Hydroxyurea at escalated dose versus fixed low-dose hydroxyurea in adults with sickle cell disease.

Hydroxyurea reduces the frequency of vaso-occlusive complications, increases hemoglobin, and decreases mortality in sickle cell disease (SCD). Although current guidelines recommend escalation to maximum tolerated dose (MTD), the use of fixed low-dose hydroxyurea is common in low-resource countries. We conducted a systematic review and meta-analysis to evaluate the efficacy of escalated doses versus fixed low-dose of hydroxyurea in adults with SCD. Nine studies were included in the quantitative synthesis, four evaluating fixed low-dose and five evaluating escalated doses of hydroxyurea. Average daily doses of hydroxyurea in the fixed low-dose and escalated dose studies were ~10 and 22 mg/kg, respectively. There was no difference in the estimate of vaso-occlusive crisis rate between escalated and fixed low-dose studies (p = .73). The mean difference in hemoglobin from baseline to follow-up was greater for fixed low-dose than escalated dose studies (1.07 g/dL vs. 0.54 g/dL, p = .01). No difference was seen in the mean estimate of fetal hemoglobin. Despite limited eligible studies and substantial heterogeneity of effect between the studies for several outcomes, there appears to be clinical equipoise regarding the most appropriate hydroxyurea dosing regimen in adults with SCD. Controlled studies of hydroxyurea at MTD versus fixed low-dose in adults with SCD are required.

Urinary angiotensinogen is associated with albuminuria in adults with sickle cell anaemia.

We explored the association of novel urinary biomarkers with albumin-creatinine ratio (ACR) in adults with sickle cell anaemia. Of 37 participants, 13 (35.2%) had persistent albuminuria (PA). Urinary levels of clusterin (p = 0.002), retinol-binding protein 4 (p = 0.008), alpha-1 microglobulin (p = 0.002) and angiotensinogen (p = 0.006) were significantly higher in participants with PA than in those without PA. Although univariate analysis showed significant associations between both alpha-1 microglobulin (p = 0.035) and angiotensinogen (p = 0.0021) with ACR, only angiotensinogen was associated with ACR in multivariable analysis (p = 0.04). Our results suggest that urinary angiotensinogen may identify sickle cell anaemia patients at risk for kidney disease.

MicroRNA29B induces fetal hemoglobin via inhibition of the HBG repressor protein MYB in vitro and in humanized sickle cell mice.

Introduction: Therapeutic strategies aimed at reactivating HBG gene transcription and fetal hemoglobin (HbF) synthesis remain the most effective strategy to ameliorate the clinical symptoms of sickle cell disease (SCD). We previously identified microRNA29B (MIR29B) as a novel HbF inducer via targeting enzymes involved in DNA methylation. We provided further evidence that the introduction of MIR29B into KU812 leukemia cells significantly reduced MYB protein expression. Therefore, the aim of this study was to determine the extent to which MIR29B mediates HbF induction via targeting MYB in KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF induction in vivo in the humanized Townes SCD mouse model. Materials and methods: Human KU812 were cultured and normal CD34 cells (n = 3) were differentiated using a two-phase erythropoiesis culturing system and transfected with MIR29B (50 and 100 nM) mimic or Scrambled (Scr) control in vitro. A luciferase reporter plasmid overexpressing MYB was transfected into KU812 cells. Luciferase activity was quantified after 48 h. Gene expression was determined by quantitative real-time PCR. In vivo studies were conducted using Townes SCD mice (6 per group) treated with MIR29B (2, 3, and 4 mg/kg/day) or Scr control by 28-day continuous infusion using subcutaneous mini osmotic pumps. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes at weeks 0, 2, and 4. Flow cytometry was used to measure the percentage of HbF-positive cells. Results: In silico analysis predicted complementary base-pairing between MIR29B and the 3'-untranslated region (UTR) of MYB. Overexpression of MIR29B significantly reduced MYB mRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-length MYB 3'-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-length MYB 3'-UTR as compared to cells that only expressed MYB 3'-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpress MYB on HBG activation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B and MYB in both cell types further demonstrated the inhibitory effect of MIR29B on MYB expression, resulting in HBG reactivation by real-time PCR, Western blot, and flow cytometry analysis. Finally, we confirmed the ability of MIR29B to reduce sickling and induce HbF by decreasing expression of MYB and DNMT3 gene expression in the humanized Townes sickle cell mouse model. Discussion: Our findings support the ability of MIR29B to induce HbF in vivo in Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulate HBG transcription by MYB gene silencing in vivo. Our research highlights a novel MIR-based epigenetic approach to induce HbF supporting the discovery of new drugs to expand treatment options for SCD.

Sickle cell disease in the new era: advances in drug treatment.

Sickle cell disease is an inherited blood disorder afflicting an estimated 100,000 individuals in the United States and over 20 million people worldwide. The disease is heralded as the first molecular disease. However, despite its genetic simplicity, the pathophysiologic processes leading to its clinical sequelae are complex, heterogeneous and interrelated, making drug development to treat the disease challenging. For over two decades only one drug, hydroxyurea, had been used as disease-modifying therapy. New pharmacologic agents are rapidly evolving with three new drugs, with different mechanisms of action, approved by the United States Food and Drug Administration in recent years (L-glutamine, crizanlizumab and voxelotor). Several therapeutic approaches targeting different pathways in the disease pathophysiology are being investigated. These include inhibition of hemoglobin S polymerization such as by fetal hemoglobin induction or by increasing hemoglobin oxygen affinity, as well as intervention of downstream pathways including inhibiting cellular adhesion, reducing inflammation and oxidant stress, modulating platelet activation and coagulation abnormalities, and targeting nitric oxide signaling. This review will provide an overview of these therapeutic strategies, discuss the four currently approved drugs in detail, and summarize ongoing clinical trials of new drugs or drug indications for the treatment of sickle cell disease in different phases of development excluding those related to cellular therapies.

Hydroxyurea and fetal hemoglobin effect on leg ulcers in patients with sickle cell disease.

The presence of leg ulcers in individuals with sickle cell disease often represents an early sign of vasculopathy and future end organ damage. Pathophysiological mechanisms of formation and evolution of leg ulcers are poorly understood; nevertheless, HbF has been associated with lower incidence of leg ulcers, while hydroxyurea has been correlated with high risk of leg ulcers. As a result, there is hesitation regarding hydroxyurea use in patients with SCD and leg ulcers. In this study, we aim to define (1) a target of HbF that offers protection against leg ulcer development and (2) the impact of hydroxyurea therapy on leg ulcer prevalence. Our study demonstrated that in order to reduce leg ulcer incidence by one-third, a HbF > 25% is needed, a threshold not commonly reached and maintained in the adult SCD population. Importantly, leg ulcer incidence appears to be independent of HU use (p = 0.50). Our interpretation of this data is that the use of HU in a patient with SCD and leg ulcers should be guided by a careful assessment of risks and benefits of this therapeutic modality.

First Report of Compound Heterozygosity for Hb S (HBB: c.20A>T) and Hb Haringey (HBB: c.131A>G).

Sickle cell disease variants include hemoglobinopathies that result from inheritance of the sickle cell globin mutation with another globin mutation. The most common variants include the homozygous disease state (Hb SS disease), Hb S (HBB: c.20A>T)/Hb C (HBB: c.19G>A) disease and Hb S/ß-thalassemia (Hb S/ß-thal). Other rare/less common variants such as Hb S/Hb E (HBB: c.79G>A) and Hb S/HPFH [hereditary persistence of fetal hemoglobin (Hb)] disease exist. We report the first case of compound heterozygosity for Hb S and Hb Haringey (HBB: c.131A>G) in a 35-year-old male following a positive sickle screen test on hospital admission for pancreatitis. Ion exchange high performance liquid chromatography (HPLC), Hb electrophoresis and genetic sequencing were utilized to identify a new sickle Hb variant: Hb S/Hb Haringey. Hb S/Hb Haringey is a newly discovered sickle cell variant which seems to portray a mild/benign clinical phenotype of sickle cell disease.

Management of Sickle Cell Disease Complications Beyond Acute Chest Syndrome.

Sickle cell disease results in numerous complications that can lead to significant morbidity and mortality. Amongst them, acute chest syndrome is the leading cause of mortality. As a result, most providers are in tune with this complication and well versed with management. As sickle cell patients now live longer, they face a multitude of other complications that if left unattended, can lead to significant morbidity and mortality as well. It is critical to look beyond acute chest syndrome and adopt a more comprehensive approach to the management of the sickle cell patient.

Local Anesthesia With General Anesthesia for Pediatric Bone Marrow Procedures.

BACKGROUND: Pediatric patients with cancer undergo repeated painful procedures, including bone marrow aspirations and biopsies (BMABs). Optimal management of procedure-related pain can reduce discomfort, anxiety, and distress. METHODS: Children with neuroblastoma were randomly assigned to 1 of 2 arms on a prospective, single-blind, crossover trial conducted at Memorial Sloan Kettering Cancer Center from October 2016 to January 2018 (www.clinicaltrials.gov, identifier NCT02924324). Participants underwent 2 sequential BMABs: one with general anesthesia (GA) alone, the other with GA plus local anesthesia (LA) (GA + LA). The objective was to assess procedure-related pain and its interference with quality of life (QoL) with GA versus GA + LA. Primary outcome was percentage of participants requiring postprocedural opioids. Secondary outcomes were total opioid and nonopioid analgesics, pain scores, time to first analgesic, QoL, and toxicity. Management of postprocedural pain was standardized. RESULTS: Of 56 participants randomly assigned (3-16.5 years old), 46 completed both procedures. There was no significant difference in percentage of participants requiring opioids with GA versus GA + LA (24% vs 20%, P = .5). Pain scores in the recovery room were significantly lower for GA + LA versus GA (median [IQR]: 0 [0-2] vs 2 [0-4], P = .002). There were no statistically significant differences in total opioid or nonopioid analgesic, 6- and 24-hour pain scores, median time to first analgesic, or pain interference. No adverse events occurred. CONCLUSIONS: LA was associated with significant improvement in pain scores in the immediate recovery period. LA did not reduce postprocedural opioid use, nor did it improve QoL for patients undergoing BMAB with GA.

Successful Management of Blue Rubber Bleb Nevus Syndrome (BRBNS) with Sirolimus.

Blue rubber bleb nevus syndrome (BRBNS) is a rare disease with vascular malformations in several systems of the body, most commonly the skin and gastrointestinal tract. Bleeding from the gastrointestinal (GI) tract is a major complication, which may lead to chronic iron deficiency anemia and the need for frequent blood transfusions due to ongoing gastrointestinal blood loss. In this case report, we describe a now 19-year-old female with BRBNS who required six blood transfusions per year and after starting sirolimus is symptom- and transfusion-free.

Reduced blood pressure of CFTR-F508del carriers correlates with diminished arterial reactivity rather than circulating blood volume in mice.

The F508del mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common cause of cystic fibrosis (CF). Both CF patients and F508del carriers have decreased blood pressure. While this has been attributed to salt depletion, recent studies have shown F508del expression interferes with smooth muscle cell calcium mobilization. We tested the hypothesis that carriers of the F508del mutation have lower adult blood pressures and reduced aortic contractility without a reduction in circulating blood volume. By radiotelemetry, F508del heterozygous mice had significantly lower arterial pressures than wild-type C57BL/6 controls, with the greatest effect seen at the time of dark-to-light cycle transition (mean difference of 10 mmHg). To replicate the vascular effects of sympathetic arousal, isoproterenol and epinephrine were co-infused, and F508del mice again had significantly reduced arterial pressures. Aortas isolated from F508del heterozygous mice had significantly decreased constriction to noradrenaline (0.9 ± 0.2 versus 2.9 ± 0.7 mN). Inhibition of wild-type CFTR or the inositol triphosphate receptor replicated the phenotype of F508del aortas. CFTR carrier status did not alter circulating blood volume. We conclude the CFTR-F508del mutation decreases aortic contractility and lowers arterial pressures. As a cAMP-activated chloride channel that facilitates calcium mobilization, we speculate wild-type CFTR co-activation during adrenergic receptor stimulation buffers the vasodilatory response to catecholamines, and loss of this compensatory vasoconstrictor tone may contribute to the lower arterial pressures seen in heterozygote carriers of a CFTR-F508del mutation.