Cell-specific mitotic defect and dyserythropoiesis associated with erythroid band 3 deficiency.

Most eukaryotic cell types use a common program to regulate the process of cell division. During mitosis, successful partitioning of the genetic material depends on spatially coordinated chromosome movement and cell cleavage. Here we characterize a zebrafish mutant, retsina (ret), that exhibits an erythroid-specific defect in cell division with marked dyserythropoiesis similar to human congenital dyserythropoietic anemia. Erythroblasts from ret fish show binuclearity and undergo apoptosis due to a failure in the completion of chromosome segregation and cytokinesis. Through positional cloning, we show that the ret mutation is in a gene (slc4a1) encoding the anion exchanger 1 (also called band 3 and AE1), an erythroid-specific cytoskeletal protein. We further show an association between deficiency in Slc4a1 and mitotic defects in the mouse. Rescue experiments in ret zebrafish embryos expressing transgenic slc4a1 with a variety of mutations show that the requirement for band 3 in normal erythroid mitosis is mediated through its protein 4.1R-binding domains. Our report establishes an evolutionarily conserved role for band 3 in erythroid-specific cell division and illustrates the concept of cell-specific adaptation for mitosis.

Potentiation of erythroid abnormalities following macrophage depletion in aged rats.

OBJECTIVES: The effects of prolonged macrophage depletion on haematological parameters were investigated in aged rats and compared with those in young ones. METHODS: Four weekly i.v. injections of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-CL) were employed to achieve a prolonged depletion of bone marrow (BM) and spleen macrophages. The number of BM macrophages was then assessed by flow cytometry, whereas the spleen clearance function was judged by the elimination of oxidised red blood cells (RBC). Haematological parameters and signs of RBC ageing (reduced MCV, increased density and augmented 4.1a/4.1b membrane protein ratio) were determined. Finally, the recovery from phlebotomy-induced acute anaemia was investigated. RESULTS: Following the Cl2MDP-CL treatment, in comparison with young rats, the aged animals showed: (i) reduced numbers of BM macrophages; (ii) greater impairment of spleen clearance function; (iii) similar anaemic condition and signs of RBC ageing; (iv) greater increase in white blood cell (WBC) numbers (mainly neutrophils). In addition, whereas aged control rats showed a recovery from phlebotomy-induced acute anaemia which was similar to that of the untreated young animals, in the aged-treated rats, a significantly diminished/delayed restoration of RBC, Hb and reticulocyte to normal values was observed, accompanied by a significantly higher increase in WBC numbers than in the other groups of animals. CONCLUSION: Haematological abnormalities because of Cl2MDP-CL-induced macrophage depletion are potentiated in aged rats in which the BM regenerative potential of the erythroid lineage as well as the clearance function of the spleen appear compromised. Thus, in aged rats, macrophage dysfunction is likely to interfere with erythroid homeostasis particularly during haemopoietic stress.

Advances in the understanding of the congenital dyserythropoietic anaemias.

The congenital dyserythropoietic anaemias (CDAs) are a heterogeneous group of diseases in which the anaemia is predominantly caused by dyserythropoiesis and marked ineffective erythropoiesis; three major (types I, II and III) and several minor subgroups have been identified. Additional information on the natural history of these conditions, the beneficial role of splenectomy in CDA type II and efficacy of interferon-alpha in type I have recently been reported. A disease gene has been localised to a chromosomal segment in the three major types and in CDA type I, a disease gene has been identified (CDANI). Mutations have been detected in both familial and sporadic cases but the predicted protein structure gives few clues as to its function. In both type I and II, there are cases unlinked to the identified localisations, suggesting genetic heterogeneity.

Aging of red blood cells and impaired erythropoiesis following prolonged administration of dichloromethylene diphosphonate containing liposomes in rats.

OBJECTIVES: To investigate whether macrophage-depleted rats may serve as a model for studying red blood cell (RBC) aging. METHODS: Rats were macrophage-depleted by 4 weekly injections of dichloromethylene diphosphonate-containing liposomes (Cl2MDP-CL). The macrophage content of spleens and bone marrows (BMs) was investigated by immunohistochemistry and light microscopy and by flow cytometry, respectively, after staining with macrophage-specific monoclonal antibodies. In addition, the ultrastructure of residual BM macrophages and their ability to phagocytose zymosan was studied. BM was also studied for apoptosis (by the TUNEL reaction) and for erythroid progenitor cell content. Furthermore, RBC indices, morphology, life span (by 51Cr labeling) and aging features (MCV, density, 4.1a/4.1b membrane protein ratio, anti-spectrin IgG binding, microvesiculation) were investigated. Serum TNF-alpha, iron, total iron-binding capacity (TIBC) and ferritin were also determined. RESULTS: Prolonged treatment with Cl2MDP-CL caused an almost complete depletion of macrophages in the spleen and a 58% reduction of those in the BM; the residual BM macrophages were activated as judged by their ultrastructure and phagocytic capacity in vitro. These alterations were accompanied by an increase in RBC life span and age-related RBC changes, as well as by mild anemia associated with a reduced reticulocyte count, reduced BM erythroid progenitors, increased numbers of apoptotic cells in the BM, low serum iron, high TIBC and increased serum TNF-alpha levels. CONCLUSIONS: Rats subjected to prolonged macrophage depletion showed an increased prevalence of senescent RBC in the circulation due to their impaired clearance by macrophages. Hence, these animals provide a model system in which mechanisms of RBC aging can be delineated. They also showed impaired erythropoiesis, presumably related to a reduction in BM macrophages and increased production of proinflammatory cytokines by residual activated marrow macrophages and other cells.

Long-term ovo-lacto vegetarian diet impairs vitamin B-12 status in pregnant women.

A well-planned vegetarian diet has been stated to be adequate during pregnancy. The aim of the present study was to compare serum vitamin B-12 and homocysteine concentrations in pregnant women (n = 109) consuming vegetarian and Western diets and to evaluate the adequacy of current dietary reference intakes of vitamin B-12 for these women. Pregnant women adhering to vegetarian diets for at least 3 y, with subgroups of ovo-lacto vegetarians (OLVs; n = 27), low-meat eaters (LME, n = 43), and women eating an average Western diet (control group, n = 39), were recruited. Dietary vitamin B-12 intake, serum vitamin B-12, and plasma total homocysteine (tHcy) concentrations were measured in wk 9-12, 20-22, and 36-38 of pregnancy. During pregnancy serum vitamin B-12 concentrations of ovo-lacto vegetarians (P < 0.001) and low-meat eaters (P = 0.050) were lower than those of the control group. We observed the combination of low serum vitamin B-12 concentrations and elevated plasma tHcy in 22% of ovo-lacto vegetarians, in 10% of low-meat eaters, and in 3% of controls (P = 0.003). In OLVs, serum vitamin B-12 predicted 60% of the plasma tHcy variation (P < 0.001), but in LMEs and controls only <10% (NS). Serum vitamin B-12 concentrations increased and plasma tHcy decreased sharply with increasing dietary intake of vitamin B-12 toward a cutoff point of 3 mug/d. Pregnant women consuming a long-term predominantly vegetarian diet have an increased risk of vitamin B-12 deficiency. Current recommended dietary intakes urgently need reevaluation.

Longitudinal concentrations of vitamin B(12) and vitamin B(12)-binding proteins during uncomplicated pregnancy.

BACKGROUND: Because reference values for vitamin B(12) concentrations and vitamin B(12)-binding capacities for pregnant women have not been established, the reference values for nonpregnant women are often applied to assess vitamin B(12) status. The aim of the present study was to describe ranges of biochemical indices of vitamin B(12) status, including red blood cell (RBC) vitamin B(12), saturated and unsaturated cobalamin-binding proteins, and binding capacities in all trimesters of uncomplicated pregnancy. METHODS: A total of 39 healthy pregnant women with long-term daily intake of vitamin B(12) >2.6 microg/day and uncomplicated pregnancies participated in the study throughout their pregnancies. RBCs and serum vitamin B(12), holo-haptocorrin, unsaturated cobalamin-binding proteins, unsaturated and total vitamin B(12)-binding capacities, total homocysteine (tHcy), and RBC count were assessed in weeks 9-12, 20-22, and 36-38 of gestation. RESULTS: Significant changes in vitamin B(12) status occurred in the course of pregnancy. Serum vitamin B(12) concentrations and percentage of saturation of vitamin B(12)-binding proteins decreased steadily throughout pregnancy. In the third trimester, 35% of the participants had serum vitamin B(12) concentrations <150 pmol/L and 68.6% had <15% saturation of total vitamin B(12)-binding capacities, but no women had RBC vitamin B(12) concentrations <148 pmol/L. However, the decrease in these indices was not associated with reduced hemoglobin concentrations or RBC count or with increased tHcy concentrations. CONCLUSIONS: Our findings suggest that the reference values for vitamin B(12) status in nonpregnant women may not be applicable to pregnant women.

Congenital dyserythropoietic anemia, type 1, in a polynesian patient: response to interferon alpha2b.

The authors attempted to assess the utility of interferon alpha2b treatment in a Polynesian girl with a relatively severe form of congenital dyserythropoietic anemia, type 1. The diagnosis was established using routine hematologic and biochemical tests, light and electron microscopy, and electrophoresis of red cell membrane proteins. Response to the treatment was monitored using the blood count and reticulocyte count. The patient was age 14 when interferon treatment was started. Previously, she had been partially dependent on transfusions, and gallstones and iron overload had developed. The dose of interferon alpha2b was initially 3 x 10 units three times a week for 1 year and 3 x 10 units twice a week thereafter. On this treatment, hemoglobin and reticulocytes increased and transfusions became unnecessary. In keeping with a few previous reports, interferon alpha2b proved to be effective in congenital dyserythropoietic anemia, type 1. The patient became transfusion-independent. More cases need to be studied to optimize the dosage of interferon alpha2b and determine how long the treatment can be tolerated.