Search

SUMOylation is a highly conserved and dynamic post-translational mechanism primarily affecting nuclear programs for adapting organisms to stressful challenges. Alteration of SUMOylation cycles leads to severe developmental and homeostatic defects and malignancy, but signals coordinating SUMOylation are still unidentified. The adrenal cortex is a zonated endocrine gland that controls body homeostasis and stress response. Here, we show that in human and in mouse adrenals, SUMOylation follows a decreasing centripetal gradient that mirrors cortical differentiation flow and delimits highly and weakly SUMOylated steroidogenic compartments, overlapping glomerulosa, and fasciculata zones. Activation of PKA signaling by acute hormonal treatment, mouse genetic engineering, or in Carney complex results in repression of small ubiquitin-like modifier (SUMO) conjugation in the inner cortex by coordinating expression of SUMO pathway inducers and repressors. Conversely, genetic activation of canonical wingless-related integration site signaling maintains high SUMOylation potential in the outer neoplastic cortex. Thus, SUMOylation is tightly regulated by signaling pathways that orchestrate adrenal zonation and diseases.-Dumontet, T., Sahut-Barnola, I., Dufour, D., Lefrancois-Martinez, A.-M., Berthon, A., Montanier, N., Ragazzon, B., Djari, C., Pointud, J.-C., Roucher-Boulez, F., Batisse-Lignier, M., Tauveron, I., Bertherat, J., Val, P., Martinez, A. Hormonal and spatial control of SUMOylation in the human and mouse adrenal cortex.

International audience; Daily synchronous rhythms of cell division at the tissue or organism level are observed in many species and suggest that the circadian clock and cell cycle oscillators are coupled. For mammals, despite known mechanistic interactions, the effect of such coupling on clock and cell cycle progression, and hence its biological relevance, is not understood. In particular, we do not know how the temporal organization of cell division at the single-cell level produces this daily rhythm at the tissue level. Here we use multispectral imaging of single live cells, computational methods, and mathematical modeling to address this question in proliferating mouse fibroblasts. We show that in unsynchronized cells the cell cycle and circadian clock robustly phase lock each other in a 1:1 fashion so that in an expanding cell population the two oscillators oscillate in a synchronized way with a common frequency. Dexamethasone-induced synchronization reveals additional clock states. As well as the low-period phase-locked state there are distinct coexisting states with a significantly higher period clock. Cells transition to these states after dexamethasone synchronization. The temporal coordination of cell division by phase locking to the clock at a single-cell level has significant implications because disordered circadian function is increasingly being linked to the pathogenesis of many diseases, including cancer.

Inhibitors of histone deacetylases (HDACi's) are promising novel tools for cancer therapy. We have compared the growth inhibitory and apoptogenic potential of the pan-HDACi SAHA and the sub-class I selective HDAC inhibitor MS275, as well as valproic acid (VPA) on glucocorticoid sensitive and resistant B (B-ALL) and T (T-ALL) cell acute lymphoblastic leukemia cells and patients blasts. In contrast, to our previous results with U937 acute myeloid leukemia (AML) cells which showed a similar activity of MS275 and SAHA in growth inhibition and apoptosis induction, both B and T-ALL cells were much more efficiently killed by SAHA and VPA than by MS275. The same relative potency was observed with some patient ALL blasts treated ex vivo. SAHA displayed similar efficacy on glucocorticoid-sensitive and insensitive ALL cells but did not synergize with dexamethasone. In studying mediators of apoptosis we found that the TRAIL receptor DR5 is constitutively expressed in glucocorticoid-sensitive CEM-C7 cells which are also TRAIL sensitive. In contrast, glucocorticoid-insensitive CEM-C1 cells do not express DR5 and are insensitive to TRAIL. However, SAHA induces, in addition to p21(WAF1/CIP1) also re-expression of DR5. Importantly, SAHA-induced apoptosis of CEM-C7 cells operates through initiator caspase 10, while it induces apoptosis of CEM-C1 cells through the intrinsic, as well as through caspase-independent death pathways. Our data suggest that the generation of resistance to glucocorticoids has dramatically altered death signaling in these cells and that SAHA overcomes these restrictions by inducing alternative death pathways.

International audience; The primary cilium is an organelle present in most of the cells of the organism. Ciliopathies are genetic disorders of the primary cilium and can be associated with obesity. We have studied the primary cilium during adipocyte differentiation of human adipose stem cells (hASC). We show here that the size of the primary cilium follows several modifications during adipocyte differentiation. It is absent in growing cells and appears in confluent cells. Interestingly, during the first days of differentiation, the cilium undergoes a dramatic elongation that can be mimicked by dexamethasone alone. Thereafter, its size decreases. It can still be detected in cells that begin to accumulate lipids but is absent in cells that are filled with lipids. The cilium elongation does not seem to affect the localization of proteins associated with the cilium such as Kif3-A or Smoothened. However, Hedgehog signaling, an anti-adipogenic pathway dependent on the primary cilium, is inhibited after three days of differentiation, concomitantly with the cilium size increase. Together, these results shed new light on the primary cilium and could provide us with new information on adipocyte differentiation under normal and pathological conditions.

International audience; In chickens, leptin is expressed mainly in the liver, where its receptor gene expression has also been reported, and in adipose tissue. In view of the key role played by the liver in lipogenesis in avian species, the hepatic expression of leptin may have physiological significance. In this study, we showed that leptin is constitutively expressed and secreted in a chicken-derived hepatoma cell line (LMH). Although insulin regulates leptin expression in vivo, incubation of LMH cells in the presence of 100 nM insulin for 24 or 48 h had no effect on leptin expression or its secretion in the culture medium. In addition, we developed a specific chicken leptin receptor real-time reverse transcription (RT)-PCR, and downregulation of leptin receptor gene expression by homologous and heterologous signals was demonstrated, as relative leptin receptor mRNA levels were significantly decreased after exposure of LMH cells to recombinant chicken leptin or porcine insulin. In conclusion, our results indicate that leptin is probably able to desensitize its own response in the chicken liver. Finally, the ability of insulin and leptin to regulate chicken leptin receptor gene expression suggests a direct role of leptin in the control of hepatic metabolism.

International audience; Corticosteroids have been shown to reduce short-term mortality during septic shock and therefore recommended in the most severe patients as adjuvant therapy. Until recently, recombinant human activated protein C (APC) was also considered in the management of more severe cases. As myocardial depression has long been recognized as a manifestation of organ dysfunction during septic shock, we examined whether corticosteroids (dexamethasone, 150 µg/kg per hour) and/or APC (33 µg/kg per hour) treatments improve sepsis-induced cardiac dysfunction during cecal ligature and puncture-induced septic shock in Wistar rats. All rats received intravenous saline resuscitation (10 mL/kg per hour) and antibiotics. Eighteen hours after surgery, anesthesia was performed (isoflurane), and myocardial function was assessed using a conductance catheter introduced into the left ventricle. Rats were then killed; blood and heart were harvested for biological analysis, including radical oxygen species determination. Cecal ligature and puncture induced hypotension, depression of myocardial systolic performance (demonstrated by significant decreases in dP/dtmax [first derivative of maximal developed pressure during isovolumetric contraction], end-systolic pressure-volume relationship, and preload recruitable stroke work) and alteration of diastolic function (dP/dtmin [first derivative of minimal developed pressure during isovolumetric relaxation]), whereas dexamethasone, APC, and their combination thereof allowed correction of hemodynamic disorders and improved myocardial mechanical efficiency. Cecal ligature and puncture was associated with higher levels of nitric oxide and superoxide anion (O2) in heart (electron paramagnetic resonance studies) and consequently peroxynitrite. Dexamethasone and APC also improved cardiac dysfunction by downregulating the inducible nitric oxide synthase pathway and reducing myocardial oxidative stress.

Differentiation of cholinergic cell line NG108-15 induced by a combination of dibutyryl cyclic AMP (dbcAMP) and dexamethasone enhances the cholinergic phenotype of the cells more than that induced by either agent alone. We investigated the effect of treatment with dbcAMP and dexamethasone on potassium depolarization-evoked influx of calcium and its regulation by the muscarinic agonist carbachol. Depolarization of control cells and of cells differentiated in the presence of dbcAMP or dexamethasone alone, or in the combined presence of dbcAMP and dexamethasone induced, respectively, 2.2-, 4.3-, 2.7- and 10.7-fold increases of the resting [Ca(2+)](i). Dexamethasone alone and the combination of dbcAMP and dexamethasone augmented the number of muscarinic receptors by 25 and 40%, respectively. Inhibitors of N (omega-conotoxin GVIA) or P/Q (omega-agatoxin TK) calcium channels had no effect on Ca(2+) influx in control cells, whereas in cells differentiated in the combined presence of dbcAMP and dexamethasone they significantly diminished the influx of Ca(2+) by 20 and 5%, respectively. Carbachol attenuated calcium influx in differentiated cells in an atropine-insensitive manner if it was present during stimulation. This effect of carbachol was probably due to an open-channel block of L type channels. In the presence of nifedipine, carbachol attenuated the influx of Ca(2+) into cells differentiated with dbcAMP and dexamethasone by 20% in an atropine-sensitive way. Data show that differentiation of NG108-15 cells by dbcAMP and dexamethasone promotes the expression of functional nifedipine-insensitive N and P/Q types of Ca(2+) channels and that the nifedipine-insensitive calcium influx becomes subject to inhibitory regulation by muscarinic receptors.

The vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT) are encoded by genes organized in a single gene locus, and coregulation of the transcription of the two genes has been repeatedly reported in cholinergic tissues. In the present study, different stimuli were used to induce the differentiation of the hybridoma cells NG108-15 and we examined their effects on the modulation of VAChT and ChAT expression at the mRNA and protein levels. All agents upregulated the VAChT and ChAT mRNA levels, but to a different extent. ChAT activity was increased by retinoic acid, dexamethasone, and dibutyrylcyclic AMP (dbcAMP), and a synergistic effect was observed with a combined dexamethasone and dbcAMP treatment. Nonetheless, no changes in the VAChT protein level could be observed, as judged from ligand binding studies as well as from immunochemical detection. Hemicholinium-3-sensitive choline uptake, hemicholinium-3 binding, and acetylcholine content were increased by differentiating agents, with a rank order of potency comparable to their effects on ChAT activity. Prominent changes were observed in the expression of vesicular protein markers, particularly with the associated treatment dexamethasone and dbcAMP. Thus, it appears that although the different stimuli we have been using are able to stimulate neuronal features and activate the transcription of cholinergic genes, they did not contrive to increase the level of VAChT protein in these cells.

We have recently shown that stable expression of an epitope-tagged cDNA of the hepatocyte- enriched transcription factor, hepatocyte nuclear factor (HNF)4, in dedifferentiated rat hepatoma H5 cells is sufficient to provoke reexpression of a set of hepatocyte marker genes. Here, we demonstrate that the effects of HNF4 expression extend to the reestablishment of differentiated epithelial cell morphology and simple epithelial polarity. The acquisition of epithelial morphology occurs in two steps. First, expression of HNF4 results in reexpression of cytokeratin proteins and partial reestablishment of E-cadherin production. Only the transfectants are competent to respond to the synthetic glucocorticoid dexamethasone, which induces the second step of morphogenesis, including formation of the junctional complex and expression of a polarized cell phenotype. Cell fusion experiments revealed that the transfectant cells, which show only partial restoration of E-cadherin expression, produce an extinguisher that is capable of acting in trans to downregulate the E-cadherin gene of well-differentiated hepatoma cells. Bypass of this repression by stable expression of E-cadherin in H5 cells is sufficient to establish some epithelial cell characteristics, implying that the morphogenic potential of HNF4 in hepatic cells acts via activation of the E-cadherin gene. Thus, HNF4 seems to integrate the genetic programs of liver-specific gene expression and epithelial morphogenesis.

International audience; The capacity of the liver-enriched transcription factor hepatocyte nuclear factor 4 (HNF4) to direct redifferentiation of dedifferentiated rat hepatoma cells was investigated by stable transfection of epitope-tagged HNF4 cDNA into H5 variant cells. HNF4-producing cells expressed the previously silent HNF1 gene and showed activation of some hepatic functions, including alpha1-antitrypsin, beta-fibrinogen, and transthyretin, but not of the endogenous HNF4 gene. Expression of the other hepatocyte-enriched transcription factors was not modified. Treatment of the HNF4tag-expressing cells with dexamethasone induced expression of the transgene by 10-fold, resulting in enhanced expression of target genes of both glucocorticoid hormones and HNF4. The set of activated hepatic genes was extended by treatment of cells with the demethylating agent 5-azacytidine followed by selection in dexamethasone-containing glucose-free medium. Some of the colonies that developed reexpressed the entire set of hepatic functions tested. Fusion of HNF4tag-producing H5 cells with well-differentiated Fao cells showed that only those hybrids which maintained expression of HNF4tag were protected from complete extinction, including that of the Fao HNF4 gene. Thus, H5 cells must produce an extinguisher of the HNF4 gene. In addition, this result implies that HNF4 itself, or its target HNF1, is a positive regulator of HNF4. In conclusion, HNF4tag expression overcomes repression of the hepatic phenotype of the H5 cell without abolishing its potential to extinguish an active genome. Taken together, these results predict that expression of HNF4 should be sufficient to establish heritable expression of many parameters of the hepatic differentiated state.

International audience; The estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined.

Purpose To identify the criteria for hematologic and cardiac response to treatment in immunoglobulin light chain (AL) amyloidosis based on survival analysis of a large patient population. Patients and Methods We gathered for analysis 816 patients with AL amyloidosis from seven referral centers in the European Union and the United States. A different cohort of 374 patients prospectively evaluated at the Pavia Amyloidosis Research and Treatment Center was used for validation. Data was available for all patients before and 3 and/or 6 months after initiation of first-line therapy. The prognostic relevance of different criteria for hematologic and cardiac response was assessed. Results There was a strong correlation between the extent of reduction of amyloidogenic free light chains (FLCs) and improvement in survival. This allowed the identification of four levels of response: amyloid complete response (normal FLC ratio and negative serum and urine immunofixation), very good partial response (difference between involved and uninvolved FLCs [dFLC] < 40 mg/L), partial response (dFLC decrease > 50%), and no response. Cardiac involvement is the major determinant of survival, and changes in cardiac function after therapy can be reliably assessed using the cardiac biomarker N-terminal natriuretic peptide type B (NT-proBNP). Changes in FLC and NT-proBNP predicted survival as early as 3 months after treatment initiation. Conclusion This study identifies and validates new criteria for response to first-line treatment in AL amyloidosis, based on their association with survival in large patient populations, and offers surrogate end points for clinical trials.

International audience; POEMS syndrome is a rare disorder characterized by polyneuropathy, monoclonal gammopathy, multiorgan involvement, and elevated vascular endothelial growth factor levels. Localized bone lesions require irradiation, whereas young patients with disseminated disease receive intensive treatment with stem cell support. Treatment of older and non responding patients is not yet standardized. We report the use of a combination of lenalidomide and dexamethasone in 20 patients with POEMS syndrome. Four patients were newly diagnosed, and 16 had relapsed or progressed after treatment. All but one of the patients responded: clinical improvements were noted in neuropathies (16/20) organomegaly (13/13), peripheral edema (14/15), and pulmonary hypertension (5/5). At least a very good partial response was noted in 68% of patients, with partial responses in 26%. Serum VEGF levels fell markedly in all 17 patients with available values. Twelve patients had 18-FDG-PET/CT at diagnosis (11 with positive findings), and nine patients during follow-up. The number of lesions fell markedly in five cases and remained stable in two cases, while two patients became negative. During a median follow-up of 22 months, four patients relapsed. Toxicity, predominantly hematological, was mild and manageable. Lenalidomide thus appears to be effective in POEMS syndrome, inducing high rate of clinical and biological responses.

International audience; BACKGROUND: There is no consensual first-line chemotherapy for elderly patients with mantle cell lymphoma (MCL). The GOELAMS (Groupe Ouest-Est des Leucémies Aiguës et Maladies du Sang) group previously developed the (R)VAD+C regimen (rituximab, vincristine, doxorubicin, dexamethasone and chlorambucil), which appeared as efficient as R-CHOP (rituximab, cyclophosphamide, doxorubicine, vincristine, prednisone) while less toxic. Based on this protocol, we now added bortezomib (RiPAD+C: rituximab, bortezomib, doxorubicin, dexamethasone and chlorambucil) given its efficacy in relapsed/refractory MCL patients. The goal of the current phase II trial was to evaluate the feasibility and efficacy of the RiPAD+C regimen as frontline therapy for elderly patients with MCL. PATIENTS AND METHODS: Patients between 65 and 80 years of age with newly diagnosed MCL received up to six cycles of RiPAD+C. RESULTS: Thirty-nine patients were enrolled. Median age was 72 years (65-80). After four cycles of RiPAD+C, the overall response rate was 79%, including 51% complete responses (CRs). After six cycles, CR rate increased up to 59%. After a 27-month follow-up, median progression-free survival (PFS) is 26 months and median overall survival has not been reached. Four patients (10%) discontinued the treatment because of a severe toxicity and seven patients (18%) experienced grade 3 neurotoxicity. CONCLUSION: The bortezomib-containing RiPAD+C regimen results in high CR rates and prolonged PFS with predictable and manageable toxic effects in elderly patients with MCL.

Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2) (PGI(2)) may fully substitute for the cAMP-elevating agent isobutylmethylxanthine (IBMX). Moreover, selective activation of Epac-dependent signaling promoted adipocyte differentiation when the Rho-associated kinase (ROCK) was inhibited. Unlike the case for murine preadipocytes cell lines, long-chain fatty acids, like arachidonic acid, did not promote adipocyte differentiation of hMADS cells in the absence of a PPARγ agonist. However, prolonged treatment with the synthetic PPARδ agonist L165041 promoted adipocyte differentiation of hMADS cells in the presence of IBMX. Taken together our results emphasize the need for cAMP signaling in concert with treatment with a PPARγ or PPARδ agonist to secure efficient adipocyte differentiation of human hMADS mesenchymal stem cells.

Viguerie N, Picard F, Hul G, Roussel B, Barbe P, Iacovoni JS, Valle C, Langin D, Saris WHM. Multiple effects of a short-term dexamethasone treatment in human skeletal muscle and adipose tissue. Physiol Genomics 44: 141-151, 2012. First published November 22, 2011; doi:10.1152/physiolgenomics.00032.2011.-Glucocorticoids are frequently prescribed drugs with important side-effects such as glucose intolerance and tissue remodeling. The goal was to explore the molecular basis of the response of skeletal muscle and adipose tissue during a short-term dexamethasone treatment to better understand the induction of side-effects of glucocorticoids on these metabolic tissues. Fifteen healthy male subjects were assigned to a 4-day treatment with dexamethasone at 4 mg/day. The primary outcome measures were changes in gene expression profiling of subcutaneous skeletal muscle and adipose tissue. Urinary cortisol, plasma, and metabolic biochemistry were also assessed. In both tissues the prominent observation was a response to stress and increased inflammatory responses. An upregulation of the serum amyloid A was detected in skeletal muscle, adipose tissue, and plasma, whereas circulating levels of C reactive protein, another acute phase protein, decreased along with a worsened insulin sensitivity index. As tissue-specific features, tissue remodeling was shown in skeletal muscle while the adipose tissue exhibited a decreased energy metabolism. Several limitations might be raised due to the small number of subjects investigated: a possible cross talk with the mineralocorticoid receptor, and a single time point may not identify regulations occurring during longitudinal treatment. In line with the known physiological effect of glucocorticoids the early modulation of stress response genes was observed. An unexpected feature was the upregulation of the inflammatory and immune pathways. The identification of novel impact on two glucocorticoid target tissues provides a molecular basis for the design of more specific glucocorticoids devoid of adverse effects.

International audience; Various renal disorders are associated with monoclonal gammopathies, secondary to tissue deposition or precipitation of a monoclonal immunoglobulin (Ig) or a fragment thereof (isolated Ig light chain or heavy chain). They are classified according to the localization of renal lesions, either glomerular or tubular and to the pattern of ultrastructural organization of Ig deposits. Renal disease in monoclonal gammopathies may be isolated, or associated with various systemic symptoms particularly in AL amyloidosis, Randall-type monoclonal Ig deposition disease and monoclonal cryoglobulinemias. Except for myeloma cast nephropathy, which occurs in the setting of high-mass myeloma and is recognized after electrophoretic analysis of proteinuria and AL amyloidosis, which diagnosis is usually made after pathological examination of non-invasive tissue specimens (i.e. abdominal fat or minor salivary glands), a kidney biopsy is required to identify the other types of renal disorders associated with monoclonal gammopathies and to estimate renal prognosis. Renal pathological diagnosis is difficult and relies on careful examination of kidney biopsy samples, by light microscopy, immunofluorescence studies using conjugates specific for Ig light and heavy chains, IgG sub-classes and heavy chain constant domains and by electron microscopy. In some cases, additional studies are required to identify the nature of deposits, such as immuno-electron microscopy or mass spectrometric-based proteomic analysis after laser dissection. In patients with renal disorders related to Ig light chain precipitation or deposition (myeloma cast nephropathy, AL amyloidosis, Randall-type light chain deposition disease), measurement of serum free light chains at baseline and throughout follow-up is mandatory to evaluate clonal response to chemotherapy. A more than or equal to 50% decrease in serum free light chain levels is associated with increased renal and patient survival. In AL amyloidosis, serum levels of markers of cardiac disease (NT-proBNP and troponin) are also closely associated with prognosis. Efficient chemotherapy, tailored to the underlying plasma cell or lymphoproliferative disorder and adapted to renal function, should be promptly introduced, even in the absence of overt malignant haematological disease. Renal prognosis and patient survival (particularly in AL amyloidosis and cast nephropathy) are closely associated with the rapid achievement of an haematological response. The combination of melphalan plus dexamethasone (MDex) is currently used as first-line chemotherapy in systemic AL amyloidosis. Bortezomib-based regimens are commonly employed as first-line treatment in myeloma cast nephropathy and Randall-type monoclonal Ig deposition disease and as second line therapy in AL amyloidosis patients with advanced cardiomyopathy or refractory to previous chemotherapy. Solid organ transplantation (heart and kidney) should be considered in patients with AL amyloidosis or Randall-type monoclonal Ig deposition disease and advanced cardiac or renal failure. Prolonged graft and patient survival may be obtained, providing that recipients do not have other severe organ involvement or symptomatic myeloma and that haematological remission has been achieved with chemotherapy before or after organ transplantation.

International audience; Systemic AL amyloidosis is a rare complication of monoclonal gammopathies. Renal manifestations are frequent, mostly characterized by heavy proteinuria, with nephrotic syndrome and renal failure in more than half of the patients at diagnosis. Without treatment, median survival does not exceed 12 months. Amyloid heart disease and diffusion of amyloid deposits are associated with reduced survival. Treatment of systemic AL amyloidosis has been profoundly modified with the introduction of international criteria for the definition of organ involvement and hematologic response, and with the use of sensitive tests for the measurement of serum-free light chain levels. Melphalan plus dexamethasone is now established as the gold standard for first line treatment of systemic AL, with similar efficacy and reduced treatment-related mortality compared to high-dose therapy. Modern chemotherapy regimens, based on the use of novel agents such as bortezomib and lenalidomide, might further improve patient survival.

International audience; Recent multicenter randomized studies about persistent macular edema in venous occlusions provided us with interesting results. Until now, the standard of care treatment of macular edema due to branch vein occlusion remained grid laser in contrast with central vein occlusion where the absence of treatment was still recommended. Score, Geneva, Bravo and Cruise studies recently provided us with the following results. Score study found triamcinolone to be interesting to treat macular edema due to central vein occlusion but not from branch occlusion. Geneva study assessed the effect of a delivery system of dexamethasone to treat macular edema due to venous occlusion whatever the clinical form with an improvement of visual acuity. Cruise and Bravo studies assessed the effect of ranubizumab, which was found to improve the visual acuity of macular edema due to either central or branch vein occlusions. At this stage we need comparative studies to precise the indication of these different approaches that remain perhaps complementary of laser treatment.

International audience; Neorogioltriol is a tricyclic brominated diterpenoid isolated from the organic extract of the red algae Laurencia glandulifera. In the present study, the anti-inflammatory effects of neorogioltriol were evaluated both in vivo using carrageenan-induced paw edema and in vitro on lipopolysaccharide (LPS)-treated Raw264.7 macrophages. The in vivo study demonstrated that the administration of 1 mg/kg of neorogioltriol resulted in the significant reduction of carregeenan-induced rat edema. In vitro, our results show that neorogioltriol treatment decreased the luciferase activity in LPS-stimulated Raw264.7 cells, stably transfected with the NF-κB-dependent luciferase reporter. This effect on NF-κB activation is not mediated through MAPK pathways. The inhibition of NF-κB activity correlates with decreased levels of LPS-induced tumor necrosis factor-alpha (TNFα) present in neorogioltriol treated supernatant cell culture. Further analyses indicated that this product also significantly inhibited the release of nitric oxide and the expression of cyclooxygenase-2 (COX-2) in LPS-stimulated Raw264.7 cells. These latter effects could only be observed for neorogioltriol concentrations below 62.5 μM. To our knowledge, this is the first report describing a molecule derived from Laurencia glandulifera with anti-inflammatory activity both in vivo and in vitro. The effect demonstrated in vitro may be explained by the inhibition of the LPS-induced NF-κB activation and TNFα production. NO release and COX-2 expression may reinforce this effect.

International audience; INTRODUCTION: With their ability to migrate into the cornea and release toxins, caterpillar hairs can induce different clinical presentations such as conjunctivitis, keratoconjunctivitis, uveitis, and less frequently vitreoretinal inflammation (hyalitis, papillitis, macular edema). OBSERVATION: We report a case that occurred in Alsace (France) in a 13-years-old boy presenting with keratitis caused by caterpillar hairs. We localized them in the cornea, for the first time, using confocal microscopy and anterior segment spectral optical coherence tomography. CONCLUSION: Confocal microscopy and spectral optical coherence tomography can be useful for diagnosis and follow-up of this disease.

International audience; CONTEXT: Ectopic ACTH syndrome (EAS) in youngsters has seldom been reported and is poorly known. SETTING: We conducted a multicenter retrospective study involving 18 French tertiary hospitals. Cases of EAS presenting Cushing's syndrome before the age of 20 during the period from 1985 to 2008 were analyzed. PATIENTS: Ten patients aged 14 to 20 yr were identified and compared to 20 age-matched patients with Cushing's disease diagnosed during the same period. MAIN OUTCOME MEASURES: Etiologies, clinical, biochemical and radiological features, prognosis, and treatment were described. RESULTS: Seven patients had well-differentiated neuroendocrine tumors (five bronchial carcinoids, one mediastinal lymph node, and one thymic), one had a poorly differentiated thymic carcinoma, one had a pleural Ewing's sarcoma, and one had a liver nested stromal epithelial tumor. At presentation, seven tumors were identified with computed tomography scanning and somatostatin receptor scintigraphy, and one with fluoro-18-L-dihydroxyphenylalanine positron emission tomography scan. Two carcinoids were occult and were identified during follow-up. Cushing's syndrome was more intense in EAS, but the clinical and biological spectrum overlapped with that of Cushing's disease. No dynamic test achieved 100% accuracy, whereas petrosal sinus sampling provided correct diagnosis in all patients tested. Medical treatment of hypercortisolism was successful in six of the eight patients with whom it was attempted, and bilateral adrenalectomy had to be performed in only two cases. Prognosis was good; nine patients with curative resection of the tumor were alive and cured (median follow-up, 6.5 yr), whereas one patient died. CONCLUSIONS: EAS in youngsters displays many similarities to that described in adults. The diagnostic and therapeutic algorithms recommended in adults can be used in this population.

International audience; BACKGROUND: The prognosis of acute lymphoblastic leukemia in the elderly is poor. The GRAALL-SA1 phase II, randomized trial compared the efficacy and toxicity of pegylated liposomal doxorubicin versus continuous-infusion doxorubicin in patients 55 years or older with Philadelphia chromosome-negative acute lymphoblastic leukemia. DESIGN AND METHODS: Sixty patients received either continuous-infusion doxorubicin (12 mg/m(2)/day) and continuous-infusion vincristine (0.4 mg/day) on days 1-4 or pegylated liposomal doxorubicin (40 mg/m(2)) and standard vincristine (2 mg) on day 1, accompanied by dexamethasone, followed at day 28 by a second cycle, reinforced by cyclophosphamide. End-points were safety, outcome and prognostic factors. RESULTS: Myelosuppression was reduced in the pegylated liposomal doxorubicin arm with shorter severe neutropenia (P=0.05), shorter severe thrombocytopenia (P=0.03), and fewer red blood cell transfusions (P=0.04). Grade 3/4 infections and Gram-negative bacteremia were reduced in the pegylated liposomal doxorubicin arm (P=0.04 and P=0.02, respectively). There was a trend towards fewer cardiac events among the patients who received pegylated liposomal doxorubicin (1/29 versus 6/31). The complete remission rate was 82% and, with a median follow-up of 4 years, median event-free survival and overall survival were 9 and 10 months, respectively. Despite the better tolerance of pegylated liposomal doxorubicin, no differences in survival were observed between the two arms, due to trends towards more induction refractoriness (17 versus 3%, P=0.10) and a higher cumulative incidence of relapse (52% versus 32% at 2 years, P=0.20) in the pegylated liposomal doxorubicin arm. CONCLUSIONS: With the drug schedules used in this study, pegylated liposomal doxorubicin did not improve the outcome of elderly patients with acute lymphoblastic leukemia despite reduced toxicities.

International audience; INTRODUCTION: Lipiodol is used as a vector for chemoembolization or internal radiotherapy in unresectable hepatocellular carcinomas (HCCs). The aim of this study is to improve the tumoral uptake of Lipiodol by modulating membrane fluidizing agents to optimize the effectiveness of Lipiodol vectorized therapy. METHODS: The effect of dexamethasone and tamoxifen on membrane fluidity was studied in vitro by electron paramagnetic resonance applied to rat hepatocarcinoma cell line N1S1. The tumoral uptake of Lipiodol was studied in vivo on rats with HCC, which had been previously treated by dexamethasone and/or tamoxifen, after intra-arterial administration of (99m)Tc-SSS-Lipiodol. RESULTS: The two molecules studied here exhibit a fluidizing effect in vitro which appears dependent on time and dose, with a maximum fluidity obtained after 1 hr at concentrations of 20 μM for dexamethasone and 200 nM for tamoxifen. In vivo, while the use of dexamethasone or tamoxifen alone tends to lead to increased tumoral uptake of Lipiodol, this effect does not reach levels of significance. On the other hand, there is a significant increase in the tumoral uptake of (99m)Tc-SSS-Lipiodol in rats pretreated by both dexamethasone and tamoxifen, with a tumoral uptake (expressed in % of injected activity per g of tumor) of 13.57 ± 3.65% after treatment, as against 9.45 ± 4.44% without treatment (P

International audience

Purpose The prognostic impact of complete response (CR) achievement in multiple myeloma (MM) has been shown mostly in the context of autologous stem-cell transplantation. Other levels of response have been defined because, even with high-dose therapy, CR is a relatively rare event. The purpose of this study was to analyze the prognostic impact of very good partial response (VGPR) in patients treated with high-dose therapy. Patients and Methods All patients were included in the Intergroupe Francophone du Myelome 99-02 and 99-04 trials and treated with vincristine, doxorubicin, and dexamethasone (VAD) induction therapy followed by double autologous stem-cell transplantation (ASCT). Best post-ASCT response assessment was available for 802 patients. Results With a median follow-up of 67 months, median event-free survival (EFS) and 5-year EFS were 42 months and 34%, respectively, for 405 patients who achieved at least VGPR after ASCT versus 32 months and 26% in 288 patients who achieved only partial remission (P = .005). Five-year overall survival (OS) was significantly superior in patients achieving at least VGPR (74% v 61% P = .0017). In multivariate analysis, achievement of less than VGPR was an independent factor predicting shorter EFS and OS. Response to VAD had no impact on EFS and OS. The impact of VGPR achievement on EFS and OS was significant in patients with International Staging System stages 2 to 3 and for patients with poor-risk cytogenetics t(4;14) or del(17p). Conclusion In the context of ASCT, achievement of at least VGPR is a simple prognostic factor that has importance in intermediate and high-risk MM and can be informative in more patients than CR.

International audience; Stathmin-like 2 (STMN2) protein, a neuronal protein of the stathmin family, has been implicated in the microtubule regulatory network as a crucial element of cytoskeletal regulation. Herein, we describe that STMN2 expression increases at both mRNA and protein levels during osteogenesis of human mesenchymal stem cells derived from adipose tissue (hMADS cells) and bone marrow (hBMS cells), whereas it decreases to undetectable levels during adipogenesis. STMN2 protein is localized in both Golgi and cytosolic compartments. Its expression appears modulated in osteoblasts by nerve growth factor, dexamethasone or RhoA kinase inhibitor Y-27632 which are known effectors of osteogenesis. Thus STMN2 appears a novel marker of osteogenesis and osteoblast per se, that could play a role in the regulation of the adipocyte/osteoblast balance.

Summary Background Immunoglobulinic (AL) amyloidosis is a complication of plasma cell dyscrasia, characterized by widespread deposition of amyloid fibrils derived from monoclonal light chains. Cardiac amyloid is the main prognostic factor, with a median survival of six months. Cardiac transplantation in AL amyloidosis is associated with high mortality, due to disease recurrence in the allograft and systemic progression. Suppression of light chain (LC) production with chemotherapy by melphalan plus dexamethasone (MD) or high dose melphalan followed by autologous stem cell transplantation (HDM/ASCT) improves survival. However, both the indications and results of chemotherapy in patients transplanted for cardiac AL amyloidosis remain unclear. Aims To assess the outcome of cardiac transplantation and haematological therapy in patients with cardiac AL amyloidosis. Methods Eight French patients, who underwent heart transplantation for cardiac AL amyloidosis between 2001 and 2006 were studied retrospectively. Results Before transplantation, six patients received MD (n = 5) or HDM/ASCT (n = 1). Haematological remission was obtained in three patients treated with MD. In the three remaining patients, postoperative HDM/ASCT (n = 2) or allogeneic bone marrow transplantation (n = 1) resulted in haematological remission in one patient. In 2 patients not treated before transplantation, post-operative treatment with MD resulted in complete hematological remission in one. After a median follow-up of 26 months from cardiac transplantation, six patients were alive and four had sustained haematological remission, as indicated by normal serum free LC levels. Conclusion Appropriate haematological therapy, including MD, may result in a survival benefit in AL amyloidosis patients with advanced heart failure requiring transplantation.

International audience; Recurrence in the allograft and progression in other organs increase mortality after cardiac transplantation in AL amyloidosis. Survival may be improved after suppression of monoclonal light chain (LC) production following high dose melphalan and autologous stem cell transplantation (HDM/ASCT). However, because of high treatment related mortality, this tandem approach is restricted to few patients without significant extra-cardiac involvement. A diagnosis of systemic AL amyloidosis was established in a 45-year old patient with congestive heart failure related to restrictive cardiomyopathy, nephrotic syndrome, peripheral neuropathy, postural hypotension, macroglossia, and lambda LC monoclonal gammopathy. After melphalan and dexamethasone (M-Dex) therapy, which resulted in 80% reduction of serum free lambda LC, he underwent orthotopic cardiac transplantation. Two years later, he remains in a sustained hematologic remission, with no evidence of allograft or extra-cardiac amyloid accumulation. M-Dex should be considered as an alternative therapy in AL amyloid heart transplant recipients ineligible for HDM/ASCT.

International audience; Unlike classical protein extraction techniques, proteomic mapping using a selective subcellular extraction kit revealed S100A11 as a new member of the S100 protein family modulated by glucocorticoids in keratinocytes. Glucocorticoids (GC)-induced S100A11 redistribution in the "organelles and membranes" compartment. Microscopic examination indicated that glucocorticoids specifically routed cytoplasmic S100A11 toward perinuclear compartment. Calcium, a key component of skin terminal differentiation, directed S100A11 to the plasma membrane as previously reported. When calcium was added to glucocorticoids, minor change was observed at the proteomic level while confocal microscopy revealed a rapid and dramatic translocation of S100A11 toward plasma membrane. This effect was accompanied by strong nuclear condensation, loss of mitochondrial potential and DNA content, and increased high molecular weight S100A11 immunoreactivity, suggesting corticoids accelerate calcium-induced terminal differentiation. Finally, our results suggest GC-induced S100A11 relocalization could be a key step in both keratinocyte homeostasis and glucocorticoids side effects in human epidermis.

International audience

International audience; BACKGROUND: Glucocorticoids (GCs) decrease tissue mast cell (MC) number and prevent their activation via their high-affinity IgE receptor. Glucocorticoid-induced leucine zipper (GILZ) is one of the GC-induced genes, which inhibits the functions of the transcriptional activators AP-1 and NF-kappaB. GILZ appears to be a critical actor in the anti-inflammatory and immunosuppressive effects of GCs in human T lymphocytes, macrophages and dendritic cells. AIMS OF THE STUDY: We investigated whether GILZ was produced by human MCs and whether GILZ synthesis was stimulated by GCs. We also investigated whether GILZ production was modulated by (i) IL-10, because of its common immunosuppressive properties with GCs, (ii) histamine because of its pro-inflammatory properties and (iii) IL-4 and IL-5 because of their ability to favour MC survival and proliferation with SCF. METHODS: The human MC lines HMC-1 5C6 and LAD-2, and cord blood-derived MCs (CB-MCs) were cultured alone or in the presence of GCs, IL-10, histamine, IL-4 or IL-5. The expression of GILZ was evaluated by using RT-PCR, Western blotting or immunocytochemistry. RESULTS: We found that human MC lines and CB-MCs constitutively produce GILZ. We also show that GCs and IL-10 stimulate GILZ production by human MCs. Our present results indicate that histamine, IL-4 and IL-5 alone or in combination with SCF do not downregulate GILZ production by MCs. CONCLUSIONS: These results show that GCs and IL-10 stimulate GILZ production by human MCs. As GILZ mediates anti-inflammatory effects of GCs in immune cells, we speculate that GILZ could account for the deactivation of MCs by GCs and IL-10.

International audience; We report on a series of 24 patients with newly diagnosed mantle cell lymphoma treated with four to six courses of DHAP-rituximab followed by autologous stem cell transplantation for patients

International audience; Dysregulation of Hedgehog signaling can lead to several pathologies such as congenital defects and cancer. Here, we show that Hedgehog signaling is active in undifferentiated 3T3-L1 cells and decreases during adipocyte differentiation. Interestingly, this is paralleled by a decrease in Indian Hedgehog expression. We then tested if this down-regulation was sufficient to induce adipocyte differentiation. To this end, we demonstrate that the well-characterized Hedgehog inhibitor cyclopamine induced a decrease in Hedgehog signaling, similar to the one observed during adipocyte differentiation. However, cyclopamine did not induce nor potentiate adipocyte differentiation, as monitored by triglyceride staining and by the expression of several adipocyte markers: aP2, adipsin, C/EBPalpha, and Pref-1. Moreover, cyclopamine cannot substitute for other components of the differentiation medium: insulin, dexamethasone or IBMX. These results indicate that although Hedgehog signaling decreases during adipocyte differentiation, this down-regulation is not sufficient to trigger adipocyte differentiation. This suggests that Hedgehog signaling is an inadequate pharmacological target for patient suffering from syndromes associated with a decrease in fat mass, such as the ones observed in lipodystrophies.

The present work aimed to study in rats whether substitution of a low level of fish oil (FO; 2.2% of calories) into a low-fat diet (6.6% of calories from fat as peanut-rape oil or control diet) 1) has a tissue-specific effect on insulin signaling pathway and 2) prevents dexamethasone-induced alteration of insulin signaling in liver, muscle, and adipose tissue. Sixteen rats were used for study of insulin signaling, and sixteen rats received an oral glucose load (3 g/kg). Eight rats/group consumed control diet or diet containing FO over 5 wk. Four rats from each group received a daily intraperitoneal injection of saline or dexamethasone (1 mg·kg−1·day−1) for the last 5 days of feeding. In liver, FO decreased phosphatidylinositol 3′-kinase (PI 3′-kinase) activity by 54% compared with control diet. A similar result was obtained in muscle. In both liver and muscle, FO clearly amplified the effect of dexamethasone. FO did not alter early steps of insulin signaling, and in muscle GLUT4 protein content remained unaltered. In adipose tissue, FO increased PI 3′-kinase activity by 74%, whereas dexamethasone decreased it by 65%; inhibition of PI 3′-kinase activity by dexamethasone was similar in rats fed FO or control diet, and GLUT4 protein content was increased by 61% by FO. Glycemic and insulinemic responses to oral glucose were not modified by FO. In conclusion, FO increased PI 3′-kinase activity in adipose tissue while inhibiting it in liver and muscle. The maintenance of whole body glucose homeostasis suggests an important role of adipose tissue for control of glucose homeostasis.

International audience; Interleukin-6 (IL-6) is a major survival factor for multiple myeloma (MM) cells preventing apoptosis induced by dexamethasone (DEX) or chemotherapy. In all, 24 consecutive patients with MM in first-line therapy received DEX for 4 days, followed by melphalan (HDM: 140 mg/m2) and autologous stem cell transplantation (ASCT). The anti-IL-6 monoclonal antibody (mAb) (B-E8) was given till haematological recovery, starting 1 day before DEX. Results were historically compared to MM patients treated with HDM 140 and 200 mg/m2. Our results show (1) that B-E8 was able to fully neutralize IL-6 activity in vivo before and after HDM as shown by inhibition of C reactive protein (CRP) production; (2) no haematological toxicity; (3) a significant reduction of mucositis and fever; (4) a median event-free survival of 35 months and an overall survival of 68.2% at 5 years with a median follow-up of 72 months; and (5) the overall daily IL-6 production progressively increased on and after 7 days post-HDM, with the increased serum CRP levels. In the 5/24 patients with uncontrolled CRP production, a large IL-6 production was detected (320 microg/day) that could not possibly be neutralized by B-E8. These data show the feasibility to neutralize IL-6 in vivo with anti-IL-6 mAb in the context of HDM.

The original publication is available at www.springerlink.com; AIMS/HYPOTHESIS: Glucocorticoid treatments are associated with increased whole-body oxygen consumption. We hypothesised that an impairment of muscle energy metabolism can participate in this increased energy expenditure. METHODS: To investigate this possibility, we have studied muscle energetics of dexamethasone-treated rats (1.5 mg kg(-1) day(-1) for 6 days), in vivo by (31)P NMR spectroscopy. Results were compared with control and pair-fed (PF) rats before and after overnight fasting. RESULTS: Dexamethasone treatment resulted in decreased phosphocreatine (PCr) concentration and PCr:ATP ratio, increased ADP concentration and higher PCr to gamma-ATP flux but no change in beta-ATP to beta-ADP flux in gastrocnemius muscle. Neither 4 days of food restriction (PF rats) nor 24 h fasting affected high-energy phosphate metabolism. In dexamethasone-treated rats, there was an increase in plasma insulin and non-esterified fatty acid concentration. CONCLUSIONS/INTERPRETATION: We conclude that dexamethasone treatment altered resting in vivo skeletal muscle energy metabolism, by decreasing oxidative phosphorylation, producing ATP at the expense of PCr.

International audience; Terminal B-cell differentiation is a multi-step process, from short-lived plasmablasts to mature long-lived plasma cells (PC). The anti-apoptotic Bcl-2 family member Bfl-1/A1 plays a critical role in the survival of mature B cells. However, its potential involvement at the later stages of B-cell development remains highly controversial. Our aim was thus to clarify the place of Bfl-1/A1 in the biology of normal PC and in the pathogenesis of multiple myeloma (MM), the major PC dyscrasia. Using gene expression profiling and quantifiable reverse transcription polymerase chain reaction experiments, we found a similar down-regulation of Bfl-1/A1 in both normal immature plasmablasts and mature PC when compared with B cells. In myeloma cells, the level of Bfl-1/A1 was low and Bfl-1/A1 was not a nuclear factor kappaB-inducible gene. Collectively, these data demonstrate that Bfl-1/A1 is not involved in the prolonged survival of normal mature PC, and that Bfl-1/A1 deregulation is not a common oncogenic event in MM. However, overexpression of Bfl-1/A1 by retroviral transduction promoted autonomous survival of an interleukin-6-dependent myeloma cell line and rendered it less sensitive to dexamethasone. Thus, Bfl-1/A1 transduction could be an interesting tool to obtain myeloma cell lines from primary samples and to favour the in vitro generation of antibody-secreting, long-lived normal PC.

The pluripotent mesoblastic C1 cell line was used under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to drive chondrogenesis. Sequential addition of two systemic hormones, dexamethasone and triiodothyronine, permits full chondrogenic differentiation. The cell intrinsic activation of the BMP signaling pathway and Sox9 expression occurring on mesoblastic condensation is insufficient for recruitment of the progenitors. Dexamethasone-dependent Sox9 upregulation is essential for chondrogenesis. Introduction: Differentiation of lineage stem cells relies on cell autonomous regulations modulated by external signals. We used the pluripotent mesoblastic C1 cell line under serum-free culture conditions to investigate how paracrine and autocrine signals cooperate to induce differentiation of a precursor clone along the chondrogenic lineage. Materials and Methods: C1 cells, cultured as aggregates, were induced toward chondrogenesis by addition of 10−7 M dexamethasone in serum-free medium. After 30 days, dexamethasone was replaced by 10 nM triiodothyronine to promote final hypertrophic conversion. Mature and hypertrophic phenotypes were characterized by immunocytochemistry using specific antibodies against types II and X collagens, respectively. Type II collagen, bone morphogenetic proteins (BMPs), BMP receptors, Smads, and Sox9 expression were monitored by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blot, and/or Western blot analysis. Results and Conclusions: Once C1 cells have formed nodules, sequential addition of two systemic hormones is sufficient to promote full chondrogenic differentiation. In response to dexamethasone, nearly 100% of the C1 precursors engage in chondrogenesis and convert within 30 days into mature chondrocytes, which triggers a typical cartilage matrix. On day 25, a switch in type II procollagen mRNA splicing acted as a limiting step in the acquisition of the mature chondrocyte phenotype. On day 30, substitution of dexamethasone with triiodothyronine triggers the final differentiation into hypertrophic chondrocytes within a further 15 days. The chondrogenic process is supported by intrinsic expression of Sox9 and BMP family genes. Similarly to the in vivo situation, activation of Sox9 expression and the BMP signaling pathway occurred on mesoblastic condensation. After induction, BMP-activated Smad nuclear translocation persisted throughout the process until the onset of hypertrophy. After dexamethasone addition, Sox9 expression was upregulated. Dexamethasone withdrawal reversed the increase in Sox9 expression and stopped differentiation. Thus, Sox9 seems to be a downstream mediator of dexamethasone action.

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor–like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells, we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma, particularly by monocytes and stromal cells, but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration, HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines, through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti–IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether, these data identify ErbB receptors as putative therapeutic targets in multiple myeloma.

International audience; Tissue distribution of the cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (cGPDH and mGPDH) activities in jerboa (Jaculus orientalis), a hibernator, shows the highest level of enzyme activity in skeletal muscle and brown adipose tissue, respectively. The effect of hibernation on cGPDH indicates an increase of activity in all tissues examined. In contrast, hibernation decreases mGPDH activity in all tissues, except skeletal muscle. The effect of thyroid hormones on GPDH activity was tissue specific: in kidneys, cGPDH activity doubled in euthermic jerboas treated with T4. In contrast, 6-n-propyl-2-thiouracil treatment provokes an increase of enzyme activity in brown adipose tissue, liver and brain. T4 treatment leads to a 2.7-fold increase in liver mGPDH activity. 6-n-propyl-2-thiouracil treatment decreases mGPDH activity in the skeletal muscle whereas the opposite effect was observed in brain. Dexamethasone stimulates cGPDH in all tissues examined, except skeletal muscle and kidneys. In the case of mGPDH activity, this increase was observed only for brown adipose tissue and brain. Our results suggest that hibernation, thyroid hormones and dexamethasone probably play a role in the regulation of cGPDH and mGPDH activities in jerboa. Our findings confirm that these enzymes are involved in metabolic adaptation to thermal stress in Jaculus orientalis.

International audience; Peptidylglycine alpha-amidating monooxygenase (PAM), which catalazyes the two-step formation of bioactive alpha-amidated peptides from their glycine-extended precursors, has been found in H9c2 myoblasts. The expression of PAM has been evaluated in H9c2 cells. Northern blot analysis and amplification of fragments derived from rat PAM by the reverse transcription/polymerase chain reaction method has demonstrated the presence of rPAM-1, -2, -3, -3a and -3b mRNA transcripts. These forms of PAM mRNA may be generated by alternative splicing. PAM mRNA levels are increased to 160 +/- 12% of control values by treatment with dexamethasone but are unchanged during triiodothyronine incubation of the cells. PAM activity is very low, which is not comparable to the high levels found in adult atrium tissue. Western blot analysis has demonstrated 86-, 76-, and 46-kDa PAM proteins in the particulate fraction. The soluble fraction contains major PAM proteins of 110, 86, and 46 kDa. In situ hybridization studies with 35S-labeled full length RNA antisense transcripts of rat PAM-1 cDNA have localized autoradiographic grains around the nucleus. Our data clearly demonstrate PAM expression in H9c2 rat heart cells, suggesting the ability of these cardiac cells to make bioactive alpha-amidated hormones and/or neuropeptides.

International audience; Interleukin-1 (IL-1) receptors have been characterized in mouse pituitary and brain. Previous studies have demonstrated that IL-1 receptor density is high in the dentate gyrus in the hippocampus and that lipopolysaccharide (LPS) injection caused an 80% decrease in the number of hippocampal IL-1 receptors, while pituitary receptors in the anterior pituitary were unaffected. In order to investigate on the role of glucocorticoids (GC) in the control of IL-1 receptor expression in the brain and pituitary, the effect of stress, exogenous GC administration or adrenalectomy (ADX) on IL-1 receptor density was determined. Assays were achieved under basal and LPS-stimulated conditions by in situ quantitative autoradiography technique using human recombinant 125I-IL-1 alpha as a tracer. An increase of GC concentration in serum, following immobilization stress or dexamethasone (DEX) treatment (short and long term), did not modify, in the hippocampus, the density of IL-1 receptors under basal conditions or after peripheral LPS injection. On the contrary, ADX significantly decreased IL-1 receptor density in LPS-treated animals. In the anterior pituitary, a significant increase in the density of basal IL-1 receptors was observed 6 h following immobilization stress or after 7 days of DEX treatment while short-term DEX treatments are ineffective. In contrast to what was observed in the hippocampus, no changes in pituitary receptor densities were observed in ADX mice. These results indicate that hippocampal and pituitary IL-1 receptor expressions are differentially regulated by GC. Therefore, this report constitutes the first demonstration of an in vivo regulation of IL-2 receptors in the pituitary.

International audience; Glucocorticoids are known to inhibit the induced differentiation of murine erythroleukemia cells. I show here that the binding of glucocorticoid receptors to the intact nuclei of these cells is reduced by exogenous extracellular pyridoxine (1-4 mM). This reduction of glucocorticoid-receptor binding did not abrogate the inhibition by dexamethasone of the induced differentiation.

Subclones of the murine erythroleukemia cell line DS 19 with different sensitivities towards inhibition by dexamethasone (DEX) of the hexamethylene bisacetamide induced differentiation have been obtained. Scatchard analysis of DEX binding to whole cells and the time course of DEX binding to nuclei show that resistance to inhibition of erythrodifferentiation by DEX is neither associated with a reduction in specific binding of the hormone nor in a lack of activation or translocation to the nuclei of hormone receptor complexes. Hence resistance in these subclones must result from a defect at an event after binding of hormone-receptor complexes to chromatin if inhibition of differentiation is mediated by these complexes.

Summary The hexamethylene bisacetamide(HMBA) induced differentiation of murine erythroleukemia cells is inhibited by dexamethasone (DEX). We show here that addition of hemin (100 pM) to cells cultured with HMBA (4mM) and DEX (100 μM) leads to an appearance of hemoglobin containing cells. However these cells cultured with HMBA, DEX, and hemin are not reprogrammed to limited growth. DEX does not inhibit the synthesis of the nuclear protein IP25. Hence commitment to terminal differentiation is controlled by at least one heme-independant event (other than the induction of IP25) which is inhibited by DEX.

International audience; A transitory increase in ornithine decarboxylase (ODC) activity is shown not to be a prerequisite for the differentiation induced by hexamethylene bisacetamide (HMBA) in murine erythroleukemic (MEL) cells. On the contrary, conditions are described, where inhibition of the ODC activity with alpha-difluoromethyl ornithine (DFMO) stimulated the induced differentiation. Polyamine analysis demonstrated that a reduction in intracellular putrescine and spermidine occurred in MEL cells before commitment to erythrodifferentiation. The presence of DFMO increased the rapidity and the amplitude of these changes. No effect of dexamethasone on these changes in ODC activity or intracellular polyamines was observed.

International audience

International audience; Friend erythroleukemic cells can be induced to differentiate by chemicals such as dimethyl sulfoxide and hexamethylene bisacetamide (HMBA) in a dose-dependent manner. Others have shown that dexamethasone and other steroid hormones can inhibit the differentiation induced by dimethyl sulfoxide. We show here that dexamethasone has a dual mode of action on the HMBA-induced differentiation of a Friend cell line, DS19. At concentrations above 10(-10) M, dexamethasone inhibits the HMBA-induced differentiation in DS19 cells. We further found that as the concentration of HMBA is reduced, the amount of dexamethasone required to inhibit differentiation increases. Such inhibition seems to act primarily by decreasing the probability that a cell will become committed to differentiate. Besides, dexamethasone does not inhibit hemoglobin synthesis of cells which are committed in its absence. In addition, we show that sensitivity to the inhibition by dexamethasone is inversely related to the inducibility of cell populations. The second action of dexamethasone, observed at concentrations between 10(-10) and 10(-13) M, is to increase the proportion of hemoglobin-containing cells relative to that for cells cultured in its absence. The degree of this synergistic effect for induced differentiation is inversely related to the level of induction in the absence of dexamethasone and thus is best observed in the cells cultured at low levels of HMBA. We present evidence that this synergistic effect may be due to an increased viability of the induced cells and possibly an increase in the proliferative capacity of these cells.