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The objective of the present study was to compare recent methods for characterizing cell modifications. We studied the effect of extracellular reduced glutathione (GSH) on an olfactory neuroblast cell line (13s24). Three methods were used to monitor, in label-free, noninvasive real-time experiments, cell surface occupancy by measuring impedance (xCELLigence), cell behavior (HoloMonitor cytometry), cell ultrastructure by measuring refractive index (3D Nanolive microscopy). Reduced glutathione dose-dependently increased cell volume and motility and decreased cell adhesion. Cell sorting analyses revealed that after short-term exposure (6 h), GSH reduced F-actin polymerization and extracellular glycoproteins leading to adhesion strength loss. Results support the hypothesis that excreted GSH could modulate disulfide bound-dependent integrin conformations involved in neurogenesis and/or neuronal plasticity. This is the first evidence of a causal link between GSH and changes in cell volume and motility required for cell division, migration, and/or differentiation. Results show the importance of real-time analysis methods, without labelling, in the study of cell responses under culture conditions. The present findings highlight important criteria in the choice of methods, beyond the parameters studied, such as cell preparation time, plate filling time, number of cells studied, friendly use of the devices, and the complexity of data processing.

Aim: Serum calprotectin (SC), a novel biomarker of inflammatory bowel diseases (IBD), has been recently investigated with conflicting results. The purpose of this study was to assess the ability of SC to predict relapse in IBD patients treated by biologic therapies, and to evaluate the correlation between SC, clinical and endoscopic relapse and other biomarkers as fecal calprotectin (FC) and C-reactive protein (CRP). Methods: All consecutive IBD patients in deep remission (clinical, endoscopic or imaging remission) were followed 12 months in this prospective study. Blood and stool samples were collected for SC, serum CRP and FC. SC was measured the day of inclusion (baseline, D0), 3 months (M3) and at 6 months (M6) or during the study period for clinical relapse. Relapse was defined as clinical, biomarkers, or endoscopic/imaging activities. Evolution of SC was quantified before relapse to analyze a predicting value of loss of response (LOR). SC for patients with active IBD and those with symptoms without inflammation were also compared. Results: Among the 119 patients included, 54 (46.4%) patients experienced a disease relapse during follow-up. Median SC levels did not increase in patients with clinical relapse (3.15 µg/ml at baseline, 3.38 µg/ml at M3, 3.33 µg/ml at M6 and 3.99 µg/ml in case of relapse (p = 0.63)). SC were compared during relapse in patients with endoscopic remission but clinical symptoms defined as secondary Irritable Bowel Syndrome (IBS). SC levels were higher in active IBD and similar between the groups of patients with IBS or deep remission (3.05 µg/ml IBS vs 2.99 µg/ml remission vs 5.1 µg/ml for clinical relapse, p = 0.04). In patients with clinical symptoms, SC presents a good predictive value for relapse (AUROC 0.764, IC95: 0.68–0.88), with a sensitivity of 72%, a specificity of 77%, using a cut-off value of 4.45 µg/ml. A weak, but significant correlation was found between SC and FC levels (r = 0.35, P = 0.001). A combined score with CRP, FC and SC is not efficient to improve IBD diagnostic. Conclusion: SC was significantly higher in patients with clinical relapse compared to those with endoscopic remission with or without clinical symptoms. SC allow to discriminate patients with active IBD or with IBS but failed to predict relapse.

Metabolic disorders related to obesity are largely dependent on adipose tissue hypertrophy, which involves adipocyte hypertrophy and increased adipogenesis. Adiposize is regulated by lipid accumulation as a result of increased lipogenesis (mainly lipid uptake in mature adipocytes) and reduced lipolysis. Using realtime 2D cell culture analyses of lipid uptake, we show (1) that high glucose concentration (4.5 g/L) was required to accumulate oleic acid increasing lipid droplet size until unilocularization similar to mature adipocytes in few days, (2) oleic acid reduced Peroxisome-Proliferator Activated Receptor Gamma (PPARG) gene transcription and (3) insulin counteracted oleic acid-induced increase of lipid droplet size. Although the lipolytic activity observed in high versus low glucose (1 g/L) conditions was not altered, insulin was found to inhibit oleic acid induced gene transcription required for lipid storage such as Cell Death Inducing DFFA Like Effectors (CIDEC) and G0S2 (G0 switch gene S2), possibly through PPARA activity. Although this signalling pathway requires more detailed investigation, the results point out the differential mechanisms involved in the pro-adipogenic effect of insulin in absence versus its protective effect on adiposity in presence of oleic acid uptake.Abbreviations: AICAR, 5-Aminoimidazole-4-carboxamide-1-D-ribofuranoside; AMPK, AMP-Activated protein kinase, ASCs, adipose stem cell; ATGL, adipose triglyceride lipase; BSA, Bovine serum albumin; CEBPA, CCAAT enhancer binding protein alpha; CIDEs, Cell Death Inducing DFFA Like Effectors; dA, differentiated adipocyte; DMEM, Dulbecco’s Modified Eagle’s Medium; FABPs, Fatty Acid Binding Proteins; FAT/CD36, Fatty acid translocase; FCS, Foetal calf serum; FN1, fibronectin 1; FFA, free fatty acid; G0S2, G0 switch gene S2; GLUTs, Glucose transporters; GPR120, G protein-coupled receptor 120; HG, high glucose; HSL, hormone sensitive lipase; INSR, insulin receptor; LG, low glucose; OA, oleic acid; PBS, Phosphate buffer saline; PPARs, Peroxisome-Proliferator Activated Receptors; PKA, Protein kinase cyclic AMP-dependent; PKG, Protein kinase cyclic GMP dependent; PTGS2, cytochrome oxidase 2; RTCA, realtime cell analysis; TG, triglyceride.

Abstract Background ABP501 is a biosimilar to Reference Adalimumab (HUMIRA®) produced by AMGEN. Adalimumab (ADA) has a marketing authorization for Crohn's disease, ulcerative colitis and other inflammatory or autoimmune diseases. The aim of this study was to evaluate the LISA-TRACKER assays developed by Theradiag (France), for the monitoring of ABP501 and anti-ABP501 antibodies in human serum. Results 68 ABP501 clinical samples were measured with the LISA TRACKER Duo Adalimumab assay. LISA TRACKER has been validated as suitable for quantification of ABP501 in human serum samples. Accuracy of the LISA-TRACKER was measured using 3 human serum matrices spiked with known levels of biosimilar, 3 levels spanning the dynamic range. Percentages of recovery were ranged from 90 to 120% for biosimilar batch1, and between 93 and 105% for biosimilar batch2. The acceptance criteria (CV 50% for specificity. Specificity was tested with Biosimilar spiked samples, Biosimilar with Humira® spiked samples, and clinical samples from patients treated with adalimumab biosimilar. All of these samples were spiked with polyclonal antibodies directed against Humira®. Specificity inhibition and specificity detection steps were also part of the validation parameters. Reagents made with ABP501 gave similar results than reagents made with Humira® meeting acceptance criteria. Conclusions LISA-TRACKER ADA and anti-ADA assays are reliable for the monitoring of patients treated with ABP501.

Abstract We have determined the lipid, protein and miRNA composition of skeletal muscle (SkM)-released extracellular vesicles (ELVs) from Ob/ob (OB) vs wild-type (WT) mice. The results showed that atrophic insulin-resistant OB-SkM released less ELVs than WT-SkM, highlighted by a RAB35 decrease and an increase in intramuscular cholesterol content. Proteomic analyses of OB-ELVs revealed a group of 37 proteins functionally connected, involved in lipid oxidation and with catalytic activities. OB-ELVs had modified contents for phosphatidylcholine (PC 34-4, PC 40-3 and PC 34-0), sphingomyelin (Sm d18:1/18:1) and ceramides (Cer d18:1/18:0) and were enriched in cholesterol, likely to alleviated intracellular accumulation. Surprisingly many ELV miRNAs had a nuclear addressing sequence, and targeted genes encoding proteins with nuclear activities. Interestingly, SkM-ELV miRNA did not target mitochondria. The most significant function targeted by the 7 miRNAs altered in OB-ELVs was lipid metabolism. In agreement, OB-ELVs induced lipid storage in recipient adipocytes and increased lipid up-take and fatty acid oxidation in recipient muscle cells. In addition, OB-ELVs altered insulin-sensitivity and induced atrophy in muscle cells, reproducing the phenotype of the releasing OB muscles. These data suggest for the first time, a cross-talk between muscle cells and adipocytes, through the SkM-ELV route, in favor of adipose tissue expansion.

Summary: Background: Severe COVID-19 is associated with a high circulating level of calprotectin, the S100A8/S100A9 alarmin heterodimer. Baseline calprotectin amount measured in peripheral blood at diagnosis correlates with disease severity. The optimal use of this biomarker along COVID-19 course remains to be delineated. Methods: We focused on patients with a WHO-defined moderate COVID-19 requiring hospitalization in a medical ward. We collected plasma and serum from three independent cohorts (N = 626 patients) and measured calprotectin amount at admission. We performed longitudinal measures of calprotectin in 457 of these patients (1461 samples) and used a joint latent class mixture model in which classes were defined by age, body mass index and comorbidities to identify calprotectin trajectories predicting the risk of transfer into an intensive care unit or death. Findings: After adjustment for age, sex, body mass index and comorbidities, the predictive value of baseline calprotectin in patients with moderate COVID19 could be refined by serial monitoring of the biomarker. We discriminated three calprotectin trajectories associated with low, moderate, and high risk of poor outcome, and we designed an algorithm available as online software (https://calpla.gustaveroussy.fr:8443/) to monitor the probability of a poor outcome in individual patients with moderate COVID-19. Interpretation: These results emphasize the clinical interest of serial monitoring of calprotectin amount in the peripheral blood to anticipate the risk of poor outcomes in patients with moderate COVID-19 hospitalized in a standard care unit. Funding: The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

We have determined whether orange juice-derived nanovesicles (ONVs) could be used for the treatment of obesity-associated intestinal complications. ONVs were characterized by lipidomic, metabolomic, electron microscopy. In vitro, intestinal barriers (IBs = Caco-2+HT-29-MTX) were treated with ONVs and co-cultured with adipocytes to monitor IB fat release. In vivo, obesity was induced with a high-fat, high-sucrose diet (HFHSD mice) for 12 weeks. Then, half of HFHSD mice were gavaged with ONVs. One-month ONV treatment did not modify HFHSD-induced insulin resistance but reversed diet-induced gut modifications. In the jejunum, ONVs increased villi size, reduced triglyceride content, and modulated mRNA levels of genes involved in immune response (tumor necrosis factor [TNF]-α and interleukin [IL]-1β), barrier permeability (CLDN1, OCLN, ZO1), fat absorption, and chylomicron release. ONVs targeted microsomal triglyceride transfer protein (MTP) and angiopoietin-like protein-4 (ANGPTL4), two therapeutic targets to reduce plasma lipids and inflammation in gastrointestinal diseases. Interestingly, ONV treatment did not aggravate liver steatosis, as MTP mRNA was increased in the liver. Therefore, ONVs protected both intestine and the liver from fat overload associated with the HFHSD. As ONVs concentrated amino acids and bioactive lipids versus orange juice, which are deficient in obese patients, the use of ONVs as a dietary supplement could bring physiological relevant compounds in the jejunum to accelerate the restoration of intestinal functions during weight loss in obese patients.

Antibody-dependent enhancement (ADE) can increase the rates and severity of infection with various viruses, including coronaviruses, such as MERS. Some in vitro studies on COVID-19 have suggested that prior immunization enhances SARS-CoV-2 infection, but preclinical and clinical studies have demonstrated the contrary. We studied a cohort of COVID-19 patients and a cohort of vaccinated individuals with a heterologous (Moderna/Pfizer) or homologous (Pfizer/Pfizer) vaccination scheme. The dependence on IgG or IgA of ADE of infection was evaluated on the serum samples from these subjects (twenty-six vaccinated individuals and twenty-one PCR-positive SARS-CoV-2-infected patients) using an in vitro model with CD16- or CD89-expressing cells and the Delta (B.1.617.2 lineage) and Omicron (B.1.1.529 lineage) variants of SARS-CoV-2. Sera from COVID-19 patients did not show ADE of infection with any of the tested viral variants. Some serum samples from vaccinated individuals displayed a mild IgA-ADE effect with Omicron after the second dose of the vaccine, but this effect was abolished after the completion of the full vaccination scheme. In this study, FcγRIIIa- and FcαRI-dependent ADE of SARS-CoV-2 infection after prior immunization, which might increase the risk of severe disease in a second natural infection, was not observed.

(1) Background: The link between periodontal disease and rheumatoid arthritis (RA) is now widely reported. Several studies suggest the role of Porphyromonas gingivalis (P. gingivalis) in the pathophysiology of RA and some observations highlight the improvement of the disease activity induced by therapies against P. gingivalis. We have very little data on the prevalence of P. gingivalis carriage in patients with juvenile idiopathic arthritis (JIA) and its possible involvement in the pathophysiology of inflammatory joint diseases in children. (2) Methods: The specific IgG responses against P. gingivalis and Prevotella intermedia (P. intermedia) were determined in a cohort of 101 patients with JIA and 19 patients with other autoimmune diseases (inflammatory bowel disease and type 1 diabetes). (3) Results: Specific anti-P. gingivalis and anti-P. intermedia IgG titers were higher in JIA group than in control groups. These differences were mainly observed in the oligoarthritis group. The same pattern was observed in enthesitis-related arthritis (ERA). (4) Conclusions: Children with oligoarticular and ERA subsets had higher IgG titers to P. gingivalis and P. intermedia. These results suggest involvement of an oral dysbiosis in the occurrence of JIA in these subgroups.

Adipose tissue function in the regulation of lipemia is highly dependent on intestinal absorption of nutrients. Therefore the aim of the present study was the development and validation of an in vitro multiculture model allowing to measure intestinal absorption using adipocytes as lipid sensors. We previously described (1) novel methods to study oleic acid induction of adipogenesis and lipogenesis and (2) a functional reconstituted intestinal barrier using human cell lines Caco-2/HT29-MTX (9:1). In the present study we develop a co-culture model with either adipocytes or hepatocytes as sensors for intestinal lipid absorption. This model was validated using oleic acid (OA) pre-absorbed onto the intestinal barrier. Optimized experimental conditions were obtained with partially differentiated 3T3L1-MBX adipocytes sensing up to 5 μM OA in solution or 40 μM OA pre-absorbed by Caco2/HT29-MTX intestinal barriers. Metabolism including glycemia and insulinemia greatly influenced the ability to TG accumulation in adipocytes. By comparison AML12 hepatocytes found less sensitive to OA (up to 1 μM). The present study demonstrates a much better functionality for fatty acid uptake and release in Caco2/HT29-MTX versus Caco-2 intestinal barriers. Taken together these results open new opportunities to study in vitro lipid transfer between intestinal barriers and either adipocytes or hepatocytes. Abbreviations: BSA: Bovine serum albumin; CIDEs: Cell Death Inducing DFFA Like Effectors; DMEM, Dulbecco’s Modified Eagle’s Medium; FABPs: Fatty Acid Binding Proteins; FAT/CD36: Fatty acid translocase; FCS: Fetal calf serum; GLP2: Glucagon-like peptide-2; NAFLD: Nonalcoholic fatty liver disease; OA: oleic acid; PBS: Phosphate buffer saline; PPARs: Peroxisome-Proliferator Activated Receptors; RTCA: realtime cell analysis; TG: triglyceride

In 2018, 3.8 million premature deaths were attributed to exposure to biomass burning nanoparticles from wood combustion. The objective of this study was to investigate and compare the toxic effect of wood-combustion-related biomass burning nanoparticles from three different combustion stages (i.e., flaming, smoldering, and pyrolysis) on alveolar lung cells, by studying cell proliferation, and structural and behavioral parameters. A549 lung epithelial cells were treated with 31, 62, 125, 250, and 500 µg/mL of water-soluble particulate pollutants from wood burning, and measured by means of real-time cell analysis, cell imaging, and phase imaging microscopy. At low concentrations (31 and 62 µg/mL), all three types of wood burning samples exhibited no toxicity. At 125 µg/mL, they caused decreased cell proliferation compared to the control. Exposure to higher concentrations (250 and 500 µg/mL) killed the cells. Cell physical parameters (area, optical volume, eccentricity, perimeter, and optical thickness) and behavioral parameters (migration, motility, and motility speed) did not change in response to exposure to wood burning materials up to a concentration of 125 µg/mL. Exposure to higher concentrations (250 and 500 µg/mL) changed cell perimeter, optical thickness for smoldering and flaming particles, and led to decreased migration, motility, and motility speed of cells. In conclusion, all three of the combustion water-soluble organic pollutants were identified as equally toxic by real-time cell analysis (RTCA) results. The parameters describing cell structure suggest that pyrolysis particles were slightly less toxic than others.

The measuring of nanoparticle toxicity faces an important limitation since it is based on metrics exposure, the concentration at which cells are exposed instead the true concentration inside the cells. In vitro studies of nanomaterials would benefit from the direct measuring of the true intracellular dose of nanoparticles. The objective of the present study was to state whether the intracellular detection of nanodiamonds is possible by measuring the refractive index. Based on optical diffraction tomography of treated live cells, the results show that unlabeled nanoparticles can be detected and localized inside cells. The results were confirmed by fluorescence measurements. Optical diffraction tomography paves the way to measuring the true intracellular concentrations and the localization of nanoparticles which will improve the dose-response paradigm of pharmacology and toxicology in the field of nanomaterials.

In 2018, seven million people died prematurely due to exposure to pollution. Polycyclic aromatic hydrocarbons (PAHs) are a significant source of secondary organic aerosol (SOA) in urban areas. We investigated the toxic effects of by-products of naphthalene SOA on lung cells. These by-products were 1,4-naphthoquinone (1,4-NQ), 2-hydroxy-1,4-naphthoquinone (2-OH-NQ), phthalic acid (PA) and phthaldialdehyde (OPA). Two different assessment methodologies were used to monitor the toxic effects: real-time cell analysis (RTCA) and the Holomonitor, a quantitative phase contrast microscope. The chemicals were tested in concentrations of 12.5 to 100 µM for 1,4-NQ and 1 to 10 mM for 2-OH-NQ, PA and OPA. We found that 1,4-NQ is toxic to cells from 25 to 100 µM (EC50: 38.7 µM ± 5.2); 2-OH-NQ is toxic from 1 to 10mM (EC50: 5.3 mM ± 0.6); PA is toxic from 5 to 10 mM (EC50: 5.2 mM ± 0.3) and OPA is toxic from 2.5 to 10 mM (EC50: 4.2 mM ± 0.5). Only 1,4-NQ and OPA affected cell parameters (migration, motility, motility speed and optical volume). Furthermore, 1,4-NQ is the most toxic by-product of naphthalene, with an EC50 value that was one hundred times higher than those of the other compounds. RTCA and Holomonitor analysis showed a complementarity when studying the toxicity induced by chemicals.

In “The Ends of an Idiom: ‘Sexual Difference’ in Translation,” Anne Emmanuelle Berger reflects on the different uses and meanings of the notion of “sexual difference” in the Francophone and Anglophone worlds. “Sexual difference” was elevated to the status of a quasi-concept by psychoanalysis and became subsequently one of the key terms in thinking about sex and gender. Looking at the pluralization of its uses and the inflection of its meaning in North-American gender and queer theory, the author stresses its “becoming-queer” and ponders over the contemporary queering of feminist thought. Through her examination of the unending differentiations of the meaning of “sexual difference”, she seeks to bring out the conceptual heterogeneity – in her eyes as irreducible as it is productive – of the theoretical field of gender studies. This is also a way of interrogating the modes of constitution of the Franco-Anglo-American theoretical axis, which has dominated feminist thought in the 20th century.