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International audience; The imaging of different intercellular regions is attracting growing interest in the fields of biosensing, drug delivery, and gene therapy for cancer treatment. We present the synthesis of luminescent gold nanoclusters (AuNCs), grafted with a PEGylated U 11 peptide derivative, for tumor recognition and their capacity to label the nucleus of pancreatic cancer cells. The short peptide named C 3 E 6 U 11 , used to synthesize and functionalize gold nanoclusters, is composed of a tricysteine sequence with sidechain thiol residues as peptide anchors for the Au nanoclusters together with the recognition peptide domain (U 11). The active targeting red-or blue-emitting AuNCs can label pancreatic cancer cells and localize preferentially in the nucleus. The presence of U 11 at their surface facilitates their penetration into pancreatic cell nuclei. Finally, in vivo imaging experiments in early embryos and freeswimming juvenile zebrafish are also performed to evaluate their biodistribution.

International audience; Antibody-secreting cells (ASC) are divided into two principal subsets, including the long-lived plasma cell (PC) subset residing in the bone marrow and the short-lived subset, also called plasmablast (PB). PB are described as a proliferating subset circulating through the blood and ending its differentiation in tissues. Due to their inherent heterogeneity, the molecular signature of PB is not fully established. The purpose of this study was to decipher a specific PB signature in humans and mice through a comprehensive meta-analysis of different data sets exploring the PB differentiation in both species and across different experimental conditions. The present study used recent analyses using whole RNA sequencing in prdm1-GFP transgenic mice to define a reliable and accurate PB signature. Next, we performed similar analysis using current data sets obtained from human PB and PC. The PB-specific signature is composed of 155 and 113 genes in mouse and human being, respectively. Although only nine genes are shared between the human and mice PB signature, the loss of B-cell identity such as the down-regulation of PAX5, MS4A1, (CD20) CD22 and IL-4R is a conserved feature across species and across the different experimental conditions. Additionally, we observed that the IRF8 and IRF4 transcription factors have a specific dynamic range of expression in human PB. We thus demonstrated that IRF4/IRF8 intranuclear staining was useful to define PB in vivo and in vitro and able to discriminate between atypical PB populations and transient states.

Regulatory B cells (Bregs) have been highlighted in very different pathology settings including autoimmune diseases, allergy, graft rejection, and cancer. Improving tools for the characterization of Bregs has become the main objective especially in humans. Transitional, mature B cells and plasma cells can differentiate into IL-10 producing Bregs in both mice and humans, suggesting that Bregs are not derived from unique precursors but may arise from different competent progenitors at unrestricted development stages. Moreover, in addition to IL-10 production, regulatory B cells used a broad range of suppressing mechanisms to modulate the immune response. Although Bregs have been consistently described in the literature, only a few reports described the molecular aspects that control the acquisition of the regulatory function. In this manuscript, we detailed the latest reports describing the control of IL-10, TGFβ, and GZMB production in different Breg subsets at the molecular level. We focused on the understanding of the role of the transcription factors STAT3 and c-MAF in controlling IL-10 production in murine and human B cells and how these factors may represent an important crossroad of several key drivers of the Breg response. Finally, we provided original data supporting the evidence that MAF is expressed in human IL-10- producing plasmablast and could be inducedin vitrofollowing different stimulation cocktails. At steady state, we reported that MAF is expressed in specific human B-cell tonsillar subsets including the IgD+CD27+unswitched population, germinal center cells and plasmablast.

International audience; The Transient Receptor Potential Vanilloid type 2 (TRPV2) channel is highly selective for Ca2+ and can be activated by lipids, such as LysoPhosphatidylCholine (LPC). LPC analogues, such as the synthetic alkyl-ether-lipid edelfosine or the endogenous alkyl-ether-lipid Platelet Activating Factor (PAF), modulates ion channels in cancer cells. This opens the way to develop alkyl-ether-lipids for the modulation of TRPV2 in cancer. Here, we investigated the role of 2-Acetamido-2-Deoxy-l-O-Hexadecyl-rac-Glycero-3-PhosphatidylCholine (AD-HGPC), a new alkyl-ether-lipid (LPC analogue), on TRPV2 trafficking and its impact on Ca2+ -dependent cell migration. The effect of AD-HGPC on the TRPV2 channel and tumour process was further investigated using calcium imaging and an in vivo mouse model. Using molecular and pharmacological approaches, we dissected the mechanism implicated in alkyl-ether-lipids sensitive TRPV2 trafficking. We found that TRPV2 promotes constitutive Ca2+ entry, leading to migration of highly metastatic breast cancer cell lines through the PI3K/Akt-Girdin axis. AD-HGPC addresses the functional TRPV2 channel in the plasma membrane through Golgi stimulation and PI3K/Akt/Rac-dependent cytoskeletal reorganization, leading to constitutive Ca2+ entry and breast cancer cell migration (without affecting the development of metastasis), in a mouse model. We describe, for the first time, the biological role of a new alkyl-ether-lipid on TRPV2 channel trafficking in breast cancer cells and highlight the potential modulation of TRPV2 by alkyl-ether-lipids as a novel avenue for research in the treatment of metastatic cancer.

Ciguatera fish poisoning (CFP) and neurotoxic shellfish poisoning syndromes are induced by the consumption of seafood contaminated by ciguatoxins and brevetoxins. Both toxins cause sensory symptoms such as paresthesia, cold dysesthesia and painful disorders. An intense pruritus, which may become chronic, occurs also in CFP. No curative treatment is available and the pathophysiology is not fully elucidated. Here we conducted single-cell calcium video-imaging experiments in sensory neurons from newborn rats to study in vitro the ability of Pacific-ciguatoxin-2 (P-CTX-2) and brevetoxin-1 (PbTx-1) to sensitize receptors and ion channels, (i.e., to increase the percentage of responding cells and/or the response amplitude to their pharmacological agonists). In addition, we studied the neurotrophin release in sensory neurons co-cultured with keratinocytes after exposure to P-CTX-2. Our results show that P-CTX-2 induced the sensitization of TRPA1, TRPV4, PAR2, MrgprC, MrgprA and TTX-r NaV channels in sensory neurons. P-CTX-2 increased the release of nerve growth factor and brain-derived neurotrophic factor in the co-culture supernatant, suggesting that those neurotrophins could contribute to the sensitization of the aforementioned receptors and channels. Our results suggest the potential role of sensitization of sensory receptors/ion channels in the induction or persistence of sensory disturbances in CFP syndrome.

Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-α) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD- CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population (referred to as NARB+) characterized by the expression of marginal zone B-cell markers CD45RB and CD1c. Similar to memory B cells, NARB+ cells were in a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals while maintaining their susceptibility to IL-21 activation-induced apoptosis as observed for the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil and gut-associated lymphoid tissues, supported that NARB+ are uncommitted precursors of human marginal zone B cells.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1–sarco/endoplasmic reticulum calcium transport ATPase (SERCA) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In pancreatic AR42J cells expressing the p.E152K variant, Ca2+ signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation. This article has an associated First Person interview with the first author of the paper.

Immunological memory is essential for effective immune protection upon antigen rechallenge. Memory B cells encompass multiple subsets, heterogeneous in terms of phenotypes, origins and precursors, anatomical localization, and functional responses. B-cell responses are conditioned by micro-environmental signals, including cytokines. Here, we analyzed in vitro the effects of two cytokines implicated in B-cell differentiation, interferon-alpha (IFN-α) and interleukin (IL)-21, on the early functional response of four different mature B-cell subsets (IgD+ CD27- naive, IgD+ CD27+ unswitched, IgD- CD27+ switched and double-negative B cells). The dual response of naive and memory B cells to IL-21 allowed us to uncover a unique IgD+ CD27- CD10- B-cell population characterized by the expression of marginal zone B-cell markers CD45RB and CD1c (referred to as NARB+). Similar to memory B cells, NARB+ cells displayed a pre-activated state, allowing them to rapidly differentiate into plasmablasts upon innate signals. However, this population also maintained its susceptibility to IL-21 activation-induced apoptosis similarly to the naive compartment. Both in-depth phenotypic analysis of circulating B cells, and identification of these cells in spleen, tonsil, and gut-associated lymphoid tissues, suggested that NARB+ could be uncommitted precursors of different developmental pathways including human marginal zone-like B cells (MZB). In this regard, we showed that patients with primary Sjogren’s syndrome exhibited a severe reduction of IgD+CD27+ MZB-like cells associated with a diminution of the NARB+ subset suggesting a developmental relationship.

Red tides involving Karenia brevis expose humans to brevetoxins (PbTxs). Oral exposition triggers neurotoxic shellfish poisoning, whereas inhalation induces a respiratory syndrome and sensory disturbances. No curative treatment is available and the pathophysiology is not fully elucidated. Protease-activated receptor 2 (PAR2), cathepsin S (Cat-S) and substance P (SP) release are crucial mediators of the sensory effects of ciguatoxins (CTXs) which are PbTx analogs. This work explored the role of PAR2 and Cat-S in PbTx-1-induced sensory effects and deciphered the signaling pathway involved. We performed calcium imaging, PAR2 immunolocalization and SP release experiments in monocultured sensory neurons or co-cultured with keratinocytes treated with PbTx-1 or P-CTX-2. We demonstrated that PbTx-1-induced calcium increase and SP release involved Cat-S, PAR2 and transient receptor potential vanilloid 4 (TRPV4). The PbTx-1-induced signaling pathway included protein kinase A (PKA) and TRPV4, which are compatible with the PAR2 biased signaling induced by Cat-S. Internalization of PAR2 and protein kinase C (PKC), inositol triphosphate receptor and TRPV4 activation evoked by PbTx-1 are compatible with the PAR2 canonical signaling. Our results suggest that PbTx-1-induced sensory disturbances involve the PAR2-TRPV4 pathway. We identified PAR2, Cat-S, PKA, and PKC that are involved in TRPV4 sensitization induced by PbTx-1 in sensory neurons.

Objective Pain, temperature, and itch are conventionally thought to be exclusively transduced by the intraepidermal nerve endings. Although recent studies have shown that epidermal keratinocytes also participate in sensory transduction, the mechanism underlying keratinocyte communication with intraepidermal nerve endings remains poorly understood. We sought to demonstrate the synaptic character of the contacts between keratinocytes and sensory neurons and their involvement in sensory communication between keratinocytes and sensory neurons. Methods Contacts were explored by morphological, molecular, and functional approaches in cocultures of epidermal keratinocytes and sensory neurons. To interrogate whether structures observed in vitro were also present in the human epidermis, in situ correlative light electron microscopy was performed on human skin biopsies. Results Epidermal keratinocytes dialogue with sensory neurons through en passant synaptic-like contacts. These contacts have the ultrastructural features and molecular hallmarks of chemical synaptic-like contacts: narrow intercellular cleft, keratinocyte synaptic vesicles expressing synaptophysin and synaptotagmin 1, and sensory information transmitted from keratinocytes to sensory neurons through SNARE-mediated (syntaxin1) vesicle release. Interpretation By providing selective communication between keratinocytes and sensory neurons, synaptic-like contacts are the hubs of a 2-site receptor. The permanent epidermal turnover, implying a specific en passant structure and high plasticity, may have delayed their identification, thereby contributing to the long-held concept of nerve endings passing freely between keratinocytes. The discovery of keratinocyte-sensory neuron synaptic-like contacts may call for a reassessment of basic assumptions in cutaneous sensory perception and sheds new light on the pathophysiology of pain and itch as well as the physiology of touch. ANN NEUROL 2020;88:1205-1219.

International audience

The intracellular Ca

Since deregulation of intracellular Ca

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.Summary statementp.E152K-STIM1 variant found in pancreatitis patients leads to intracellular changes in calcium homeostasis through SERCA interaction, enabling intracellular trypsin activation and pancreatic acinar cell death.

The intracellular Ca2+ concentration is mainly controlled by Ca2+ channels. These channels form complexes with K+ channels, which function to amplify Ca2+ flux. In cancer cells, voltage-gated/voltage-dependent Ca2+ channels and non-voltage-gated/voltage-independent Ca2+ channels have been reported to interact with K+ channels such as Ca2+-activated K+ channels and voltage-gated K+ channels. These channels are activated by an increase in cytosolic Ca2+ concentration or by membrane depolarization, which induces membrane hyperpolarization, increasing the driving force for Ca2+ flux. These complexes, composed of K+ and Ca2+ channels, are regulated by several molecules including lipids (ether lipids and cholesterol), proteins (e.g. STIM), receptors (e.g. S1R/SIGMAR1), and peptides (e.g. LL-37) and can be targeted by monoclonal antibodies, making them novel targets for cancer research.

Background Dysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. Methods An extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. Results Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup. Conclusions These data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane. Electronic supplementary material The online version of this article (10.1186/s40425-019-0591-3) contains supplementary material, which is available to authorized users.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

La temperature, la douleur et le prurit sont classiquement considerees comme etant exclusivement percus par les fibres nerveuses intra-epidermiques. Alors que des etudes recentes ont demontre que les keratinocytes epidermiques participent egalement a la transduction sensorielle cutanee, le mecanisme qui sous-tend la communication entre les keratinocytes et les fibres nerveuses intra-epidermiques reste meconnu. Nous demontrons, dans cette etude, que les kerationocytes dialoguent avec les neurones sensoriels par l’intermediaire de contacts « en passant » de type synaptique. Ces contacts ont les caracteristiques ultrastructurales et les caracteristiques moleculaires de synapses chimiques, vehiculant des informations sensorielles des keratinocytes vers les neurones sensoriels par l’intermediaire d’une exocytose mediee par des complexes SNAREs. Le renouvellement permanent de l’epiderme, qui implique une structure specifique de type « en passant » ainsi qu’une grande plasticite, pourrait avoir retarde leur identification, contribuant par consequent a la perennite du concept de fibres nerveuses intra-epidermiques cheminant librement entre les keratinocytes. En assurant une communication selective entre les keratinocytes et les neurones sensoriels, ces contacts synaptiques pourraient etre le pivot d’un recepteur a deux sites, l’un keratinocytaire et l’autre neuronal. La decouverte de ces contacts neuro-keratinocytaires de type synaptique pourrait amener a une reconsideration des concepts etablis au sujet de la perception sensorielle cutanee, et invite a porter un regard nouveau sur la physiopathologie de la douleur et du prurit, aussi bien que de la physiologie du toucher.

International audience; Biases in the distribution and phenotype of T, B, and antigen-presenting cell populations are strongly connected to mechanisms of disease development in mouse models of systemic lupus erythematosus (SLE). Here, we describe longitudinal changes in lymphoid and antigen-presenting cell subsets in bone marrow, blood and spleen from two lupus-prone strains (MRL/lpr and B6.Sle1.Sle2.Sle3 tri-congenic mice), and how they integrate in our present understanding of the pathogenesis of the disease. In particular, we focus on (autoreactive) T cell activation patterns in lupus-prone mice. Break of T cell tolerance to chromatin constituents (histone peptides) is key to the development of the disease and is related to T cell intrinsic defects, contributed by genetic susceptibility factors and by extrinsic amplificatory mechanisms, in particular over-stimulation by antigen-presenting cells. We also describe shifts in B cell sub-populations, going from skewed immature B cell populations as an indication of disturbed central and peripheral tolerance checkpoints, to enriched long-lived plasma cells, which are key to persistent autoantibody production in the disease. B cell activation mechanisms in SLE are both T cell-dependent (break of tolerance and production of specific autoantibodies) and -independent (polyclonal B cell activation, production of autoantibodies by long-lived plasma cells). By providing a comprehensive evaluation of B and T cell surface markers in two major mouse models of SLE and a description of their changes before and after disease onset, this review illustrates how the study of lymphoid cell phenotype delivers key information regarding pathogenic pathways and supplies tools to assess the beneficial effects of novel therapeutic interventions.

International audience; Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformational changes accompanying homo- or heterodimerization of STIM1. For this purpose, the ER luminal portion of STIM1 (residues 58-236), which includes the sterile alpha motif (SAM) domain plus the calcium-binding EF-hand domains 1 and 2 attached to the STIM1 transmembrane region (TM), was modeled and embedded in a virtual membrane. Next, molecular dynamics simulations were performed to study the conformational changes that take place during STIM1 activation and subsequent protein-protein interactions. Indeed, the simulations revealed exposure of residues in the EF-hand domains, which may be important for dimerization steps. Altogether, understanding conformational changes in STIM1 can help in drug discovery when targeting this key protein in intracellular calcium functions.

International audience; Calumenin is a secretory pathway protein regulating different endoplasmic reticulum (ER) proteins such as the sarco-endoplasmic reticulum calcium ATPase (SERCA) pumps. Combined with its diverse cellular distribution, its calcium-binding ability, and its interaction with proteins involved in calcium signaling, it is easy to speculate on future description of important roles of calumenin in calcium homeostasis in many cell types, as it was initially observed in muscle cells. In this chapter, we describe basic techniques to modulate calumenin expression and detect its impact on ER calcium content using classic transfection and Western blot techniques, as well as ER calcium measurement using microplate reader.

BACKGROUND Several studies had suggested the potential role of calcium signaling in prostate cancer (PCa) prognosis and agressiveness. We aimed to investigate selected proteins contributing to calcium (Ca2+ ) signaling, (Orai, stromal interaction molecule (STIM), and transient receptor potential (TRP) channels) and involved in cancer hallmarks, as independent predictors of systemic recurrence after radical prostatectomy (RP). METHODS A case-control study including 112 patients with clinically localized PCa treated by RP between 2002 and 2009 and with at least 6-years' follow-up. Patients were divided into two groups according to the absence or presence of systemic recurrence. Expression levels of 10 proteins involved in Ca2+ signaling (TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, STIM1, STIM2, Orai1, Orai2, and Orai3), were assessed by immunohistochemistry using tissue microarrays (TMAs) constructed from paraffin-embedded PCa specimens. The level of expression of the various transcripts in PCa was assessed using quantitative polymerase chain reaction (qPCR) analysis. RNA samples for qPCR were obtained from fresh frozen tissue samples of PCa after laser capture microdissection on RP specimens. Relative gene expression was analyzed using the 2-▵▵Ct method. RESULTS Multivariate analysis showed that increased expression of TRPC1, TRPC4, TRPV5, TRPV6, TRPM8, and Orai2 was significantly associated with a lower risk of systemic recurrence after RP, independently of the prostate-specific antigen (PSA) level, percentage of positive biopsies, and surgical margin (SM) status (P = .007, P = .01, P

The racemic synthesis in four steps of pyrazole SKF-96365 analogues without substituent (CF3 group) on the pyrazole platform was realized in moderate to good yields. The separation of (±) hydroxyl enantiomers 4 was developed successfully using the method of "half-concentration" with commercial (+)-(1S)- and (-)-(1R)-10-camphorsulfonic acid (CSA) followed by neutralization of diastereomers with MeONa in dry MeOH solution. With the pure enantiomers (-)-(1S)-4b and (+)-(1R)-4b, initial attempts to obtain the crystallized (-)-(1S)-7d and (+)-(1R)-7d after treatment of intermediate 6d with a solution of 1M HCl (for precipitation of hydrochloride salt 7d) failed. We have also investigated the effects of compounds 7(a-d) on endoplasmic reticulum (ER) Ca2+ and SOCE on PLP-B lymphocyte cell line and compound 7d was identified as a better SOCE inhibitor than SKF-96365. This preliminary SAR study showed that the MeO group in para-position of the phenethyl-1H-pyrazolium skeleton or for the Cbeta-phenylpropoxy side chain of 7 influenced the SOCE activity.

Introduction Les ciguatoxines (CTXs) et les brevetoxines (PbTxs) sont des neurotoxines impliquees respectivement dans la ciguatera et l’intoxication neurologique par les fruits de mer, deux syndromes consecutifs a l’ingestion de poissons ou de mollusques contamines. Les symptomes communs mais d’intensite plus marquee dans la ciguatera, sont des troubles neuro-cutanes (prurit, paresthesies et dysesthesies au froid) qui peuvent persister plusieurs semaines a plusieurs annees. Les cibles primaires de ces toxines sont le canal sodium dependant du potentiel de membrane (Nav) dont elles entrainent l’ouverture. Il s’ensuit une hyperexcitabilite membranaire des neurones peripheriques. Cependant, les evenements moleculaires en aval restent peu connus et aucun traitement n’existe pour soulager ces troubles neuro-cutanes persistants et invalidants. Recemment, nous avons mis en evidence que la Pacific-ciguatoxine-2 (P-CTX-2), appliquee a une co-culture de keratinocytes/neurones sensoriels, induit la liberation des neuropeptides de l’inflammation neurogene (CGRP et substance P). Chez la souris, il a ete montre que l’allodynie au froid induite par les CTXs implique la sensibilisation (i.e. l’abaissement du seuil d’activation) Nav-dependante de TRPA1. Nos travaux ont pour but d’etudier la sensibilisation de plusieurs recepteurs sensoriels cutanes par ces neurotoxines. Materiel et methodes Des cultures de neurones sensoriels de rats nouveaux-nes ont ete incubes pendant 24 h avec la P-CTX-2 ou la brevetoxine-1 (PbTx-1). A l’aide de la video-microscopie en fluorescence sur cellule unique, nous avons etudie la modulation par ces toxines de la reponse calcique a un agoniste specifique du recepteur sensoriel d’interet. Dans un second temps, nous avons dose dans le surnageant de co-cultures de keratinocytes et neurones sensoriels traitee par la P-CTX-2 plusieurs mediateurs susceptibles de contribuer a la sensibilisation de recepteurs sensoriels. Resultats Nos resultats montrent que la P-CTX-2 et la PbTx-1 sensibilisent differents recepteurs sensoriels de la peau. De plus, ils mettent en evidence que la P-CTX-2 augmente, dans la coculture la liberation de plusieurs mediateurs, suggerant leur role dans la sensibilisation des recepteurs sensoriels cutanes induite par les neurotoxines. Discussion Ce travail permet de mieux comprendre la physiopathologie des troubles sensoriels induits par les CTXs et les PbTxs et le prurit de maniere generale. Cette etude suggere que le mecanisme de sensibilisation mis en evidence pourrait participer a l’initiation et la persistance des troubles neuro-cutanees induits par la P-CTX-2 et la PbTx-1. Nos resultats et la caracterisation des mecanismes impliques dans la sensibilisation pourraient permettre d’identifier des voies therapeutiques ciblees. Ce travail a ete realise avec le soutien de la Societe francaise de dermatologie.

Cystic fibrosis (CF), one of the most common fatal hereditary disorders, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene product is a multidomain adenosine triphosphate-binding cassette (ABC) protein that functions as a chloride (Cl(-)) channel that is regulated by intracellular magnesium [Mg(2+)]i. The most common mutations in CFTR are a deletion of a phenylalanine residue at position 508 (ΔF508-CFTR, 70-80 % of CF phenotypes) and a Gly551Asp substitution (G551D-CFTR, 4-5 % of alleles), which lead to decreased or almost abolished Cl(-) channel function, respectively. Magnesium ions have to be finely regulated within cells for optimal expression and function of CFTR. Therefore, the melastatin-like transient receptor potential cation channel, subfamily M, member 7 (TRPM7), which is responsible for Mg(2+) entry, was studies and [Mg(2+)]i measured in cells stably expressing wildtype CFTR, and two mutant proteins (ΔF508-CFTR and G551D-CFTR). This study shows for the first time that [Mg(2+)]i is decreased in cells expressing ΔF508-CFTR and G551D-CFTR mutated proteins. It was also observed that the expression of the TRPM7 protein is increased; however, membrane localization was altered for both ΔF508del-CFTR and G551D-CFTR. Furthermore, both the function and regulation of the TRPM7 channel regarding Mg(2+) is decreased in the cells expressing the mutated CFTR. Ca(2+) influx via TRPM7 were also modified in cells expressing a mutated CFTR. Therefore, there appears to be a direct involvement of TRPM7 in CF physiopathology. Finally, we propose that the TRPM7 activator Naltriben is a new potentiator for G551D-CFTR as the function of this mutant increases upon activation of TRPM7 by Naltriben.

International audience; From a series of (1, 1)-1[β-(phenylalkoxy)-(phenetyl)]-1-pyrazolium hydrochloride as new analogues of SKF-96365, one has an interesting effect for endoplasmic reticulum (ER) Carelease and store-operated Caentry (SOCE) (IC25 μM) on the PLP-B lymphocyte cell line. A successful resolution of (±) 1-phenyl-2-(1-pyrazol-1-yl)ethan-1-ol has been developed by using the method of "half-concentration" in the presence of (+)-(1)- or (-)-(1)-CSA.

The stinging test is an in vivo protocol that evaluates sensitive skin using lactic acid (LA). A soothing sensation of cosmetics or ingredients can be also appreciated through a decrease in stinging score. To predict the soothing sensation of a product before in vivo testing, we developed a model based on an LA test and substance P (SP) release using a co-culture of human keratinocytes and NGF-differentiated PC12 cells. A bacterial fucose-rich polysaccharide present in Fucogel® was evaluated as the soothing molecule in the in vivo stinging test and our in vitro model. Excluding toxic concentrations, the release of SP was significant from 0.2% of lactic acid for the PC12 cells and from 0.1% of lactic acid for the keratinocytes. When the pH was adjusted to approximately 7.4, LA did not provoke SP release. At these concentrations of LA, 0.1% of polysaccharide showed a significant decrease in SP release from the two cellular types and in co-cultures without modifying the pH of the medium. In vivo, a stinging test using the polysaccharide showed a 30% decrease in prickling intensity vs the placebo in 19 women between the ages of 21 and 69. Our in vitro model is ethically interesting and is adapted for cosmetic ingredients screening because it does not use animal experimentation and limits human volunteers. Moreover, Fucogel® reduced prickling sensation as revealed by the in vivo stinging test and inhibits the neurogenic inflammation as showed by our new in vitro stinging test based on SP release.

The remodeling of calcium homeostasis contributes to the cancer hallmarks and the molecular mechanisms involved in calcium channel regulation in tumors remain to be characterized. Here, we report that SigmaR1, a stress-activated chaperone, is required to increase calcium influx by triggering the coupling between SK3, a Ca2+-activated K+ channel (KCNN3) and the voltage-independent calcium channel Orai1. We show that SigmaR1 physically binds SK3 in BC cells. Inhibition of SigmaR1 activity, either by molecular silencing or by the use of sigma ligand (igmesine), decreased SK3 current and Ca2+ entry in breast cancer (BC) and colorectal cancer (CRC) cells. Interestingly, SigmaR1 inhibition diminished SK3 and/or Orai1 levels in lipid nanodomains isolated from BC cells. Analyses of tissue microarray from CRC patients showed higher SigmaR1 expression levels in cancer samples and a correlation with tumor grade. Moreover, the exploration of a cohort of 4937 BC patients indicated that high expression of SigmaR1 and Orai1 channels was significantly correlated to a lower overall survival. As the SK3/Orai1 tandem drives invasive process in CRC and bone metastasis progression in BC, our results may inaugurate innovative therapeutic approaches targeting SigmaR1 to control the remodeling of Ca2+ homeostasis in epithelial cancers.

International audience; Maintenance of self-tolerance of auto-reactive lymphocytes is a fundamental mechanism to prevent the onset of autoimmune diseases. Deciphering the mechanisms involved in the deregulations leading to tolerance disruption and autoimmunity is still a major area of interest to identify new therapeutic targets and options. Ca(2+) signaling plays a major role in B cell normal development and is therefore finely tuned by B cell receptor (BCR)-dependent and independent pathways. Developmental changes in the characteristics of BCR-dependent Ca(2+) signals as well as the modulation of basal intracellular concentration ([Ca(2+)]i) contribute strongly to self-tolerance maintaining mechanisms responsible for the physical or functional elimination of autoreactive B cells such as clonal deletion, receptor editing, and anergy. Implication of Ca(2+) signals in B tolerance mechanisms mainly occurs through the specific activation of transcriptional programs depending on the amplitude, shape, and duration of Ca(2+) signals. A large number of studies reported Ca(2+) signaling defects in autoimmune pathology such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary Sjӧgren's syndrome (pSS). However, the precise nature of the molecular events responsible for these deregulations is not fully understood. Moreover, the demonstration of a direct correlation between Ca(2+) signaling defects and tolerance disruption is still lacking. The recent identification of proteins involved in B cell Ca(2+) signals such as ORAI, stromal interaction molecule and transient receptor potential is opening new horizons for understanding Ca(2+) signaling defects observed in autoimmune diseases and for proposing potentially new therapeutic solutions. This review aims to present an overview of the developmental evolution of BCR dependent Ca(2+) signaling and to place this signaling pathway in the context of mechanisms involved in tolerance maintenance and breakdown.

International audience; Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca(2+)) homeostasis. In this study, we discovered that the Ca(2+) binding protein Calumenin (CALU) is a key regulator in the maintenance of ER-Ca(2+) calcium homeostasis in both wild type and F508del-CFTR expressing cells. Calumenin modulates SERCA pump activity without drastically affecting ER-Ca(2+) concentration. In addition, reducing Calumenin expression in CF cells results in a partial restoration of CFTR activity, highlighting a potential function of Calumenin in CFTR maturation. These findings demonstrate a pivotal role for Calumenin in CF cells, providing insights into how modulation of Calumenin expression or activity may be used as a potential therapeutic tool to correct defects in F508del-CFTR.

Cystic Fibrosis (CF) is the most frequent fatal genetic disease in Caucasian populations. Mutations in the chloride channel CF Transmembrane Conductance Regulator (CFTR) gene are responsible for functional defects of the protein and multiple associated dysregulations. The most common mutation in patients with CF, F508del-CFTR, causes defective CFTR protein folding. Thus minimal levels of the receptor are expressed at the cell surface as the mutated CFTR is retained in the endoplasmic reticulum (ER) where it correlates with defective calcium (Ca

Skin-derived precursor cells (SKPs) have been shown as a population of stem cells that express some markers common to the neural crest stem cells (NCSCs) as progenitor neural nestin, the low affinity neurotrophin receptor p75 and transcription factors as Sox9 and 10 and Pax3, Slug, Snail. SKPs are NCSCs that persist in certain adult tissues, particularly in the skin, which can generate a large part of the peripheral nervous system, particularly sensory neurons (SN). In this study, we reported a protocol to obtain SNs from SKPs. For this, we rely on a protocol already established by Reinhardt et al. in 2013 from Embryonic stem cells (ESCs) and induced Pluripotent stem cells (iPSCs). The SKPs were induced to generate SNs by treating cells with trophic factors and / or chemicals that had previously been shown to be important for the generation of SNs: Purmorphamine, CHIR99021, BMP4 and GDNF, BDNF, NGF. We show that the differentiation of SKPs into SNs was regulated by neurogenins 1, 2 and 3. At the end of the differentiation, the protocol allowed gene transcription of BRN3A and PRDM12 (marker of pain-sensing nerve cells). Expression was from 1000 to 2500 times higher for PRDM12 and BRN3A respectively versus undifferentiated SKP. Using immunostaining we showed that 65% and 80% of cells expressed BRN3A and peripherin, marker for peripheral neurons. Furthermore, cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, H1, B3-tubulin. Using calcium imaging, a proportion of cells was responding for histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKP are NCSC, that able to differentiate directly into functional sensory neuron.