Regeneration of erythroid committed precursor cells in polycythaemic mice treated with cyclophosphamide.

The recovery of erythroid stem cells and pluripotent haemopoietic stem cells (CFUS) was estimated in mice treated with cyclophosphamide (Cy) by measuring CFUS, the erythroid repopulating ability (ERA) of bone marrow cells and erythropoietin responsive cells (ERC). The experiments were performed in hypertransfused CBA mice previously injected with 200 mg/kg Cy. CFUS, ERA and response to a single dose of erythropoietin-Ep (5 U) were measured at 24 h time intervals for 7, 4 and 8 days respectively. The proliferation rate of CFUS and erythroid repopulating cells was estimated on the basis of cytosine arabinoside (AraC) suicide data. The results obtained demonstrate that CFUS and ERA do not regenerate at the same rate. ERA recovery preceded CFUS regeneration at the time when the Ep response was depressed. The proliferation rate of CFUS decreased with regeneration of CFUS while AraC suicide of erythroid repopulating cells remained high at the time when ERA had recovered to 80% of control values.

Method for growing primitive erythroid progenitors (BFU-E) from rat bone marrow.

A method for growing erythroid bursts from rat bone marrow cells is presented. The methylcellulose culture technique with rat spleen-conditioned medium was used. Successful growth of rat BFU-E was obtained with 20% fetal bovine serum when anemic rat plasma was used as the source of erythropoietin. Plating efficiency was 60 BFU-E per 2 x 10(5) nucleated cells seeded. The proliferative activity determined by cytosine arabinoside (Ara-C) suicide was about 22%. The effect of chronic hypertransfusion on erythroid colony formation is presented.

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.

Erythropoietin and anemia in chronic renal failure.

Serum erythropoietin levels were measured by radioimmunoassay and compared to the severity of anemia in patients with end stage renal disease of different etiology, on chronic hemodialysis. It was demonstrated that the difference in severity of anemia in those patients is a consequence of a difference in erythropoietin production, rather than due to a difference in the level of erythropoiesis inhibitors. It was stressed that in patients with polycystic kidney disease the kidney tissue kept its endocrine function although it had no residual excretory renal function. The positive correlation between hematocrit values and erythropoietin levels indicates that in these patients erythropoietin synthesis is not regulated by general hypoxia. It is suggested that control of erythropoietin production in diseased kidney differs from normal physiological control.

Granulopoiesis in anemic Belgrade laboratory (b/b) rats.

The aim of this study was to clarify the nature of the abnormalities of granulopoiesis in anemic b/b rats. The size of different granulocytic cell compartments (granulocytic and macrophage precursor cells, proliferative and nonproliferative morphologically recognizable granulocytic cells) in the bone marrow, spleen, and peripheral blood was determined, and activity of granulocytic stimulators (colony-stimulating activities derived from lung-conditioned medium of endotoxin-treated rats and from spleen cells stimulated in vitro with pokeweed mitogen) was investigated. Depressed bone marrow granulopoiesis and expanded granulopoiesis in the spleen, together with the sustained production of granulocytic stimulators, was found in anemic b/b rats. It is suggested that the changes within the bone marrow microenvironment are the essential cause of disturbances of granulopoiesis in anemic b/b rats. The increased proliferation of granulocytic cells in iron-treated b/b rats indicates a role of iron in the cellular proliferation.

Effects of prostaglandin synthetase inhibitors, salt overload and renomedullary dissection on the hypoxia stimulated erythropoietin production in rats.

The effect of prostaglandin synthetase inhibitors, aspirin and indomethacin, salt overload and salt depletion, as well as renomedullary dissection on serum Ep level in rats exposed to acute hypoxia was studied. Male rats were given aspirin or saline for seven days prior to the exposure to hypoxia. Other group of animals was fed salt free diet for the same period of time. Indomethacin was given on two consecutive days and renomedullary dissection was performed three weeks before exposure to hypoxia. Ep level was indirectly determined by measuring the 48 h 59Fe incorporation into RBC of mice with posthypoxic polycythemia. Hypoxia induced Ep production was diminished after blocking the PG synthesis using PG synthesis inhibitors. Salt overload, procedure known to decrease PG synthesis, had the same effect. Further on, dissection of the renal medulla, the main source of renal PG, decreased Ep production. The results indicate that PG are part of the mechanism which controls Ep production.

Erythroid progenitors in anemic Belgrade laboratory (b/b) rats.

The anemia of Belgrade b/b rats has been shown to be due to intracellular iron deficiency. The aim of this study of erythropoiesis at the progenitor cell level in these rats was to determine if a defect is present in the early phase of red cell production. Both erythroid colony-forming unit (CFU-E)- and erythroid burst-forming unit (BFU-E)-derived colonies were found to be few in untreated b/b rats and made up of a small number of poorly hemoglobinized erythroblasts of different size and irregular cell shape. Following treatment with iron, the anemia of the rats improved, and the number of CFU-E-derived colonies and the number of cells per colony increased, but the peculiar erythroblast morphology persisted. The high serum level of biologically active erythropoietin (Ep) in b/b rats rules out inadequate Ep production as a cause of their anemia. The results presented indicate, in addition to the earlier described defective transmembrane iron transport, a defect in erythroid progenitor cells. The effect of iron treatment in these rats detected in vitro on erythroid progenitors confirms the importance of iron for cellular proliferation.

Megakaryocytopoiesis in experimentally induced chronic normobaric hypoxia.

It was recently proposed that prolonged hypoxia produces hypomegakaryocytic thrombocytopenia by reducing the pool of committed megakaryocyte progenitor cells at the expense of a greatly expanded erythroid progenitor pool. In order to test this hypothesis we have studied the relationship between megakaryocytopoiesis, erythropoiesis, and granulopoiesis at the level of progenitor cells (megakaryocyte colony-forming unit, CFU-Mk; erythroid CFU, CFU-E; erythroid burst-forming units; BFU-E; and granulocyte-macrophage CFU, CFU-GM) in the marrow of rats exposed for 4 weeks to normobaric hypoxia. We have found that hypomegakaryocytic thrombocytopenia was accompanied by decreased CFU-Mk, increased CFU-E, and a normal number of BFU-E and CFU-GM. These results support the hypothesis that prolonged hypoxia reduces the precursor cell commitment to differentiate into the megakaryocyte series by enhancing demand for differentiation into the erythroid cell line. However, the underlying mechanism needs further investigation.

Erythrocytosis in spontaneously hypertensive rats.

Spontaneously hypertensive rats (SH) with an increased number of red blood cells (RBC), microcytosis, and normal hemoglobin (Hb) concentration were used to study the effect of different manipulations of the erythron on erythropoietin production and on erythroid progenitor proliferation by bone marrow cells in order to gain insight regarding the regulation of erythropoiesis. The serum erythropoietin (Ep) level was increased in untreated SH rats. After stimulation by either bleeding, hemolysis, or acute hypoxia, both the erythropoietin level and erythroid colony-forming unit (CFU-E) proliferation by bone marrow cells increased in SH rats to levels that were similar to those of normotensive Wistar (W) rats. Exposure to chronic hypoxia induced an increase in Hb concentration in SH rats concomitantly with the increase in RBC. The results obtained in SH rats raise the possibility of a defect in nonEp stimulators of erythropoiesis that may alter Hb synthesis.

Hematopoietic growth factors in anemia of Belgrade laboratory (b/b) rats.

In this study, the extent to which growth factor production and microenvironment might be responsible for defective erythropoiesis and granulopoiesis in anemic b/b rats is investigated. Radioimmunoassay-determined serum erythropoietin (Epo) levels are high in b/b rats and closely related to degree of anemia. The low number of erythroid progenitors in b/b rats despite a high Epo level suggested that the defective erythropoiesis could be due to a low level of burst-promoting activity (BPA). A pokeweed mitogen-stimulated medium (PWM-SCM) was prepared with b/b rat spleen cells and used in normal and anemic rat bone marrow and spleen cultures to determine BPA and other growth factor levels. No erythroid burst-forming unit-derived colonies were found but granulocyte-macrophage colony-forming units were counted in significant number, suggesting that the production of growth factors that supports the growth of granulopoietic progenitors is not significantly disturbed. Because BPA is produced mainly by T-lymphocytes, the low BPA level in b/b rat PWM-SCM raised the question of the functional capacity of T-lymphocytes. Investigations showed a decrease in the proliferative activity of b/b rat spleen mitogen-activated T-lymphocytes to about 20% of controls as well as a decrease in interleukin-2 activity in b/b rat spleen cell supernatants. These results point to defective T-lymphocytes. A study of bone marrow fibroblastoid cell colonies (CFU-F) revealed significantly lower CFU-F counts in the b/b rats. This finding is indicative of a disturbed microenvironment, which could also to some extent be responsible for decreased growth factor production and depressed hematopoiesis in the b/b rat.

Acute erythremic myelosis (DiGuglielmo) following atypical aplastic anemia.

A patient presented with acute erythromyelosis (DiGuglielmo) which was developed after 3 yr of aplastic anemia. Aplastic anemia differed from the classical form, since erythroid cells and megakaryocytes were relatively preserved in the bone marrow. Treatment with androgens induced the increase of hematocrit and reticulocyte as well as general improvement. The sudden appearance of hemorrhagic syndrome due to thrombocytopenia was associated with aggravation of anemia and granulocytopenia. In the bone marrow, giant multinuclear proerythroblasts with bizarre nuclear morphology and PAS positivity with coarse granules was found. Serum erythropoietin (Ep) level was high. Bone marrow cells culture in vitro revealed two types of erythroid colonies: typical and giant multinuclear cells, both benzidine-positive. The number of colonies was irrespective to the Ep dose. "Autonomous" Ep independent growth of these colonies was also demonstrated. The number of colonies was more than 3 times higher per number of cells seeded when compared to normals, which indicated malignant proliferation and Ep independent growth. Treatment with 6-mercaptopurine and transfusions was without effect and the patient died after 15 days with signs of cerebral bleeding.

Anemia in Balkan endemic nephropathy.

The severity of anemia in patients at different stages of the evolution of two tubulointerstitial nephropathies, Balkan endemic nephropathy and chronic pyelonephritis, was compared to clarify the previous observations that anemia appears earlier and is more severe in Balkan endemic nephropathy than in other renal diseases. The role of erythropoietin insufficiency as the cause of anemia in endemic nephropathy was studied as well. The severity of anemia increased with the impairment of renal function in endemic nephropathy and was similar to anemia in chronic pyelonephritis. However, in patients with endemic nephropathy at the initial stage of renal insufficiency significantly lower red cell concentrations were found compared with control subjects from the endemic region. In contrast, patients with pyelonephritis did not have decreased red cell concentrations at the early phase of their renal failure, suggesting that earlier appearance of anemia is characteristic for endemic nephropathy. To confirm this finding a study involving larger number of patients would be necessary. The serum erythropoietin levels, inappropriately low for the degree of anemia in patients with renal failure, were unrelated to the type of tubulointerstitial nephropathy.

Megakaryocytopoiesis in spontaneously hypertensive rats (SHR).

Bone marrow megakaryocytes and their progenitors were studied in SHR in order to obtain more information about megakaryocytopoiesis in hypertension since it is known that various anomalies of platelet function occur in hypertension. Megakaryocytopoiesis under steady state conditions and following stimulated erythropoiesis and thrombocytopenia was not found to be significantly different in SHR from that in normotensive Wistar controls.

The effect of serum containing granulocytic stimulating activity and colony stimulating activity on regenerating hemopoiesis in mice.

The influence of postinflammatory rat serum containing granulocytic stimulating activity (GSA) on the process of spontaneous regeneration of hemopoietic tissue was studied and compared to the effect of colony stimulating activity (CSA) in CBA mice in the regenerative phase of hemopoiesis after aplasia induced by a sublethal dose of cyclophosphamide (Cy). One hour after Cy application, mice were injected with 0.2 ml of GSA or CSA. Then 6, 12, 24 and 48 hr later the changes within bone marrow and spleen CFU - GM, morphologically recognizable hemopoietic cells and white blood cells (WBC) were followed. GSA stimulated spontaneous regeneration of granulocytic cells, specifically increasing the number of morphologically recognizable proliferative granulocytes continuously during 48 hr. The number of bone marrow (at the 6th hr) and splenic (until the 24th hr) CFU - GM were also increased under the influence of GSA. Under the same experimental conditions in vivo, CSA did not induce any changes within bone marrow granulocytic cells, but the increase in splenic CFU - GM was observed until the 12th hr. The differences in the action of GSA and CSA on the process of spontaneous regeneration of granulocytic cells in vivo in Cy treated mice indicate that these 2 factors play different roles in the regulation of granulopoiesis.

Serum erythropoietin levels in hemodialysed patients after administration of recombinant human erythropoietin.

In anemic patients on regular hemodialysis (HD), correction of anemia with recombinant human erythropoietin (rHuEpo) administered intravenously (iv) or subcutaneously (sc) was followed over a 2-month period. Monitoring serum Epo post-dose concentrations after the first iv rHuEpo injection and following another regular injection after 2 months of therapy with rHuEpo iv in 9 patients showed that the Epo elimination half-life was reduced from 7.48 h to 4.68 h. In the same patients the initially low percentage of erythroblasts and mature erythroid progenitors increased during 2 months of rHuEpo therapy. Because Epo molecules bound to Epo receptors are internalized in target cells we suggest that the expansion of the Epo responsive cell pool could explain the shorted Epo elimination time after 2 months of rHuEpo treatment. By monitoring serum Epo concentration following sc rHuEpo injection in 7 HD patients it was found that the modest increase in serum Epo levels (30-60 mU/ml) was sufficient to correct anemia.

Erythropoietin & erythroid progenitors in rats exposed to chronic hypoxia.

In order to better understand the mechanisms affecting erythropoietin (Epo) synthesis and red cell mass increase under chronic hypoxia, we examined Epo production and erythroid progenitors (CFU-E) in rats exposed to normobaric hypoxia for four weeks. Hypoxia induced the rise of hematocrit (Htc), hemoglobin (Hb) concentration and the red blood cell (RBC) number with a plateau in hematocrit values after two weeks. After 24 h of hypoxia, Epo levels were increased 20 fold, followed by a significant decrease. After the first week of hypoxia, the values were still higher than in the controls, but after two weeks Epo levels did not differ significantly from the normal values. The fall of Epo levels coincided with the plateau values of hematocrit. The changes in the CFU-E number followed the changes in Epo concentration: a two fold increase after 24 h of hypoxia; a further increase during the next two weeks reaching a peak on day 14, and then a progressive decrease at the time when Epo concentration was at a normal level. Although decreased, but still higher than normal, the CFU-E number during the last two weeks of hypoxia could be necessary for the maintenance of an achieved steady state under persistent hypoxic conditions with normal Epo concentration sufficient to maintain the existing rate of erythropoiesis.

Erythropoiesis after kidney transplantation: the role of erythropoietin, burst promoting activity and early erythroid progenitor cells.

Successful kidney transplantation is followed by the anemia correction due to re-establishment of normal erythropoietin secretion. The possible role of growth factors and cytokines regulating hematopoiesis in this anemia correction are not completely understood. The aim of this study was to investigate the role of erythropoietin and other stimulators in the regulation of erythropoiesis after kidney transplantation. Thirty-six kidney graft recipients with stable graft function for more than 12 months were studied. According to the hemoglobin levels they were divided into: group 1 (12 patients) with normal graft function (sCr = 145.2 +/- 15.8 micromol/l) and normal hemoglobin (12.7+/-0.3 g/dl), group 2 (11 patients) with normal graft function (sCr = 135 +/- 6.5 miromol/l) and posttransplant erythrocytosis (Hb = 18.1 +/- 0.2 g/dl) and group 3 (13 patients) with chronic graft failure (sCr = 223.7 +/- 28.9 micromol/l, range 181-294) and anemia (Hb = 9.0 +/- 0.8 g/dl). Early erythroid progenitors (BFU-E) from peripheral blood, serum immunoreactive Epo and burst promoting activity (BPA) in PHA-LCM prepared from patients' peripheral blood mononuclear cells were measured in all studied patients. The expected Epo for Hb was found normal in patients with normal graft function, 10 times higher in patients with PTE and low in patients with anemia. BPA in PHA-LCM prepared from PTE was increased in 4/6 patients, normal in 4/6 anemic patients, but it was decreased in 5 patients with normal Hb. The mean values were 20.8 +/- 6.3 in PTE group and 16.2 +/- 6.8 in anemic group, and 4.1 +/- 1.8 (at the level of normal controls) in group 1. The number of BFU-E derived colonies was low in most patients with normal hemoglobin and anemia, and increased in most patients with PTE. Spontaneous BFU-E colonies i.e. without Epo added to the cultures were found in 7 of 12 patients with PTE. The mean values of BFU-E showed significant differences between patients with PTE (17.43 +/- 7.3), and patients with normal hemoglobin and anemia (4.39 +/- 1.2 vs. 6.5 +/- 1.1). The results presented suggest that inappropriate Epo secretion depends on the graft function and is the primarily important regulator that caused PTE or anemia after kidney transplantation. Synergistic action of BPA with Epo as well as increased sensitivity of early erythroid precursors to these stimulators could explained sustained erythropoiesis in PTE patients. The high BPA levels in anemic transplant patients with moderate chronic graft failure could be beneficial if rHuEpo treatment is applied in this patient group.

Factors inducing posttransplant erythrocytosis.

In this study factors possibly contributing to the development of erythrocytosis after renal transplantation (PTE) were analyzed. Out of 131 transplanted patients nine developed PTE (mean hemoglobin 17. 9 +/- 0.3 g/dl) 2 to 27 months after transplantation (group 1) and were compared to the nine with normal hemoglobin concentration (mean hemoglobin 12.4 +/- 0.2 g/dl, control group 2). The study was performed about two years after transplantation (25 +/- 3.9 months group 1 and 23.7 +/- 2.6 months group 2). Immunosuppressive therapy given in standard doses consisted of cyclosporine, azathioprine and prednisone. At the onset of the study no difference in renal graft function was noted between the groups (for group 1 sCr = 111.7 +/- 10.4 micromol/l and for group 2 sCr = 154.6 +/- 27.6 micromol/l). The mean serum immunoreactive erythropoietin (Epo) levels were significantly higher in PTE patients compared to control group of patients (33.9 +/- 4.6 mU/ml vs 21.6 +/- 2.5 mU/ml, p = 0.03). In addition, the ratio between observed to expected (O/E) Epo, a useful index in assessing Epo secretion in renal transplant patients, was ten times higher for group 1 than for group 2 (Median value 10.0 vs. 1.05). Spontaneous growth of Burst-forming unit- erythroid (BFU-E) in peripheral blood was detected in 5 out of 9 patients from group 1 and none in patients from group 2 (p = 0.04). Burst Promoting Activity (BPA) in Phytohemagglutinine Stimulated Leukocytes Condition Medium (PHA-LCM) from patients blood were higher in the PTE patients than in controls. Whole blood cyclosporine levels were higher in group 1 than in group 2 throughout the first 30 weeks after transplantation. It was concluded that sustained erythropoiesis after correction of renal anemia by kidney transplantation, leading to PTE could be explained as a consequence of increased levels of Epo and BPA and increased sensitivity of early erythroid progenitors to these stimulators induced by high cyclosporine levels.

In vivo effects of recombinant human erythropoietin on bone marrow hematopoiesis in patients with chronic renal failure.

Studies of hemopoietic progenitors and precursors in bone marrow before and after two months of recombinant human erythropoietin (rhEpo) therapy in 12 patients with uremic anemia are the subject of this investigation caried out in order to have a better insight into the effect of Epo in vivo. Eight patients were on hemodialysis and four others were predialysis patients with chronic renal failure. The starting dose of rhEpo was 30-50 U/kg bw and was increased by 50 percent every four weeks. The mean hemoglobin values rose from 6.08 +/- 1.03 to 9.8 +/- 1.98 g/dl at the time of study. The number of bone marrow derived erythroid colonies, both early (BFU-E) and late (CFU-E) were found to be higher than subnormal values, found before the therapy. The percentage of erythroid progenitors in cell cycle increased to higher than normal values for BFU-E and to normal values for CFU-E. At the same time granulocytic progenitors (CFU-GM) decreased to the range of normal values (67.3 per 10 superset5 cells). Slightly increased Epo levels (approx. 30mU/ml) during the replacement therapy were optimal for correction of anemia. The rhEpo therapy induced an increase of percentage of erythroblasts and the decrease of myeloid to erythroid ratio (M/E) in the bone marrow. Only in predialysis patients in whom the target hemoglobin values were achieved by rhEpo therapy at the time of the study the percentage of erythroblasts in the bone marrow increased to normal values. Increase of erythroblasts in bone marrow in patients under two months of substitutive therapy with rhEpo with the increase of both, early and late erythroid progenitors we have observed, is significant indicating the stimulative effect of rhEpo on all subsets of erythropoiesis leading to normalization of erythropoiesis at all levels. No stimulative effect of rhEpo replacement therapy on granulopoiesis was observed.