Identification of Aptamers That Bind to Sickle Hemoglobin and Inhibit Its Polymerization.

The pathophysiology of sickle cell disease (SCD) is dependent on the polymerization of deoxygenated sickle hemoglobin (HbS), leading to erythrocyte deformation (sickling) and vaso-occlusion within the microvasculature. Following deoxygenation, there is a delay time before polymerization is initiated, during which nucleation of HbS monomers occurs. An agent with the ability to extend this delay time or slow polymerization would therefore hold a therapeutic, possibly curative, potential. We used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method to screen for HbS-binding RNA aptamers modified with nuclease-resistant 2'-fluoropyrimidines. Polymerization assays were employed to identify aptamers with polymerization-inhibitory properties. Two noncompeting aptamers, DE3A and OX3B, were found to bind hemoglobin, significantly increase the delay time, and reduce the rate of polymerization of HbS. These modifiable, nuclease-resistant aptamers are potential new therapeutic agents for SCD.

Association between baseline fetal hemoglobin levels and incidence of severe vaso-occlusive pain episodes in children with sickle cell anemia.

The ameliorating effect of high fetal hemoglobin (HbF) levels on the incidence of pain episodes in sickle cell anemia (SCA) is well-known; however, in children this relationship is less clearly established. We hypothesized that higher HbF levels in children with SCA are associated with fewer severe pain episodes. A meta-analysis of data from the Silent Infarct Transfusion Trial (n = 456) and the Cooperative Study of Sickle Cell Disease (n = 764), demonstrated that baseline HbF levels were associated with the incidence of severe pain, commonly defined across studies as an event requiring hospitalization (P-value = 0.02).

A phase 1/2 trial of HQK-1001, an oral fetal globin inducer, in sickle cell disease.

Therapeutics which reduce the pathology in sickle cell syndromes are needed, particularly noncytotoxic therapeutics. Fetal hemoglobin (HbF, α(2) γ(2) ) is established as a major regulator of disease severity; increased HbF levels correlate with milder clinical courses and improved survival. Accordingly, sodium dimethylbutyrate (HQK-1001), an orally-bioavailable, promoter-targeted fetal globin gene-inducing agent, was evaluated in a randomized, blinded, dose-ranging Phase I/II trial in 24 adult patients with HbSS or S/ß thalassemia, to determine safety and tolerability of three escalating dose levels. The study therapeutic was administered once daily for two 6-week cycles, with a two-week interim dose holiday. Twenty-one patients completed the study. Five patients received study drug at 10 or 20 mg/kg doses, seven patients received study drug at 30 mg/kg/dose, and 4 patients received placebo. HQK-1001 was well-tolerated with no unexpected drug-related adverse events; a dose-limiting toxicity was not identified. Plasma drug levels were sustained above targeted levels for 24 hr. Increases in HbF above baseline were observed particularly with 30 mg/kg/day doses; in five of seven treated patients, a mean absolute increase in HbF of 0.2 g/dl and a mean increase in total hemoglobin (Hgb) of 0.83 g/dl above baseline were observed, whereas no increases occurred in placebo-treated controls. These findings of favorable PK profiles, tolerability, early rises in HbF, and total Hgb indicate that trials of longer duration appear warranted to more definitively evaluate the therapeutic potential of HQK-1001 in sickle cell disease.

α-Thalassemia impairs the cytoadherence of Plasmodium falciparum-infected erythrocytes.

BACKGROUND: α-Thalassemia results from decreased production of α-globin chains that make up part of hemoglobin tetramers (Hb; α(2)ß(2)) and affects up to 50% of individuals in some regions of sub-Saharan Africa. Heterozygous (-α/αα) and homozygous (-α/-α) genotypes are associated with reduced risk of severe Plasmodium falciparum malaria, but the mechanism of this protection remains obscure. We hypothesized that α-thalassemia impairs the adherence of parasitized red blood cells (RBCs) to microvascular endothelial cells (MVECs) and monocytes--two interactions that are centrally involved in the pathogenesis of severe disease. METHODS AND FINDINGS: We obtained P. falciparum isolates directly from Malian children with malaria and used them to infect αα/αα (normal), -α/αα and -α/-α RBCs. We also used laboratory-adapted P. falciparum clones to infect -/-α RBCs obtained from patients with HbH disease. Following a single cycle of parasite invasion and maturation to the trophozoite stage, we tested the ability of parasitized RBCs to bind MVECs and monocytes. Compared to parasitized αα/αα RBCs, we found that parasitized -α/αα, -α/-α and -/-α RBCs showed, respectively, 22%, 43% and 63% reductions in binding to MVECs and 13%, 33% and 63% reductions in binding to monocytes. α-Thalassemia was associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite's main cytoadherence ligand and virulence factor, on the surface of parasitized RBCs. CONCLUSIONS: Parasitized α-thalassemic RBCs show PfEMP1 display abnormalities that are reminiscent of those on the surface of parasitized sickle HbS and HbC RBCs. Our data suggest a model of malaria protection in which α-thalassemia ameliorates the pro-inflammatory effects of cytoadherence. Our findings also raise the possibility that other unstable hemoglobins such as HbE and unpaired α-globin chains (in the case of ß-thalassemia) protect against life-threatening malaria by a similar mechanism.

Subtotal splenic embolization is a safe and effective treatment for isolated splenic vascular tumors associated with consumptive coagulopathy.

Consumptive coagulopathy is a known complication of large vascular tumors. We describe 2 episodes of consumptive coagulopathy in young children, which were secondary to isolated splenic vascular tumors. One child was successfully treated by subtotal embolization of the spleen, whereas the second child required splenectomy after an initial embolization improved--but did not fully control--his consumptive coagulopathy.

A role for fetal hemoglobin and maternal immune IgG in infant resistance to Plasmodium falciparum malaria.

BACKGROUND: In Africa, infant susceptibility to Plasmodium falciparum malaria increases substantially as fetal hemoglobin (HbF) and maternal immune IgG disappear from circulation. During the first few months of life, however, resistance to malaria is evidenced by extremely low parasitemias, the absence of fever, and the almost complete lack of severe disease. This resistance has previously been attributed in part to poor parasite growth in HbF-containing red blood cells (RBCs). A specific role for maternal immune IgG in infant resistance to malaria has been hypothesized but not yet identified. METHODS AND FINDINGS: We found that P. falciparum parasites invade and develop normally in fetal (cord blood, CB) RBCs, which contain up to 95% HbF. However, these parasitized CB RBCs are impaired in their binding to human microvascular endothelial cells (MVECs), monocytes, and nonparasitized RBCs--cytoadherence interactions that have been implicated in the development of high parasite densities and the symptoms of malaria. Abnormal display of the parasite's cytoadherence antigen P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on CB RBCs accounts for these findings and is reminiscent of that on HbC and HbS RBCs. IgG purified from the plasma of immune Malian adults almost completely abolishes the adherence of parasitized CB RBCs to MVECs. CONCLUSIONS: Our data suggest a model of malaria protection in which HbF and maternal IgG act cooperatively to impair the cytoadherence of parasitized RBCs in the first few months of life. In highly malarious areas of Africa, an infant's contemporaneous expression of HbC or HbS and development of an immune IgG repertoire may effectively reconstitute the waning protective effects of HbF and maternal immune IgG, thereby extending the malaria resistance of infancy into early childhood.

Evaluation of safety and pharmacokinetics of sodium 2,2 dimethylbutyrate, a novel short chain fatty acid derivative, in a phase 1, double-blind, placebo-controlled, single-dose, and repeat-dose studies in healthy volunteers.

Pharmacologic induction of fetal globin synthesis is an accepted therapeutic strategy for treatment of the beta hemoglobinopathies and thalassemias, as even small increases in hemoglobin F (HbF) levels reduce clinical severity in sickle cell disease (SCD) and reduce anemia in beta thalassemia. Prior generation short chain fatty acid therapeutics, arginine butyrate (AB), and phenylbutyrate, increased fetal and total hemoglobin levels in patients, but were limited by high doses or intravenous (IV) infusion. A fetal globin-inducing therapeutic with convenient oral dosing would be an advance for these classic molecular diseases. Healthy adult human subjects were treated with a novel short chain fatty acids (SCFA) derivative, sodium 2,2 dimethylbutyrate (SDMB), or placebo, with 1 of 4 single dose levels (2, 5, 10, and 20 mg/kg) or daily doses (5, 10, or 15 mg/kg) over 14 days, and monitored for adverse clinical and laboratory events, drug levels, reticulocytes, and HbF assays. SDMB was well-tolerated with no clinically significant adverse events related to study medication. The terminal half-life ranged from 9 to 15 hours. Increases in mean absolute reticulocytes were observed at all dose levels in the 14-day study. The favorable pharmacokinetics (PK) profiles and safety findings indicate that SDMB warrants further investigation for treatment of anemic subjects with beta hemoglobinopathies.

Genome-wide association study identifies genetic variants influencing F-cell levels in sickle-cell patients.

Fetal hemoglobin (HbF) level has emerged as an important prognostic factor in sickle-cell disease (SCD) and can be measured by the proportion of HbF-containing erythrocytes (F-cells). Recently, BCL11A (zinc-finger protein) was identified as a regulator of HbF, and the strongest association signals were observed either directly for rs766432 or for correlated single-nucleotide polymorphisms (SNPs). To identify additional independently associated genetic variants, we performed a genome-wide association study (GWAS) on the proportion of F-cells in individuals of African ancestry with SCD from the Silent Infarct Transfusion (SIT) Trial cohort. Our study not only confirms the association of rs766432 (P-value <3.32 × 10(-13)), but also identifies an independent novel intronic SNP, rs7606173, associated with F-cells (P-value <1.81 × 10(-15)). The F-cell variances explained independently by these two SNPs are ∼13% (rs7606173) and ∼11% (rs766432), whereas, together they explain ∼16%. Additionally, in men, we identify a novel locus on chromosome 17, glucagon-like peptide-2 receptor (GLP2R), associated with F-cell regulation (rs12103880; P-value <3.41 × 10(-8)). GLP2R encodes a G protein-coupled receptor and involved in proliferative and anti-apoptotic cellular responses. These findings highlight the importance of denser genetic screens and suggest further exploration of the BCL11A and GLP2R loci to gain additional insight into HbF/F-cell regulation.

Corticosteroids and increased risk of readmission after acute chest syndrome in children with sickle cell disease.

BACKGROUND: Acute chest syndrome (ACS) is a frequent cause of hospitalization and mortality in children with sickle cell disease. Transfusion is often required to prevent respiratory failure and treatment with dexamethasone may reduce the length of admission and the need for transfusions. We performed a retrospective cohort study to evaluate risk factors for readmission and prolonged hospitalization after different treatments for ACS. PROCEDURE: We identified patients <22 years of age hospitalized with ACS at Johns Hopkins Hospital from January 1998 to April 2004 using the hospitals discharge database and by reviewing dictated summaries. RESULTS: We identified 65 patients with 129 episodes of ACS (mean age 12.5 years, range 1.2-21.9 years). Thirty-nine episodes were treated with corticosteroids and 51 with transfusions. Patients were readmitted within 14 days after 23 episodes (18%). Readmission was strongly associated with report of an inhaler or nebulizer at home [odds ratio (OR) 6.0, P < 0.05], diastolic BP at 48 hr (OR 1.8 per 10 mm increase, P<0.01), corticosteroids (OR 20, P < 0.005), or transfusion (OR 0.03, P < 0.05). Treatment with corticosteroids alone (P < 0.05) and older age (P < 0.001) were associated with longer hospitalization. CONCLUSIONS: These results demonstrate a greatly elevated independent risk of readmission after ACS in children with asthma and after treatment with corticosteroids and a protective effect of transfusion. Although dexamethasone has documented efficacy for reducing the duration of ACS, the substantial risk of readmission for pain should limit its use.

Role of cyclic nucleotides in fetal hemoglobin induction in cultured CD34+ cells.

OBJECTIVE: In vivo, several drugs have been shown to increase fetal hemoglobin (HbF), including 5-azacytidine (AZA), sodium butyrate (SB), and hydroxyurea (HU). Studies in K562 cells suggest that cyclic guanosine monophosphate (cGMP) is required for HbF induction; however, the role of cyclic nucleotides in HbF induction in primary erythroid cultures has not been established. METHODS: CD34-selected peripheral blood monocytes cultured in a semi-solid serum-free system that mimics in vivo F-cell production are utilized to explore the role of cyclic adenosine monophosphate (cAMP) and cGMP in HbF induction in response to HU, AZA, and SB. RESULTS: In serum-free CD34 cultures, HU, SB, and AZA all markedly stimulate FNRBC production up to 30-fold, associated with induction of gamma-globin mRNA and total HbF protein. Guanylate cyclase inhibition results in only minimal blunting of HbF induction by each agent. In contrast, adenylate cyclase inhibition markedly reduces HU, SB, and AZA-mediated FNRBC induction and gamma-globin mRNA induction. The adenylate cyclase activator forskolin modestly induces FNRBC production and augments the action of standard induction agents. HU, AZA, and SB, however, fail to significantly stimulate adenylate cyclase themselves. CONCLUSIONS: In human CD34(+) cultures, cAMP production is required for full induction of HbF by HU, SB, and AZA, while perturbation of cGMP production has only minimal effects. These findings are in marked contrast to data in K562 cells where cGMP production is critical for HbF induction while cAMP stimulation blunts HbF response, and suggest that these agents may share a common induction pathway.

Regulation of granulocyte and monocyte differentiation by CCAAT/enhancer binding protein alpha.

CCAAT/enhancer binding protein alpha (C/EBPalpha)-ER induces 32Dcl3 neutrophilic differentiation and inhibits 32DPKCdelta maturation to macrophages in response to phorbol ester. In 32Dcl3 cells, C/EBPalpha-ER rapidly induces the PU.1 and C/EBPalpha RNAs even in the presence of cycloheximide, suggesting that these are direct C/EBPalpha genetic targets. C/EBPalpha strongly binds and modestly activates the murine PU.1 promoter via an evolutionarily conserved binding site. C/EBPalpha-ER variants incapable of binding DNA still slow G1 progression but do not induce differentiation. N-terminally truncated C/EBPalpha variants, including the p30 isoform expressed in a subset of AMLs, also retain the ability to slow 32D cl3 proliferation, whereas the C/EBPalpha(BRM2)-ER variant does not slow G1 progression, has a reduced capacity to induce early granulocytic markers, and does not induce terminal maturation. In 32DPKCdelta cells, C/EBPalpha-ER strongly inhibits endogenous or exogenous JunB induction, dependent upon the outer surface of the C/EBPalpha basic region, but does not inhibit c-Jun, PU.1, or C/EBPbeta expression. Exogenous JunB restores AP-1 DNA binding but does not overcome inhibition of monopoiesis by C/EBPalpha-ER. In summary, we propose that while C/EBPalpha is required for development of immature granulocyte-monocyte progenitors, C/EBPalpha subsequently inhibits monopoiesis, via inhibition of JunB express and via additional activities, and induces granulopoiesis, via induction of PU.1, C/EBPepsilon, and cell cycle arrest.

Reciprocal effects of C/EBPalpha and PKCdelta on JunB expression and monocytic differentiation depend upon the C/EBPalpha basic region.

Monocytic differentiation of 32DPKCdelta cells in response to activation of protein kinase C delta (PKCdelta) by phorbol 12-myristate 13-acetate (PMA) was inhibited by exogenous CCAAT/enhancer binding protein alpha-estradiol receptor (C/EBPalpha-ER), which impeded morphologic maturation and induction of macrosialin mRNA. Inhibition of monopoiesis was also evident in 32DPKCdelta subclones expressing C/EBPalphaLeu12Val-ER, which cannot dimerize or bind DNA because of mutation of the leucine zipper, C/EBPalphaGZ-ER, in which the leucine zipper has been replaced by the GCN4 zipper, or C/EBPalphaDelta3-8-ER, lacking the C/EBPalpha transactivation domains. In contrast, C/EBPalphaBR3-ER, containing a mutant basic region, did not inhibit monocytic differentiation. C/EBPalpha-ER strongly inhibited endogenous AP-1 DNA-binding. Supershift analysis revealed that the major AP-1 complex contains JunB. Activation of C/EBPalpha-ER specifically reduced endogenous JunB RNA and protein and exogenous JunB levels without affecting endogenous or exogenous c-Jun. The stability of PMA-induced JunB was not affected. Thus, C/EBPalpha-ER suppresses both JunB transcription and posttranscriptional protein generation or induction. PU.1 levels and activity were increased. The Leu12Val, GZ, and Delta3-8 mutants also inhibited JunB expression, whereas the BR3 mutant was ineffective, indicating that inhibition of JunB expression and monocytic differentiation by C/EBPalpha-ER depends upon an interaction mediated by its basic region. Exogenous JunB restored AP-1 DNA-binding but did not prevent inhibition of macrosialin expression by C/EBPalpha-ER, indicating that JunB is not the only target relevant to inhibition of monopoiesis by C/EBPalpha.

Morphological and biochemical strategies for monitoring trafficking of epitope-tagged G protein-coupled receptors in agonist-naive and agonist-occupied states.

Epitope tagged alpha 2-AR subtypes have been used to address a variety of cell biological questions, and the strategies used are readily applicable to all GPCR as well as other cell surface proteins. We have provided detailed protocols for successful utilization of the epitope-tagged receptor in the studies of protein localization and trafficking in epithelial cells, and the mechanisms by which this is achieved. We have also described reversible biotinytion strategies to examine agonist-dependent (and independent) receptor turnover at the cell surface.