Comparative proximity biotinylation implicates the small GTPase RAB18 in sterol mobilization and biosynthesis.

Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.

Effects of Repeated High-Intensity Effort Training or Repeated Sprint Training on Repeated High-Intensity Effort Ability and In-Game Performance in Professional Rugby Union Players.

ABSTRACT: Glaise, P, Rogowski, I, and Martin, C. Effects of repeated high-intensity effort training or repeated sprint training on repeated high-intensity effort ability and in-game performance in professional rugby union players. J Strength Cond Res 38(5): 932-940, 2024-This study investigated the effects of repeated high-intensity efforts (RHIE) training compared with repeated sprint exercise (RSE) training on RHIE ability (RHIEa) and in-game performance in professional rugby union players. Thirty-nine, male, professional, rugby union players were randomly assigned to 3 training groups (RHIE training, RSE training, and control). Repeated high-intensity effort ability and high-intensity effort characteristics (including sprints, acceleration, and contact efforts) during official games were measured before and after a 10-week specific (RHIE, RSE, or control) training period. The results of this study showed that concerning RHIEa, both the RHIE and RSE training significantly increased the players' average sprint velocity ( p < 0.001, d = -0.39 and p < 0.001, d = -0.53 respectively), average sled push velocity (ASPV; p < 0.001, d = -0.81 and p = 0.017, d = -0.48 respectively), and RHIE score ( p < 0.001, d = -0.72 and p < 0.001, d = -0.60 respectively). Repeated high-intensity effort training trended in a smaller increase in average sprint velocity than RSE training, a larger increase in ASPV, and a similar increase in RHIE score. Concerning in-game high-intensity efforts, both the RHIE and RSE training produced significant improvements in the number of sprints ( p = 0.047, d = -0.28 and p < 0.001, d = -0.47 respectively), total distance ( p < 0.001, d = -0.50 and p = 0.002, d = -0.38 respectively), the number of accelerations ( p < 0.001, d = -0.37 and p = 0.003, d = -0.32 respectively), and contact rate ( p < 0.001, d = -0.97 and p = 0.020, d = -0.28 respectively). Conversely, the magnitude of the increase in contact rate was almost twice as high in RHIE compared with RSE training. To conclude, the findings of this study were that both RSE and RHIE training are effective methods for developing RHIEa and in-game high-intensity efforts in professional rugby union. In practical applications, as the gains in certain abilities and game performance data differed depending on the training method chosen, we suggest that coaches choose the most appropriate method according to the profile of the players, their position, and the style of play they want to develop.

High levels of eicosanoids and docosanoids in the lungs of intubated COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 is responsible for coronavirus disease 2019 (COVID-19). While COVID-19 is often benign, a subset of patients develops severe multilobar pneumonia that can progress to an acute respiratory distress syndrome. There is no cure for severe COVID-19 and few treatments significantly improved clinical outcome. Dexamethasone and possibly aspirin, which directly/indirectly target the biosynthesis/effects of numerous lipid mediators are among those options. Our objective was to define if severe COVID-19 patients were characterized by increased bioactive lipids modulating lung inflammation. A targeted lipidomic analysis of bronchoalveolar lavages (BALs) by tandem mass spectrometry was done on 25 healthy controls and 33 COVID-19 patients requiring mechanical ventilation. BALs from severe COVID-19 patients were characterized by increased fatty acids and inflammatory lipid mediators. There was a predominance of thromboxane and prostaglandins. Leukotrienes were also increased, notably LTB4 , LTE4 , and eoxin E4 . Monohydroxylated 15-lipoxygenase metabolites derived from linoleate, arachidonate, eicosapentaenoate, and docosahexaenoate were also increased. Finally yet importantly, specialized pro-resolving mediators, notably lipoxin A4 and the D-series resolvins, were also increased, underscoring that the lipid mediator storm occurring in severe COVID-19 involves pro- and anti-inflammatory lipids. Our data unmask the lipid mediator storm occurring in the lungs of patients afflicted with severe COVID-19. We discuss which clinically available drugs could be helpful at modulating the lipidome we observed in the hope of minimizing the deleterious effects of pro-inflammatory lipids and enhancing the effects of anti-inflammatory and/or pro-resolving lipid mediators.

Effect of hip and knee joint angles on resting hamstring muscles rigidity in men and women.

PURPOSE: Hamstring muscle strains are one of the most common injuries in sports practice, for both men and women. However, sex disparities in the rate of muscle injuries have been observed. As these muscular injuries usually occur at long muscle length, this study aimed to determine the effect of sex on hamstring muscles' resting rigidity under different stretching conditions. METHODS: The shear wave speed (SWS) of resting hamstring muscles was measured in 12 men and 12 women in different hip and knee positions (hip extended with knee flexed, hip flexed with knee extended, both joints extended and both joints flexed). RESULTS: Combining all the positions, the SWS of the semitendinosus was higher in men than in women (2.96 vs. 2.71 m.s-1). Regardless of sex, a significant rise in SWS was systematically observed when the semimembranosus was stretched (1.86, 2.37, 2.76 and 4.39 m.s-1) but it was neither the case for the semitendinosus (p = 0.82) nor for the biceps femoris (p = 0.50). Finally, differences in SWS among the hamstring muscles were only observed at the longest muscle length, with greater SWS values for the semimembranosus and semitendinosus in comparison with the biceps femoris (4.39 and 4.12 vs. 3.38 m.s-1 respectively). CONCLUSION: In conclusion, a sex difference was only observed in the resting semitendinosus rigidity. Independently of sex, the increase in resting hamstring muscles SWS with stretch was muscle specific.

Endocannabinoid hydrolysis inhibition unmasks that unsaturated fatty acids induce a robust biosynthesis of 2-arachidonoyl-glycerol and its congeners in human myeloid leukocytes.

The endocannabinoid (eCB) 2-arachidonoyl-gycerol (2-AG) modulates immune responses by activating cannabinoid receptors or through its multiple metabolites, notably eicosanoids. Thus, 2-AG hydrolysis inhibition might represent an interesting anti-inflammatory strategy that would simultaneously increase the levels of 2-AG and decrease those of eicosanoids. Accordingly, 2-AG hydrolysis inhibition increased 2-AG half-life in neutrophils. Under such setting, neutrophils, eosinophils, and monocytes synthesized large amounts of 2-AG and other monoacylglycerols (MAGs) in response to arachidonic acid (AA) and other unsaturated fatty acids (UFAs). Arachidonic acid and UFAs were ~1000-fold more potent than G protein-coupled receptor (GPCR) agonists. Triascin C and thimerosal, which, respectively, inhibit fatty acyl-CoA synthases and acyl-CoA transferases, prevented the UFA-induced MAG biosynthesis, implying glycerolipid remodeling. 2-AG and other MAG biosynthesis was preceded by that of the corresponding lysophosphatidic acid (LPA). However, we could not directly implicate LPA dephosphorylation in MAG biosynthesis. While GPCR agonists poorly induced 2-AG biosynthesis, they inhibited that induced by AA by 25%-50%, suggesting that 2-AG biosynthesis is decreased when leukocytes are surrounded by a pro-inflammatory entourage. Our data strongly indicate that human leukocytes use AA and UFAs to biosynthesize biologically significant concentrations of 2-AG and other MAGs and that hijacking the immune system with 2-AG hydrolysis inhibitors might diminish inflammation in humans.

Intuitive eating is associated with elevated levels of circulating omega-3-polyunsaturated fatty acid-derived endocannabinoidome mediators.

The regulation of food intake and eating behaviours involves interactions between different systems. The endocannabinoidome, comprising several fatty acid-derived mediators, plays a central role in the regulation of food intake. Alterations of this system have been suggested to intervene in the aetiology of eating disorders. This study aimed to examine the associations between non-pathological eating behaviours and circulating endocannabinoidome mediators in a heterogeneous human population. Plasma 2-monoacyl-glycerol and N-acyl-ethanolamine congeners were measured by LC-MS/MS in a sample of 190 men and women. Eating behaviours were assessed using the Three-Factor Eating Questionnaire (TFEQ) and the Intuitive Eating Scale-2 (IES-2). Following adjustment for body mass index and age, plasma levels of omega-3 polyunsaturated fatty acid-derived 2-monoacyl-glycerols, 2-eicosapentaenoyl-glycerol (2-EPG) and 2-docosapentaenoyl-glycerol (2-DPG), were associated with higher intuitive eating scores (0.15 ≤ rho ≤ 0.20; p < 0.05). These associations were independent of the dietary intake of the fatty acid precursors of these 2-monoacyl-glycerols. However, almost no association was found between plasma levels of N-acyl-ethanolamine congeners and the TFEQ or the IES-2 scores. The results of the present study suggest the association of 2-monoacyl-glycerols, especially 2-EPG and 2-DPG, in the regulation of intuitive eating and the potential implication therein of bioactive lipids.

Assessment of the Effects of Dietary Vitamin D Levels on Olanzapine-Induced Metabolic Side Effects: Focus on the Endocannabinoidome-Gut Microbiome Axis.

Vitamin D deficiency is associated with poor mental health and dysmetabolism. Several metabolic abnormalities are associated with psychotic diseases, which can be compounded by atypical antipsychotics that induce weight gain and insulin resistance. These side-effects may be affected by vitamin D levels. The gut microbiota and endocannabinoidome (eCBome) are significant regulators of both metabolism and mental health, but their role in the development of atypical antipsychotic drug metabolic side-effects and their interaction with vitamin D status is unknown. We studied the effects of different combinations of vitamin D levels and atypical antipsychotic drug (olanzapine) exposure on whole-body metabolism and the eCBome-gut microbiota axis in female C57BL/6J mice under a high fat/high sucrose (HFHS) diet in an attempt to identify a link between the latter and the different metabolic outputs induced by the treatments. Olanzapine exerted a protective effect against diet-induced obesity and insulin resistance, largely independent of dietary vitamin D status. These changes were concomitant with olanzapine-mediated decreases in Trpv1 expression and increases in the levels of its agonists, including various N-acylethanolamines and 2-monoacylglycerols, which are consistent with the observed improvement in adiposity and metabolic status. Furthermore, while global gut bacteria community architecture was not altered by olanzapine, we identified changes in the relative abundances of various commensal bacterial families. Taken together, changes of eCBome and gut microbiota families under our experimental conditions might contribute to olanzapine and vitamin D-mediated inhibition of weight gain in mice on a HFHS diet.

Germ-free mice exhibit profound gut microbiota-dependent alterations of intestinal endocannabinoidome signaling.

The gut microbiota is a unique ecosystem of microorganisms interacting with the host through several biochemical mechanisms. The endocannabinoidome (eCBome), a complex signaling system including the endocannabinoid system, approximately 50 receptors and metabolic enzymes, and more than 20 lipid mediators with important physiopathologic functions, modulates gastrointestinal tract function and may mediate host cell-microbe communications there. Germ-free (GF) mice, which lack an intestinal microbiome and so differ drastically from conventionally raised (CR) mice, offer a unique opportunity to explore the eCBome in a microbe-free model and in the presence of a reintroduced functional gut microbiome through fecal microbiota transplant (FMT). We aimed to gain direct evidence for a link between the microbiome and eCBome systems by investigating eCBome alterations in the gut in GF mice before and after FMT. Basal eCBome gene expression and lipid profiles were measured in various segments of the intestine of GF and CR mice at juvenile and adult ages using targeted quantitative PCR transcriptomics and LC-MS/MS lipidomics. GF mice exhibited age-dependent modifications in intestinal eCBome gene expression and lipid mediator levels. FMT from CR donor mice to age-matched GF male mice reversed several of these alterations, particularly in the ileum and jejunum, after only 1 week, demonstrating that the gut microbiome directly impacts the host eCBome and providing a cause-effect relationship between the presence or absence of intestinal microbes and eCBome signaling. These results open the way to new studies investigating the mechanisms through which intestinal microorganisms exploit eCBome signaling to exert some of their physiopathologic functions.

Effects of hypoxia-reoxygenation stimuli on renal redox status and nuclear factor erythroid 2-related factor 2 pathway in sickle cell SAD mice.

NEW FINDINGS: What is the central question of this study? What are the effects of repeated subclinical vaso-occlusions on nuclear factor erythroid 2 related factor 2 (Nrf2) and oxidative stress balance regulation in the kidney of transgenic SAD mice? What is the main finding and its importance? In response to hypoxia-reoxygenation, nuclear Nrf2 protein expression decreased in the kidney of SAD mice while haem oxygenase transcripts were increased. This suggest that in SAD mice, other transcription factors than Nrf2 could be involved in renal antioxidant gene regulation in response to hypoxia-reoxygenation. ABSTRACT: Hypoxia-reoxygenation (H/R) stress is known to increase oxidative stress in transgenic sickle mice and can cause organ failure. Here we described the effects of H/R on nuclear factor erythroid 2-related factor 2 (Nrf2) as a putative regulator of redox status in the kidneys of SAD mice investigating Nrf2-regulated antioxidant enzymes. Transgenic SAD mice and healthy C57Bl/6J mice were exposed to 4 h of hypoxia followed by various times of reoxygenation at ambient air (2 or 6 h). Regardless of the conditions (i.e. normoxia or H/R), SAD mice expressed higher renal oxidative stress levels. Nuclear Nrf2 protein expression decreased after 2 h post-hypoxia only in the medulla region of the kidney and only in SAD mice. Simultaneously, haem oxygenase transcripts were affected by H/R stimulus with a significant enhancement after 2 h post-hypoxia. Similarly, hypoxia inducible factor-1α staining increased after 2 h post-hypoxia in SAD mice in both cortex and medulla areas. Our data confirm that the kidneys are organs that are particularly sensitive to H/R stimuli in sickle cell SAD mice. Also, these results suggest an effect of the duration of recovery period (short vs. long) and specific responses according to kidney areas, medulla vs. cortex, on Nrf2 expression in response to H/R stimuli in SAD mice.

Altered blood rheology and impaired pressure-induced cutaneous vasodilation in a mouse model of combined type 2 diabetes and sickle cell trait.

OBJECTIVE: Type 2 diabetes (T2D)-related vascular dysfunction and hemorheological abnormalities could possibly be amplified by sickle cell trait (SCT). These alterations could potentially increase the risk of vascular complications in individuals with combined T2D and SCT. Therefore, this study used a mouse model to determine whether vascular function and blood rheology were more severely altered in combined T2D and SCT than in T2D or SCT alone. METHODS: Townes transgenic mice with or without SCT received a 12-week high fat high sucrose or standard diet to create models of combined T2D-SCT, T2D, SCT, and controls. Pressure-induced vasodilation (PIV) and sodium nitroprusside (SNP)-mediated vasodilation in-vivo, and hemorheological parameters were measured. RESULTS: No significant differences in blood viscosity, hematocrit, erythrocyte deformability, or PIV were observed between the control and T2D mice, or the control and SCT mice. However, blood viscosity, erythrocyte deformability, and PIV were significantly altered in the T2D-SCT mice compared to the control mice. There were no differences in SNP response between the groups. CONCLUSIONS: Although neither T2D nor SCT alone had significant effects on blood rheology parameters or vascular function, combined T2D-SCT mice had significantly altered blood rheology and significantly impaired vascular function.

Combined and differential effects of alpha-thalassemia and HbF-quantitative trait loci in Senegalese hydroxyurea-free children with sickle cell anemia.

BACKGROUND: Our objective was to investigate the combined and differential effects of alpha-thalassemia -3.7 kb deletion and HbF-promoting quantitative trait loci (HbF-QTL) in Senegalese hydroxyurea (HU)-free children and young adults with sickle cell anemia (SCA). PROCEDURE: Steady-state biological parameters and vaso-occlusive crises (VOC) requiring emergency admission were recorded over a 2-year period in 301 children with SCA. The age of the first hospitalized VOC was also recorded. These data were correlated with the alpha-globin and HbF-QTL genotypes. For the latter, three different genetic loci were studied (XmnI, rs7482144; BCL11A, rs1427407; and the HBS1L-MYB region, rs28384513) and a composite score was calculated, ranging from zero (none of these three polymorphisms) to six (all three polymorphisms at the homozygous state). RESULTS: A positive clinical impact of the HbF-QTL score on VOC rate, HbF, leucocytes, and C-reactive protein levels was observed only for patients without alpha-thalassemia deletion. Conversely, combination of homozygous -3.7 kb deletion with three to six HbF-QTL was associated with a higher VOC rate. The age of the first hospitalized VOC was delayed for patients with one or two alpha-thalassemia deletions and at least two HbF-QTL. CONCLUSION: Alpha-thalassemia -3.7 kb deletion and HbF-QTL are modulating factors of SCA clinical severity that interact with each other. They should be studied and interpreted together and not separately, at least in HU-free children.

The Endocannabinoid Metabolite Prostaglandin E2 (PGE2)-Glycerol Inhibits Human Neutrophil Functions: Involvement of Its Hydrolysis into PGE2 and EP Receptors.

The endocannabinoids 2-arachidonoyl-glycerol and N-arachidonoyl-ethanolamine mediate an array of pro- and anti-inflammatory effects. These effects are related, in part, to their metabolism by eicosanoid biosynthetic enzymes. For example, N-arachidonoyl-ethanolamine and 2-arachidonoyl-glycerol can be metabolized by cyclooxygenase-2 into PG-ethanolamide (PG-EA) and PG-glycerol (PG-G), respectively. Although PGE2 is a recognized suppressor of neutrophil functions, the impact of cyclooxygenase-derived endocannabinoids such as PGE2-EA or PGE2-G on neutrophils is unknown. This study's aim was to define the effects of these mediators on neutrophil functions and the underlying cellular mechanisms involved. We show that PGE2-G, but not PGE2-EA, inhibits leukotriene B4 biosynthesis, superoxide production, migration, and antimicrobial peptide release. The effects of PGE2-G were prevented by EP1/EP2 receptor antagonist AH-6809 but not the EP4 antagonist ONO-AE2-227. The effects of PGE2-G required its hydrolysis into PGE2, were not observed with the non-hydrolyzable PGE2-serinol amide, and were completely prevented by methyl-arachidonoyl-fluorophosphate and palmostatin B, and partially prevented by JZL184 and WWL113. Although we could detect six of the documented PG-G hydrolases in neutrophils by quantitative PCR, only ABHD12 and ABHD16A were detected by immunoblot. Our pharmacological data, combined with our protein expression data, did not allow us to pinpoint one PGE2-G lipase, and rather support the involvement of an uncharacterized lipase and/or of multiple hydrolases. In conclusion, we show that PGE2-G inhibits human neutrophil functions through its hydrolysis into PGE2, and by activating the EP2 receptor. This also indicates that neutrophils could regulate inflammation by altering the balance between PG-G and PG levels in vivo.

Moderate exercise training decreases inflammation in transgenic sickle cell mice.

Chronic systemic inflammation is a pathophysiological feature of sickle cell disease (SCD). Considering that regular exercise exerts multiple beneficial health effects including anti-inflammatory actions, we investigated whether a treadmill training program could minimize the inflammatory state in transgenic sickle cell (SS) mice. To test this hypothesis, SS mice were subjected to a treadmill training protocol of 1h/day, 5days a week for 8weeks. Exercise training increased the percent of venous oxyhemoglobin and sharply decreased the percent of carboxyhemoglobin suggesting that exercise training may limit the proportion of erythrocytes that were deoxygenated in the venous circulation. Exercise training attenuated systemic inflammation as attested by a significant drop in white blood cell (WBC) count and plasma Th1/Th2 cytokine ratio. There was reduction in interleukin-1ß and endothelin-1 mRNA expression in trained sickle mice. The spleen/body mass ratio was significantly decreased in trained sickle mice and there was a strong correlation between the magnitude of congestion and the relative spleen mass in all animals (trained and untrained). We conclude that moderate intensity exercise training, without any noticeable complications, may be associated with limited baseline blood deoxygenation and inflammation in sickle cell mice, and reduce sequestration of sickle erythrocytes/congestion in the spleen.

Hydroxyurea therapy modulates sickle cell anemia red blood cell physiology: Impact on RBC deformability, oxidative stress, nitrite levels and nitric oxide synthase signalling pathway.

Hydroxyurea (HU) has been suggested to act as a nitric oxide (NO) donor in sickle cell anemia (SCA). However, little is known about the HU NO-related effects on red blood cell (RBC) physiology and NO signalling pathway. Thirty-four patients with SCA (22 under HU treatment (HU+) and 12 without (HU-)) and 17 healthy subjects (AA) were included. RBC nitrite content, deformability and reactive oxygen species (ROS) levels were measured. RBC NO-synthase (RBC-NOS) signalling pathway was assessed by the measurement of RBC-NOS serine1177 and RBC-AKT serine473 phosphorylation. We also investigated the in vitro effects of Sodium Nitroprusside (SNP), a NO donor, on the same parameters in SCA RBC. RBC nitrite content was higher in HU+ than in HU- and AA. RBC deformability was decreased in SCA patients compared to AA but the decrease was more pronounced in HU-. RBC ROS level was increased in SCA compared to AA but the level was higher in HU- than in HU+. RBC-NOS serine1177 and RBC-AKT serine473 phosphorylation were decreased in HU+ compared to HU- and AA. SCA RBC treated with SNP showed increased deformability, reduced ROS content and a decrease in AKT and RBC-NOS phosphorylation. Our study suggests that HU, through its effects on foetal hemoglobin and possibly on NO delivery, would modulate RBC NO signalling pathway, RBC rheology and oxidative stress.

ANISEED 2015: a digital framework for the comparative developmental biology of ascidians.

Ascidians belong to the tunicates, the sister group of vertebrates and are recognized model organisms in the field of embryonic development, regeneration and stem cells. ANISEED is the main information system in the field of ascidian developmental biology. This article reports the development of the system since its initial publication in 2010. Over the past five years, we refactored the system from an initial custom schema to an extended version of the Chado schema and redesigned all user and back end interfaces. This new architecture was used to improve and enrich the description of Ciona intestinalis embryonic development, based on an improved genome assembly and gene model set, refined functional gene annotation, and anatomical ontologies, and a new collection of full ORF cDNAs. The genomes of nine ascidian species have been sequenced since the release of the C. intestinalis genome. In ANISEED 2015, all nine new ascidian species can be explored via dedicated genome browsers, and searched by Blast. In addition, ANISEED provides full functional gene annotation, anatomical ontologies and some gene expression data for the six species with highest quality genomes. ANISEED is publicly available at: http://www.aniseed.cnrs.fr.

Does physical activity increase or decrease the risk of sickle cell disease complications?

Sickle cell disease (SCD) is the most common inherited disease in the world. Red blood cell sickling, blood cell-endothelium adhesion, blood rheology abnormalities, intravascular haemolysis, and increased oxidative stress and inflammation contribute to the pathophysiology of SCD. Because acute intense exercise may alter these pathophysiological mechanisms, physical activity is usually contra-indicated in patients with SCD. However, recent studies in sickle-cell trait carriers and in a SCD mice model show that regular physical activity could decrease oxidative stress and inflammation, limit blood rheology alterations and increase nitric oxide metabolism. Therefore, supervised habitual physical activity may benefit patients with SCD. This article reviews the literature on the effects of acute and chronic exercise on the biological responses and clinical outcomes of patients with SCD.

Association between oxidative stress and vascular reactivity in children with sickle cell anaemia and sickle haemoglobin C disease.

Oxidative stress and haemolysis-associated nitric oxide (NO) depletion plays a crucial role in the development of vasculopathy in sickle cell anaemia (SS). However it remains unknown whether oxidative stress and haemolysis levels influence vascular function in patients with sickle haemoglobin C disease (SC). Microvascular response to heat (using Laser Doppler flowmetry on finger), oxidative stress biomarkers, NO metabolites, endothelin-1 and haematological parameters were compared between patients with SS and SC. Vascular function, oxidative and nitrosative markers were also measured in healthy (AA) children. SS and SC had increased plasma advanced oxidation protein products (AOPP), malondialdehyde, plasma antioxidant activities and NO end products, compared to AA. SC had lower catalase activity compared to AA and SS. Haemolytic rate, glutathione peroxidase and nitrotyrosine concentrations were significantly increased in children with SS compared to SC and AA. SS and SC had impaired microvascular reactivity compared to AA. In SS, the plateau phase of the response to local thermal heating was negatively associated with nitrotyrosine and AOPP. No association between vascular function parameters and oxidative stress markers was observed in SC. Mild haemolysis in SC, compared to SS, may limit oxidative and nitrosative stress and could explain the better preserved microvascular function in this group.

UGT1A1 (TA)n genotype is not the major risk factor of cholelithiasis in sickle cell disease children.

OBJECTIVES: Because of the increased hemolytic rate, a significant proportion of patients with sickle cell disease (SCD) are prone to develop cholelithiasis. The present study investigated the role of several genetic factors (UGT1A1 promoter (TA)n repeat polymorphism, alpha-globin status), hematological parameters, clinical severity, and hydroxyurea (HU) therapy on the occurrence of cholelithiasis in SCD. METHODS: One hundred and fifty-eight children (2-18 yr old) regularly followed at the University Hospital of Lyon (France) were included. A multivariate Cox model was used to test the associations between cholelithiasis and the different parameters analyzed. RESULTS: We confirmed that alpha-thalassemia and low basal reticulocyte (RET) count were independent protective factors for cholelithiasis while 7/7, 8/8 and 7/8 UGT1A1 (TA)n genotypes were independent predisposing factors for this complication. We also showed for the first time that HU treatment decreased the risk for cholelithiasis while frequent vaso-occlusive crises and acute chest syndrome events increased that risk. CONCLUSIONS: Our findings demonstrate that UGT1A1 (TA)n polymorphism is not the only factor triggering gallstone formation in SCD. Cholelithiasis is also modulated by RET count, the number of deleted alpha-genes, HU therapy and the frequency of vaso-occlusive events.

Alpha-thalassaemia promotes frequent vaso-occlusive crises in children with sickle cell anaemia through haemorheological changes.

BACKGROUND: Sickle cell anaemia (SCA) is a severe hereditary haemoglobinopathy characterised by haemorheological abnormalities, which play a role in the occurrence of several acute and chronic clinical complications. While ßS -haplotypes and alpha-thalassaemia modulate SCA clinical severity, their effects on blood rheology have been incompletely described. The aim of this study was to test the effects of these genetic modifiers on the haemorheological properties and clinical complication of children with SCA. PROCEDURE: Steady-state haemorheological profile, biological parameters, ßS -haplotypes, alpha-globin status, vaso-occlusive crisis (VOC) and acute chest syndrome frequencies were analysed in 128 children (aged 5 to 18 years) with SCA. RESULTS: Patients with alpha-thalassaemia showed increased red blood cell (RBC) deformability and aggregation compared to those without. Median VOC rate was higher in patients with homozygous alpha-thalassaemia compared to those with a normal alpha genotype. Conversely, the haemorheological profile and clinical complications were not influenced by the ßS -haplotypes in our study. CONCLUSION: Our results demonstrate that alpha-thalassaemia is associated with higher risk for VOC events in children with SCA, which may be due in part to its effects on RBC deformability and aggregation.

Genetic Background of the Sickle Cell Disease Pediatric Population of Dakar, Senegal, and Characterization of a Novel Frameshift ß-Thalassemia Mutation [HBB: c.265_266del; p.Leu89Glufs*2].

Sickle cell disease is a genetic disorder with a large variability in the pattern and severity of clinical manifestations. Different genetic modulators have been identified but very few epidemiologic data are available on these modifier genes in Senegal. This study aimed to determine their prevalence in a Senegalese sickle cell disease pediatric population. The following genetic parameters were genotyped in 295 sickle cell disease children of the Dakar pediatric hospital: sickle cell disease genotype [ßS/ßS (HBB: c.20A>T), ßS/ßC (HBB: c.19G>A), ßS/ß0-thalassemia (ß0-thal)], XmnI polymorphism, the five most common α-thalassemia (α-thal) deletions and the A(-) and Betica glucose-6-phosphate-dehydrogenase (G6PD) deficient variants. Despite very few ßS/ßC and ßS/ß0-thal children (1.0% each), a novel frameshift ß0-thal mutation was characterized: HBB: c.265_266del; p.Leu89Glufs*2. The -α3.7 (rightward) deletion was the only α-thal deletion identified in this cohort (12.0% allelic frequency). Most of ßS/ßS patients (61.9%) were homozygous for the XmnI polymorphism and assumed to carry a Senegal/Senegal ßS haplotype. The remaining haplotypes were predominantly of the Benin type. While the Betica G6PD variant was quite frequent (13.0%), a low frequency of the A(-) variant was detected (1.0-2.0%). The systematic genotyping of the -α3.7 deletion and of the G6PD Betica variant in sickle cell disease patients from Senegal could be useful to identify patients at risk for several complications, such as cerebral vasculopathy, where it has been demonstrated that a normal α-globin genotype and G6PD deficiency are predisposing factors. These patients should be eligible for a transcranial Doppler examination that is not routinely offered in Senegal.