Cheryl A. Hillery

Hydroxyurea therapy in children severely affected with sickle cell disease

HYPOTHESIS The major clinical manifestations of sickle cell disease (SCD) are hemolytic anemia, predisposition to infection, and recurrent vaso-occlusive episodes resulting in pain, organ dysfunction, or both. There has been no satisfactory treatment for children with recurrent severe painful episodes caused by SCD. Hydroxyurea is an antimetabolite drug shown in adults with SCD to increase fetal hemoglobin levels and reduce the symptoms of SCD. We hypothesized that hydroxyurea therapy in children with severe (defined as > or = 3 vaso-occlusive events per year) SCD could improve hematologic parameters and reduce vaso-occlusive events. OBJECTIVE To assess the safety and efficacy of hydroxyurea for the treatment of severe SCD in children. STUDY DESIGN After obtaining informed consent, we initiated hydroxyurea therapy at a dosage of 10 to 20 mg/kg per day in 15 patients with severe SCD (hemoglobin SS, hemoglobin SS-alpha thalassemia, or hemoglobin S-beta0-thalassemia). Doses were escalated as tolerated. Patients were monitored with bimonthly physical examinations and monthly laboratory measures. To assess the impact of hydroxyurea on clinical symptoms, we recorded the number of inpatient days for each patient during the period of treatment and compared it with the number of inpatient days for the 12- to 24-month period before institution of hydroxyurea therapy, using the subject as his or her own control subject. RESULTS Thirteen patients received hydroxyurea for a median of 24 months (range, 6 to 39 months). The mean dose of hydroxyurea was 21.4 +/- 5.2 mg/kg. Treatment with hydroxyurea induced statistically significant increases in the total hemoglobin concentration, mean corpuscular volume, and percentage of hemoglobin F, and a decrease in the serum concentration of bilirubin. Toxic effects included three episodes of a reversible myelotoxic reaction, two of which required transfusion of packed erythrocytes. For the entire group, hospitalization decreased from a prehydroxyurea median of 3.9 +/- 2.0 days per month to 1.1 +/- 2.1 days per month of therapy (p = 0.09). For those subjects (n = 10) completing at least 1 year of treatment, the decrease in hospitalization from 4.1 +/- 2.2 to 1.0 +/- 1.7 days per month was significant (p = 0.03). CONCLUSIONS In this pilot trial, hydroxyurea treatment of severe SCD in children was associated with improved hematologic parameters, acceptable toxic effects, and a trend to reduced hospitalization. Hydroxyurea appears to be a safe and potentially effective agent for the treatment of severe SCD in children. A prospective, controlled trial to investigate the efficacy of hydroxyurea in children is therefore warranted.

L-4F, an Apolipoprotein A-1 Mimetic, Dramatically Improves Vasodilation in Hypercholesterolemia and Sickle Cell Disease

Background—Hypercholesterolemia and sickle cell disease (SCD) impair endothelium-dependent vasodilation by dissimilar mechanisms. Hypercholesterolemia impairs vasodilation by a low-density lipoprotein (LDL)–dependent mechanism. SCD has been characterized as a chronic state of inflammation in which xanthine oxidase (XO) from ischemic tissues increases vascular superoxide anion (O2·−) generation. Recent reports indicate that apolipoprotein (apo) A-1 mimetics inhibit atherosclerosis in LDL receptor–null (Ldlr−/−) mice fed Western diets. Here we hypothesize that L-4F, an apoA-1 mimetic, preserves vasodilation in hypercholesterolemia and SCD by decreasing mechanisms that increase O2·− generation. Methods and Results—Arterioles were isolated from hypercholesterolemic Ldlr−/− mice and from SCD mice that were treated with either saline or L-4F (1 mg/kg per day). Vasodilation in response to acetylcholine was determined by videomicroscopy. Effects of L-4F on LDL-induced increases in endothelium-dependent O2·− generation were determined on arterial segments via the hydroethidine assay and on stimulated endothelial cell cultures via superoxide dismutase–inhibitable ferricytochrome c reduction. Effects of L-4F on XO bound to pulmonary arterioles and content in livers of SCD mice were determined by immunofluorescence. Hypercholesterolemia impaired vasodilation in Ldlr−/− mice, which L-4F dramatically improved. L-4F inhibited LDL-induced increases in O2·− in arterial segments and in stimulated cultures. SCD impaired vasodilation, increased XO bound to pulmonary endothelium, and decreased liver XO content. L-4F dramatically improved vasodilation, decreased XO bound to pulmonary endothelium, and increased liver XO content compared with levels in untreated SCD mice. Conclusions—These data show that L-4F protects endothelium-dependent vasodilation in hypercholesterolemia and SCD. Our findings suggest that L-4F restores vascular endothelial function in diverse models of disease and may be applicable to treating a variety of vascular diseases.

Coagulation activation and inflammation in sickle cell disease-associated pulmonary hypertension.

Patients with sickle cell disease-associated pulmonary hypertension have increased endothelial dysfunction, coagulation activation and inflammation compared with patients without pulmonary hypertension. Endothelial dysfunction and coagulation activation appear to be the result of chronic hemolysis. See related perspective on page 1. Background Pulmonary hypertension (PHT) is common in sickle cell disease (SCD). The purpose of this study was to determine whether markers of coagulation activation and inflammation are associated with PHT in SCD. Design and Methods This cross-sectional study was performed using a cohort of patients followed at an adult Sickle Cell Clinic. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age, sex and body mass index-adjusted reference ranges. Clinical laboratory examinations, including hematologic studies and biochemical tests, as well as various measures of coagulation activation, endothelial activation and inflammation, were conducted on SCD subjects and on healthy, race-matched control subjects without SCD. Results Patients with SCD (n=76) had higher plasma levels of markers of coagulation (thrombin-antithrombin complex, prothrombin fragment F1+2, D-dimer) and endothelial (soluble vascular endothelial cell adhesion molecule, sVCAM) activation compared with control subjects (n=6). SCD patients with PHT (n=26) had significantly higher levels of sVCAM compared with those patients without PHT (n=50). Although PHT patients showed increased plasma measures of coagulation activation, the differences were not statistically significant when compared to those of patients without PHT. HbSS patients with PHT also had a trend towards higher levels of other inflammatory cytokines (interleukins 6, 8 and 10) than HbSS patients without PHT. There was a modest negative correlation between hemoglobin and plasma measures of coagulation and endothelial activation, and modest positive correlations between markers of hemolysis and plasma measures of coagulation and endothelial activation. Conclusions SCD patients with PHT have higher levels of markers of endothelial activation and other inflammatory markers than patients without PHT. A trend towards an increased level of markers of coagulation activation was observed in SCD patients with PHT compared with that in patients without PHT. Markers of hemolysis are associated with coagulation activation and endothelial dysfunction in SCD patients. Clinical trials of anticoagulants and anti-inflammatory agents are warranted in SCD patients with PHT.

Hydroxyurea therapy decreases the in vitro adhesion of sickle erythrocytes to thrombospondin and laminin

The adhesion of sickle erythrocytes to the vascular endothelium and subendothelial matrix probably contributes to the pathogenesis of vaso‐occlusive disease. The chemotherapeutic agent hydroxyurea (HU) decreases the frequency of vaso‐occlusive crises in patients with sickle cell disease. However, the exact mechanism(s) of HUs effect on vaso‐occlusive crises is not fully understood. The goal of this study was to determine the effect of HU therapy on the adhesion of sickle erythrocytes to the subendothelial matrix proteins thrombospondin (TSP) and laminin under conditions of flow in vitro. Erythrocytes from patients with severe sickle cell disease on HU therapy (n = 14) had significantly less adhesion to TSP (687 ± 92 erythrocytes/mm2, mean ± SE) than untreated patients with severe disease (n = 18, 1176 ± 117 erythrocytes/mm2, P = 0·003). In addition, there was significantly less adhesion of erythrocytes to immobilized laminin in patients treated with HU (1695 ± 293 erythrocytes/mm2) than in the untreated patients (2590 ± 296 erythrocytes/mm2, P = 0·02). Erythrocytes from an additional nine patients with severe sickle cell disease were studied both before and after initiation of HU therapy. Erythrocytes from these patients became less adhesive to both TSP (P = 0·001) and laminin (P = 0·01), a change that was sustained in most patients throughout the duration of the study (2 months to > 12 months). This study suggests that HU modulates the adhesive phenotype of sickle erythrocytes, an effect that may be in addition to, or independent of, other known effects of HU, such as an increase in fetal haemoglobin level.

Clinical and Hematologic Benefits of Partial Splenectomy for Congenital Hemolytic Anemias in Children

ObjectiveTo assess the role of partial splenectomy for symptomatic children with various congenital hemolytic anemias. Summary Background DataThe use of total splenectomy for symptomatic children with congenital hemolytic anemias is restricted by concern of postsplenectomy sepsis. A partial splenectomy is an alternative procedure, although its utility remains incompletely defined. MethodsThis longitudinal cohort study followed 25 symptomatic children with various congenital anemias who underwent partial splenectomy. Sixteen children had hereditary spherocytosis (HS), and nine children had other erythrocyte disorders. Outcome measures were clinical and laboratory hemolysis, splenic phagocytic and immune function, and splenic regrowth as measured by ultrasonography. Discrete parameters were compared using the Student t test. ResultsPartial splenectomy was successful in all 25 children, with minimal morbidity. Follow-up ranged from 7 months to 6 years (mean 2.3 ± 1.5 years). Following surgery, children with HS had increased hemoglobin values, decreased reticulocyte and bilirubin levels, and preserved splenic function. Most children without HS had decreased symptoms of hypersplenism and splenic sequestration. Over time, variable rates of splenic regrowth were noted, although regrowth did not necessarily correlate with recurrent hemolysis. ConclusionsIn children with hereditary spherocytosis, a partial splenectomy appears to control hemolysis while retaining splenic function. In children with other congenital hemolytic anemias, a partial splenectomy appears to control symptoms of hypersplenism and splenic sequestration.

Transient receptor potential vanilloid 1 mediates pain in mice with severe sickle cell disease.

Pain is the leading cause of emergency department visits, hospitalizations, and daily suffering in individuals with sickle cell disease (SCD). The pathologic mechanisms leading to the perception of pain during acute RBC sickling episodes and development of chronic pain remain poorly understood and ineffectively treated. We provide the first study that explores nociceptor sensitization mechanisms that contribute to pain behavior in mice with severe SCD. Sickle mice exhibit robust behavioral hypersensitivity to mechanical, cold, and heat stimuli. Mechanical hypersensitivity is further exacerbated when hypoxia is used to induce acute sickling. Behavioral mechanical hypersensitivity is mediated in part by enhanced excitability to mechanical stimuli at both primary afferent peripheral terminal and sensory membrane levels. In the present study, inhibition of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) with the selective antagonist A-425619 reversed the mechanical sensitization at both primary afferent terminals and isolated somata, and markedly attenuated mechanical behavioral hypersensitivity. In contrast, inhibition of TRPA1 with HC-030031 had no effect on mechanical sensitivity. These results suggest that the TRPV1 receptor contributes to primary afferent mechanical sensitization and a substantial portion of behavioral mechanical hypersensitivity in SCD mice. Therefore, TRPV1-targeted compounds that lack thermoregulatory side effects may provide relief from pain in patients with SCD.

Low-molecular-weight heparin in thrombotic disease in children and adolescents.

PURPOSE The use of unfractionated heparin (UFH) in children is problematic. In adults, subcutaneous low-molecular-weight heparin (LMWH) is as effective as UFH in the treatment of thrombosis. Because pediatric data are limited, the authors studied the use of enoxaparin in children. PATIENTS AND METHODS Nineteen children (ages 18 days to 19 years; median age, 40 months) with indications for thrombosis treatment or prophylaxis were studied. Six patients (median age, 33 months), treated on a protocol that included pharmacokinetic studies, initially received enoxaparin 1 mg/kg subcutaneously every 12 hours; doses then were adjusted until target plasma levels of 0.5 to 1.2 anti-Xa U/mL were achieved. The records of 13 additional patients treated with enoxaparin off study were reviewed. RESULTS In the first six patients, enoxaparin pharmacokinetics was found to be similar to that in adults; once targeted levels were achieved, these remained stable. Among all 19 subjects, 14 had treatment of active thrombosis and 5 underwent thrombosis prophylaxis. For treatment of thrombosis, enoxaparin 1 mg/kg initially was administered subcutaneously every 12 hours. Target anti-Xa levels were achieved with 0.55 to 1.5 mg/kg every 12 hours (mean, 0.98 mg/kg; median, 1.0 mg/kg) in 1 to 7 days (median, 1 day). All patients in the treatment group had clinical improvement within 2 to 5 days, and 12 had follow-up radiological studies that confirmed this. For prophylaxis, enoxaparin was given at 1 mg/kg subcutaneously every 24 hours. No new thrombi were clinically evident in this group. There was no major bleeding with enoxaparin; one patient had transient mild mucosal oozing. CONCLUSION In this limited population, enoxaparin seems to be a safe, effective, and convenient alternative to UFH in children and adolescents. The adult therapeutic target range of 0.5 to 1.2 anti-Xa U/mL is readily achievable with a starting dose of 1 mg/kg every 12 hours in most children. Initial close monitoring with plasma anti-Xa activity should be done and doses adjusted to achieve target range, particularly in neonates. In the population of this study, enoxaparin seems as effective as UFH in the period immediately thrombotic episode. These results should be confirmed in the ongoing randomized trial comparing LMWH with UFH in children.

Pulmonary hypertension and NO in sickle cell.

To the editor: We are honored that our work merits the attention of Dr Bunn and colleagues[1][1] but disagree with some of their conclusions. Traditional risk factors for vaso-occlusive pain crisis, such as leukocytosis and high hemoglobin levels, incompletely predict vasculopathic events and

D-4F, an apoA-1 mimetic, decreases airway hyperresponsiveness, inflammation, and oxidative stress in a murine model of asthma

Asthma is characterized by oxidative stress and inflammation of the airways. Although proinflammatory lipids are involved in asthma, therapies targeting them remain lacking. Ac-DW F KA F YDKVAEK F KEA F NH2 (4F) is an apolipoprotein (apo)A-I mimetic that has been shown to preferentially bind oxidized lipids and improve HDL function. The objective of the present study was to determine the effects of 4F on oxidative stress, inflammation, and airway resistance in an established murine model of asthma. We show here that ovalbumin (OVA)-sensitization increased airway hyperresponsiveness, eosinophil recruitment, and collagen deposition in lungs of C57BL/6J mice by a mechanism that could be reduced by 4F. OVA sensitization induced marked increases in transforming growth factor (TGF)β-1, fibroblast specific protein (FSP)-1, anti-T15 autoantibody staining, and modest increases in 4-hydroxynonenal (4-HNE) Michaels adducts in lungs of OVA-sensitized mice. 4F decreased TGFβ-1, FSP-1, anti-T15 autoantibody, and 4-HNE adducts in the lungs of the OVA-sensitized mice. Eosinophil peroxidase (EPO) activity in bronchial alveolar lavage fluid (BALF), peripheral eosinophil counts, total IgE, and proinflammatory HDL (p-HDL) were all increased in OVA-sensitized mice. 4F decreased BALF EPO activity, eosinophil counts, total IgE, and p-HDL in these mice. These data indicate that 4F reduces pulmonary inflammation and airway resistance in an experimental murine model of asthma by decreasing oxidative stress.

Phosphatidylethanolamine is externalized at the surface of microparticles.

Microparticles (MPs) are membrane-bound vesicles shed normally or as a result of various (pathological) stimuli. MPs contain a wealth of bio-active macromolecules. Aminophospholipid phosphatidylserine (PS) is present on the surface of many MPs. As PS and phosphatidylethanolamine (PE) are related, yet distinct aminophospholipids, the purpose of this study was to systematically and directly assess PE exposure on MPs. We examined MPs from various human cellular sources (human breast cancer, endothelial, red and white blood cells) by flow cytometry using a PE-specific probe, duramycin, and two PS-specific probes, annexin V and lactadherin. PS and PE exposure percentage was comparable on vascular and blood cell-derived MPs (80-90% of MP-gated events). However, the percentage of malignant breast cancer MPs exposing PE (~90%) was significantly higher than PS (~50%). Thus, while PS and PE exposure can result from a general loss of membrane asymmetry, there may also be distinct mechanisms of PE and PS exposure on MPs that vary by cellular source.