High-mobility group box 1 increases platelet surface P2Y12 and platelet activation in sickle cell disease.

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of numerous thromboinflammatory diseases, including sickle cell disease (SCD). While platelets are central to thrombogenesis and inflammation, the molecular mechanisms of crosstalk between the 2 remain elusive. High-mobility group box 1 (HMGB1) regulates inflammation and stimulates platelet activation through Toll-like receptor 4. However, it remains unclear whether HMGB1 modulates other thrombotic agonists to regulate platelet activation. Herein, using human platelets, we demonstrate that HMGB1 significantly enhanced ADP-mediated platelet activation. Furthermore, inhibition of the purinergic receptor P2Y12 attenuated HMGB1-dependent platelet activation. Mechanistically, we show that HMGB1 stimulated ADP secretion, while concomitantly increasing P2Y12 levels at the platelet membrane. We show that in SCD patients, increased plasma HMGB1 levels were associated with heightened platelet activation and surface P2Y12 expression. Treatment of healthy platelets with plasma from SCD patients enhanced platelet activation and surface P2Y12, and increased sensitivity to ADP-mediated activation, and these effects were linked to plasma HMGB1. We conclude that HMGB1-mediated platelet activation involves ADP-dependent P2Y12 signaling, and HMGB1 primes platelets for ADP signaling. This complementary agonism between ADP and HMGB1 furthers the understanding of thromboinflammatory signaling in conditions such as SCD, and provides insight for therapeutic P2Y12 inhibition.

Sterile inflammation induces vasculopathy and chronic lung injury in murine sickle cell disease.

Murine sickle cell disease (SCD) results in damage to multiple organs, likely mediated first by vasculopathy. While the mechanisms inducing vascular damage remain to be determined, nitric oxide bioavailability and sterile inflammation are both considered to play major roles in vasculopathy. Here, we investigate the effects of high mobility group box-1 (HMGB1), a pro-inflammatory damage-associated molecular pattern (DAMP) molecule on endothelial-dependent vasodilation and lung morphometrics, a structural index of damage in sickle (SS) mice. SS mice were treated with either phosphate-buffered saline (PBS), hE-HMGB1-BP, an hE dual-domain peptide that binds and removes HMGB1 from the circulation via the liver, 1-[4-(aminocarbonyl)-2-methylphenyl]-5-[4-(1H-imidazol-1-yl)phenyl]-1H-pyrrole-2-propanoic acid (N6022) or N-acetyl-lysyltyrosylcysteine amide (KYC) for three weeks. Human umbilical vein endothelial cells (HUVEC) were treated with recombinant HMGB1 (r-HMGB1), which increases S-nitrosoglutathione reductase (GSNOR) expression by ∼80%, demonstrating a direct effect of HMGB1 to increase GSNOR. Treatment of SS mice with hE-HMGB1-BP reduced plasma HMGB1 in SS mice to control levels and reduced GSNOR expression in facialis arteries isolated from SS mice by ∼20%. These changes were associated with improved endothelial-dependent vasodilation. Treatment of SS mice with N6022 also improved vasodilation in SS mice suggesting that targeting GSNOR also improves vasodilation. SCD decreased protein nitrosothiols (SNOs) and radial alveolar counts (RAC) and increased GSNOR expression and mean linear intercepts (MLI) in lungs from SS mice. The marked changes in pulmonary morphometrics and GSNOR expression throughout the lung parenchyma in SS mice were improved by treating with either hE-HMGB1-BP or KYC. These data demonstrate that murine SCD induces vasculopathy and chronic lung disease by an HMGB1- and GSNOR-dependent mechanism and suggest that HMGB1 and GSNOR might be effective therapeutic targets for reducing vasculopathy and chronic lung disease in humans with SCD.

The Effect of an Adapted Digital Mental Health Intervention for Sickle Cell Disease on Engagement: A Pilot Randomized Controlled Trial.

INTRODUCTION: Despite promising outcomes, lack of engagement and poor adherence are barriers to treating mental health using digital CBT, particularly in minority groups. After conducting guided focus groups, a current mental health app was adapted to be more inclusive for minorities living with SCD. METHODS: Patients between the ages of 16-35 with SCD who reported experiencing anxiety or depression symptoms were eligible for this study. Once enrolled, participants were randomly assigned to receive one of two versions of a mental health app 1) the current version designed for the general population and 2) the adapted version. Baseline measures for depression, anxiety, pain and self-efficacy were completed at the start of the study and again at postintervention (minimum 4 weeks). RESULTS: Compared to baseline, pain (p = 0.03), self-efficacy (p = 0.007) and depression symptoms (p = 0.016) improved for the group that received the adapted app. Regardless of group assignment, a positive relationship (r = 0.47) was shown between app engagement and a change in depression symptoms (p = 0.042). DISCUSSION: Target enrollment for this study sought to enroll 40 participants. However, after difficulties locating qualified participants, enrollment criteria were adjusted to expand the population pool. Regardless of these efforts, the sample size for this study was still smaller than anticipated (n = 21). Additionally, irrespective of group approximately 40% of participants did not engage with the app. However, despite a small sample size and poor engagement, participants in the intervention group displayed better outcomes and showed trends for greater app interaction. CONCLUSION: These promising results should encourage future researchers to continue exploring ideal adaptations for implementing digital CBT in minority populations. Future studies should also consider implementing post-intervention surveys to help identify common factors relating to a lack of engagement.

Centrifugation Removes a Population of Large Vesicles, or "Macroparticles," Intermediate in Size to RBCs and Microvesicles.

Microparticles or microvesicles (MPs/MVs) are sub-cellular vesicles with a growing number of known biological functions. Microvesicles from a variety of parent cells within the vascular system increase in numerous pathological states. Red blood cell-derived MVs (RMVs) are relatively less studied than other types of circulating MVs despite red blood cells (RBCs) being the most abundant intravascular cell. This may be in part due the echoes of past misconceptions that RBCs were merely floating anucleate bags of hemoglobin rather than dynamic and responsive cells. The initial aim of this study was to maximize the concentration of RMVs derived from various blood or blood products by focusing on the optimal isolation conditions without creating more MVs from artificial manipulation. We found that allowing RBCs to sediment overnight resulted in a continuum in size of RBC membrane-containing fragments or vesicles extending beyond the 1 µm size limit suggested by many as the maximal size of an MV. Additionally, dilution and centrifugation factors were studied that altered the resultant MV population concentration. The heterogeneous size of RMVs was confirmed in mice models of hemolytic anemia. This methodological finding establishes a new paradigm in that it blurs the line between RBC, fragment, and RMV as well as suggests that the concentration of circulating RMVs may be widely underestimated given that centrifugation removes the majority of such RBC-derived membrane-containing particles.

IL-18 mediates sickle cell cardiomyopathy and ventricular arrhythmias.

Previous reports indicate that IL18 is a novel candidate gene for diastolic dysfunction in sickle cell disease (SCD)-related cardiomyopathy. We hypothesize that interleukin-18 (IL-18) mediates the development of cardiomyopathy and ventricular tachycardia (VT) in SCD. Compared with control mice, a humanized mouse model of SCD exhibited increased cardiac fibrosis, prolonged duration of action potential, higher VT inducibility in vivo, higher cardiac NF-κB phosphorylation, and higher circulating IL-18 levels, as well as reduced voltage-gated potassium channel expression, which translates to reduced transient outward potassium current (Ito) in isolated cardiomyocytes. Administering IL-18 to isolated mouse hearts resulted in VT originating from the right ventricle and further reduced Ito in SCD mouse cardiomyocytes. Sustained IL-18 inhibition via IL-18-binding protein resulted in decreased cardiac fibrosis and NF-κB phosphorylation, improved diastolic function, normalized electrical remodeling, and attenuated IL-18-mediated VT in SCD mice. Patients with SCD and either myocardial fibrosis or increased QTc displayed greater IL18 gene expression in peripheral blood mononuclear cells (PBMCs), and QTc was strongly correlated with plasma IL-18 levels. PBMC-derived IL18 gene expression was increased in patients who did not survive compared with those who did. IL-18 is a mediator of sickle cell cardiomyopathy and VT in mice and a novel therapeutic target in patients at risk for sudden death.

Impaired Bile Secretion Promotes Hepatobiliary Injury in Sickle Cell Disease.

BACKGROUND AND AIMS: Hepatic crisis is an emergent complication affecting patients with sickle cell disease (SCD); however, the molecular mechanism of sickle cell hepatobiliary injury remains poorly understood. Using the knock-in humanized mouse model of SCD and SCD patient blood, we sought to mechanistically characterize SCD-associated hepato-pathophysiology applying our recently developed quantitative liver intravital imaging, RNA sequence analysis, and biochemical approaches. APPROACH AND RESULTS: SCD mice manifested sinusoidal ischemia, progressive hepatomegaly, liver injury, hyperbilirubinemia, and increased ductular reaction under basal conditions. Nuclear factor kappa B (NF-κB) activation in the liver of SCD mice inhibited farnesoid X receptor (FXR) signaling and its downstream targets, leading to loss of canalicular bile transport and altered bile acid pool. Intravital imaging revealed impaired bile secretion into the bile canaliculi, which was secondary to loss of canalicular bile transport and bile acid metabolism, leading to intrahepatic bile accumulation in SCD mouse liver. Blocking NF-κB activation rescued FXR signaling and partially ameliorated liver injury and sinusoidal ischemia in SCD mice. CONCLUSIONS: These findings identify that NF-κB/FXR-dependent impaired bile secretion promotes intrahepatic bile accumulation, which contributes to hepatobiliary injury of SCD. Improved understanding of these processes could potentially benefit the development of therapies to treat sickle cell hepatic crisis.

Redox Signaling in Sickle Cell Disease.

Sickle cell disease (SCD) is characterized by chronic hemolysis and repeated episodes of vascular occlusion leading to progressive organ injury. SCD is characterized by unbalanced, simultaneous pro-oxidant and anti-oxidant processes at the molecular, cellular and tissue levels, with the majority of reactions tipped in favor of pro-oxidant pathways. In this brief review we discuss new findings regarding how oxidized hemin, hemolysis, mitochondrial dysfunction and the innate immune system generate oxidative stress while hemopexin, haptoglobin, heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) may provide protection in human and murine SCD. We will also describe recent clinical trials showing beneficial effects of antioxidant therapy in SCD.

Chemokine (c-c motif) receptor 2 mediates mechanical and cold hypersensitivity in sickle cell disease mice.

Approximately one-third of individuals with sickle cell disease (SCD) develop chronic pain. This debilitating pain is inadequately treated because the underlying mechanisms driving the pain are poorly understood. In addition to persistent pain, patients with SCD are also in a tonically proinflammatory state. Previous studies have revealed that there are elevated plasma levels of many inflammatory mediators including chemokine (c-c motif) ligand 2 (CCL2) in individuals with SCD. Using a transgenic mouse model of SCD, we investigated the contributions of CCL2 signaling to SCD-related pain. Inhibition of chemokine receptor 2 (CCR2), but not CCR4, alleviated the behavioral mechanical and cold hypersensitivity in SCD. Furthermore, acute CCR2 blockade reversed both the behavioral and the in vitro responsiveness of sensory neurons to an agonist of TRPV1, a neuronal ion channel previously implicated in SCD pain. These results provide insight into the immune-mediated regulation of hypersensitivity in SCD and could inform future development of analgesics or therapeutic measures to prevent chronic pain.

Characterization of a mouse model of sickle cell trait: parallels to human trait and a novel finding of cutaneous sensitization.

Sickle cell trait (SCT) has classically been categorized as a benign condition except in rare cases or upon exposure to severe physical conditions. However, several lines of evidence indicate that individuals with SCT are not always asymptomatic, and additional physiological changes and risks may remain unexplored. Here, we utilized mice harbouring one copy of normal human ß globin and one copy of sickle human ß globin as a model of SCT to assess haematological, histopathological and somatosensory outcomes. We observed that SCT mice displayed renal and hepatic vascular congestion after exposure to hypoxia. Further, we observed that SCT mice displayed increased cold aversion as well as mechanical and heat sensitivity, though to a lesser degree than homozygous sickle cell disease mice. Notably, mechanical hypersensitivity increased following hypoxia and reoxygenation. Overall our findings suggest that SCT is not entirely benign, and further assessment of pain and cutaneous sensitization is warranted both in animal models and in clinical populations.

Determining the longitudinal validity and meaningful differences in HRQL of the PedsQL™ Sickle Cell Disease Module.

BACKGROUND: Detecting change in health status over time and ascertaining meaningful changes are critical elements when using health-related quality of life (HRQL) instruments to measure patient-centered outcomes. The PedsQL™ Sickle Cell Disease module, a disease specific HRQL instrument, has previously been shown to be valid and reliable. Our objectives were to determine the longitudinal validity of the PedsQL™ Sickle Cell Disease module and the change in HRQL that is meaningful to patients. METHODS: An ancillary study was conducted utilizing a multi-center prospective trial design. Children ages 4-21 years with sickle cell disease admitted to the hospital for an acute painful vaso-oclusive crisis were eligible. Children completed HRQL assessments at three time points (in the Emergency Department, one week post-discharge, and at return to baseline (One to three months post-discharge). The primary outcome was change in HRQL score. Both distribution (effect size, standard error of measurement (SEM)) and anchor (global change assessment) based methods were used to determine the longitudinal validity and meaningful change in HRQL. Changes in HRQL meaningful to patients were identified by anchoring the change scores to the patient's perception of global improvement in pain. RESULTS: Moderate effect sizes (0.20-0.80) were determined for all domains except the Communication I and Cognitive Fatigue domains. The value of 1 SEM varied from 3.8-14.6 across all domains. Over 50% of patients improved by at least 1 SEM in Total HRQL score. A HRQL change score of 7-10 in the pain domains represented minimal perceived improvement in HRQL and a HRQL change score of 18 or greater represented moderate to large improvement. CONCLUSIONS: The PedsQL™ Sickle Cell Disease Module is responsive to changes in HRQL in patients experiencing acute painful vaso-occlusive crises. The study data establish longitudinal validity and meaningful change parameters for the PedsQL™ Sickle Cell Disease Module. TRIAL REGISTRATION: ClinicalTrials.gov (study identifier: NCT01197417 ). Date of registration: 08/30/2010.

Substance P is increased in patients with sickle cell disease and associated with haemolysis and hydroxycarbamide use.

Sickle cell disease (SCD) pain transitions from acute to chronic for unknown reasons. Chronic elevation of the pain neurotransmitter substance P (SP) sensitizes pain nociceptors. We evaluated SP levels in controls and SCD patients during baseline and acute pain and investigated associations between SP and age, gender, pain history, haemolysis and hydroxycarbamide (also termed hydroxyurea) use. Plasma SP levels were measured using enzyme-linked immunosorbent assay. Independent samples t-test compared SP levels between: (i) SCD baseline and controls, and (ii) SCD baseline and acute pain. Multivariate linear regression determined associations between SP and age, gender, pain history and hydroxycarbamide use. Spearman correlation determined an association between SP and haemolysis. We enrolled 35 African American controls, 25 SCD baseline and 12 SCD pain patients. SCD patients were 7-19 years old. Mean ± standard deviation SP level (pg/ml) in SCD baseline was higher than controls (32·4 ± 11·6 vs. 22·9 ± 7·6, P = 0·0009). SP in SCD pain was higher than baseline (78·1 ± 43·4 vs. 32·4 ± 11·6, P = 0·004). Haemolysis correlated with increased SP: Hb (r = -0·7, P = 0·0002), reticulocyte count (r = 0·61, P = 0·0016), bilirubin (r = 0·68, P = 0·0216), lactate dehydrogenase (r = 0·62, P = 0·0332), aspartate aminotransferase (r = 0·68, P = 0·003). Patients taking hydroxycarbamide had increased SP (ß = 29·2, P = 0·007). SP could be a mediator of or marker for pain sensitization in SCD and a biomarker and/or target for novel pain treatment.

R-Ras Regulates Murine T Cell Migration and Intercellular Adhesion Molecule-1 Binding.

The trafficking of T-lymphocytes to peripheral draining lymph nodes is crucial for mounting an adaptive immune response. The role of chemokines in the activation of integrins via Ras-related small GTPases has been well established. R-Ras is a member of the Ras-subfamily of small guanosine-5'-triphosphate-binding proteins and its role in T cell trafficking has been investigated in R-Ras null mice (Rras-/-). An examination of the lymphoid organs of Rras-/- mice revealed a 40% reduction in the cellularity of the peripheral lymph nodes. Morphologically, the high endothelial venules of Rras-/- mice were more disorganized and less mature than those of wild-type mice. Furthermore, CD4+ and CD8+ T cells from Rras-/- mice had approximately 42% lower surface expression of L-selectin/CD62L. These aberrant peripheral lymph node phenotypes were associated with proliferative and trafficking defects in Rras-/- T cells. Furthermore, R-Ras could be activated by the chemokine, CCL21. Indeed, Rras-/- T cells had approximately 14.5% attenuation in binding to intercellular adhesion molecule 1 upon CCL21 stimulation. Finally, in a graft-versus host disease model, recipient mice that were transfused with Rras-/- T cells showed a significant reduction in disease severity when compared with mice transplanted with wild-type T cells. These findings implicate a role for R-Ras in T cell trafficking in the high endothelial venules during an effective immune response.

A multicenter randomized controlled trial of intravenous magnesium for sickle cell pain crisis in children.

Magnesium, a vasodilator, anti-inflammatory, and pain reliever, could alter the pathophysiology of sickle cell pain crises. We hypothesized that intravenous magnesium would shorten length of stay, decrease opioid use, and improve health-related quality of life (HRQL) for pediatric patients hospitalized with sickle cell pain crises. The Magnesium for Children in Crisis (MAGiC) study was a randomized, double-blind, placebo-controlled trial of intravenous magnesium vs normal saline placebo conducted at 8 sites within the Pediatric Emergency Care Applied Research Network (PECARN). Children 4 to 21 years old with hemoglobin SS or Sß(0) thalassemia requiring hospitalization for pain were eligible. Children received 40 mg/kg of magnesium or placebo every 8 hours for up to 6 doses plus standard therapy. The primary outcome was length of stay in hours from the time of first study drug infusion, compared using a Van Elteren test. Secondary outcomes included opioid use and HRQL. Of 208 children enrolled, 204 received the study drug (101 magnesium, 103 placebo). Between-group demographics and prerandomization treatment were similar. The median interquartile range (IQR) length of stay was 56.0 (27.0-109.0) hours for magnesium vs 47.0 (24.0-99.0) hours for placebo (P = .24). Magnesium patients received 1.46 mg/kg morphine equivalents vs 1.28 mg/kg for placebo (P = .12). Changes in HRQL before discharge and 1 week after discharge were similar (P > .05 for all comparisons). The addition of intravenous magnesium did not shorten length of stay, reduce opioid use, or improve quality of life in children hospitalized for sickle cell pain crisis. This trial was registered at www.clinicaltrials.gov as #NCT01197417.

Dietary supplementation with docosahexanoic acid (DHA) increases red blood cell membrane flexibility in mice with sickle cell disease.

Humans and mice with sickle cell disease (SCD) have rigid red blood cells (RBCs). Omega-3 fatty acids, such as docosahexanoic acid (DHA), may influence RBC deformability via incorporation into the RBC membrane. In this study, sickle cell (SS) mice were fed natural ingredient rodent diets supplemented with 3% DHA (DHA diet) or a control diet matched in total fat (CTRL diet). After 8weeks of feeding, we examined the RBCs for: 1) stiffness, as measured by atomic force microscopy; 2) deformability, as measured by ektacytometry; and 3) percent irreversibly sickled RBCs on peripheral blood smears. Using atomic force microscopy, it is found that stiffness is increased and deformability decreased in RBCs from SS mice fed CTRL diet compared to wild-type mice. In contrast, RBCs from SS mice fed DHA diet had markedly decreased stiffness and increased deformability compared to RBCs from SS mice fed CTRL diet. Furthermore, examination of peripheral blood smears revealed less irreversibly sickled RBCs in SS mice fed DHA diet as compared to CTRL diet. In summary, our findings indicate that DHA supplementation improves RBC flexibility and reduces irreversibly sickled cells by 40% in SS mice. These results point to potential therapeutic benefits of dietary omega-3 fatty acids in SCD.

Association of pro-inflammatory high-density lipoprotein cholesterol with clinical and laboratory variables in sickle cell disease.

Background Although cholesterol levels are known to be decreased in sickle cell disease (SCD), the level of pro-inflammatory high-density lipoprotein cholesterol (proHDL) and its association with clinical complications and laboratory variables has not been evaluated. Design and methods Plasma levels of total cholesterol, high-density lipoprotein cholesterol (HDL), proHDL, and selected clinical and laboratory variables were ascertained in a cohort of SCD patients and healthy African American control subjects in this single-center, cross-sectional study. Results Although total cholesterol was significantly lower in SCD patients compared with control subjects, HDL and proHDL levels were similar in both the SCD and control groups. In univariate analyses, proHDL was correlated with echocardiography-derived tricuspid regurgitant jet velocity. ProHDL was higher in SCD patients with suspected pulmonary hypertension (PHT) compared to patients without suspected PHT. ProHDL was positively correlated with lactate dehydrogenase, total bilirubin, direct bilirubin, indirect bilirubin, prothrombin fragment 1+2, D-dimer, and thrombin-antithrombin complexes. In multivariable analyses, only higher lactate dehydrogenase and direct bilirubin levels were associated with higher levels of proHDL. Conclusions SCD is characterized by hypocholesterolemia. Although proHDL is not increased in SCD patients compared with healthy controls, it is significantly associated with markers of liver disease. In addition, proHDL is associated with tricuspid regurgitant jet velocity and markers of coagulation, although these associations are not significant in multivariable analyses.

Sickle cell disease increases high mobility group box 1: a novel mechanism of inflammation.

High mobility group box 1 (HMGB1) is a chromatin-binding protein that maintains DNA structure. On cellular activation or injury, HMGB1 is released from activated immune cells or necrotic tissues and acts as a damage-associated molecular pattern to activate Toll-like receptor 4 (TLR4). Little is known concerning HMGB1 release and TLR4 activity and their role in the pathology of inflammation of sickle cell disease (SCD). Circulating HMGB1 levels were increased in both humans and mice with SCD compared with controls. Furthermore, sickle plasma increased HMGB1-dependent TLR4 activity compared with control plasma. HMGB1 levels were further increased during acute sickling events (vasoocclusive crises in humans or hypoxia/reoxygenation injury in mice). Anti-HMGB1 neutralizing antibodies reduced the majority of sickle plasma-induced TLR4 activity both in vitro and in vivo. These findings show that HMGB1 is the major TLR4 ligand in SCD and likely plays a critical role in SCD-mediated inflammation.

Cold hypersensitivity increases with age in mice with sickle cell disease.

Sickle cell disease (SCD) is associated with acute vaso-occlusive crises that trigger painful episodes and frequently involves ongoing, chronic pain. In addition, both humans and mice with SCD experience heightened cold sensitivity. However, studies have not addressed the mechanism(s) underlying the cold sensitization or its progression with age. Here we measured thermotaxis behavior in young and aged mice with severe SCD. Sickle mice had a marked increase in cold sensitivity measured by a cold preference test. Furthermore, cold hypersensitivity worsened with advanced age. We assessed whether enhanced peripheral input contributes to the chronic cold pain behavior by recording from C fibers, many of which are cold sensitive, in skin-nerve preparations. We observed that C fibers from sickle mice displayed a shift to warmer (more sensitive) cold detection thresholds. To address mechanisms underlying the cold sensitization in primary afferent neurons, we quantified mRNA expression levels for ion channels thought to be involved in cold detection. These included the transient receptor potential melastatin 8 (Trpm8) and transient receptor potential ankyrin 1 (Trpa1) channels, as well as the 2-pore domain potassium channels, TREK-1 (Kcnk2), TREK-2 (Kcnk10), and TRAAK (Kcnk4). Surprisingly, transcript expression levels of all of these channels were comparable between sickle and control mice. We further examined transcript expression of 83 additional pain-related genes, and found increased mRNA levels for endothelin 1 and tachykinin receptor 1. These factors may contribute to hypersensitivity in sickle mice at both the afferent and behavioral levels.

Circulating membrane-derived microvesicles in redox biology.

Microparticles or microvesicles (MVs) are subcellular membrane blebs shed from all cells in response to various stimuli. MVs carry a battery of signaling molecules, many of them related to redox-regulated processes. The role of MVs, either as a cause or as a result of cellular redox signaling, has been increasingly recognized over the past decade. This is in part due to advances in flow cytometry and its detection of MVs. Notably, recent studies have shown that circulating MVs from platelets and endothelial cells drive reactive species-dependent angiogenesis; circulating MVs in cancer alter the microenvironment and enhance invasion through horizontal transfer of mutated proteins and nucleic acids and harbor redox-regulated matrix metalloproteinases and procoagulative surface molecules; and circulating MVs from red blood cells and other cells modulate cell-cell interactions through scavenging or production of nitric oxide and other free radicals. Although our recognition of MVs in redox-related processes is growing, especially in the vascular biology field, much remains unknown regarding the various biologic and pathologic functions of MVs. Like reactive oxygen and nitrogen species, MVs were originally believed to have a solely pathological role in biology. And like our understanding of reactive species, it is now clear that MVs also play an important role in normal growth, development, and homeostasis. We are just beginning to understand how MVs are involved in various biological processes-developmental, homeostatic, and pathological-and the role of MVs in redox signaling is a rich and exciting area of investigation.

Anion exchange HPLC isolation of high-density lipoprotein (HDL) and on-line estimation of proinflammatory HDL.

Proinflammatory high-density lipoprotein (p-HDL) is a biomarker of cardiovascular disease. Sickle cell disease (SCD) is characterized by chronic states of oxidative stress that many consider to play a role in forming p-HDL. To measure p-HDL, apolipoprotein (apo) B containing lipoproteins are precipitated. Supernatant HDL is incubated with an oxidant/LDL or an oxidant alone and rates of HDL oxidation monitored with dichlorofluorescein (DCFH). Although apoB precipitation is convenient for isolating HDL, the resulting supernatant matrix likely influences HDL oxidation. To determine effects of supernatants on p-HDL measurements we purified HDL from plasma from SCD subjects by anion exchange (AE) chromatography, determined its rate of oxidation relative to supernatant HDL. SCD decreased total cholesterol but not triglycerides or HDL and increased cell-free (cf) hemoglobin (Hb) and xanthine oxidase (XO). HDL isolated by AE-HPLC had lower p-HDL levels than HDL in supernatants after apoB precipitation. XO+xanthine (X) and cf Hb accelerated purified HDL oxidation. Although the plate and AE-HPLC assays both showed p-HDL directly correlated with cf-Hb in SCD plasma, the plate assay yielded p-HDL data that was influenced more by cf-Hb than AE-HPLC generated p-HDL data. The AE-HPLC p-HDL assay reduces the influence of the supernatants and shows that SCD increases p-HDL.

Intravenous magnesium for pediatric sickle cell vaso-occlusive crisis: methodological issues of a randomized controlled trial.

Multiple recent Sickle Cell Disease studies have been terminated due to poor enrollment. We developed methods to overcome past barriers and utilized these to study the efficacy and safety of intravenous magnesium for vaso-occlusive crisis (VOC). We describe the methods of the Intravenous Magnesium in Sickle Vaso-occlusive Crisis (MAGiC) trial and discuss methods used to overcome past barriers. MAGiC was a multi-center randomized double-blind placebo-controlled trial of intravenous magnesium versus normal saline for treatment of VOC. The study was a collaboration between Pediatric Hematologists and Emergency Physicians in the Pediatric Emergency Care Applied Research Network (PECARN). Eligible patients were randomized within 12 hours of receiving intravenous opioids in the Emergency Department (ED) and administered study medication every 8 hours. The primary outcome was hospital length of stay. Associated plasma studies elucidated magnesium's mechanism of action and the pathophysiology of VOC. Health-related quality of life was measured. Site-, protocol-, and patient-related barriers from prior studies were identified and addressed. Limited study staff availability, lack of collaboration with the ED, and difficulty obtaining consent were previously identified barriers. Leveraging PECARN resources, forging close collaborations between Sickle Cell Centers and EDs of participating sites, and approaching eligible patients for prior consent helped overcome these barriers. Participation in the PECARN network and establishment of collaborative arrangements between Sickle Cell Centers and their affiliated EDs are major innovative features of the MAGiC study that allowed improved subject capture. These methods could serve as a model for future studies of VOCs.