Circulating Iron in Patients with Sickle Cell Disease Mediates the Release of Neutrophil Extracellular Traps.

Introduction: Neutrophils promote chronic inflammation and release neutrophil extracellular traps (NETs) that can drive inflammatory responses. Inflammation influences progression of sickle cell disease (SCD), and a role for NETs has been suggested in the onset of vaso-occlusive crisis (VOC). We aimed to identify factors in the circulation of these patients that provoke NET release, with a focus on triggers associated with hemolysis. Methods: Paired serum and plasma samples during VOC and steady state of 18 SCD patients (HbSS/HbSß0-thal and HbSC/HbSß+-thal) were collected. Cell-free heme, hemopexin, and labile plasma iron have been measured in the plasma samples of the SCD patients. NETs formation by human neutrophils from healthy donors induced by serum of SCD patients was studied using confocal microscopy and staining for extracellular DNA using Sytox, followed by quantification of surface coverage using ImageJ. Results: Eighteen patients paired samples obtained during VOC and steady state were available (11 HbSS/HbSß0-thal and 7 HbSC/HbSß+-thal). We observed high levels of systemic heme and iron, concomitant with low levels of the heme-scavenger hemopexin in sera of patients with SCD, both during VOC and in steady state. In our in vitro experiments, neutrophils released NETs when exposed to sera from SCD patients. The release of NETs was associated with high levels of circulating iron in these sera. Although hemin triggered NET formation in vitro, addition of hemopexin to scavenge heme did not suppress NET release in SCD sera. By contrast, the iron scavengers deferoxamine and apotransferrin attenuated NET formation in a significant proportion of SCD sera. Discussion: Our results suggest that redox-active iron in the circulation of non-transfusion-dependent SCD patients activates neutrophils to release NETs, and hence, exerts a direct pro-inflammatory effect. Thus, we propose that chelation of iron requires further investigation as a therapeutic strategy in SCD.

Prospective evaluation of chronic organ damage in adult sickle cell patients: A seven-year follow-up study.

Organ damage in sickle cell disease (SCD) is a crucial determinant for disease severity and prognosis. In a previous study, we analyzed the prevalence of SCD-related organ damage and complications in adult sickle cell patients. We now describe a seven-year follow-up of this cohort.All patients from the primary analysis in 2006 (n = 104), were included for follow-up. Patients were screened for SCD-related organ damage and complications (microalbuminuria, renal failure, elevated tricuspid regurgitation flow velocity (TRV) (≥2.5 m/seconds), retinopathy, iron overload, cholelithiasis, avascular osteonecrosis, leg ulcers, acute chest syndrome (ACS), stroke, priapism and admissions for vaso-occlusive crises (VOC) biannually. Upon 7 years of follow-up, progression in the prevalence of avascular osteonecrosis (from 12.5% to 20.4%), renal failure (from 6.7% to 23.4%), retinopathy (from 39.7% to 53.8%) was observed in the whole group. In HbSS/HbSß0 -thal patients also progression in microalbuminuria (from 34% to 45%) and elevated TRV (from 40% to 48%) was observed while hardly any progression in the prevalence of cholelithiasis, priapism, stroke or chronic ulcers was seen. The proportion of patients with at least one episode of ACS increased in the group of HbSS/HbSß0 -thal patients from 32% to 49.1%. In conclusion, 62% of the sickle cell patients in this prospective cohort study developed a new SCD-related complication in a comprehensive care setting within 7 years of follow-up. Although the hospital admission rate for VOC remained stable, multiple forms of organ damage increased substantially. These observations underline the need for continued screening for organ damage in all adult patients with SCD.

Nucleosomes and neutrophil activation in sickle cell disease painful crisis.

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/HbSß(0)-thalassemia (Sr=0.55, P<0.001) and HbSC/HbSß(+-)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.

[An infant with prolonged jaundice]. / Een zuigeling met persisterende icterus.

BACKGROUND: In every neonate presenting with prolonged jaundice persisting beyond day 21 of life, neonatal cholestasis should always be excluded even if the infant is breast fed. Pale stools are an alarm symptom and additional tests for neonatal cholestasis should be carried out directly. CASE DESCRIPTION: We describe the case of a five-week-old girl of Chilean origin who was referred with conjugated hyperbilirubinaemia. The jaundice had possibly arisen directly after birth, but due to the dark skin colour of the neonate the jaundice was not recognized as such, although her scleras were yellow. According to the stool colour card, her stools were pale. The findings of a histological examination of a liver biopsy confirmed the diagnosis of biliary atresia, for which a Kasai hepatoportoenterostomy was performed. CONCLUSION: Neonatal cholestasis is always pathological and requires further investigation. In infants with dark skin, jaundice is sometimes difficult to see and inspection of the scleras should give the definitive answer.

[A pregnant woman without fetal heartsounds]. / Geen foetale harttonen te vinden bij zwangere vrouw.

A 31-year-old woman reported to a Malawian district hospital. She was pregnant of twins and in labour, and had received no prenatal care. There were no fetal heartsounds on examination. The patient spontaneously delivered 2 macerated stillbirths. A monochorionic placenta in 1 amniotic sack was seen with 2 entangled umbilical cords, a feared complication in monoamniotic monochorionic twin pregnancy.