[PREVALENCE OF SNUFF IN PATIENTS TREATED IN HOSPITAL CONSULTATION DAY AND RHEUMATOLOGY NURSING]. / Prevalencia de consumo de tabaco en pacientes tratados en el Hospital de Día y Consulta de Enfermería de Reumatología.

OBJECTIVES: To assess the prevalence of tobacco consumption in patients with inflammatory arthritis treated in our department and to raise awareness against tobacco in order to reduce the maximum number of active smokers. METHOD: A tobacco consumption survey was conducted to patients with inflammatory arthritis treated at the department of Rheumatology. Variables assessed: demographics, diagnosis, treatment, and current smoking. In smokers and former smokers patients: onset age of smoking, number of cigarettes per day, time exposure to tobacco and if they were active smokers before the diagnosis of their disease. All patients were also asked if received information about tobacco as a risk factor for inflammatory disease; and current to the active smokers if they wanted to stop. Awareness activities against tobacco were conducted. RESULTS: Patients were included 198. The most prevalent diagnosis was rheumatoid arthritis (58.1%). Most patients were treated with biological therapy. Fifty percent of patients were non-smokers, 31% former smokers and 19% active smokers. Ninety-two percent of smokers and 89% former smokers already smoked before diagnosis of the disease. Thirty-five percent of all patients had received information about the risks of tobacco. Eighty percent of current smokers wanted to stop smoking. CONCLUSIONS: Active smoking was reported in 19% of patients with inflammatory arthropathies visited in our Arthritis. Department patients were willing to receive tobacco education. These results indicate the need to provide advice against tobacco in a systematic and structured manner.

Prevalencia de consumo de tabaco: en pacientes tratados en el hospital de día y consulta de enfermería de reumatología / Prevalence of snuff in patients treated in hospital consultation day and rheumatology nursing

Objetivos. Evaluar la prevalencia del consumo de tabaco de los pacientes con artropatías inflamatorias tratados en nuestro servicio. Realizar educación antitabáquica con la finalidad de reducir al máximo el número de fumadores activos. Método. Se realizó una encuesta de consumo de tabaco a los pacientes con artropatías inflamatorias tratados en el servicio de Reumatología. Variables evaluadas: demográficas, diagnóstico, tratamiento actual y hábito tabáquico. En los pacientes fumadores y exfumadores: edad inicio tabaco, número de cigarrillos día, tiempo exposición al tabaco y si eran fumadores activos antes del diagnóstico de su enfermedad. Asimismo, se preguntó a todos los pacientes si tenían información de que el tabaco era un factor de riesgo para su enfermedad, EN PACIENTES TRATADOS EN EL HOSPITAL DE DÍA Y CONSULTA DE ENFERMERÍA DE REUMATOLOGÍA y a los fumadores activos si querían dejar de fumar. Se realizó educación antitabáquica. Resultados. Se incluyeron 198 pacientes. El diagnóstico más prevalente fue la artritis reumatoide (58.1 %). La mayoría estaban tratados con terapia biológica. El 50 % de los pacientes no era fumador, el 31 % exfumador y el 19 % fumador activo. El 92 % de los pacientes fumadores y el 89 % de los exfumadores fumaba antes del diagnóstico de la enfermedad. El 35 % refería haber recibido información sobre el riesgo del hábito tabáquico y el 81 % de los fumadores activos quería dejar de fumar. Conclusiones. Un 19 % de pacientes con artropatías inflamatorias visitados en la Unidad de Artritis son fumadores activos. La mayoría de los pacientes han sido receptivos a la educación antitabáquica. Los resultados justifican la indicación de realizar consejo antitabáquico de forma sistémica y estructurada (AU)

Objectives. To assess the prevalence of tobacco consumption in patients with inflammatory arthritis treated in our department and to raise awareness against tobacco in order to reduce the maximum number of active smokers. Method. A tobacco consumption survey was conducted to patients with inflammatory arthritis treated at the department of Rheumatology. Variables assessed: demographics, diagnosis, treatment, and current smoking. In smokers and former smokers patients: onset age of smoking, number of cigarettes per day, time exposure to tobacco and if they were active smokers before the diagnosis of their disease. All patients were also asked if received information about tobacco as a risk factor for inflammatory disease; and current to the active smokers if they wanted to stop. Awareness activities against tobacco were conducted. Results. Patients were included 198. The most prevalent diagnosis was rheumatoid arthritis (58.1 %). Most patients were treated with biological therapy. Fifty percent of patients were nonsmokers, 31 % former smokers and 19 % active smokers. Ninety-two percent of smokers and 89 % former smokers already smoked before diagnosis of the disease. Thirty-five percent of all patients had received information about the risks of tobacco. Eighty percent of current smokers wanted to stop smoking. Conclusions. Active smoking was reported in 19 % of patients with inflammatory arthropathies visited in our Arthritis. Department patients were willing to receive tobacco education. These results indicate the need to provide advice against tobacco in a systematic and structured manner (AU)

Dietary exposure to a low dose of pesticides alone or as a mixture: the biological metabolic fingerprint and impact on hematopoiesis.

Consumers are exposed to a mixture of pesticides through their food intake. These compounds are considered risk factors for human health, and the impact of dietary exposure to low doses of pesticide mixtures remains poorly understood. For this study we developed a mouse model to mimic consumer exposure in order to compare the effect of pesticides both alone or combined at doses corresponding to their Acceptable Daily Intake value. Female mice were exposed to pesticides throughout gestation and lactation. After weaning pups were fed the same pesticide-enriched diet their mothers had received for an additional 11 weeks. A metabonomic approach using (1)H NMR-based analysis of plasma showed that exposure to each pesticide produced a specific metabolic fingerprint in adult offspring. Discriminant metabolites between groups were glucose or lactate, choline, glycerophosphocholine and phosphocholine. Interestingly, metabolite differences were observed as early as weaned animals that had not yet been directly exposed themselves. Studies of the hematopoietic system revealed that dietary exposure to one particular pesticide, endosulfan, produced a significant decrease in red blood cell and hemoglobin levels, consistent with hemolytic anemia. Moreover, cell signaling profiles of bone marrow progenitors were also clearly affected. Expression of cell signaling proteins such as P35, CDC27, FAK, P38 MAP kinase, calcineurin and caspase as well as proteins involved in the stability or structure of the cytoskeleton (vinculin, MAP2) was changed upon dietary exposure to pesticides. Finally, we found that dietary exposure to a mixture of pesticides had effects that differed and were often lesser or equal to that of the most efficient pesticide (endosulfan), suggesting that the effect of pesticide mixtures cannot always be predicted from the combined effects of their constituent compounds.

A phase Ib GOELAMS study of the mTOR inhibitor RAD001 in association with chemotherapy for AML patients in first relapse.

The mTORC1 signaling pathway is constitutively activated in almost all acute myelogenous leukemia (AML) patients. We conducted a phase Ib trial combining RAD001 (everolimus), an allosteric inhibitor of mTORC1, and conventional chemotherapy, in AML patients under 65 years of age at first relapse (clinical trial NCT 01074086). Increasing doses of RAD001 from 10-70 mg were administrated orally on days 1 and 7 (d1 and d7) of a 3+7 daunorubicin+cytarabine conventional induction chemotherapy regimen. Twenty-eight patients were enrolled in this trial. The treatment was well tolerated with <10% toxicity, mainly involving the gastrointestinal tract and lungs. In this phase Ib trial, the RAD001 maximum tolerated dose was not reached at 70 mg. Sixty-eight percent of patients achieved CR, of which 14 received a double induction. Eight subsequently were intensified with allogeneic-stem cell transplant. Strong plasma inhibition of P-p70S6K was observed after RAD001 administration, still detectable at d7 (d7)at the 70 mg dosage. CR rates in patients with RAD001 areas under or above the curve median were 53% versus 85%. A 70 mg dose of RAD001 at d1 and d7 of an induction chemotherapy regimen for AML has acceptable toxicity and may improve treatment.

The short form of RON is expressed in acute myeloid leukemia and sensitizes leukemic cells to cMET inhibitors.

Several receptor tyrosine kinases (TKs) are involved in the pathogenesis of acute myeloid leukemia (AML). Here, we have assessed the expression of the Recepteur d'Origine Nantais (RON) in leukemic cell lines and samples from AML patients. In a series of 86 AML patients, we show that both the full length and/or the short form (sf) of RON are expressed in 51% and 43% of cases, respectively. Interestingly, sfRON is not expressed in normal CD34+ hematopoietic cells and induces part of its oncogenic signaling through interaction with the Src kinase Lyn. sfRON-mediated signaling in leukemic cells also involves mTORC1, the proapoptotic bcl2-family member, BAD, but not the phosphatidylinositol 3-kinase/Akt pathway. Furthermore, the expression of sfRON was specifically downregulated by 5-azacytidine (AZA). Conversely, AZA could induce the expression of sfRON in sfRON-negative leukemic cells suggesting that the activity of this drug in AML and myelodysplastic syndromes could involve modulation of TKs. cMET/RON inhibitors exhibited an antileukemic activity exclusively in AML samples and cell lines expressing sfRON. These results might support clinical trials evaluating cMET/RON inhibitors in AML patients expressing sfRON.

Specific small nucleolar RNA expression profiles in acute leukemia.

Apart from microRNAs, little is known about the regulation of expression of non-coding RNAs in cancer. We investigated whether small nucleolar RNAs (snoRNAs) accumulation displayed specific signatures in acute myeloblastic and acute lymphoblastic leukemias. Using microarrays and high-throughput quantitative PCR (qPCR), we demonstrate here that snoRNA expression patterns are negatively altered in leukemic cells compared with controls. Interestingly, a specific signature was found in acute promyelocytic leukemia (APL) with ectopic expression of SNORD112-114 snoRNAs located at the DLK1-DIO3 locus. In vitro experiments carried out on APL blasts demonstrate that transcription of these snoRNAs was lost under all-trans retinoic acid-mediated differentiation and induced by enforced expression of the PML-RARalpha fusion protein in negative leukemic cell lines. Further experiments revealed that the SNORD114-1 (14q(II-1)) variant promoted cell growth through cell cycle modulation; its expression was implicated in the G0/G1 to S phase transition mediated by the Rb/p16 pathways. This study thus reports three important observations: (1) snoRNA regulation is different in normal cells compared with cancer cells; (2) a relationship exists between a chromosomal translocation and expression of snoRNA loci; and (3) snoRNA expression can affect Rb/p16 cell cycle regulation. Taken together, these data strongly suggest that snoRNAs have a role in cancer development.

RET fusion genes are associated with chronic myelomonocytic leukemia and enhance monocytic differentiation.

Myeloproliferative neoplasms are frequently associated with aberrant constitutive tyrosine kinase (TK) activity resulting from chimaeric fusion genes or point mutations such as BCR-ABL1 or JAK2 V617F. We report here the cloning and functional characterization of two novel fusion genes BCR-RET and FGFR1OP-RET in chronic myelomonocytic leukemia (CMML) cases generated by two balanced translocations t(10;22)(q11;q11) and t(6;10)(q27;q11), respectively. The two RET fusion genes leading to the aberrant activation of RET, are able to transform hematopoietic cells and skew the hematopoietic differentiation program towards the monocytic/macrophage lineage. The RET fusion genes seem to constitutively mimic the same signaling pathway as RAS mutations frequently involved in CMML. One patient was treated with Sorafenib, a specific inhibitor of the RET TK function, and demonstrated cytological and clinical remissions.

Sex differences in the GSK3ß-mediated survival of adherent leukemic progenitors.

Therapeutic resistance of acute myeloid leukemia stem cells, enriched in the CD34(+)38(-)123(+) progenitor population, is supported by extrinsic factors such as the bone marrow niche. Here, we report that when adherent onto fibronectin or osteoblast components, CD34(+)38(-)123(+) progenitors survive through an integrin-dependent activation of glycogen synthase kinase 3ß (GSK3ß) by serine 9-dephosphorylation. Strikingly, GSK3ß-mediated survival was restricted to leukemic progenitors from female patients. GSK3ß inhibition restored sensitivity to etoposide, and impaired the clonogenic capacities of adherent leukemic progenitors from female patients. In leukemic progenitors from female but not male patients, the scaffolding protein RACK1, activated downstream of α(5)ß(1)-integrin engagement, was specifically upregulated and controlled GSK3ß activation through the phosphatase protein phosphatase 2A (PP2A). In a mirrored manner, survival of adherent progenitors (CD34(+)38(-)) from male but not female healthy donors was partially dependent on this pathway. We conclude that the GSK3ß-dependent survival pathway might be sex-specific in normal immature population and flip-flopped upon leukemogenesis. Taken together, our results strengthen GSK3ß as a promising target for leukemic stem cell therapy and reveal gender differences as a new parameter in anti-leukemia therapy.

Gender-linked haematopoietic and metabolic disturbances induced by a pesticide mixture administered at low dose to mice.

Defining the impact on health of exposure to a low-dose pesticide mixture via food intake is a topical question since epidemiological studies suggest that this may increase the risk of pathologies and particularly haematopoietic malignancies. Here we investigated on the haematopoietic system of mice, the effect of a mixture of six pesticides frequently ingested through the intake of fruits and vegetables produced in France (alachlor, captan, diazinon, endosulfan, maneb, mancozeb). The mixture was administered repeatedly by gavage to mice for 4 weeks at levels derived from the human Acceptable Daily Intake (ADI) level adapted to the mean weight of mice. Using a NMR-based metabonomic approach, we show that this treatment led to specific gender-linked variations in the level of hepatic metabolites involved in oxidative stress and in the regulation of glucose metabolism, indicating a metabolic signature for this repeated administration. Interestingly, exposure to the low-dose pesticide mixture induced significant changes in the blood cell counts with modifications in the clonogenic and differentiating capacities of haematopoietic progenitors showing abnormalities in the granulocytic and monocytic lineages in female and male mice, respectively. From a molecular point of view, the changes induced by the pesticide treatment correlated with modifications of the PI 3-kinase/Akt signalling pathway, the tyrosine kinase Pyk2 and the c-Myc transcription factor, which are involved in the balance between self-renewal and differentiation of haematopoietic stem cells. Our results point to a significant effect of a very low dose of a mixture of commonly used pesticides on mice metabolism and haematopoietic system with major differences between males and females.

The level of AKT phosphorylation on threonine 308 but not on serine 473 is associated with high-risk cytogenetics and predicts poor overall survival in acute myeloid leukaemia.

The phosphoinositide 3-kinase/Akt pathway is an important signalling pathway governing cell survival and proliferation in acute myeloid leukaemia (AML). As full activation of Akt requires phosphorylation on both threonine 308 (Thr308) and serine 473 (Ser473) residues, we studied the level of phosphorylation on the both sites in 58 AML samples by flow cytometry. The ratio of the mean fluorescence intensity of Thr308 and Ser473 represented a continuum ranging from 0.3 to 5.6 and from 0.4 to 2.87, respectively. There were no significant correlations between age, gender, French-American-British classification, leukocytosis, FLT3-ITD and Akt phosphorylation. However, the level of phosphorylation on Thr308, but not on Ser473, was significantly correlated with high-risk karyotype. Thr308(high) patients had significantly shorter overall survival (11 vs 47 months; P=0.01), event-free survival (9 vs 26 months; P=0.005) and relapse-free survival (10 months vs not reached; P=0.02) than Thr308(low) patients. Neither screening for AKT1 E17K mutation nor changes in the level of PTEN expression and phosphorylation could be linked to increased phosphorylation on Thr308 in high-risk cytogenetic AML cells. However, PP2A activity was significantly reduced in high-risk samples compared with intermediate-risk samples. Moreover, the specific Akt inhibitor, Akti-1/2, inhibited cell proliferation and clonogenic properties, and induced apoptosis in AML cells with high-risk cytogenetics, suggesting that Akt may represent a therapeutic target in high-risk AML.

G2/M checkpoint stringency is a key parameter in the sensitivity of AML cells to genotoxic stress.

Acute myeloid leukemia (AML) cells exposed to genotoxic agents arrest their cell cycle at the G2/M checkpoint and are inherently chemoresistant. To understand the mechanism of this chemoresistance, we compared the ability of immature CD34+ versus mature CD34- AML cell lines (KG1a and U937, respectively) to recover from a DNA damage-induced cell cycle checkpoint in G2. Here, we report that KG1a cells have a more stringent G2/M checkpoint response than U937 cells. We show that in both cell types, the CDC25B phosphatase participates in the G2/M checkpoint recovery and that its expression is upregulated. Furthermore, we show that CHK1 inhibition by UCN-01 in immature KG1a cells allows checkpoint exit and induces sensitivity to genotoxic agents. Similarly, UCN-01 treatment potentializes genotoxic-induced inhibition of colony formation efficiency of primary leukemic cells from AML patients. Altogether, our results demonstrate that checkpoint stringency varies during the maturation process and indicate that targeting checkpoint mechanisms might represent an attractive therapeutic opportunity for chemoresistant immature AML cells.

Human telomerase is regulated by erythropoietin and transforming growth factor-beta in human erythroid progenitor cells.

Telomerase catalytic subunit (hTERT) exerts important cellular functions including telomere homeostasis, genetic stability, cell survival and perhaps differentiation. However, the nature of external or internal signals, which regulate hTERT expression in tissues, remains poorly understood. Thus, whereas it has been described that hTERT gene is regulated along the differentiation of primitive myeloid progenitors, the effect of specific cytokines on telomerase expression in each myeloid lineage is currently unknown. Based on these considerations, we have investigated hTERT expression in erythroid cells treated with erythropoietin (EPO) and transforming growth factor beta (TGFbeta), as putative positive and negative regulators, respectively. We describe here that EPO activates hTERT gene transcription in in vitro-expanded primary erythroid precursors as well as in UT7 erythroleukemia cells. In UT7 cells, this study shows also that EPO acts through a JAK2/STAT5/c-myc axis. In contrast, TGFbeta blocks EPO signaling downstream of c-myc induction through a Smad3-dependent mechanism. Finally, hTERT appears to be efficiently regulated by EPO and TGFbeta in an opposite way in erythropoietic cells, arguing for a role of telomerase in red blood cell production.

Expression of beta-catenin by acute myeloid leukemia cells predicts enhanced clonogenic capacities and poor prognosis.

Activation of the Wnt/beta-catenin pathway has recently been shown to be crucial to the establishment of leukemic stem cells in chronic myeloid leukemia. We sought to determine whether beta-catenin was correlated to clonogenic capacity also in the acute myeloid leukemia (AML) setting. Eighty-two patients were retrospectively evaluated for beta-catenin expression by Western blot. beta-Catenin was expressed (although at various protein levels) in 61% of patients, and was undetectable in the remaining cases. In our cohort, beta-catenin expression was correlated with the clonogenic proliferation of AML-colony forming cells (AML-CFC or CFU-L) in methylcellulose in the presence of 5637-conditioned medium, and more strikingly with self-renewing of leukemic cells, as assessed in vitro by a re-plating assay. In survival analyses, beta-catenin appeared as a new independent prognostic factor predicting poor event-free survival and shortened overall survival (both with P<0.05). Furthermore, variations in beta-catenin protein levels were dependent on post-transcriptional mechanisms involving the Wnt/beta-catenin pathway only in leukemic cells. Indeed, beta-catenin negative leukemic cells were found to increase beta-catenin in response to Wnt3a agonist in contrast to normal counterparts. Altogether, our data pave the way to the evaluation of Wnt pathway inhibition as a new rationale for eradicating the clonogenic pool of AML cells.

A crosstalk between the Wnt and the adhesion-dependent signaling pathways governs the chemosensitivity of acute myeloid leukemia.

Relapses following chemotherapy are a major hindrance to patients' survival in acute myeloid leukemia (AML). To investigate the role of the hematopoietic niche in the chemoresistance of leukemic cells, we examined two pathways: one mediated by adhesion molecules/integrins, and the other by soluble factors of the morphogen Wnt pathway. In our study, both the adhesion of leukemic blasts to fibronectin and the addition of Wnt antagonists induced, independently, resistance of AML cells to daunorubicin in a cell survival assay. Using pharmacological inhibitors and siRNA, we showed that both resistance pathways required the activity of the glycogen synthase kinase 3beta (GSK3beta). Moreover, the AML cell protection downstream of GSK3beta was mediated by NF-kappaB. A link between the adhesion and the Wnt pathway was found, as adhesion of U937 on human osteoblasts, a component of the hematopoietic niche, triggered the secretion of the Wnt antagonist sFRP-1 and supported resistance to daunorubicin. The osteoblast-conditioned medium could also confer chemoresistance to U937 cells cultured in suspension, and this cell protective effect was abrogated after depletion of sFRP-1. In the context of this potential double in vivo resistance, modulators of the common signal GSK3beta and of its target NF-kappaB could represent important novel therapeutic tools.

TNFalpha stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells.

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.

[Hemophagocytic syndrome associated with neutropenia after chemotherapy]. / Syndromes hémophagocytaires en phase d'aplasie post chimiothérapie.

MATERIAL AND METHODS: This retrospective study reports 15 cases of hemophagocytic syndrome in children treated in our department during a eight-year period. RESULTS: Underlying diseases were acute lymphoblastic leukemia (n = 8) acute myeloblastic leukemia (n = 6) and Burkitt lymphoma (n = 1). Hemophagocytic syndrome was suspected after chemotherapy, in case of an unusual prolonged febrile neutropenia (n = 14) or isolated thrombocytopenia (n = 1). That fever was associated with cutaneous, pulmonary, hematologic, digestive and cardiac signs. Biological disorders included hypoprotidemia, hyponatremia, increased liver enzymes and fibrinopenia. Thrombocytopenia was observed in all patients and was associated with neutropenia for 14 of them. Diagnosis of hemophagocytic syndrome was always confirmed by bone marrow aspiration (infiltration with activated macrophages). Infection was documented in eight children. The treatment of hemophagocytic syndrome relied on steroids and resolution of symptoms occurred within three days of therapy. No recurrence of hemophagocytic syndrome was observed with a median follow up of two years and a half. CONCLUSION: Such complication should be suspected in cases of prolonged febrile neutropenia and/or thrombocytopenia, and confirmed by bone marrow aspiration. Indeed, steroid therapy is effective and chemotherapy can be then pursued.

Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I-T-cell interactions.

Neonatal injection of semiallogeneic spleen cells in BALB/c mice induces a self-limited state of chimerism that promotes the differentiation of donor-specific CD4 T cells toward the Th2 phenotype. Here we show that injection of spleen cells from beta2-microglobulin-deficient (BALB/c x C57BL/6) F1 mice into BALB/c newborns with a disrupted beta2-microglobulin (beta2m) gene results in a lethal lymphoproliferative disorder associated with uncontrolled Th2 response, long-term persistence of donor B cells, and sustained blood eosinophilia. Autoimmune manifestations are also enhanced and characterized by a severe autoantibody-mediated glomerulonephritis. Histological examination of the spleen shows a hyperplasia of periarteriolar lymphoid sheaths, with accumulation of eosinophils and basophils, and variable degree of fibrosis. Perivascular lymphoid infiltrates with eosinophils are also found in the lung and are correlated with disease severity. Such abnormalities are almost absent using beta2m-sufficient mice. These data demonstrate that induction of lymphoid chimerism in the absence of MHC class I-T-cell interactions results in a lethal form of host-versus-graft disease that represents a unique model of Th2-dependent chronic inflammatory disease associated with an hypereosinophilic syndrome in mice.