Stem Cell Migration Induced by Erythropoietin or Haemolytic Anaemia: the Effects of Actinomycin and Endotoxin Contamination of Erythropoietin Preparations

The injection of erythropoietin or the induction of anaemia with phenylhydrazine leads to changes in murine pluripotent and granulocyte-macrophage stem cells indicating migration from marrow to spleen. In order to evaluate the interrelationship between erythroid differentiation and stem cell migration we have selectively suppressed erythroid differentiation with actinomycin D. Anaemia or EP injection resulted in stem cell changes consistent with migration; actinomycin blocked these changes in anaemic but not EP injected mice while blocking erythropoiesis in both groups. The erythropoietin contained from 0.01 to 1000 microgram/ml of endotoxin as defined by the limulus test; it decreased marrow erythropoiesis and stimulated marrow granulopoiesis. Adsorption of the erythropoietin preparation with limulus lysate removed endotoxin without decreasing erythropoietin activity. Adsorbed erythropoietin stimulated erythropoiesis and not granulopoiesis, and stem cell changes induced by its administration were largely blocked by actinomycin, suggesting that endotoxin in the non-adsorbed erythropoietin caused the actinomycin resistant stem cell changes. The observation that actinomycin blocks both erythroid differentiation and stem cell migration suggests that these two physiologic events are closely linked. The effects of injected erythropoietin on murine haemopoietic stem cells may, to a significant extent, be secondary to the presence of endotoxin in the erythropoietin preparations.     

Effects of actinomycin D in vivo on murine erythroid stem cells

Low-dose actinomycin D (Acto) selectively suppresses murine erythropoiesis without decreasing erythropoietin (Ep) production. We used the plasma clot system to determine the stage of erythroid differentiation at which this inhibition occurs. Late erythroid precursors, CFU-E, and less differentiated committed erythroid stem cells, BFU-E, were assayed in CF1 mice given Acto 75-82 microgram/kg/day or saline subcutaneously for 5 days. We also assayed pluripotent (CFU-S) and committed granulocyte-monocyte (CFU-C) stem cells. Reticulocytes and marrow and spleen nucleated erythroid precursors were decreased by 99% in the Acto-treated mice; tibial marrow CFU-E were decreased by 97% and splenic CFU-E by 99%. Tibial BFU- E were not decreased by Acto, although there was a 66% diminution in splenic BFU-E. Acto increased tibial CFU-S, but splenic CFU-S and tibial and splenic CFU-C were unchanged. Thus Acto inhibits erythropoiesis by suppressing the ability of immediate committed erythroid precursors of CFU-E or CFU-E themselves to differentiate further in response to Ep. Acto does not affect survival or proliferation of the less differentiated cells--CFU-C, CFU-S, and marrow BFU-E. The suppression of splenic BFU-E in Acto-treated mice may indicate that marrow and splenic BFU-E are basically different stem cells. Alternatively, Acto treatment may impair migration of BFU-E from marrow to spleen.     

Renal Mechanisms Underlying Cyclic AMP Action on Erythropoiesis

S ummary . Dibutyryl cyclic AMP (dbc-AMP) was injected into ex-hypoxic polycythaemic mice either alone or with anti-erythropoietin (anti-Ep) serum. Anti-Ep totally abolishes the wave of erythropoiesis evoked by dbc-AMP. These results might indicate either that the action of this agent is totally Ep-dependent, or that a residual amount of endogenous Ep is necessary to allow dbc-AMP to exert a direct effect at the marrow level. The latter mechanism, however, is precluded by experiments indicating that administration of moderate amounts of anti-Ep, although abolishing totally the erythroid response to dbc-AMP, do not induce complete suppression of endogenous Ep activity and erythropoiesis. Furthermore, a significant rise of Ep plasma levels is observed in rats receiving dbc-AMP. Since this agent does not apparently modify the kinetics of endogenous Ep, it is postulated that dbc-AMP induces a rise in Ep production. This phenomenon, although unmodified in ureter-ligated animals, is completely abolished by bilateral nephrectomy. It is therefore concluded that dbc-AMP induces in vivo a stimulatory effect on erythropoiesis via increased production of Ep, via a renal mechanism possibly represented by elevated levels of the renal erythropoietic factor.     

Regeneration of erythroid progenitor cells in polycythemic mice treated with cyclophosphamide

Mice with posthypoxic polycythemia treated with a sublethal dose of cyclophosphamide (Cy) were used as a model to investigate, by in vitro methods, the kinetics of regeneration of erythroid committed precursors (ECP) and to study the influence of erythropoietin (Ep) on those precursor cells. The results demonstrated that erythroid burst-forming units (BFU-E), early (d10) and late (d4), and erythroid colony-forming units (CFU-E) recover at different rates after Cy. Early BFU-E recovery was not Ep dependent and closely resembled regeneration of pre-erythropoietin-responsive cells (pre-ERC) found previously using the same experimental model. The absence of spontaneous recovery of mature BFU-E and CFU-E in the bone marrow and spleen of Cy-treated polycythemic mice, which is contrary to the findings in normal mice treated with Cy, indicates the importance of Ep for BFU-E (d4) and CFU-E regeneration. This was confirmed when exogenous Ep was injected. The effect on BFU-E (d4) of exogenous Ep injected into the polycythemic Cy-treated mice at the time when primitive BFU-E have regenerated considerably suggested an influence of Ep on the transition of BFU-E (d10) to BFU-E (d4). The fast regeneration of CFU-E in the spleen of normal mice and after Ep injection in polycythemic Cy-treated mice confirms the well-known and significant role of the spleen in mouse erythropoiesis under stress conditions. It could be suggested that the patterns of BFU-E (d4) and CFU-E recovery as well as Ep responsiveness closely resemble the findings observed earlier for ERC in the same experimental model.     

Chronic administration of transforming growth factor-beta suppresses erythropoietin-dependent erythropoiesis and induces tumour necrosis factor in vivo

Summary. Transforming growth factor beta is a known inhibitor of the proliferation and differentiation of early haematopoietic progenitors but has no effect on mature erythroid cells in vitro. Mice injected with rhTGFβ₁ exhibited severe and progressive suppression of erythropoiesis manifested by a decline of reticulocyte count, marrow erythroblasts and marrow and spleen CFU-E, which could be prevented by administration of erythropoietin. This suppression of erythropoiesis was associated with the appearance of tumour necrosis factor in the blood, development of pronounced cachexia and depression of serum erythropoietin levels. TGFβ induces TNF in vivo that leads to cachexia, decrease of serum erythropoietin levels and suppression of erythropoietin dependent erythropoiesis.     

Erythropoiesis during an erythroblastic phase of chronic myeloproliferative disorder associated with monosomy 7

A chronic myeloproliferative disorder associated with monosomy 7 is described in a 3 1/2-year-old boy. His presenting features closely resembled those of juvenile chronic myeloid leukaemia (JCML). Cytogenetic study of bone marrow cells showed that all of the metaphases examined had chromosome 7 deletions. He developed an erythroblastic phase, characterized by anaemia, marked erythroid hyperplasia of bone marrow and the appearance of nucleated red blood cells in the peripheral blood. During the erythroblastic phase, blood transfusion resulted in a suppression of erythropoiesis as evidenced in both the peripheral blood and bone marrow. The in vitro culture studies showed that the erythroid precursor was dependent upon erythropoietin (Ep) for differentiation and proliferation during the erythroblastic phase. However, the Ep dose-response curve showed that a peak of erythroid colony formation occurred at a lower concentration than in the healthy controls. These findings suggest that although the erythroid precursor remains under the control of Ep, it has an increased sensitivity to Ep during the erythroblastic phase of monosomy 7.     

Variations in the Relationships Between Total 59Fe Uptake and the Uptakes Into Heme and Nonheme Fractions of Spleen and Bone Marrow in Irradiated Mice and Mouse Radiation Chimeras

A chemical method for the separation ofheme and nonheme iron-containing fractions in mouse tissues has been used tostudy the uptake of ⁵⁹Fe into both theheme and nonheme iron fractions inspleen and femoral bone marrow, in orderto investigate the possible relationshipsbetween the total uptake into spleen tissue and the rate of uptake into the hemefractions. In control mice, only aboutone third of the ⁵⁹Fe is directly associated with heme during the first dayafter administration, and this fraction decreases as radioactive red cells are released from the spleen into blood. Tendays after X-irradiation of mice, the proportion of ⁵⁹Fe in the spleen heme fraction 6 hr after intraperitoneal administration decreased from about 30 to 10%of the total splenic activity as the radiation dose was increased from 500 to 750rads. A similar alteration in the proportion of ⁵⁹ Fe in the spleen haem fractionoccurred in mouse radiation chimeras asthe dose of injected syngeneic bonemarrow cells was reduced from 5 x 10⁵to zero. Similar results were found infemoral bone marrow. These results indicate that in this system the rate of uptake into whole tissue is not a measureof the uptake into heme; it is necessary,therefore, to use the method involvingthe measurement of iron uptake intoheme, rather than the total tissue uptake,when estimating hemoglobin synthesisor erythropoiesis.

Submitted on February 15, 1971 Revised on April 5, 1971 Accepted on April 8, 1971     

Effects of an aplastic anemia urinary extract on mouse erythroid progenitor cells in vivo

We investigated the in vivo effects of a crude extract from the urine of aplastic anemia patients (AA urinary extract) on erythroid precursor cells in the femoral bone marrow and spleens of normal adult mice. A single intraperitoneal injection of AA urinary extract induced a significant increase in the number of splenic erythroid burst-forming units (BFU-e) and erythroid colony-forming units (CFU-e) within 24 h after injection. We then injected pure recombinant erythropoietin (Epo) equivalent to the amount present in the urinary extract. This addition increased the number of splenic CFU-e by almost the same degree as the amount induced by the AA urinary extract 24 h after injection, but failed to elicit any change in the number of splenic BFU-e. In other studies, mice were injected with the same amount of lipopolysaccharide (LPS) and/or pure Epo as that present in the AA urinary extract. Experiments with Limulus amebocyte lysate-adsorbed (endotoxin-depleted) or nonadsorbed (endotoxin-containing) AA urinary extracts showed that endotoxin contamination interfered with the increase in numbers of marrow CFU-e and enhanced the increase in splenic CFU-e numbers induced by pure Epo or Epo activity in the AA urinary extract. The number of splenic BFU-e, however, was not affected by administration of LPS and/or Epo or by adsorbed endotoxin. These data suggest that AA urinary extract contains a stimulating activity for mouse splenic BFU-e, and that this activity is not attributable to the Epo activity or endotoxin contamination within the urinary extract.     

Interaction of stem cell factor and its receptor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen

The phenotypes of mice that harbor a defect in the genes encoding either stem cell factor (SCF) or its receptor, c-kit, indicate that this ligand/receptor pair is necessary for maintenance of normal hematopoiesis in the adult. Our objective was to determine whether SCF, like erythropoietin, is necessary for acute erythroid expansion during recovery from hemolytic anemia. Monoclonal antibody ACK2, which recognizes the murine c-kit receptor, was used to selectively block the hematopoietic growth-promoting effects of SCF. Mice were treated with phenylhydrazine on day 0 and day 1 to induce hemolytic anemia and also received no antibody, control IgG, or ACK2 on day 0. The mice were killed on day 3 and the hematocrit (Hct), reticulocyte count, and numbers of erythroid and myeloid hematopoietic progenitor cells (colony- forming unit-erythroid [CFU-E], burst-forming unit [BFU]-E, and CFU- granulocyte-macrophage [GM]) were quantitated in the femoral marrow and spleen using hematopoietic colony-forming assays. Induction of hemolytic anemia with phenylhydrazine resulted in a drop in the Hct from approximately 50% to 30%, and an approximate 8- to 10-fold increase in the reticulocyte count. The numbers of CFU-E increased modestly in the femur, and approximately 25- to 50-fold in the spleen, in comparison with normal mice. BFU-E and CFU-GM values did not increase in the femur but expanded 6- to 10-fold in the spleen, in comparison with normal mice. This confirms that much of the erythroid expansion in response to hemolytic anemia occurs in the murine spleen. Neutralizing quantities of the ACK2 antibody reduced femoral CFU-E, BFU- E, and CFU-GM content to less than half that found in phenylhydrazine- treated control mice and nearly totally ablated splenic hematopoiesis. These results suggest that c-kit receptor function may be required for optimal response to acute erythropoietic demand and that erythropoiesis in the splenic microenvironment is more dependent on SCF/c-kit receptor interaction than is erythropoiesis in the marrow microenvironment. Because expansion of late erythropoiesis in the spleen was preferentially blocked, we tested the hypothesis that homing of more primitive hematopoietic cells to the spleen was dependent on c-kit receptor function. Lethally irradiated mice were injected with marrow cells obtained from mice that had received phenylhydrazine plus control IgG or with marrow cells obtained from mice that had received phenylhydrazine plus ACK2. In parallel experiments, normal murine marrow cells were treated in vitro with control IgG or with ACK2 and were injected into lethally irradiated mice. The fraction of BFU-E and CFU-GM retrieved from the marrow and spleen of the recipient mice 4 hours later was reduced by approximately 75% when progenitor cells had been exposed to ACK2, in comparison with control IgG. These data suggest that interaction of SCF with the c-kit receptor affects the homing behavior of hematopoietic progenitor cells in the adult animal.     

Long-term recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment severely depresses murine marrow erythropoiesis without causing an anemia.

We hereby report profound effects of long-term granulocyte colony-stimulating factor (G-CSF) administration on murine erythropoiesis. Recombinant human (rh)G-CSF (150 micrograms/kg body weight/day) was administered over 24 days to female C57Bl mice. Marrow erythroid colony-forming units (CFU-E) and erythroblast numbers declined to less than 5% of normal, whereas splenic erythropoiesis simultaneously increased. Splenic erythropoiesis effectively compensated for the loss of marrow erythropoiesis as indicated by the maintenance of a normal hematocrit. In the marrow the numbers of spleen colony-forming units (CFU-S) and erythroid burst-forming units (BFU-E) declined as well. Simultaneously, however, these numbers increased both in the spleen and in the peripheral blood by a factor of 20 to 30. These findings suggest a continuous migration of stem cells and progenitor cells out of the marrow and an efficient seeding in the spleen, directly or indirectly induced by G-CSF. In addition the differentiation and/or amplification of BFU-E to CFU-E was impaired in the marrow but not in the spleen. The marrow and splenic microenvironment also behaved differently with respect to granulopoiesis. G-CSF did not lead to an enhanced granuloid amplification in the spleen but exerted its proliferation activity mainly in the marrow. These findings imply that prolonged G-CSF treatment might cause erythroid depression in animals and humans when spleen erythropoiesis is less efficient than in mice.     

The anaemia of chronic renal failure in sheep: studies in vitro

S ummary . The presence of inhibitors which accumulate during uraemia has been postulated as a significant factor in the development of anaemia in chronic renal failure (CRF). To determine whether factors in uraemic serum depress erythropoiesis, samples were obtained from sheep prior to and after surgical induction of CRF. The sera were tested in vitro for their effect on erythroid colony growth. The sheep sera were substituted for fetal calf serum (30% concentration) in cultures of serotype-matched or autologous sheep marrow cells at optimal doses of erythropoietin (Ep). Forty-two paired sera from five animals were tested against normal (22) and uraemic marrow (20). In 7/42 random pairs, erythroid colony growth was decreased by 20% in the presence of uraemic serum when compared to a normal sample from the same animal. In the remainder of the cultures, uraemic sera stimulated or supported erythroid colony growth as well as normal sera. When the results were analysed individually, serum from only one of five animals showed minimal (10%) in vitro inhibition of erythroid colony growth. This study, performed in a prospective manner utilizing compatible target cells, disputes the hypothesis that uraemic toxins significantly inhibit in vitro erythropoiesis. These data correlate with the in vivo response to Ep in this sheep model, and suggest Ep would be effective in treating the anaemia of CRF.     

T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro.

The pathogenesis of the anemia associated with malignancy was investigated in a patient with T cell chronic lymphocytic leukemia. The plasma clot culture system was used as a measure in vitro of erythropoiesis. The patient's peripheral blood and marrow T lymphocytes obtained both before and after transfusion therapy suppressed erythroid colony formation by normal human bone marrow cells. Pretreatment of the patient's bone marrow T cells by antithymocyte globulin (ATG) and complement reversed this suppression. In addition, pretreatment of the patient's marrow cells with ATG and complement markedly augmented erythropoiesis in vitro. The expression of erythroid activity caused by the selective destruction of the suppressor T lymphocytes in the patient's bone marrow with ATG and the suppression of normal erythropoiesis by the patient's bone marrow and peripheral blood lymphocytes suggest that interaction between the malignant T cell and the erythropoietin-responsive stem cell is important in production of anemia in this patient.     

Evidence for a Physiologic Role of Erythropoietin in Fetal Erythropoiesis

Administration of antibody prepared inrabbits against human urinary Ep to fetalsheep during the last third of the gestation period resulted in a suppression oferythropoiesis in the fetus. This wasevidenced by a sharp decrease in the numbers of circulating reticulocytes and a reduced rate of ⁵⁹Fe incorporation into RBC,spleen, and bone marrow, effects ascribable to a neutralization of endogenous Ep.In addition, there was a significant decrease in the numbers of nucleated erythroid cells in the bone marrows of thesefetuses. These results point to a physiologic role for Ep in fetal erythropoiesis.

Submitted on December 17, 1973 Revised on February 19, 1974 Accepted on February 20, 1974     

In vitro culture of proerythroblasts: characterization of proliferative response to erythropoietin and steroids

Summary This study characterized variables affecting the in vitro liquid culture of proerythroblasts. When bone marrow from mice depleted of haemoglobin containing cells, was cultured in vitro in the presence of human urinary erythropoietin (Ep) a significant degree of erythroid cell proliferation and maturation occurred as measured directly by ³H-thymidine (³H-TdR) incorporation into DNA (autoradiographical measurement). Proliferation increased indirect proportion to the dose of Ep added to the culture. We also demonstrated a highly significant positive correlation between proliferation measured directly by ³H-TdR incorporation into DNA or indirectly by ⁵⁹Fe incorporation into haem. Ep was a potent stimulator of proerythroblast proliferation. We also examined the role of the androgenic and non-androgenic steroids on in vitro proliferation. All the hormones tested were stimulatory but only in the presence of Ep. The androgenic steroids primarily affected the more mature erythroid precursors whereas the glucocorticoids were more general growth promoters. Their addition in physiologic concentration to liquid culture reduced Ep requirements. Thus when both testosterone and hydrocortisone were added to culture the Ep concentration that produced the same degree of proliferation as a culture containing Ep alone was decreased by 90%. This finding is important as it indicates that in vitro culture conditions can be created that more closely mimic in vivo erythropoiesis where Ep requirements are far less.     

The effects of therapeutic dialysis and renal transplantation on uraemic serum inhibitors of erythropoiesis in vitro

The methyl cellulose culture system using human bone marrow and erythropoietin (Ep) at different concentrations was used to examine the effects of uraemic sera on erythropoiesis in vitro.
Sera from undialysed patients with advanced uraemia (plasma creatinine ± 900 μmol/l) when added to cultures at 10% with Ep at 2-0 u/ml were consistently inhibitory to the growth of erythroid burst-forming units (BFU-E). No inhibition of erythroid colony-forming units (CFU-E) was observed at this Ep concentration but inhibition was consistently demonstrated with Ep at 0-2 u/ml. Sera from undialysed patients with less severe uraemia (plasma creatinine < 900 μmiol/1) were not inhibitory to BFU-E or CFU-E at Ep 2-0 u/ml.
Sera from patients with stable, functioning renal transplants were stimulatory to erythropoiesis in vitro with Ep at 2 -0 u/ml. This finding is consistent with the normal or increased haematocrits often found following renal transplantation. Sera from patients on maintenance haemodialysis and continuous ambulatory peritoneal dialysis (CAPD) were not significantly different from normal in their effect on BFU-E growth in vitro and were slightly but significantly stimulatory to the growth of CFU-E. This suggests that these two forms of dialysis are equally effective in reducing the activity of uraemic inhibitors in serum and that inhibition of the marrow response to Ep and/or burst-promoting activity (BPA) is unlikely to be a major factor in the continued anaemia of dialysis patients.     

Changes in murine bone marrow macrophages and erythroid burst-forming cells following the intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP)

In order to explore the effect on bone marrow macrophages of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP), mice were injected intravenously with a preparation of such liposomes at a dose known to deplete spleen and liver macrophages. Two days later, the macrophages in the marrow of the femoral bones were quantified by flow cytometry using a macrophage-specific monoclonal antibody (F4/80), and their ultrastructure and phagocytic activity towards zymosan particles was assessed. To determine the effect on erythropoiesis of liposome-encapsulated Cl2MDP-induced changes in bone marrow macrophages, red blood cell parameters and the formation of erythroid burst-forming unit (BFU-E)-derived colonies in vitro were evaluated. In mice injected with liposome-encapsulated Cl2MDP, there was a 54% and 67% decrease in the total number of bone marrow macrophages as compared to uninjected controls and mice treated with empty liposomes, respectively. Moreover, residual macrophages showed an abnormal ultrastructure, with reduced numbers of crystalloid inclusions and increased numbers of large myelin figures. However, the phagocytic activity of these cells was unimpaired or slightly enhanced. In mice injected with liposome-encapsulated Cl2MDP there was an approximately 60% decrease in the percentage and total number of circulating reticulocytes and a 54% reduction in the BFU-E number, demonstrating deregulation of erythropoiesis under conditions of macrophage loss and impairment. The results suggest that mice treated with liposome-encapsulated Cl2MDP are a model for studying the role of macrophages in erythropoiesis.     

Normal and friend virus specific cell compartments in friend leukemia characterized by their sensitivity to actinomycin D in vivo

Summary DBA/2 mice were infected with the polycythemia inducing Friend Virus (F-MuLV-P) and treated with different doses of Actinomycin D (Act D) when the whole erythroid cell system, as measured by the CFU-E technique with and without addition of erythropoietin (Ep), had been transformed into Ep-independence. During and after this therapy the different stem cell pools CFU-S, CFU-C, BFU-E and CFU-E (with and without Ep) were studied and their sensitivity to Act D in bone marrow and spleen was compared to that of normal mice, recently published by other authors. There seemed to be no difference in the Act D sensitivity between normal erythropoiesis and Ep-independent erythropoiesis caused by F-MuLV-P. Furthermore a cell called ICPC (infectious centers producing cell) was studied. This cell system, detected by spleen colony formation due to high local virus production in an unirradiated host, proved to be Act D sensitive in the spleen but not in the marrow. When the erythroid cell system regenerated after Act D chemotherapy, all erythroid colony growth was Ep-independent. This means that Act D did not induce normal erythropoiesis as seen with hydroxyurea treatment.Supported by Deutsche Forschungsgemeinschaft, (SFB 112) and Stiftung VolkswagenwerkWith assistance of E. Barthel, M. Grünefeld, and K. Steinhoff     

Suppression of mouse erythroid colony formation by L1210 leukemia cells.

The effect of L1210 transplantable leukemic cells on in vitro formation of erythroid colonies from CD2F1 mouse bone marrow progenitor cells (CFU-E) was investigated. Clonal cell culture was carried out by a methylcellulose technique. Human urinary erythropoietin served as the stimulator. After 44 hours of incubation aggregates of eight or more erythroid cells were scored as colonies. The number of CFU-E which could be demonstrated in marrow cells from mice that had been injected intravenously 6 days before with 5 x 10(4) L1210 cells was far below that obtained from normal marrow cells. When 1.3 x 10(5) marrow cells from leukemic mice or L1210 ascites cells were cultured with an equal amount of normal cells, the number of CFU-E expressed was reduced by 51% and by 86%, respectively, relative to controls with normal cells only. Neither lethally irradiated L1210 cells (4500 rad) nor L1210 cell conditioned media suppressed erythroid colony formation. It is suggested that in L1210 leukemia erythropoiesis is decreased because of a cell-to-cell inhibitory action of the leukemia cells on CFU-E.     

Influence of the joint treatment with granulocyte colony-stimulating factor and drugs elevating extracellular adenosine on erythropoietic recovery following 5-fluorouracil-induced haematotoxicity in mice

The presented data address the problem of pleiotropic effects of granulocyte colony-stimulating factor (G-CSF) and suggest the ability of drugs increasing the level of extracellular adenosine to activate erythropoiesis when given jointly with G-CSF. To demonstrate these interactions, the effects of the drugs on the recovery from erythropoietic damage induced in mice by a single dose of 5-fluorouracil (5-FU) were investigated. Elevation of extracellular adenosine and thus activation of adenosine receptors was induced by joint administration of dipyridamole (DP), a drug inhibiting the cellular uptake of adenosine, and adenosine monophosphate (AMP), an adenosine prodrug. The drugs were injected in a 4-d treatment regimen starting 2 h after 5-FU injection. Both DP+AMP and G-CSF alone induced only weak effects. However, the combination of the three drugs produced significant elevation of erythrocytes in the peripheral blood which pertained in the posttreatment period. Stimulation of proliferation of erythroid progenitor cells (BFU-E) in femoral bone marrow and increased levels of reticulocytes in the peripheral blood were observed in the course of the 4-d treatment regimen. In addition, significantly decreased mean cell haemoglobin accompanying the elevated numbers of erythrocytes in the combination-treated mice was found. This effect could be interpreted as the result of a sublethal 5-FU-induced damage to erythroid progenitor and precursor cells forced to proliferate intensively by the combination therapy. The observed additivity and synergism of G-CSF with elevated extracellular adenosine in terms of erythropoiesis is an interesting finding with potential implications in clinical practice.     

Erythropoietic effects of triiodothyronine on the anemia of renal failure in rats: comparative studies with testosterone and erythropoietin

A pronounced and significant stimulatory effect on erythropoiesis was observed in anemic uremic rats receiving either T3 (50 micrograms/kg/day) or Ep (7.5 and 15 U[units]/day) for ten days. A lack of erythropoietic response was seen after the administration of testosterone (5 mg/kg/day) for the same period of time. Renal failure and anemia were studied in partially nephrectomized rats that had received nephrotoxic doses of kanamycin (500 mg/kg/day). The marked increase in red blood cell production produced by T3 and Ep in anemic uremic rats was evident, not only from increased hemoglobin and hematocrit values in peripheral blood, but also from an elevated number of circulating reticulocytes and generally increased absolute counts of nucleated erythroid cells per milligram of bone marrow. The effects of T3 on erythropoiesis in anemic rats with renal insufficiency are in accordance with our previous report demonstrating the direct effect of thyroid hormones on marrow erythroid precursors. This effect can occur only when high levels of the free active forms of T3 are present in plasma, as can happen in the uremic rats receiving daily doses of T3. Since the possibility of producing large amounts of Ep for the treatment of the anemia associated with chronic renal failure is unlikely in the near future, utilization of T3, mainly compounds without calorigenic effects, may be a potential therapeutic alternative.