Impact of Next-Generation Sequencing in Diagnosis, Prognosis and Therapeutic Management of Acute Myeloid Leukemia/Myelodysplastic Neoplasms

International audienceFor decades, the diagnosis, prognosis and thus, the treatment of acute myeloblastic leukemias and myelodysplastic neoplasms has been mainly based on morphological aspects, as evidenced by the French-American-British classification. The morphological aspects correspond quite well, in a certain number of particular cases, to particular evolutionary properties, such as acute myelomonoblastic leukemias with eosinophils or acute promyelocytic leukemias. Advances in biology, particularly “classical” cytogenetics (karyotype) and molecular cytogenetics (in situ hybridization), have made it possible to associate certain morphological features with particular molecular abnormalities, such as the pericentric inversion of chromosome 16 and translocation t(15;17) in the two preceding examples. Polymerase chain reaction techniques have made it possible to go further in these analyses by associating these karyotype abnormalities with their molecular causes, CBFbeta fusion with MYH11 and PML-RAR fusion in the previous cases. In these two examples, the molecular abnormality allows us to better define the pathophysiology of leukemia, to adapt certain treatments (all-transretinoic acid, for example), and to follow up the residual disease of strong prognostic value beyond the simple threshold of less than 5% of marrow blasts, signaling the complete remission. However, the new sequencing techniques of the next generation open up broader perspectives by being able to analyze several dozens of molecular abnormalities, improving all levels of management, from diagnosis to prognosis and treatment, even if it means that morphological aspects are increasingly relegated to the background

Comparison of high-temperature conversion and equilibration methods for the determination of d31-palmitic acid oxidation in man using continuous-flow isotope ratio mass spectrometry

International audienceDuring nutritional interventions, the ingestion of d31-palmitic acid and H218O allows assessment of dietary fatty acid oxidation from cumulative 2H recovery in urine and estimation of the total body water pool (TBW) from 18O dilution. Continuous flow – isotope ratio mass spectrometry (CF-IRMS) coupled to either equilibration or high temperature conversion techniques (HTC) permits 2H and 18O enrichment measurements in biologicalfluids. Thus it was of great interest to compare these methods applied to the determination of dietary fatty acid oxidation. Linearity, accuracy and correlation between CF-equilibration and CF-HTC were first checked using 2H- and 18O- enriched water and urine samples. Urine samples from 14 subjects were then measured with both methods. 2H- and 18O- raw data were normalised against calibration lines. The final aim was to study the impact of normalised raw results on physiological data (i.e. TBW and d31-palmitate recovery). No significant difference was observed between δ18O‰ and δ2H‰ enrichment measurements depending on the analytical method used. Volumes of TBW calculated from δ18O‰ enrichments measured either with CF-equilibration or CF-HTC were not significantly different with respectively 45.2±1.0L or 45.7±1.0L (mean±sem, p=0.09). Palmitic acidoxidation results obtained from δ2H‰ enrichment measurements and TBW from CFequilibration vs CF-HTC were not significantly different (p≥0.26) with respectively 16.2±1.6% vs 16.2±1.1% at 8h, 18.7±2.0% vs 17.6±1.3% at 12h and 21.7±1.9% vs 21.5±1.3% at 3 days post-dose (mean ± sem).Thus, even if CF-HTC was preferred because it was more practical to carry out, both methods allow study of dietary lipid oxidation in man and generate similar result

B Cell Receptors and Complement Receptors Target the Antigen to Distinct Intracellular Compartments

International audienceAbstract The processing of exogenous Ags is an essential step for the generation of immunogenic peptides that will be presented to T cells. This processing relies on the efficient intracellular targeting of Ags, because it depends on the content of the compartments in which Ags are delivered in APCs. Opsonization of Ags by the complement component C3 strongly enhances their presentation by B cells and increases their immunogenicity in vivo. To investigate the role of C3 in the targeting of Ags, we compared the intracellular traffic of proteins internalized by complement receptor (CR) and B cell receptor (BCR) in B lymphocytes. Whereas both receptors are able to induce efficient Ag presentation, their intracellular pathways are different. CR ligand is delivered to compartments containing MHC class II molecules (MHC-II) but devoid of transferrin receptor and Lamp-2, whereas BCR rapidly targets its ligand toward Lamp-2-positive, late endosomal MHC-II-enriched compartments through intracellular vesicles containing transferrin receptor. CR and BCR are delivered to distinct endocytic pathways, and the kinetic evolution of the protein content of these pathways is very different. Both types of compartments contain MHC-II, but CR-targeted compartments receive less neosynthesized MHC-II than do BCR-targeted compartments. The targeting induced by CR toward compartments that are distinct from BCR-targeted compartments probably participates in C3 modulation of Ag presentation

Relation entre composition salivaire, obésité et métabolisme lipidique postprandial chez l'Homme

Relation entre composition salivaire, obésité et métabolisme lipidique postprandial chez l'Homme. 12. Journées Francophones de Nutrition (JFN

Altered postprandial lipemia and fatty acid handling in obese vs. Normal-weight men is rectified by reducing dietary fat load: A dose-response trial

International audienceBackground Postprandial hyperlipemia and altered dietary lipid beta-oxidation are now recognized as metabolic risk factors in obesity that are directly associated with dietary fat intake. The amount and size of chylomicrons are known to impact their clearance and thus possibly modulate the final partitioning of dietary fatty acids (FA) between β-oxidation and storage, which is altered in obesity. However, the detailed impact of ingested fat amount on postprandial lipemia and dietary FA fate in obese subjects remains to be elucidated. Methods In a randomized crossover study, eighteen healthy normal-weight (NW) and obese men ingested meals containing 10g or 40g of fat (typical breakfast fat contents) labeled with a mix of 13C-triglyceride tracers. Chylomicron triglyceride content and size were measured during 8h of digestion. Plasma concentrations of 13C-palmitate and 13C-oleate were also measured during 8h in parallel with their fecal loss (72h-stool collection). 13CO2 breath-test coupled to indirect calorimetry measures during 8h allowed to calculate exogenous lipid fate. Results Chylomicron triglycerides increased in all subjects according to ingested fat amount (P<0.01). After 40g of fat, obese men had delayed postprandial lipemia compared to NW men (PtimexBMI<0.0001) especially at 5–8h post-breakfast (P<0.01), with different variations in chylomicron size (PtimexBMI<0.01). This was associated with a lower appearance of tracers in plasma in obese men (P<0.01 for AUC 0–5h vs. NW), and a tendency to higher fecal excretion of 13C-oleate (P=0.1 vs. NW) after 40g of fat. However after 10g of fat, chylomicron TG and size, and tracer kinetics and fecal loss, were similar regardless of BMI. Finally, the impact of fat amount on exogenous lipid β-oxidation was different according to BMI (PdosexBMI<0.01): 39.6% of ingested lipids were β-oxidized after 40g of fat in obese (vs. 45.1% in NW) but this was increased to 53.1% in obese for 10g of fat (vs. 49.7% in NW subjects; P<0.001 vs. 40g). Conclusions Postprandial lipemia profile after a realistic high-fat load is altered in obese subjects. Although the reduction of ingested fat amount seems to normalize the postprandial fate of dietary lipids in obese men, further research is needed to understand mechanisms of altered postprandial lipid metabolism in obesity

Exercise training improves fat metabolism independent of total energy expenditure in sedentary overweight men, but does not restore lean metabolic phenotype

International audienceBACKGROUND: Obesity is a dietary fat storage disease. Although exercise prevents weight gain, effects of chronic training on dietary fat oxidation remains understudied in overweight adults. OBJECTIVE: We tested whether 2 months of training at current guidelines increase dietary fat oxidation in sedentary overweight adults like in sedentary lean adults. DESIGN: Sedentary lean (n = 10) and overweight (n = 9) men trained on a cycle ergometer at 50% VO2peak, 1 h day(-1), four times per week, for 2 months while energy balance was clamped. Metabolic fate of [d(31)] palmitate and [1-C-13] oleate mixed in standard meals, total substrate use, total energy expenditure (TEE), activity energy expenditure (AEE) and key muscle proteins/enzymes were measured before and at the end of the intervention. RESULTS: Conversely to lean subjects, TEE and AEE did not increase in overweight participants due to a spontaneous decrease in non-training AEE. Despite this compensatory behavior, aerobic fitness, insulin sensitivity and fat oxidation were improved by exercise training. The latter was not explained by changes in dietary fat trafficking but more likely by a coordinated response at the muscle level enhancing fat uptake, acylation and oxidation (FABPpm, CD36, FATP1, ACSL1, CPT1, mtGPAT). ACSL1 fold change positively correlated with total fasting (R-2 = 0.59, P<0.0001) and post-prandial (R-2 = 0.49, P = 0.0006) fat oxidation whereas mtGPAT fold change negatively correlated with dietary palmitate oxidation (R-2 = 0.40, P = 0.009), suggesting modified fat trafficking between oxidation and storage within the muscle. However, for most of the measured parameters the post-training values observed in overweight adults remained lower than the pre-training values observed in the lean subjects. CONCLUSION: Independent of energy balance and TEE, exercise training at current recommendations improved fitness and fat oxidation in overweight adults. However the improved metabolic phenotype of overweight adults was not as healthy as the one of their lean counterparts before the 2-month training, likely due to the spontaneous reduction in non-training AEE

Salivary composition in obese vs normal-weight subjects: towards a role in postprandial lipid metabolism?

In the pathophysiological context of obesity, oral exposure to dietary fat can modulate lipid digestion and absorption but underlying in-mouth mechanisms have not been clearly identified. Therefore we tested the hypothesis that salivary components related to dietary fat sensitivity would differ according to BMI and postprandial lipid metabolism in young men. Saliva was collected from 9 normal-weight (BMI=22.3±0.5 kg/m2) and 9 non-morbid obese (BMI=31.7±0.3 kg/m2) men before a 8 h-postprandial metabolic exploration test involving the consumption of a 40 g fat-meal, in which obese subjects revealed a delayed postprandial lipid metabolism. Nine salivary characteristics (flow, protein content, lipolysis, amylase, proteolysis, total antioxidant status, lysozyme, lipocalin 1, carbonic anhydrase-VI) were investigated. We show that under fasting conditions, salivary lipolysis was lower in obese vs normal-weight subjects, while proteolysis and CAVI were higher. We reveal through multivariate and Mann-Whitney analysis that differences in fasting salivary lipolysis and proteolysis between both groups are related to differences in postprandial lipid metabolism including exogenous fatty acid absorption and beta-oxidation. These results suggest a potential role of salivary composition on postprandial lipid metabolism and bring novel causal hypotheses on the links between salivary composition, sensitivity to dietary fat oral income and postprandial lipid metabolism according to BMI.International Journal of Obesity accepted article preview online, 28 April 2015. doi:10.1038/ijo.2015.71

Exercise performed immediately after fructose ingestion enhances fructose oxidation and suppresses fructose storage

Background: Exercise prevents the adverse effects of a high-fructose diet through mechanisms that remain unknown. Objective: We assessed the hypothesis that exercise prevents fructose-induced increases in very-low-density lipoprotein (VLDL) triglycerides by decreasing the fructose conversion into glucose and VLDL-triglyceride and fructose carbon storage into hepatic glycogen and lipids. Design: Eight healthy men were studied on 3 occasions after 4 d consuming a weight-maintenance, high-fructose diet. On the fifth day, the men ingested an oral 13C-labeled fructose load (0.75 g/kg), and their total fructose oxidation (13CO2 production), fructose storage (fructose ingestion minus 13C-fructose oxidation), fructose conversion into blood 13C glucose (gluconeogenesis from fructose), blood VLDL-13C palmitate (a marker of hepatic de novo lipogenesis), and lactate concentrations were monitored over 7 postprandial h. On one occasion, participants remained lying down throughout the experiment [fructose treatment alone with no exercise condition (NoEx)], and on the other 2 occasions, they performed a 60-min exercise either 75 min before fructose ingestion [exercise, then fructose condition (ExFru)] or 90 min after fructose ingestion [fructose, then exercise condition (FruEx)]. Results: Fructose oxidation was significantly (P < 0.001) higher in the FruEx (80% ± 3% of ingested fructose) than in the ExFru (46% ± 1%) and NoEx (49% ± 1%). Consequently, fructose storage was lower in the FruEx than in the other 2 conditions (P < 0.001). Fructose conversion into blood 13C glucose, VLDL-13C palmitate, and postprandial plasma lactate concentrations was not significantly different between conditions. Conclusions: Compared with sedentary conditions, exercise performed immediately after fructose ingestion increases fructose oxidation and decreases fructose storage. In contrast, exercise performed before fructose ingestion does not significantly alter fructose oxidation and storage. In both conditions, exercise did not abolish fructose conversion into glucose or its incorporation into VLDL triglycerides. This trial was registered at clinicaltrials.gov as NCT01866215

13C tracer recovery in human stools after digestion of a fat-rich meal labelled with [1,1,1-13C3]tripalmitin and [1,1,1-13C3]triolein

International audienceLipid metabolism studies focus mainly on oxidation and storage but rarely on faecal elimination, which is needed to assess total lipid distribution during the postprandial period. The purpose of the present work was to set up and validate the analysis of lipid tracers in stools, with an aim of later using this methodology in studies of postprandial lipid tracer metabolism. Eight subjects received a mixture of [1,1,1-(13)C3]tripalmitin and [1,1,1-(13)C3]triolein with a fat-rich meal. The nature and amounts of (13)C lipids excreted in stools during 3 days post-dose were determined by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analysis of fatty acid methyl esters (FAMEs) from total fatty acid (TFA), free fatty acid (FFA) and triacylglycerol (TAG) fractions. The results were expressed as the Cumulative Tracer Recovery of the administered dose (CTR%). The quantities and labelling of FAMEs were higher in FFA than in TAG, indicating that label loss was not due to a lack of digestive lipase activity. The labelling was higher for C16:0 than for C18:1. The CTRs were 7.03 ± 0.77% and 6.87 ± 0.91%, respectively, in TFA and FFA for [1-(13)C] C16:0, while they were 0.60 ± 0.15% and 0.51 ± 0.11% for [1-(13)C] C18:1 (mean ± sem). By studying the kinetics of lipid excretion from subjects, two groups emerged. The first one showed rapid excretion in stool #1, whereas the second showed slower excretion in stools #2-#3. A significant difference was found in the FFA in stool #1 for C16:0 (p < 0.01) and C18:1 (p < 0.05). Individual excretion kinetics showed marked variability. Nevertheless, the CTR over the 3-day study period was substantial and homogenous for all subjects. These results confirm that the assessment of faecal elimination is of great importance when establishing total lipid distribution during the postprandial period and validate the analysis of cumulative tracer loss during 72 h post-tracer ingestion