Plasma erythropoiesis inhibitors in humans during hyperbaric hyperoxia]

Before exposure of man to hyperbaric hyperoxia the blood plasma possessed marked erythropoietic activity. Erythropoietins disappeared completely and erythropoiesis inhibitors appeared in the plasma of persons untrained to hyperbaric hyperoxia after a 24-hour exposure to hyperoxic conditions (25% of oxygen in the inhaled air) in the high pressure chamber corresponding to the depth of 63 metres. A reduction of the peripheral red blood indices was observed by that time.     

In inherited porphyrias, lead intoxication is a toxogenetic disorder

1. delta-Aminolevulinic acid dehydratase (ALA-D), blood lead and several enzymes and metabolites of the heme biosynthetic pathway were measured in a number of symptomatic porphyric patients, 22 with acute intermittent porphyria, three with hereditary hepatic coproporphyria, 10 with hereditary porphyria cutanea tarda, two with erythropoietic protoporphyria and two with congenital erythropoietic porphyria and in 84 lead intoxicated persons. 2. In the 39 individuals suffering from the inherited porphyrias and in 32 lead poisoned patients with a 30-50% reduced deaminase, blood lead content was not sufficiently increased (average 28 micrograms%) to account for the greatly decreased activity of ALA-D (average 36% of controls). 3. After a relatively trifling lead exposure they developed the signs of acute lead intoxication. 4. A second group of lead intoxicated patients showing low ALA-D activity and corresponding high concentration of lead in blood, exhibited no other physiologic deviation in the enzymes and metabolites of porphyrin biosynthesis. 5. Individuals with inherited porphyrias are ultrasensitive to low level lead exposure and that lead would also act as a triggering factor. In these patients, lead intoxication can be considered a toxogenetic disorder. 6. An inversely linear correlation between ALA-D activity and blood lead content was obtained for both groups of lead intoxicated patients, however, a different constant (k) for each was obtained, which we have taken as a measure of lead toxogeneticity: k = 10 +/- 1 for lead intoxicated individuals with otherwise normal heme metabolism and k = 5 +/- 0.5 for lead intoxicated symptomatic porphyric patients. 7. Determination of erythrocytic ALA-D, besides blood lead, will be a valuable indicator for preventive medical care for these patients, when they are expected to be exposed to lead either environmentally or in their professional life.     

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.     

Effect of bleeding on irradiation-induced erythropoiesis in the spleen of AKR mice

The effect of erythropoietin, increased by bleeding, on the erythropoiesis induced by irradiation in the spleen of AKR mice, has been studied. The following parameters were measured to quantify the erythropoietic activity: the number and size of hematopoietic nodules (colonies) and proerythroblasts in the spleen, the spleen, blood and red-cell 59Fe uptake and the hematocrit and reticulocytes in the blood. Under erythropoietic stimulus an increase in the number and size of colonies was observed and these colonies were observed sooner because of their more rapid growth. The proerythroblasts in the spleen appeared earlier, and there were increases in the spleen, blood and red-cell 59Fe uptake and in the hematocrit and reticulocytes in the blood.     

Periodic changes in the organs involved in the erythropoiesis of anemic newts

In Triturus cristatus carnifex (Laurenti) newts rendered totally anemic by treatment with acetylphenylhydrazine (APH) diluted in their tank water (25 mg/liter for 48 hours, with four changes) the recovery of erythron occurs through periodic cycles of mitotic activity in the erythropoietic tissue. These cycles determine a marked increase in blood erythrocyte concentration at regular intervals of about 1 month. The consequence of this trend is the alternation of ferritin and hemosiderin accumulation phases during periods of stasis with iron mobilization phases during periods of erythropoietic activity, which is particularly evident in the Kupffer cells of the liver. Iron mobilization and erythropoietic activity are strictly related to the periodic hypertrophy of some Bowman's capsule cells in the renal corpuscle, which were previously denominated "lactate sensitive cells" (LSC). The histochemistry, location, and behavior of LSC indicate that they are probably the site of erythropoietin production in the newt.     

Isolation of an erythropoietic stimulating factor from the serum of anemic chicks

A factor stimulating fowl erythropoiesis in vitro has been isolated from the blood of anemic chick by gel filtration and ion exchange chromatography. This purification procedure yields a preparation whose specific activity is enriched 44 times and which has been relieved of toxic factors present in the crude anemic serum. In vitro erythropoiesis requires addition into the culture medium of erythropoietic stimulating factor and of another factor present in the serum of anemic as well as non-anemic chick. This last factor is lost during purification of the erythropoietic stimulating factor and is not present in fetal calf serum.     

Preliminary observation of blood cells and hematopoietic organs in Lutjanus herenbergi and Lutjanus flaviflammus

The morphology of differentiated and differentiating cells of the red and white series in Lutjanus herenbergi and in Lutjanus flaviflammus is described. Early stages of red and white blood cells may be found only in smears of hemopoietic organs. Polychromatic erythroblasts, myelocytes and lymphoblasts may also occasionally be found in blood smears. Mature blood cells may be found both in blood smears and in hemopoietic organs. Differential white cell counts seem to demonstrate that the granulocytic series elements are the most common leukocytes in blood smears. Almost all granulocytes may be classified in the first three Arneth classes. An analysis of hemopoietic organs in these species was also performed. It was found that the only organs carrying on a hemopoietic function are the kidney and the spleen. The kidney is essentially a site of granulocytic differentiation while the spleen is a lymphopoietic organ. An erythropoietic activity may generally be observed in the kidney although weak erythropoietic activity may at times be found in the spleen.     

Transient erythropoietic spleen colonies: effects of erythropoietin in normal and genetically anemic W/Wv mice.

Properties of the cells (TE-CFU) that give rise within four to six days to transient endogenous erythropoietic spleen colonies in irradiated mice have been investigated. The results obtained indicate that (1) erythropoietic maturation within such colonies is highly erythropoietin-dependent, (2) the population size of TE-CFU is not erythropoietin-dependent, (3) initial exposure to a high dose of erythropoietin followed by continuing exposure to lower doses is required for maximal efficiency of colony formation by TE-CFU, (4) successful transplantation of TE-CFU has not been achieved, but they appear among the progeny of transplanted hemopoietic cells, (5) TE-CFU are defective in mice of genotype W/Wv. These findings are consistent with the view that the TE-CFU assay detects a class of early erythropoietin-sensitive progenitor cells committed to erythropoietic diffferentiation, rather than "abortive" colony formation by pluripotent stem cells.     

Effect of acute hypoxia on the renin and erythropoietin activities of the blood serum in rats

A comparative study of renin and erythropoietin content in the blood serum of rats with "endocrine" kidneys and changes in their activity following the action of specific erythropoietic stimulus (4-hour hypoxia) has been conducted. The presence of "endocrine" kidneys increased renin and erythropoietin activity in such animals. Acute hypoxia produced further increase in erythropoietin titre in the blood serum, with renin remaining at the same level. Possible differences in the mechanisms of renin and erythropoietin biogenesis in the kidneys are considered.     

Uroporphyrinogen III cosynthetase in asymptomatic carriers of congenital erythropoietic porphyria

Mean activity of the enzyme uroporphyrinogen III cosynthetase in hemolysates from five asymptomatic carriers of bovine erythropoietic porphyria was intermediate to the means of the activities in normal controls and affected animals. Similarly, mean cosynthetase activity in hemolysates from eight presumed carriers of human congenital erythropoietic porphyria was lower than the mean for 38 nonporphyric individuals and higher than the activity in hemolysates from patients. These results are consistent with the known autosomal recessive mode of inheritance of the disorder in cattle and the presumed autosomal recessive inheritance in man. The activity of cosynthetase is higher in young red cells than in older ones, which explains the relatively high enzyme activities in blood from human patients with nonporphyric hemolytic disorders accompanied by reticulocytosis. It also explains why cosynthetase activity is higher in newborn porphyric calves than in older porphyric animals, because the former have a period of accelerated erythropoiesis and a high percentage of very young red cells in the circulation.Supported in part by U.S. Public Health Service Grants NB-05367 and AM-10858.     

Microcythemia from anemic hypoxia and normal erythropoiesis in the newt

Specimens of the newt, Triturus cristatus carnifex (Laurenti), rendered totally anemic, restore erythron by cyclic waves of erythropoietic activity that alternate with intervals of stasis. Hemolysis is obtained by administering 25 mg/liter of acetylphenylhydrazine in the breeding water for 36 h. The first cycle of erythropoietic activity produces microcytes, which have completely differentiated by 8 weeks after treatment. However, if the animals are raised in a hyperbaric chamber at a pressure of 1.5 atmospheres, in order to compensate for hypoxia, normocytes are produced. In both cases the hematocrit and hematic concentration of hemoglobin reach analogous values, so microcythemia appears to be the only effect of hypoxia. The hemoglobin, hematocrit values, and normocyte counts in hyperbaric animals are about one-half those of the controls newts. These data, together with those on the life span of red blood cells (RBC) and time span between two successive erythropoietic cycles (2 months and 1 month, respectively), indicate that the newts normally keep only two sets (one new, one old) of RBC in circulation, whose approximate parameters can be defined as RBC count: 60,000/mm3, hematocrit: 17%, and hemoglobin: 5.4 g/100 ml.     

Role of the spleen in the exaggerated polycythemic response to hypoxia in chronic mountain sickness in rats.

In a rat model of chronic mountain sickness, the excessive polycythemic response to hypoxic exposure is associated with profound splenic erythropoiesis. We studied the uptake and distribution of radioactive iron and red blood cell (RBC) morphology in intact and splenectomized rats over a 30-day hypoxic exposure. Retention of (59)Fe in the plasma was correlated with (59)Fe uptake by both spleen and marrow and the appearance of (59)Fe-labeled RBCs in the blood. (59)Fe uptake in both the spleen and the marrow paralleled the production of nucleated RBCs. Splenic (59)Fe uptake was approximately 10% of the total marrow uptake under normoxic conditions but increased to 60% of the total marrow uptake during hypoxic exposure. Peak splenic (59)Fe uptake and splenomegaly occurred at the most intense phase of erythropoiesis and coincided with the rapid appearance of (59)Fe-labeled RBCs in the blood. The bone marrow remains the most important erythropoietic organ under both resting and stimulated states, but inordinate splenic erythropoiesis in this rat strain accounts in large measure for the excessive polycythemia during the development of chronic mountain sickness in chronic hypoxia.     

Effect of liposomal muramyl tripeptide phosphatidylethanolamine and indomethacin on hematopoietic recovery in irradiated mice

The effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV, radioprotective immunomodulator; 10 mg/kg) and indomethacin (INDO, inhibitor of prostaglandin production; 2 mg/kg) on post-irradiation recovery of hematopoietic functions in mice were investigated. Two agents with distinct radioprotective mechanisms were administered alone or in combination 24 h and 3 h before exposure to 7 Gy (60)Co radiation. In the post-irradiation period (3-14 days) combined pre-treatment of mice accelerated recovery of bone marrow cellularity, weight of spleen and myelopoietic and erythropoietic activity in both hematopoietic organs, compared to treatment with MTP-PE/MLV or indomethacin alone. In the peripheral blood, improved radioprotective effects of combined drug administration were found in the recovery of reticulocytes and platelet count. No further significant differences in the recovery of leukocyte count were observed in the examined groups until post-irradiation day 14. Within the first 3-6 post-irradiation days, the bone marrow and peripheral blood smears of mice pre-treated with indomethacin alone or its combination with MTP-PE/MLV more frequently featured blast cells and large cells with abundant cytoplasm which could be considered the hematopoietic stem cells.     

The cellular composition of the blood and haematopoietic organs of turbot Scophthalmus maximus L.

The cellular composition of the blood, anterior kidney, spleen and thymus of turbot Scophrhalmus maximus L., aged 1 + was determined. Ninety-four per cent of blood cells belonged to the erythrocyte lineage of which 82% were mature erythrocytes. The leucocytes, which represented 4.5% of the blood cells, were mainly lymphocytes (50%). The presence of crythroblasts in the anterior kidney and the spleen demonstrated an erythropoietic activity in both organs. However, this activity appeared to be prevalent in the spleen which also appeared to act as a storage zone for erythrocytes and as the centre point for thrombopoiesis. Although 96% of the anterior kidney cells were leucocytes, the number of white cells per gram of organ was higher in the spleen.     

Trypanosoma lewisi: Immunohematopoietic interrelationships of the infection in normal,hypoxic, and rebound animals

A study has been made of erythropoietic and immune responses in rats subjected to a simulated altitude of 17,000 ft and infected with Trypanosoma lewisi. Observations were also made during the period of rebound (posthypoxic polycythaemia). The erythropoietic response was evaluated on the basis of reticulocyte and hematocrit values in peripheral blood, and the immune response on the basis of the level of parasitemia. When hypoxia and infection were combined, the infection was heavier than in the nonhypoxic animal, as judged by the level of the parasitemia, whereas the erythropoietic response to hypoxia was less intense. Rebound animals, on the other hand, dealt with the infection more effectively, as indicated by the lower level of parasitemia, and the more rapid disappearance of parasites from the blood.     

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 erythropoiesis due to radiation or chemotherapy as studied by flow cytometric determination of peripheral blood reticulocytes

Flow cytometric determination of time dependent changes of numbers of reticulocytes in peripheral blood were investigated as a parameter for changes in erythropoiesis induced by radiation- or chemotherapy. Rats irradiated or treated with drugs (such as e.g. cyclophosphamide 100 mg/kg, vincristin 0.2 mg/kg, or mitomycin C 1.0 mg/kg) showed clear changes in erythropoietic activity. Reticulocyte numbers decreased rapidly until day 3-4 after treatment; this period was followed by a gradual increase and normal control values were seen at day 8-11. Radiation effects of doses as low 0.5 Gy could be detected in such a way. Similar studies were performed with patients with ovarian tumors treated with cis-platinum, a drug that may cause non-immune haemolysis. During prolonged treatment some patients showed increasing numbers of reticulocytes, measured at the first day of each hospitalization period, whereas leucocyte and platelet counts stayed more or less constant. Increasing numbers of reticulocytes generally indicates stimulation of erythropoietic activity of the bone marrow (due to increased blood loss); in this study increasing numbers often preceeded a decrease in hemoglobin values later on. Flow cytometric analysis of reticulocytes is therefore a potentially useful tool to detect changes in erythropoiesis, and considered more sensitive for the early recognition of patients that develop anemia, than hemoglobin measurements only.     

Antibody formation and B-cell migration from bone marrow to spleen in mice under conditions of stimulation and suppression of erythropoiesis]

The effect of erythropoietic stimulation and suppression on the production of antibody-forming cells (AFC) in the spleen and on the B-cell migration from the bone marrow to the spleen was investigated in the CBA mice. Erythropoiesis stimulation proved to sharply increase the AFC count in the spleen and the B-cell migration from the bone marrow to the spleen 1 and 4 days after the bleeding. Erythropoiesis suppression resulted in a slight increase of the AFC count in the spleen 4 and 7 days after the transfusion of syngeneic red blood cells. However, the erythropoietic suppression inhibited the B-cell migration from the bone marrow to the spleen. Possible mechanisms of the effect of the erythropoietic stimulation and suppression on the antibody production are discussed.     

Selective modulation of the erythropoietic and tissue-protective effects of erythropoietin: time to reach the full therapeutic potential of erythropoietin

Erythropoietin (EPO) has been used clinically both as an erythropoietic stimulating agent in the treatment of anemia and as a tissue-protective agent in diverse clinical settings including stroke, multiple sclerosis, acute myocardial infarction and others. However, use of EPO or EPO-analogues leads to simultaneous targeting of both the erythropoietic and tissue-protective properties of EPO, and this strategy has been associated with several problems. Specifically, the benefit of correction of cancer-related anemia can be offset by the tissue-protective effects of EPO, which may lead to stimulation of cancer cell proliferation. Conversely, the benefit of tissue-protection in patients with stroke or myocardial infarction can be offset by adverse effects associated with the erythropoietic effects of EPO such as elevation of red blood cell mass, hypertension and prothrombotic phenomena. The finding that the erythropoietic and tissue-protective properties of EPO are conferred via two distinct receptor systems raises the interesting possibility of discovering novel drugs that selectively stimulate either the erythropoietic or the tissue-protective activities of EPO. This article reviews the current status of the clinical use of EPO and EPO-analogues in the treatment of cancer-related anemia and for tissue protection, outlines the distinct molecular biology of the tissue-protective and erythropoietic effects of EPO and discusses strategies of selective targeting of these activities with the goal of exploiting the full therapeutic potential of EPO.     

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.