Improvement of erythropoiesis in beta-thalassemic mice by continuous erythropoietin delivery from muscle

beta-Thalassemias are highly prevalent genetic disorders that can cause severe hemolytic anemia. The main pathophysiologic feature of beta-thalassemia is the accumulation of unpaired alpha-globin chains in erythrocyte precursors and red blood cells (RBCs). This accumulation alters cell membrane function and results in early cell destruction and ineffective erythropoiesis. Correction of globin chain imbalance through the induction of fetal hemoglobin (HbF) synthesis is a tentative therapeutic approach for this class of diseases. In short-term in vitro or in vivo assays, recombinant human erythropoietin increases the frequency of erythroid precursors programmed to HbF in humans and to beta-minor globin in mice. In contrast, long-term treatment of beta-thalassemic patients did not induce HbF significantly. We took advantage of highly efficient adeno-associated virus-mediated (AAV-mediated) gene transfer into mouse muscle to induce a robust and sustained secretion of mouse erythropoietin in beta-thalassemic mice, which represent a suitable model for human beta-thalassemia intermedia. A 1-year follow-up of 12 treated animals showed a stable correction of anemia associated with improved RBC morphology, increased beta-minor globin synthesis, and decreased amounts of alpha-globin chains bound to erythrocyte membranes. More effective erythropoiesis probably accounted for a reduction of erythroid cell proliferation, as shown by decreased proportions of circulating reticulocytes and by reduced iron 59 ((59)Fe) incorporation into erythroid tissues. This study indicates that the continuous delivery of high amounts of autologous erythropoietin induced a sustained stimulation of beta-minor globin synthesis and a stable improvement of erythropoiesis in the beta-thalassemic mouse model. (Blood. 2000;95:2793-2798)     

Preleukaemia and Reticulocytosis: A Case Report with in Vitro Evidence for Abnormal Reticulocyte Maturation

A case of preleukaemia with marked reticulocytosis and a slightly reduced red cell ⁵¹Cr survival is presented. An in vitro ‘reticulocyte survival’ test suggested that the apparent reticulocytosis was due to delayed maturation of reticulocytes. The globin synthesis ratio suggested an abnormal ribonucleic acid. At autopsy, in addition to the findings of acute leukaemia, mesenteric lipodystrophy was discovered. This abnormality has been seen in patients with malignant diseases, especially lymphomas, but heretofore has not been reported in acute leukaemia.     

X-linked syndrome of platelet dysfunction, thrombocytopenia, and imbalanced globin chain synthesis with hemolysis

An unusual family is described with a congenital bleeding disorder present in four males belonging to three generations. Of the three surviving affected males, all had splenomegaly and petechiae. The three had moderate thrombocytopenia (55-90 X 10(9)/liter) and markedly prolonged Ivy-template bleeding times (greater than 30 min). They were also noted to have reticulocytosis and, upon further investigation, imbalanced globin chain synthesis resembling that of beta-thalassemia minor. Studies on nine additional family members in four generations were normal except for slight elevations of reticulocyte counts in female members, one of whom had the abnormal globin chain synthesis ratio. In male members, the bleeding tendency and clinical signs always occurred in the presence of the globin chain synthesis defect and reticulocytosis. This previously undescribed condition was apparently transmitted as an X-linked disorder.     

Fetal hemoglobin in acute and chronic states of erythroid expansion

Physiologic principles underlying the differences in fetal hemoglobin (HbF) induction between acute and chronic states of erythroid expansion are poorly understood. Whereas abrupt erythroid expansion is characterized by a high proportion of reticulocytes coexpressing adult and fetal globin (F reticulocytes), HbF levels wane with chronic erythropoietic stimulation. To investigate this phenomenon, we used various schedules of erythropoietin (epo) administration in primates. Acute intravenous epo administration promoted a 2- to 10-fold preferential induction of F reticulocytes compared with total reticulocytes. Total reticulocyte and F reticulocyte production were significantly correlated (correlation coefficient .41 to .74). With chronic epo administration, preferential F reticulocyte production was lost, and there was no correlation between reticulocyte and F reticulocyte production (correlation coefficient -.03). The mean percentage of F reticulocytes did not change between acute and chronic schedules of epo administration. The subcutaneous route of high-dose (3,000 U/kg) epo administration was as effective as intravenous administration in the induction of HbF. Reticulocyte and F reticulocyte responses to increasing epo doses were found to be saturable. These results suggest that the kinetics rather than absolute levels of reticulocyte and F reticulocyte response form the basis for preferential F reticulocyte induction with acute erythropoietic stimulation, and they support the hypothesis that F reticulocytes arise from a relatively rapid pathway of erythroid maturation.     

Perturbations in the erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine

In vivo observations on the kinetics of F cells and of fetal hemoglobin (HbF) synthesis and in vitro studies of erythroid progenitors, their number, and the gamma-gene expression in their progeny were carried out in baboons (Papio cynocephalus) treated with 5-azacytidine. Maximum effect on the increase of HbF production in vivo was observed only when an expanded erythroid marrow population was present. In these animals, as well as in normal animals, treatment resulted in a significant reduction of the late erythroid progenitor cell pools (erythroid clusters and erythroid colony-forming units, CFU-E) in the marrow. This reduction was more pronounced among those progenitors grown in the absence of added erythropoietin, and it was followed by a rebound a few days after treatment cessation, reflecting the accumulation of regenerating progenitors. An early increase in the in vitro synthesis of HbF in erythroid clusters and CFU-E colonies was observed. This increase was further documented at the cellular level, with immunofluorescent labeling of colonies with monoclonal anti-gamma- globin chain antibodies. In contrast to the findings in late progenitors, the number of erythroid burst-forming unit (BFU-E) colonies and the synthesis of HbF in these colonies was not influenced significantly by 5-azacytidine treatment. It is proposed that the toxic effects of 5-azacytidine on late progenitors, leading to faster mobilization of earlier progenitors to the next more mature compartment, play a role in the in vivo augmentation of HbF synthesis by this drug. This perturbation in the progenitor cell population kinetics and the presumed hypomethylation of the surviving differentiating cells may act synergistically to produce a maximum HbF response after 5-azacytidine treatment.     

Iron metabolism in the Belgrade rat

Iron metabolism in the Belgrade rat was examined in the intact animal and in the reticulocyte suspensions. The plasma iron turnover was increased. However, when allowance was made for the effect of the elevated plasma iron concentration, erythroid marrow capacity for iron uptake was at basal levels. Numbers of erythroid cells in marrow and spleen measured by the radioiron dilution technique were increased. Thus iron uptake was not proportionate to the erythroid hyperplasia in the b/b rat, despite a more than adequate plasma iron supply. This relative deficiency in iron uptake was reflected in a severe microcytosis and elevated red cell protoporphyrin. Reticulocyte incubation studies demonstrated an unimpaired uptake of the transferrin- iron-receptor complex but a marked reduction in iron accumulation. The diferric transferrin molecule, when it did give up iron within the cell, released both of its iron atoms so that only apotransferrin was returned to the media. In contrast to the nearly complete release of iron within the normal reticulocyte, the major portion of iron taken up by the Belgrade reticulocyte was returned to the plasma. The release mechanism that can be impaired in iron-deficient reticulocytes by EDTA or cadmium was shown to be affected by lower concentrations of these substances in the Belgrade reticulocyte. It is concluded that the Belgrade rat has an abnormality of iron release within the absorptive vacuole that is responsible for a state of intracellular iron deficiency, involving the erythron and other body tissues.     

Effects of hydroxurea, stem cell factor, and erythropoietin in combination on fetal hemoglobin in the baboon

OBJECTIVE: Augmentation of the level of fetal hemoglobin (HbF) is considered therapeutic for patients with sickle cell disease. The objective of this study was to determine the effect of treatment with a combination of erythropoietin (Epo), stem cell factor (SCF), and hydroxyurea (HU) on HbF levels. MATERIALS AND METHODS: The effect of treatment with a combination of Epo, SCF, and HU on HbF, F-cell numbers, and globin chain synthesis was evaluated in a baboon model. RESULTS: Treatment with a combination of SCF+Epo resulted in a two-fold increase in HbF, F-cells, and F-reticulocytes compared to Epo alone. The combination of SCF+Epo+HU resulted in an additional two-fold increase in HbF, whereas F-cells and F-reticulocytes increased only 25% compared to the SCF+Epo regimen. Measurement of differential globin chain synthesis indicated that the SCF+Epo+HU treatment also increased the I gamma/V gamma (homologous to human G gamma and A gamma) synthetic ratio toward the fetal ratio. CONCLUSIONS: HU can effectively augment growth factor-induced HbF synthesis in vivo. Because I gamma/V gamma ratios are unaffected by erythropoietic stress and similar increases in this ratio have only been observed following administration of 5-azacytidine, we suggest that these two agents may share a common mechanism of action involving the recruitment of a similar target cell population to terminal erythroid differentiation.     

Effects of dexamethasone on fetal hemoglobin synthesis in peripheral blood erythroid burst-forming units

Colonies derived from erythroid burst-forming units (BFU-E) synthesize fetal hemoglobin (HbF) in amounts that far exceed in vivo levels. There is some evidence that HbF synthesis is controlled at the level of a primitive erythroid precursor cell. Dexamethasone may potentiate the development of BFU-E. Since a means of augmenting HbF production in sickle cell anemia or severe β-thalassemia would be of great therapeutic value, we studied the effects of dexamethasone on HbF and γ-globin chain synthesis in BFU-E from patients with sickle cell anemia and controls. HbF was measured by radioimmunoassay of BFU-E lysate and γ-chain synthesis by the incorporation of ³H-leucine into globin, which was then purified by gel filtration and column chromatography. Dexamethasone (10^?9 M) produced an increase in the number of BFU-E in 16 of 19 subjects when compared with numbers of BFU-E cultured with only erythropoietin. The individual BFU-E were larger and contained more subcolonies. Dexamethasone did not increase HbF or γ-chain synthesis, and there was no relationship between increased proliferation of BFU-E and augmented HbF production. Thus, although dexamethasone augmented the development of erythroid bursts, there was no increment in HbF.     

Haemoglobin Biosynthesis during in Vitro Maturation of the Rat Reticulocyte

Summary. The relationship of changes in cellular iron uptake, haem and globin synthesis in the maturing reticulocyte was studied during in vivo maturation of rat reticulocytes produced after phlebotomy. Cellular iron uptake and haem synthesis declined at a more rapid rate than did globin synthesis. During maturation there was a uniform loss of all ribosomal classes and [¹⁴C]leucine incorporation studies indicated a decreased time for polypeptide chain synthesis per polysome. These observations support the postulate that globin synthesis is controlled by the availability of haem, the latter in turn being dependent on the availability of iron. The following sequence of events in the maturing reticulocyte is suggested: first, there is a loss of the capacity of the reticulocyte to take up iron leading to cessation of haem synthesis. Depletion of available haem then results in either an accumulation of globin, which by a feedback mechanism suppresses further globin synthesis, or an activation of reticulocyte ribonuclease which breaks down the polyribosome-mRNA complex, making further protein synthesis impossible.     

Fetal hemoglobin induction with butyric acid: efficacy and toxicity

Butyric acid induces fetal hemoglobin (HbF), a property of potential therapeutic advantage in patients with disorders of globin chain synthesis. We performed dose escalation studies of this compound in baboons to assess whether clinically significant increases in HbF are achievable, and to define the associated toxicities. Additionally, the effect of butyrate in combination with erythropoietin on HbF induction was assessed. HbF induction in response to butyrate was dependent on the dose and duration of treatment. Doses of butyrate less than 4 g/kg/d were associated with minimal toxicity (hypokalemia) and significant HbF induction in these nonanemic animals, with 1 g/kg/d producing an increase in HbF-containing reticulocytes (F reticulocytes) from 0.9% to 8.7% and an increase in HbF from 0.8% to 1.4%. A dose of 2 g/kg/d resulted in an increase in F reticulocytes from 2.1% to 27.8% and an increase in HbF from 0.7% to 2.2%. Doses of 4 g/kg/d in another animal produced an increase in F reticulocytes from 1% to 21.6% and in HbF from 1.9% to 5.3%. Infusions in excess of 4 g/kg/d were complicated (after a variable amount of time) by a decreased level of alertness (caused by hyperosmolality or butyrate itself) and hematologic toxicity (with declines in reticulocyte, white blood cell, and platelet counts). Prolonged infusions of high doses of butyrate (8 to 10 g/kg/d) were associated with peak F reticulocyte percentages reaching 38% to 64.5% and HbF reaching levels in excess of 20%. These high doses (8 to 10 g/kg/d) were complicated in two animals with a striking and unique neuropathologic picture and, in one animal, multiorgan system failure. Erythropoietin in combination with butyrate, induced F reticulocytosis in an additive manner. We conclude that butyric acid is a strong inducer of HbF, particularly when administered in combination with erythropoietin. As chronic toxicities remain undefined, patients in future clinical trials of this and similar compounds should be monitored closely for evidence of neurologic toxicity.     

On the induction of fetal hemoglobin by butyrates: in vivo and in vitro studies with sodium butyrate and comparison of combination treatments with 5-AzaC and AraC

To obtain information on the cellular mechanism of induction of fetal hemoglobin (HbF) by sodium butyrate (NaB), we treated adult baboons with NaB and assessed its effects on HbF expression. Infusion of NaB increased F reticulocytes and F-positive CFUe and e-cluster colonies without induction of reticulocytosis or increase in progenitor cell numbers. Addition of NaB in bone marrow cultures increased the frequency of F-positive CFUe and e-clusters without increasing progenitor cell numbers. NaB induced HbF in human adult BFUe cultures and increased the gamma/gamma + beta globin chain and mRNA ratios in short-term incubations of culture-derived erythroblasts. There was a synergistic induction of HbF by NaB and 5-azacytidine (5-azaC), but not when the animal was treated with NaB and cytarabine (AraC). Our results suggest that the activation of gamma-globin expression by NaB reflects an action of this compound on globin genes or globin chromatin.     

Fetal Haemoglobin in Early Malignant Osteopetrosis

The characteristics of the fetal haemoglobin (HbF) in two children with osteopetrosis and high levels of HbF have been studied. The structural analysis of the gamma chains demonstrated a fetal Ggamma/Agamma ratio. HbF was distributed inside only 30% of the peripheral red blood cells. In vitro globin chain synthesis studies showed that there was balanced globin chain production, despite the increased level of HbF.     

Balanced bone marrow globin synthesis in mideastern alpha-thalassemia

Hematological data and globin synthesis studies in 8 alpha-thalassemic children whose parents are from various regions in the Middle East are reported. All patients were devoid of Hb H. 5 of them had mild anemia, hypochromia and microcytosis and their blood alpha-/non-alpha-globin radioactivity ratios ranged between 0.56 and 0.75. The other 3 patients were hematologically normal with blood radioactivity ratios between 0.77 and 0.88. Bone marrow studies showed balanced globin chain synthesis for 6 of the patients and relatively more alpha-globin synthesis than in the blood for the remaining 2. The decrease in the relative synthesis of alpha-globin on erythroid cell maturation may relate to the molecular basis of alpha-thalassemia in the Middle East.     

Isolation and in vitro differentiation of human erythroid precursor cells.

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.     

Bone marrow sinus cell packing: a determinant of cell release.

Ultrastructural studies of erythropoietin effects on the bone marrow of control and hypertransfused (65 hct) mice revealed a decrease in adventitial cell cover of the sinus apertures in erythropoietin-treated animals. A more striking finding, however, was the marked inhibition of erythropoietin-induced reticulocytosis by hypertransfusion itself. Hypertransfusion of the erythropoietin-treated animals appeared to decrease the reticulocyte response by inhibiting reticulocyte response by marrow cords in addition to inhibiting erythroid proliferation. This inhibition of reticulocyte response was associated with clustering of reticulocytes around the marrow sinuses which were packed with red cells. Acute lowering of the hematocrit of erythropoietin-treated, hypertransfused animals to normal at the time of maximal reticulocyte response in control animals resulted in more than a twofold increase in reticulocytosis with 2 hr. It is suggested that (1) elevated levels of erythropoietin are associated with a diminution of the normal marrow-peripheral blood barrier, thereby contributing to the premature release of marrow elements and (2) the hematocrit is an important determinant of cell release from the marrow into the peripheral circulation.     

Microcytic anemia with iron malabsorption: An inherited disorder of iron metabolism

Two siblings were identified with severe hypoproliferative microcytic anemia and iron malabsorption, in the absence of any gastrointestinal disorder or blood loss. These children had severe microcytosis (MCV 48 fl, hemoglobin 7.5 g/dl) with decreased serum iron, elevated serum TIBC, and decreased serum ferritin, despite prolonged treatment with oral iron. An iron challenge study with an oral dose of 2 mg/kg elemental iron as ferrous sulfate documented iron malabsorption. After treatment with intravenous iron dextran, there was an absence of the expected reticulocytosis and only a partial correction of the hemoglobin, hematocrit, and microcytosis. The bone marrow was hypocellular with abnormal iron incorporation into erythroid precursor cells. This appears to be a rare form of inherited anemia characterized by iron malabsorption and disordered iron metabolism that only partially corrects after the administration of parenteral iron. These features resemble those found in the microcytic mouse (mk/mk), which also has severe microcytic anemia and iron malabsorption that partially responds to parenteral iron. © 1996 Wiley-Liss, Inc.