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Monitoring of anti-drug antibodies in patients on ustekinumab is not routinely recommended in patients with inflammatory bowel disease (IBD) due to low rates of immunogenicity. Aim of study: The purpose of this study was to investigate the relationship between anti-drug antibodies detected by a drug-tolerant assay and loss of response (LOR) to therapy in a cohort of patients with IBD being treated with ustekinumab. Patients and Methods: This retrospective study consecutively enrolled all adult patients with moderate to severe active IBD who had at least 2 years of follow-up after ustekinumab was initiated. LOR was defined as CDAI > 220 or HBI > 4 for Crohn’s disease (CD) and partial Mayo subscore > 3 for ulcerative colitis (UC) and with a modification in disease management. Results: Ninety patients were included (78 CD and 12 UC; mean age 37 years). Median levels of anti-ustekinumab antibodies (ATU) were significantly higher in patients with LOR compared to those with ongoing clinical response (15.2 µg/mL-eq CI (7.9–21.5) and 4.7 µg/mL-eq CI (2.1–10.5), respectively; p = 0.04). The area under the ROC curve (AUROC) for ATU in predicting LOR was 0.76. The optimal cut-off point for identifying patients with LOR was 9.5 µg/mL-eq with a sensitivity of 80% and specificity of 85%. Uni- and multivariate analyses showed that serum ATU ≥ 9.5 µg/mL-eq (hazard ratio (HR) 2.54, 95%CI (1.80–5.93)), p = 0.022, prior vedolizumab (HR 2.78, 95%CI (1.09–3.34), p = 0.019) and prior azathioprine (HR 0.54, 95%CI (0.20–0.76), p = 0.014) exposures were the only factors independently associated with LOR to UST. Conclusion: In our real-life cohort, ATU was identified as an independent predictor of LOR to ustekinumab in patients with IBD.

The human leukocyte antigen B27 (HLA-B27), associated with chronic inflammatory diseases is thus widely performed for diagnostic purposes. Genotyping by molecular biology is the current gold standard for HLA-B27 typing, but cheaper and faster flow cytometry methods have been developed.In this report, we compare analytical performances of three CE-IVD flow cytometry kits: IOTest™ and DuraClone B27™ from Beckman Coulter and BD HLA-B27™ from BD Biosciences on a Navios™ cytometer as compared to molecular biology.Routine analyses could conclude for HLA-B27 in197 patients (23.2%) and was not conclusive for 66 patients (7%, 8%). The experience revealed the needs to complete IOTest™ with a lineage marker (like CD3-APC) and a standardization procedure in detection of fluorescence. Comparative analysis considering 23/44 non-conclusive samples as negative, pointed out a 100% sensitivity and specificity of IOTest™ in determining genetically proved HLA-B27. Specificity of the BD HLA-B27The DuraClone B27™ specificity was poor using the manufacturer cutoff but raised to 100% with a 8.0 cutoff instead of 15.The three flow cytometry kits displayed satisfying performances but need adjustments. The DuraClone B27™ kit seems to be the best while the IOTest™ kit is not conclusive in 8% of cases. In most cases the use of molecular biology can be spared, but genotyping remains essential for patients whose HLA-B27 status cannot be determined with confidence by flow cytometry.

(1) Background: The link between periodontal disease and rheumatoid arthritis (RA) is now widely reported. Several studies suggest the role of

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.

Glutathione is the most abundant thiol in animal cells. Reduced glutathione (GSH) is a major intracellular antioxidant neutralizing free radicals and detoxifying electrophiles. It plays important roles in many cellular processes, including cell differentiation, proliferation, and apoptosis. In the present study we demonstrate that extracellular concentration of reduced glutathione markedly increases cell volume within few hours, in a dose-response manner. Pre-incubation of cells with BSO, the inhibitor of γ-glutamylcysteine synthetase, responsible for the first step in intracellular glutathione synthesis did not change the effect of reduced glutathione on cell volume suggesting a mechanism limited to the interaction of extracellular reduced glutathione on cell membrane. Similarly, inhibition of γ-glutamylcyclotransferase involved in intracellular glutamate production had no effect on the action of reduced glutathione. Oxidized glutathione exerted no effect on cell volume. Results show that reduced GSH decreases cell adhesion resulting in an increased cell volume. Since many cell types are able to export GSH, the present results suggest that this could be a fundamental self-regulation of cell volume, giving the cells a self-control on their adhesion proteins.

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.

Adipose tissue hypertrophy during obesity plays pleiotropic effects on health. Adipose tissue expandability depends on adipocyte size and number. In mature adipocytes, lipid accumulation as triglycerides into droplets is imbalanced by lipid uptake and lipolysis. In previous studies, we showed that adipogenesis induced by oleic acid is signed by size increase and reduction of FAT/CD36 (SR-B2) activity. The present study aims to decipher the mechanisms involved in fat mass regulation by fatty acid/FAT-CD36 signalling. Human adipose stem cells, 3T3-L1, and its 3T3-MBX subclone cell lines were used in 2D cell cultures or co-cultures to monitor in real-time experiments proliferation, differentiation, lipolysis, and/or lipid uptake and activation of FAT/CD36 signalling pathways regulated by oleic acid, during adipogenesis and/or regulation of adipocyte size. Both FABP4 uptake and its induction by fatty acid-mediated FAT/CD36-PPARG gene transcription induce accumulation of intracellular FABP4, which in turn reduces FAT/CD36, and consequently exerts a negative feedback loop on FAT/CD36 signalling in both adipocytes and their progenitors. Both adipocyte size and recruitment of new adipocytes are under the control of FABP4 stores. This study suggests that FABP4 controls fat mass homeostasis.

J’ai participe a l’automne dernier a une journee d’etudes organisee par une jeune equipe de l’ENS de Lyon qui portait sur le statut du corps dans les « Gender Studies » (ce sont les organisateurs qui avaient choisi la formule anglaise plutot que sa « traduction » francaise). M’ayant invitee en ma qualite de specialiste de ce champ, elles et ils m’ont pose diverses questions sur le sujet. Mais leur toute premiere question a porte sur la « diversite » – c’etait leur mot, sans doute un euphemism...

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.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.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.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.The study received support (research grants) from ThermoFisher immunodiagnostics (France) and Gustave Roussy Foundation.

Anti-drug antibodies develop mostly during the induction therapy with anti-tumour necrosis factor (TNF) drugs and can be revealed by means of a drug-tolerant assay.To investigate whether the early detection of anti-drug antibodies during the induction therapy was predictive of treatment discontinuation.In a prospective study, consecutive patients with inflammatory bowel disease (IBD), who should start an anti-TNF, were enrolled and followed regularly during 24 months or less in case of non- or loss of response (LOR) or adverse events requiring treatment discontinuation. Anti-TNF levels and anti-drug antibodies were measured at week 2 for adalimumab (ADA) and weeks 2 and 6 for infliximab (IFX) using a drug-tolerant assay.One hundred and eight patients were enrolled (54 under ADA). At week 2, antibodies to ADA and to IFX were detected in 76% and 67% of patients. Time to treatment discontinuation was significantly shorter (P 0.001) in patients with antibodies to ADA ≥2.0 µg/mL-eq (6.0 vs 24 months, HR = 18.51, 95% CI [4.35-78.71]) or with antibodies to IFX ≥4.0 µg/mL-eq (5.5 vs24 months, HR = 13.89, 95% CI [4.08-47.31]) at week 2 compared to patients without positive antibodies. Antibodies to ADA and to IFX were predictive of treatment failure within 24 months with a sensitivity of 79% and 62%, and specificities and positive predictive values of 100%. In multivariate analysis, antibodies to ADA or to IFX at week 2 were the only factors associated with treatment discontinuation.The prevalence of antibodies to anti-TNF is high when detected early using a drug-tolerant assay, and their appearance predicts further treatment discontinuation.

From its founding, women’s and gender studies developed along a transatlantic epistemological and geopolitical axis. An interdisciplinary field, it fostered difficult conversations among disciplines that had each developed its own conceptual language. Women’s and gender studies is thus centrally concerned with crossing(s), whether crossing(s) functions as a political goal, a meta-metaphor for the field’s variegated theoretical endeavor, or as the name of a multifaceted epistemological problem. This essay focuses on the problem of translation as a form and act of crossing in the geopolitical context of globalization. It asks whether translation, a neohumanist practice of transnational exchange premised on the irreducibility of idioms and the hospitality to differences, can withstand the homogenizing pull of globalization. And it asks what the collapse of differences might do to an intellectual, political, and social field whose very raison d’être has been and continues to be the excavation of unrecognized or unwanted differences and the promotion of plurality.

The objective of the study was to evaluate whether Point-of-Care (POC) assays are equivalent to ELISAs for measuring residual trough levels of adalimumab (ADA) in a cohort of Inflammatory Bowel Disease (IBD) patients. ADA trough levels obtained by POC assays were used to optimize patients in daily clinical practice. Different assays (three ELISAs (Enzyme-Linked ImmunoSorbent Assay) from two different suppliers and two POC assays) were compared to measure ADA trough levels in a first cohort of 31 IBD patients. All assays revealed a high correlation within the assays, ranging from 0.86 to 0.99. Cut-off values were always higher with ELISAs than with POC assays. Then, a small prospective clinical study with a second cohort of 37 IBD patients was performed to compare POC assays and ELISAs for their ability to optimize patients on the basis of the measured ADA trough levels. The use of a POC assay to monitor ADA trough levels did not improve the follow-up of patients with loss of response, as they were always optimized whatever their ADA residual rate. For patients in clinical remission, a POC assay can be useful in some clinical situations to maintain or de-escalate ADA doses according to the measured trough levels. In conclusion, different assays for ADA monitoring are quite equivalent. A POC assay could be only useful for a proactive strategy for asymptomatic patients with a sub-therapeutic dose of ADA, but new therapeutic thresholds need to be identified.

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, Graphical Abstract

Background Two scores have shown their interest in evaluating the luminal activity of CD in magnetic resonance (MR), the MARIA and Clermont scores, but their calculation can be complicated in clinical practice. Recently, the Spanish authors validated a new simplified MARIA score (MARIAs) and retrospectively showed a very good correlation with the initial MARIA score (1). The purpose of this study was to validate this correlation in an independent cohort and to compare its feasibility with other scores. Methods This was a retrospective analysis of all MR performed in our department to evaluate the luminal activity of CD. Two independent radiologists each calculated the MARIA, Clermont and MARIAs scores (wall thickness > 3mm, oedema, comb sign, ulcerations), each blind to the other and blind to clinical activity. In case of disagreement between the two radiologists, a third reader analyzed MR activity. Correlations were calculated between these three tests for the entire cohort. In addition, concordance tests were performed for predetermined thresholds of 7 to 11 for the MARIA score, 8.5 to 12.5 for the Clermont score and 1 to 2 for the MARIAs score. The duration of the analysis was reported for each reading. Isolated colonic CD were excluded from the study. Results One hundred and twenty-one CD were included (65 in clinical and biological activity, 33% ileal phenotype, mean age: 38.5 years, sex ratio M/F = 54.5%). The agreement between the MARIA score between 7 and 11, the Clermont score between 8.5 and 12.5 and the MARIAs score between 1 and 2 was 0.92 (Fleiss kappa test). The correlation between the MARIA score and its simplified score was very strong (r = 0.93; 95% CI: 0.9–0.95) as well as between the Clermont score and the MARIAs score (r = 0.92 95% CI: 0.89–0.95). Inter-observer agreement was significantly higher in the calculation of the MARIAs score (96%) compared with 80% for the MARIA and Clermont scores (p = 0.04). Reading time was significantly faster for the MARIAs score (3.45 ± 0.4 min) compared with the MARIA (10.05 ± 1.2 min) and Clermont (13.5 ± −3.5 min) scores (p < 0.01). Positive predictive values for deep remission (clinical remission and calprotectin < 250 µg/g stool) were 95% at thresholds Conclusion In this independent cohort, we confirm the very strong correlation between the simplified MARIA score and the two reference scores (MARIA and Clermont) in the analysis of MC luminal activity as well as its rapid calculation and strong inter-observer agreement.

International audience; Background: Vedolizumab (VDZ), a humanized monoclonal antibody targeting α4β7 integrin, is effective in induction and maintenance therapy in patients with inflammatory bowel disease (IBD) who have not adequately responded to standard therapies, and high levels of vedolizumab trough levels (VTL) have been associated with clinical remission. The α4β7 integrin binds to endothelial MAdCAM-1 and is up-regulated by retinoic acid (RA). Aim: To determine the relations between soluble MAdCAM-1 (sMAdCAM-1) and RA concentrations with clinical remission during VDZ maintenance therapy. Methods: In a retrospective study performed in IBD patients treated with VDZ we measured VTL, sMAdCAM-1 and RA concentrations. Results: Among the 62 included patients (38 Crohn's disease) 24 relapsed and 38 stayed in remission between weeks 10 to 30 after VDZ initiation. During this maintenance therapy, median values of VTL and RA were 15.4 \textmug/mL and 0.97 ng/mL, whereas sMAdCAM-1 was undetectable (\textless 0.41 ng/mL) in 67.3% of samples. The positive predictive value (PPV) of undetectable sMAdCAM-1 for clinical remission was 80.0% with a corresponding sensitivity of 74.6%. On multivariate analysis undetectable sMAdCAM-1 and high VTL (\textgreater 19 \textmug/mL) were independently associated with clinical remission (OR=7.5, p=0.006 and OR=2.2, p=0.045, respectively). The combination of sMAdCAM-1 \textless 0.41 ng/mL and VTL \textgreater 19 \textmug/mL was the best pharmacokinetic profile with a PPV of 95.2%. Median values of sMAdCAM-1 and RA were significantly higher (p=0.0001) before VDZ therapy than during the follow-up (sMAdCAM-1: 40.5 vs \textless 0.41 ng/mL; RA: 1.7 vs 0.97 ng/mL). Only RA \textgreater 1.86 ng/mL before VDZ therapy was predictive of clinical remission during the follow-up (AUROC=80.7%). Conclusions: Undetectable sMAdCAM-1 appears strongly associated with clinical remission during VDZ maintenance therapy. Combination of undetectable sMAdCAM-1 with high VTL is also potentially interesting for therapeutic drug monitoring. Baseline RA concentrations are predictive of clinical remission. These findings need to be confirmed in further prospective studies.

International audience; Gastrointestinal epithelium is the unique route for nutrients and for many pharmaceuticals to enter the body. The present study aimed to analyze precisely whether co-culture of two colon cancer cell lines, mucus-producing cells HT29-MTX and enterocyte-like Caco-2 cells, ameliorate differentiation into an in vitro intestinal barrier model and the signaling pathways involved. Differentiated Caco-2 cells gene datasets were compared first to intestinal or cancer phenotypes and second to signaling pathway gene datasets. Experimental validations were performed in real-time experiments, immunochemistry, and gene expression analyses on Caco-2 versus co-cultures of Caco-2 and HT29-MTX (10%) cells. Partial maintenance of cancer-cell phenotype in differentiated Caco-2 cells was confirmed and fatty acids merged as potential regulators of cancer signaling pathways. HT29-MTX cells induced morphological changes in Caco-2 cells, slightly increased their proliferation rate and profoundly modified gene transcription of phenotype markers, fatty acid receptors, intracellular transporters, and lipid droplet components as well as functional responses to oleic acid. In vitro, enterocyte phenotype was rescued partially by co-culture of cancer cells with goblet cells and completed through oleic acid interaction with signaling pathways dysregulated in cancer cells.

Background Nearly 40% of patients who failed on ADA have therapeutic residual trough levels (pharmacodynamic failure). It is then recommended to change the therapeutic class to a non-anti-TNF biological. In this situation, however, a quarter of patients respond to an ADA dose optimisation. The purpose of this work was to compare these two strategies and identify predictors of response for each strategy. Methods This is a bi-centre, retrospective study based on a clinical and biological database that included patients followed from 2015 to 2016 with an IBD in maintenance treatment with ADA as monotherapy (40 mg/sc/14 days) and in loss of response (CDAI > 220 with faecal calprotectin > 250 µg/g stool for CD and Mayo score > 6 with endoscopic sub-score > 1 for UC). All patients included had residual trough ADA levels > 5 µg/ml. Relapsed patients were treated either with dose optimisation (ADA: 40 mg/sc/7 days) or other biotherapy (ustekinumab or vedolizumab, according to classic regimens and dosages). The primary endpoint was the relapse-free survival rate (defined as clinical and biological or endoscopic activity requiring therapeutic change). The survival curve analysis was performed using the Kaplan–Meier method and the log-rank test. Results In total, 125 patients (mean age: 38 years, 75% CD, sex ratio M/F = 57%, previous infliximab = 42%) were included and 50 were treated with another biological (Vedolizumab: 30 patients, Ustekinumab: 20 patients). The characteristics of the two groups were comparable except for significantly higher levels of CRP and faecal calprotectin at inclusion in the biological change arm. The duration without relapse was significantly longer after the class change (p = 0.0005; HR = 0.39[0.23–0.68]). The relapse-free survival rates at 54 weeks were 35% for the optimised arm and 66% for a class change (p = 0.04). In multivariate analysis, the only long-term response predictor was biotherapy class change vs. ADA dose optimisation (p = 0.042; HR = 0.3[0.02–0.95]). Previous use of IFX or type of IBD were not associated with relapse. For the dose optimisation strategy, in multivariate analysis, only age < 40 years was associated with a progression without relapse (p = 0.007; HR = 0.09[0.01–0.52]). Residual ADA levels were not predictive of response to optimisation (p = 0.73) and no ADA quartile was significantly associated with a more favourable clinical response. For the class change strategy, no relapse predictors were isolated in univariate analysis. Conclusion In patients on ADA who are losing response despite therapeutic rates, the change of class to non-anti-TNF biotherapy allows a significantly greater lasting response than a dose optimisation strategy for ADA.

Background Several studies have shown a link between circulating anti-TNF levels during induction and clinical progression during IBD. There are less data on the detection of anti-drug antibodies at induction because it requires the use of so-called ‘drug-tolerant’ techniques. The purpose of our study was to investigate in two distinct cohorts (IBD patients on IFX or ADA) whether the early appearance of anti-drug antibodies measured using a drug-tolerant test was associated with clinical progression. Methods In a retrospective study, we included all patients followed in our department for at least 3 months and having had an anti-TNF dosage from W2 of induction under ADA or IFX. At follow-up, therapeutic failure was defined by the obligation to optimise, a change in biotherapy, the use of corticosteroids or surgery. IFX and ADA levels were measured routinely (Lisa-Tracker; Theradiag) and anti-medicine antibodies were detected by ‘drug-sensitive’ ELISA technique (Lisa-Tracker; Theradiag) or ‘drug-tolerant’ as previously described [1]. Patients with a positive rheumatoid factor were excluded. Results 54 patients were included in ADA (mean age: 36 years, sex ratio M/F = 54%, CD: 65%, combotherapy: 10%) and 54 treated with IFX (mean age: 35 years, sex ratio M/F: 46%, CD: 62%, combotherapy: 65%). The search for anti-drug antibodies to W2 using a ‘drug-sensitive’ technique did not reveal any antibodies, whereas 74% of patients on ADA (AAA > or = 2.5 µg/ml eq) and 55% of patients on IFX (ATI > or = 4µg/ml eq). Under ADA, the duration of treatment until loss of response was significantly shorter in the presence of AAA at W2: 6 months vs. 24 months (HR = 18.51; 95CI = 4.35–78.11; p < 0.001). In multivariate analysis, the only independent factor associated with failure was the rate of ADA antibodies to W2 (HR = 18.17; 95CI: 4.27–77.45; p < 0.001). Under IFX, the treatment time to loss of response was significantly shorter in the presence of ATI to W2 (ATI > or = 4µg/ml eq): 5.5 months vs. >24 months (HR = 13.89; 95CI = 4.08–47.31; p < 0.001). In multivariate analysis, the only independent factor associated with failure was the level of anti-IFX antibodies to W2 (> or = 4 µg/ml eq) (HR = 8.07; 95CI: 3.13–20.82; p < 0.001). The higher quartiles of ATI and AAA were significantly associated with poor outcomes (p < 0.001 for each anti-TNF). Conclusion The early onset of anti-drug antibodies in the induction phase as early as W2 is the only isolated independent factor associated with loss of response. Anti-drug antibody frequencies are very high when measured with a drug-tolerant test and these Abs may be more neutralising with ADA than with IFX.

Background In a recent prospective, randomised trial, we showed that the rates of clinical failure and occurrence of unfavourable pharmacokinetics using a drug-sensitive assay after a switch of anti-TNF were significantly higher in monotherapy compared with combination therapy (Log-rank test p < 0.0001), whatever the anti-TNF used in IBD patients. We aimed in a post hoc analysis to assess the time-course of antidrug antibodies in the two groups of patients using a drug-tolerant assay previously described (1). Methods After switching of anti-TNF under mono or combotherapy, blood samples were uptake at various time-points (6, 12, 18 and 24 months). Using a drug-tolerant assay, we defined pharmacokinetic failure when antidrug antibodies were detected (> 2.5 μg/ml Eq). Transient anti-drug antibodies were defined as antibodies that appeared during the course of anti-TNF therapy with no clinical worsening, and finally that disappeared after 2 consecutive measurements. Results Ninety patients were analysed. According to a drug-tolerant assay, the occurrence of antibodies against anti-TNF was significantly higher in patients under monotherapy (log-rank test p < 0.0001 (HR = 3.36 [1.94–5.86]) than under combination therapy. The rates of anti-drug antibody were significantly lower under IFX-AZA combotherapy compared with ADA-AZA combotherapy or under monotherapy. Monotherapy ADA (HR = 5.55[2.46–12.34]) or IFX (HR = 6.05[2.67–13.70]) and combination therapy ADA-AZA (HR = 3.34 [1.407.98]) increased significantly the rates of pharmacokinetic failure using a drug-tolerant assay compared with IFX-AZA combotherapy. The time-course without pharmacokinetic failure was significantly different between the 4 groups of patients from 15 months (p = 0.04) but was not significantly different at 6 months (p = 0.12). Overall, 9% of patients developed transient antibodies. Detection of antibodies against anti-TNF at 6 months using a drug-tolerant assay predicted significantly an immunogenic failure assessed previously by a drug-sensitive assay with a sensitivity of 57.1%, a specificity of 92.7%, a positive predictive value of 90.3%. In contrary to the association between clinical failure and immunogenic failure using drug-sensitive assay, the association of detectable drug and positive antibodies using a drug-tolerant assay was not significantly associated with clinical failure (HR = 1.3[0.3–6.8], p = 0.51). Conclusion Pharmacokinetic failure using a drug-tolerant assay was significantly lower under combotherapy, especially for IFX-AZA. Detection of anti-drug antibodies could predict pharmacokinetic failure. Reference

Objective: This study was performed to analyze the modifications within adipose tissue during calorie restriction and more specifically to state whether hysteresis occurs during fat mass reduction. Method: Rats male Wistar increased their body weight by 130 g under control conditions and were then submitted to a calorie restriction (CR) at 30% or 60% of control. Experiment has been stopped when the body weight of the group CR60% returned back to its initial value. Samples of retroperitoneal adipose tissue were collected by biopsies along the study. Adipose cell size was analyzed using multisizer IV (Beckman Coulter) to determine the size distribution curves during natural growth and after calorie restriction. Results: After CR60%, body weights and adipose tissue masses were similar to the ones at the beginning of the experiment. Adipose cell size distribution curve was shifted to the left compared to the one of initial control. Adipose cell sizes were significantly lower after CR60% than those of control at the beginning of the experiment. Conclusions: These results state for the first time that hysteresis occurs in white adipose tissue after calorie restriction. The composition of adipose tissue after calorie restriction was significantly different than the one of initial control. After significant weight loss, organisms must be considered as different from the initial controls, they are most likely governed by different regulations which will have to be identified.

Obesity is linked to adipose tissue hypertrophy (increased adipocyte cell size) and hyperplasia (increased cell number). Comparative analyses of gene datasets allowed us to identify 1426 genes which may represent common adipose phenotype in humans and mice. Among them we identified several adipocyte-specific genes dysregulated in obese adipose tissue, involved in either fatty acid storage (acyl CoA synthase ACSL1, hormone-sensitive lipase LIPE, aquaporin 7 AQP7, perilipin PLIN) or cell adhesion (fibronectin FN1, collagens COL1A1, COL1A3, metalloprotein MMP9, or both (scavenger receptor FAT/CD36). Using real-time analysis of cell surface occupancy on xCELLigence system we developed a new method to study lipid uptake and differentiation of mouse 3T3L1 fibroblasts and human adipose stem cells. Both processes are regulated by insulin and fatty acids such as oleic acid. We showed that fatty acid addition to culture media increased the differentiation rate and was required for full differentiation into unilocular adipocytes. Significant activation of lipogenesis, i.e. lipid accumulation, by either insulin or oleic acid was monitored in times ranging from 1 to 24 h, depending on differentiation state, whereas significant effects on adipogenesis, i.e., surperimposed lipid accumulation and gene transcriptional regulations were measured after 3 to 4 d. Combination of selected times for analysis of lipid contents, cell counts, size fractionations, and gene transcriptional regulations showed that FAT/CD36 specific inhibitor AP5258 significantly increased cell survival of oleic acid-treated mouse and human adipocytes, and partially restored the transcriptional response to oleic acid in the presence of insulin through JNK pathway. Taken together, these data open new perspectives to study the molecular mechanisms commonly dysregulated in mouse and human obesity at the level of lipogenesis linked to hypertrophy and adipogenesis linked to hyperplasia.

International audience; The delivery of SiC-based nanoparticles (SiC-NPs) into living eukaryotic cells is facilitated in the presence of cell-penetrating peptides, both cationic (SAP) and anionic (SAP-E). The SiC-NP surface functional group modification combined with rational CPP selection introduces an additional mode of delivery control.

Cancer progression may be affected by metabolism. In this study, we aimed to analyze the effect of glucose on the proliferation and/or survival of human hepatocellular carcinoma (HCC) cells. Human gene datasets regulated by glucose were compared to gene datasets either dysregulated in HCC or regulated by other signaling pathways. Significant numbers of common genes suggested putative involvement in transcriptional regulations by glucose. Real-time proliferation assays using high (4.5 g/L)versuslow (1 g/L) glucose on two human HCC cell lines and specific inhibitors of selected pathways were used for experimental validations. High glucose promoted HuH7 cell proliferation but not that of HepG2 cell line. Gene network analyses suggest that gene transcription by glucose could be mediated at 92% through ChREBP in HepG2 cells, compared to 40% in either other human cells or rodent healthy liver, with alteration of LKB1 (serine/threonine kinase 11) and NOX (NADPH oxidases) signaling pathways and loss of transcriptional regulation of PPARGC1A (peroxisome-proliferator activated receptors gamma coactivator 1) target genes by high glucose. Both PPARA and PPARGC1A regulate transcription of genes commonly regulated by glycolysis, by the antidiabetic agent metformin and by NOX, suggesting their major interplay in the control of HCC progression.

Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3 beta, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.

International audience; Introduction: Adipocyte size and body fat distribution are strongly linked to the metabolic complications of obesity. The aim of the present study was to test the plasticity of white adipose tissue in response to insulin deprivation and replacement. We have characterized the changes of adipose cell size repartition and gene expressions in type 1 diabetes Sprague-Dawley rats and type 1 diabetic supplemented with insulin. Methods: Using streptozotocin (STZ)-induced diabetes, we induced rapid changes in rat adipose tissue weights to study the changes in the distribution of adipose cell sizes in retroperitoneal (rWAT), epididymal (eWAT) and subcutaneous adipose tissues (scWAT). Adipose tissue weights of type 1 diabetic rats were then rapidly restored by insulin supplementation. Cell size distributions were analyzed using multisizer IV (Beckman Coulter). Cell size changes were correlated to transcriptional regulation of genes coding for proteins involved in lipid and glucose metabolisms and adipocytokines. Results: The initial body weight of the rats was 46565.2 g. Insulin privation was stopped when rats lost 100 g which induced reductions in fat mass of 68% for rWAT, 42% for eWAT and 59% for scWAT corresponding to decreased mode cell diameters by 31.1%, 20%, 25.3%, respectively. The most affected size distribution by insulin deprivation was observed in rWAT. The bimodal distribution of adipose cell sizes disappeared in response to insulin deprivation in rWAT and scWAT. The most important observation is that cell size distribution returned close to control values in response to insulin treatment. mRNAs coding for adiponectin, leptin and apelin were more stimulated in scWAT compared to other depots in diabetic plus insulin group. Conclusion: Fat depots have specific responses to insulin deprivation and supplementation. The results show that insulin is a major determinant of bimodal cell repartition in adipose tissues.

International audience; Exosomes are nanometer-sized microvesicles formed in multivesicular bodies (MVBs) during endosome maturation. Exosomes are released from cells into the microenvironment following fusion of MVBs with the plasma membrane. During the last decade, skeletal muscle-secreted proteins have been identified with important roles in intercellular communications. To investigate whether muscle-derived exosomes participate in this molecular dialog, we determined and compared the protein contents of the exosome-like vesicles (ELVs) released from C2C12 murine myoblasts during proliferation (ELV-MB), and after differentiation into myotubes (ELV-MT). Using a proteomic approach combined with electron microscopy, western-blot and bioinformatic analyses, we compared the protein repertoires within ELV-MB and ELV-MT. We found that these vesicles displayed the classical properties of exosomes isolated from other cell types containing components of the ESCRT machinery of the MVBs, as well as numerous tetraspanins. Specific muscle proteins were also identified confirming that ELV composition also reflects their muscle origin. Furthermore quantitative analysis revealed stage-preferred expression of 31 and 78 proteins in ELV-MB and ELV-MT respectively. We found that myotube-secreted ELVs, but not ELV-MB, reduced myoblast proliferation and induced differentiation, through, respectively, the down-regulation of Cyclin D1 and the up-regulation of myogenin. We also present evidence that proteins from ELV-MT can be incorporated into myoblasts by using the GFP protein as cargo within ELV-MT. Taken together, our data provide a useful database of proteins from C2C12-released ELVs throughout myogenesis and reveals the importance of exosome-like vesicles in skeletal muscle biology.

International audience; Hepatocellular carcinoma (HCC) represents one of the most frequently diagnosed human cancers; however, there are currently few treatment alternatives to surgical resection. In this study we performed bioinformatic analysis of previously published transcriptomic data in order to characterize liver specific networks, including biological functions, signaling pathways and transcription factors, potentially dysregulated in HCC. By incorporating specific signaling inhibitors into real-time proliferation assays using HepG2 cells, we then validated these in silico results. We found that G protein subunits Gi/G0, protein kinase C, Mek1/2, and Erk1/2 (P42/44), JAK1, PPARA and NFκB p65 subunit were the major signaling molecules required for survival and proliferation of human HCC cell lines. We also found that these pathways regulate the expression of key hepatic transcription factors involved in cell differentiation, such as CEBPA, EGR1, FOXM1 and PPARs. By combining bioinformatic and functional analyses, major signaling pathways related to tumorigenicity in HCC are revealed, thereby elucidating potential targets for drug therapies.

Berger E, Rome S, Vega N, Ciancia C, Vidal H. Transcriptome profiling in response to adiponectin in human cancer-derived cells. Physiol Genomics 42A: 61-70, 2010. First published June 22, 2010; doi:10.1152/physiolgenomics.00013.2010.-The adipocyte-derived hormone adiponectin exerts protective actions in several disorders, including some cancers. However, while growing data suggest that adiponectin could be an effective anticancer agent, its mechanism of action in cancer cells is still poorly known. Here, using microarrays, we identified a set of 1,301 genes commonly modulated in three cancer-derived cell lines in response to short-term stimulation with full-length recombinant human adiponectin. Most of these genes are involved in translation regulation, immune or stress responses, and cell proliferation. Furthermore, among genes linked to disease that were retrieved by functional enrichment tests using text mining based on PubMed analysis, we found that 66% are involved in malignant neoplasms, further supporting the link between adiponectin and cancer mechanisms. Bioinformatic analysis demonstrated the diversity of signaling pathways and transcription factors potentially mediating adiponectin effects on gene expression, illustrating the complexity of adiponectin mechanisms of action in cancer cells.