bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of testosterone and erythropoietin on erythroid colony formation in human bone marrow cultures
Authors: Moriyama Y , Fisher JW
Published: October 2019

The effects of testosterone and erythropoietin (ESF) on erythroid colony formation in normal human bone marrow cultures were studied in vitro using a methyl cellulose gel system. Testosterone was found to produce a significant increase in erythroid colony formation at concentrations of 10–4–10-4M in vitro. In this system, the numbers of erythroid colonies formed per plate increased in direct proportion to the increase in the number of erythroid precursors inoculated as well as to the increase in the dose of ESF in vitro. In addition, a synergistic effect of a combination of testosterone and ESF on erythroid colony formation was seen when ESF was present at high concentrations. These data suggest that a greater number of erythropoietin-responsive cells are available for ESF to differentiate into the nucleated erythroid cell line in the presence of testosterone, indicating that the effect of a combination of testosterone and ESF is greater in enhancing erythropoiesis than the additive effects of either agent alone.
Direct effects of thyroid hormones on bone marrow erythroid cells of rats
Authors: Malgor LA , Blanc CC , Klainer E , Irizar SE , Torales PR , Barrios L
Published: October 2019

A stimulatory effect on bone marrow cellularity was observed in normal and nephrectomized rats continuously infused with T3 and T4. Results of bone marrow studies are expressed in absolute numbers of total nucleated erythroid cells per milligram of femoral marrow at the beginning and after 8 hr of continuous intravenous infusions. Administration of T3 and T4 to nephrectomized rats produced a marked and significant increase in total erythroid cells counted. After differential analyses of the nucleated erythroid elements, a significant increase in all erythroid cell types was also observed. Similar results were seen in a control group of rats in which both ureters have been previously ligated and in groups of nephrectomized rats receiving rabbit antiserum against erythropoietin before starting the intravenous infusions of T3 and T4. These results indicate that stimulation of marrow erythropoiesis produced by thyroid hormones in our system is not dependent on renal or extra-renal production of erythropoietin. The progressive introduction of T3 and T4 into the circulation of rats with bilateral nephrectomy or ureter-ligated normal rats, may overload the mechanism of transport of these hormones in plasma. As a consequence, a progressive increase in free active forms of T3 and T4 in plasma may occur. Our interpretation of the present findings is that thyroid hormones stimulate directly bone marrow erythropoiesis. This stimulation is clearly evident when high levels of free active forms of thyroid hormones are present in plasma.
Metabolic effects of antisickling amounts of nitrogen and nor-nitrogen mustard on rabbit and human erythrocytes
Authors: Roth EF Jr , Nagel RL , Neuman G , Vanderhoff G , Kaplan BH , Jaffe ER
Published: October 2019

Nitrogen mustard (NH2) and Nor-nitrogen mustard (Nor-HN2) both inhibit the polymerization of deoxyhemoglobin S in solution and in intact erythrocytes. Metabolic studies were undertaken to determine the feasability of an extracorporeal treatment with these or related agents. Glucose utilization, hexose monophosphate shunt activity, methemoglobin reduction, and incubation with acetylphenylhydrazine for Heinz body formation were performed, as well as specific assays for hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glutathione reductase, ATP, reduced glutathione (GSH), and survival of autologous mustard-treated cells in rabbits. HN2 was found to enter red cells rapidly and bind to intracellular contents. Metabolic studies revealed no significant inhibition or alteration of function by Nor-HN2 at 10 mg/ml of whole blood. Rabbit red cell survival was also normal. HN2, however, inhibited glutathione reductase and blocked the free sulfhydryl group of GSH by forming serveral addition products of alkylated GSH. Heinz body test with acetylphenylhydrazine became positive in HN2-treated cells, and rabbit red cell survival was shortened considerably in the concentration range used to inhibit sickling. Ascorbic acid stimulation of the hexose shunt pathway was inhibited by HN2, but methylene blue stimulation remained unaffected. 14-C-HN2 remains bound to red cells in vivo, and the disappearance of radioactivity is similar to that found with 14-C-DFP (disopropylfluorophosphate). Oxygen affinity of both HN2 and Nor-HN2 treated human red cells remains virtually the same as that found in control samples. It is concluded that Nor-HN2 may be a suitable agent for an extracorporeal therapy, and that each mustard needs to be evaluated individually for its antisickling effects and its suitability for extracorporeal use.
Defective mononuclear leukocyte chemotaxis in the Chediak-Higashi syndrome of humans, mink, and cattle
Authors: Gallin JI , Klimerman JA , Padgett GA , Wolff SM
Published: October 2019

Chemotaxis of mononuclear leukocytes from humans, mink, and cattle was evaluated in vitro using a morphologic Boyden chamber technique and a new 51-Cr-labeled mononuclear radioassay with a double micropore filter system. Significantly decreased mononuclear leukocyte chemotactic response were noted when human, mink, or cattle Chediak-Higashi cells were tested using autologous serum or endotoxin-activated autolotous serum. A similar Chediak-Higashi mononuclear leukocyte defect was noted in humans when kallikrein or dialyzable transfer factor were used as the chemotactic stimulus. Studies using smaller pore filters in the chemotactic chamber exaggerated the chemotactic defect. Serum from Chediak-Higashi subjects had normal chemotactic activity. Additional studies on the spontaneous (random) locomotion of Chediak-Higashi mononuclear leukocytes revealed normal results when a capillary tube assay system was used, but abnormal results were obtained when a Boyden chamber micropore filter assay was used, demonstrating fundamental differences in these two assays of random locomotion. It is clear from these studies that defective mononuclear leukocyte chemotaxis is another feature of the imparied host defenses in the Chediak-Higashi syndrome that may contribute to the marked susceptibility to pyogenic infections so characteristic of this dease.
The effect of Hydrocortisone on the kinetics of normal human lymphocytes
Authors: Fauci AS , Dale DC
Published: October 2019

Lymphocyte kinetic studies employing 51-chromium-labeled autologous lymphocytes were performed in nine normal volunteers in order to determine the effects of hydrocortisone administration on the recirculating versus the nonrecirculating intravascular lymphocyte pools. Following infusion of labeled cells, the recirculating portion of the labeled cells rapidly equilibrated with the total intravascular lymphocyte pool and the vastly larger total-body recirculating lymphocyte pool, so that by 1 hr following infusion 21.8% plus or minus 3.2% of the labeled lymphocytes were left in the circulation. Four hundred milligrams of intravenous hydrocortisone administered 24 hr after infusion of labeled cells caused a profound but transient lymphocytopenia which was maximal at 4 hr with return of lymphocyte counts to normal by 24 hr after injection. Concomitant with the lymphocytopenia there was a dramatic increase in lymphocyte specific activity (cpm per 10–6 lymphocytes), while the total lymphocyte- associated radioactivity remaining in the circulation was unchanged, indicating that corticosteroid administration depleted the unlabeled recirculating cells. As the lymphocyte counts returned to normal following hydrocortisone, the specific activity also returned to normal. These studies indicated that hydrocortisone administration caused a transient lymphocytopenia by a preferential depletion of the recirculating portion of the intravascular lymphocyte pool.
Energy metabolism in human erythrocytes: the role of phosphoglycerate kinase in cation transport
Authors: Segel GB , Feig SA , Glader BE , Muller A , Dutcher P , Nathan DG
Published: October 2019

Three models of disturbed erythrocyte metabolism, triose-depleted normal, phosphoglycerate kinase (PGK)-deficient, and pyruvate kinase (PK)-deficient cells, have been studied to examine further the role of PGK in erythrocyte cation transport. Sodium (Na-+) and potassium (K-+) transport were reduced only in cells fully depleted of triose. In such cells the PGK step presumably was inoperative due to total lack of substrate; 2,3-diphosphoglycerate (2,3-DPG) then became the sole substrate source for remaining steps in glycolysis. At increased intracellular Na-+ concentrations which normally stimulate transport and glycolysis, triose-depleted cells had marked impairment of cation transport and ouabain-inhibitable lactate and pyruvate production from 2,3-DPG. PGK-deficient cells and normal cells with high intracellular Na-+ concentrations had similar increases in transport and ouabain- inhibitable lactate production. PK-deficient cells with high intracellular Na-+ concentrations showed an appropriate increase in transport but less stimulation of lactate production. Transport was not related to total cellular adenosine triphosphate (ATP) concentration. These data suggested that normal coupled cation transport occurred despite diminished metabolite flow through PGK, as in PGK- or PK- deficient cells. Transport was diminished only in triose-depleted cells where metabolite flow through PGK was presumably absent. These data, therefore, support the concept that transport and glycolysis interact at the PGK step, although impairment of PGK must be profound before its effect on transport is eviden.
The effects of ATP on platelets: evidence against the central role of released ADP in primary aggregation
Authors: Macfarlane DE , Mills DC
Published: October 2019

The influence of freshly purified ATP on the effects of aggregating agents on human platelets was studied. ATP inhibited aggregation induced by ADP competitively (Ki = 20 muM) and immediately without need for prior incubation. ATP had no effect on primary aggregation induced by adrenaline, thrombin, vasopressin, or 5-hydroxytryptamine (5HT). ATP inhibited the shape change and the consumption of metabolic ATP induced by ADP but did not inhibit these effects when induced by thrombin, vasopressin, or 5HT. ATP counteracted the inhibition by ADP of PGE1- stimulated cyclic AMP production in platelets but did not reduce inhibition by adrenaline. It is concluded that adrenaline, thrombin, 5HT, and vasopressin each can induce primary aggregation of human platelets by a mechanism independent of extracellular ADP.
The resistance of fibrinogen and soluble fibrin monomer in blood to degradation by a potent plasminogen activator derived from cadaver limbs
Authors: Gurewich V , Hyde E , Lipinski B
Published: October 2019

The effect of a cadaver-derived vascular plasminogen activator (VA) on the degradation of fibrinogen, soluble fibrin monomer, and fibrin was studied and compared with the effect of equivalent fibrinolytic potencies of streptokinase (SK), urokinase (UK), and plasmin. The proteolytic activity of the three activators and plasmin was determined by a standard fibrin plate assay and was expressed in CTA units from a UK reference curve. Fibrinogen degradation was measured by clottable protein determinations and by an electrophoretic technique sensitive to small changes in the molecular weight of fibrinogen. When VA was incubated in plasma, no degradation of fibrinogen occurred, whereas rapid fibrinolysis took place after the plasma was clotted. By contrast, equivalent potencies of SK, UK, and plasmin caused extensive fibrinogenolysis. Since the plasmin added and that formed by the three activators had equivalent fibrinolytic activity, the failure of VA to induce fibrinogen degradation was attributed to antiactivators rather than antiplasmins. VA activity in plasma was consumed by clotting, whereas the antiactivator activity remained in the serum, suggesting dissociation of the VA-antiactivator complex on the fibrin clot. Fibrinogen and its soluble derivatives resisted degradation by VA in plasma because a solid phase appeared necessary for the complex to dissociate. The findings indicated that the degradation of fibrinogen or soluble fibrin in blood as a result of plasminogen activation by VA was unlikely to occur due to a large excess of antiactivator activity. Alternative pathways for their catabolism are discussed.
Iron metabolism, sickle cell disease, and response to cyanate
Authors: Peterson CM , Graziano JH , de Ciutiis A , Grady RW , Cerami A , Worwood M , Jacobs A
Published: October 2019

In an attempt to understand the variability of the hematologic response to oral sodium cyanate, iron metabolism was studied in a group of 39 patients with sickel cell disease. Eleven of the 39 patients were found to have no stainable iron in the marrow despite the fact that patients with sickle cell disease are generally considered to have hemosiderosis. The mean per cent saturation and total iron-binding capacity were in the low-normal range in sickle cell patients whether or not stainable iron was present in the bone marrow aspirate. Serum ferritin concentrations, on the other hand, were found to be high in both groups (greater than 500 mu g/liter) when compared to controls (60 mu g/liter). The high serum ferritin levels denoted significant total- body iron deposition which may be unavailable for normal metabolic processes. One patient with no stainable iron in the bone marrow aspirate did respond to iron therapy alone with an increase in hemoglobin concentration. Serum ceruloplasmin levels were also found to be high in sickle cell disease patients. The ability to respond to oral cyanate therapy was correlated with the amount of stainable iron in the bone marrow aspirate. These studies emphasize the necessity of a reevaluation of iron metabolism in the pathophysiology and treatment of sickle cell disease.
Glucose 6-phosphate dehydrogenase deficiency and sickle cell anemia: frequency and features of the association in an African community
Authors: Bienzle U , Sodeinde O , Effiong CE , Luzzatto L
Published: October 2019

The glucose 6-phosphate dehydrogenase (G6PD) genotype was determined in 100 male patients with homozygous sickle cell anemia (SS) by a combination of quantitative assay, cytochemical testing, and starch-gel electrophoresis. Of the 100 patients tested, 16 were found to be G6PD deficient (GdA-), AND 84 G6PD normal (22GsA and 62 GdB). This distribution of G6PD genotypes did not differ significantly from that observed in the general population. The level of G6PD activity in GdA- SS patients was nearly always higher than in G6PD-deficient subjects who did not have an associated hemolytic state, but it was nearly always lower than in G6PD-normal subjects. The clinical course of sickle cell disease, including the degree of anemia, was not milder in GdA- than in G6PD-normal patients but could not be proved to be significantly more severe. It was concluded that in this community the incidence of G6PD deficiency in sickle cell anemia was not greater than would be expected by chance, and there was no evidence that the coexistence of the GdA- gene in SS patients ameliorated their disease.
Limitation of splenic growth as studied by heterotopic splenic implants
Authors: Tavassoli M
Published: October 2019

Multiple spleens were implanted in the subcutaneous tissue or the peritoneal cavity of inbred rats which had undergone splenectomy. The total weight of regenerated implants was compared to the weight of implanted tissue. The weight of recovered tissue was not a function of implanted spleen mass; rather, it approximated the unit ratio of the animal's own spleen. Those animals receiving more than ten spleens died by days 3–6 postimplantation, probably as a result of massive necrosis that occurred during the process of regeneration. When implants were made in a temporal sequence of two spleens every 3 days, the first set was uniformly successful, whereas regeneration was aborted in subsequent sets of implants. These observations are consistent with the conclusion that certain factors control the total mass of spleen in any one animal.
Osmotic behavior of normal and leukemic lymphocytes
Authors: Ben-Sasson S , Shaviv R , Bentwich Z , Slavin S , Doljanski F
Published: October 2019

The response of normal human peripheral blood lymphocytes to a hypotonic environment may be divided into two phases: the cells first exhibit rapid osmotic swelling, followed by a slower shrinking phase, during which they regain their initial physiologic volume. This osmotic behavior is characteristic of most mammalian and avian nucleated cells so far examined. The normal human blood lymphocyte, however, shows the most rapid recovery phase (5 min). Lymphocytes from chronic lymphatic leukemia patients, in comparison, show a strikingly slower rate of return to their initial isotonic volumes. The mechanism underlying osmotic cell volume regulation and its significance are discussed.
Granule enzymes of polymorphonuclear neutrophils: A phylogenetic comparison
Authors: Rausch PG , Moore TG
Published: October 2019

The functional significance of granule enzymes in polymorphonuclear leukocytes (PMN) is not fully understood because of the multiplicity of the enzymes and the rare occurrence of deficiencies in man. In order to select appropriate laboratory animals for functional studies, a phylogenetic comparison of enzyme levels in animal and human PMN was undertaken. Neutrophils were obtained from a variety of laboratory animals and man; the activities of alkaline phosphatase, lysozyme, myeloperoxidase, and beta-glucuronidase were determined by histochemical and analytical techniques. Marked interspecies differences in enzyme activity were found; many species were deficient in alkaline phosphatase or lysozyme. Differences in pH optima and metal requirements of alkaline phosphatase were not of sufficient magnitude to explain the variations of this enzyme.
Growth stimulation of human bone marrow cells in agar culture by vascular cells
Authors: Knudtzon S , Mortensen BT
Published: October 2019

Human vascular cells are capable of stimulating granulopoiesis in agar culture of human bone marrow cells. This effect was obtained by including vein fragments in the culture or by using endothelial cells separated from the vein of human umbilical cords as feeder cells. Furthermore, the stimulatory capacity of conditioned medium obtained from cord veins was found to be highly active in comparison to that obtained from peripheral leukocytes. Endothelial cells within the bond marrow cavity are suggested as a local source of factors regulating granulopoiesis in humans in addition to the monocyte.
Micropinocytosis of transferrin by developing red cells: an electron- microscopic study utilizing ferritin-conjugated transferrin and ferritin-conjugated antibodies to transferrin
Authors: Sullivan AL , Grasso JA , Weintraub LR
Published: October 2019

Electron-microscopic examination of rat reticulocytes and normoblasts incubated with transferrin conjugated to ferritin or ferritin-labeled antitransferrin revealed binding of ferritin conjugates to the surface membrane, and uptake of ferritin conjugates in micropinocytotic vesicles. No binding or endocytosis of ferritin was visualized when rat reticulocytes or normoblasts were incubated with ferritin alone or ferritin conjugated to nonspecific rabbit IgG. These observations support the concept that transferrin binds to a surface membrane receptor and is subsequently internalized by the developing red cell. Time course and temperature dependence studies suggest the endocytosis of transferrin may be an important mechanism in delivery of iron to the developing red cell.
The binding of human and bovine thrombin to human platelets
Authors: Shuman MA , Tollefsen DM , Majerus PW
Published: October 2019

Human thrombin binds to specific receptors on the surface of human platelets in a manner analogous to bovine thrombin. Thus, two classes of binding are observed--high affinity with a dissociation constant (Kdiss) of 0.02 U/ml and low affinity with a Kdiss of 5 U/ml. Bovine and human thrombin bind to the same platelet receptors, although bovine thrombin binds with slightly greater affinity. When the amount of thrombin bound to platelets is related to the extent of 14C-serotonin release, bovine and human thrombin are equally effective. Antibodies to human and bovine thrombin were found to differ markedly in their ability to precipitate thrombin of the two species. Thus, antibovine thrombin precipitated eightfold more bovine thrombin than human thrombin, while antihuman thrombin precipitated tenfold more human thrombin than bovine thrombin. Similar differences were found in the ability of Fab fragments of these antibodies to block the interaction of thrombin of each species with human platelets. The finding that both species of thrombin, despite significant evolutionary differences in primary structure, retain essentially identical binding sites to platelets suggests that this part of the thrombin molecule is physiologically important and supports our hypothesis of a role for thrombin binding to platelets in platelet function and hemostasis.
A new method for studying splenic reticuloendothelial dysfunction in sickle cell disease patients and its clinical application: a brief report
Authors: Casper JT , Koethe S , Rodey GE , Thatcher LG
Published: October 2019

Differential interference contrast (DIC) microscopy (Nomarsky optics) readily demonstrates the formation of “pits” or crater-like depressions in red cell membranes of splenectomized individuals. Splenic reticuloendothelial dysfunction characteristic of many patients with sickle cell disease (SCD) can be demonstrated by technetium spleen scans, but this technique is expensive, requires injection of radioactive material into children, and is cumbersome to perform at regular intervals. However, pit formation in red cells, which also appears to reflect splenic dysfunction, can readily be quantitated in a finger-stick blood sample using DIC microscopy. In this study, the degree of red cell pitting was compared with results of technetium spleen scans and measurements of Howell-Jolly bodies in individuals with sickle cell disease. The average pitted cell percentage in the control population was 0.5% +/- 0.5 (range 0.0–2.6) and 30.5% +/- 13.9 in the SCD population (range 2.4-71.1) (less than 0.001). Of the individuals studied with SCD, 12 also had technetium (99mTc) sulfur colloid scans and measurements of Howell-Jolly bodies. The percentage of Howell-Jolly bodies was low and did not correlate well with the degree of splenic visualization. However, there was an excellent correlation between pit count and splenic dysfunction as measured by spleen scan. Determination of red cell pitting, therefore, appears to offer a simple means for clinical evaluation of splenic reticuloendothelial function in patients with SCD.
Preleukemia and leukemia in polycythemia vera
Authors: Meytes D , Katz D , Ramot B
Published: October 2019

A group of 76 polycythemia vera patients was followed prospectively with bone marrow examinations at regular intervals. Six terminated in acute leukemia, preceded by an indolent preleukemic phase succeeded by a very fulminant leukemia. At autopsy, all patients had extramedullary hematopoiesis. In one of them, only the bone marrow was leukemic, suggesting that it was the primary site of this transformation. In three with leukemic involvement of nonhematopoietic organs, the infiltrate consisted of myelo- or myelomonoblasts and normoblasts. The preleukemic phase was characterized by a rapidly evolving spontaneous pancytopenia, with a drop from polycythemic blood counts within 2 mo. In the bone marrow, megaloblastoid erythropoiesis with ring sideroblasts was prominent. A similar preleukemic phase evolving in other potentially leukemic conditions, has been described. Therefore, in patients at risk, the appearance of ring sideroblasts may be regarded as an early indicator of imminent acute leukemic transformation.
Platelet release abnormality associated with a variant of von Willebrand's disease
Authors: Dowling SV , Muntz RH , D'Souza S , Ekert H
Published: October 2019

A family with a platelet release abnormality (PRA) is described. The only son also showed a reduced rate of platelet aggregation in response to ristocetin, markedly reduced levels of von Willebrand's factor (vWf, ristocetin cofactor), and increased mobility of factor VIII-like antigen, features which were suggestive of von Willebrand's disease (vWd). No inhibition of vWf was found in his plasma. Family studies showed no evidence of vWd in the mother. The father's investigations showed a low rate of ristocetin aggregation on one of the two occasions when it was tested and low vWf on two of four occasions. Despite repeated testing, the findings in the father did not conclusively rule out the possibility of mild vWd, and it was impossible to determine whether the vWd in the son was inherited or arose as a mutation. The findings in this family suggest a possible relationship between abnormalities of the factor VIII complex and defective platelet function.
Human bone marrow lymphocytes: B and T cell precursors and subpopulations
Authors: Abdou NL , Alavi JB , Abdou NI
Published: October 2019

Characterization of the different lymphocyte populations in normal human bone marrow (BM) was attempted and compared to that in the peripheral blood (PB). B cells comprised 34% +/- 11% of lymphocytes in BM and 23% +/- 9% in PB. The majority of B cells carried IgM in BM and IgG in the PB. In the BM, cells carrying complement or Fc receptors were fewer than cells carrying Ig, but in the PB they were equal. T cells comprised 6% +/- 4% of lymphocytes in the BM and 62% +/- 7% in the PB. The majority of BM lymphocytes did not have B or T cell markers; these probably included B and T cell precursors. BM lymphocytes carrying surface Ig increased in a 7-day culture, whereas those of the PB decreased. Pokeweed mitogen induced Ig synthesis in B cells of PB but not those of BM. BM-T cells were more efficient than PB- T cells in inhibiting Ig synthesis of PB-B cells. These results indicate that the BM compartment contains immature B cells that are capable of partial differentiation and maturation in vitro. BM-B lymphocytes are probably not involved in the effector phase of the immune response since they are unable to synthesize Ig and because they carry few receptors for complement of Fc, BM-T lymphocytes are very few and have suppressor capability and therefore may play an essential role in regulation of Ig synthesis by B cells.
Lipid membrane peroxidation in beta-thalassemia major
Authors: Rachmilewitz EA , Shohet SB , Lubin BH
Published: October 2019

The composition of membrane lipids was studied in 17 splenectomized and eight unsplenectomized patients with beta-thalassemia major and compared to normal controls. The results showed a nearly twofold increase in total cell lipids; a reduction in the percentage, but not the absolute amount of phosphatidylethanolamine, and a corresponding increase in phosphatidylcholine in the lipids; a considerable increase in the percentage of the saturated fatty acid, palmitic acid, and a reciprocal decrease in the polyunsaturated fatty acid, arachidonic acid; a twofold increase in the amount of malonyldialdehyde (MDA) generated after peroxide threat to the RBC when calculated either per gram hemoglobin or per cell; no change in the amount of MDA generated when calculated per microgram of membrane phosphorus at risk per cell; and a considerable decrease in serum alpha-tocopherol (vitamin E) levels. Thalassemic erythrocytes contain more lipid per cell which is susceptible to peroxidation. In addition, the distribution of fatty acids in these cells suggests that autooxidation of that lipid may have occurred. Autooxidation may be initiated by free radicals, which are constantly formed in the normal red cell, and may be especially prevalent when unstable hemoglobins are present. The low MCHC or some other intracellular defect of thalassemic cells may allow such potent oxidants to find their way to the cell membrane. Vitamin E, a biologic antioxidant is decreased in these patients, and clinical supplementation may be indicated to prevent some of the membrane damage in thalassemia.
Activation of the coagulation system in polycythemia vera
Authors: Carvalho A , Ellman L
Published: October 2019

Thrombosis is one of the major complications of polycythemia vera. Seventeen patients with polycythemia vera in good hematologic control were evaluated for abnormalities of the coagulation system. Activation of the intrinsic coagulation cascade was suggested by low levels of factor XII, prekallikrein, and kallikrein inhibitors in 12 of 17 patients. The group also demonstrated a significant increase in soluble fibrin complexes using plasma gel filtration on 4% agarose. Fibrin degradation products were normal and antithrombin III levels were slightly elevated. It appears that patients with polycythemia vera have chronic activation of the coagulation system, probably initiated by activation of factor XII. No correlation between the degree of coagulation abnormalities and thromboembolic complications was evident in this group of patients.
Hemoglobin pyrgos alpha2 beta2 83 (EF7) Gly leads to Asp: a new hemoglobin variant in double heterozygosity with hemoglobin S
Authors: Tatsis B , Sofroniadou K , Stergiopoulos CI
Published: October 2019

An electrophoretically fast-moving hemoglobin variant was found to be present together with hemoglobin S, in the hemolysate of the rythrocytes of at 3-yr-old Greek boy. Electrophoresis of the parents' erythrocyte hemolysates revealed that the father was an AS heterozygote, while the mother was a carrier of the variant hemoglobin. A sibling was also found to be a carrier. The amount of the mutant hemoglobin in the peripheral blood of the propositus, his mother, and his brother was 62.2%, 52.5%, and 51.1%, respectively, as determined by column chromatography. The patients peripheral blood smear showed mild anisocytosis, microcytosis, and hypochromia. Similar but less pronounced red cell abnormalities were found in the other two carriers. Structural analysis of the variant hemoglobin revealed substitution of an aspartic acid for the glycine residue at the beta83 (EF7) position. This new hemoglobin was named hemoglobin Pyrgos. All the carriers of hemoglobin Pyrgos are clinically healthy, and there seems to be no interaction between hemoglobin Pyrgos and hemoglobin S as manifested clinically.
Colony-stimulating factor in patients with chronic neutropenia
Authors: Wewerka JR , Dale DC
Published: October 2019

Urinary and serum colony-stimulating factor (CSF) levels were measured in 11 patients with chronic idiopathic neutropenia without infections and in 10 normal individuals. Urinary CSF output was determined using mouse marrow target cells, and serum CSF activity was assayed with human marrow target cells by the double agar layer technique. Using these methods, there was no significant difference between CSF levels of neutropenic and normal subjects. These data indicate that CSF levels are not inversely related to the blood neutrophil count in chronic idiopathic neutropenia and suggest that CSF is not a hormone regulating the blood neutrophil count in a manner analogous to the erythropoietin regulation of circulating erythrocyte levels.
Experimental Pseudomonas pneumonia in leukopenic dogs: comparison of therapy with antibiotics and granulocyte transfusions
Authors: Dale DC , Reynolds HY , Pennington JE , Elin RJ , Herzig GP
Published: October 2019

Pseudomonas aeruginosa pneumonia was produced in dogs with radiation- induced leukopenia to study the comparative efficacy of several different therapies. In a randomized control trial, five treatment regimens were compared: no antibiotics or granulocytes (controls), gentamicin (5 mg/kg/day), carbenicillin (500 mg/kg/day), gentamicin and carbenicillin (same dosages), and daily granulocyte transfusion (minimum 5 x 10(9) cells/day) plus gentamicin (5 mg/kg/day). The most effective therapy was gentamicin plus granulocyte transfusions. Gentamicin alone was not significantly better than no specific therapy. Carbenicillin with or without gentamicin gave intermediate results. This study further supports the utility of granulocyte replacement therapy of infections in severely granulocytopenic subjects. The results also indicate that the relative value of granulocyte transfusions depends upon the specific antibiotic regimen with which these transfusions are compared.
Assessment of erythrocytic and granulocytic colony formation in an in vivo plasma clot diffusion chamber culture system
Authors: Steinberg HN , Handler ES , Handler EE
Published: October 2019

Normal rat bone marrow cells seeded into a plasma clot diffusion chamber culture developed into erythrocytic and granulocytic colonies in vivo. Chambers implanted into the peritoneal cavity of normal hosts showed erythrocytic colony numbers reaching an initial peak on day 2, declining on days 3--5, and increasing in a secondary growth phase on day 7. Day 2 colonies were evenly dispersed; day 7 colonies were grouped into discrete areas of bursts. Granulocytic colony numbers reached a peak on day 4 and gradually declined through day 7. Cells in various stages of differentiation could be detected in both colony types. Colony numbers were proportional to the number of marrow cells seeded into the chamber. Host animals treated with phenylhydrazine induced a marked increase in erythrocytic colony numbers and size and a decrease in granulocytic colony formation. Host animals treated with endotoxin suppressed erythrocytic colonies while increasing granulocytic colony size. This method may prove advantageous for the study of hematopoietic colony formation in a physiologic environment.
Decreased life span and membrane damage of carbamylated erythrocytes in vitro
Authors: Lane TA , Burka ER
Published: October 2019

Red blood cells exposed to cyanate (CNO) in vitro have a concentration- dependent decreased cell survival time associated with an inhibition of the ability of the cell membrane to synthesize lipids. The t1/2 of rabbit erythrocytes exposed to 30 mM or 50 mM cyanate for 1 hr at 37 degrees C is reduced from the normal 24 days to 15 and 9 days, respectively. The cyanate-induced defect in membrane lipid metabolism is irreversible. Carbamylation of membrane proteins and damage to metabolism are minimized by limiting exposure in vitro to 15 mM cyanate at 4 degrees C for 30 min. Cells carbamylated under these conditions do not have a shortened life span. Levels of globin carbamylation of 0.5 moles CNO/mole hemoglobin, shown to be clinically effective in prolonging the life span of sickle erythrocytes, are obtained under these conditions and reach maximal levels after only 30 min of incubation. Carbamylation of blood in CPD anticoagulant is inferior to either ACD or heparin. The findings indicate that adequate carbamylation of sickle erythrocytes with minimal red cell membrane damage can be achieved without significant modification of the standard plasmapheresis procedure utilized by the working blood bank.
Complement-mediated granulocyte dysfunction in paroxysmal nocturnal hemoglobinuria
Authors: Craddock PR , Fehr J , Jacob HS
Published: October 2019

In paroxysmal nocturnal hemoglobinuria (PNH), infection, both viral and bacterial, disproportionate to the mild neutropenia seen in many such patients is responsible for significant morbidity. We report impaired granulocyte chemotaxis efficiency which may contribute to the problems induced by bacterial infections. PNH (but not normal) granulocytes, after exposure to very small concentrations of activated serum complement components, migrate poorly, as documented by their inhibited chemotaxis toward bacterial products or activated complement components in Boyden chambers. The granulocytes remain intact, excluding trypan blue, phagocytosing, and killing bacteria, despite this activated complement exposure. It is also suggested that this chemotactic defect may involve only a clone of cells, analogous to the clonal lysis of PNH erythrocytes; those few granulocytes capable of migration after exposure to activated complement contain normal quantities of leukocyte alkaline phosphatase (LAP), in contrast to the LAP deficiency of the overall PNH granulocyte population. Since bacterial infection may initiate or potentiate hemolysis, one of the major symptoms of the disease, these results could explain much of the morbidity of PNH.
Detection of soluble intermediates of the fibrinogen-fibrin conversion using erythrocytes coated with fibrin monomers
Authors: Largo R , Heller V , Straub PW
Published: October 2019

The presence of minimal amounts of fibrinogen-fibrin intermediates in human plasma was visualized by an agglutination reaction of glutaraldehyde-treated human erythrocytes coated with purified fibrin monomers. A degree of monomer coating was established which produced erythrocytes not agglutinated by normal plasma but by plasma containing minimal amounts of soluble complexes of fibrinogen with fibrin monomers. Under standardized conditions of coating, erythrocyte concentration, temperature, pH, and incubation time, the agglutination time varied with the ratio of soluble fibrin to fibrinogen in plasma. The test was sensitive down to a soluble fibrin concentration of 0.675% of the plasma fibrinogen concentration. Early fibrinogen and fibrin degradation products (FDP) in the plasma led to a prolongation of the agglutination time at a concentration of more than 16 mg/100 ml. Late FDP in a concentration of 100 mg/100 ml did not convert a positive test to negative. The test was not affected by heparin and protamine at concentrations of up to 12.5 and 50 NIH units/ml, respectively.
Identification of monocytes in suspensions of mononuclear cells
Authors: Rothbarth PH , Hendriks-Sturkenboom I , Ploem JS
Published: October 2019

A new histochemical technique for morphological studies of mononuclear cells and granulocytes, based on fluorescent staining with methyl green, pyronine Y, and stilbene-isothiocyanato disulfonic acid (MPS stain) is described. The method was applied to mononuclear cells isolated from the blood of normal human subjects by Ficoll-Isopaque density centrifugation. Three cell populations were distinguished, mainly on the basis of differences in morphology and cytochemistry, utilizing the MPS stain. One of the cell types had many of the morphological characteristics of the monocyte. This technique, augmented by lysosomal content and endocytosis capacity studies, revealed contimination of the cell suspension with a larger percentage of monocytes than has usually been reported in the literature.
Degranulation and abnormal bactericidal function of granulocytes procured by reversible adhesion to nylon wool
Authors: Klock JC , Bainton DF
Published: October 2019

Granylocyte bactericidal capacity, chemotaxis, hexose monophosphate shung activity (before and after phagocytic stimulus), and quantitative nitroblue tetrazolium reduction and enzyme content were examined in cells obtained by filtration leukaphresis (FL) and continuous-flow centrifugation (CFC). A decrease in the bactericidal efficiency of FL- produced cells compared to that of both normal and CFC-procured granulocytes was found; the decrease was 17% with a cell-to-bacteria ratio of 5:1, and 55% with a 1:1 ratio. Moreover, FL-acquired cells were often vacuolated and consistently contained less acid phosphatase and beta-glucuronidase than did normal granulocytes. When normal cells were incubated for 1–2 hr with nylon wool, 30% of the total acid phosphatase and beta-glucuronidase was released, with no evidence of cell death, thus suggesting degranulation. Similar results were obtained with glass, cotton, or polysulfone plastic fibers. Electron microscopic and peroxidase cytochemical studies of the adherence of normal granulocytes to nylon fibers were also carried out. After 30 min of incubation, cell-to-fiber attachment and cellular aggregation had occurred, although the cells per se appeared normal. After 60 and 120 min, other changes became apparent: (1) a decrease in the amount of cytoplasmic granules; (2) large, intracytoplasmic vaculoles; and (3) extracellular peroxidase on fiber surfaces. We conclude that granulocytes obtained by adherence to nylon fibers show both morphological and biochemical evidence of degranulation and diminished bactericidal capacity, and that these abnormalities may be causally related to decreased granulocyte survival in transfusion recipients.
Severe aplastic anemia: a prospective study of the effect of early marrow transplantation on acute mortality
Authors: Camitta BM , Thomas ED , Nathan DG , Santos G , Gordon-Smith EC , Gale RP , Rappeport JM , Storb R
Published: October 2019

A prospective randomized trial of therapy for severe aplastic anemia was designed to compare early bone marrow transplantation with conventional treatments. All patients with a sibling matched at the major histocompatibility region were transplanted. Transplantation was performed with 17–100 (median 33) days of original diagnosis. Conventional treatments included transfusion support with or without androgens. Twenty-four of 36 patients intered on the transplant arm are alive after 4–20 (median 9) mo with full marrow reconstitution. Only two are limited by chronic graft-versus-host disease. In contrast only 12 of 31 conventionally treated patients are alive. Six of these survivors have improved, five incompletely. The 19 nontransplant deaths have occurred within 1–11 (median 3) mo of diagnosis. Compared to nontransplant regimens, early transplantation more effectively restores normal marrow function and decreases the acute mortality of severe marrow aplasia (p = 0.006). Pending longer follow-up, early marrow transplantation appears to be the most effective available treatment for severe aplastic anemia.
Role of copper in mitochondrial iron metabolism
Authors: Williams DM , Loukopoulos D , Lee GR , Cartwright GE
Published: October 2019

Heme synthesis by copper-deficient cells was investigated to elucidate the nature of the defect in intracellular iron metabolism. Iron uptake from transferrin by copper-deficient reticulocytes was 52% of normal, and the rate of heme synthesis was 33% of normal. Hepatic mitochondria isolated from copper-deficient animals were deficient in cytochrome oxidase activity and failed to synthesize heme from ferric iron (Fe III) and protoporphyrin at the normal rate. The rate of heme synthesis correlated with the cytochrome oxidase activity. Heme synthesis from Fe(III) and protoporphyrin by normal mitochondria was enhanced by succinate and inhibited by malonate, antimycin A, azide, and cyanide. It is proposed that an intact electron transport system is required for the reduction of Fe(III), thereby providing a pool of ferrous iron (Fe II) for protoheme and heme a synthesis.
Potassium uptake and release by human blood platelets
Authors: Wiley JS , Kuchibhotla J , Shaller CC , Colman RW
Published: October 2019

Thrombin is known to reduce the K+ content of human platelets, but the subcellular origin of the lost K+ is not known. The effect of aggregating agents on K+ release was studied in platelets labeled in plasma by preincubation with 42KCI. Platelets were separated from plasma by gel filtration through Sepharose 2B equilibrated with K+ - free Tyrode's buffer. Platelet K+ was 116nEq/10(8) platelets, of which 23% was found to be extracellular immediately after gel filtration. K+ influx was 65 nEq/10(8) platelets/hr at pH 7.5 and was more rapid at pH 7.9. About 70% of cell K+ exchanged with plasma in 4 hr with first- order kinetics, while a minor fraction of about 30% exchanged with a slower time course. This slowly exchanging fraction of platelet K+ was thought to arise from heterogeneity in the platelet population. Epinephrine and ADP aggregated gel-filtered platelets and released serotonin, but with loss of only 5%-10% of cell K+ and no beta- glucuronidase. In contrast, thrombin released up to 30% of platelet K+, whether aggregation occurred or was prevented by not stirring the cells. The specific activity of K+ released by all aggregating agents was identical to the specific activity of total platelet K+. Thrombin (0.01–0.2 NIH U/ml) released serotonin and also beta-glucuronidase (an enzyme of the alpha-granule), and there was a linear relation between release of K+ and this enzyme (r = 0.88). No lysis of platelets occurred, since lactic dehydrogenase was not detected. Pretreatment of platelets with aspirin in vitro inhibited thrombin-induced release of serotonin but had no effect on the loss of K+ or beta-glucuronidase. In contrast, the ingestion of aspirin by mouth inhibited the release of serotonin, beta-glucuronidase, and K+ by thrombin. The data suggested that the K+ loss induced by thrombin was primarily derived from release of alpha-granules and that these organelles contained about 20% of the total platelet K+ in a freely exchangeable and nonsequestered state.
Phospholipid transfer between plasma and platelets in vitro
Authors: Joist JH , Dolezel G , Lloyd JV , Mustard F
Published: October 2019

Washed rabbit platelets were resuspended in plasma in which all of the major phospholipids had been isotopically labeled by injection of 32PO4 into rabbits. At certain time intervals during a 6-hr incubation at 37 degrees C, aliquots were removed from the incubation mixture and the platelets were isolated and subjected to lipid extraction and phospholipid analysis. A continuous rise in platelet non-lipid-bound and lipid-bound radioactivity was observed through-out the incubation period. Two platelet phospholipids, lecithin and lysolecithin, were significantly labeled, whereas little or no labeling of the other phospholipids was found. There was no detectable change in total or individual platelet phospholipid content. At 6 hr, 4% of total platelet phospholipid, 43% of platelet lysolecithin, and 7% of platelet lecithin were labeled. Platelets incubated in plasma from rabbits with diet- induced hyperlipidemia took up and incorporated significantly more label into their phospholipids than did platelets in normal plasma. Labeling of both platelet lysolecithin and lecithin could be due to uptake and metabolism of plasma lysolecithin by platelets. However, labeling of platelet lecithin could at least in part be the result of direct exchange of this phospholipid with the plasma. Uptake and incorporation of endogenous plasma lysolecithin by platelets and, possibly, direct exchanged of platelet lecithin may be important mechanisms in the modification by plasma lipids of platelet membrane phospholipid fatty acid composition and platelet function.
Spinal cord disease in hereditary spherocytosis: report of two cases with a hypothesized common mechanism for neurologic and red cell abnormalities
Authors: McCann SR , Jacob HS
Published: October 2019

Two unrelated patients with hereditary spherocytosis developed idiopathic spinal cord dysfunction. This coincidence, combined with similar individual case reports in the older European literature, suggests that abnormalities may exist in constituents common to red cell membranes and nerve tissue. A similar conclusion has been proposed to explain red cell membrane abnormalities in some of the muscular dystrophies.