Hemoglobin switching: a cellular model.

Regulation of hemoglobin synthesis depends in part on the population of cells available for erythroid differentiation. Mouse erythroleukemia cells were cloned, and the clones were induced with dimethyl sulfoxide to test the relative induction of beta minor and beta major synthesis. Cells of line 745 produced approximately 35 percent beta minor after induction, and 39 clones of line 745 produced from 23 to 61 percent beta minor. Further subcloning of the clone that produced 61 percent beta minor led to three subclones, all of which produced more than 90 percent beta minor. Thus one kind of hemoglobin regulation occurs at the cellular level.

Deletion of the A gamma-globin gene in G gamma-delta beta-thalassemia.

In an individual homozygous for G gamma-delta beta-thalassemia, a physical alteration in gamma-globin gene organization was detected by restriction enzyme mapping. The data indicated that the absence of A gamma-globin chains resulted from extension of the DNA deletion from the delta beta-globin gene region into the gamma-globin gene region rather than a functional disturbance of gamma-gene expression.

Erythroid precursors in congenital hypoplastic (Diamond-Blackfan) anemia.

To explore the etiology of congenital hypoplastic anemia (CHA) or the Diamond-Blackfan anemia, erythropoietin responsive committed erythroid precursors were enumerated by the plasma clot method. These included blood and marrow erythroid burst-forming units (BFU-E) and marrow erythroid colony-forming units (CFU-E). The peripheral blood nucleated cells of 11 patients and the marrow cells of seven of these patients were examined. Studies were repeated in several patients during relapse and after induction of remission. BFU-E were undetectable in the marrow and blood of all but one relapsed patient, and the numbers of marrow CFU-E were depressed in all relapsed patients. Blood BFU-E remained low in all of the patients in remission. No evidence was obtained for suppression of normal CFU-E or BFU-E by CHA lymphocytes. Erythropoietin dose-response curves performed in two patients revealed a 10-fold increase in erythropoietin requirement for marrow CFU-E colony growth. This marked unresponsiveness to erythropoietin was strikingly improved by steroid therapy in one patient. We suggest that CHA is the result of a qualitative and/or quantitative deficiency of BFU-E. If BFU-E are produced, they must be relatively unresponsive to erythropoietin. The abnormal BFU-E give rise to erythropoietin unresponsive CFU-E and, thence, to proerythroblasts that are, in turn, trapped in that early stage of development because of their poor erythropoietic response. Hence, red cell production is deficient. Steroids appear to improve the erythropoietin response of CHA erythroid precursors.

Switch from fetal to adult hemoglobin is associated with a change in progenitor cell population.

To examine the switch from fetal to adult hemoglobin at the cellular level, erythroid progenitor cells from newborn infants and adults were cultured in methyl cellulose with erythropoietin. Individual erythroid colonies were labeled with [3H]leucine at various times, and globin synthesis patterns examined by gel electrophoresis and fluorography. The percent gamma- or beta-globin synthesis was determined from the total of gamma + beta, and the percent G gamma from the total of G gamma + A gamma. The nonparametric correlation coefficients of percent G gamma with percent gamma or beta were obtained. Each group of colonies at each time point was examined separately. In colonies from adult blood, the proportion of G gamma-synthesis did not correlate with the proportion of gamma-synthesis. Colonies from newborn blood fell into two groups. Those that developed from relatively mature progenitor cells, and were seen on day 14, showed a strong negative correlation of G gamma with beta-globin synthesis. However, those newborn colonies that developed from immature progenitors, and were seen later in culture (days 17 and 21), showed no correlation of G gamma with beta-synthesis. These findings are compatible with a clonal model for hemoglobin switching. Fetal progenitors, in which G gamma- and beta-syntheses are negatively correlated, are gradually replaced during ontogeny by adult progenitors. The adult progenitors produce more beta (less gamma), and the proportions of G gamma- and gamma- or beta-synthesis are not correlated.

Control of the simian fetal hemoglobin switch at the progenitor cell level.

This investigation was designed to define the cellular level at which the gamma to beta globin switch is established in the developing simian fetus in order to determine whether the switch is controlled by environmental influences within differentiating erythroid precursors or predetermined by the genetic program of erythroid progenitors. Samples of marrow and liver were obtained from rhesus fetuses throughout the switch period, and marrow was obtained from adult rhesus monkeys. Globin chain synthesis was then measured in differentiated erythroblasts and in erythroid progenitor-derived colonies grown in semisolid media. The relative rates of synthesis of gamma and beta chains were determined by the uptake of [(3)H]leucine into the respective chains separated by Triton gel electrophoresis and in some cases by urea carboxymethyl cellulose chromatography. Four periods of the switch were defined during fetal development. In the preswitch period both erythroblasts and progenitor-derived colonies produced <5% beta globin. In the early switch erythroblasts produced 5-15% beta globin, while progenitor-derived colonies produced 10-35% beta globin. In mid-switch erythroblasts synthesized 50% beta globin, whereas progenitor-derived colonies produced only 15-35% beta. At the completion of the switch erythroblasts produced 100% beta globin, while progenitor-derived colonies produced as little as 40% beta chains. We conclude that the program of globin synthesis that characterizes the fetal switch is established at the level of erythroid progenitors. Fetal erythroid burst-forming units (BFU-E) dominate the marrow prior to the switch. The early switch period is heralded by the appearances of adult erythroid burst-forming units programmed to express increasing beta chain synthesis in colonies. By mid-switch a second class of adult erythroid progenitors capable of giving rise to fetal and adult hemoglobin synthesis in in vitro colonies becomes apparent. These shifting populations of erythroid progenitors with unique globin synthesis programs give rise to the erythroblasts that create the sigmoid pattern of the fetal to adult hemoglobin switch in the developing simian fetus.

Investigations of the simian ontogenic switch from fetal to adult hemoglobin at the progenitor cell level.

The ontogenic switch from fetal to adult hemoglobin could result from discontinuous events, such as replacement of fetal erythroid progenitor cells by adult ones, or gradual modulation of the hemoglobin program of a single progenitor cell pool. The former would result in progenitors at midswitch with skewed fractional beta-globin synthesis programs, the latter in a Gaussian distribution. For these studies, we obtained bone marrow from rhesus monkey fetuses at 141-153 d (midswitch). Mononuclear cells were cultured in methyl cellulose with erythropoietin, and single BFU-E-derived colonies were removed and incubated with [3H]leucine. Globin synthesis was examined by gel electrophoresis and fluorography. The beta-globin synthesis pattern of single fetal colonies was skewed, and did not fit a normal distribution. The fetal pattern resembled the pattern of an artificial mixture of fetal and adult progenitors, suggesting that the fetal progenitor pool could contain populations with different beta-globin programs. This non-Gaussian distribution in the progenitors of midswitch fetuses is consistent with a discontinuous model for hemoglobin switching during ontogeny.

Effects of treatment with 5-azacytidine on the in vivo and in vitro hematopoiesis in patients with myelodysplastic syndromes.

The myelodysplastic syndrome (MDS) comprises a group of clonal hematopoietic disorders derived from an abnormality affecting a multipotent hematopoietic stem cell. Despite trials testing numerous agents in patients with MDS, no single drug has yet emerged as the accepted standard of treatment. Observation and supportive care with blood products and antibiotics, when necessary, continue to be the mainstays of therapy. We administered 5-azacytidine, a cell-cycle specific ring analog of the pyrimidine nucleoside cytosine, as a continuous intravenous infusion, 75 mg/m2 per day for 7 days every 4 weeks. Patients had refractory anemia with excess blasts (RAEB) or refractory anemia with excess blasts in transformation (RAEB-T). Responses were seen in 21 (49%) of 43 evaluable patients: five (12%) in complete remission (CR, complete normalization of bone marrow and peripheral blood counts); 11 (25%) in partial remission (PR, > or = 50% restoration of the deficit from normal of all three peripheral blood cell lines, elimination of transfusion requirements, and a decrease in percentage bone marrow blasts by > or = 50% from prestudy values); five (12%) improved (> or = 50% restoration in the deficit from normal of one or more peripheral blood cell lines and/or a > or = 50% decrease in transfusion requirements). A trilineage improvement (CR and PR) occurred in 37% of the patients. The median survival for all patients was 13.3 months and the median duration of remission for those with CR and PR was 14.7 months. Mild to moderate nausea and/or vomiting was the most common side effect (63%). Myelosuppression, either bone marrow hypoplasia or drug related cytopenias requiring a reduction in the dose of azacitidine, occurred in only 33% of the patients. Prior to treatment, bone marrow erythroid progenitor cells were assayed in vitro. Colonies derived from erythroid burst-forming units (BFU-e) were undetectable in one patient and reduced in two. The number of colonies derived from erythroid colony-forming units (CFU-e)) were also reduced in two of the three patients. In the two patients with detectable colony growth prior to treatment, colony number decreased by day 8 of the first cycle, followed by a subsequent increase. Continued treatment with azacitidine led to normalization of the number of CFU-e derived colonies as well as an increase in the number of BFU-e derived colonies. This improvement in erythroid colony number correlated with the spontaneous rise in hemoglobin levels and red cell transfusion independence.(ABSTRACT TRUNCATED AT 400 WORDS)

Fetal erythropoiesis in stress hematopoiesis.

Fetal erythropoiesis occurs during chronic bone marrow failure, or during recovery from marrow suppression. Fetal erythrocytes have HB F, with more G gamma than A gamma chains, "i" antigen, large MCV, characteristic enzyme levels, low carbonic anhydrase, low HB A2, and short life span. Many of these fetal characteristics are present in the red cells of patients with temporary or chronic hematopoietic stress. In those in whom normal hematopoiesis ensues, the fetal erythrocytes disappear. The fetal phase of recovery may be with homologous stem cells after bone marrow engraftment, or with autologous cells. Chronic fetal erythropoiesis is seen in patients with constitutional aplastic anemia, such as Fanconi's anemia or Diamond-Blackfan anemia. In one patient with the latter disorder, fetal erythropoiesis accompanied chronic anemia, and actually decreased slightly during the acute phase of clinical improvement. Culture of late erythroid precursor cells (CFU-Es) from a patient with transient erythroblastopenia of childhood led to in vitro development of colonies with HB F, an event not seen in normal marrow cultures. Thus fetal erythropoiesis occurs during hematopoietic stress, whether chronic or transient, if there is some marrow activity, and may be due to expansion of fetal clones.

Effects of low oxygen tension and antioxidants on human erythropoiesis in vitro.

Cultured cells grow better in low oxygen than in room air, and the addition of a sulfhydryl-reducing agent is usually routine. It has been thought that such reagents protect cells from free-radical toxicity in atmospheric oxygen, which is higher than in physiological oxygen tension. Previous data demonstrating the efficacy of the usual antioxidant, 2-mercaptoethanol (2ME), were obtained in murine studies. We have compared the effect of 2ME and other antioxidants on human erythropoiesis using neonatal (cord) and adult blood. Without antioxidants, erythroid colony numbers were higher in 5% oxygen than in air, while colony formation could be improved even in 5% oxygen by the addition of antioxidants. In dose-response curves of each drug, the maximum increase in colony number was achieved with 0.1 microM 2ME, 10 microM reduced glutathione (GSH), 1 U/mL catalase (CAT), and 1 U/mL superoxide dismutase (SOD). Erythroid colony growth in air and in 5% oxygen was identical with 10 microM GSH and higher than in any other conditions, including with combinations of GSH and any other antioxidant. For human erythroid progenitor cell cultures, 10 microM GSH appears to be more appropriate than the usual 100 microM 2ME. Antioxidants are necessary, however, even in low oxygen tension. The mechanism of the GSH action is incompletely understood.

Increased synthesis of mouse minor hemoglobin in erythroid colonies: a cellular model for hemoglobin regulation.

Globin synthesis was examined in mouse erythroid colonies. Bone marrow cells from DBA/2J adults were cultured in methylcellulose and labeled with 3H-leucine; globin synthesis ratios were determined following electrophoresis of lysates on polyacrylamide gels containing urea, acid, and Triton X-100. Colonies derived from the immature progenitor cells, BFU-E and CFU-E, produced close to 40% beta mi of total beta, while cluster-forming units, erythroblasts, and reticulocytes synthesized approximately 30% beta mi. Thus, beta mi synthesis decreased with increasing maturity of the erythroid compartment being examined, qualitatively resembling the decrease in fetal hemoglobin between BFU-E and erythrocytes in human adults. The mouse system described here thus provides a small animal model for studies of changes in hemoglobin expression during erythroid development.

Increased mouse minor hemoglobin during erythroid stress: a model for hemoglobin regulation.

In mice with "diffuse" hemoglobin (Hb), the decrease in the proportion of minor Hb during ontogeny qualitatively resembles the decline observed in human Hb F. Since Hb F reappears during some forms of erythroid stress, we investigated the effect of hematopoietic stress on minor Hb in DBA/2 mice. The stresses were acetlyphenylhydrazine-induced hemolysis, phlebotomy, or infection with Friend erythroleukemia virus. Recovery from anemia was associated with a transient increase in the synthesis of minor Hb similar to the reappearance of Hb F in man. Minor Hb synthesis also increased during the evolution of erythroleukemia induced by both the anemic and the polycythemic strains of virus. Thus, the mouse model can be used to study Hb regulation, since changes in the modulation of minor Hb synthesis occur under conditions which are associated with alterations in Hb F synthesis in humans.

Platelet number and function in Diamond-Blackfan anemia.

Congenital red cell aplasia (Diamond-Blackfan anemia) is occasionally associated with hematologic defects other than a deficiency of red blood cell progenitors, but such alterations have not been well studied. The frequency and magnitude of abnormalities in platelet count and platelet function were therefore examined in 38 patients. Thrombocytosis was seen in 21 patients, and 12 had mild thrombocytopenia on at least one occasion. Elevated platelet counts were demonstrated repeatedly in nine children. The three patients with the lowest platelet counts also had leukopenia. Platelet aggregation was normal in all 16 patients in whom it was studied, and bleeding times were within the normal range in 14 of them. Bleeding signs and symptoms were not observed. It is concluded that thrombocytosis or thrombocytopenia often occurs but that platelet function is normal in patients with Diamond-Blackfan anemia.

Comparison of erythroid progenitor cell growth in vitro in polycythemia vera and chronic myelogenous leukemia: only polycythemia vera has endogenous colonies.

The ability of erythroid cultures to distinguish among myeloproliferative disorders was examined. We studied 14 patients with polycythemia vera (PV), 11 with chronic myelogenous leukemia (CML), four with non-PV erythrocytosis, two with agnogenic myeloid metaplasia, as well as three normal fetuses and greater than 25 normal adults. Endogenous, i.e. grew without added erythropoietin, bone marrow CFU-E-derived colonies were observed in all but one PV patient. However, endogenous blood BFU-E-derived bursts were observed in only eight of 14 PV patients. Endogenous erythroid colonies were not seen in cultures from any normal adults or fetuses, or patients with CML, erythrocytosis, or myeloid metaplasia. In PV, relative HbF synthesis was always greater in cultures without erythropoietin, while in cultures from all other patients relative HbF synthesis was similar to that observed in cultures from normal individuals. We conclude that PV and CML are distinguishable in culture since CML patients do not have endogenous growth. Most important, endogenous bone marrow CFU-E-derived colonies are the only consistently unique observation in patients with PV, and endogenous CFU-E- and BFU-E-derived colonies and bursts are not uniformly observed in PV blood cultures. In-vitro studies of erythropoiesis to confirm the diagnosis of PV, therefore, require marrow when endogenous colonies and bursts are absent from blood cultures.

Antenatal diagnosis of thalassemia: a review.

The overall results of the WHO International Registry for the Prenatal Monitoring of Hereditary Anemias are summarized in Tables 11 and 12. Comparison of data regarding adequacy of samples, and freedom from errors and from fetal losses show that the chance of a couple obtaining a useful result is greater than 90%, whether fetal flood is sampled because the fetus is at risk for a hemoglobinopathy or another disease, or fetal DNA is obtained for globin gene analysis. Each approach serves as a model for the others. Fetal blood sampling technology has been improved because of the need for entirely pure samples for diseases requiring specimens other than red cells. The advances in obtaining fetal cells early in pregnancy and extraction of DNA will soon be applied to diseases other than hemoglobinopathies (such as hemophilias and muscular dystrophies) as molecular probes become available. More than 6000 fetuses have now been examined in utero, 5617 for all hemoglobinopathies using fetal blood or DNA, and 5921 for all disorders using fetal blood (Table 12). The total reported to the Registry by the end of 1983 was 6282 cases. At the moment, the only choices when an affected fetus is detected are termination of the pregnancy, or delivery of a child known to have a serious and sometimes life-threatening illness. However, early diagnosis will lead to early treatment of such infants, thereby offering a better prognosis. When specific treatment such as gene therapy becomes available, fetal diagnosis will identify the appropriate cases in utero. Although this approach is currently speculative, it is an area of great interest and endeavor. Thus, prenatal diagnosis of hemoglobinopathies has led to the development of fetal diagnosis of many genetic diseases, and resulted in techniques for obtaining fetal blood or DNA specimens. In addition to these scientific advances, it has also led to the control of thalassemia in certain geographic areas in which the public health burden involved in the management of such cases is overwhelming. Reduction of the number of newborn thalassemics is a necessity in some places, because appropriate care has not been possible, and the lifespan of affected individuals is significantly shortened or at least uncertain. All approaches to management of this disease are relevant, including improved treatment, specific therapy, and prenatal diagnosis. Each country and each family must determine for itself where to place the emphasis.

Fanconi anemia: myelodysplasia as a predictor of outcome.

The adverse potential of the development of myelodysplastic syndrome (MDS) in Fanconi anemia (FA) was examined in a retrospective study of 41 FA patients who had bone marrow morphology and chromosomes reviewed by a single group. Thirty-three patients had adequate cytogenetic studies, and 16 (48%) had one or more abnormal studies: nine initially, and seven more on follow-up. Cytogenetic clonal variation was frequent, including disappearance of clones, clonal evolution, and appearance of new clones. The estimated five-year survival with a cytogenetic clone is 0.40, compared to 0.94 without a clone. Morphologic myelodysplasia (MDS), independent of a cytogenetic clone, was found in 13/41 patients (32%). The estimated five-year survival with MDS is 0.09, versus 0.92 without MDS. Leukemia developed in three patients whose initial cytogenetic clones prior to leukemia were t(1;18), t(5;22) and monosomy 7; the one with t(1;18) also had MDS. Our results focus on marrow morphology, and suggest that morphologic MDS may be more important than classical cytogenetics in prediction of an adverse outcome.

Bone marrow failure syndromes.

Laboratory diagnosis of inherited bone marrow failure syndromes includes general evaluations, such as blood counts, examination of the peripheral blood smear for morphology, and bone marrow aspirates and biopsies, which may help the clinician classify the patient, particularly if there are no characteristic physical anomalies. Specific diagnoses require unique tests that are only available for a few of the diagnoses. The most useful is chromosome breakage in the diagnosis of FA, with gene mutation analysis or mapping about to become the gold standard when all of the FA genes have been cloned. The diagnosis of DC remains clinical at this time, although linkage to Xq28 and skewed maternal X inactivation may be helpful in some families. Laboratory proof of SD may be provided by decreased serum trypsinogen or other evidence of exocrine pancreatic insufficiency. CHH is substantiated when absent central pigment in hair is found and when it is mapped to 9p21-p13. The only mitochondrial syndrome, PS, is proved with demonstration of deleted mitochondrial DNA. RD is diagnosed from blood and marrow studies that demonstrate lack of lymphoid as well as myeloid activity. Amega requires absent or abnormal marrow megakaryocytes; if radii are also absent, the diagnosis is TAR. DBA usually has elevated red-cell ADA, and the DBA locus may map to 19q13. KS is diagnosed in patients who have congenital nonimmune severe neutropenia. Clinical suspicion of particular diagnoses can often be substantiated by laboratory tests of varying specificity.

Dyskeratosis congenita.

Dyskeratosis congenita is a rare genodermatosis. Malignant deterioration and hematologic complications are well-described features of this syndrome. Correct recognition is essential for proper management. A review of diagnostic considerations and treatment guidelines is presented.

Pure red cell aplasia associated with hepatitis C infection.

We report the case of a 34-year-old woman with recurrent pure red cell aplasia and evidence of hepatitis B and C infection. Review of the English literature identified 19 prior cases in which pure red cell aplasia was associated with hepatitis. This case is the first in which serologic evidence of hepatitis C infection was documented. This patient also had porphyria cutanea tarda and marked hepatic siderosis but no active hepatitis or cirrhosis. Treatment with cyclophosphamide and prednisone produced complete remission of the pure red cell aplasia. Erythroid colony formation (colony-forming unit-erythroid and erythroid burst-forming unit) was reduced in cultures of bone marrow obtained during relapse but was normal in remission marrow. However, addition of the patient serum, whether collected during relapse or remission, inhibited erythroid colony formation by her bone marrow. These observations, and the known extrahepatic immunologic manifestations of hepatitis C infection, suggest that the pure red cell aplasia occurred because of autoimmune mechanism provoked by the infection.

Effects of hemin on erythropoiesis.

It is clear that in vitro hemin increases the number of blood BFU-E derived colonies from normal donors. This occurs with sickle donors as well, despite the increased levels of hemin in vivo in these patients. The effect of hemin on relative gamma globin synthesis is inconsistent, however. In a few cases, delayed addition of hemin led to increased gamma globin synthesis. In time course studies of cultures from normal donors, hemin added on day 0 shifted the day of peak colony number from 13-14 to 16-20 days. The temporal decline in gamma globin synthesis was not altered. In cultures from sickle donors we found that the time for maximal colony number was later than in normals, occurring at 16-20 days even without hemin, and was not further delayed by hemin. The relative proportion of gamma globin synthesis was higher on day 14 in the sickle than the normal cultures, and the temporal decline was somewhat slowed in the sickle cultures by hemin. The elevated gamma synthesis and the later time for peak colony growth in the sickle cultures suggest that the erythroid progenitors in the blood of the sickle patients are less mature than those from normal individuals. There are several possible explanations for the detection of increased numbers of colonies in cultures containing hemin. Hemin may delay the final maturation of erythroblasts within erythroid colonies, thus shifting the time of maximal growth. It may also increase the extent of final maturation, leading to more complete hemoglobinization of the erythroblasts within the colonies, and thus increasing the number of colonies that are eventually recognized as erythroid.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital hypoplastic anemia.

CHA, a rare failure of erythrogenesis in infancy and childhood, has been re-examined on the basis of a review of 42 cases of our own and 133 others. Although the disease may have its onset late in gestation, severe anemia usually is not found at birth but, in 75% of cases, becomes apparent within the first 4 months. Associated congenital anomalies include abnormal thumbs, webbed neck and growth retardation. Treatment with corticosteroids as soon as the diagnosis is established induces a remission in 80% of patients. Thereafter, this therapy may need to be continued, often at an extremely low dosage level, sometimes intermittently, for many years before the hemoglobin value maintains itself without medication. Spontaneous remission has occurred in a few cases after years of transfusions. Although it has long been considered that the disease was a failure of normal erythrogenesis manifest by a normocytic normochromic anemia, more exact measurements reveal that the anemia is macrocytic, with MCV usually ranging between 100 and 115 mu3. Other abnormalities of the red cells are present; in general, these resemble characteristics of fetal erythrocytes rather than normal adult cells. These red cell changes may persist even after the patient has been in remission for years, thus representing a permanent abnormality in erythrogenesis.