Combining NK cells and mAb9.2.27 to combat NG2-dependent and anti-inflammatory signals in glioblastoma

Glioblastoma is a deadly brain cancer with limited treatment options. Targeting chondroitin sulfate proteoglycan 4 (CSPG4, best known as NG2) with the monoclonal antibody mAb9.2.27 and activated natural killer (NK) cells abrogated the tumor growth and prolonged the survival of glioblastoma-bearing animals by favoring the establishment of a pro-inflammatory microenvironment. The combination of NK cells and mAb9.2.27 recruited ED1+CCR2low macrophages that stimulated ED1+ED2lowMHCIIhigh microglial cells to exert robust cytotoxicity. Our findings demonstrate the therapeutic potential of targeting salient tumor associated-antigens.publishedVersio

Elucidating tumour-associated microglia/macrophage diversity along glioblastoma progression and under ACOD1 deficiency

In glioblastoma (GBM), tumour-associated microglia/macrophages (TAMs) represent the major cell type of the stromal compartment and contribute to tumour immune escape mechanisms. Thus, targeting TAMs is emerging as a promising strategy for immunotherapy. However, TAM heterogeneity and metabolic adaptation along GBM progression represent critical features for the design of effective TAM-targeted therapies. Here, we comprehensively study the cellular and molecular changes of TAMs in the GL261 GBM mouse model, combining single-cell RNA-sequencing with flow cytometry and immunohistological analyses along GBM progression and in the absence of Acod1 (also known as Irg1), a key gene involved in the metabolic reprogramming of macrophages towards an anti-inflammatory phenotype. Similarly to patients, we identify distinct TAM profiles, mainly based on their ontogeny, that reiterate the idea that microglia- and macrophage-like cells show key transcriptional differences and dynamically adapt along GBM stages. Notably, we uncover decreased antigen-presenting cell features and immune reactivity in TAMs along tumour progression that are instead enhanced in Acod1-deficient mice. Overall, our results provide insight into TAM heterogeneity and highlight a novel role for Acod1 in TAM adaptation during GBM progression.publishedVersio

Association of the PHACTR1/EDN1 genetic locus with spontaneous coronary artery dissection

Background: Spontaneous coronary artery dissection (SCAD) is an increasingly recognized cause of acute coronary syndromes (ACS) afflicting predominantly younger to middle-aged women. Observational studies have reported a high prevalence of extracoronary vascular anomalies, especially fibromuscular dysplasia (FMD) and a low prevalence of coincidental cases of atherosclerosis. PHACTR1/EDN1 is a genetic risk locus for several vascular diseases, including FMD and coronary artery disease, with the putative causal noncoding variant at the rs9349379 locus acting as a potential enhancer for the endothelin-1 (EDN1) gene. Objectives: This study sought to test the association between the rs9349379 genotype and SCAD. Methods: Results from case control studies from France, United Kingdom, United States, and Australia were analyzed to test the association with SCAD risk, including age at first event, pregnancy-associated SCAD (P-SCAD), and recurrent SCAD. Results: The previously reported risk allele for FMD (rs9349379-A) was associated with a higher risk of SCAD in all studies. In a meta-analysis of 1,055 SCAD patients and 7,190 controls, the odds ratio (OR) was 1.67 (95% confidence interval [CI]: 1.50 to 1.86) per copy of rs9349379-A. In a subset of 491 SCAD patients, the OR estimate was found to be higher for the association with SCAD in patients without FMD (OR: 1.89; 95% CI: 1.53 to 2.33) than in SCAD cases with FMD (OR: 1.60; 95% CI: 1.28 to 1.99). There was no effect of genotype on age at first event, P-SCAD, or recurrence. Conclusions: The first genetic risk factor for SCAD was identified in the largest study conducted to date for this condition. This genetic link may contribute to the clinical overlap between SCAD and FMD

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Targeting the anti-inflammatory interplay promoting glioblastoma progression with combined natural killer cells and mab9.2.27 against NG2/CSPG4. A novel strategy to reveal glioblastoma to the immune system

Glioblastoma (GBM) is the most frequent and aggressive brain tumor in adults. The patients’ median survival is only 14.6 months despite multimodal treatment, including surgery and concomitant radiation and chemotherapy. Thus, there is a stark need for the development of new, potent therapeutic strategies and targets. For a decade our group investigated the implication of the cell surface neuroglial-2 proteoglycan, NG2/CSPG4, in the development and growth of glioma. These studies revealed that NG2/CSPG4 is critical in multiple mechanisms that favor tumor survival and expansion. Indeed, GBM cells expressing NG2/CSPG4 were characterized to have a higher proliferation rate and migratory capacity in vitro. In addition, in vivo implantation of GBM cell lines expressing this proteoglycan resulted in a bigger tumor mass, with a higher neovascularization compared to its negative counterpart. Furthermore, these NG2/CSPG4 positive neoplastic cells were more resistant to chemotherapy and radiotherapy in vitro and in vivo. We further demonstrated that 50 % of GBM patients expressed NG2/CSPG4, and that this expression independently correlated with a poorer survival. Taken together, this proteoglycan might be an amenable target for immunotherapy. On the other hand, our research team has high expertise on natural killer (NK) cell biology. We observed that despite the promising results described for NK cell based immunotherapy to treat hematological malignancies, there is sparse data on their use to treat GBM. The main goal of this thesis was the study and the validation of a novel combination treatment using mAb9.2.27 targeted against NG2/CSPG4 and activated NK cells to treat GBM in rat orthotopic xenograft models. Thus, we first investigated the presence and phenotype of NK cells in the healthy brain. We further characterized the immunological status of GBM patients, both within the tumor microenvironment and their peripheral blood. Then we designed a novel purification method to obtain highly pure “untouched” NK cells from rat. This last method was developed to be able to finally investigate the efficacy of the combined NK+mAb9.2.27 treatment in GBM -bearing rats. All this work led to 4 original publications. The first publication of this thesis revealed that NK cells could be found in the brain of naïve mice as well as in the cerebrospinal fluid obtained from non-pathological brain of patients. Phenotypically, brain NK cells displayed immature CD11blowCD27+ phenotypes in mice and they were mostly CD56bright in patients. In this publication we discussed these new findings in relation to the existing literature on systemic and brain NK cells in the context of central nervous system (CNS) disorders, such as brain tumors, infections, neurodegenerative diseases and mental disorders. We observed that the knowledge on brain NK cells is limited in the context of CNS disorders, though more information about the presence and role of NK cells in the peripheral blood of patients with such disorders is readily available. Nevertheless, this work distinguished that NK cells could be either implicated in neuroprotection or neurodegeneration. This review highlights the potential of a better comprehension of NK cells in brain pathogenesis that could further help to delineate new therapeutic targets for the treatment of CNS disorders. In the second paper, we performed the characterization of the immunological status of GBM patients, in order to delineate the interplay between the immune system and tumor that can favor tumor growth. Immunohistochemistry staining on 65 GBM biopsies revealed that the patients’ survival correlated with their infiltration by CD3+ cells as well as CD8+ cells. We further observed that following tumor infiltration the T lymphocytes as well as antigen presenting cells down modulated their activation molecules and up regulated their inhibitory molecules. Furthermore, we characterized for the first time the presence of infiltrated regulatory T cells with CD8+CD28-Foxp3+ phenotype that may further propagate the anti-inflammatory environment created by the tumor. Despite these findings, further analyses indicated potential for immunomodulatory therapies for GBM management. The third paper of this thesis detailed a novel protocol to obtain highly pure “untouched” rat NK cells, based on magnetic-bead purification method. As currently no commercial kits to get untouched rat NK cells exist, many research teams used positive selection to purify them. Our method permits the separation of NK cells from athymic nude, Lewis and Fisher rat strains with high purity, ease, and cost affectivity and without bias of method-related activation artifacts. Indeed, we observed that positive selection of NK cells modified their proliferative and functional capacities. We conclude that for future fundamental studies, negative purification should be preferred to limit activation bias induced by separation methods. This method was further applied to purify NK cells in order to investigate their efficacy as monotherapy or in combination with mAb9.2.27 against NG2/CSPG4 as a new strategy to treat GBM. This NK+mAb9.2.27 treatment was evaluated in several orthotropic models of rats bearing GBM tumors. We observed that this combined therapy prolonged animal survival compared to monotherapy controls. This was associated with a diminution of tumor mass, associated with increased cellular apoptosis and diminished proliferation in the tumor bed. The levels of pro-inflammatory IFN-g and TNF-a, were increased in the brain of treated animals, while in contrast anti-inflammatory IL-10 molecules were reduced. We further identified tumor recruited pro-inflammatory macrophages as the mechanism that mediated the anti-tumor immune responses as their depletion by clodronate abolished tumor destruction. All together, we propose that targeting the anti-inflammatory interplay promoting GBM progression with combined NK cells and mAb9.2.27 against NG2/CSPG4 could be amenable to treat GBM patients, by reversing the anti-inflammatory tumor microenvironment to a proinflammatory one in order to reveal GBM to the immune system

Polymérisation radicalaire contrôlée d'esters et d'amides de vinyle (études expérimentales et théoriques)

Ces travaux de thèse portent sur la polymérisation radicalaire contrôlée (PRC) des esters et amides de vinyle. L une des possibilités de contrôle est le piégeage dynamique réversible des chaînes radicalaires croissantes (P ) par un agent de contrôle (T) formant une espèce dormante (P T ). La concentration en radicaux dans le milieu peut alors diminuer dramatiquement de sorte que les réactions indésirables de terminaisons soient négligeables et que le contrôle de la masse molaire des polymères soit atteint avec un faible indice de dispersité. L utilisation de complexes métalliques, pouvant s oxider et former une liaison métal-carbone, comme agent de piégeage des radicaux est une manière de réaliser ce contrôle. La PRC est alors appelée Polymérisation Radicalaire Contrôlée par voie Organométallique (OMRP). A ce jour, plusieurs métaux de transitions ont été utilisés avec plus ou moins de succès en OMRP. Lors de cette étude, nous avons synthétisé des complexes de cuivre(I) et testé leurs performances pour l OMRP de l acétate de vinyle et de l éthylène. Nous avons également utilisé des outils de chimie théorique pour mieux comprendre pourquoi le cobalt(II) acétylacétonate est, jusqu à aujourd hui, le meilleur agent de contrôle pour la polymérisation de l acétate de vinyle et des amides de vinyle. Grâce à la théorie de la fonctionnelle de densité (DFT), nous avons mis en lumière le rôle crucial de la coordination sur le cobalt des groupements carbonyles des monomères étudiés.This thesis focus on Controlled Radical Polymerization (CRP) of vinyl esters and vinyl amides. One of the possibilities to achieve this control is a dynamic reversible trapping of the growing radical chains (P ) by a controlling agent (T) to form a dormant species (P T ). The radical concentration in the medium can be dramatically reduced so that the unwanted terminations are disfavored and polymers with controlled molecular weights and low dispersity can be obtained. A way to achieve this control is the use of metallic complexes, which can oxidize and form a metal-carbon bond, as trapping agent in the so-called Organometallic Mediated Radical Polymerization (OMRP). So far, different transition metals have been used with gretaer or smaller success. In this study, the synthesis of copper(I) complexes and their investigation for the vinyl acetate and ethylene polymerization under OMRP conditions were performed. We also used computational chemistry as a tool to better understand why the cobalt(II) acetylacetonate (Co(acac)2) has, so far, given the best results for either vinyl acetate or vinyl amides polymerization. Thanks to Density Functional Theory (DFT), the crucial role of the monomer carbonyl group coordination to cobalt was pointed out.TOULOUSE-INP (315552154) / SudocSudocFranceF

Combining NK cells and mAb9.2.27 to combat NG2-dependent and anti-inflammatory signals in glioblastoma

Glioblastoma is a deadly brain cancer with limited treatment options. Targeting chondroitin sulfate proteoglycan 4 (CSPG4, best known as NG2) with the monoclonal antibody mAb9.2.27 and activated natural killer (NK) cells abrogated the tumor growth and prolonged the survival of glioblastoma-bearing animals by favoring the establishment of a pro-inflammatory microenvironment. The combination of NK cells and mAb9.2.27 recruited ED1+CCR2low macrophages that stimulated ED1+ED2lowMHCIIhigh microglial cells to exert robust cytotoxicity. Our findings demonstrate the therapeutic potential of targeting salient tumor associated-antigens

Effect of Head-to-Head Addition in Vinyl Acetate Controlled Radical Polymerization: Why Is Co(acac) 2 -Mediated Polymerization so Much Better?

International audienceThe controlled polymerization of vinyl acetate has been recently achieved by several techniques, but PVAc with targeted Mn and low dispersity up to very high monomer conversions and high degrees of polymerization was only obtained with Co(acac)2 as controlling agent in the so-called CMRP, a type of organometallic mediated radical polymerization (OMRP). Other techniques (including ATRP, ITP, TERP, and RAFT/MADIX) have shown a more or less pronounced slowdown in the polymerization kinetics, which was attributed to the higher strength of the C–X bond between the radical PVAc chain and the trapping agent (X) in the dormant species and to a consequent slower reactivation after a less frequent head-to-head monomer addition. The reason for the CMRP exception is clarified by the present contribution. First, a detailed investigation by 1H, 13C and multiplicity-edited HSQC and DEPT-135 NMR of the PVAc obtained by CMRP, in comparison with a regular polymer made by free radical polymerization under the same conditions, has revealed that Co(acac)2 does not significantly alter the fraction of head-to-head sequences in the polymer backbone and that there is no accumulation of Co(acac)2-capped chains with a head-to-head ω end. Hence, both dormant chains (following the head-to-head and the head-to-tail monomer additions) must be reactivated at similar rates. A DFT study shows that this is possible because the dormant chains are stabilized not only by the C–Co σ bond but also by formation of a chelate ring through coordination of the ω monomer carbonyl group. The head-to-head dormant chain contains an inherently stronger C–Co bond but forms a weaker 6-membered chelate ring, whereas the weaker C–Co bond in the head-to-tail dormant chain is compensated by a stronger 5-membered chelate ring. Combination of the two effects leads to similar activation enthalpies, as verified by DFT calculations using a variety of local, gradient-corrected, hybrid and “ad hoc” functionals (BPW91, B3PW91, BPW91*, M06 and M06L). While the BDE(C–X) of model H-VAc–X molecules [X = Cl, I, MeTe, EtOC(S)S and Co(acac)2] are functional dependent, the BDE difference between head-to-head and head-to-tail dormant chain models is almost functional insensitive, with values of 5–9 kcal/mol for the ATRP, ITP and TERP models, 3–6 for the RAFT/MADIX model, and around zero for CMRP

Key role of intramolecular metal chelation and hydrogen bonding in the cobalt-mediated radical polymerization of N-vinyl amides

This work reveals the preponderance of an intramolecular metal chelation phenomenon in a controlled radical polymerization system involving the reversible trapping of the radical chains by a cobalt complex, i.e. the bis(acetylacetonato)cobalt(II). The cobalt-mediated radical polymerization (CMRP) of a series of N-vinyl amides was considered in order to evidence the effect of the cobalt chelation by the amide moiety of the last monomer unit of the chain. The latter reinforces the cobalt-polymer bond in the order N-vinylpyrrolidone < N-vinyl caprolactam < N-methyl-N-vinyl acetamide, and is responsible for the optimal control of the polymerizations observed for the last two monomers. Such a double linkage between the controlling agent and the polymer, via a covalent bond and a dative one, is unique in the field of controlled radical polymerization and represents a powerful opportunity to fine tune the equilibrium between latent and free radicals. The possible hydrogen bond formation is also taken into account in the case of N-vinyl acetamide and N-vinyl formamide. These results are essential for understanding factors influencing a Co-C bond strength in general, and the CMRP mechanism in particular, but also for developing a powerful platform for the synthesis of new precision poly(N-vinyl amide)s, an important class of polymers which sustains numerous applications today