Increase In Erythropoiesis Research Articles

Circulating Erythroid Progenitors and Predominant Contribution of Liver to Erythropoiesis in Adult Xenopus laevis.

Cross-species comparisons of hematopoietic systems will elucidate the conservation and diversity among species such as zebrafish, Xenopus, chick and mouse, which are not only of interest but different approaches would contribute to general hematology. To begin to understand their hematopoietic systems, particularly the whole animal-physiology, across non-mammalian vertebrates, we have focused on amphibian hematopoiesis. We tried to clarify the localization of definitive hematopoietic progenitors in adult Xenopus laevis, which is still to be determined. When Xenopus was induced acute hemolytic anemia by intraperitoneal phenylhydrazine (PHZ) administration, immature erythroblasts emerging in the circulation and notable increase in erythropoiesis within the liver were observed. We first screened putative hematopoietic tissues, liver, spleen, bone marrow and kidney, for erythroid progenitors using polyclonal antibodies to putative Xenopus erythropoietin receptor (xlEPOR) that we recently identified. MACS and FACS sorting and analysis revealed the existence of xlEPOR expressing cells in both liver and anemic peripheral blood. These xlEPOR positive cells were hemoglobin-positive with o-dianisidine staining, and had typical blastic morphology with high nucleus-to-cytoplasm ratio. We next developed and established an in vitro colony assay system to identify and score the hematopoietic progenitors retrospectively. The method enabled the identification and quantification of erythroid progenitors. Briefly, cells were prepared from liver, spleen, bone marrow and kidney followed by placing in semi-solid culture medium (α-MEM containing 0.8% methylcellulose, 20% FCS with appropriate hematopoietic stimulators), and cultured at 23°C with 5% CO2. The anemic serum exhibited the apparent erythropoietic stimulating activity toward the formation of remarkable number of colonies derived from anemic peripheral blood cells, resembling typical mammalian hematopoietic colony formation. Most of the colonies consisted of hemoglobin-expressing erythroids after two days culture, indicating that colony-forming units-erythroid (CFU-e) appeared in anemic blood. The normal and anemic liver also contained CFU-e, resulting in the formation of mixed and pure hematopoietic colonies. This also proved to be a useful in vitro assay system for identifying and quantifying various hematopoietic progenitors and activities of related cytokines. Figure shows the number of erythroid colonies derived from PHZ-induced anemic peripheral blood and liver stimulated with anemic serum. We furthermore examined spleen and bone marrow side-by-side, since amphibian hematopoietic system is known to unique as erythropoiesis, granulopoiesis, and thrombopoiesis occur at distinct organs. The results demonstrated the direct evidences of predominant contribution of adult liver to erythropoiesis rather than bone marrow or spleen. A new animal model developed here should provide new insights into the basis of hematopoietic regulations. [Display omitted]

Erythrocyte Zinc Protoporphyrin Is Elevated With Prematurity and Fetal Hypoxemia

To examine the utility of red blood cell (RBC) zinc protoporphyrin/heme ratio (ZnPP/H) as an indicator of fetal iron status, because unfavorable neurodevelopmental outcomes have been associated with poor iron status at birth, as indicated by low serum ferritin, and because few reliable indicators of fetal and early neonatal iron status exist. Consecutively studied preterm and term fetuses at delivery included the following groups: (1) control nonhypoxic, (2) fetuses with intrauterine growth retardation (IUGR), and (3) fetuses of insulin-treated mothers (FDM). We hypothesized (1) that rapid growth velocity associated with an accelerated erythropoiesis among normal fetuses will lead to reduced iron delivery to a rapidly expanding RBC mass and higher umbilical cord blood RBC ZnPP/H and (2) that fetuses that are exposed to pathologic hypoxemia will experience an additional increase in erythropoiesis and higher cord ZnPP/H. ZnPP/H was determined on saline-washed cord blood erythrocytes by hematofluorometry and was examined for its relationship with clinical factors and cord blood laboratory measurements indicative of tissue oxygenation (plasma erythropoietin [EPO] and reticulocyte count) and iron status (plasma ferritin and erythrocyte indices). Statistical testing included 1-way analysis of variance, 2-way analysis of variance with covariates, simple linear regression, and multiple regression analysis. Among control group subjects, gestational age at birth was inversely correlated with RBC ZnPP/H and reticulocyte count and positively correlated with ferritin and EPO. Relative to control subjects, IUGR and FDM fetuses at specified gestational age groupings had higher ZnPP/H, lower plasma ferritin, and higher plasma EPO. Statistical modeling of the relationship between ZnPP/H and plasma ferritin among all study groups demonstrated significant impacts of gestational age, plasma EPO, maternal hypertension, and maternal smoking. The inverse association of fetal ZnPP/H with gestational age at birth among control subjects is attributable to erythropoietic stimulation likely as a result of increasing growth velocity at the earliest gestational ages. The relatively higher ZnPP/H observed among fetuses in the IUGR and FDM groups likely is attributable to increased erythropoietic activity secondary to pathologic hypoxemia. Decreased placental iron transfer may also have limited iron availability and contributed to elevated ZnPP/H in the IUGR group. These data support the concept that increased erythropoietic activity and/or limited iron transport may place infants of diabetic mothers and infants with growth retardation at risk for developing systemic iron deficiency later in infancy and in early childhood.

Haematopoietic Indicators of Fetal Metabolic Acidosis

SUMMARY We aimed to study the haematopoietic response in normal and acidotic deliveries following vaginal and abdominal delivery and to compare this to the surrogate markers of perinatal acidosis. Blood gas analyses, complete blood pictures and erythropoietin assays were performed on umbilical or early neonatal blood samples. Placental sections were examined for the presence of nucleated red blood cells. Perinatal clinical risk factors and major neonatal outcomes were collected. The control population was 78 deliveries where the cord arterial pH was > 7.10. Controls born after labour were compared to those born prior to the onset of labour and to 14 acidotic infants born after labour. Nucleated red blood cells did not increase with labour in the control groups but were significantly higher (p < 0.05) in the acidotic group. Erythropoietin did not significantly change with either labour or acidosis. The predictive values from nucleated red blood cell counts were higher than those from low Apgar scores, atypical cardiotocograph traces, meconium-stained amniotic fluid, erythropoietin and the presence of nucleated red blood cells in placental sections. Nucleated red blood cell counts may be a useful surrogate marker of acidosis where blood gas analysis is unavailable. Further studies are required to examine the timing of the increase of erythropoiesis to help define the onset of the stimulus.

Bone Marrow Response to Acute and Chronic Trypanosoma congolense Infection in Multimammate Rats (Mastomys coucha)

The femoral bone marrow of multimammate rats (n=90), aged 3–8 weeks, experimentally infected with different doses of Trypanosoma congolense was examined by light and electron microscopy. Some animals died from trypanosomosis, but groups of 10 were killed at 4–8, 9–16, 20–24, 30, 40, 50 and 60 days post-infection (dpi). In the acute stage of infection (4–8 dpi) the bone marrow invariably showed a striking increase in erythropoiesis, characterized by an increase in the number of mitotic figures and erythroblastic islands and by a marked decrease in the myeloid:erythroid cell ratio. Later in the infection, erythropoietic activity decreased, while erythrophagocytosis, granulopoiesis, megakaryopoiesis and plasma cell population increased. In chronic infection (16–60 dpi), erythropoietic activity decreased, while intra- and extra-vascular erythrophagocytosis greatly increased. There was also an increase in the bone marrow stroma cells. Excessive erythrophagocytosis by these cells led to the formation of myelin figures and cytoplasmic telephagolysosomes. Degeneration and necrosis of neutrophils lining the adluminal surfaces of the blood sinuses were observed. It is concluded that in the acute stage of the infection, the bone marrow is responsive to the anaemia and that in the chronic stage, dyserythropoiesis and increased erythrophagocytosis by the expanded and activated cells of the mononuclear phagocytic system play an important role in the production of anaemia.

Haematopoietic indicators of fetal metabolic acidosis

We aimed to study the haematopoietic response in normal and acidotic deliveries following vaginal and abdominal delivery and to compare this to the surrogate markers of perinatal acidosis. Blood gas analyses, complete blood pictures and erythropoietin assays were performed on umbilical or early neonatal blood samples. Placental sections were examined for the presence of nucleated red blood cells. Perinatal clinical risk factors and major neonatal outcomes were collected. The control population was 78 deliveries where the cord arterial pH was > 7.10. Controls born after labour were compared to those born prior to the onset of labour and to 14 acidotic infants born after labour. Nucleated red blood cells did not increase with labour in the control groups but were significantly higher (p < 0.05) in the acidotic group. Erythropoietin did not significantly change with either labour or acidosis. The predictive values from nucleated red blood cell counts were higher than those from low Apgar scores, atypical cardiotocograph traces, meconium-stained amniotic fluid, erythropoietin and the presence of nucleated red blood cells in placental sections. Nucleated red blood cell counts may be a useful surrogate marker of acidosis where blood gas analysis is unavailable. Further studies are required to examine the timing of the increase of erythropoiesis to help define the onset of the stimulus.

Plasma transferrin receptor levels and indices of erythropoiesis and iron status in healthy term infants.

The goal of this study was to determine if the postnatal changes in plasma transferrin receptor (TfR) levels in healthy infants were associated with changes in erythropoiesis or iron status. Longitudinal blood samples were obtained monthly from healthy term infants fed iron-fortified formula for the first 7 months and analyzed for plasma TfR and indices of erythropoiesis and iron status. Plasma TfR level rose during the first 2 months of life (p < 0.002). When examined for its association with indices of erythropoiesis, plasma TfR was negatively associated with hemoglobin (Hb) (p < 0.01), and positively associated with plasma erythropoietin (EPO) concentration (p < 0.005) and absolute reticulocyte count (p < 0.005). Plasma TfR was not associated with erythrocyte protoporphyrin. Although indices of iron status were not suggestive of iron deficiency, plasma TfR was negatively associated with plasma ferritin, Tf saturation, and plasma iron, and positively associated with total iron binding capacity (TIBC) (p < 0.0001 for all). Increases in plasma TfR levels were observed during normal infancy. The increases in plasma TfR levels correlate with increases in erythropoiesis without evidence for functional iron deficiency.

Iron metabolism in transgenic mice with hypoplastic anaemia due to incomplete deficiency of erythropoietin.

Iron overload is a serious complication of many forms of anaemia, arising in part from mechanisms associated with compensatory increases in erythropoiesis. To investigate other mechanisms by which anaemia itself may perturb iron metabolism, without the confounding effects of compensatory erythropoiesis, we studied transgenic mice with a partially disabling insertion in the erythropoietin gene, which manifested as incomplete erythropoietin deficiency. Mice were studied aged 7-8 weeks. Haemoglobin concentrations were 6.6 +/- 0.8 g/dl in mice homozygous for the modified erythropoietin gene, 12.9 +/- 2.2 g/dl in heterozygous mice and 14.1 +/- 1.0 g/dl in controls. Homozygous mice showed significant hepatic iron loading (2-fold increase in liver non-haem iron, compared with heterozygous mice and normal controls, with iron staining principally in the periportal hepatocytes). Absorption studies using 59Fe showed increased uptake from the lumen of an in vivo isolated duodenal segment in homozygous mice, although at this point in time overall transfer of radioiron to the circulation and other tissues (mucosal transfer) was not different from controls. These observations demonstrate that anaemia can lead to hepatic iron loading even in the absence of increased erythropoiesis, and are consistent with the possibility that anaemic hypoxia can enhance mucosal iron uptake by the duodenal enterocyte.

Pernicious anaemia patients should be searched for iron deficiency during follow up

Pernicious anaemia (PA) is an athropic gastropathy with a deficiency of intrinsic factor secretion by parietal cells. Parietal cells are also the source of hydrochloric acid, which is necessary for the reduction of iron from the ferric to the ferrous form. It is in this ferrous state that iron absorption in the proximal ileum is made possible.1 Iron deficiency may, therefore, accompany PA.2 To investigate whether simultaneous iron deficiency occurs in PA, we studied 84 PA patients (48 men, 36 women; median age 51 years; range 19-82 years). Before starting cyanocobalamin therapy, iron stores were evaluated by bone marrow iron staining, and simultaneous iron deficiency was detected in 18 of the patients (21.4%). After a median follow-up of 30 months (range 16-64 months), 8 additional patients (9.5%) developed iron deficiency, giving a total of 26 patients (31%) with iron deficiency. To determine whether older patients are more likely to be iron deficient, we divided the patients into two groups: one group younger than 65 years (50 patients) and the other 65 years and over (34 patients). At the beginning, 7 out of the 50 and 11 out of the 34 patients were shown to be iron deficient. During follow-up, 3 additional patients in the younger and 5 in the older group developed iron deficiency. This made a total of 10 out of 50 and 16 out of 34 patients with simultaneous iron deficiency; this difference between the groups was significant (p<0.05). No cause could be identified to account for the iron deficiency despite close examination. Simultaneous iron deficiency is often observed in PA patients.2 and this may occur at any time. Iron deficiency may not be a problem initially, but increase in erythropoiesis with cyanocobalamin treatment may deplete iron which is already poorly absorbed and whose stores are inadequate.3 Iron deficiency might, therefore, accompany PA years after the diagnosis, and evaluation of body iron status at the beginning cannot guarantee that iron deficiency will not develop in the future. Iron deficiency might, therefore, be a problem in PA patients, particularly in the elderly, as many of them consume less iron than the recommended daily allowance.4 We recommend that PA patients, particularly those older than 65 years, should be evaluated for iron stores during-follow-up.

Bioavaliability of Fe, Ca, P and Mg in Fe-deficient rats treated with different sources of dietary iron

Iron deficiency (D0) markedly decreases the concentration of iron and calcium in the liver, femur and sternum. We investigated the effect of the source of dietary iron [ferric citrate alone (citrate diet), or combined with bovine blood at a proportion of 1:1 (citrate + heme diet), and ferric protein (protein-Fe diet)] on the nutritive utilization of Fe, Ca, P and Mg in anemic and healthy rats. All diets contained the same amount of Fe (40 mg/kg diet). In Fe-deficient rats (DT) fed citrate, citrate + heme or protein-Fe diet the apparent digestibility coefficient (ADC) of Fe were 39%, 37% and 94% greater than in the respective control groups (C); hemoglobin regeneration efficiencies (HRE) were respectively 47%, 51%, and 172% greater than in the control groups. The ADC of Ca was not affected by Fe deficiency, whereas the ADC of Mg decreased significantly, and that of P increased under our experimental conditions. In Fe-deficient rats fed Fe for 10 days (DT), the concentrations of hemoglobin approached normal values. However, serum Fe remained low, indicating that Fe reserves were still depleted. Hepatic and femoral Fe concentrations were also lower in all DT groups, whereas Fe concentrations in the sternum increased significantly, suggesting an increase in erythropoiesis. After supply of dietary Fe for 10 days in iron-deficient groups (DT), Ca and P concentrations increased in sternum, a site assumed to have higher requirements for these minerals. In the liver these values tended to normalize, whereas in the femur Ca and P stores remained depleted. Mg concentrations after the replenishment period generally approached normality in all three organs.

Control of erythropoiesis in humans during prolonged exposure to the altitude of 6,542 m

Altitude hypoxia induces an increase in erythropoiesis. Some of the factors involved in the control of altitude polycythemia were studied. Ten subjects (4 women, 6 men) were exposed for 3 wk to extreme altitude (6,542 m). Blood was withdrawn in normoxia (N) and after 1 wk (H1), 2 wk (H2), or 3 wk (H3) at 6,542 m for the measurement of serum erythropoietin (EPO), blood hemoglobin (Hb), hematocrit (Hct), intraerythrocyte folate (Fol), and plasma ferritin (Fer) concentrations. Renal blood flow (RBF) and absolute proximal reabsorption rate (APR) were measured by the p-aminohippuric acid and lithium clearance, respectively, in N and H2 conditions. O2 supply to the kidneys was calculated using RBF and arterial O2 content (CaO2). After an initial sharp increase in EPO, it decreased at H2 and H3. Hct and Hb increased from N to H1 and H2 and then unexpectedly decreased from H2 to H3. Mean corpuscular Hb content (MCHC = Hb/Hct) was lower in all H than in N conditions. Increase in EPO at H1 varied from 3- to 134-fold among individuals. Women showed a smaller increase in Hct and Hb and a greater decrease in MCHC. Two women showed a large increase in EPO without increase in Hb. Fol was not modified by altitude hypoxia. Fer showed a marked decrease in H1 and H3 compared with N. Hb was positively related to Fer in hypoxia. Iron intake in food was markedly decreased during the 2-wk ascent to 6,542 m. EPO was inversely related to CaO2 and positively related to APR.(ABSTRACT TRUNCATED AT 250 WORDS)

Negligible contribution of splenic erythropoiesis to recovery from anemia in rats.

The contribution of extramedullary erythropoiesis in the spleen during recovery from anemia was examined in rats. Six- and 18-week-old female Wistar rats were subjected to a total splenectomy or sham-splenectomy, and anemia was induced by bleeding the next day. Removing approximately 20% of the total volume of blood in the body resulted in a 9-26% reduction in erythrocyte count, hematocrit, and hemoglobin 3 days after bleeding, but the values returned to normal within 8 days of bleeding in young rats and within 14 days of bleeding in adult rats. Histology, image analysis, and differential cell counts revealed a marked increase in erythropoiesis in the spleen and femoral bone marrow in shamsplenectomy rats after bleeding; however, there were no statistical differences in hematology or bone marrow histology between the splenectomy and sham-splenectomy groups. These results indicate that the contribution of splenic erythropoiesis to the recovery from anemia was negligible in both young and adult rats.

High-Altitude Training

Physical training at high altitude improves performance at high altitude. However, studies assessing performance improvements at sea level after training at higher altitudes have produced ambiguous and inconclusive results. Hypoxia-induced secondary polycythemia is a major contributor to increased work capacity at altitude. The common finding upon exposure to hypoxia is a transient increase in haemoglobin concentration and haematocrit because of a rapid decrease in plasma volume followed by an increase in erythropoiesis per se. Both nonathletes and elite endurance athletes have maximal reticulocytosis after about 8 to 10 days at moderate altitude. Training periods of 3 weeks at moderate altitudes result in individual increase of haemoglobin concentration of about 1 to 4%. A more accentuated increase in haemoglobin can be obtained with longer sojourns at moderate altitude. The normal erythropoietin reaction upon exposure to hypoxia comprises initially increased levels followed by a decrease after about 1 week. Thus, the maintenance of a high erythropoietin concentration is not a prerequisite for a sustained increase in erythrocyte formation at high altitude. The main pharmacological modulator of erythropoietin production seems to be adenosine. But modulators such as growth hormone and catecholamines may also potentiate the effect of hypoxia per se on erythropoietin production. On the other hand, there is a risk that the stress hormones may induce a relative depression of the bone marrow particularly in the early phase of altitude training when the adaptation is minimal and the stress reaction is most accentuated. The most important 'erythropoiesis-specific' nutrition factor is iron availability which can modulate erythropoiesis over a wide range in humans. Adequate iron stores are a necessity for haematological adaptation to hypoxia. However, at moderate altitude, there is a need for rapid mobilisation of iron and even if the stores are normal there is a risk that they cannot be mobilised fast enough for an optimal synthesis of haemoglobin. Data from healthy athletes training at moderate altitudes suggest a true increase in haemoglobin concentration of about 1% per week. Complete haematological adaptation occurred when sea level residents have similar haemoglobin concentrations at moderate altitude compared with residents. The normal difference in haemoglobin concentrations can be estimated to be about 12% between permanent residents at sea level and at 2500m above sea level. This difference indicates a necessary adaptation time of about 12 weeks. If the training period at moderate altitude must be shorter, several sojourns at short intervals are recommended. The important factor in haematological adaptation in athletes at moderate altitude is hypoxia.(ABSTRACT TRUNCATED AT 400 WORDS)

Serial immunoreactive erythropoietin levels in autologous blood donors

The variations in plasma erythropoietin (EPO) concentration during preoperative deposit of autologous blood were studied in 12 patients (8 men, 4 women). Four donations were scheduled at weekly intervals. A predonation hemoglobin concentration of 11 g per dL (110 g/L) was required. Hemoglobin concentration decreased from 14.3 +/- 1.1 g per dL (143 +/- 11 g/L) (mean +/- SD) before the first donation to 11.7 +/- 0.7 g per dL (117 +/- 7 g/L) on Day 22 (p less than or equal to 0.0001). Reticulocyte counts increased from a median of 31,800 (range, 4900-95,000) per microL (median, 32 x 10(9)/L [range, 5-95 x 10(9)/L]) to 93,800 (16,800-194,900) per microL (median, 94 x 10(9)/L [range, 17-195 x 10(9)/L]) on Day 28 (p less than or equal to 0.01). Plasma EPO concentration was 17.8 +/- 5.1 mU per mL prior to the first donation and displayed a small and transient peak after each donation. A sustained elevation followed each peak. Although plasma EPO concentration differed significantly from the baseline value after the first donation, only the peak concentrations after the second (35.5 +/- 15.5 mU/mL), third (38.0 +/- 14.5 mU/mL), and fourth (36.1 +/- 11.0 mU/mL) donations exceeded the normal range. The moderate, biphasic increase in plasma EPO concentration and the moderate increase in erythropoiesis suggest two strategies in autologous blood donation that should be investigated with respect to efficiency and safety: 1) more aggressive donation schemes, which reduce donation intervals and/or the minimum hemoglobin concentration and 2) the administration of recombinant human EPO.

Effect of subcutaneously administered human recombinant erythropoietin on erythropoiesis in patients with myelodysplasia

In a phase II study, 12 patients with a myelodysplastic syndrome (MDS) and anaemia (nine transfusion-dependent) were treated with recombinant human erythropoietin (rHuEpo) to assess the therapeutic effect on erythropoiesis and on transfusion requirement. Patients with a low risk of developing acute leukaemia were included, i.e. refractory anaemia (RA), RA with ringed sideroblasts (RARS) and RA with excess blasts (RAEB), providing the percentage of myeloblasts in the bone marrow did not exceed 10%. Recombinant HuEpo treatment was initiated at a dose of 50 units/kg body weight and administered subcutaneously three times weekly. At 3-week intervals the dose was increased with 50 units/kg per injection, until after 15 weeks a maximum dose of 250 units/kg three times weekly was reached. All patients completed the study. Recombinant HuEpo was well tolerated and no serious side effects were seen. There was no evidence of the emergence of a new malignant clone in response to rHuEpo as shown by sequential karyotyping. In none of the patients was an increase in haemoglobin level or a diminished red blood cell transfusion requirement seen. In four out of 10 evaluable sequential bone marrow smears, an increase in erythropoiesis was seen, suggesting stimulation of ineffective red cell production. One of these patients also showed a rise in reticulocyte count. The number of erythroid progenitor cells (BFU-E and CFU-E) in blood and bone marrow was not affected by rHuEpo treatment. Also no change in the number of myeloid progenitor cells (CFU-GM) in blood and bone marrow was noted. In conclusion, subcutaneous treatment with rHuEpo at dosages up to 250 units/kg body weight (three times weekly) fails to increase the haemoglobin level or to diminish the transfusion requirement in patients with MDS and anaemia. It is unclear whether higher doses of rHuEpo are effective or whether patients with less severe anaemia who are transfusion independent, have a higher likelihood of response.

Neutropenia and thrombocytopenia in infants with Rh hemolytic disease

To assess the incidence and mechanisms of thrombocytopenia and neutropenia in neonates with Rh hemolytic disease, we studied 20 consecutive patients with this condition who were born at our hospital. All five patients with severe disease (hydrops) had neutropenia and thrombocytopenia before and after exchange transfusions. Two of six patients with moderately severe disease (not hydropic but requiring exchange transfusion) had neutropenia; all six had thrombocytopenia. Of nine patients with mild disease (not treated with exchange transfusions), two had neutropenia but none had thrombocytopenia. The mean platelet volume was low or normal (7.5 +/- 0.2 ft) in the patients with thrombocytopenia, and the neutropenia was not accompanied by a "left shift" (ratio of immature to total neutrophils 0.26 +/- 0.03, mean +/- SEM). In two severely affected patients, erythroid progenitor levels were elevated and their proliferative rates (tritiated thymidine suicide) were increased, whereas their granulocyte-macrophage progenitor levels and the proliferative rates of those progenitors were diminished. In a severely affected patient, the in vitro maturation of multipotent progenitors was altered, with production of a greater than normal proportion of normoblasts (p less than 0.01) but fewer neutrophils (p less than 0.02) and megakaryocytes (p less than 0.03). It appears that the marked increase in erythropoiesis in fetuses with Rh hemolytic disease can be accompanied by a down-modulation of neutrophil and platelet production.

Dietary copper deficiency and autoimmunity in the NZB mouse

NZB mice were exposed from birth to a diet either adequate or deficient in copper. By age 6 wk the mice exposed to the copper-deficient diet showed symptoms characteristic of copper deficiency (anemia, hypoceruloplasminemia, and achromatrichia). The splenic lymphocytes from the copper-deficient group had reduced numbers of cells expressing the following surface markers: Ly-5, Ly-1, B-220, and sIg. Less than 10% of the splenic lymphocytes in this group were cycling, as determined by flow cytometry analysis. The spontaneous 96-h anti-ss-DNA levels in the copper-deficient group were lower than those in the control group. The exogenous colony-forming units (CFUs) were significantly enhanced in the copper-deficient mice. The decreased splenic lymphoid populations, decreased anti-ss-DNA titers, and increased exogenous CFUs in the copper-deficient mice appear to be due to an increase in erythropoiesis at the expense of lymphopoiesis.

Quantitative assessment of erythropoiesis in bone marrow expansion areas using 52Fe.

Quantitative 52Fe scans were performed in 180 patients. Expansion of bone marrow was observed in 70. This bone marrow expansion was a nearly constant feature in haemolytic anaemia and in sideroblastic anaemia. It occurred in a third of the patients with myelofibrosis. In patients with polycythaemia rubra vera, expansion was noticed in only two out of seven. Erythropoiesis in expansion areas occurred despite persistence of fat in the iliac crest bone marrow biopsy. It could exist with a slight increase in erythropoiesis and might develop only after a long period of erythropoietic stimulation. Increased marrow activity can take place without erythropoietic expansion in long bones. The fraction of iron uptake in expansion areas did not exceed a third of total marrow iron uptake. With increasing erythropoiesis, the increase in iron uptake in expansion areas was less marked than the increase in the central areas. Erythropoiesis in expansion areas was usually not of major quantitative importance but could nevertheless reach the erythropoiesis of a normal adult.

Contamination of erythropoietin by endotoxin: in vivo and in vitro effects on murine erythropoiesis

Endotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU- e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10(-5)-10(-3) mug of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.

Contamination of Erythropoietin by Endotoxin: In Vivo and In Vitro Effects on Murine Erythropoiesis

AbstractEndotoxin was detected in all erythropoietin preparations tested and was removed from four lots, without loss of erythropoietic activity, by adsorption with limulus amebocyte lysate. Comparison of adsorbed (endotoxin-depleted) and nonadsorbed (endotoxin-containing) erythropoietin preparations demonstrated significant inhibition of CFU-e and BFU-e in vitro by nonadsorbed erythropoietin at concentrations higher than 0.25 U/ml and 2.0 U/ml, respectively. CFU-e and BFU-e were inhibited significantly by readdition in vitro of 10-5-10-3 μg of endotoxin per unit of limulus-adsorbed erythropoietin. Administration of saline or 6 U of nonadsorbed or adsorbed erythropoietin twice a day for 4 days of CF1 mice resulted in reticulocyte counts of 2.1%, 9.9%, and 15.9%, respectively. Nonadsorbed erythropoietin resulted in a 29% decrease in erythropoiesis, a 42% decrease in CFU-e, and a 16% increase in granulopoiesis in the marrow, whereas adsorbed erythropoietin caused a 28% increase in erythropoiesis, no significant change in CFU-e and a 19% decrease in granulopoiesis in the marrow. Both preparations resulted in marked increases in splenic erythropoiesis and granulopoiesis. The effects of adsorbed erythropoietin are similar to those produced following stimulation of hematopoiesis by endogenous erythropoietin. Hemopoietic changes induced by nonadsorbed erythropoietin in vivo and in vitro are affected substantially by contamination of the erythropoietin preparations with endotoxin.

Effects of androgens on burst forming units (BFU-E) in normal rabbit bone marrows

The mechanism of action of androgenic steroids on erythropoiesis is not well understood. In order to assess whether the site of action of androgens is on the early erythroid committed stem cell compartment, the in vitro effects of testosterone (T), 5α-dihydrotestosterone (5α-DHT) and 5β-dihydrotestosterone (5β-DHT) on the so-called erythropoietic burst forming unit (BFU-E) in normal rabbit bone marrows were studied. Even though all of the steroids studied increased the number of BFU-E in the presece of Ep, 5β-DHT was the most potent in stimulating BFU-E. Testosterone was moderately effective in increasing BFU-E. Even though 5α-DHT produced a significant increase in BFU-E, it was the least effective of the 3 steroids studied. Preincubation (2 hrs) of normal rabbit bone marrow cells with testosterone followed by removal of T from the culture system resulted in a significant increase in BFU-E when compared with that of non-treated marrow cells in the presence of Ep. These data suggest that testosterone and 5β-DHT and possibly 5α-DHT act on an early uncommitted stem cell, perhaps the CFU-S, to increase the numbers of erythroid committed stem cells to eventually cause an increase in erythropoiesis in combination with Ep.