Treatment of sickle cell anemia with hydroxyurea and erythropoietin.

BACKGROUND: Hydroxyurea increases the production of fetal hemoglobin (hemoglobin F) in patients with sickle cell anemia and therefore has the potential for alleviating both the hemolytic and vaso-occlusive manifestations of the disease. There is preliminary evidence that recombinant human erythropoietin may also increase hemoglobin F production. METHODS AND RESULTS: We treated five patients with sickle cell disease with escalating doses of intravenous erythropoietin for eight weeks. Three of these patients were subsequently treated with daily doses of oral hydroxyurea. After the optimal dose was determined, erythropoietin was then given along with hydroxyurea for four weeks. Treatment with erythropoietin, either alone or in combination with hydroxyurea, had no significant effect on the percentage of hemoglobin F-containing reticulocytes (F reticulocytes) or red cells (F cells). In contrast, hydroxyurea treatment was associated with a 3-to-25-fold increase in F reticulocytes, a 1.6-to-7-fold increase in F cells, and a 2.3-to-16-fold increase in the percentage of hemoglobin F. In all three patients given hydroxyurea, treatment with this drug was associated with reduced hemolysis, shown by decreases in serum bilirubin and lactic dehydrogenase and prolongation of red-cell survival. Hydroxyurea treatment also resulted in a decrease in the percentage of irreversibly sickled cells and sickling at partial oxygen saturation, an increase in oxygen affinity and total red-cell cation content, and a reduction in potassium-chloride cotransport. All three patients had a decrease in the number of pain crises. CONCLUSIONS: This study confirms that hydroxyurea therapy increases hemoglobin F production and provides objective evidence that hydroxyurea reduces the rate of hemolysis and intracellular polymerization of hemoglobin S. In contrast, recombinant human erythropoietin, whether alone or in combination with hydroxyurea, offers no measurable benefit.

Increase of CALLA-positive stimulated lymphoid cells in transient erythroblastopenia of childhood.

In 1986 and 1987 11 children with TEC (transient erythroblastopenia of childhood) were referred to our hospital. Bone marrow aspirations were performed to exclude haematological malignancy. There was a marked reduction of erythropoiesis in 9 cases (1%-8%), two children had already recovered (33% and 44% erythropoiesis). Eight patients exhibited high percentages of stimulated lymphoid cells. The subsequent immunotyping revealed the expression of CALLA (common acute lymphoblastic leukaemia antigen) on these cells but there was no other sign for malignancy. The patients recovered without any specific treatment except transfusions of packed red cells. Eight patients were followed up 11-18 months after initial presentation and were all found to be in good health. A prominent increase of CALLA-positive stimulated lymphoid cells has also been found in other haematological diseases such as neutropenia and immune thrombocytopenia. The expression of CALLA in bone marrow lymphocytes is a general reactive change to various alterations.

Ankyrin and the hemolytic anemia mutation, nb, map to mouse chromosome 8: presence of the nb allele is associated with a truncated erythrocyte ankyrin.

Mice with normoblastosis, nb/nb, have a severe hemolytic anemia. The extreme fragility and shortened lifespan of the mutant erythrocytes result from a defective membrane skeleton. Previous studies in our laboratory indicated a 50% deficiency of spectrin and an absence of normal ankyrin in erythrocyte membranes of nb/nb mice. We now report genetic mapping data that localize both the nb and erythroid ankyrin (Ank-1) loci to the centromeric end of mouse chromosome 8. Using immunological and biochemical methods, we have further characterized the nature of the ankyrin defect in mutant erythrocytes. We do not detect normal sized (210 kDa) erythroid ankyrin by immunoblot analysis in nb/nb reticulocytes. However, nb/nb reticulocytes do contain a 150-kDa ankyrin immunoreactive protein. The 150-kDa protein is present with normal-sized ankyrin in nb/+ reticulocytes but is not found in +/+ reticulocytes. Our genetic and biochemical data indicate that the nb mutation results from a defect in the erythroid ankyrin gene. A human hereditary spherocytosis putatively resulting from an ankyrin defect maps to a segment of human chromosome 8 that is homologous to the nb-ankyrin region of mouse chromosome 8. The linkage data suggest that the mouse and human diseases result from mutations in homologous loci.

High concentration of free trimethyllysine in red blood cells.

A high concentration of a basic unidentified amino compound was found in the blood of rats. It was isolated and identified as N epsilon,N epsilon,N epsilon-trimethyllysine by paper chromatography, thin-layer chromatography, high-performance liquid chromatography and amino acid analyzer. It was localized exclusively in red blood cells in the blood of rats. Free trimethyllysine was also determined in the liver, kidney, spleen, brain, muscle, heart and testis of rat. The concentration of free trimethyllysine in red blood cells was more than 10-times as high as that in the other tissues. This compound in red blood cells was found in different species of animals. The relationship between this free trimethyllysine and carnitine was discussed.

Two new sheep haemoglobins, one of which is replaced by haemoglobin C in anaemia.

Two new haemoglobin variants, provisionally named Hb G and Hb H, were found during a survey of 295 Welsh Mountain cross-bred sheep. Both haemoglobins appear to be beta chain variants controlled by genes allelic to those for the common forms Hb A and Hb B. Studies on an anaemic Hb AH and an Hb AG type sheep showed that Hb G, like Hb A, is replaced by Hb C in anaemia whereas Hb H, like Hb B, is not replaced.

Heat-stable translational inhibitor from rabbit reticulocyte lysates.

We have purified to apparent homogeneity a heat-stable (HS) factor from the postribosomal supernatant of rabbit reticulocyte lysates [(1988) FEBS Lett. 236, 479-483]. HS inhibits translation in hemin-supplemented lysates and induces phosphorylation of the alpha-subunit of the eukaryotic initiation factor 2 as does hemin deficiency. The translational inhibition produced by addition of HS to hemin-containing reticulocyte lysates and the accompanying phosphorylation of the eIF-2 alpha subunit can be prevented or reversed by NADPH generators including glucose 6-phosphate, NADPH itself, and also by dithiols, e.g., dithiothreitol, but not by fructose 1,6-bisphosphate or by monothiols, e.g., 2-mercaptoethanol. When added to crude preparations of the proinhibitor form (proHCI) of the heme-controlled translational inhibitor (HCI), HS produces a pronounced increase of the HCI to proHCI ratio. It appeared possible that HS might be oxidized glutathione (GSSG) but this is not the case, for HS is not a substrate for highly purified glutathione reductase from rabbit erythrocytes. The spectral analysis of highly purified HS is consistent with the idea that HS could be a nucleotide derivative.

Presence of contaminating mitochondrial DNA from host reticulocytes in experimental infections of Plasmodium berghei.

Superposition of two unrelated processes, namely terminal reticulocyte differentiation and synchronous plasmodial development, takes place in experimental infections of Plasmodium berghei. The first process is shown to be responsible for the appearance of some discrete restriction bands of host origin when DNA is extracted from leucocyte-free blood containing synchronous parasites at early stages of infection. These discrete DNA fragments cross-hybridize with host cell mitochondrial DNA. Purification steps are suggested to reduce this effect, which might be relevant also in the case of other plasmodial species exhibiting preference for reticulocytes as host cell.

Low molecular weight iron from guinea pig reticulocytes isolated by Sephadex G-25 chromatography.

As part of a continuing study of the low MW iron pool, guinea pig reticulocytes were incubated with 59Fe-labeled transferrin, and the reticulocyte hemolysate was chromatographed on Sephadex G-25. 59Fe, in amounts corresponding to that which was in a low MW peak eluting from an Ultrogel column and to that not precipitated by ammonium sulfate, adsorbed to the Sephadex column. The adsorbing 59Fe, on elution from the Sephadex with dilute formic acid, coeluted with phosphate and pentose. When EDTA was added to disrupt the putative iron complex, neither iron nor P adsorbed to the column, supporting the argument that they exist as a compound in the cytosol and adsorb and elute together for that reason. These observations provide additional evidence that P-containing compounds, probably originating as nucleotides, are important components of the low MW iron pool of cells.

The mRNA transcripts from a mutant beta-globin gene derived from splicing at preferential cryptic sites.

We have analyzed mRNA transcripts from beta-globin genes carrying a homozygous point mutation at the 5' splicing site of the first intron, using a method allowing in vivo analysis of mRNA transcripts. As expected, this mutation decreases normal splicing of mRNA when cryptic splicing are utilized. We have observed that, in reticulocytes, most mature mRNA transcribed from beta-globin genes derives from specific sites of abnormal splicing. Our results differ from those previously obtained using mutant beta-globin genes introduced in cultured cells and indicate a preferential processing of the abnormal globin mRNA species in red cell precursors.

Low-Mr iron isolated from guinea pig reticulocytes as AMP-Fe and ATP-Fe complexes.

Guinea pig reticulocytes were pulse-labelled with 59Fe bound to transferrin. Haemolysates prepared from these reticulocytes were subjected to rapid (NH1)2SO1 precipitation and then chromatography on an anion-exchange resin. ATP-bound 59Fe was the dominant species in the reticulocyte cytosol; 2,3-bisphosphoglycerate and GTP iron complexes were not detected despite the fact that these were stable with (NH1)2SO1 precipitation and readily detected with anion-exchange chromatography. AMP-bound Fe was a minor component of the cytosol following rapid (NH1)2SO4 precipitation, and the major component when iron was released from transferrin by haemolysates. We speculate that ATP-Fe may be degraded in the cell to permit utilization of its iron for haem synthesis.

Chronic exposure to tumor necrosis factor in vivo preferentially inhibits erythropoiesis in nude mice.

The anemia of chronic disease (ACD) is associated with conditions in which macrophage activation occurs. Activated marrow macrophages suppress erythropoiesis in vitro and produce tumor necrosis factor (TNF). Therefore, we tested the effects of chronic in vivo exposure to TNF to determine if it was a candidate for a mediator of ACD. Nude mice were inoculated with Chinese hamster ovary (CHO) cells expressing the human TNF gene or with control cells containing the transfection vector alone. The TNF mice promptly became reticulocytopenic, and after 3 weeks their corrected reticulocytes were 2.6% +/- 0.7% as compared with 7.3% +/- 4% in control mice. The hematocrit at 3 weeks was 28.4% +/- 1.7% in TNF mice as compared with 46% +/- 0.8% in control mice. This anemia was also associated with low serum iron and normal iron stores and increased erythropoietin (Epo) levels. The TNF mice showed an absolute monocytosis with twice the number of circulating monocytes as control mice and had M-colony-stimulating factor (CSF) activity in their serum. The TNF mice also became mildly thrombocytopenic. Marrow CFU-E and BFU-E were profoundly decreased (1.2 +/- 0.2 x 10(3) v 8.6 +/- 0.2 x 10(4) CFU-E per femur, and 6.5 +/- 1 x 10(2) v 8.5 +/- 0.2 x 10(4) BFU-E per femur). Splenic CFU-E and BFU-E were similarly depressed. In contrast, marrow CFU-GM and CFU-GEMM were not affected. The residual BFU-E in TNF mice were relatively resistant to TNF as compared with control mice. These data demonstrate that TNF preferentially inhibits erythropoiesis in vivo and may be important in the pathogenesis of ACD.

Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index.

Flow cytometric reticulocyte quantification with thiazole orange has been reported to be of potential utility in a clinical hematology laboratory. We have instituted this technique into routine clinical testing for 18 months and we describe this experience. Flow cytometric analysis provided not only reproducible, cost-effective reticulocyte quantification, but a quantitative reticulocyte maturity index proportional to the amount of RNA in the reticulocytes. The reticulocyte maturity index measurement represents an independent parameter of erythropoiesis, which provided clinically valuable information regarding bone marrow engraftment in patients following autologous bone marrow transplantation. The findings of this study demonstrate the clinical utility of thiazole orange reticulocyte analysis and indicate the diagnostic importance of the reticulocyte maturity index measurement in the evaluation of erythropoietic activity.

1,3'-Diethyl-4,2'-quinolylthiacyanine iodide as a "thiazole orange" analogue for nucleic acid staining.

Flow cytometric evaluation of reticulocytes has become a valuable and more precise alternative for the microscopically determined reticulocyte count. Until now, the most promising results are obtained with the "thiazole orange" (TO) nucleic acid dye. We report on the use of a "thiazole orange" analogue, 1,3'-diethyl-4,2'-quinolylthiacyanine iodide (DEQTC), which gives comparable results and is commercially available at low cost.

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II.

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.

[Erythropoiesis and serum erythropoietin concentrations before and after kidney allotransplantation]. / Erythropoiese und Serumerythropoietinkonzentration vor und nach Nierenallotransplantation.

Hematological parameters and serum erythropoietin (EPO) levels were measured before and sequentially after grafting in 50 consecutive cadaver renal transplant recipients. EPO was estimated using a sensitive radioimmunoassay. Values for nonanemic controls were 15-25 mU/ml. Mean hematological values before transplantation were as follows: hemoglobin 9.7 +/- 2.4 g/dl; hematocrit 29 +/- 8%; corrected reticulocytes count 15 +/- 8% and EPO 29 +/- 23 (11-131) mU/ml. In the entire studied population, 35 patients had inadequate low EPO levels for their degree of anemia. In the whole population, there was a significant positive exponential correlation between EPO and hematocrit (r = 0.31; p less than 0.05). In the subset of patients with underlying cystic kidney disease and in hemodialysis patients treated with recombinant human EPO, hemoglobin, hematocrit and EPO levels were higher when compared to hemodialysis or CAPD patients with other kidney diseases. Following successful renal transplantation, EPO increased to 45 +/- 31 mU/ml at 1 month and then decreased to 25 +/- 18, 18 +/- 7 and 19 +/- 4 mU/ml at 3,6 and 9 months, respectively. Within the 1st month after transplantation there was a 4-fold increase in reticulocytes from 9 +/- 5 to 38 +/- 14%, followed by a slow decrease over the next several months to 23 +/- 11% at 9 months. In contrast, the hematocrit level rose more gradually from 28 +/- 7 to 44 +/- 6% at 9 months. In 25 of 36 patients with a functioning graft who were followed for more than 6 months, anemia was corrected and 11 patients remained slightly anemic with a mean hematocrit level of 36 +/- 4%.

A sensitive method for the determination of the mitochondrial susceptibility factor (MSF).

A sensitive method for the determination of the activity of MSF in the cytosol of reticulocytes is described. It is based on the fact that proteolysis products from the mitochondria containing stroma of immature reticulocytes (fraction I) are only liberated if MSF is added. The reproducibility of the method is documented with partly purified MSF. A drawback is the limited range of MSF which can be tested.

Erythrocyte carnitine: a study of erythrocyte fractions isolated by discontinuous density gradient centrifugation.

Erythrocyte fractions of varying density were isolated by discontinuous density gradient centrifugation of washed erythrocytes of five subjects (three adults and two cord bloods). Free and total carnitine concentrations were determined in each gradient fraction to compare the carnitine content of less dense with more dense erythrocytes. Erythrocyte, leukocyte, and reticulocyte counts and hemoglobin were measured on all fractions of each gradient. The density gradient studies showed that the highest proportion of reticulocytes were associated with the least dense gradient fractions of all five subjects. Linear regression analyses revealed significant positive correlations (r = 0.94 to 0.99, P less than 0.02 to P less than 0.001) between the number of reticulocytes per fraction and the total or free carnitine concentrations per fraction for all subjects. No correlation was found between free or total carnitine and hemoglobin, number of erythrocytes, or number of leukocytes per fraction. It appears that erythrocyte carnitine is localized in circulating reticulocytes which have mitochondria and carnitine-dependent fatty acid metabolism.

Quantification and characterization of regulin, a Mr-230,000 highly elongated protein of rabbit reticulocytes.

Procedures are described by which regulin in rabbit reticulocytes was quantified and isolated in relatively large amounts. In these cells the protein occurs at a ratio of about 1.1-1.6 regulin monomers/spectrin tetramer, corresponding to 80,000-100,000 molecules of Mr-230,000 regulin/cell. Erythrocytes contain less than 12% of the amount of regulin in reticulocytes and the protein has not been detected in non-erythroid cells. Regulin was found primarily in the cytosolic fraction of lysed reticulocytes. It appears to be unusually sensitive to proteolysis by Ca2+-activated thiol proteases. Isolation of Mr-230,000 undegraded regulin was accomplished by the use of protease inhibitors including N-ethylmaleimide. A striking characteristic of regulin is its tendency to aggregate in neutral solution of low ionic strength. Physical studies of the isolated protein indicate that it has a highly elongated form in solution. The protein has no known enzymatic activity but was shown previously to interact with and increase the enzymatic activity of a protein phosphatase. The properties of regulin suggest that it may have a structural function but it appears to be physically and immunologically distinct from known proteins. It is suggested that regulin may contribute to a gel matrix within the cytoplasm of reticulocytes.