High mTORC1 activity drives glycolysis addiction and sensitivity to G6PD inhibition in acute myeloid leukemia cells.

Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.

Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes.

OBJECTIVE: Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. METHODS: Human cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-ß (IL-1ß), tumour necrosis factor α (TNF-α) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. ·NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2(·-) production, NOX subunit expression and nitrosylation were also assessed. RESULTS: iNOS expression and nitrite (but not peroxynitrite) production were ~0.20 to ~0.12 nmol min(-1) mg proteins(-1) (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2(·-) production was restored at normoxic levels (0.21 nmol min(-1) mg of proteins(-1)). CONCLUSIONS: Our results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia.

[Evaluation of analytical and diagnostic performances of the anti-cyclic citrullinated peptides (anti-CCP) automated assay on Elecsys analyzer]. / Dosage automatisé des anticorps anti-peptides cycliques citrullinés (anti-CCP) sur l'analyseur Elecsys: validation analytique et valeur diagnostique.

Anti-cyclic citrullinated peptides (anti-CCP) are highly characteristics of rheumatoid arthritis (RA). Since 2006, anti-CCP assays have been included in both French and European recommendations. We have evaluated the analytical and clinical performances of the anti-CCP assay on the Elecsys analyzer (Roche Diagnostics). Two plasma pools (target values: 17.2 and 363.0 U/mL) and two quality controls (target values: 24.5 and 157.0 U/mL) were tested; we also analyzed three hundred plasma samples from healthy subjects (n = 86) and diseased patients (presenting with RA, non rheumatoid disorders, or undifferentiated arthritis: n = 214). Analytical performances (intra- and inter-assay precisions) and clinical performances (ROC analysis and method comparison) were evaluated. Elecsys assay was compared to Immunoscan RA(R) assay using contingency tables. Intra- and inter-assay precisions showed coefficients of variation less than 5%. ROC analysis showed an area under the curve at 0.886. Considering the value of 17 U/mL as the optimal cut-off, we found sensibility and specificity at 75% and 95%, respectively. Comparison of the Elecsys anti-CCP assay with the Immunoscan RA(R) assay showed an overall agreement of 98,3%. We conclude that the the Elecsys anti-CCP assay displayed a high precision and clinical performances comparable to that of the efficient anti-CCP assay Immunoscan RA(R).

Differential diagnosis of acute dyspnea: the value of B natriuretic peptides in the emergency department.

Congestive heart failure (CHF) is the main cause of acute dyspnea in patients presenting to an emergency department (ED) and is associated with high morbidity and mortality. B-type natriuretic peptide (BNP) is a polypeptide, released by ventricular myocytes in direct proportion to wall tension, which lowers renin-angiotensin-aldosterone activation. For the diagnosis of CHF, both BNP and the biologically inactive NT-proBNP have similar accuracy. Threshold values are higher in an elderly population, and in patients with renal dysfunction. They might also have a prognostic value. Studies have demonstrated that the use of BNP or NT-proBNP in dyspneic patients early following admission to the ED, reduced the time to discharge and total treatment cost. BNP and NT-proBNP should be available in every ED 24 h a day, because the literature strongly suggests the beneficial impact of an early appropriate diagnosis and treatment in dyspneic patients. The purpose of this review is to indicate recent developments in biomarkers of heart failure and to evaluate their impact on clinical use in the emergency setting.

Useulness of B Natriuretic Peptides and Procalcitonin in Emergency Medicine.

Congestive heart failure (CHF) is the main cause of acute dyspnea in patients presented to an emergency department (ED), and it is associated with high morbidity and mortality. B-type natriuretic peptide (BNP) is a polypeptide, released by ventricular myocytes directly proportional to wall tension, for lowering renin-angiotensin-aldosterone activation. For diagnosing CHF, both BNP and the biologically inactive NT-proBNP have similar accuracy. Threshold values are higher in elderly population, and in patients with renal dysfunction. They might have also a prognostic value. Studies demonstrated that the use of BNP or NT-proBNP in dyspneic patients early in the ED reduced the time to discharge, total treatment cost. BNP and NT-proBNP should be available in every ED 24 hours a day, because literature strongly suggests the beneficial impact of an early appropriate diagnosis and treatment in dyspneic patients.Etiologic diagnosis of febrile patients who present to an ED is complex and sometimes difficult. However, new evidence showed that there are interventions (including early appropriate antibiotics), which could reduce mortality rate in patients with sepsis. For diagnosing sepsis, procalcitonin (PCT) is more accurate than C-reactive protein. Thus, because of its excellent specificity and positive predictive value, an elevated PCT concentration (higher than 0.5 ng/mL) indicates ongoing and potentially severe systemic infection, which needs early antibiotics (e.g. meningitis). In lower respiratory tract infections, CAP or COPD exacerbation, PCT guidance reduced total antibiotic exposure and/or antibiotic treatment duration.

[The N-terminal pro-brain natriuretic peptide (NT-proBNP) assay with the Stratus CS analyzer]. / Evaluation des performances analytiques du dosage du NT-proBNP sur analyseur semi-automatisé Stratus CS.

BACKGROUND: NT-proBNP is an efficient biomarker for the evaluation, management and prognosis of patients with heart failure. METHODS: We evaluated the analytical performance of the NT-proBNP immunoenzymatic assay with the Stratus CS semi-automated analyzer in two hospital laboratories. The characteristics assessed included imprecision, functional sensitivity, linearity/recovery, interferences study, high-dose hook effect and a comparison of Acute Care(TM) pPBNP (on Stratus)CS) versus PBNP (on Dimension HM) results on patient heparinized plasma samples. RESULTS: Total imprecision reached < 5% coefficient of variation at NT-proBNP concentrations of 186-19,649 ng/L; recovery values for diluted samples were between 89.0 and 110.0 %; functional sensitivity reached 21 ng/L; there was no high-dose hook effect at concentrations up to 400,000 ng/L; hemaoglobin affected negatively but <10% the NT-proBNP assay, while bilirubin and triglycerides did not affected it more than 5%; Stratus CS results were strongly correlated with Dimension HM results (R(2)=1,00). CONCLUSION: The Stratus CS Acute Care pPBNP assay demonstrated excellent analytical performance which agreed with the Dimension HM PBNP assay. This analyzer is therefore suitable for use by low NT-proBNP test volume laboratories, and also by Emergency departments and Intensive care units.

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1beta and tumour necrosis factor-alpha.

OBJECTIVES: to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha), and to study the NADPH oxidase involvement in this production. MATERIAL AND METHODS: Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1beta and TNF-alpha, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. RESULTS: Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. CONCLUSIONS: NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4).

Serum protein oxidation in patients with rheumatoid arthritis and effects of infliximab therapy.

OBJECTIVE: To examine protein oxidation in rheumatoid arthritis (RA) and evaluate its evolution after infliximab therapy in a subgroup of patients. METHODS: Seventy-one consecutive patients with RA were included. Among them, 30 patients refractory to conventional therapy were treated with infliximab. Serum markers of oxidative stress were determined at baseline and before the infusions of infliximab at weeks 6 and 30. Baseline values were compared with those in 30 healthy volunteers. RESULTS: Mean levels of serum carbonyl groups were significantly higher in RA patients than in controls (1.29+/-0.76 versus 0.58+/-0.39 nmol/mg of protein, p<0.0001), whereas thiol levels were found to be lower (238.3+/-61.6 versus 316.5+/-54.8 micromol/L, p<0.0001). Thiol levels inversely correlated with the disease activity score (r=-0.42, p=0.004), and with CRP values (r=-0.45, p=0.001). Immunoblots showed that albumin and heavy chain immunoglobulin were oxidized more markedly than in healthy volunteers. Significantly lower levels of thiol groups were detected in patients with refractory RA disease (208.9+/-66.8 versus 264.2+/-43.0 micromol/L, p<0.0004) but concentrations of carbonyl groups were similar. Short-term treatment with infliximab significantly decreased carbonyl groups (0.97+/-0.47 nmol/mg protein, p=0.02) and increased thiol (231.2+/-48.7 micromol/L, p=0.02) levels. CONCLUSION: Our results highlight free radical protein damage in RA and a link with inflammation, as underlined by the beneficial effects of infliximab.