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A N Schechter

Publications and source records attributed to A N Schechter.

At least 55 records · Page 3Linked to original sources
PubMed1995

Hydroxyurea therapy in beta-thalassaemia intermedia: improvement in haematological parameters due to enhanced beta-globin synthesis.

The beta-thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid beta-globin mRNA expression and imbalanced alpha/beta-globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating gamma-globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in beta-thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese beta-thalassaemia patients we unexpectedly found that two unrelated patients with beta-thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4.1 to 6.3 g/dl, patient 1; from 6.5 to 9.7 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300d of low-dosage HU treatment. These effects, however, appear to be due to increased beta-globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the beta/alpha ratio (from 0.301 to 0.581, patient 1; from 0.348 to 0.487, patient 2) with minimal changes in gamma-globin biosynthesis. We conclude that in addition to its known effects in stimulating gamma-globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including beta-globin in some thalassaemia intermedia patients who maintain the capacity to express normal beta-globin chains.

Female↗
PubMed1995

Contributions of sickle hemoglobin polymer and sickle cell membranes to impaired filterability.

Sickle cell anemia is a disease of abnormal rheology caused by acute and reversible, as well as chronic and irreversible, changes in the properties and deformability of sickle erythrocytes. Deformability is determined by several factors, including intracellular sickle hemoglobin polymerization, the abnormal membrane properties of sickle cells, and the abnormal rheological properties of the soluble concentrated hemoglobin solution within dense sickle red blood cells. In this study, we used a 5-microns pore nickel mesh filter to evaluate quantitatively the effects of these factors on the filterability of erythrocytes containing sickle hemoglobin. We used sickle trait and sickle/beta(+)-thalassemia cells, because they have minimal membrane abnormalities or density heterogeneity, to investigate the effects of polymer formation on rheological properties. We found that filterability of these cells is sensitive to small amounts of intracellular polymer and that impaired filtration is linearly related to oxygen-dependent polymer formation, up to a polymer fraction of 0.3. By increasing the proportion of dense cells in populations of normal cells or cells from individuals with sickle syndromes and equilibrating these cells with gas ligands, we estimate that polymerization, even at 95% saturation, contributes twice as much to impaired filterability of sickle erythrocytes as the abnormal membranes in homozygous sickle cell disease. At lower saturation values, the effects of polymer are even greater. The viscosity of the concentrated hemoglobin in dense cells had the smallest effect, over physiologically relevant saturation values. These results emphasize the importance of sickle hemoglobin polymerization in the pathogenesis of sickle cell disease and should help define its pathophysiology and responses to therapy in quantitative terms.

Anemia, Sickle Cell↗
PubMed1994

Variation in fetal hemoglobin parameters and predicted hemoglobin S polymerization in sickle cell children in the first two years of life: Parisian Prospective Study on Sickle Cell Disease.

Intracellular hemoglobin S (HbS) polymerization is most likely to be the primary determinant of the clinical and biologic manifestations of sickle cell disease (SCD). Fetal hemoglobin (HbF) does not enter the HbS polymer and its intracellular expression in sickle erythrocytes inhibits polymerization. HbF levels, high at birth but decreasing thereafter, protect the newborn from the clinical manifestations of this hemoglobinopathy. We have measured the sequential changes in HbF, F reticulocytes, and F cells in the first 2 years of life in 25 children with SCD and compared the results with those obtained in 30 normal children (AA). We have also calculated HbF per F cell (F/F cell), the preferential survival of F cells versus non-F cells, as measured by the ratio F cells versus F reticulocytes (FC/FR) and polymer tendency at 40% and 70% oxygen saturation. HbF levels decreased from about 80.4% +/- 4.0% at birth to 9.2% +/- 2.9% at 24 months. During this time, we observed a regular decrease of the F reticulocytes and the F cells. The kinetics of the decline of F/F cell was comparable with the decline of HbF, rapid from birth (mean, 27.0 +/- 3.6 pg) to 12 months of age (mean, 8.5 +/- 1.5 pg) and then slower from 12 to 24 months of age (mean, 6.2 +/- 1.0 pg) in the SCD children. In the AA children, the decrease in HbF, due to changes in both numbers of F cells and F/F cell, was more precipitous, reaching steady-state levels by 10 months of age. Calculated values for mean polymer tendency in the F-cell population showed that polymerization should begin to occur at 40% oxygen saturation at about 3 months and increase progressively with age, whereas polymerization at 70% oxygen saturation would not occur until about 24 months. These values correspond to HbF levels of 50.8% +/- 10.8% and 9.2% +/- 2.9%, respectively, and F/F cell levels of 15.6 +/- 4.5 pg and 6.2 +/- 1.0 pg, respectively. In the non--F-cell population, polymerization was expected at birth at both oxygen saturation values. Three individuals had significantly greater predicted polymerization tendency than the remainder of the group because of early decreases in HbF. These individuals in particular, the remainder of the cohort, as well as other recruited newborns, will be studied prospectively to ascertain the relationship among hematologic parameters, which determine polymerization tendency and the various clinical manifestations of SCD.

Anemia, Sickle Cell↗
PubMed1994

Reducing erythropoietin in cultures of human erythroid precursors elevates the proportion of fetal haemoglobin.

In order to clarify the mechanism of the effect of erythropoietin (Epo) on the fetal haemoglobin (HbF) phenotype of peripheral erythrocytes, we studied the dose-response effect of Epo on HbF production by erythroid precursors derived from the peripheral blood of normal adult individuals and grown in a two-phase liquid culture system. The proportion of HbF out of the total haemoglobin (Hb) content (%HbF) was dependent on the duration of exposure to Epo; on day 6 it comprised up to 15%, but dropped to < 2% on day 14. Both cell yield and cellular Hb content were markedly increased by high (1 U/ml) Epo, compared to normal physiological (20-50 mU/ml) levels, but neither the initial nor final %HbF were dependent on the increased Epo dose. However, when cells grown with high Epo were transferred on day 7 to low Epo, their progeny contained by day 14 a higher %HbF as compared to cells that were continuously exposed to high Epo. This was accompanied by acceleration and synchronization of their maturation process, as evidenced by their morphology, density and size, and restriction on cell multiplication, as indicated by the lower cell yield. These results are consistent with the following model. As early erythroid precursors, with relatively high HbF, mature under steady-state levels of Epo, HbA production predominates and HbF is diluted. However, when such precursors are switched from high to low levels of Epo they undergo a synchronized, accelerated maturation which shortens the period of HbA production, leading to a decreased Hb content and a relatively high proportion of HbF. This mechanism may contribute to the elevated HbF observed following Epo administration (due to short half-life of Epo in vivo), and might also explain the HbF-augmenting effect of Epo administered together with hydroxyurea observed in patients with sickle cell anaemia.

Cell Division↗
PubMed1994

RNA transcripts of the beta-thalassaemia allele IVS-2-654 C-->T: a small amount of normally processed beta-globin mRNA is still produced from the mutant gene.

IVS-2-654 C-->T is a common Chinese beta-thalassaemia mutation. Previous studies report that this mutation resulted in the formation of an abnormally spliced mRNA and the absence of detectable normal beta-globin mRNA, hence the mutation was considered to cause beta o-thalassaemia. We recently used the method of PCR amplified cDNA copies of circulating erythroid cell mRNA to analyse the mutant gene transcripts and found that this IVS-2-654 mutation does not abolish normal RNA processing entirely, but that a significant amount (over 15%) of normally processed beta-globin mRNA is produced. Microglobin chain biosynthetic analysis using the HPLC method showed that beta-globin chain was also present in the blood of patients with IVS-2-654 C-->T mutation. Accordingly, this mutant allele leads to a beta (+)-thalassaemia. Further, the methodology described in this paper provides a new approach towards the detection of RNA transcripts of beta-thalassaemia alleles as well as the study of gene expression in beta-thalassaemia and other genetic diseases.

Alleles↗
PubMed1994

Treatment of beta-thalassemia with hydroxyurea (HU)--effects of HU on globin gene expression.

A newly developed method of RT-PCR/competitive PCR for measuring the relative and absolute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study the alterations of globin gene expressions in the patients with beta-thalassemia pre- and post-hydroxyurea (HU) treatment. It was found for the first time that HU had the effect of enhancing beta-globin gene expression in some patients. Two cases with beta-thalassemia who were subjected to HU treatment for over two years showed a marked increase in beta-globin mRNA level and beta-globin chain synthesis, resulting in more effective erythropoiesis and the alleviation of clinical symptoms.

Adult↗
PubMed1993

Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S at physiologic ligand saturations.

Recent interest in therapies for sickle cell anemia based on elevating fetal Hb has made accurate estimates of the sparing effect of fetal Hb (Hb F) and other non-sickle Hbs on sickle Hb (Hb S) polymerization essential. We have developed a technique, using HbCO as surrogate for HbO2, that enables us to assess the solubility of Hb S as a function of ligand saturation under conditions that mimic those of the sickling disorders. Equimolar mixtures of unliganded Hb S with Hb F or normal Hb A2 were isosoluble. Solubilities for equimolar mixtures with normal (Hb A) or abnormal (Hb C) Hbs were also identical but were lower than in the prior case. Thus, the sparing effect of both Hb F and Hb A2 should be considered in therapeutic strategies designed to modify Hb S polymerization. Hemolysates, stripped of 2,3-bisphosphoglycerate, from sickle cell disease patients with Hb (F + A2) levels varying from 6 to 25%, as well as from a sickle trait individual, were used to evaluate equilibrium solubility as a function of ligand saturation over the range of pathophysiologic interest (25-70%). Our results show that the sparing effect of Hb (F + A2) increases relative to that of Hb A as ligand saturation increases, and that in the absence of ligand, approximately 30% Hb (F + A2) is essentially isosoluble with the 60% Hb A of sickle trait. Although detailed knowledge of expected therapeutic benefits is confounded by the heterogeneity of Hb F distribution and other variables, these data should provide a framework for estimating likely clinical benefit from pharmacologic efforts to modulate globin gene expression.

Anemia, Sickle Cell↗
PubMed1993

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

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.

Anemia, Sickle Cell↗
PubMed1993

Augmentation by erythropoietin of the fetal-hemoglobin response to hydroxyurea in sickle cell disease.

BACKGROUND: Hydroxyurea increases the production of fetal hemoglobin in patients with sickle cell anemia, inhibiting the polymerization of hemoglobin S and potentially improving vaso-occlusive manifestations and hemolysis. Recombinant erythropoietin increases the number of reticulocytes containing fetal hemoglobin in laboratory animals and in humans. We studied whether hydroxyurea and erythropoietin might have a potentiating effect on the production of fetal hemoglobin in patients with sickle cell disease. METHODS: We treated four patients who were receiving hydroxyurea for sickle cell disease (three who were homozygous for sickle cell anemia and one with sickle beta zero-thalassemia) with escalating doses of intravenous erythropoietin for seven weeks, along with oral iron sulfate. Doses of hydroxyurea on four consecutive days were alternated with doses of erythropoietin on three consecutive days. RESULTS: There was a 28 percent increase in the number of reticulocytes containing fetal hemoglobin and a 48 percent increase in the percentage of fetal hemoglobin, as compared with the maximal values obtained with hydroxyurea alone. The percentage of erythrocytes containing fetal hemoglobin (F cells) increased from 64 to 78 percent. As compared with hydroxyurea alone, treatment with hydroxyurea and erythropoietin decreased the mean (+/- SD) serum indirect bilirubin level from 0.8 +/- 0.2 to 0.5 +/- 0.1 mg per deciliter (13.3 +/- 2.9 to 8.9 +/- 2.2 mumol per liter) (P = 0.02), suggesting a further decrease in hemolysis. Red-cell filterability improved. CONCLUSIONS: Intravenous recombinant erythropoietin with iron supplementation alternating with hydroxyurea elevates fetal-hemoglobin and F-cell levels more than hydroxyurea alone. Such increases decrease intracellular polymerization of hemoglobin S and improve the overall rheologic characteristics of erythrocytes. A reduced dosage of hydroxyurea alternating with erythropoietin may prove less myelotoxic than hydroxyurea given daily or in pulsed-dose regimens. It may also increase levels of fetal hemoglobin in patients with sickle cell disease who have not been helped by hydroxyurea alone.

Adult↗
PubMed1993

Sickle cell disease pathophysiology.

The primary pathophysiological event in the erythrocytes of individuals with the various sickle syndromes is the intracellular aggregation or polymerization of sickle haemoglobin (HbS). The extent of polymerization is determined by the intracellular haemoglobin composition (% HbS and % HbS A, A2 and F), concentration (MCHC and % of dense cells) and oxygen saturation, as well as minor factors such as intracellular pH and DPG concentration. Intracellular HbS polymerization leads to a marked decrease in the flexibility or rheological properties of the sickle erythrocytes and obstruction in various microcirculatory beds, as well as chronic anaemia. Other abnormalities in the properties of the sickle erythrocytes, including membrane abnormalities, changes in ion fluxes and volume and endothelial adhesion, result from acute and chronic oxygen-linked polymerization events and may, in turn, modify polymerization. However, within a good approximation, many aspects of sickle cell disease pathophysiology--for example variations in anaemia among the different sickle syndromes--can be explained in terms of differences in polymerization tendency. Thus, the effects of alpha-thalassaemia can be explained with reference to changes in MCHC and syndromes with high HbF are understandable in terms of the sparing effect of HbF on polymerization. Recent therapeutic approaches to sickle cell disease focus on attempts to reduce intracellular HbS polymerization by altering the haemoglobin molecules, erythrocyte properties, or the distribution of intracellular haemoglobin species. The last, through pharmacological elevation of HbF, has become the central focus of much laboratory and clinical research in recent years. Agents such as hydroxyurea (with or without recombinant erythropoietin) and butyrate compounds elevate HbF (and reduce HbS) in a majority of sickle erythrocytes, thus decreasing intracellular polymerization. Current prospective protocols are designed to see if these changes cause clinical improvement at acceptable doses. Other treatment strategies, including bone marrow transplantation and possible gene replacement therapies, are also under active clinical or laboratory investigation.

Anemia, Sickle Cell↗
PubMed1992

DNA sequence variation in a negative control region 5' to the beta-globin gene correlates with the phenotypic expression of the beta s mutation.

The clinical diversity of sickle cell anemia is strongly related to the degree of intracellular hemoglobin S (Hb S) polymerization, which in turn is dependent on the intracellular concentration of Hb S. We have recently defined a region of DNA approximately 500 bp 5' to the human beta-globin gene that acts as a silencer for the transcription of this gene and have shown that a polymorphism in this sequence is associated with a thalassemic phenotype of the beta-globin gene. In this work we have examined the correlation of DNA sequence polymorphisms in this silencer with binding of a previously identified putative repressor protein, BP1, and with the expression of Hb S in individuals heterozygous for the beta s allele. It was found that specific configurations of the motif, (AT)x(T)y, are homogeneous for the major haplotypes of the beta-globin gene cluster described on beta s chromosomes. Binding of BP1 was measured to DNA of three haplotypes: Indian, Benin, and Bantu. BP1 binds most tightly to DNA of the Indian haplotype, and these patients produce less beta s protein than Benin patients, whose DNA exhibits weaker affinity for BP1. Binding of BP1 is the weakest to DNA of the Bantu haplotype, which is associated with clinically more severe sickle cell symptoms. These data are consistent with the hypothesis that these polymorphisms may not be neutral and that the DNA sequence at this site may affect the expression of the beta s gene. Such an effect may be synergistic with other genetic variables, such as fetal hemoglobin levels, F-cell numbers, and the number of alpha-globin genes, in determining intracellular polymerization and, thus, the severity of the sickle cell syndromes.

Africa↗
PubMed1992

Binding of erythroid and non-erythroid nuclear proteins to the silencer of the human epsilon-globin-encoding gene.

To clarify the molecular mechanisms involved in the developmental control of hemoglobin-encoding genes we have been studying the expression of these genes in human cells in continuous culture. We have previously reported the presence of a transcriptional control element with the properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin-encoding gene [Cao et al., Proc. Natl. Acad. Sci. USA 86 (1989) 5306-5309]. We also showed that this silencer has stronger inhibitory activity in HeLa cells, as compared to K562 human erythroleukemia cells. Using deletion mutants and cis-cloned synthetic oligodeoxyribonucleotides in transient expression assays, nucleotide sequences responsible for this effect have now been further delimited to 44 bp located from -294 to -251 bp. Gel electrophoresis mobility shift assays and DNaseI footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. Two binding proteins are detected in K562 nuclear extracts, while only one is found in extracts from HeLa cells. Possible mechanisms by which these proteins may regulate transcription of the epsilon-globin-encoding gene in erythroid and non-erythroid cells are discussed.

Base Sequence↗