qui laisse entrevoir la presence d'un mode d'action medie par les Ob-R En conclusion, la leptine a eu un effet contractile sur le muscle lisse des voies respiratoires qui etait potentialise par la denudation de l'endothelium ou par la sensibilisation, et le mode d'action lie au recepteur Ob-R constituait un mediateur de l'effet possible. [Traduit par la Redaction] Mots-des: muscle lisse des voies respiratoires, cobaye, leptine, Ob-R., Introduction Leptin is a pro-inflammatory cytokine protein synthesized by human adipocytes with the serum levels being directly linked to the mass of the adipose tissue (Paz-Filho et al. 2015). Generated [...]

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte.sup.1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy.sup.3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release.sup.4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE1.sup.1, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy. FERONIA prevents polyspermy in Arabidopsis by enabling pectin-stimulated nitric oxide accumulation at the filiform apparatus after the first pollen tube arrives, which disengages LURE1 chemoattraction to prevent late-arriving pollen tubes from entering the ovule., Author(s): Qiaohong Duan [sup.1] [sup.2] , Ming-Che James Liu [sup.1] [sup.3] , Daniel Kita [sup.1] [sup.4] [sup.7] , Samuel S. Jordan [sup.1] , Fang-Ling Jessica Yeh [sup.1] , Robert Yvon [...]

Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males.sup.1-3. However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174--an X-chromosome-encoded G-protein-coupled receptor--suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell-B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more G[alpha]i protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174-G[alpha]i association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity. Male and female B cells show differing abilities to localize and contribute to germinal centres, in a way that depends on the G-protein-coupled guidance receptor GPR174 and its chemokine ligand CCL21., Author(s): Ruozhu Zhao [sup.1] [sup.2] [sup.3] [sup.4] , Xin Chen [sup.1] [sup.2] [sup.3] [sup.4] , Weiwei Ma [sup.1] [sup.2] [sup.3] , Jinyu Zhang [sup.2] [sup.3] [sup.5] , Jie Guo [sup.6] [...]

The tricarboxylic acid cycle intermediate succinate is involved in metabolic processes and plays a crucial role in the homeostasis of mitochondrial reactive oxygen species.sup.1. The receptor responsible for succinate signalling, SUCNR1 (also known as GPR91), is a member of the G-protein-coupled-receptor family.sup.2 and links succinate signalling to renin-induced hypertension, retinal angiogenesis and inflammation.sup.3-5. Because SUCNR1 senses succinate as an immunological danger signal.sup.6--which has relevance for diseases including ulcerative colitis, liver fibrosis.sup.7, diabetes and rheumatoid arthritis.sup.3,8--it is of interest as a therapeutic target. Here we report the high-resolution crystal structure of rat SUCNR1 in complex with an intracellular binding nanobody in the inactive conformation. Structure-based mutagenesis and radioligand-binding studies, in conjunction with molecular modelling, identified key residues for species-selective antagonist binding and enabled the determination of the high-resolution crystal structure of a humanized rat SUCNR1 in complex with a high-affinity, human-selective antagonist denoted NF-56-EJ40. We anticipate that these structural insights into the architecture of the succinate receptor and its antagonist selectivity will enable structure-based drug discovery and will further help to elucidate the function of SUCNR1 in vitro and in vivo. High-resolution crystal structures of the rat succinate receptor SUCNR1 in an inactive confirmation, and the humanized rat SUCNR1 bound to an antagonist, provide insights into the structure of these receptors and the species selectivity of antagonist binding., Author(s): Matthias Haffke [sup.1] [sup.2] , Dominique Fehlmann [sup.3] , Gabriele Rummel [sup.1] , Jacques Boivineau [sup.1] , Myriam Duckely [sup.1] , Nina Gommermann [sup.2] , Simona Cotesta [sup.2] , [...]

The causative agent of plague, Yersinia pestis, uses a type III secretion system to selectively destroy immune cells in humans, thus enabling Y. pestis to reproduce in the bloodstream and be transmitted to new hosts through fleabites. The host factors that are responsible for the selective destruction of immune cells by plague bacteria are unknown. Here we show that LcrV, the needle cap protein of the Y. pestis type III secretion system, binds to the N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors. Plague infection in mice is characterized by high mortality; however, Fpr1-deficient mice have increased survival and antibody responses that are protective against plague. We identified FPR1.sup.R190W as a candidate resistance allele in humans that protects neutrophils from destruction by the Y. pestis type III secretion system. Thus, FPR1 is a plague receptor on immune cells in both humans and mice, and its absence or mutation provides protection against Y. pestis. Furthermore, plague selection of FPR1 alleles appears to have shaped human immune responses towards other infectious diseases and malignant neoplasms. The receptor FPR1 on human immune cells interacts with Yersinia pestis, mutations in this receptor provide resistance against plague in humans and Fpr1 deficiency enhances survival in mice., Author(s): Patrick Osei-Owusu [sup.1] [sup.2] , Thomas M. Charlton [sup.1] [sup.2] , Hwan Keun Kim [sup.1] [sup.2] , Dominique Missiakas [sup.1] [sup.2] , Olaf Schneewind [sup.1] [sup.2] Author Affiliations: (1) [...]

Patients with acute myeloid leukaemia (AML) often achieve remission after therapy, but subsequently die of relapse.sup.1 that is driven by chemotherapy-resistant leukaemic stem cells (LSCs).sup.2,3. LSCs are defined by their capacity to initiate leukaemia in immunocompromised mice.sup.4. However, this precludes analyses of their interaction with lymphocytes as components of anti-tumour immunity.sup.5, which LSCs must escape to induce cancer. Here we demonstrate that stemness and immune evasion are closely intertwined in AML. Using xenografts of human AML as well as syngeneic mouse models of leukaemia, we show that ligands of the danger detector NKG2D--a critical mediator of anti-tumour immunity by cytotoxic lymphocytes, such as NK cells.sup.6-9--are generally expressed on bulk AML cells but not on LSCs. AML cells with LSC properties can be isolated by their lack of expression of NKG2D ligands (NKG2DLs) in both CD34-expressing and non-CD34-expressing cases of AML. AML cells that express NKG2DLs are cleared by NK cells, whereas NKG2DL-negative leukaemic cells isolated from the same individual escape cell killing by NK cells. These NKG2DL-negative AML cells show an immature morphology, display molecular and functional stemness characteristics, and can initiate serially re-transplantable leukaemia and survive chemotherapy in patient-derived xenotransplant models. Mechanistically, poly-ADP-ribose polymerase 1 (PARP1) represses expression of NKG2DLs. Genetic or pharmacologic inhibition of PARP1 induces NKG2DLs on the LSC surface but not on healthy or pre-leukaemic cells. Treatment with PARP1 inhibitors, followed by transfer of polyclonal NK cells, suppresses leukaemogenesis in patient-derived xenotransplant models. In summary, our data link the LSC concept to immune escape and provide a strong rationale for targeting therapy-resistant LSCs by PARP1 inhibition, which renders them amenable to control by NK cells in vivo. Leukaemic stem cells in acute myeloid leukaemia are defined by a lack of expression of NKG2D ligands, which mediates their ability to evade surveillance by NK cells., Author(s): Anna M. Paczulla [sup.1] , Kathrin Rothfelder [sup.2] [sup.3] [sup.4] , Simon Raffel [sup.5] [sup.6] [sup.7] , Martina Konantz [sup.1] , Julia Steinbacher [sup.2] [sup.3] , Hui Wang [sup.1] [...]

Germinal centres are important sites for antibody diversification and affinity maturation, and are also a common origin of B cell malignancies. Despite being made up of motile cells, germinal centres are tightly confined within B cell follicles. The cues that promote this confinement are incompletely understood. P2RY8 is a G[alpha].sub.13-coupled receptor that mediates the inhibition of migration and regulates the growth of B cells in lymphoid tissues.sup.1,2. P2RY8 is frequently mutated in germinal-centre B cell-like diffuse large B cell lymphoma (GCB-DLBCL) and Burkitt lymphoma.sup.1,3-6, and the ligand for this receptor has not yet been identified. Here we perform a search for P2RY8 ligands and find P2RY8 bioactivity in bile and in culture supernatants of several mouse and human cell lines. Using a seven-step biochemical fractionation procedure and a drop-out mass spectrometry approach, we show that a previously undescribed biomolecule, S-geranylgeranyl-l-glutathione (GGG), is a potent P2RY8 ligand that is detectable in lymphoid tissues at the nanomolar level. GGG inhibited the chemokine-mediated migration of human germinal-centre B cells and T follicular helper cells, and antagonized the induction of phosphorylated AKT in germinal-centre B cells. We also found that the enzyme gamma-glutamyltransferase-5 (GGT5), which was highly expressed by follicular dendritic cells, metabolized GGG to a form that did not activate the receptor. Overexpression of GGT5 disrupted the ability of P2RY8 to promote B cell confinement to germinal centres, which indicates that GGT5 establishes a GGG gradient in lymphoid tissues. This work defines GGG as an intercellular signalling molecule that is involved in organizing and controlling germinal-centre responses. As the P2RY8 locus is modified in several other types of cancer in addition to GCB-DLBCL and Burkitt lymphoma, we speculate that GGG might have organizing and growth-regulatory roles in multiple human tissues. S-geranylgeranyl-l-glutathione (GGG) is identified as a cell signalling molecule that interacts with the receptor P2RY8 to mediate migration inhibition and growth regulation of germinal-centre B cells., Author(s): Erick Lu [sup.1] , Finn D. Wolfreys [sup.1] , Jagan R. Muppidi [sup.1] [sup.2] , Ying Xu [sup.1] , Jason G. Cyster [sup.1] Author Affiliations: (1) Howard Hughes Medical [...]

In high-fat diet (HFD) induced nonalcoholic fatty liver disease (NAFLD), there is an increase in the endocannabinoid system activity, which significantly contributes to steatosis development. The aim of our study was to investigate the effects of cannabinoid receptor type 1 blockade on adipokine and proinflammatory cytokine content in adipose and hepatic tissue in mice with NAFLD. Male mice C57BL/6 were divided into a control group fed with a control diet for 20 weeks (C, n = 6) a group fed with a HFD for 20 weeks (HF, n = 6), a group fed with a control diet and treated with rimonabant after 18 weeks (R, n = 9), and a group fed with HFD and treated with rimonabant after 18 weeks (HFR, n = 10). Rimonabant significantly decreased leptin, resistin, apelin, visfatin, interleukin 6 (IL-6), and interferon-[gamma] (IFN-[gamma]) concentration in subcutaneous and visceral adipose tissue in the HFR group compared to the HF group (p < 0.01). Rimonabant reduced hepatic IL-6 and IFN-[gamma] concentration as well as plasma glucose and insulin concentration and the homeostatic model assessment index in the HFR group compared to the HF group (p < 0.01). It can be concluded that the potential usefulness of CB1 blockade in the treatment of HFD-induced NAFLD is due to modulation of the adipokine profile and proinflammatory cytokines in both adipose tissues and liver as well as glucose metabolism.Key words: NAFLD, endocannabinoid system, CB1 receptor blockade, adipokines, proinflammatory cytokines, adipose tissue, liver.Dans la steatose hepatique non alcoolique (SHNA) provoquee par un regime alimentaire a haute teneur en matieres grasses (RHG), on observe une augmentation de l'activite du systeme endocannabinoide, ce qui contribue clairement a l'apparition de la steatose. Nos travaux avaient pour but d'etudier les effets de l'inhibition des recepteurs cannabinoides de type 1 sur les concentrations d'adipokines et de cytokines pro-inflammatoires dans les tissus adipeux et hepatique de souris atteintes de SHNA. Nous avons reparti des souris C57BL/6 males dans les groupes suivants : temoin avec un regime alimentaire temoin pendant 20 sem (T, n = 6), RHG pendant 20 sem (HG, n = 6), regime alimentaire temoin avec administration de rimonabant apres 18 sem (R, n = 9) et RHG avec rimonabant apres 18 sem (HGR, n = 10). Le rimonabant entrainait une diminution notable des concentrations de leptine, de resistine, d'apeline, de visfatine, d'interleukine 6 (IL-6) et d'interferon-[gamma] (IFN-[gamma]) dans les tissus adipeux sous-cutane et visceral des souris HGR comparativement au groupe HG (p < 0,01). Le rimonabant entrainait une diminution des concentrations hepatiques d'IL-6 et d'IFN-[gamma], ainsi que de la glycemie et des concentrations plasmatiques d'insuline, et de l'indice du modele d'evaluation de l'homeostasie chez les souris HGR comparativement au groupe HG (p < 0,01). On peut en arriver a la conclusion que l'inhibition des recepteurs CB1 pourrait etre efficace dans le traitement de la SHNA provoquee par le RHG, en raison de la modulation du profil des adipokines et des cytokines pro-inflammatoires dans les tissus adipeux comme hepatiques, ainsi que du metabolisme du glucose. [Traduit par la Redaction]Mots-cles : steatose hepatique non alcoolique, systeme endocannabinoide, inhibition des recepteurs CB1, adipokines, cytokines pro-inflammatoires, tissu adipeux, foie., IntroductionNonalcoholic fatty liver disease (NAFLD) is widely accepted as a hepatic manifestation of the metabolic syndrome (MS). NAFLD is frequently associated with obesity, insulin resistance (IR), hyperglycaemia, dyslipidaemia, increased production [...]

Although depression and cardiovascular diseases are related, the role of antidepressants such as fluoxetine (increasing serotonin levels) within cardiac regulation remains unclear. We aimed to determine whether fluoxetine modifies the pharmacological profile of serotonergic influence on vagal cardiac outflow. Rats were treated with fluoxetine (10 mg/kg per day; p.o.) for 14 days or equivalent volumes of drinking water (control group); then, they were pithed and prepared for vagal stimulation. Bradycardic responses were obtained by electrical stimulation of the vagal fibers (3, 6, and 9 Hz) or i.v. acetylcholine (ACh; 1, 5, and 10 [micro]g/kg). The i.v. administration of 5-hydroxytryptamine (5-HT; 10 and 50 [micro]g/kg) inhibited the vagally induced bradycardia. 5-CT (5-[HT.sub.1/7] agonist) and L-694,247 (5-[HT.sub.1D] agonist) mimicked the serotonin inhibitory effect while [alpha]-methyl-5-HT (5-[HT.sub.2] agonist) was devoid of any action. SB269970 (5-[HT.sub.7] antagonist) did not abolish 5-CT inhibitory action on the electrically induced bradycardia. Pretreatment with LY310762 (5-[HT.sub.1D] antagonist) blocked the effects induced by L-694,247 and 5-CT. 5-HT and 5-CT failed to modify the bradycardia induced by exogenous ACh. Our outcomes suggest that fluoxetine treatment modifies 5-HT modulation on heart parasympathetic neurotransmission in rats, evoking inhibition of the bradycardia via prejunctional 5-[HT.sub.1D] in pithed rats.Key words: 5-HT, 5-[HT.sub.1D] receptor, bradycardia, cardiac parasympathetic neurotransmission, fluoxetine.Bien qu'il existe un lien entre la depression et les maladies cardiovasculaires, le role d'antidepresseurs comme la fluoxetine (augmentation des taux de serotonine) demeure a clarifier en termes de regulation de la fonction cardiaque. Nous visions a etablir si la fluoxetine entraine des modifications dans le profil pharmacologique de l'influence serotoninergique sur le flux vagal efferent ver le coeur. Nous avons administre a des rats de la fluoxetine (10 mg/kg par jour; p.o.) pendant 14 jours ou des volumes equivalents d'eau a boire (groupe temoin); puis, nous les avons demedulles et prepares a une stimulation vagale. Nous avons obtenu des reactions de bradycardie a la suite de la stimulation electrique des fibres vagales (3, 6 et 9 Hz) ou de l'injection i.v. d'acetylcholine (ACh; 1,5 et 10 [micro]g/kg). L'administration i.v. de 5-hydroxytryptamine (5-HT; 10 et 50 [micro]g/kg) a entraine une inhibition de la bradycardie engendree par l'intermediaire des nerfs vagues. Le 5-CT (agoniste 5-[HT.sub.1/7]) et le L-694,247 (agoniste 5-[HT.sub.1D]) mimaient l'effet inhibiteur de la serotonine tandis que l'[alpha]-methyle-5-HT (agoniste 5-[HT.sub.2]) etait exempt de toute activite. Le SB269970 (antagoniste 5-[HT.sub.7]) n'a pas contre l'action inhibitrice du 5-CT sur la bradycardie engendree par la stimulation electrique. L'administration prealable de LY310762 (antagoniste 5-[HT.sub.1D]) entrainait une inhibition des effets produits par le L-694,247 et le 5-CT. Le 5-HT et le 5-CT n'ont pas entraine de modification de la bradycardie engendree par l'ACh exogene. Nos resultats laissent entendre que l'administration de fluoxetine modifierait la modulation par le 5-HT de la neurotransmission parasympathique cardiaque chez le rat, ce qui susciterait l'inhibition de la bradycardie par l'intermediaire du recepteur prejonctionnel 5-[HT.sub.1D] chez le rat demedulle. [Traduit par la Redaction]Mots-cles : 5-HT, recepteur 5-[HT.sub.1D], bradycardie, neurotransmission parasympathique cardiaque, fluoxetine., Introduction and backgroundDepression and cardiovascular diseases are closely associated pathologies and 2 of the world's leading health problems. The former of which may cause and worsen the other. Major depression [...]

Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.Key words: HD-PTP, ESCRT, endosomal sorting, receptor trafficking, tumorigenesis.Les recepteurs de la surface cellulaire declenchent l'activation des voies de signalisation qui regulent des processus cellulaires cles, dont la survie et la proliferation cellulaire. L'internalisation, le tri et le trafic des recepteurs actives jouent ainsi un role important dans la regulation et l'attenuation de la signalisation cellulaire. Le tri efficace des recepteurs internalises par endocytose est realise par le complexe ESCRT. Ces proteines ciblent les recepteurs en vue de leur degradation par l'etablissement sequentiel de complexes proteiques. Ces evenements sont etroitement regules, et le fonctionnement defectueux des composantes de ESCRT peut conduire a un trafic anormal et une signalisation prolongee et favorisant la formation ou la progression tumorale. Dans cette synthese, les auteurs analysent l'organisation du domaine modulaire de HD-PTP, une proteine associee a ESCRT, et son role dans le trafic des recepteurs et la tumorigenese. [Traduit par la Redaction]Mots-cles : HD-PTP, ESCRT, tri endosomal, trafic des recepteurs, tumorigenese., IntroductionCell surface receptors are activated by the binding of their cognate ligands. This event leads to receptor ubiquitylation, internalization, and sorting to be either recycled back to the plasma membrane [...]

Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-[kappa]B signalling.sup.1. Recent studies indicate that the bacterial metabolite d-glycero-[beta]-d-manno-heptose 1,7-bisphosphate (HBP) can activate NF-[kappa]B signalling in host cytosol.sup.2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-[beta]-d-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-[kappa]B activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-[kappa]B. A CRISPR-Cas9 screen showed that activation of NF-[kappa]B by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.The bacterial metabolite ADP-heptose activates NF-[kappa]B in host cells via alpha-kinase 1 and the TIFA-TRAF signalling pathway., Author(s): Ping Zhou [sup.1] , Yang She [sup.1] [sup.2] [sup.3] , Na Dong [sup.4] , Peng Li [sup.1] , Huabin He [sup.1] , Alessio Borio [sup.5] , Qingcui Wu [sup.1] [...]

The prelimbic cortex (PrL) as a part of the medial prefrontal cortex (mPFC) plays a crucial role in drug addiction. Previous studies have shown that glutamatergic transmission through the NMDA and AMPA receptors plays an important role in morphine rewarding properties. In this study, we evaluated the effect of glutamate receptors blockade within the PrL on morphine self-administration. Male Wistar rats were randomly selected and divided into 7 groups. Trained rats were placed in self-administration apparatus, where they pressed an active lever for receiving morphine (5 mg/mL) in test groups and saline in saline group during 11 consecutive days for 2 h per session. The effects of intra-prelimbic AMPA receptor antagonist (CNQX; 0.5 and 2.5 [micro]g/0.5 [micro]L) and the NMDA antagonist (AP5; 0.1 and 1 [micro]g/0.5 [micro]L) on self- administration were tested. Our results demonstrated that intra-prelimbic injection of different doses of CNQX and AP5, and co-administration of these 2 drugs before self-administration significantly decreased active lever pressing compared with morphine group (p < 0.001). Also, the number of self-infusion significantly decreased in test groups compared with morphine group (p < 0.001). These findings suggest that a reduction in PrL glutamatergic output can modulate morphine reinforcement. Key words: morphine, prelimbic cortex, glutamate receptors, reinforcing effect. Le cortex prelimbique (PrL) en tant que secteur du cortex prefrontal median (CPFm) joue un role central dans la dependance aux drogues. Des etudes anterieures ont montre que la transmission glutamatergique par l'intermediaire des recepteurs NMDA et AMPA joue un role important dans les proprietes de recompense de la morphine. Dans cette etude, nous avons evalue l'effet de l'inhibition des recepteurs du glutamate dans le PrL sur l'auto-administration de morphine. Nous avons selectionne aleatoirement des rats Wistar males, que nous avons repartis dans sept groupes. Nous avons place les rats conditionnes dans un appareil d'auto-administration ou ils devaient appuyer sur une manette active pour recevoir de la morphine (5 mg/mL) dans les groupes test et une solution saline dans le groupe saline au cours de seances de 2 h pendant 11 jours consecutifs. Nous avons etudie les effets d'un antagoniste des recepteurs AMPA intralimbiques (CNQX; 0,5 et 2,5 [micro]g/0,5 [micro]L), ainsi que d'un antagoniste des recepteurs NMDA (AP5; 0,1 et 1 [micro]g/0,5 [micro]L) sur l'auto-administration. Nos resultats ont montre que l'injection intralimbique de differentes doses de CNQX et d'AP5, ainsi que l'administration concomitante de ces deux medicaments avant l'auto-administration entrainaient une diminution plus marquee de l'actionnement de la manette que dans le groupe morphine (p < 0,001). Par ailleurs, le nombre d'autoperfusions diminuait nettement plus dans les groupes test que dans le groupe morphine (p < 0,001). Ces observations laissent entrevoir qu'une diminution de la production glutamatergique dans le PrL peut participer a la modulation du renforcement de l'utilisation de la morphine. [Traduit par la Redaction] Mots-cles: morphine, cortex prelimbique, recepteurs du glutamate, effet de renforcement., Introduction Drug addiction is a chronic relapsing disorder, characterized by compulsive drug seeking and periods of repeated drug use. It has been evidenced that the medial prefrontal cortex (mPFC) plays [...]

Anticonvulsant effects of minocycline have been explored recently. This study was designed to examine the anticonvulsant effect of acute administration of minocycline on pentylenetetrazole-induced seizures in mouse considering the possible role of the nitric oxide/N-methyl-D-aspartate (NMDA) pathway. We induced seizure using intravenous administration of pentylenetetrazole. Our results showed that acute administration of minocycline increased the seizure threshold. Furthermore, co- administration of sub-effective doses of the nonselective nitric oxide synthase (NOS) inhibitor [N.sup.G]-L-arginine methyl ester (10 mg/kg) and the neuronal NOS inhibitor 7-nitroindazole (40 mg/kg) enhanced the anticonvulsant effect of subeffective doses of minocycline (40 mg/kg). We found that inducible NOS inhibitor aminoguanidine (100 mg/kg) had no effect on the antiseizure effect of minocycline. Moreover, L-arginine (60 mg/kg), as a NOS substrate, reduced the anticonvulsant effect of minocycline. We also demonstrated that pretreatment with the NMDA receptor antagonists ketamine (0.5 mg/kg) and MK-801 (0.05 mg/kg) increased the anticonvulsant effect of subeffective doses of minocycline. Results showed that minocycline significantly decreased the hippocampal nitrite level. Furthermore, co-administration of a neuronal NOS inhibitor like NMDA receptor antagonists augmented the effect of minocycline on the hippocampal nitrite level. In conclusion, we revealed that anticonvulsant effect of minocycline might be, at least in part, due to a decline in constitutive hippocampal nitric oxide activity as well as inhibition of NMDA receptors. Key words: minocycline, anticonvulsant, nitric oxide, NO, neuronal nitric oxide synthase, nNOS, N-methyl- Daspartate, NMDA. On a recemment explore les effets anticonvulsivants de la minocycline. Cette etude a ete concue en vue d'examiner l'effet anticonvulsivant de l'administration aigue de minocycline sur des convulsions provoquees par le pentylenetetrazole chez la souris, en tenant compte du role eventuel de la voie de signalisation oxyde nitrique/N-methyl-D-aspartate (NMDA). Nous avons provoque des convulsions par l'administration intraveineuse de pentylenetetrazole. Nos resultats ont montre que l'administration aigue de minocycline augmentait le seuil des convulsions. En outre, l'administration concomitante de doses sous-efficaces de <> (inhibiteur non selectif de l'oxyde nitrique synthase (NOS); 10 mg/kg) et de 7-nitroindazole (inhibiteur de la NOS neuronale; 40 mg/kg) entramait une augmentation de l'effet anticonvulsivant de doses sous efficaces de minocycline (40 mg/kg). Nous avons observe que l'aminoguanidine (inhibiteur de la NOS inductible; 100 mg/kg) n'avait aucun effet sur les effets anticonvulsivants de la minocycline. De surcroit, la L-arginine (60 mg/kg), en tant que substrat de la NOS, entramait une reduction de l'effet anticonvulsivant de la minocycline. Nous avons aussi montre que l'administration prealable d'antagonistes des recepteurs du NMDA, de ketamine (0,5 mg/kg) et de MK-801 (0,05 mg/kg), entramait une augmentation de l'effet anticonvulsivant de doses sous-efficaces de minocycline. Les resultats ont montre que la minocycline entramait une diminution marquee des concentrations de nitrite dans l'hippocampe. En outre, l'administration concomitante d'inhibiteurs de la NOS neuronale comme des antagonistes des recepteurs du NMDA entramait une augmentation de l'effet de la minocycline sur les taux de nitrite dans l'hippocampe. En conclusion, nous avons revele que l'effet anticonvulsivant de la minocycline pourrait etre, au moins en partie, cause par la diminution de l'activite de l'oxyde nitrique constitutif dans l'hippocampe, ainsi que par l'inhibition des recepteurs du NMDA. [Traduit par la Redaction] Mots-cles: minocycline, anticonvulsivant, oxyde nitrique, NO, oxyde nitrique synthase neuronale, nNOS, N-methyl-D-aspartate, NMDA., Introduction Epilepsy arises due to abnormal and excessive neuronal activity of the brain and is recognized as a syndrome characterized by additional co-occurring conditions (Fisher et al. 2014). Regardless of [...]

The antinociceptive effects of cannabinoids and opioids have been known for centuries. Serotonin and its receptors are also known to play important roles in nociception. However, the contribution of spinal [5-HT.sub.5A] receptors in antinociceptive effects of cannabinoids and opioids has not been studied. We conducted this study to clarify spinal mechanisms of the actions of the antinociceptive effects of cannabinoids and opioids. Hot plate and tail flick tests were used to assess the antinociceptive activity in Balb/c mice. WIN 55,212-2, a nonselective CB1 and CB2 agonist, and morphine exerted significant antinociceptive effects at 1, 3, and 10 mg/kg doses administered intraperitoneally in both hot plate and tail flick tests. The selective [5-HT.sub.5A] receptor antagonist SB-699551 (10 nmol/mouse) was administered intrathecally 10 min before the agonists. SB-699551 significantly reduced the antinociceptive effect of both WIN 55,212-2 and morphine. In the rotarod test, WIN 55,212-2 disrupted the motor coordination at a dose of 10 mg/kg, while morphine did not affect this function at any dose. Our findings show that spinal 5-HT5A receptors are involved in the antinociceptive effects of WIN 55,212-2 and morphine. Key words: antinociception, morphine, SB-699551, spinal 5-HT5A receptors, WIN 55,212-2. L'effet antinociceptif des cannabinoides et des opioides est connu depuis des siecles. On sait aussi que la serotonine et ses recepteurs jouent un role important dans la nociception. Cependant, la contribution des recepteurs [5-HT.sub.5A] de la moelle epiniere aux effets antinociceptifs des cannabinoides et des opioides n'a pas ete etudiee. Nous avons mene cette etude en vue de clarifier les modes d'action de l'action des effets antinociceptifs des cannabinoides et des opioides dans la moelle epiniere. A l'aide des tests de la plaque chauffante et de retraction de la queue, nous avons evalue l'activite antinociceptive chez des souris Balb/c. Le WIN 55,212-2, un agoniste non selectif des recepteurs CB1 et CB2, ainsi que la morphine ont exerce des effets antinociceptifs importants avec l'administration intraperitoneale de doses de 1, de 3 et de 10 mg/kg pour les tests de la plaque chauffante et de retraction de la queue. Dix minutes avant l'administration des agonistes, nous avons administre par voie intrathecale le SB-699551, un antagoniste selectif des recepteurs [5-HT.sub.5A] (10 nmol/souris). Le SB-699551 entrainait une diminution notable de l'effet antinociceptif du WIN 55,212-2 comme de celui de la morphine. Avec le test de la tige tournante (<< rotarod >>), le WIN 55,212-2 perturbait la coordination motrice a la dose de 10 mg/kg, alors que la morphine n'avait aucun effet sur ce fonctionnement, quelle que soit la dose. Nos resultats montrent que les recepteurs [5-HT.sub.5A] de la moelle epiniere jouent un role dans les effets antinociceptifs du WIN 55,212-2 et de la morphine. [Traduit par la Redaction] Mots-cles: antinociception, morphine, SB-699551, recepteurs 5-HT5A de la moelle epiniere, WIN 55,212-2., Introduction Opioids and cannabinoids are 2 different groups of analgesics that have comparable pharmacological characteristics, including analgesia, hypoactivity, sedation, and hypothermia. Likewise, tolerance and physical dependence develop with long-term use [...]

Nonalcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. Coagonists of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) are under clinical investigation for the treatment of obesity and type 2 diabetes. In this study, we have demonstrated the effect of a balanced coagonist in the treatment of NAFLD using mouse models. GLP-1R agonist exendin-4, glucagon, and coagonist (Aib2 C24 chimera2) were administered to C57BL6/J mice, in which NAFLD was induced by carbon tetrachloride ([CCl.sub.4]) treatment after high-fat diet (HFD) feeding, and choline-deficient, Lamino-acid-defined HFD (CDAHFD) feeding. Repeated dose administration of coagonist significantly attenuated liver inflammation and steatosis induced by acute and long-term treatment with [CCl.sub.4] in HFD-fed mice. Coagonist markedly attenuated the CDAHFDinduced expression of TIMP-1, MMP-9, TNF-[alpha], MCP-1, COL1A1, and [alpha]-SMA. It also inhibited progression of hepatic steatosis and fibrosis in mice. Exendin-4 was better than glucagon, but coagonist was most effective in reduction of hepatic inflammation as well as steatosis. Coagonist of GLP-1R and GCGR improved NAFLD in C57BL6/J mice. This effect is mediated by reduction in lipotoxicity and inflammation in liver. Key words: coagonist of GLP-1 and glucagon receptors, GLP-1, glucagon, inflammation, NAFLD. Resume: La steatose hepatique non alcoolique (SHNA) est souvent associee a l'obesite et au diabete de type 2. Les coagonistes des recepteurs GLP-1R (pour << glucagon-like peptide-1 receptor >>) et du glucagon (GCGR) sont a l'etude clinique du traitement de l'obesite et du diabete de type 2. Dans cette etude, nous avons montre l'effet d'un coagoniste equilibre dans des modeles de traitement de la SHNA chez la souris. Nous avons administre de l'exendine-4 (un agoniste du GLP-1R), du glucagon et un coagoniste (Aib2 C24 chimera2) a des souris C57BL6/J chez lesquelles nous avions provoque une SHNA en administrant de tetrachlorure de carbone ([CCl.sub.4]) apres un regime alimentaire a haute teneur en matieres grasses (HMG) et un regime alimentaire du type HMG avec carence en choline, acides L-amines definis (HMGCCA). L'administration de doses reiterees du coagoniste a nettement attenue l'inflammation et la steatose hepatiques engendrees par l'administration a long terme de [CCl.sub.4] chez les souris du groupe HMG. Le coagoniste a permis d'attenuer nettement l'augmentation de l'expression de TIMP-1, de MMP-9, de TNF-[alpha], de MCP-1, de COL1A1 et d' [alpha]-SMA que le regime alimentaire HMGCCA provoquait. Il permettait aussi d'attenuer revolution de la steatose et de la fibrose hepatiques chez les souris. L'exendine-4 etait plus efficace que le glucagon, mais le coagoniste etait la substance la plus efficace dans la reduction de l'inflammation et de la steatose hepatiques. Le coagoniste des recepteurs GLP-1R et GCGR permettait d'attenuer la SHNA chez les souris C57BL6/J. Cet effet est medie par la reduction de la lipotoxicite et de l'inflammation dans le foie. [Traduit par la Redaction] Mots-cles: coagoniste des recepteurs de GLP-1 et du glucagon, GLP-1, glucagon, inflammation, SHNA., Introduction Nonalcoholic fatty liver disease (NAFLD) is caused by deposition of lipids in liver (Brea and Puzo 2013). NAFLD may start from only steatosis, and can worsen to nonalcoholic steatohepatitis [...]

[beta]-arrestins are critical regulator and transducer proteins for G-protein-coupled receptors (GPCRs). [beta]-arrestin is widely believed to be activated by forming a stable and stoichiometric GPCR-[beta]-arrestin scaffold complex, which requires and is driven by the phosphorylated tail of the GPCR. Here we demonstrate a distinct and additional mechanism of [beta]-arrestin activation that does not require stable GPCR-[beta]-arrestin scaffolding or the GPCR tail. Instead, it occurs through transient engagement of the GPCR core, which destabilizes a conserved inter-domain charge network in [beta]-arrestin. This promotes capture of [beta]-arrestin at the plasma membrane and its accumulation in clathrin-coated endocytic structures (CCSs) after dissociation from the GPCR, requiring a series of interactions with membrane phosphoinositides and CCS-lattice proteins. [beta]-arrestin clustering in CCSs in the absence of the upstream activating GPCR is associated with a [beta]-arrestin-dependent component of the cellular ERK (extracellular signal-regulated kinase) response. These results delineate a discrete mechanism of cellular [beta]-arrestin function that is activated catalytically by GPCRs.Transient engagement of the G protein-coupled receptor core can act as a catalyst to activate cellular [beta]-arrestin function after dissociation from the receptor., Author(s): Kelsie Eichel [sup.1] [sup.2] , Damien Jullié [sup.1] [sup.2] , Benjamin Barsi-Rhyne [sup.1] [sup.2] , Naomi R. Latorraca [sup.3] [sup.4] [sup.5] [sup.6] , Matthieu Masureel [sup.5] , Jean-Baptiste Sibarita [...]

Targeting peroxisome proliferator-activated receptor-gamma (PPAR-[gamma]) is an approved strategy in facing insulin resistance (IR) for diabetes mellitus (DM) type 2. The PPAR-[gamma] modulators display improvements in the insulin-sensitizing and adverse effects of the traditional thiazolidinediones. Nitazoxanide (NTZ) is proposed as a PPAR-[gamma] receptor ligand with agonistic posttranscriptional effects. Currently, NTZ antidiabetic activities versus pioglitazone (PIO) in a high-fat diet/streptozotocin rat model of type 2 diabetes was explored. Diabetic adult male Wistar rats were treated orally with either PIO (2.7 mg x [kg.sup.- 1] x day-1) or NTZ (200 mg x [kg.sup.-1] x [day.sup.-1]) for 14, 21, and 28 days. Body masses, fasting blood glucose, IR, lipid profiles, and liver and kidney functions of rats were assayed. Hepatic glucose metabolism and PPAR-[gamma] protein expression levels as well as hepatic, pancreatic, muscular, and renal histopathology were evaluated. Significant time-dependent euglycemic and insulin-sensitizing effects with preservation of liver and kidney functions were offered by NTZ. Higher hepatic levels of glucose-6-phosphatase and glucose-6- phosphate dehydrogenase enzymes and PPAR-[gamma] protein expressions were acquired by NTZ and PIO, respectively. NTZ could be considered an oral therapeutic strategy for DM type 2. Further systematic NTZ/PPAR-[gamma] receptor subtype molecular activations are recommended. Simultaneous use of NTZ with other approved antidiabetics should be explored. Key words: high-fat diet/streptozotocin-induced diabetes, insulin resistance, nitazoxanide, peroxisome proliferator activated receptor-gamma, pioglitazone. Le recepteur gamma active par les proliferateurs de peroxysomes (PPAR-[gamma] pour peroxisome << proliferator- activated receptor-gamma >>) est une cible homologuee pour contrer la resistance a l'insuline (RI) dans le diabete de type 2 (DT2). Les modulateurs du PPAR-[gamma] permettent d'obtenir des ameliorations sur le plan des effets indesirables et de la sensibilisation a l'insuline lies aux thiazolidinediones classiques. On propose d'utiliser la nitazoxanide (NTZ), dotee d'une activite posttranscriptionnelle agoniste, comme ligand des PPAR-[gamma]. Dans les presents travaux, nous avons compare l'activite antidiabetique de la NTZ a celle de la pioglitazone (PIO) dans un modele de DT2 engendre par un regime alimentaire a teneur elevee en matieres grasses et la streptozotocine chez le rat. Chez des rats Wistar males adultes diabetiques, nous avons administre par voie orale de la PIO (2,7 mg x [kg.sup.-1] x [jour.sup.-1]) ou de la NTZ (200 mg-[kg.sup.-1]-jour-1) pendant 14, 21 et 28 jours. Nous avons mesure le poids corporel, la glycemie a jeun, la RI, le profil lipidique et les fonctions renale et hepatique des rats. Nous avons evalue le metabolisme hepatique du glucose, les taux d'expression proteique du PPAR-[gamma], ainsi que l'histopathologie du foie, du pancreas, des muscles et des reins. La NTZ permettait d'obtenir des effets marques et proportionnels a la duree d'euglycemie, de sensibilisation a l'insuline et de preservation des fonctions renale et hepatique. Avec la NTZ et la PIO, nous avons observe respectivement une augmentation des concentrations hepatiques des enzymes glucose-6-phosphatase et glucose-6-phosphate deshydrogenase, ainsi que de l'expression proteique du PPAR-[gamma]. On pourrait considerer la NTZ comme constituant une strategie therapeutique orale contre le DT2. Nous recommandons d'etudier plus avant l'activation moleculaire de sous-types du PPAR-[gamma] par la NTZ de maniere systematique. Il conviendrait aussi d'etudier l'utilisation simultanee de NTZ avec d'autres antidiabetiques homologues. [Traduit par la Redaction] Mots-cles : diabete engendre par un regime alimentaire a teneur elevee en matieres grasses et la streptozotocine, resistance a l'insuline, nitazoxanide, recepteur gamma active par les proliferateurs de peroxysomes, pioglitazone., Introduction Insulin resistance (IR) is a disease where cellular failure to insulin responses (Kubota et al. 2017; Sam et al. 2017) and obesity are the main contributors of diabetes mellitus [...]

Cumulative data suggest the significant role of the renin-angiotensin system in the development of the pathological consequences of diabetes mellitus (DM). Newly synthesized AT2 receptor agonists gained importance as a target for creating new antihypertensives. The aim of the present work was to study the effects of peptide AT2 agonist novokinin, infused intracerebroventricularly, on the consequences of the streptozotocin-induced type 1 DM (T1DM) in Wistar rats. Food and water consumption, body mass, urine excretion (metabolic cages), motor activity (open-field test), anxiety (elevated plus maze), nociception (paw pressure analgesimeter test), spatial memory (T-maze alternation test), and plasma levels of glucose and corticosterone (ELISA) were assessed 2 weeks after the T1DM induction. Novokinin increased water and food consumption, as well as urine output, and reduced mass gain in the control rats. Diabetic rats demonstrated hyperalgesia, increased level of plasma corticosterone, decreased motor and exploratory activity, and impaired spatial memory. Novokinin infusion increased water intake, diuresis, and mortality rate, decreased food intake, exacerbated diabetes-induced hyperalgesia, and provoked anxiety-like behavior but improved spatial memory in diabetic rats. These initial data suggest that angiotensin AT2 receptors participate in the pathogenesis of TIDM-induced complications in the function of the nervous system. Key words: angiotensin AT2 receptors, diabetes mellitus, behavior, corticosterone, rats. Le cumul des donnees laisse entrevoir un role important pour le systeme renine-angiotensine dans l'apparition de consequences pathologiques du diabete sucre (DS). De nouveaux antagonistes synthetiques des recepteurs de l'AT2 ont acquis une certaine importance en tant que cible en vue de creer de nouveaux antihypertenseurs. Les presents travaux avaient pour but d'etudier les effets de la perfusion a l'interieur des ventricules cerebraux de novokinine, un peptide agoniste AT2, sur les consequences du DS de type 1 provoque par la streptozotocine chez le rat Wistar. Deux semaines apres la formation du DS de type 1, nous avons evalue la consommation d'eau et de nourriture, le poids corporel, l'excretion d'urine (cages metaboliques), l'activite motrice (test en terrain libre), l'anxiete (labyrinthe en croix sureleve), la nociception (test de pression de la patte avec analgesimetrie), la memoire spatiale (test d'alternance dans le labyrinthe en T), ainsi que les taux plasmatiques de glucose et de corticosterone (ELISA). Chez les rats temoins, la novokinine entrainait une augmentation de la consommation d'eau et de nourriture, ainsi que de la production d'urine, avec une diminution de la prise de poids. Chez les rats atteints de diabete, nous avons observe une hyperalgesie et une augmentation des taux plasmatiques de corticosterone, avec une diminution de l'activite motrice et exploratoire, ainsi qu'une deterioration de la memoire spatiale. Chez les rats atteints de diabete, la perfusion de novokinine entrainait une augmentation de la consommation d'eau, de la diurese et du taux de mortalite, avec une diminution de la consommation de nourriture, une exacerbation de l'hyperalgesie provoquee par le diabete, ainsi que des comportements semblables a l'anxiete, mais une amelioration de la memoire spatiale. Ces donnees initiales laissent entendre que les recepteurs de l'AT2 participeraient a la pathogenese des complications provoquees par le DS de type 1 quant au fonctionnement du systeme nerveux. [Traduit par la Redaction] Mots-cles : recepteurs de l'AT2, diabete sucre, comportement, corticosterone, rats., Introduction Diabetes mellitus (DM) is accompanied by chronic complications, such as neuropathic pain, loss of sensation, and impaired motor coordination (Feldman et al. 2017; Timar et al. 2016). In some [...]