Erythropoietin-like factor production by young chick blastoderms.

When grown in liquid culture, chick blastoderm cells release an erythropoietic stimulating factor (ESF) whose properties in vitro and in vivo are very similar to those of erythropoietin found in the serum of anemic adult chick. The role of this factor in the induction of the first blood islands of the chick embryo is discussed.     

In vitro development of chicken erythropoietin-sensitive cells

Addition of anemic chick serum to cultures of chicken marrow in plasma clots induces the development of erythrocytic colonies. Mammal erythropoietin shows no effect on cultures of chicken marrow. These observations confirm the existence of an erythropoiesis stimulating factor specific for birds, with properties similar to those of mammalian erythropoietin.     

Studies on the erythropoietic effect of plasma from anemic toads both with and without testis.

Injection of plasma from experimentally induced anemic toad with intact testis increases erythropoiesis in starved toads evidenced by the increase of red blood cell, hemoglobin and hematocrit, whereas the plasma of castrated and phenylhydrazine-HCl treated anemic toad failed to do so. It can be suggested that the erythropoiesis stimulating factor (ESF) is produced from the testis of toad and the production of this factor (ESF) was found to increase during anemia in an attempt to correct the anemic condition.     

Effect of Trypanosoma brucei brucei on erythropoiesis in infected rats

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of β-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.     

Studies of the development of frog hemopoietic tissue in vitro. I. Spleen culture assay of an erythropoietic factor in anemic frog blood.

A new in vitro technique has been described for demonstrating the presence of an erythropoietic factor in the circulating blood of frogs. The assay system consisted of MC33 medium, erythropoietically active spleen cells from Rana pipiens, and plasma or serum from frogs made anemic via phenylhydrazine or bleeding. The spleen cells, which remain erythropoietically active for up to nine days, were found to incorporate 59Fe, [3H]thymidine, [3H]uridine, and [3H]leucine at a greater rate in the presence of plasma or serum from anemic versus normal frogs. The hormones triiodothyronine, prolactin, and erythropoietin were not effective in eliciting an hemopoietic response. The data presented suggest that the spleen from that adult frog is a major site of erythroid differentiation and maturation.     

The anemia of thermal injury: mechanism of inhibition of erythropoiesis

The anemia of thermal injury is a multifactorial process and includes hemorrhage and hemolysis. Much evidence suggests that a reduced rate of erythropoiesis contributes to this anemia. Prior studies show that this anemia is temporally related to the appearance in burn patients sera of a substance(s) capable of inhibiting erythropoiesis in vitro. Four experiments were done to elucidate the mechanism of action of this inhibitor. In all experiments sera from burn patients previously shown to be inhibitory to erythropoiesis in vitro were studied. In the first, inhibitory sera were exposed to erythropoietin solutions without loss of erythropoietic activity. Second, mouse marrow cells were preincubated with serum without loss of their ability to form erythroid colonies. Third, the inhibitory effect could not be overcome with increasing amounts of erythropoietin. Finally, erythroid colony formation was effected only if the inhibitory serum was present during the first 8 to 12 hr of culture. The data suggest that the erythropoietic inhibitor in these sera acts directly on erythroid stem cells in vitro and not by inactivating or interference with erythropoietin.     

Insulin-like growth factor I and erythropoiesis.

The bone marrow, the primary site of hematopoiesis, is a self-renewing system consisting of a unique micro-environment that promotes the differentiation and proliferation of the various hematopoietic cell lines. While many critical factors necessary for red cell production have been identified, the regulation of erythropoiesis has not been completely elucidated. In addition to multi-lineage growth factors (e.g. interleukin 3 or 4) and lineage-specific hematopoietic growth factors (e.g. erythropoietin), several lines of evidence suggest a key role for insulin-like growth factor I (IGF-I). First, growth hormone stimulates erythropoiesis and IGF-I is known to mediate many of growth hormone's actions (somatomedin hypothesis). Second, factors in bovine serum and in serum from an anephric human with erythropoietic activity distinct from erythropoietin have been identified as IGFs. Third, IGF receptors are found on both erythrocyte precursors as well as mature erythrocytes. Fourth, in vitro IGF-I stimulates erythropoiesis in bone marrow cultures. Fifth, IGF-I administration to neonatal or hypophysectomized animals results in increased erythropoiesis in vivo. Recent studies indicate that IGF-I at physiologic concentrations stimulates erythropoiesis and that growth hormone's action is indirect, occurring via IGF-I. The physiologic source of IGF-I for the bone marrow may be delivery from the serum (an endocrine mechanism) or synthesis within the bone marrow by stromal or other cells (a paracrine mechanism). Our recent studies have shown that mouse bone marrow stromal cells secrete both IGF-I and IGF binding proteins (IGFBPs). The role of IGFBPs in erythropoiesis is not known, but they might modulate the local concentration of IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of mammary stimulating factor, a protein which promotes proliferation of mammary epithelium.

This report describes the partial purification and characterization of mammary stimulating factor (MSF), a mitogenic peptide isolated from serum which initiates growth in mouse mammary epithelium. By using ion-exchange chromatography, gel filtration, and isoelectric focusing, MSF was purified 250-fold from porcine serum. It is a heat-stable protein of molecular weight 10,100--10,400 with an isoelectric point of 5.5--6.0. MSF initiates DNA synthesis in vitro in mammary epithelium to a greater extent than in mouse mammary tumor cells (CZF), 3T3 cells, or chick embryo cells. Comparison of the biological, physical, and immunological properties of MSF with other established growth-promoting peptides suggests that MSF is a unique serum factor.     

Sites and trend of erythropoiesis in anemic, normal, and splenectomized newts

Newts, Triturus cristatus carnifex (Laurenti), were anesthetized by submersion in 2% chlorbutol in tap water for 15 min, splenectomized and then rendered totally anemic two months later by treatment with acetylphenylhydrazine (APH) diluted in their tanks (25 mg/liter for 36 h, changing the solution every 12 h). In the 14 weeks following hemolysis, erythron restoration occurred with the same intermittence as it did in whole animals rendered anemic by APH treatment: Beginning the second week the red blood cell count progressively increases for about one month, followed by a period of stasis which lasts about three weeks, then by a new increase, and then by a final period of stasis. Histological examination shows that erythropoietic activity occurs partly in the circulating blood and partly in erythroblasts nestled in the crypts between the muscular trabeculae of the ventricle as well as in the atrial walls. These cells, which are not part of the freely circulating elements in the blood stream, become very abundant in both whole and splenectomized anemic newts but are also present in normal animals. Newts, thus, have three sites for erythropoiesis: the spleen, the blood stream, and the heart. The other components compensate for the elimination of the spleen without determining any lack of, or delay in, erythropoietic response.     

17-beta-Hydroxysteroid dehydrogenase in rabbit bone marrow

Because of the general presence of 17-B-hydroxysteroid dehydrogenase (17-B-HSD) in animals and human erythrocytes, it is very likely that this enzyme is also present in the erythrocyte precursor cells in bone marrow. Adult male white New Zealand rabbits were used to demonstrate the existence of the 17-B-HSD in bone marrow cells and to evaluate the biological function of 17-B-HSD in the erythropoietic system in bone marrow. Anemia was artificially induced in these animals in order to stimulate the proliferation of erythropoietic cells. This was accomplished by bleeding or by intra-peritoneal injection of 40 mg/kg of phenylhydrazine. The 17-B-HSD activities were then examined by histochemical and biochemical assays. The results showed a two to three-fold increase of the enzyme activities in both peripheral erythrocytes and bone marrow cells of anemic animals. Our experiments further demonstrate the fine control of erythropoiesis. The enzyme, which favors the conversion of oestradiol to oestrone, is itself boosted by an anemic state. As a result, the inhibitory action of hemopoiesis by oestradiol was relieved.     

Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.

The erythropoietin receptor (EpoR) is essential for production of red blood cells; a principal function of EpoR is to rescue committed erythroid progenitors from apoptosis. Stat5 is rapidly activated following EpoR stimulation, but its function in erythropoiesis has been unclear since adult Stat5a-/-5b-/- mice have normal steady-state hematocrit. Here we show that Stat5 is essential for the high erythropoietic rate during fetal development. Stat5a-/-5b-/- embryos are severely anemic; erythroid progenitors are present in low numbers, show higher levels of apoptosis, and are less responsive to Epo. These findings are explained by a crucial role for Stat5 in EpoR's antiapoptotic signaling: it mediates the immediate-early induction of Bcl-X(L) in erythroid cells through direct binding to the Bcl-X promoter.     

Intermittent erythropoiesis in anemic newts

A group of 88 newts, Triturus cristatus carnifex (Laurenti), was rendered totally anemic by administering acetylphenylhydrazine (APH) in the breeding water for 48 h at a concentration of 25 mg/liter. The course of erythron restoration was followed for 5 months, sacrificing four specimens per week and analyzing the blood and spleen hemopoietic tissue. The return to the normal values of the red blood cell count occurred through marked increases in concentration at fairly regular intervals, which is best explained by a discontinuous, rhythmic erythropoiesis. This fact is strictly correlated with the intermittent mitotic activity observed in the spleen and with the periodic appearance of large quantities of immature elements in the blood smears. The APH-induced synchronization of newt erythropoietic activity revealed the approximate length of each erythropoietic cycle to be 4 to 5 weeks and the erythropoietic life span to be 50 to 60 days.     

An erythropoietic stimulating factor similar to erythropoietin released by macrophages after treatment with silica

An erythropoietic stimulating factor (ESF) can be detected in the supernatant from fetal liver and adult bone marrow and spleen cells when preincubated with the macrophage-specific cytotoxic agent, silica. Stimulation is observed in 12-day fetal liver CFU-E cultures in the absence of added erythropoietin (Ep). The concentration of ESF in the supernatant added to CFU-E cultures is dependent on the preincubated cell dose and the volume added. The stimulating activity is abolished when mice are hypertransfused and increased above normal values when mice are bled. A concentrated silica-treated spleen supernatant was able to stimulate erythropoiesis in the polycythemic mouse bioassay. It is concluded that the ESF is similar, if not identical, to Ep.     

Identification of ovotransferrin as a heme-, colony- and burst-stimulating factor in chick erythroid cell cultures

Clonal growth of erythroid cells from bone marrow of 2-day-old chicks in fibrin clots under different culture conditions has been used as independent assays for heme-, colony- and burst-stimulating activities found in anemic chicken plasma. The properties of the heme-stimulating activity analyzed by gel filtration, isoelectrofocusing, resistance to heat denaturation, ammonium sulfate precipitation, DEAE-chromatography or HAP-chromatography, suggested serum transferrin as the heme-stimulating factor(s). Heme-, colony- and burst-stimulating factor(s) from anemic chicken plasma did not separate from each other by gel filtration or isoelectrofocusing. Ovotransferrin from egg white also showed heme-, colony- and burst-stimulating activities by the assay employed even after further purification by limited trypsin digestion, electrophoresis, hydroxylapatite chromatography or fractionation by ConA-Sepharose chromatography.     

Isotopic evidence of unaccounted for Fe and Cu erythropoietic pathways

Despite its potential importance for understanding perturbations in the Fe-Cu homeostatic pathways, the natural isotopic variability of these metals in the human body remains unexplored. We measured the Fe, Cu, and Zn isotope compositions of total blood, serum, and red blood cells of ~50 young blood donors by multiple-collector ICP-MS after separation and purification by anion exchange chromatography. Zinc shows much less overall isotopic variability than Fe and Cu, which indicates that isotope fractionation depends more on redox conditions than on ligand coordination. On average, Fe in erythrocytes is isotopically light with respect to serum, whereas Cu is heavy. Iron and Cu isotope compositions clearly separate erythrocytes of men and women. Fe and Cu from B-type men erythrocytes are visibly more fractionated than all the other blood types. Isotope compositions provide an original method for evaluating metal mass balance and homeostasis. Natural isotope variability shows that the current models of Fe and Cu erythropoiesis violate mass balance requirements. It unveils unsuspected major pathways for Fe, with erythropoietic production of isotopically heavy ferritin and hemosiderin, and for Cu, with isotopically light Cu being largely channeled into blood and lymphatic circulation rather than into superoxide dismutase-laden erythrocytes. Iron isotopes provide an intrinsic measuring rod of the erythropoietic yield, while Cu isotopes seem to gauge the relative activity of erythropoiesis and lymphatics.     

Changes in erythropoietic activity of the rat plasma in the course of posttransfusion polycythaemia

The aim of this study was to determine the erythropoietic activity of the plasma of polycythaemic rats and of one of its protein fractions playing a role in erythropoiesis regulation. The erythropoietin activity and that of the erythropoiesis inhibitor varied in the examined plasma samples at definite time periods after induction of polycythaemia. It was demonstrated that the most suitable time of plasma collection for the inhibitor investigation is the period between 115 and 187 h after the first transfusion, and that in some cases separation of this factor from erythropoietin present simultaneously in the plasma is indispensable in order to reveal the inhibitory activity. The erythropoiesis inhibitor administered jointly with erythropoietin was found to exert no influence on erythropoiesis either in normal or in polycythaemic recipients of the tested plasma.     

Testosterone action on erythropoiesis does not require its aromatization to estrogen: Insights from the testosterone and estrogen treatment of two aromatase-deficient men

Androgens act on erythropoiesis, but the relative role of testosterone (T) and estradiol (E₂) on erythropoietic parameters in men is a poorly investigated issue. In order to evaluate separately the effects on erythropoiesis of high-dose T administration alone and of physiological dose of E₂ administration alone two adult men with aromatase deficiency were assessed before and during each treatment. Blood cell count, hemoglobin (Hb), hematocrit (Hct), erythrocyte mean cell volume (MCV), erythrocyte mean corpuscular hemoglobin (MCH), erythrocyte mean corpuscular hemoglobin concentration (MCHC), serum ferritin, iron and total iron-binding capacity (TIBC), serum erythropoietin, serum total testosterone and estradiol were evaluated. Hb, Hct and red cell count rose during testosterone treatment, consistently with the increase in circulating testosterone, but failed to increase during estradiol treatment. A decrease in Hb, Hct and red cell count was recorded in one of the two subjects during estradiol treatment, with a concomitant decrease in serum testosterone. Circulating T alone is capable of and sufficient to influence erythropoiesis, especially at supraphysiological dosage, while circulating E₂ have not the same effect on erythropoietic parameters, suggesting the hypothesis that the erythropoietic changes induced by androgens are not mediated via its aromatization to estrogens.     

The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis. The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval-specific alpha-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes. Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.     

The regenerating liver: a site of erythropoiesis in the adult Long-Evans rat

Erythropoiesis, which is primarily hepatic in the rat during fetal and early neonatal life, shifts almost entirely to the bone marrow in the neonatal-adolescent stage of development. In the adult, extramedullary erythropoiesis has been demonstrated in the liver and spleen under certain pathological conditions when bone marrow red cell production is insufficient. In the present study, erythropoietic foci have been found in young-adult rat liver regenerating 24-72 hr after subtotal hepatectomy. This erythropoiesis is both extravascular and sinusoidal, with some erythroblastic islands noted. The centrolobular hepatic area contains the highest concentration of erythroblasts. Peripheral blood reticulocytosis coincides with the appearance of these cells and this is considered as an indicator of effective erythropoiesis. Liver regenerating after partial hepatectomy produces significant quantities of erythropoietin (Ep) in response to hypoxia. Subtotal hepatectomy may confer upon the adult liver the ability to revert to a fetal-like condition both in its ability to produce Ep and to function as a hematopoietic inductive microenvironment for erythropoiesis.     

Action of human serum on differentiation of skin in vitro. Morphological data and study of the incorporation of labeled amino acids

Epithelial-mesodermal tissue interactions have been shown to be required for normal cytodifferentiation of chick embryo skin. Six-day limb skin does not develop in a protein free chemically defined medium, but keratinization has been observed in medium containing chicken serum. In the present study the authors show that the addition of human serum may stimulate the in vitro differentiation of explants of six-day chick embryo skin. Human serum is able to support skin keratinization and this finding has been confirmed by histological and histochemical criteria. Synthesis of proteins in tissue cultures supplemented with human serum has been studied by use of labeled amino acids such as H3-Leucine and C14-Cystine. These incorporation studies show the existence of macromolecular factors in human serum which may be responsible for the observed skin differentiation.