Hydroxyurea-Mediated Reactivation of Fetal Genes in Primary Erythroid Cell Cultures from Beta-Thalassemia Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3809-3809
Author(s):  
Roberta Calzolari ◽  
Aurelio Maggio ◽  
Alice Pecoraro ◽  
Vito Borruso ◽  
Antonio Troia ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Sickle Cell ◽  
Cell Cultures ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Alternative Mechanism ◽  
Rna Fish ◽  
F Cells

Abstract Fetal hemoglobin (HbF) is frequently increased in the hemoglobinopathies such as sickle cell anemia and b-thalassemia. Epidemiological studies have indicated that an increase in HbF ameliorates the clinical symptoms of these diseases (Rodgers and Rachmilewitz - Bri J. Haemat. 91: 263 -1995). In sickle cell anemia, HbF containing red blood cells have a lower concentration of sickle hemoglobin (HbS), and the HbF itself inhibits HbS polymerization, decreasing cell sickling process (Eaton and Hofrichter - Science 268:1142 -1995). In b-thalassemia patients, HbF partially compensate HbA deficiency and could potentially improve RBC survival resulting in an increase of hemoglobin levels. Hydroxyurea (HU) is one of the pharmacological agents currently used to stimulate HbF synthesis in patients with sickle cell anemia and more recently has been tested in clinical trials for b-thalassemia patients too (Olivieri et al. Ann. NY Acad Sci850:100-1998; Rigano et al. Hemogl.21(3): 219- 1997; Dixit et al. Ann. Haematol. 84: 441 -2005). The mechanism involved in the HU-mediated changes is still unclear. It may involve a selection of a minor pre-existing subpopulation of F-cells that has a growth and/or survival advantage (cellular mechanism). This mechanism may be particularly effective for cells derived from patients with hemoglobinopathies, where F-cells may be resistant to “ineffective erythropoiesis”. An alternative mechanism could involve stimulation of HbF in all or the majority of cell-population by direct induction of g genes (molecular mechanism). Here we report the analysis on thalassemia patients homozygoutes for Lepore genotype that present high levels of fetal hemoglobin. We combined the use of primary erythroid cell culture from peripheral blood stem cells of these patients, with primary transcript in situ hybridization (RNA-FISH) of the g and b globin genes to investigate the mechanism of action of hydroxyurea in adult erythroid cells. RNA-FISH on erythroid cell cultures from these patients reveals that the majority of cells express one g allele only (g: 75.2 %, g:g 19.6%). The analysis in hydroxyurea-treated cultures shows the increase of cells transcribing both g-alleles, indicating the reactivation of fetal genes (g: 58.1%, g:g 40%). This evidence suggests that the molecular mechanism is involved directly on fetal genes reactivation to increase fetal hemoglobin production in HU-treated patients.

Absolute Reticulocyte Count Is Negatively Correlated with Fetal Hemoglobin during Infancy and Early Childhood in Patients with Sickle Cell Anemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4081-4081
Author(s):  
Emily R. Meier ◽  
Colleen Byrnes ◽  
Y. Terry Lee ◽  
Maxine Weissman ◽  
Jeffery L. Miller
Keyword(s):  
Steady State ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Conflicts Of Interest ◽  
Fetal Hemoglobin ◽  
Convenience Sample ◽  
Risk Of Death ◽  
Increased Risk ◽  
F Cells ◽  
Analytical Approaches

Abstract Hemoglobin switching is largely complete in healthy infants by 6 months of age. In infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). Previous studies demonstrated that patients with an ARC greater than 200 K/uL during early infancy (60-196 days of age) were at the highest risk for SCA-associated events. 1,2 The objective of this study was to determine if ARC is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study [42 (39.6%) less than 1 year of age (28-362 days old), 46 (43.4%) between the ages of 1 and 10 years, and 18 (17.0%) between 10 and 20 years old]. After consent and assent were provided, discarded peripheral blood was obtained during routine clinic visits at steady state and analyzed within 48 hours of collection and storage at 40C. Steady state was defined as a sample drawn at least 30 days following an acute event and at least 60 days following a blood transfusion. Hematologic data, including ARC and HbF levels, were measured using CLIA approved methods. F-cells were enumerated by flow cytometry following intracellular staining with a fluorescent antibody directed against HbF. Correlations were calculated to determine the relationships of ARC with HbF, F-cells, and other hematologic data, while two-tailed t tests were used to compare means. Initial studies compared groups based upon ARC greater than or equal to 200 K/uL (ARC≥200) during infancy because of the previously reported utility of this threshold as a predictive marker for SCA severity.1 Over one third of the infants less than 1 year of age (n=16) had an ARC≥200. Mean HbF and F-cell levels were significantly lower in the ARC≥200 group when compared to the ARC<200 group (HbF: 29.9±10.9% vs. 53.5±17.6%, respectively, p=2.2E-05; F-cells: 83.5±13.2% vs. 96.6±5.7%, p=6.2E-05). Mean hemoglobin levels were also lower in the ARC≥200 group [8.1±1.4 g/dL vs. 9.5±1.6 g/dL (ARC<200), p=0.005]. Of the 22 (52.4%) infants who had a HbF level greater than 40%, only 2 (9.1%) had an ARC greater than 200K/uL. Enrolled patients were also grouped according to age and comparisons were made between ARC and HbF or F-cell levels. HbF and F-cell levels were negatively correlated to ARC in the infant subgroup (r=-0.696, p=3.1E-07 and r=-0.795, p=0.000, respectively). HbF and F-cell levels from children between the ages of 1 and 10 years were inversely related to the ARC, but the correlation was less significant (r=-0.626, p=3.3E-06 and r=-0.538, p=1.2E-04, respectively). The inverse relationship was no longer present in the oldest group of patients (HbF vs. ARC r=-0.203, p=0.420 and F-cells vs. ARC, r=-0.258, p=0.302). According to both analytical approaches described here, increased ARC is associated with decreased HbF and F-cell levels in infants with SCA. Less robust negative correlations are maintained through age 10 years, but no significant correlation was identified in adolescence and young adulthood. Overall, the data suggest that increased ARC levels may identify SCA infants who manifest a more rapid or greater loss of fetal hemoglobin during the later stages of the HbF-to-HbS switching phenomenon. Meier ER, Byrnes C, Lee YT, et al. Increased reticulocytosis during infancy is associated with increased hospitalizations in sickle cell anemia patients during the first three years of life. PLoS One 2013; 8(8):e70794. doi: 10.1371/journal.pone.0070794.Meier ER, Wright EC, Miller JL. Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease. Am J Hematol 2014 May 31; doi: 10.1002/ajh.23777. [Epub ahead of print] Disclosures No relevant conflicts of interest to declare.

2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2379-2384
Author(s):  
Mabel Koshy ◽  
Louise Dorn ◽  
Linda Bressler ◽  
Robert Molokie ◽  
Donald Lavelle ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Complete Blood Count ◽  
Fetal Hemoglobin ◽  
Therapeutic Benefit ◽  
Additional Treatment ◽  
Acute Chest Syndrome ◽  
F Cells ◽  
Pain Crisis ◽  
Globin Synthesis

Augmentation of the fetal hemoglobin (HbF) levels is of therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute chest syndrome, and requirements for blood transfusions. For patients with no HbF elevation after HU treatment, augmentation of HbF levels by 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) could serve as an alternate mode of treatment. Eight adult patients participated in a dose-escalating phase I/II study with 5-aza-CdR at doses ranging from 0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F cell, γ-globin synthesis ratio, complete blood count, and chemistry were measured. The average γ-globin synthesis relative to non-α-globin synthesis prior to therapy was 3.19% ± 1.43% and increased to 13.66% ± 4.35% after treatment. HbF increased from 3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from 21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17% to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all patients tolerated the drug. Maximum HbF was attained within 4 weeks of treatment and persisted for 2 weeks before falling below 90% of the maximum. Therefore 5-aza-CdR could be effective in increasing HbF in patients with sickle cell anemia who failed to increase HbF with HU. Demonstration of sustained F levels with additional treatment cycles without toxicity is currently being performed.

scholarly journals High prevalence of individuals with low concentration of fetal hemoglobin in F-cells in sickle cell anemia in Tanzania

2016 ◽  
Vol 91 (8) ◽  
pp. E323-E324 ◽  
Author(s):  
Florence Urio ◽  
Magdalena Lyimo ◽  
Siana Nkya Mtatiro ◽  
Sharon E. Cox ◽  
Bruno p. Mmbando ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Low Concentration ◽  
High Prevalence ◽  
F Cells

scholarly journals Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or beta-thalassemia

Blood ◽  
1993 ◽  
Vol 81 (6) ◽  
pp. 1630-1635
Author(s):  
E Fibach ◽  
LP Burke ◽  
AN Schechter ◽  
CT Noguchi ◽  
GP Rodgers
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Fold Increase ◽  
Cell Number ◽  
Model Systems ◽  
Beta Thalassemia ◽  
Mean Corpuscular Hemoglobin ◽  
Normal Individuals ◽  
Culture Procedure

Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood- derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5- fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta- thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.

scholarly journals Increased HbF in sickle cell anemia is determined by a factor linked to the beta S gene from one parent

Blood ◽  
1984 ◽  
Vol 63 (1) ◽  
pp. 64-72 ◽  
Author(s):  
PF Milner ◽  
JD Leibfarth ◽  
J Ford ◽  
BP Barton ◽  
HE Grenett ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Sickle Cell Trait ◽  
Fetal Hemoglobin ◽  
S Gene ◽  
Cell Counts ◽  
F Cells ◽  
Hereditary Persistence ◽  
Large Families ◽  
Cell Gene

Abstract Members of 7 large families, containing 20 patients with sickle cell anemia (SS) characterized by high levels of fetal hemoglobin (HbF), were studied using immunofluorescence to count F cells and a radioimmunoassay to measure small amounts of HbF. In five of these families, one of the sickle cell trait (AS) parents had a much higher HbF and F-cell count than the other; in one family, both parents had a marked increase in HbF and F cells; in the remaining family, HbF and F cells were at borderline values in both parents. Seven of 14 AS siblings, but only 1 of 8 normal hemoglobin (AA) siblings, also had HbF and F-cell counts above the “normal” range. It seems that a factor for increased F cells, linked to the beta S gene of one parent, is segregating in these families and is responsible for the greatly increased HbF and F cells in the SS subjects. HbF per F cell in AS parents and siblings was the same as that of normal AA subjects, whereas in the SS offspring it was greatly increased, suggesting that it was the result of marrow hyperplasia associated with their hemolytic anemia. The similarity of this “increased F-cell gene” to heterocellular hereditary persistence of fetal hemoglobin (HPFH). Swiss type, is discussed, and it is suggested that it may control the persistent synthesis of HbF in sickle cell anemia by its presence in early infancy.

Activity of CNTO 530 in a Murine Model of Sickle Cell Anemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4606-4606
Author(s):  
Dorie Makropoulos ◽  
Ram Achuthanandam ◽  
Patricia Rafferty ◽  
Denise Chroscinski ◽  
Kerry Brosnan ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Erythropoietin Receptor ◽  
Beta Thalassemia ◽  
Cell Phenotype ◽  
Employment Equity ◽  
Epoetin Alpha ◽  
F Cells ◽  
Darbepoetin Alpha ◽  
Equity Ownership

Abstract Abstract 4606 CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia) and in a model of beta-thalassemia. Here we report on the activity of CNTO 530 in murine globin KO-human HbS transgenic (sickle cell) mice. Berkeley mice, as originally described by Pászty et al (Pászty et al. Science 1997), are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Thus, these mice express exclusively human hemoglobin A (HbAsickle) (or sickle hemoglobin, HbS) and can also express human fetal hemoglobin (HbF). To test the effectiveness of CNTO 530 in this model, mice received a single subcutaneous (s.c.) dose of CNTO 530 (0.3 mg/kg), epoetin-alpha (0.1 mg/kg) or darbepoetin-alpha (0.03 mg/kg) (as comparators) at 10,000 U/kg, a dose previously shown to increase hemoglobin in normal mice. Hematology, histology of the spleen, F-cells and expression of HbF were then evaluated over time. We show that these mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. In addition, splenomegaly and extramedullary erythropoiesis were observed. Ten days after dosing, CNTO 530 but not epoetin-alpha or darbepoetin-alpha caused an increase in reticulocytes, red blood cells (RBC), F-cells, total hemoglobin (Hgb) and HbF. In conclusion, these results demonstrate that a single dose of CNTO 530 can increase expression of HbF in sickle cell mice and suggest that human treatment with CNTO 530 may have beneficial effects in sickle cell anemia patients. Disclosures: Makropoulos: Centocor, R&D, Inc: Employment, Equity Ownership. Achuthanandam:Centocor, R&D, Inc: Employment, Equity Ownership. Rafferty:Centocor, R&D, Inc: Employment, Equity Ownership. Chroscinski:ACE Animals, Inc: CRO. Brosnan:Centocor, R&D, Inc: Employment, Equity Ownership. Ogunkua:Chroscinski: Employment, Equity Ownership. Miller:Centocor, R&D, Inc: Employment, Equity Ownership. Avery:Centocor, R&D, Inc: Employment, Equity Ownership. Walker:Centocor, R&D, Inc: Employment, Equity Ownership. Bugelski:Centocor, R&D, Inc: Employment, Equity Ownership.

scholarly journals Hydroxyurea-induced augmentation of fetal hemoglobin production in patients with sickle cell anemia

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 109-116 ◽  
Author(s):  
S Charache ◽  
GJ Dover ◽  
MA Moyer ◽  
JW Moore
Keyword(s):  
Clinical Improvement ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Fetal Hemoglobin ◽  
Pharmacokinetic Studies ◽  
Cell Responses ◽  
Small Risk ◽  
F Cells ◽  
Hemoglobin Production

Five patients with sickle cell anemia were treated with hydroxyurea (HU), in hopes of augmenting their production of fetal hemoglobin. Laboratory responses in two patients treated for more than 2 years were encouraging and there were suggestions of clinical improvement. Long- term HU therapy should be considered for severely affected adults with sickle cell anemia who are willing to accept what is probably a small risk of carcinogenesis. Preliminary chromosomal analysis and knowledge of the clastogenic properties of HU suggest that conception and pregnancy should be avoided. Pharmacokinetic studies will probably be necessary to adjust individual dosage schedules so that cytotoxicity is avoided. F cell responses can be seen in 2 to 3 weeks if the HU dose is optimal, but establishment of a large number of F cells in the circulation may take a month or longer.

Fetal hemoglobin in sickle cell anemia: a glass half full?

Blood ◽  
2014 ◽  
Vol 123 (4) ◽  
pp. 481-485 ◽  
Author(s):  
Martin H. Steinberg ◽  
David H. K. Chui ◽  
George J. Dover ◽  
Paola Sebastiani ◽  
Abdulrahman Alsultan
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Severe Disease ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Sickle Hemoglobin ◽  
F Cells ◽  
The Mean ◽  
Number Of Cells

Abstract Fetal hemoglobin (HbF) modulates the phenotype of sickle cell anemia by inhibiting deoxy sickle hemoglobin (HbS) polymerization. The blood concentration of HbF, or the number of cells with detectable HbF (F-cells), does not measure the amount of HbF/F-cell. Even patients with high HbF can have severe disease because HbF is unevenly distributed among F-cells, and some cells might have insufficient concentrations to inhibit HbS polymerization. With mean HbF levels of 5%, 10%, 20%, and 30%, the distribution of HbF/F-cell can greatly vary, even if the mean is constant. For example, with 20% HbF, as few as 1% and as many as 24% of cells can have polymer-inhibiting, or protective, levels of HbF of ∼10 pg; with lower HbF, few or no protected cells can be present. Only when the total HbF concentration is near 30% is it possible for the number of protected cells to approach 70%. Rather than the total number of F-cells or the concentration of HbF in the hemolysate, HbF/F-cell and the proportion of F-cells that have enough HbF to thwart HbS polymerization is the most critical predictor of the likelihood of severe sickle cell disease.

2-deoxy 5-azacytidine and fetal hemoglobin induction in sickle cell anemia

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2379-2384 ◽  
Author(s):  
Mabel Koshy ◽  
Louise Dorn ◽  
Linda Bressler ◽  
Robert Molokie ◽  
Donald Lavelle ◽  
...  
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Complete Blood Count ◽  
Fetal Hemoglobin ◽  
Therapeutic Benefit ◽  
Additional Treatment ◽  
Acute Chest Syndrome ◽  
F Cells ◽  
Pain Crisis ◽  
Globin Synthesis

Abstract Augmentation of the fetal hemoglobin (HbF) levels is of therapeutic benefit in patients with sickle cell anemia. Hydroxyurea (HU), by increasing HbF, lowers rates of pain crisis, episodes of acute chest syndrome, and requirements for blood transfusions. For patients with no HbF elevation after HU treatment, augmentation of HbF levels by 5-aza-2′-deoxycytidine (5-aza-CdR, decitabine) could serve as an alternate mode of treatment. Eight adult patients participated in a dose-escalating phase I/II study with 5-aza-CdR at doses ranging from 0.15 to 0.30 mg/kg given 5 days a week for 2 weeks. HbF, F cell, F/F cell, γ-globin synthesis ratio, complete blood count, and chemistry were measured. The average γ-globin synthesis relative to non-α-globin synthesis prior to therapy was 3.19% ± 1.43% and increased to 13.66% ± 4.35% after treatment. HbF increased from 3.55% ± 2.47% to 13.45% ± 3.69%. F cells increased from 21% ± 14.8% to 55% ± 13.5% and HbF/F cell increased from 17% to 24%. In the HU nonresponders HbF levels increased from 2.28% ± 1.61% to 2.6% ± 2.15% on HU, whereas on 5-aza-CdR HbF increased to 12.70% ± 1.81%. Total hemoglobin increased by 1 g/dL in 6 of 8 patients with only minor reversible toxicities, and all patients tolerated the drug. Maximum HbF was attained within 4 weeks of treatment and persisted for 2 weeks before falling below 90% of the maximum. Therefore 5-aza-CdR could be effective in increasing HbF in patients with sickle cell anemia who failed to increase HbF with HU. Demonstration of sustained F levels with additional treatment cycles without toxicity is currently being performed.

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