Plasma microparticles of sickle patients during crisis or taking hydroxyurea modify endothelium inflammatory properties.

Microparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS), detected at high concentration in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs on endothelial cells (ECs) from 60 sickle cell disease (SCD) patients. MPs were collected from SCD patients and compared with MPs isolated from healthy individuals (AA). Other plasma MPs were purified from SS patients before and 2 years after the onset of hydroxyurea (HU) treatment or during a vaso-occlusive crisis and at steady-state. Compared with AA MPs, SS MPs increased EC ICAM-1 messenger RNA and protein levels, as well as neutrophil adhesion. We showed that ICAM-1 overexpression was primarily caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MP PS capping using annexin V. MPs from SS patients treated with HU were less efficient to induce a proinflammatory phenotype in ECs compared with MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared with MPs collected at steady-state. Furthermore, neutrophil adhesion was abolished by a blocking anti-ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a proinflammatory phenotype of ECs. We also uncover a new mode of action for HU and identify potential therapeutics: annexin V and anti-ICAM-1 antibodies.

Oxidative stress, inflammation, blood rheology, and microcirculation in adults with sickle cell disease: Effects of hydroxyurea treatment and impact of sickle cell syndrome.

Inflammation and oxidative stress play a key role in the pathophysiology of sickle cell disease (SCD). However, the potential influence of different sickle genotypes, or hydroxyurea (HU) treatment, on these factors remains poorly documented. The present study compared several plasma markers of inflammation and oxidative stress, as well as microvascular function, between patients with sickle SC disease (HbSC, n = 19) and patients with sickle cell anemia (HbSS) under hydroxyurea (HU) treatment (n = 16), or not (n = 13). Hemorheological parameters and levels of inflammatory (IL-6, IL-8, IFN-γ, MCP-1, MIP-1ß, TNF-α) and oxidative stress (AOPP, MDA, MPO) markers were determined. Peripheral microcirculatory cutaneous blood flow and immediate microvascular response to local heat were evaluated using laser Doppler flowmetry. Oxidative stress and inflammation were lower in HbSC patients and HbSS patients under HU therapy compared to HbSS patients not treated with HU. Blood viscosity was higher in HbSC than in HbSS patients treated with or not with HU. Vasodilation response of the cutaneous microcirculation to heat stress was higher in HbSS patients receiving HU treatment. Our results clearly established that both sickle cell genotype and HU treatment modulate inflammation and oxidative stress.

Point-of-care screening for sickle cell disease in low-resource settings: A multi-center evaluation of HemoTypeSC, a novel rapid test.

Sickle cell disease (SCD) is a common, life-threatening genetic disorder that is best managed when diagnosed early by newborn screening. However, SCD is most prevalent in low-resource regions of the world where newborn screening is rare and diagnosis at the point-of-care is challenging. In many such regions, the majority of affected children die, undiagnosed, before the age of 5 years. A rapid and affordable point-of-care test for SCD is needed. The diagnostic accuracy of HemoTypeSC, a point-of-care immunoassay, for SCD was evaluated in individuals who had SCD, hemoglobin C disease, the related carrier (trait) states, or a normal hemoglobin phenotype. Children and adults participated in low-, medium- and high-resource environments (Ghana [n = 383], Martinique [n = 46], and USA [n = 158]). Paired blood specimens were obtained for HemoTypeSC and a reference diagnostic assay. HemoTypeSC testing was performed at the site of blood collection, and the reference test was performed in a laboratory at each site. In 587 participants, across all study sites, HemoTypeSC had an overall sensitivity of 99.5% and specificity of 99.9% across all hemoglobin phenotypes. The test had 100% sensitivity and specificity for sickle cell anemia. Sensitivity and specificity for detection of normal and trait states were >99%. HemoTypeSC is an inexpensive (<$2 per test), accurate, and rapid point-of-care test that can be used in resource-limited regions with a high prevalence of SCD to provide timely diagnosis and support newborn screening programs.

Metabolic response to oral glucose tolerance test performed in neutral and warm environmental temperature.

Objective: Hot environments are associated with impaired glucose metabolism at rest in healthy humans. The purpose of this study was to explore the contribution of key glucoregulatory hormones and biomarkers to this altered glucose tolerance. Methods: The effects of ambient temperature on glucose tolerance and its determinants were assessed with a 3-hr oral glucose tolerance test (OGTT) administered to 19 healthy young men and women at 22 °C and 31 °C. Results: The glucose response amplitude was greater in warm environment (AUC 904 ± 151 vs. 721 ± 89 mmol/l·180 min at 31 °C and 22 °C, respectively, p < .001). There was no significant effect of environmental temperature on insulin, growth hormone or pancreatic polypeptide concentrations (all p > .17). The cortisol response to the glucose load was reduced 30, 60, 90 and 120 minutes postload at 31 °C compared with 22 °C (p = .001). The interleukin-6 concentration was also lower in the session at 31 °C (p = .043). Conclusion: We conclude that the effects of environmental temperature on the glucoregulatory hormones and biomarkers reported in this study do not explain the exaggerated increase in blood glucose after a glucose load taken in a warm environmental temperature. Precis statement: This work demonstrates in healthy men and women that the ingestion of glucose elicits an exaggerated increase in blood glucose when the environmental temperature is warm.

Lipid profiles in French West Indies sickle cell disease cohorts, and their general population.

BACKGROUND: The pathophysiology of sickle cell disease (SCD) and the variability of its clinical expression remain not fully understood, whether within or between different SCD genotypes. Recent studies have reported associations between lipid levels and several SCD complications. If lipid levels have been previously described as low in sickle cell anemia (SCA), few data have been provided for sickle cell SC disease (SCC). We designed our epidemiological study to isolate lipid levels and profiles by genotype in Guadeloupian cohorts of SCA and SCC adult patients, at steady state. We compared SCD lipid levels with those of the Guadeloupian general population (GGP), and analyzed potential associations between lipid levels and SCD complications (vaso-occlusive crises, acute chest syndrome and osteonecrosis). METHODS: Lipids, apolipoproteins, biological variables and anthropometric evaluation, were collected at steady state from medical files for 62 SCC and 97 SCA adult patients. Clinical SCD complications were collected from the clinical files. Analysis was conducted by genotype for all variables. RESULTS: Different SCC and SCA lipid profiles, both distinct from their GGP's, were identified. Compared to SCC and GGP, higher triglyceride (TG) levels were observed in SCA patients, independent of hydroxyurea, hemolysis, gender, age, body mass index (BMI), abdominal obesity and clinical nutritional status. Our survey highlights also subsequent anthropometrical phenotypes, with an over-representation of abdominal obesity with normal BMI in SCA patients, and affecting almost exclusively females in both genotypes. Moreover, more frequent positive history of acute chest syndrome (ACS) was observed in SCA patients with TG level higher than 1.50 g/l, and of osteonecrosis in SCC patients having non high-density lipoprotein-cholesterol level (Non HDL-C) higher than 1.30 g/l. CONCLUSIONS: This study reveals that SCA and SCC patients exhibit distinct lipid profiles and suggests that high TG and Non HDL-C levels are associated with past histories of ACS and osteonecrosis in SCA and SCC patients, respectively.

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.

Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study.

Painful vaso-occlusive crisis, a hallmark of sickle cell anaemia, results from complex, incompletely understood mechanisms. Red blood cell (RBC) damage caused by continuous endogenous and exogenous oxidative stress may precipitate the occurrence of vaso-occlusive crises. In order to gain insight into the relevance of oxidative stress in vaso-occlusive crisis occurrence, we prospectively compared the expression levels of various oxidative markers in 32 adults with sickle cell anaemia during vaso-occlusive crisis and steady-state conditions. Compared to steady-state condition, plasma levels of free haem, advanced oxidation protein products and myeloperoxidase, RBC caspase-3 activity, as well as the concentrations of total, neutrophil- and RBC-derived microparticles were increased during vaso-occlusive crises, whereas the reduced glutathione content was decreased in RBCs. In addition, natural anti-band 3 autoantibodies levels decreased during crisis and were negatively correlated with the rise in plasma advanced oxidation protein products and RBC caspase-3 activity. These data showed an exacerbation of the oxidative stress during vaso-occlusive crises in sickle cell anaemia patients and strongly suggest that the higher concentration of harmful circulating RBC-derived microparticles and the reduced anti-band 3 autoantibodies levels may be both related to the recruitment of oxidized band 3 into membrane aggregates.

Micro- and macrovascular function in children with sickle cell anaemia and sickle cell haemoglobin C disease.

It is unclear whether vascular function is affected similarly in children with sickle cell anaemia (SS) and children with sickle haemoglobin C (SC) disease. Therefore, we compared micro and macrovascular functions in healthy (AA) children, children with SS and SC disease, and assessed their association with physical activity. Participants (24 SS, 22 SC and 16 AA), were compared in terms of 1) thermal hyperaemic response (finger pad warming to 42°C) measured by Laser Doppler techniques, 2) arterial stiffness determined by pulse wave velocity, 3) daily energy expenditure related to moderate and intense physical activities estimated by questionnaire and 4) fitness level, evaluated by the six-minute walk test. Response to heating differed between SS, SC and controls. Peripheral microvascular reactivity was lower and pulse wave velocity higher in SS compared to AA. SC had blunted microvascular reactivity in response to heating compared to AA but pulse wave velocity was not different within the two groups. Physical activity and fitness levels were markedly lower in sickle cell patients compared to healthy controls but no association was observed with vascular function. Microvasodilatory reserve is decreased in both SS and SC patients but only SS patients were also characterised by impaired macrovascular function.

Cerebral and muscle microvascular oxygenation in children with sickle cell disease: Influence of hematology, hemorheology and vasomotion.

The present study investigated cerebral and muscle hemoglobin oxygen saturation (tissue oxygen index, TOI) in children with sickle cell anemia (SS), sickle cell hemoglobin C disease (SC) and healthy children (AA). TOI was measured by near-infrared spectroscopy (NIRS) and spectral analysis of the TOI variability was used to assess flowmotion and vasomotion. Arterial oxyhemoglobin saturation (SpO2), hemorheological and hematological parameters were also measured in SS and SC children. Both TOI were lower in SS compared to both AA and SC children, with SC exhibiting lower values than AA children. Cerebral vasomotion expressed in absolute values was enhanced in SS compared to AA and SC children. Muscle vasomotion did not differ between the three groups. Hematocrit, SpO2 and red blood cell deformability were positively associated with cerebral TOI in SS children. We demonstrated that 1) cerebral and muscle TOI were markedly decreased in SS children while the decrease of TOI was milder in SC children, 2) cerebral TOI level was associated with several biological markers in SS children only and 3) cerebral vasomotion was enhanced in SS, possibly to counterbalance the effects of chronic cerebral hypoxia.

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.

Which side of the balance determines the frequency of vaso-occlusive crises in children with sickle cell anemia: Blood viscosity or microvascular dysfunction?

Vascular resistance and tissue perfusion may be both affected by impaired vascular function and increased blood viscosity. Little is known about the effects of vascular function on the occurrence of painful vaso-occlusive crises (VOC) in children with sickle cell anemia (SCA). The aim of the present study was to determine which side of the balance (blood viscosity or vascular function) is the most deleterious in SCA and increases the risk for frequent hospitalized VOC. Microvascular function, microcirculatory oxygenation and blood viscosity were determined in a group of 22 SCA children/adolescents at steady state and a group of 13 healthy children/adolescents. Univariate analyses demonstrated blunted microvascular reactivity during local thermal heating test and decreased microcirculatory oxygenation in SCA children compared to controls. Multivariate analysis revealed that increased blood viscosity and decreased microcirculatory oxygenation were independent risk factors of frequent VOC in SCA. In contrast, the level of microvascular dysfunction does not predict VOC rate. In conclusion, increased blood viscosity is usually well supported in healthy individuals where vascular function is not impaired. However, in the context of SCA, microvascular function is impaired and any increase of blood viscosity or decrease in microcirculatory oxygenation would increase the risks for frequent VOC.

Impaired glucose tolerance after brief heat exposure: a randomized crossover study in healthy young men.

A high demand on thermoregulatory processes may challenge homoeostasis, particularly regarding glucose regulation. This has been understudied, although it might concern millions of humans. The objective of this project was to examine the isolated and combined effects of experimental short-term mild heat exposure and metabolic level on glucoregulation. Two experimental randomized crossover studies were conducted. Ten healthy young men participated in study A, which comprises four sessions in a fasting state at two metabolic levels [rest and exercise at 60% of maximal oxygen uptake (O2) for 40 min] in two environmental temperatures (warm: 31°C and control: 22°C). Each session ended with an ad libitum meal, resulting in similar energy intake across sessions. In study B, 12 healthy young men underwent two 3 h oral glucose tolerance tests (OGTTs) in warm and control environmental temperatures. Venous blood was sampled at several time points. In study A, repeated measure ANOVAs revealed higher postprandial serum glucose and insulin levels with heat exposure. Glycaemia following the OGTT was higher in the warm temperature compared with control. The kinetics of the serum glucose response to the glucose load was also affected by the environmental temperature (temperature-by-time interaction, P=0.030), with differences between the warm and control conditions observed up to 90 min after the glucose load (all P<0.033). These studies provide evidence that heat exposure alters short-term glucoregulation. The implication of this environmental factor in the physiopathology of Type 2 diabetes has yet to be investigated.

CAREST--Multilingual Regional Integration for Health Promotion and Research on Sickle Cell Disease and Thalassemia.

Sickle cell disease (SCD) is a significant problem in the Caribbean, where many individuals have African and Asian forebears. However, reliable prevalence data and specific health care programs for SCD are often missing in this region. Closer collaboration between Caribbean territories initiated in 2006 to set up strategies to promote better equity in the health care system for SCD patients led to the formation of CAREST: the Caribbean Network of Researchers on Sickle Cell Disease and Thalassemia. We present the effectiveness of collaborations established by CAREST to promote SCD newborn screening programs and early childhood care, to facilitate health worker training and approaches for prevention and treatment of SCD complications, and to carry out inter-Caribbean research studies.

High red blood cell nitric oxide synthase activation is not associated with improved vascular function and red blood cell deformability in sickle cell anaemia.

Human red blood cells (RBC) express an active and functional endothelial-like nitric oxide (NO) synthase (RBC-NOS). We report studies on RBC-NOS activity in sickle cell anaemia (SCA), a genetic disease characterized by decreased RBC deformability and vascular dysfunction. Total RBC-NOS content was not significantly different in SCA patients compared to healthy controls; however, using phosphorylated RBC-NOS-Ser(1177) as a marker, RBC-NOS activation was higher in SCA patients as a consequence of the greater activation of Akt (phosphorylated Akt-Ser(473) ). The higher RBC-NOS activation in SCA led to higher levels of S-nitrosylated α- and ß-spectrins, and greater RBC nitrite and nitrotyrosine levels compared to healthy controls. Plasma nitrite content was not different between the two groups. Laser Doppler flowmetric experiments demonstrated blunted microcirculatory NO-dependent response under hyperthermia in SCA patients. RBC deformability, measured by ektacytometry, was reduced in SCA in contrast to healthy individuals, and pre-shearing RBC in vitro did not improve deformability despite an increase of RBC-NOS activation. RBC-NOS activation is high in freshly drawn blood from SCA patients, resulting in high amounts of NO produced by RBC. However, this does not result in improved RBC deformability and vascular function: higher RBC-NO is not sufficient to counterbalance the enhanced oxidative stress in SCA.

Physical activity level is not a determinant of autonomic nervous system activity and clinical severity in children/adolescents with sickle cell anemia: A pilot study.

BACKGROUND: Autonomic nervous system (ANS) activity has been suggested to modulate the clinical severity of sickle cell anemia (SCA) by increasing the risk for vaso-occlusive events. Regular physical activity (PA) is known to improve ANS activity and health status in several cardiovascular and metabolic diseases. Whether regular PA improves the health status of SCA patients remains unknown. PROCEDURE: Twenty-two patients with SCA and 15 healthy (AA) children/adolescents participated to the study. Heart rate variability was measured in supine position and after a tilt-test to quantify the ANS activity. PA energy expenditure (PAEE) was assessed with questionnaire. RESULTS: 1) PAEE was lower in SCA compared to AA (190 ± 152 vs. 432 ± 277 kcal · d(-1), respectively, P < 0.01), 2) overall ANS activity was lower in SCA compared to AA, 3) parasympathetic withdrawal was observed in SCA with aging, 4) ANS reactivity was slightly impaired in SCA compared to AA (reduction in HFnu: -38 ± 27 vs. -58 ± 14%, respectively, P < 0.05), 5) ANS indices, PAEE, and rates of clinical events were not correlated. CONCLUSION: Both the level of PA and ANS activity are reduced in SCA compared to AA children/adolescents, particularly in those older than 15 years. Neither PAEE, nor ANS activity seem to influence the clinical severity of children/adolescents with SCA.

Effects of oxidative stress on red blood cell rheology in sickle cell patients.

Sickle cell anaemia (SS) and sickle cell-haemoglobin C disease (SC) patients exhibit severe red blood cell (RBC) rheological alterations involved in the development of several complications. The contribution of oxidative stress in these haemorheological abnormalities is still unknown. We compared RBC reactive oxygen species (ROS) and glutathione (GSH) content, and the haemorheological profile of SS (n = 11), SC (n = 11) and healthy subjects (n = 12) at baseline and after in-vitro treatment with t-butyl hydroperoxide (TBHP). We showed: (i) higher RBC ROS content in SS and SC patients, with the highest level observed in SS patients; (ii) lower RBC GSH content in sickle syndrome patients, especially in SS patients; (iii) TBHP increased RBC ROS production and decreased RBC GSH content in all groups; (iv) TBHP decreased RBC aggregation and increased the strength of RBC aggregates in all groups but the increase in RBC aggregates strength was greater in sickle cell patients; (v) TBHP decreased RBC deformability in the three groups but with a higher magnitude in sickle cell patients. These data suggest that RBCs from sickle cell patients have an exaggerated response to oxidative stress, which is accompanied by a profound abnormal haemorheological profile, with greater alterations in SS than in SC patients.

Haemolysis and abnormal haemorheology in sickle cell anaemia.

Although pulmonary hypertension, leg ulcers, priapism, stroke and glomerulopathy in sickle cell anaemia (SCA) result from the adverse effects of chronic haemolysis on vascular function (haemolytic phenotype), osteonecrosis, acute chest syndrome and painful vaso-occlusive crises are caused by abnormal vascular cell adhesion and increased blood viscosity (viscosity-vaso-occlusion phenotype). However, this model with two sub-phenotypes does not take into account the haemorheological dimension. We tested the relationships between the biological parameters reflecting the haemolytic rate (haemolytic component) and red blood cell (RBC) rheological characteristics in 97 adults with SCA. No significant difference in the proportion of patients with low or high haemolytic component in the low and high blood viscosity groups was observed. The RBC elongation index (i.e. deformability) was negatively correlated with the haemolytic component. The RBC aggregates strength (i.e. RBC aggregates robustness) was negatively correlated with RBC elongation index. Sickle RBCs with high density had lower elongation index and higher aggregates strength. In conclusion, (i) the 'haemolytic' phenotype is characterized by decreased RBC deformability and increased RBC aggregates strength and (ii) the viscosity-vaso-occlusive phenotype is characterized by increased RBC deformability but not always by increased blood viscosity. α-thalassaemia modulates the haemorheological properties but other factors seem to be involved.

Hematologic and hemorheological determinants of resting and exercise-induced hemoglobin oxygen desaturation in children with sickle cell disease.

The aim of the study was to determine the factors associated with resting and exercise-induced hemoglobin oxygen desaturation. The well-established six-minute walk test was conducted in 107 sickle cell children (50 with sickle hemoglobin C disease and 57 with sickle cell anemia) at steady state. Hemoglobin oxygen saturation was measured before and immediately after the six-minute walk test. Blood samples were obtained on the same day to measure hematologic and hemorheological parameters. Exercise-induced hemoglobin oxygen desaturation was defined as a drop in hemoglobin oxygen saturation of 3% or more at the end of the six-minute walk test compared to resting levels. No children with sickle hemoglobin C disease, but approximately 50% of children with sickle cell anemia showed mild or moderate oxygen desaturation at rest, which was independently associated with the percentage of reticulocytes. Exercise-induced hemoglobin oxygen desaturation was observed in 18% of children with sickle hemoglobin C disease and 34% of children with sickle cell anemia, and was independently associated with the six-minute walk test, acute chest syndrome rate and the strength of red blood cell aggregates in children with sickle cell anemia. No association was found in children with sickle hemoglobin C disease between exercise-induced hemoglobin oxygen desaturation and the measured parameters. Hemoglobin oxygen desaturation at rest was common in children with sickle cell anemia but not in children with sickle hemoglobin C disease, and was mainly associated with greater hemolysis. Physiological strain during exercise and red blood cell aggregation properties may predict the occurrence of exercise-induced hemoglobin oxygen desaturation in children with sickle cell anemia.