Courtney D. Thornburg

Translocation of sickle cell erythrocyte microRNAs into Plasmodium falciparum inhibits parasite translation and contributes to malaria resistance

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.

Hydroxycarbamide in very young children with sickle-cell anaemia: a multicentre, randomised, controlled trial (BABY HUG)

BACKGROUND Sickle-cell anaemia is associated with substantial morbidity from acute complications and organ dysfunction beginning in the first year of life. Hydroxycarbamide substantially reduces episodes of pain and acute chest syndrome, admissions to hospital, and transfusions in adults with sickle-cell anaemia. We assessed the effect of hydroxycarbamide therapy on organ dysfunction and clinical complications, and examined laboratory findings and toxic effects. METHODS This randomised trial was undertaken in 13 centres in the USA between October, 2003, and September, 2009. Eligible participants had haemoglobin SS (HbSS) or haemoglobin Sβ(0)thalassaemia, were aged 9-18 months at randomisation, and were not selected for clinical severity. Participants received liquid hydroxycarbamide, 20 mg/kg per day, or placebo for 2 years. Randomisation assignments were generated by the medical coordinating centre by a pre-decided schedule. Identical appearing and tasting formulations were used for hydroxycarbamide and placebo. Patients, caregivers, and coordinating centre staff were masked to treatment allocation. Primary study endpoints were splenic function (qualitative uptake on (99)Tc spleen scan) and renal function (glomerular filtration rate by (99m)Tc-DTPA clearance). Additional assessments included blood counts, fetal haemoglobin concentration, chemistry profiles, spleen function biomarkers, urine osmolality, neurodevelopment, transcranial Doppler ultrasonography, growth, and mutagenicity. Study visits occurred every 2-4 weeks. Analysis was by intention to treat. The trial is registered with ClinicalTrials.gov, number NCT00006400. FINDINGS 96 patients received hydroxycarbamide and 97 placebo, of whom 83 patients in the hydroxycarbamide group and 84 in the placebo group completed the study. Significant differences were not seen between groups for the primary endpoints (19 of 70 patients with decreased spleen function at exit in the hydroxycarbamide group vs 28 of 74 patients in the placebo group, p=0·21; and a difference in the mean increase in DTPA glomerular filtration rate in the hydroxycarbamide group versus the placebo group of 2 mL/min per 1·73 m(2), p=0·84). Hydroxycarbamide significantly decreased pain (177 events in 62 patients vs 375 events in 75 patients in the placebo group, p=0·002) and dactylitis (24 events in 14 patients vs 123 events in 42 patients in the placebo group, p<0·0001), with some evidence for decreased acute chest syndrome, hospitalisation rates, and transfusion. Hydroxyurea increased haemoglobin and fetal haemoglobin, and decreased white blood-cell count. Toxicity was limited to mild-to-moderate neutropenia. INTERPRETATION On the basis of the safety and efficacy data from this trial, hydroxycarbamide can now be considered for all very young children with sickle-cell anaemia. FUNDING The US National Heart, Lung, and Blood Institute; and the National Institute of Child Health and Human Development.

Impact of hydroxyurea on clinical events in the BABY HUG trial

The Pediatric Hydroxyurea Phase 3 Clinical Trial (BABY HUG) was a phase 3 multicenter, randomized, double-blind, placebo-controlled clinical trial of hydroxyurea in infants (beginning at 9-18 months of age) with sickle cell anemia. An important secondary objective of this study was to compare clinical events between the hydroxyurea and placebo groups. One hundred and ninety-three subjects were randomized to hydroxyurea (20 mg/kg/d) or placebo; there were 374 patient-years of on-study observation. Hydroxyurea was associated with statistically significantly lower rates of initial and recurrent episodes of pain, dactylitis, acute chest syndrome, and hospitalization; even infants who were asymptomatic at enrollment had less dactylitis as well as fewer hospitalizations and transfusions if treated with hydroxyurea. Despite expected mild myelosuppression, hydroxyurea was not associated with an increased risk of bacteremia or serious infection. These data provide important safety and efficacy information for clinicians considering hydroxyurea therapy for very young children with sickle cell anemia. This clinical trial is registered with the National Institutes of Health (NCT00006400, www.clinicaltrials.gov).

Adherence to hydroxyurea therapy in children with sickle cell anemia.

OBJECTIVES To assess adherence to hydroxyurea therapy in children with sickle cell anemia (SCA), evaluate the association between adherence and hematologic profile, and identify barriers and facilitators of adherence. STUDY DESIGN Children with SCA (n=75) receiving hydroxyurea were recruited for a single-institution cross-sectional study. The primary outcome was association between treatment adherence and percent fetal hemoglobin (HbF). RESULTS Good adherence was estimated at 82% with visual analog scale, 84% with Morisky score, 85% with medical provider report, 77% with clinic visits, and 49% on the basis of pharmacy refills. Increase in HbF was moderately associated with good adherence as measured with the parent/proxy Morisky score (r=-0.39; 95% CI, -0.58-0.17; P < .01) and prescription refills (r=0.39; 95% CI, 0.16-0.57; P < .01). The number of pharmacy refills and the Morisky score explained 23% of the variation in HbF response. CONCLUSIONS Adherence was > or =75% with 4 of 5 measures. Pharmacy refills and the Modified Morisky Scale may be used to identify children at high risk for poor response because of non-adherence and children with good adherence with poor response because of individual pharmacodynamics. Future research should prospectively compare adherence measures and evaluate methods to improve treatment adherence.

Induction of cytotoxic T lymphocytes with dendritic cells transfected with Human papillomavirus E6 and E7 RNA : Implications for cervical cancer immunotherapy

Human papillomavirus (HPV) infection is associated with cervical cancer. The high-risk HPV E6 and E7 oncoproteins are constitutively expressed in most cervical carcinoma cells, and are, therefore, attractive antigens for cytotoxic T-lymphocyte (CTL)–mediated immunotherapy. The objective of this study was to evaluate the use of dendritic cells (DCs) transfected with RNA encoding the E6 and E7 protein for cervical cancer immunotherapy. The authors have shown that DCs transfected with RNA-encoding antigen stimulate potent antigen-specific CTL responses in vitro and in vivo. In this study, they tried to determine whether DCs transfected with E6 and E7 RNA stimulate primary, antigen-specific CTL responses in vitro. The results show that DCs pulsed with E6 or E7 RNA stimulate antigen-specific CTL responses that recognize and lyse DCs transfected with E6 and E7 RNA and human cervical carcinoma cells expressing the E6 and E7 products, and the lysis was comparable to that achieved with E6 and E7 peptide-pulsed DCs. Dendritic cells cotransfected with both E6 and E7 RNA stimulate CTLs that are more effective at lysing human cervical cancer cells. This study provides a rationale for the development of cervical carcinoma immunotherapy using DCs transfected with HPV E6 and E7 RNA.

A pilot study of hydroxyurea to prevent chronic organ damage in young children with sickle cell anemia.

Hydroxyurea improves laboratory parameters and prevents acute clinical complications of sickle cell anemia (SCA) in children and adults, but its effects on organ function remain incompletely defined.

Bleeding risks are higher in children versus adults given prophylactic platelet transfusions for treatment-induced hypoproliferative thrombocytopenia

Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 10(11), 2.2 × 10(11), or 4.4 × 10(11) platelets/m(2) per transfusion, given for morning counts of ≤ 10 000 platelets/μL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P < .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts.

Linkage analysis identifies a locus for plasma von Willebrand factor undetected by genome-wide association

The plasma glycoprotein von Willebrand factor (VWF) exhibits fivefold antigen level variation across the normal human population determined by both genetic and environmental factors. Low levels of VWF are associated with bleeding and elevated levels with increased risk for thrombosis, myocardial infarction, and stroke. To identify additional genetic determinants of VWF antigen levels and to minimize the impact of age and illness-related environmental factors, we performed genome-wide association analysis in two young and healthy cohorts (n = 1,152 and n = 2,310) and identified signals at ABO (P < 7.9E-139) and VWF (P < 5.5E-16), consistent with previous reports. Additionally, linkage analysis based on sibling structure within the cohorts, identified significant signals at chromosome 2q12–2p13 (LOD score 5.3) and at the ABO locus on chromosome 9q34 (LOD score 2.9) that explained 19.2% and 24.5% of the variance in VWF levels, respectively. Given its strong effect, the linkage region on chromosome 2 could harbor a potentially important determinant of bleeding and thrombosis risk. The absence of a chromosome 2 association signal in this or previous association studies suggests a causative gene harboring many genetic variants that are individually rare, but in aggregate common. These results raise the possibility that similar loci could explain a significant portion of the “missing heritability” for other complex genetic traits.

Differences in health-related quality of life in children with sickle cell disease receiving hydroxyurea.

Hydroxyurea is a safe and efficacious medication for children with sickle cell disease (SCD). Our objective was to compare health-related quality of life (HRQL) between children taking hydroxyurea and those not taking hydroxyurea. We conducted a retrospective cohort study of children with SCD who had completed the PedsQL 4.0 at Duke University Medical Center or the Midwest Sickle Cell Center. Our primary outcome was HRQL in children receiving hydroxyurea therapy compared with those not receiving hydroxyurea. One hundred and ninety-one children with SCD were included in the study. Children in the hydroxyurea group had higher self-reported Total PedsQL median scores than children in the no hydroxyurea group (P=0.04). Child self-reported physical functioning scores were significantly higher for children in the hydroxyurea group (P=0.01). In conclusion, children with SCD who received hydroxyurea therapy reported better overall HRQL and better physical HRQL than children who did not receive this therapy despite disease severity. Further research assessing the impact of hydroxyurea therapy on HRQL, such as prospective assessment over time, would aid in our understanding of the effectiveness of hydroxyurea for individual children. Ultimately, this may aid in decreasing the barriers to the use of hydroxyurea.

The pediatric hydroxyurea phase III clinical trial (BABY HUG): Challenges of study design

Evidence of the laboratory benefits of hydroxyurea and its clinical efficacy in reducing acute vaso‐occlusive events in adults and children with sickle cell anemia has accumulated for more than 15 years. A definitive clinical trial showing that hydroxyurea can also prevent organ damage might support widespread use of the drug at an early age. BABY HUG is a randomized, double‐blind placebo‐controlled trial to test whether treating young children ages 9–17 months at entry with a liquid preparation of hydroxyurea (20 mg/kg/day for 2 years) can decrease organ damage in the kidneys and spleen by at least 50%. Creation of BABY HUG entailed unique challenges and opportunities. Although protection of brain function might be considered a more compelling endpoint, preservation of spleen and renal function has clinical relevance, and significant treatment effects might be discernable within the mandated sample size of 200. Concerns about unanticipated severe toxicity and burdensome testing and monitoring requirements were addressed in part by an internal Feasibility and Safety Pilot Study, the successful completion of which was required prior to enrolling a larger number of children on the protocol. Concerns over recruitment of potentially vulnerable subjects were allayed by inclusion of a research subject advocate, or ombudsman. Finally, maintenance of blinding of research personnel was aided by inclusion of an unblinded primary endpoint person, charged with transmitting endpoint data and monitoring blood work locally for toxicity (ClinicalTrials.gov number, NCT00006400). Pediatr Blood Cancer 2010;54:250–255.