Marc Lombès

Hôpital Bicêtre (Hôpitaux Universitaires Paris-Sud), Lutetia Parisorum, Île-de-France, France

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Publications (249)

  • Géraldine Vitellius · Jérôme Fagart · Brigitte Delemer · [...] · Marc Lombès
    Article · Aug 2016 · Human Mutation
  • [Show abstract] [Hide abstract] ABSTRACT: Serum steroid assays are major tools in the clinical evaluation of adrenal disorders. The main adrenal steroids are routinely measured with immunoassays. However, chromatographic methods are known to offer better specificity. We report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for simultaneous quantification of 15 adrenal steroids targeting the mineralo- and gluco-corticosteroid pathways. Serum steroids combined with deuterated internal standards were extracted using successive protein precipitation and solid phase extraction steps. Cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, progesterone, 11-deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-deoxycorticosterone, aldosterone, dehydroepiandrosterone sulfate, testosterone and androstenedione were resolved in fourteen minutes using a BEH C18 column coupled to a methanol-ammonium formate gradient. Detection was performed using multiple reaction monitoring quantitation. Routinely determined steroid levels by immunoassays were compared to those measured by LC-MS/MS. This method was applied to assess steroid profiles in congenital adrenal hyperplasia (CAH) patients with 21-hydroxylase deficiency. Low quantification limits depending on each steroid (ranging from 0.015ng/mL for aldosterone to 20ng/mL for DHEAS) are adapted to the clinical use. Recoveries of steroids range from 64% for 21deoxycortisol to 101% for cortisol and are fully corrected by internal standards. A good linearity with R>0.989 is obtained for each compound. The inter-day variation coefficients ranged from 4.7% for cortisol to 16.3% for 11-deoxycorticosterone. The immunoassay for cortisol (Immulite 2000, Siemens) showed acceptable agreement with LC-MS/MS (bias +7.2%). However, Bland-Altman plots revealed large negative bias for aldosterone (-33.4%, AldoCT, CisBio international), for 17-hydroxyprogesterone at concentrations below 2ng/mL (-74.1%, OHP-CT MP Biomedical), for androstenedione (-80.3%, RIA D4, Beckman Coulter) and for 11-deoxycortisol (-125.3%, Diasource Immunoassays). Finally, the analysis of samples from 21-hydroxylase defective patients demonstrated the potential usefulness of multiplexed steroid profiling for the diagnosis and/or monitoring of different forms of congenital adrenal hyperplasia. This LC-MS/MS method provides highly sensitive and specific assessments of mineralo- and glucocorticoids pathways from a small volume sample and is therefore a promising potent tool for clinical and experimental endocrine studies.
    Article · Jun 2016 · The Journal of steroid biochemistry and molecular biology
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    [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Mitotane (o,p'-DDD) is the standard treatment for advanced adrenocortical carcinoma (ACC). Monitoring of plasma mitotane levels is recommended to look for a therapeutic window between 14 and 20mg/L, but its positive predictive value requires optimization. We report the case of an ACC patient with a history of dyslipidemia treated with mitotane in whom several plasma mitotane levels >30mg/L were found together with an excellent neurological tolerance. This observation led us to compare theoretical or measured o,p'-DDD and o,p'-DDE levels in a series of normolipidemic and dyslipidemic plasma samples to explore potential analytical issues responsible for an overestimation of plasma mitotane levels. We demonstrate an overestimation of mitotane measurements in dyslipidemic patients. Mitotane and o,p'-DDE measurements showed a mean 20% overestimation in hypercholesterolemic and hypertriglyceridemic plasma, compared with normolipidemic plasma. The internal standard p,p'-DDE measurements showed a parallel decrease in hypercholesterolemic and hypertriglyceridemic plasma, suggesting a matrix effect. Finally, diluting plasma samples and/or using phospholipid removal cartridges allowed correcting such interference. Learning points: Hypercholesterolemia (HCH) and hypertriglyceridemia (HTG) induce an overestimation of plasma mitotane measurements.We propose a routine monitoring of lipidemic status.We propose optimized methodology of measurement before interpreting high plasma mitotane levels.
    Full-text Article · Jun 2016
  • Laetitia Martinerie · Pascal Boileau · Marc Lombès
    Article · May 2016 · The Journal of Clinical Endocrinology and Metabolism
  • Géraldine Vitellius · Jérôme Fagart · Brigitte Delemer · [...] · Marc Lombes
    [Show abstract] [Hide abstract] ABSTRACT: Generalized glucocorticoid resistance is associated with glucocorticoid receptor (GR, NR3C1) mutations. Three novel heterozygous missense NR3C1 mutations (R477S, Y478C and L672P) were identified in patients presenting with adrenal incidentalomas, glucocorticoid excess without Cushing syndrome. Dexamethasone (DXM) binding studies demonstrated that the affinity of GRR477S and GRY478C mutants, located in the DNA-binding domain of GR, was similar to wild-type GR (Kd = 2-3 nM). In contrast, GRL672P mutant, located in the ligand-binding domain of GR, was unable to bind glucocorticoids and was more sensitive to protein degradation. GR subcellular distribution revealed a marked decrease in DXM-induced nuclear translocation of GRR477S and GRY478C mutants, whereas GRL672P remained exclusively cytoplasmic. Chromatin immunoprecipitation demonstrated impaired recruitment of DBD mutants onto the regulatory sequence of FKBP5. Transactivation assays disclosed the lack of transcriptional activity of GRR477S and GRL672P while GRY478C had a reduced transactivation capacity. Three-D modeling indicated that R477S lost two essential hydrogen bonds with DNA, Y478C resulted in altered interaction with surrounding amino-acids, destabilizing DBD, while L672P altered the H8 helix folding, leading to unstructured LBD. This study identifies novel NR3C1 mutations with their molecular consequences on altered GR signaling and suggests that genetic screening of NR3C1 should be conducted in patients with subclinical hypercorticism. This article is protected by copyright. All rights reserved.
    Article · Apr 2016 · Human Mutation
  • [Show abstract] [Hide abstract] ABSTRACT: Germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AhRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells over-expressing wildtype compared to cells expressing mutant AIP. Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH3 cells GH secretion. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner.
    Article · Apr 2016 · Endocrine Related Cancer
  • [Show abstract] [Hide abstract] ABSTRACT: Breast cancer is a hormone-dependent disease in which estrogen signaling targeting drugs fail in about 10 % due to resistance. Strong evidences highlighted the mitogen role of progesterone, its ligands, and the corresponding progesterone receptor (PR) isoforms in mammary carcinoma. Several PR antagonists have been synthesized; however, some of them are non-selective and led to side or toxic effects. Herein, we evaluated the anti-tumor activity of a commercially available PR modulator, ulipristal acetate (UPA), and a new selective and passive PR antagonist "APR19" in a novel preclinical approach based on patient-derived breast tumor (HBCx-34) xenografted in nude mice. As opposed to P4 that slightly reduces tumor volume, UPA and APR19 treatment for 42 days led to a significant 30 % reduction in tumor weight, accompanied by a significant 40 % retardation in tumor growth upon UPA exposure while a 1.5-fold increase in necrotic areas was observed in APR19-treated tumors. Interestingly, PR expression was upregulated by a 2.5-fold factor in UPA-treated tumors while APR19 significantly reduced expression of both PR and estrogen receptor α, indicating a potential distinct molecular mechanism among PR antagonists. Cell proliferation was clearly reduced in UPA group compared to vehicle conditions, as revealed by the significant reduction in Ki-67, Cyclin D1, and proliferating cell nuclear antigen (PCNA) expression. Likewise, an increase in activated, cleaved poly(ADP-ribose) polymerase (PARP) expression was also demonstrated upon UPA exposure. Collectively, our findings provide direct in vivo evidence for anti-progestin-mediated control of human breast cancer growth, given their anti-proliferative and pro-apoptotic activities, supporting a potential role in breast cancer therapy.
    Article · Mar 2016 · Hormones and Cancer
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    Full-text Article · Feb 2016
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    Full-text Dataset · Jan 2016
  • [Show abstract] [Hide abstract] ABSTRACT: Background & aim: Kupffer cells (KC) play a key role in the onset of inflammation in non-alcoholic steatohepatitis (NASH). The glucocorticoid receptor (GR) induces glucocorticoid-induced leucine zipper (GILZ) expression in monocytes/macrophages and is involved in several inflammatory processes. We hypothesized that the GR-GILZ axis in KC may contribute to the pathophysiology of obesity-induced liver inflammation. Methods: By using a combination of primary cell culture, pharmacological experiments, mice deficient for the GR specifically in macrophages and transgenic mice overexpressing GILZ in macrophages, we explored the involvement of the GR-GILZ axis in KC in the pathophysiology of obesity-induced liver inflammation. Results: Obesity was associated with a downregulation of the GR and GILZ, and an impairment of GILZ induction by lipopolysaccharide (LPS) and dexamethasone (DEX) in KC. Inhibition of GILZ expression in isolated KC transfected with GILZ siRNA demonstrated that GILZ dowregulation was sufficient to sensitize KC to LPS. Conversely, liver inflammation was decreased in obese transgenic mice specifically overexpressing GILZ in macrophages. Pharmacological inhibition of the GR showed that impairment of GILZ induction in KC by LPS and DEX in obesity was driven by a downregulation of the GR. In mice specifically deficient for GR in macrophages, GILZ expression was low, leading to an exacerbation of obesity-induced liver inflammation. Conclusion: Obesity is associated with a downregulation of the GR-GILZ axis in Kupffer cells, which promotes liver inflammation. The GR-GILZ axis in KC is an important target for the regulation of liver inflammation in obesity.
    Article · Nov 2015 · Journal of Hepatology
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    [Show abstract] [Hide abstract] ABSTRACT: The progesterone receptor (PR) with its isoforms and ligands are involved in breast tumorigenesis and prognosis. We aimed at analyzing the respective contribution of PR isoforms, PRA and PRB, in breast cancer cell proliferation in a new estrogen-independent cell based-model, allowing independent PR isoforms analysis. We used the bi-inducible human breast cancer cell system MDA-iPRAB. We studied the effects and molecular mechanisms of action of progesterone (P4) and ulipristal acetate (UPA), a new selective progesterone receptor modulator, alone or in combination. P4 significantly stimulated MDA-iPRA expressing cells proliferation. This was associated with P4-stimulated expression of the anti-apoptotic factor BCL2-L1 and enhanced recruitment of PRA, SRC-1 and RNA Pol II onto the +58 kb PR binding motif of the BCL2-L1 gene. UPA decreased cell proliferation and repressed BCL2-L1 expression in the presence of PRA, correlating with PRA and SRC1 but not RNA Pol II recruitment. These results bring new information on the mechanism of action of PR ligands in controlling breast cancer cell proliferation through PRA in an estrogen independent model. Evaluation of PR isoforms ratio, as well as molecular signature studies based on PRA target genes could be proposed to facilitate personalized breast cancer therapy. In this context, UPA could be of interest in endocrine therapy. Further confirmation in the clinical setting is required.
    Full-text Article · Oct 2015 · PLoS ONE
  • [Show abstract] [Hide abstract] ABSTRACT: Context: The neonatal period, notably in preterm infants, is characterized by high sodium wasting implying that aldosterone, the main hormone regulating sodium reabsorption, is unable to maintain sodium homeostasis. Objective: Assess aldosterone secretion and action in neonates according to gestational age (GA). Design: Multicenter prospective study (NCT01176162) conducted between 2011-2014. Infants were followed during their first three months (M3). Setting: Five neonatology departments in France. Participants: The 155 newborns included were classified into three groups: <33 GW (gestational weeks) = group 1 (46 patients), 33-36 GW (67 patients) = group 2 and ≥37 GW (42 patients) = group 3. Main outcome measures: Plasma aldosterone was measured in umbilical cord blood. Urinary aldosterone (UAldo) was assessed at D0, D3, M1 and M3 postnatal. The correlation between UAldo and the urinary Na/K ratio was determined as an index of renal aldosterone sensitivity. Results: UAldo significantly increased with GA: from 8.8±7.5 μg/mmol of creatinine (group 1) to 21.1±21.0 (group 3) in correlation with plasma aldosterone levels in all groups (P<0.001), demonstrating its reliability. The aldosterone/renin ratio significantly increased with GA, suggesting an aldosterone secretion defect in preterm infants. UAldo and urinary Na/K were correlated in very preterm but not in term neonates, consistent with very preterm neonates being renal aldosterone sensitive and term neonates being aldosterone resistant. Conclusions: Very preterm infants have a previously unrecognized defective aldosterone secretion but conserved renal aldosterone sensitivity in the neonatal period, which modifies the current view of sodium balance in these infants and suggests alternative management approaches.
    Article · Sep 2015 · The Journal of Clinical Endocrinology and Metabolism
  • M. Ayrout · N. Binart · M. Lombès · S. Chauvin
    Article · Sep 2015
  • F. Le Billan · J.A. Khan · K. Lamribet · [...] · M. Lombès
    Article · Sep 2015
  • G. Vitellius · J. Bouligand · J. Fagart · [...] · M. Lombès
    Article · Sep 2015
  • Article · Sep 2015
  • E. Kuhn · K. Lamribet · S. Viengchareun · [...] · M. Lombès
    Article · Sep 2015
  • Article · Sep 2015
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    Full-text Article · Sep 2015 · Endocrinology & Metabolism Clinics of North America
  • Article · Sep 2015

Publication Stats

5k Citations

Institutions

  • 2015
    • Hôpital Bicêtre (Hôpitaux Universitaires Paris-Sud)
      Lutetia Parisorum, Île-de-France, France
  • 2014-2015
    • Université Paris-Sud 11
      Orsay, Île-de-France, France
  • 2010
    • Assistance Publique – Hôpitaux de Paris
      Lutetia Parisorum, Île-de-France, France
    • Hôpital Armand-Trousseau (Hôpitaux Universitaires Est Parisien)
      Lutetia Parisorum, Île-de-France, France
  • 2006
    • Baylor College of Medicine
      • Department of Molecular & Cellular Biology
      Houston, Texas, United States
    • Unité Inserm U1077
      Caen, Lower Normandy, France