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Activated polymorphonuclear neutrophils play an important role in the pathogenesis of vaso-occlusive painful sickle cell crisis. Upon activation, polymorphonuclear neutrophils can form neutrophil extracellular traps. Neutrophil extracellular traps consist of a meshwork of extracellular DNA, nucleosomes, histones and neutrophil proteases. Neutrophil extracellular traps have been demonstrated to be toxic to endothelial and parenchymal cells. This prospective cohort study was conducted to determine neutrophil extracellular trap formation in sickle cell patients during steady state and painful crisis. As a measure of neutrophil extracellular traps, plasma nucleosomes levels were determined and polymorphonuclear neutrophil activation was assessed measuring plasma levels of elastase-α1-antitrypsin complexes in 74 patients in steady state, 70 patients during painful crisis, and 24 race-matched controls using Enzyme Linked Immunosorbent Assay. Nucleosome levels in steady state sickle cell patients were significantly higher than levels in controls. During painful crisis levels of both nucleosomes and elastase-α1-antitrypsin complexes increased significantly. Levels of nucleosomes correlated significantly to elastase-α1-antitrypsin complex levels during painful crisis, (Sr = 0.654, P<0.001). This was seen in both HbSS/HbSβ(0)-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSβ(+-)thalassemia patients (Sr=0.90, P<0.001) during painful crisis. Levels of nucleosomes showed a correlation with length of hospital stay and were highest in patients with acute chest syndrome. These data support the concept that neutrophil extracellular trap formation and neutrophil activation may play a role in the pathogenesis of painful sickle cell crisis and acute chest syndrome.

Oxidative stress is of importance in the pathophysiology of sickle cell disease (SCD). In this open label randomized pilot study the effects of oral N-acetylcysteine (NAC) on phosphatidylserine (PS) expression as marker of cellular oxidative damage (primary end point), and markers of hemolysis, coagulation and endothelial activation and NAC tolerability (secondary end points) were studied. Eleven consecutive patients (ten homozygous [HbSS] sickle cell patients, one HbSβ(0)-thalassemia patient) were randomly assigned to treatment with either 1,200 or 2,400 mg NAC daily during 6 weeks. The data indicate an increment in whole blood glutathione levels and a decrease in erythrocyte outer membrane phosphatidylserine exposure, plasma levels of advanced glycation end-products (AGEs) and cell-free hemoglobin after 6 weeks of NAC treatment in both dose groups. One patient did not tolerate the 2,400 mg dose and continued with the 1,200 mg dose. During the study period, none of the patients experienced painful crises or other significant SCD or NAC related complications. These data indicate that N-acetylcysteine treatment of sickle cell patients may reduce SCD related oxidative stress.

Erythrocytes are both an important source and target of reactive oxygen species in sickle cell disease. Levels of glutathione, a major antioxidant, have been shown to be decreased in sickle erythrocytes and the mechanism leading to this deficiency is not known yet. Detoxification of reactive oxygen species involves the oxidation of reduced glutathione (GSH) into glutathione-disulfide (GSSG) which is actively transported out of erythrocyte. We questioned whether under oxidative conditions, GSSG efflux is increased in sickle erythrocytes. Erythrocytes of 18 homozygous sickle cell patients and 9 race-matched healthy controls were treated with 2,3-dimethoxy-l,4-naphthoquinone, which induces intracellular reactive oxygen species generation, to stimulate GSSG production. Intra- and extracellular concentrations of GSH and GSSG were measured at baseline and during 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. While comparable at baseline, intracellular and extracellular GSSG concentrations were significantly higher in sickle erythrocytes than in healthy erythrocyte after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation (69.9 ± 3.7 μmol/l vs. 40.6 ± 6.9 μmol/l and 25.8 ± 2.7 μmol/l vs. 13.6 ± 1.7 μmol/l respectively, P<0.002). In contrast to control erythrocytes, where GSH concentrations remained unchanged (176 ± 8.4 μmol/l vs. 163 ± 13.6 μmol/l, NS), GSH in sickle erythrocytes decreased significantly (from 167 ± 8.8 μmol/l to 111 ± 11.8 μmol/l, P<0.01) after 210-minute 2,3-dimethoxy-l,4-naphthoquinone stimulation. Adding multidrug resistance-associated protein-1 inhibitor (MK571) to erythrocytes blocked GSSG efflux in both sickle and normal erythrocytes. GSSG efflux, mediated by multidrug resistance-associated protein-1, is increased in sickle erythrocytes, resulting in net loss of intracellular glutathione and possibly higher susceptibility to oxidative stress., (2011 Elsevier B.V. All rights reserved.)

Sickle cell disease (SCD) is a hemoglobinopathy characterized by hemolytic anemia, increased susceptibility to infections and vaso-occlusion leading to a reduced quality of life and life expectancy. Oxidative stress is an important feature of SCD and plays a significant role in the pathophysiology of hemolysis, vaso-occlusion and ensuing organ damage in sickle cell patients. Reactive oxygen species (ROS) and the (end-)products of their oxidative reactions are potential markers of disease severity and could be targets for antioxidant therapies. This review will summarize the role of ROS in SCD and their potential implication for SCD management., (Copyright © 2011 Wiley-Liss, Inc.)

The painful crisis accounts for the majority of sickle cell disease (SCD) related hospital admissions. The prototypic long pentraxin 3 (PTX3), an acute phase protein, is elevated in patients with inflammatory and ischemic states. As the sickle cell painful crisis is associated with both inflammation and tissue ischemia, we questioned whether plasma PTX3 levels are increased during and associated with painful crisis severity. Furthermore, since PTX3 up-regulates endothelial expression of tissue factor we studied PTX levels in relation to markers of endothelial and coagulation activation. Plasma levels of PTX3, ultra-sensitive C-reactive protein (US-CRP), prothrombin fragment 1+2, thrombin-antithrombin (TAT) complexes, von Willebrand Factor antigen and soluble vascular adhesion molecule-1 were determined in 105 asymptomatic sickle cell patients, 33 patients during painful crisis and 30 race matched healthy controls. Plasma PTX3 levels were comparable between patients in asymptomatic state and healthy controls, but significantly higher during painful crisis (P<0.01). US-CRP levels were higher in asymptomatic patients compared to controls (P<0.0001) and increased further during painful crisis (P<0.0001). PTX3 levels at presentation with painful crisis correlated significantly with the duration of subsequent hospital admission (r(s) = 0.43; P = 0.013), whereas US-CRP levels did not. PTX3 levels did not correlate with markers of hypercoagulability. The increase of PTX3 levels during painful crisis and their relation to the duration of subsequent hospital stay suggest that PTX3 might serve both as a diagnostic and severity marker of the painful sickle cell crisis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The painful crisis is the hallmark of sickle-cell disease (SCD). Bone resorption, as part of physiological bone turnover, results in release into the circulation with subsequent urinary excretion of the collagen cross-links pyridinoline (PYD) and deoxypyridinoline (DPD). Urinary PYD and DPD concentrations could reflect the extent of bone infarction during painful sickle-cell crisis. Urinary concentrations of PYD and DPD, adjusted for urine creatinine, were measured in sickle-cell patients (38 clinically asymptomatics and 27 during painful crisis) and healthy controls (n 5 25) using high-performance liquid chromatography(HPLC). PYD and DPD concentrations were higher in asymptomatic HbSS/HbSb0-thalassemia patients compared to controls (P <0.05) with further increments during painful crisis in both HbSS/HbSb0-thalassemia and HbSC/HbSb1-thalassemia patients (P < 0.05). In the asymptomatic HbSS/HbSb0-thalassemia patients, there was a statistically significant positive correlation between DPD and hemolytic rate.Based on urinary PYD and DPD concentrations, bone degradation is increased in asymptomatic sickle-cell patients, with further increments during painful crisis. Urinary PYD and DPD concentrations are potentially diagnostic and prognostic tools in SCD.

Oxidative stress plays an important role in the pathophysiology of sickle cell disease (SCD). Plasma levels of advanced glycation end products (AGEs) are increased under oxidative conditions and are associated with disease severity in diabetes and inflammatory diseases. We investigated whether AGEs are increased in sickle cell patients and whether they are associated with SCD-related complications. Plasma levels of the AGEs pentosidine, N(ε) -(carboxymethyl)lysine (CML) and N(ε) -(carboxyethyl)lysine (CEL) were measured using single-column high performance liquid chromatography with fluorescence detection (pentosidine) and ultra performance liquid chromatography-tandem mass spectrometry (CML and CEL). Plasma levels of pentosidine and CML were increased in HbSS/HbSβ⁰-thalassaemia (n=60) and HbSC/HbSβ(+) -thalassaemia (n=42) patients during steady state as compared to healthy HbAA controls (n=30) without increments during painful crisis. CEL levels were comparable between all groups. Pentosidine and CML levels correlated significantly to haemolytic rate during the clinically asymptomatic state while pentosidine was significantly related to the number of haemolysis-related organ complications. The increased plasma AGE levels in sickle cell patients and their association with haemolysis and haemolysis-related complications suggest AGEs might be implicated in the pathophysiology of the haemolytic phenotype of SCD. Measurement of AGEs might be useful in predicting organ complications in SCD., (© 2010 Blackwell Publishing Ltd.)