Céline Derambure

Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa.

Olfactory ensheathing cells (OEC) have the ability to promote regeneration in the nervous system. Hence, they hold promise for cell therapy. Most of the experimental studies have investigated the role of OECs taken from olfactory bulb (OB). However, for a clinical human application, olfactory mucosa (OM) seems to be the only acceptable source for OECs. Many studies have compared the distinct ability of OECs from OB and OM to improve functional nerve regeneration after lesion of the nervous system. Nevertheless, the two populations of OECs may differ in several points, which might affect all fate after transplantation in vivo. We report here the first study which compares gene expression profiling between these two populations of OECs. It appears that OB‐OECs and OM‐OECs display distinct gene expression pattern, which suggest that they may be implicated in different physiological processes. Notably, OM‐OECs overexpress genes characteristic of wound healing and regulation of extra cellular matrix. In contrast, OB‐OECs gene profile suggests a prominent role in nervous system development. Hence, OB‐OECs and OM‐OECs fundamentally differ in their gene expression pattern, which may represent a crucial point for future clinical application.

Drastic Effect of Germline TP53 Missense Mutations in Li–Fraumeni Patients

In contrast to other tumor suppressor genes, the majority of TP53 alterations are missense mutations. We have previously reported that in the Li–Fraumeni syndrome (LFS), germline TP53 missense mutations are associated with an earlier age of tumor onset. In a larger series, we observed that mean age of tumor onset in patients harboring dominant negative missense mutations and clearly null mutations was 22.6 and 37.5 years, respectively. To assess the impact of heterozygous germline TP53 mutations in the genetic context of the patients, we developed a new functional assay of the p53 pathway on the basis of induction of DNA damage in Epstein–Barr‐virus‐immortalized lymphocytes, followed by comparative gene‐expression profiling. In wild‐type lymphocytes, we identified a core of 173 genes whose expression was induced more than twofold, of which 46 were known p53 target genes. In LFS lymphocytes with canonical missense mutations, the number of induced genes and the level of known p53 target genes induction were strongly reduced as compared with controls and LFS lymphocytes with null mutations. These results show that certain germline missense TP53 mutations, such as those with dominant negative effect, dramatically alter the response to DNA damage. This probably explains why TP53 alterations are predominantly missense mutations.

Gene profiling predicts rheumatoid arthritis responsiveness to IL-1Ra (anakinra)

OBJECTIVES The overall non-response rate to biologics remains 30-40% for patients with RA resistant to MTX. The objective of this study was to predict responsiveness to the anakinra-MTX combination by peripheral blood mononuclear cell gene profiling in order to optimize treatment choice. METHODS Thirty-two patients treated with anakinra (100 mg/day s.c.) and MTX were categorized as responders when their 28-joint DAS (DAS-28) had decreased by ≥1.2 at 3 months. Pre-treatment blood samples had been drawn. RESULTS For seven responders and seven non-responders, 52 microarray-identified mRNAs were expressed as a function of the response to treatment, and unsupervised hierarchical clustering correctly separated responders from non-responders. The levels of seven of these 52 transcripts, as assessed by real-time, quantitative RT-PCR, were able to accurately classify 15 of 18 other patients (8 responders and 10 non-responders), with 87.5% specificity and 77.8% negative-predictive value for responders. Among the 52 genes, 56% were associated with IL-1β. CONCLUSION This predictive gene expression profile was obtained with a non-invasive procedure. After further validation in other cohorts of patients, it could be proposed and used on a large scale to select likely RA responders to combined anakinra-MTX. Trial registration. Clinical Trials; NCT00213538 (http://www.clinicaltrials.gov).

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

IntroductionRheumatoid arthritis (RA) is a heterogeneous disease and its underlying molecular mechanisms are still poorly understood. Because previous microarray studies have only focused on long-standing (LS) RA compared to osteoarthritis, we aimed to compare the molecular profiles of early and LS RA versus control synovia.MethodsSynovial biopsies were obtained by arthroscopy from 15 patients (4 early untreated RA, 4 treated LS RA and 7 controls, who had traumatic or mechanical lesions). Extracted mRNAs were used for large-scale gene-expression profiling. The different gene-expression combinations identified by comparison of profiles of early, LS RA and healthy synovia were linked to the biological processes involved in each situation.ResultsThree combinations of 719, 116 and 52 transcripts discriminated, respectively, early from LS RA, and early or LS RA from healthy synovia. We identified several gene clusters and distinct molecular signatures specifically expressed during early or LS RA, thereby suggesting the involvement of different pathophysiological mechanisms during the course of RA.ConclusionsEarly and LS RA have distinct molecular signatures with different biological processes participating at different times during the course of the disease. These results suggest that better knowledge of the main biological processes involved at a given RA stage might help to choose the most appropriate treatment.

Isolation, characterization, and genetic profiling of subpopulations of olfactory ensheathing cells from the olfactory bulb

Olfactory ensheathing cells (OECs) play a crucial role during neurogenesis of primary olfactory neurons. Transplantation of OECs is considered as a promising new therapy for central nervous system repair. Nevertheless, OECs are constituted of distinct subpopulations and their role during neurogenesis is not clearly understood. In particular, OECs from the olfactory bulb (OB) constitute a heterogeneous, but not yet isolated and characterized, population of cells. In our study, flow cytometry analyses of primary OB cultures, based on cell surface expression of low‐affinity nerve growth factor receptor (p75), reveal the presence of two distinct populations of OECs. Indeed, some of them express a high level of p75 (P75High) and the other a low level of p75 (P75Low). Effects of OB microenvironment were assessed, and we were able to show that fibroblasts mediate the induction of these two populations through the secretion of soluble factors. To characterize P75High and P75Low OECs, cells were sorted based on their differential expression of p75. Microarray analyses revealed that P75High OECs overexpress genes implicated in modulation of extracellular matrix and cell sorting, whereas P75Low OECs overexpress genes involved in regulation of the inflammatory response and axonal guidance. These results permit, for the first time, to isolate the two distinct subpopulations of OECs from OB, and suggest their specific role during neurogenesis.

Major remodeling of brain microvessels during neonatal period in the mouse: A proteomic and transcriptomic study

Preterm infants born before 29 gestation weeks incur major risk of subependymal/intracerebral/intraventricular hemorrhage. In mice, neonate brain endothelial cells are more prone than adult cells to secrete proteases under glutamate challenge, and invalidation of the Serpine 1 gene is accompanied by high brain hemorrhage risk up to five days after birth. We hypothesized that the structural and functional states of microvessels might account for age-dependent vulnerability in mice up to five days after birth and might represent a pertinent paradigm to approach the hemorrhage risk window observed in extreme preterms. Mass spectrometry proteome analyses of forebrain microvessels at days 5, 10 and in adult mice revealed 899 proteins and 36 enriched pathways. Microarray transcriptomic study identified 5873 genes undergoing at least two-fold change between ages and 93 enriched pathways. Both approaches pointed towards extracellular matrix, cell adhesion and junction pathways, indicating delayed microvascular strengthening after P5. Furthermore, glutamate receptors, proteases and their inhibitors exhibited convergent evolutions towards excitatory aminoacid sensitivity and low proteolytic control likely accounting for vascular vulnerability in P5 mice. Thus, age vascular specificities must be considered in future therapeutic interventions in preterms. Data are available on ProteomeXchange (identifier PXD001718) and NCBI Gene-Expression-Omnibus repository (identification GSE67870).

Soluble alpha-enolase activates monocytes by CD14-dependent TLR4 signalling pathway and exhibits a dual function

Rheumatoid arthritis (RA) is the most common form of chronic inflammatory rheumatism. Identifying auto-antigens targeted by RA auto-antibodies is of major interest. Alpha-enolase (ENO1) is considered to be a pivotal auto-antigen in early RA but its pathophysiologic role remains unknown. The main objective of this study was to investigate the in vitro effects of soluble ENO1 on peripheral blood mononuclear cells (PBMC) from healthy donors and RA patients in order to determine the potential pathogenic role of ENO1. ELISA, transcriptomic analysis, experiments of receptor inhibition and flow cytometry analysis were performed to determine the effect, the target cell population and the receptor of ENO1. We showed that ENO1 has the ability to induce early production of pro-inflammatory cytokines and chemokines with delayed production of IL-10 and to activate the innate immune system. We demonstrated that ENO1 binds mainly to monocytes and activates the CD14-dependent TLR4 pathway both in healthy subjects and in RA patients. Our results establish for the first time that ENO1 is able to activate in vitro the CD14-dependent TLR4 pathway on monocytes involving a dual mechanism firstly pro-inflammatory and secondly anti-inflammatory. These results contribute to elucidating the role of this auto-antigen in the pathophysiologic mechanisms of RA.

A2.14 Potential in Vitro Immunomodulatory Effects of the Recombinant Human Alpha-Enolase on Peripheral Blood Mononuclear Cells (PBMCs) from Healthy Donors

Background Identification of autoantibodies associated with rheumatoid arthritis has been of major interest. In this context, we have previously identified for the first time α-enolase (ENO) as a new auto-antigen in early RA. ENO is an evolutionary conserved protein involved both in glycolysis pathway and as a plasminogen receptor which confer it a role in anti-infectious inflammatory response. In vivo, preliminary studies showed that ENO had immunomodulatory effect in the collagen induced arthritis mouse model. [1] To better understand the immunological mechanisms of ENO, the aim of this in vitro study was to determine the effects of ENO on PBMCs from healthy donors. Methods In one hand, PBMCs or different cell types (monocytes, B and T cells, and immature dendritic cells [iDC]) (n = 3) were cultured with ENO (20 µg/mL) or Bovine Serum Albumin (20 µg/mL). TNFα and IL-10 production was measured in the supernatants by ELISA at different times. On the other hand, TNFα and IL-10 production were evaluated in PBMCs, monocytes or B and T cells after LPS stimulation and pre-incubation with ENO for 24 h (n = 3). Cytometric analyses have evaluated the ability of ENO to inhibit the differentiation of monocytes into iDC. Before differentiation into iDC (GM-CSF and IL-4), monocytes (1.106 cells/mL) were incubated with ENO (20 or 50 µg/mL) for 24 h. Results In cultures of PBMCs, monocytes or iDC, ENO induces, dose dependently, an early production of TNFα followed by extended secretion of IL-10. PBMCs or individual cells (monocytes, B and T cells) stimulated by LPS secreted successively TNFα and IL-10, while PBMCs or individual cells, stimulated by LPS but previously incubated with ENO for 24 h did not secrete these cytokines. In contrast to LPS, ENO did not induce differentiation of immature dendritic cells into mature cells. But ENO has not the capacity to inhibit differentiation from monocytes to iDC. Conclusions This study suggests that ENO has no pro-inflammatory effect unlike LPS. Indeed, ENO might have immunomodulatory properties via IL-10 production. Others studies focused on an extended cytokine panel and different signalling pathways are underway to better understand the immunological mechanisms induced by ENO. Reference C Guillou et al, Arthritis and Rheumatism 2011; 63:S815.

The transcriptomic analysis from pbmc is a powerful approach to identify specific signatures able to predict responses for each tnfalpha blocking agent

Background The number of RA biological treatments is increasing but 30 to 40% of patients do not respond to tumour necrosis factor α (TNFα) blocking agents. One way to optimise the drug prescription is to identify predictive markers of drug responsiveness for each biologic agent. The authors already identified a gene combination able to predict infliximab responsiveness by transcriptomic analyses. The question is to know if this approach is enough sensitive to identify specific gene expression profile for the different TNFα blocking agents. Objectives To identify specific gene expression profiles able to predict the response of RA patients treated with methotrexate (MTX)/adalimumab (ADA) or MTX/etanercept (ETA). Methods Thirty-one RA patients were randomised to receive subcutaneously either ADA (40 mg each other week) or ETA (50 mg per week). Twenty RA patients (average age: 50±16 years old (yo), MTX: 14±6 mg/week (w), initial DAS28: 5±1) received ADA while eleven RA patients (age: 55±15 yo, MTX: 18±2 mg/w, initial DAS28: 5±1) received ETA. The drug efficacy was evaluated with the DAS28 score after 3, 6 months and after one year of treatment according to the EULAR response criteria. A blood sample was carried out in patients just before the first injection of treatment in order to isolate peripheral blood mononuclear cells (PBMC) and extract total RNA. cRNAs were synthesised, amplified, labelled and purified using the Quick AMP labelling Agilent kits. Labeled cRNAs were hybridised to Agilent 4×44 K array and scanned with an Agilent Scanner. Microarray data were extracted and normalised with the Feature Extraction software. Next, a supervised analysis was performed using t-test (GeneSpring GX software) in order to identify gene expression profiles able to separate perfectly responders (R) and moderate responders (MR) to each drug. The gene expression profiles obtained for ADA and ETA were further compared to know if the authors have a specific signature for each drug. Results Demographic, clinical and biological characteristics of all the patients were comparable whatever the treatment administered. From the 20 patients treated with ADA, a combination of 25 transcripts (p<0.01) was able to separate perfectly R (11/20) and MR (9/20). Among the 11 patients who have been treated with ETA, 3/11 were classified as R and 8/11 as MR. A combination of 2074 transcripts (p<0.01) was able to separate perfectly R and MR to ETA. When the authors compared these two combinations of transcripts, just an overlap of 2 transcripts was found between them, leading us to consider that the signatures obtained for ADA and ETA are drug specific. Conclusion The authors identified a specific drug signature able to separate perfectly R and MR to either MTX/ADA or MTX/ETA. This study has shown for the first time that the transcriptomic approach is a sensitive, relevant and powerful tool to identify specific predictive markers for each molecule, not only through the different classes of immunotherapies but also in a same class of drug such as TNFα blocking agents.