ALBERT

Description: It was recently demonstrated that erythropoietin combined with stem cell factor and transforming growth factor beta (EST; EPO+SCF+TGF-B) signals a pancellular reversal of gamma-globin gene silencing among ex vivo cultures of CD34+ cells when compared to cultures supplemented with erythropoietin (EPO) alone (Bhanu et al., Blood.2005; 105(1):387–93). This primary human erythroblast culture model is now being utilized to study the molecular basis of globin gene regulation. In this study, we sought to determine whether EST-signaled effects are restricted to the gamma-globin gene(s). For this purpose, quantitative PCR (QPCR) assays were developed for the alpha-globin (zeta, mu, alpha, and theta) and beta-globin (epsilon, gamma, delta, and beta) gene clusters. EPO versus EST matched cultures from a total of 15 donors (3 single donors and two pools, 6 donors each) were investigated using culture day 7 proerythroblasts and day 14 hemoglobinized precursors. The QPCR products were quantified by comparison with plasmid-generated standard curves (20,000,000 to 200 copies). The patterns of mu-, alpha- and theta-globin gene expression were unchanged under all culture conditions. The delta- and beta-globin transcripts showed a significant reduction in levels in response to EST. In contrast, zeta-, epsilon- and gamma-globins all demonstrated significant increases in response to EST. The increases were consistently detected in the individual and pooled samples. The most dramatic increase in the zeta-, epsilon- and gamma-globins occurred at the proerythroblast stage of differentiation. On day 7, zeta-globin levels showed 190-fold increase (EPO: 6.9E+01±5.5E+01;EST: 1.3E+04±7.7E+03; p=0.01), epsilon-globin levels, a 27-fold increase (EPO: 1.0E+03±5.8E+02; EST: 2.7E+04±1.1E+04; p=0.001), and gamma-globin levels, a 13-fold increase (EPO: 9.1E+05±4.1E+05; EST: 1.2E+07±1.8E+06; p=0.001). Consistent with the 3–4 log difference between the levels of zeta- and epsilon-, versus gamma-globin copy numbers and the low sensitivity of HPLC compared with QPCR, no embryonic proteins were detected by reverse-phase HPLC (day 14 samples studied; 9 donors). However, G-gamma and A-gamma globin peaks confirmed EST-signaled activation of both gamma-globins genes (G-gamma/A-gamma= 1.0 in EPO versus 1.9 in EST; (total gamma)/(total gamma + beta-globin) = 3.7±2.4% in EPO versus 42.4 ±5.8% in EST; p=1.6E-08). These novel data suggest that cytokines can reverse the coordinated developmental silencing of human embryonic and fetal genes in both globin loci.

Description: Erythrocyte inclusion bodies that are now commonly referred to as Howell-Jolly Bodies (HJB) were originally discovered over 100 years ago by microscopy. These nuclear fragments are observed in the erythrocytes of patients lacking splenic function, but their precise genomic origins are vague. Here we report studies of the genetic content of HJB using a molecular cytogenetic method known as Comparative Genomic Hybridization (CGH). Peripheral blood samples from six splenectomized human donors were studied. All donors had undergone splenectomies prior to this study for diagnostic and therapeutic indications related to underlying neoplastic and autoimmune disease. Ultrasound testing was used to rule out the presence of accessory splenic tissue, and peripheral blood smears confirmed the presence of HJB. Nucleated cells were removed by density gradients and leukocyte filtration for use as patient-matched controls. DNA was isolated from the leukocytes and HJB-containing erythrocytes. The presence of high molecular weight HJB DNA was confirmed in each sample using gel electrophoresis. As expected, it was not possible to obtain sufficient quantities of DNA from the control erythrocytes of healthy individuals with normal splenic function. CGH was performed using equivalent amounts of reference genomic DNA co-hybridized with HJB DNA or matched donor leukocyte DNA. Agilent oligonucleotide CGH arrays were hybridized and scanned according to the manufacturer’s instructions. The control hybridizations (genomic DNA isolated from donor leukocytes) demonstrated standard patterns consistent with the donor’s karyotype. In contrast, the HJB hybridization signals demonstrated an unusual pattern of centromeric-to-telomeric enrichment on each chromatid of each chromosome. The HJB hybridization signals also increased as a function of distance from the centromere according to the chromatid size. The enhanced signal relative to centromeric distance varied considerably between chromosomes, but was remarkably consistent between donors. These data suggest Howell-Jolly Bodies contain a non-random distribution of centromere-depleted chromatin generated after DNA double-strand breakage during erythroid differentiation.

Description: Background Urinary hepcidin is a potential biomarker of renal inflammation and acute kidney injury (AKI) which is elevated in sickle cell disease (SCD). Hepcidin in circulation is filtered through glomeruli filtration barrier and reabsorbed by the renal tubules. Hepcidin can also be synthesized by the kidney tubular cells. Thus, increased urinary levels of hepcidin may reflect either a reduction in tubular uptake or an increase in renal production. Recent studies suggested that urinary hepcidin may protect against AKI by attenuating heme-mediated injury. Thus decreased hepcidin levels in SCD patients may contribute to AKI and serve as potentially informative marker of SCD-associated kidney injury. Previously, hepcidin was measured by ELISA and mass spectrometry. Immunoassays are limited due to the cross-reactivity of antibodies to prohepcidin and truncated hepcidin-20, -22, and -24 isoforms of active hepcidin-25. Mass spectrometric assays are specific for hepcidin-25 but sample preparation remains a challenge. Objective To develop a sensitive, reliable and reproducible nanoLC/FT-MS method with simplified sample preparation for measuring of hepcidin in urine samples. Also to correlate urinary hepcidin with urinary albumin and urinary protein to access the degree of kidney dysfunction. Methods Samples were enriched and purified semi-automaticaly on 10-uL ZipTip and online trap column. Stable isotope-labeled hepcidin was used as internal standard. The standard concentration range was 1.56-800 nM and quality control samples were 5 nM, 20 nM, 80 nM and 400 nM. Samples were subjected to an LC-20AD nano HPLC system coupled to an LTQ XL™ Orbitrap mass spectrometer with an in-house made nano-HPLC column. High resolution/selected reaction monitoring (HR/SRM) scan was carried out and the narrow mass range ([M+H]+ ±0.01 Da) was used to extract ion chromatograms (EICs) for quantification. Urinary samples were collected from 20 SCD patients and 13 controls. Urinary albumin, protein and creatinine were detected by ELISA. The urine hepcidin concentrations were normalized to urine creatinine (Cr) values. Results Semi-automatic approach simplified sample preparation and accelerated the analysis. At least 24 samples could be prepared and processed at the same time. Online column trapping further purified and enriched hepcidin and improved the sensitivity and specificity of this method by eliminating interferences from urine. Hepcidin showed a good linearity within the concentration range of 1.56-800 nM with an r2 value of 0.9994. The precision intraday (n = 5) and interday (n = 5) and the repeatability (n=5) of the method were good with relative standard deviations (RSDs) lower than 5%. The analyzed samples were stable for 3 days at +4°C (RSDs

Description: Sickle cell disease (SCD) is a characterized by hemolysis, vaso-occlusion and ischemia. Several previous studies pointed to a possibility that SCD patients might be protected from HIV-1 infection. These studies described low prevalence of anti-HIV-1 antibodies in SCD patients transfused with potentially HIV-1 infected blood;1 higher number of long-term non-progressors among HIV-1 infected SCD patients, 2 and a lower frequency of HIV diagnosis among SCD patients (odds ratio 0.33).3 This study aims to decipher a mechanism of HIV-1 restriction in PBMC from SCD patient infected with HIV-1 ex vivo. HIV-1 replication in SCD PBMC was inhibited at the level of reverse transcription and transcription implicating the involvement of post-entry and transcription restriction factors. SAM domain and HD domain-containing protein 1 (SAMHD1) restricts HIV-1 infection in in myeloid cells. 4,5 by reducing intracellular nucleotide pool and blocking reverse transcription. SAMHD1 phosphorylation on Thr-592 by CDK2 or CDK1 inactivates it and prevents HIV-1 inhibition. We showed that SAMHD1 phosphorylation was reduced in SCD PBMCs and in hemin-treated promonocytic THP-1 cells. Moreover, knock-down of SAMHD1 prevent hemin-mediated inhibition of HIV-1 in THP-1 cells. We also detected a reduction of CDK2 activity in SCD PBMCs and in hemin-treated THP-1 cells which can explain reduced SAMHD1 phosphorylation. Previously, we showed that CDK2 activity is inhibited when intracellular iron levels are depleted by iron chelators. We observed reduced intracellular labile iron levels and increased expression of iron export protein, ferroportin and HIF-1α in SCD PBMCs. Importantly, treatment of SCD PBMCs with hepcidin alleviated HIV-1 inhibition. Unaltered hepcidin levels in plasma of SCD patients suggest that ferroportin expression is sustained in SCD PBMC. Our study points out to ferroportin as upstream regulator of SAMHD1 and links a reduction in iron levels, inhibition of CDK2 activity and a decrease in SAMHD1 phosphorylation to the inhibition of HIV-1 infection in SCD. Acknowledgments. This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005 and 5G12MD007597. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Literature 1. Castro O, Saxinger C, Barnes S, Alexander S, Flagg R, Frederick W. Prevalence of antibodies to human immunodeficiency virus and to human T cell leukemia virus type I in transfused sickle cell disease patients. J Infect Dis. 1990;162(3):743-745. 2. Bagasra O, Steiner RM, Ballas SK, et al. Viral burden and disease progression in HIV-1-infected patients with sickle cell anemia. Am J Hematol. 1998;59(3):199-207. 3. Nouraie M, Nekhai S, Gordeuk VR. Sickle cell disease is associated with decreased HIV but higher HBV and HCV comorbidities in US hospital discharge records: a cross-sectional study. Sex Transm Infect. 2012. 4. Hrecka K, Hao C, Gierszewska M, et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature. 2011;474(7353):658-661. 5. Laguette N, Sobhian B, Casartelli N, et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature. 2011;474(7353):654-657. Disclosures No relevant conflicts of interest to declare.

Description: Iron overload causes considerable morbidity associated with thalassemia due to inappropriate suppression of the iron regulator, hepcidin. One possible explanation for this phenomenon is that protein(s) that are normally secreted into the marrow microenvironment by erythroblasts become endocrine signals in patients with thalassemia due to the myeloproliferative nature of the disease. To test this hypothesis, progenitor and precursor cell transcriptional profiles were generated with Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays using primary erythroblasts cultured from 15 healthy human donors. For this study, informatic analyses were focused upon 54 members of the TGF–B/BMP superfamily. Among the subset of genes identified by this screen, evidence for high-level expression of a gene named growth differentiation factor 15 (GDF15) was discovered in the precursor cell profiles. Quantitative PCR, Western, and ELISA analyses confirmed expression and secretion of GDF15 during erythroblast maturation. GDF15 is an apoptosis-associated protein expressed primarily by the placenta. To determine whether GDF15 serves as a regulator of hepcidin expression, hepcidin expression assays were performed using a human hepatoma cell line (HuH-7). BMP2 and BMP4 were studied for comparison. Addition of BMP2 and BMP4 (range 10,000–500,000 pg/ml) resulted in dosed increases in hepcidin mRNA (5–30 fold). At the concentrations of GDF15 normally found in human blood (500 pg/ml), a 2-fold increase in the expression of hepcidin was measured compared to matched cultures containing no supplemental GDF15. However, GDF15 dosed to levels above 5,000 pg/ml resulted in a significant reduction in hepcidin expression. Next, plasma levels of GDF15 were measured in the peripheral blood of 162 donors (donor groups included healthy controls, sickle-cell syndromes, thalassemia syndromes, and other causes of anemia) to determine whether plasma GDF15 levels are dysregulated in thalassemia. Plasma samples from 21 hereditary hemochromatosis donors provided evidence that significantly elevated GDF15 expression is not associated with iron overload in the absence of erythroid pathology. Compared with mean plasma GDF15 levels of 536±222 pg/ml among the control samples, only patients with thalassemia intermedia, thalassemia major, and HbE-beta thalassemia showed significantly elevated plasma levels of GDF15 (9 donors; mean GDF15 24,600 pg/ml; range 8,980–75,100 pg/ml; p370 ug/L) were noted in each of those patients regardless of their transfusion or chelation history. These novel findings suggest that GDF15 is secreted from human erythroblasts, released into the circulation at extremely high levels in thalassemia patients, and contributes to iron overloading in those patients by suppressing hepcidin expression.

Description: Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Description: Although the genetic processes responsible for gamma-globin gene and protein silencing are not known, the prevailing model is that gamma-globin silencing results from a gradual change within a single hematopoietic cell lineage that is governed by intrinsic properties of the cells. In order to provide a more complete characterization of the silencing phenomenon, we studied globin expression patterns directly from clinical samples using single-cell, quantitative PCR, and globin protein phenotyping. We collected blood samples from untransfused donors: umbilical cords (n=3), infants (n=11; ages 1 day to 35 months), and adults (n=3). All samples were maintained at 4°C and analyzed within 72 hours. Flow cytometry (30,000 cells per donor) and HPLC analyses were used for globin protein phenotyping. For globin gene expression, we identified reticulocytes using a strategy that required no membrane permeabilization, and sorted them as single cells directly into lysis buffer. Oligo-dT reverse transcription of mRNA was followed by real-time PCR quantitation. Globin cDNA copy numbers were calculated using standard curves from serial dilutions of a plasmid DNA. We analyzed approximately 1000 single-cell quantitative PCR amplifications for gamma- and beta-globin gene expression. In cord blood, we detected both gamma- and beta-globin gene expression in 97.4% (112/115) of the reticulocytes. The average gamma-globin cDNA copy number was 1870±1390 copies, compared with an average beta-globin cDNA copy number of 2181±2138 copies per reticulocyte. HbF and HbA were also detected in 〉95% of the cord blood erythrocytes. In the adult samples, HbF was detected in

Description: Sleep disordered breathing including transient hypoxemia and hypercarbia are reported in 60-80% of adolescents and children with sickle cell disease (SCD); oxygen desaturation 50 mm Hg during sleep are associated with increased frequency of acute vaso-occlusion events and are suspected of contributing to microvasculature alterations. To assess the prevalence and degree of sleep-related hypoxemia and potential associations with cardiovascular functions in young adults with SCD, we performed overnight sleep studies using a Type II sleep monitor NOX-T3 (Carefusion, Inc), 6-minute walk tests, echocardiograms, hematologic and chemistry panels, and PSQI questionnaires in 17 adults with SCD, ages 21-30 years. Subjects were attending a sickle cell clinic solely for routine care with no expressed complaints of SDB. Exclusion criteria included acute clinical events, hospitalizations, or red cell transfusions within 4 weeks, and chronic transfusions. AHI〉5 (significant apnea/hypopnea hypoxemic episodes) during sleep occurred in 7/17 (41%) of subjects, and these subjects had a higher median number of hypopneas (34 vs 12, p=0.005), and oxygen desaturation indices (ODI, 5.9 vs 2.0, p5 (median MV EA ratio of 2.0 vs. 1.5, p = 0.08). TR jet velocity 〉2.5 was found in 2/17 asymptomatic subjects; (both were in the AHI〉5 group). General quality of life was lower in patients with AHI〉5 (mean score of 38 vs. 48, p = 0.012). As prolonged and frequent hypoxemic episodes may increase risks for vaso-occlusive, cardiovascular, and neurologic events, these common findings of significant nocturnal hypoxemia in young adult sickle cell subjects strongly suggest that SDB should be investigated further in larger patient populations, and interventions initiated. The observations, in addition to prior reports, also strongly suggest that screening of young adult SCD patients for SDB should be performed on a routine basis. Research reported in this publication was supported by the NHLBI under Award Number P50HL118006. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Disclosures Klings: Actelion Pharmaceuticals: Research Funding; Pfizer: Consultancy.