Gene profiling in white blood cells predicts infliximab responsiveness in rheumatoid arthritis

As indicators of responsiveness to a tumour necrosis factor (TNF)α blocking agent (infliximab) are lacking in rheumatoid arthritis, we have used gene profiling in peripheral blood mononuclear cells to predict a good versus poor response to infliximab. Thirty three patients with very active disease (Disease Activity Score 28 >5.1) that resisted weekly methotrexate therapy were given infliximab at baseline, weeks 2 and 6, and every 8th week thereafter. The patients were categorized as responders if a change of Disease Activity Score 28 = 1.2 was obtained at 3 months. Mononuclear cell RNAs were collected at baseline and at three months from responders and non-responders. The baseline RNAs were hybridised to a microarray of 10,000 non-redundant human cDNAs. In 6 responders and 7 non-responders, 41 mRNAs identified by microarray analysis were expressed as a function of the response to treatment and an unsupervised hierarchical clustering perfectly separated these responders from non-responders. The informativeness of 20 of these 41 transcripts, as measured by qRT-PCR, was re-assessed in 20 other patients. The combined levels of these 20 transcripts properly classified 16 out of 20 patients in a leave-one-out procedure, with a sensitivity of 90% and a specificity of 70%, whereas a set of only 8 transcripts properly classified 18/20 patients. Trends for changes in various transcript levels at three months tightly correlated with treatment responsiveness and a down-regulation of specific transcript levels was observed in non-responders only. Our gene profiling obtained by a non-invasive procedure should now be used to predict the likely responders to an infliximab/methotrexate combination

Early and long-standing rheumatoid arthritis: distinct molecular signatures identified by gene-expression profiling in synovia

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Profils d'expression génique au cours du carcinome hépatocellulaire sur foie cirrhotique chez l'homme

Le carcinome hépatocellulaire (CHC), tumeur primitive du foie, survient dans près de 90% des cas consécutivement à une cirrhose, dont les deux principales étiologies, en France, sont l'infection virale par le virus de l'hépatite C (VHC) et la consommation excessive et chronique d'alcool. La compréhension des mécanismes moléculaires impliqués dans le développement tumoral a largement été favorisée, ces dernières années, par l'émergence des puces à ADN. Cependant, et malgré de nets besoins, aucune puce couvrant le transcriptome hépatique de façon exhaustive n'avait été réalisée. C'est pourquoi nous avons développé une puce à ADN, basée sur la technologie des micro-réseaux d'ADNc, qui permet de couvrir et d'étudier les niveaux d'expression de l'ensemble des transcrits exprimés dans le foie. Près de 12000 ADNc, à expression au moins hépatique, ont été sélectionnés in silico puis amplifiés par PCR avant d'être déposés, de façon ordonnée sur des filtres nylons. Parallèlement à l'établissement d'une telle collection de sondes, baptisée Liverpool, une base de données, LiverTools, dédiée au stockage et à la gestion de l'ensemble des informations requises par les analyses de transcriptomes, a été développée. Ces outils ont permis de confirmer la réminiscence d'un programme d'expression génique fœtal au cours du CHC, qui se caractérise par une grande proportion de gènes conjointement réprimés dans l'hépatocyte fœtal et dans l'hépatocyte cancéreux. Parmi ces gènes, nous avons mis en évidence celui codant la duduline-2. Impliquée dans l'apoptose, cette protéine semblerait être un marqueur pronostic, témoin de l'évolution cirrhotique vers une dédifférenciation tumorale ; son intérêt pronostique est en cours d'évaluation. Par ailleurs, aucune comparaison des transcriptomes du CHC d'origine virale C et d'origine alcoolique n'avait été réalisée à ce jour. Notre étude nous a également permis de mettre en évidence les gènes significativement dérégulés en fonction de l'une ou l'autre étiologie. Alors que la réponse inflammatoire prédomine dans les CHC post-alcoolisme, la réponse à l'interféron est associée aux CHC post-VHC. De plus, nous avons observé que de telles différences étaient liées à l'état tumoral et non à l'état cirrhotique qui l'accompagne.Hepatocellular carcinoma (HCC), the main primitive neoplasia of the liver, mostly occurs in a cirrhotic context. Hepatitis C infection and excessive alcohol consumption are major risk factors for HCC in France. In recent years, the comprehension of the molecular mechanisms involved in HCC development was largely supported by the emergence of DNA chip technologies. However, no chip completely covering the liver transcriptome had been developed. Based on cDNA array technology, we have developed such a chip which covers every transcript expressed in the liver. Approximately 12.000 cDNAs for mRNAs with at least an hepatic expression were selected in silico, then amplified by PCR and spotted in an ordered fashion on nylon filters. While we were establishing such a collection of probes, dubbed Liverpool, we have also developped a database, named LiverTools, that is dedicated to the storage and management of the information and data required for our transriptome analyses. These tools have enabled us to observe that the liver transcriptome in HCC mimics that seen during the embryonic development of the liver. Indeed, we have observed that a large set of transcripts is repressed in both conditions. Among them, the transcript coding for dudulin-2, an apoptosis protein, provides a marker of the evolution from cirrhosis to tumoral dedifferenciation. The interest of dudulin-2 as a prognosis tool is being evaluated. To date, no comparison of the transcriptomes of post-hepatitis C and post-alcoholism HCC had been carried out. We have identified a set of genes that are differently expressed in liver, as a function of either of these etiologies. An inflammatory response largely prevails in post-alcoholism HCC whereas an interferon-related response is associated to the post-hepatitis C HCC. Finally, we have further observed that such etiology-dependent differences are related to the tumoral state and not to cirrhosis.ROUEN-BU Sciences (764512102) / SudocSudocFranceF

Long-range identification of hepatocyte nuclear factor-3 (FoxA) high and low-affinity binding sites with a chimeric nuclease 1 1Edited by M. Yaniv

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Predictors of treatment response in rheumatoid arthritis

International audienceThe expanding array of drugs available for treating rheumatoid arthritis is creating challenges in drug selection for the individual patient. The identification of biomarkers that predict the treatment response prior to drug exposure is therefore a current priority. This new approach, known as theranostics, is a component of personalized medicine, which involves selecting the management strategies that are most effective for a given patient at a given point in time. Antibodies to citrullinated peptides, rheumatoid factor, and the interferon signature are the most robust and best validated biomarkers identified to date. Matrices containing clinical or laboratory parameters of diagnostic or prognostic relevance may help to select the best treatment for the individual patient. Furthermore, the development of large-scale approaches requiring no a priori knowledge, such as functional genomics and metabolomics, hold considerable promise, despite persistent difficulties in replicating findings. The complexity of the treatment response in a given patient and substantial variability across patients suggest that biomarkers may be more helpful in combination than singly. The objectives of this review article are to discuss the approaches used to identify theranostic biomarkers and to present an overview of currently available biomarkers and of their performance in everyday clinical practice. However, the range of biomarkers suitable for use in daily practice remains extremely narrow

Gene expression regulated by abatacept associated with methotrexate and correlation with disease activity in rheumatoid arthritis

International audienceObjectives: Abatacept acts as a competitive inhibitor of the CD28/(CD80/86) costimulation signal required for T cell activation. Mechanisms of action of abatacept have not been fully investigated. The objective of this study was to provide detailed insight into the mode of action of Abatacept based on gene expression data. Methods: In this ancillary study from the APPRAISE trial, we investigated the global molecular effects of Abatacept in whole blood samples collected prospectively in biologic naive rheumatoid arthritis patients (n = 19) at baseline and 6 months after the initiation of Abatacept therapy concomitant with methotrexate. Whole human genome microarrays (4x44K) were performed on both baseline and 6-month samples from responders and non-responders patients categorized according to EULAR criteria. T-test with Benjamini-Hochberg correction was performed to identify significant gene expression changes. Gene Ontology and Single Experiment Analysis tools allowed us to highlight specific biological mechanisms involved in methotrexate/Abatacept. Results: In methotrexate/Abatacept responders, 672 genes were significantly (q<0.05) dysregulated at 6 months compared to baseline. Correlation analysis highlighted 19 genes whose dysregulations were significantly associated with disease activity variation (p<0.05) and whose functions were associated with proliferation, apoptosis of cells and mitochondrial metabolism, suggesting a restoration of oxidative signaling. The other 653 gene expression changes were relative to direct or indirect effects of methotrexate/Abatacept treatment and were significantly (p<0.005) involved in pathways relative to mRNA processing, proteasome, angiogenesis, apoptosis and TCR signaling. This study highlights new mechanisms of action of methotrexate/Abatacept and may provide new therapeutic targets to prevent autoimmunity in rheumatoid arthritis

The inflammation-induced down-regulation of plasma Fetuin-A (α2HS-Glycoprotein) in liver results from the loss of interaction between long C/EBP isoforms at two neighbouring binding sites

Fetuin-A is an hepatic protein whose mRNA transiently falls during the inflammatory acute phase via unknown transcriptional mechanisms. Various FETUA promoter/cat constructs transiently transfected in the Hep3B hepatoma cell line allowed us to identify four NF-1 and C/EBP binding sites (N, C) arranged in a 5′-N2-C2-N1-C1-3′ order and required for basal promoter activity. Mutant constructs demonstrated that C1 and C2 but not N1 nor N2 are required for the cytokine-driven down-regulation of the promoter. A variable spacing between C2 and N1 showed that the alignment of the (C1+N1) and (C2+N2) areas is critical for the promoter activity in quiescent but not cytokine-stimulated cells. Co-transfection of a plasmid only producing either a long or short C/EBPβ isoform prevented FETUA regulation by cytokines. Electromobility shift assays with liver nuclear extracts showed that during the acute phase the complexes formed over N1 and N2 are not modified whereas short C/EBPα and -β isoforms replace the long isoforms bound to C1 and C2 in the quiescent liver. Therefore the basal promoter activity requires an interaction between the long C/EBP isoforms bound to C1 and C2 whereas the inflammation-induced down-regulation results from the loss of interaction between the cytokine-induced, short C/EBP isoforms