bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron incorporation into ferritin and hemoglobin during differentiation of murine erythroleukemia cells
Authors: Ofer S , Fibach E , Kessel M , Bauminger ER , Cohen SG , Eikelboom J , Rachmilewitz EA
Published: October 2019

Hemoglobin and ferritin iron content have been followed during differentiation in tissue cultures of murine erythroleukemia cells (MELC) using the techniques of Mossbauer spectroscopy and electron microscopy. In undifferentiated cells grown without DMSO, only iron stored in ferritin was detected. The amount of iron in a cell grown in the presence of iron citrate is approximately 1.2 X 10(-14) g, whereas in a cell grown in the presence of transferrin the amount is approximately 0.28 X 10(-14) g. These quantities do not depend on the iron concentration in the nutrition medium in a range from 0.3 to 2.0 microgram Fe/ml and are the same for growth times between 8 hr and 7 days. Cells grown with DMSO contain, in addition to ferritin, increasing concentrations of hemoglobin. Chase experiments prove that ferritin iron participates in hemoglobin synthesis. The amount of ferritin iron reaches saturation within less than 8 hr in MELC grown with or without DMSO. In differentiating cells grown with iron citrate there is a decrease with time in ferritin iron content concomitant with the increase in hemoglobin. Cells grown with transferrin incorporate additional amounts of iron, which are approximately equal to the amounts used for hemoglobin synthesis maintaining a constant ferritin iron level. In the electron microscope, iron is seen only as ferritin within lysosomes. The density of the ferritin in lysosomes correlates with the ferritin iron concentrations determined by Mossbauer spectroscopy.
The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis
Authors: Kunicki TJ , Pidard D , Rosa JP , Nurden AT
Published: October 2019

Triton X-100 soluble proteins from 125I-labeled human platelets were studied by crossed immunoelectrophoresis employing a multispecific rabbit antibody raised against whole normal platelets. Emphasis was placed upon an analysis of immunoprecipitates containing 125I-labeled major membrane glycoproteins, and in particular, a prominent immunoprecipitate containing a glycoprotein antigen (s) previously designated as protein 16. SDS-polyacrylamide gel electrophoresis of protein 16 precipitated by a monospecific alloantibody. IgG L . . . , confirmed the presence of both glycoproteins IIb and IIIa. 125I-IgG L . . . , at concentration below that capable of precipitating protein 16 by itself, bound specifically to the precipitate containing protein 16 produced by the multispecific rabbit antibody. No other precipitates formed by the rabbit antibody contained either glycoprotein IIb or IIIa. When platelet proteins, incubated with optimum concentrations of ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol bis (B- aminoethylether) NN1-tetraacetic acid (EGTA), were electrophoresed against the rabbit antibody, previously unobserved immunoprecipitates that contained either free glycoprotein IIb or free glycoprotein IIIa were detected. Upon readdition of excess Ca++, but not Mg++, to the same protein samples, a single immunoprecipitate containing both glycoproteins was once again observed. It is thus demonstrated that glycoproteins IIb and IIIa can form Ca++-dependent complexes (protein 16) in Triton X-100 extracts of normal platelets. The potential significance of the reversible association of these glycoproteins to normal platelet function is discussed.
Clonal analysis of the response of human myeloid leukemic cell lines to colony-stimulating activity
Authors: Ruscetti FW , Collins SJ , Woods AM , Gallo RC
Published: October 2019

The recent development of two continuously proliferating human myeloid leukemic cell lines (HL-60 and KG-1) that response to CSA provides an opportunity for a detailed study of the interaction of CSA with leukemic myeloid cells. Here we report on the colony-forming ability of HL-60 and KG-1 over an extended culture life of the cells. Several different sources of human CSA of different stages of purity enhanced colony formation of these cells. CSA, obtained from conditioned media from an SV-40 transformed human trophoblast, was partially purified, and its activity for normal bone marrow copurified with the activity that stimulated HL-60 colony formation. Over 100 clones of HL-60 were developed and tested for their response to CSA. All responded to CSA by showing an increase in colony size and number. However, none of the colonies formed from any of the 100 clones differentiated in response to CSA despite the fact that many chemical can induce differentiation of HL-60. since HL-60 forms spontaneous colonies without the addition of any exogenous stimulating factors, HL-60 conditioned media and cell extracts were tested for the production by these cells of their own endogenous growth-promoting activity (such as a CSA-like molecule). No growth-promoting endogenous activity was found that stimulated normal bone marrow or HL-60 colony formation even after concentration and fractionation methods were employed. These experiments suggest that: (1) the effect of CSA markedly favors proliferation over differentiation in these cell lines; (2) CSA is unlikely to suppress growth of the age of the type of leukemic myeloid cells that HL-60 and KG-1 represent; and (3) if HL-60 cells produce their own growth- promoting factor it is not detectable in the media.
Diminished osmotic fragility of human erythrocytes following the membrane insertion of oxygenated sterol compounds
Authors: Streuli RA , Kanofsky JR , Gunn RB , Yachnin S
Published: October 2019

Oxygenated sterol compounds (OSC), when incubated for 1 hr with human erythrocytes in lipoprotein-depleted medium at concentrations of 0.625- 5 X 10(-5) M, are inserted into the cell membrane and remain there despite subsequent washing of the cells. The insertion results in expansion of the surface area of the red cell ghost membrane, an increase in critical hemolytic volume, and as a consequence, in diminished osmotic fragility of the erythrocytes. This effect is seen with echinocyte-forming as well as with non-echinocyte-forming OSC. Erythrocytes treated with OSC do not differ from control cells with respect to their mean cell volume (MCV) in isotonic solution, water content, ion fluxes, and filterability through polycarbonate filters. The shift of the osmotic fragility curve toward lower NaCl concentrations is proportional to the amount of OSC inserted into the red cell membrane. 7 beta-Hydroxycholesterol, 22-ketocholesterol, and 20 alpha-hydroxycholesterol are the most potent inhibitors of osmotic lysis. The effect of OSC on osmotic fragility is diminished if the erythrocytes are incubated in a lipoprotein-containing medium; free cholesterol, however, does not change this effect. Various progesterones also protect red cell from osmotic lysis, but only if the erythrocytes are directly exposed to the compounds present in the hypotonic NaCl solutions used for measurement of their osmotic fragility. Progesterones do not remain in the membrane after the cells have been washed. The OSC are also capable of correcting the osmotic fragility curve of red cells from patients with hereditary spherocytosis. These experiments may suggest an approach to the pharmacologic treatment of hereditary spherocytosis.
Abnormal platelet function and arachidonate metabolism in chronic idiopathic thrombocytopenic purpura
Authors: Stuart MJ , Kelton JG , Allen JB
Published: October 2019

We observed several patients with chronic idiopathic thrombocytopenic purpura (ITP) whose bleeding times were more prolonged than would have been expected from their platelet counts. To investigate this further, we performed in vivo and in vitro platelet function studies, assessed arachidonate metabolism, and measured platelet-associated IgG (PAIGG) in seven patients with chronic ITP. The bleeding times of three of the patients were prolonged for greater than 7 min, and all of these patients had impaired platelet aggregation and abnormal platelet arachidonic acid metabolism as reflected by increased production of the lipoxygenase product HETE and a concomitant decrease in cyclooxygenase products, TXB2 and HHT (p less than 0.001). The abnormalities noted were not due to concomitant drug ingestion, since they were present on repeated evaluation. There was no relationship between the platelet count and the bleeding time; however, there was a significant inverse correlation between the bleeding time and TXB2 production in all patients evaluated (r = 0.81; p less than 0.05). There was no relationship between the level of platelet-associated IgG and any parameter of platelet aggregation or arachidonate metabolism. The abnormalities noted should be looked for in the individual patient with chronic ITP, since the bleeding tendency is exacerbated by the superimposed impairment of platelet function even at platelet counts of greater than 50,000/cu mm, levels generally regarded as “safe”.
Effect of multimeric structure of the factor VIII/von Willebrand factor protein on binding to platelets
Authors: Gralnick HR , Williams SB , Morisato DK
Published: October 2019

The characteristics of the intact factor VIII/von Willebrand factor protein binding to human platelets was compared to 2-mercaptoethanol- treated factor VIII/von Willebrand factor protein and to fractions of plasma factor VIII/von Willebrand factor protein that elute after the void volume. These studies indicate that the factor VIII/von Willebrand factor protein larger size oligomers bind preferentially with high affinity to low capacity sites on human platelets. The intermediate and smaller size oligomers bind with intermediate or low affinity to sites with a much greater capacity. The results from binding analysis are also paralleled by the competitive inhibition of the intact factor VIII/von Willebrand factor protein by the various 2-mercaptoethanol- treated materials. These studies indicate that the two classes of binding sites seen in previous reports of factor VII/von Willebrand factor binding reflect heterogeneity in the oligomer size of the factor VIII/von Willebrand factor protein used in these assays. This study provides a model for understanding some of the normal structure- function relationships of the normal factor VIII/von Willebrand factor protein and the defect(s) in a variant form of von Willebrand's disease. In this form of the disease, decreased factor VIII/von Willebrand factor binding to platelets is reflected in decreased von Willebrand factor activity but coagulant and/or antigen levels are normal or only slightly decreased.
Abnormal phosphoenolpyruvate transport in erythrocytes of hereditary spherocytosis
Authors: Ideguchi H , Hamasaki N , Ikehara Y
Published: October 2019

The rate of phosphoenolpyruvate transport in erythrocytes from patients with hereditary spherocytosis and from healthy individuals was examined in a 0.1 M citrate buffer (pH 6.1 at 37 degrees C) containing 10 mM phosphoenolpyruvate and 10 mM NaF. The rate in erythrocytes from patients with hereditary spherocytosis was 0.09 +/- 0.02 mumol/min/ml of cells (mean +/- SD, n = 8), whereas the rate in erythrocytes from healthy individuals was 0.23 +/k- 0.03 mumol/min/ml of cells (mean +/- SD, n = 8). The lower rate of transport in erythrocytes of hereditary spherocytosis could not be described to a different glycolytic response to NaF or to a difference in cell age distribution. This phenomenon seemed to be a reflection of some abnormality in the erythrocyte membrane from patients with hereditary spherocytosis.
Preaggregation reactions of platelets
Authors: Gear AR
Published: October 2019

Whether platelet volume increases during the morphological changes preceding aggregation has been investigated. Previous research is controversial; resistive-counting techniques reveal an increase, centrifugal methods do not. Platelets were sized with a computerized, resistive-particle counter before and after incubation with adenosine diphosphate (ADP). Resistive volume increased by 14% (p less than 0.001) in the absence of EDTA, and only 7% in its presence (ADP, 10 micro M). EDTA inhibited platelet volume changes, whether these were shrinking induced by warming or swelling by ADP. Handling of platelets, such as during centrifugation, also caused particle swelling. Particle density decreased after ADP exposure, without release of serotonin, suggesting uptake of water. Platelet shape was experimentally manipulated to test the hypothesis that resistive volume changes stem from artifacts of particle shape. Scanning electron microscopy confirmed that colchicine, chlorpromazine, and a temperature cycle of 0 degrees to 37 degrees all caused extensive alteration from the disc shape. Subsequent exposure to ADP increased resistive volume, and in the case of chlorpromazine, no long pseudopodia were extruded. It is concluded that preaggregation reactions of platelets can be associated with an increase in particle volume, and that earlier research based on centrifugation and the presence of ETA failed to reveal the increase because of inhibitory and apparent swelling effects.
Monoclonal antibodies to von Willebrand's factor: reactivity with porcine and human antigens
Authors: Katzmann JA , Mujwid DK , Miller RS , Fass DN
Published: October 2019

Monoclonal antibodies to porcine von Willebrand's factor (vWf) were obtained from hybridomas derived from the fusion of NS-1 plasmacytoma cells and spleen cells from immunized BALB/c mice. Twenty hybrids were positive in a radioimmunoassay based on the ability of immobilized hybridoma antibodies to bind porcine vWf as detected by the subsequent binding of 125I-polyclonal rabbit anti-vWf. Growth of the hybridomas as ascitic tumors yielded fluids having titers in this assay ranging from 8 x 10(7) to 8 x 10(11). All the antibodies were positive when normal, but not von Willebrand's, plasma was used as the source fo antigen in this RIA, and when normal, but not von Willebrand's, acetone-fixed platelets were used in an indirect immunofluorescence assay. Two of these antibodies inhibited both ristocetin-induced platelet aggregation and platelet aggregating factor. In a second radioimmunoassay, only 13 were of the 20 antibodies were reactive with human vWf, and two of these would not react with human antigen in the presence of 2M Nacl. Bases on these assays, six reactivity groups of antibodies could be identified.
A human b-cell lymphoma synthesizing and expressing surface mu-chain in the absence of detectable light chain
Authors: Gordon J , Hamblin TJ , Smith JL , Stevenson FK , Stevenson GT
Published: October 2019

Lymph node cells obtained from a case of non-Hodgkin's lymphoma, of follicular center cell histology, were seen by immunofluorescence to express immunoglobulin mu heavy chains but no detectable light chain on their surfaces. Antibody specific for determinants expressed only on uncombined mu-chain reacted with these cell surfaces, but not with normal or neoplastic cells expressing surface IgM. Analysis of radioiodinated surface material revealed mu-chains which, without reduction, migrated on SDS-gel electrophoresis as normal mu-chain monomers, and again failed to detect light chains. Lysates of cells incubated with 3H-leucine revealed the synthesis of mu-chains but not of light chains. The labeled mu-chains were not secreted into the culture supernatant, a finding confirmed in parallel cultures by radioimmunoassay. The findings are discussed with particular reference to current concepts of the “mu-chain only” phenotype.
The sickle gene polymorphism in North Africa
Authors: Mears JG , Beldjord C , Benabadji M , Belghiti Ya , Baddou MA , Labie D , Nagel RL
Published: October 2019

Analysis of the restriction endonuclease Hpa 1-beta globin gene linkage has been performed in a predominantly Arab population of North Africa possessing the sickle (beta A) gene is found associated with a 7.6 kilobase) or 7.0 kb Hpa 1 fragment (54/54 assignable beta A genes), whereas the beta S gene is found associated with a 13 kb Hpa 1 fragment (42/42 assignable beta S genes). The results demonstrate a very tight linkage of the beta S gene to the 13 kb Hpa 1 fragment as well as a very low probability that a beta A gene will be found on a 13 kb Hpa 1 fragment. Thus, the North African population presents a nearly ideal opportunity for prenatal diagnosis solely by Hpa 1-beta globin gene linkage analysis. Additionally, the evidence supports the hypothesis that the beta S gene flowed from West Africa rather than from Arab populations in the Middle East.
Normal pregnancy in a patient with a prior postpartum factor VIII inhibitor: with observations on pathogenesis and prognosis
Authors: Coller BS , Hultin MB , Hoyer LW , Miller F , Dobbs JV , Dosik MH , Berger ER
Published: October 2019

A 22-yr-old primigravada developed a hemorrhagic diathesis 6 days after delivering a normal female infant and was found to have an immunoglobulin inhibitor of factor VIII (15 Bethesda units). The patient was treated with prednisone and the bleeding stopped soon thereafter. Her inhibitor titer decreased over the next 8 mo, at which time no inhibitor was detectable. Nine months later she became pregnant again and proceeded to have an uneventful pregnancy and delivery of a normal female infant without evidence of a recurrence of the inhibitor. Studies of 3H-thymidine incorporation into the patient's lymphocytes in the presence of her own plasma or plasmas from her husband, normals, a von Willebrand patient, and a hemophilic patient yielded equivocal results. Analysis of VIII:CAg using 2 different antisera failed to discern immunologic differences between the VIII:CAg of the patient, her husband, or her first child. A review of the literature revealed that there were no recurrences with second pregnancies in any of the 8 patients with postpartum factor VIII inhibitors whose inhibitors had completely disappeared prior to delivery. While the pathogenesis of this disorder remains uncertain, the apparently favorable prognosis for such patients should be considered in counselling with regard to future pregnancies.
Labeled factor IX kinetics in patients with hemophilia-B
Authors: Smith KJ , Thompson AR
Published: October 2019

Labeled factor IX was infused five time into four patients with hemophilia-B. Ten-minute plasma recovery average 35% (SD +/- 2) and the mean T 1/2 beta-phase elimination was 23 hr (+/- 5). No alteration in the postinfusion 125I-factor-IX could be detected by radioautography of plasma samples run on polyacrylamide gels or on crossed- immunoelectrophoresis. Label was excreted into the urine as free 125I- iodide. Kinetics were similar when the labeled preparation was infused alone or with a commercial concentrate containing unlabeled factor IX. Infusion of factor IX in man is best described by a two-compartment open pharmacokinetic model where factor IX is distributed in a space larger than the plasma volume.
Granulopoietic differentiation in AML blasts in culture
Authors: Marie JP , Izaguirre CA , Civin CI , Mirro J , McCulloch EA
Published: October 2019

A newly described monoclonal antibody detected an antigen (My-1) that is expressed on the cell surface during normal granulopoietic differentiation. The kinetics of expression of My-1 and NASD chloracetate esterase were studied during the formation in culture of blast colonies and normal granulopoietic colonies. My-1-positive cells increased during blast colony formation in 6 of 9 experiments, while NASD esterase-positive cells decreased. In contrast, in normal granulopoiesis in culture both markers increased coherently. We suggest that components of granulopoietic differentiation programs are expressed abnormally during blast colony formation in culture.
Elevated platelet-bound IgG associated with an episode of thrombotic thrombocytopenic purpura
Authors: Sims PJ , Boswell EB
Published: October 2019

The level of platelet-associated IgG (PAIgG) were measured during the successful treatment of a patient with thrombotic thrombocytopenic purpura. Prior to therapy. PAIgG was found to be markedly elevated to 195 fg/cell (normal range 0--3.5 fg/cell). The institution of combined therapy with intensive plasma exchange transfusions, high-dose steroids, and antiplatelet drugs resulted in a complete recovery and a decline in PAIgG to the normal range. The possible role of platelet antibody in the pathogenesis of this disorder is discussed.
Chemotactic deactivation of human neutrophils: protective influence of phenylbutazone
Authors: Nelson RD , Gracyk JM , Fiegel VD , Herron MJ , Chenoweth DE
Published: October 2019

The antiinflammatory drug, phenylbutazone (PBZ), has been studied in terms of its influence on chemotactic deactivation of human neutrophils. When PBZ was present during the time of preincubation of cells with N-formyl-methionyl-phenylalanine (F-Met-Phe), loss of subsequent spontaneous mobility and chemotactic responsivity to F-Met- Phe did not occur. The action of PBZ to protect neutrophils from peptide-mediated chemotactic deactivation was found to involve in part its inhibitory influence on hexose monophosphate shunt activity and in part its antagonistic effect on interaction of peptide receptors with N- formyl peptide. Phenylbutazone interfered with binding of N-formyl- methionyl-leucyl-[3H]phenylalanine but not [125I]C5a to the neutrophil, displaced labeled tripeptide bound in the absence of PBZ, increased the dissociation constant (KD) for labeled tripeptide, and limited down regulation of peptide receptor function. These results provide an example of drug-mediated modulation of the interaction of neutrophils with N-formyl peptide and strongly support the possibility that PBZ interacts directly and specifically with the human neutrophil peptide receptor as a competitive antagonist. They also provide an additional example of a compound outside of th N-formyl peptide series that interacts with the peptide receptor.
An autoantibody reactive with nuclei of polymorphonuclear neutrophils: a cell differentiation marker
Authors: Whittingham S , Morstyn G , Wilson JW , Vadas MA
Published: October 2019

An autoantibody that reacted with nuclei of polymorphonuclear neutrophils (PMN) was detected at titers of greater than 10 in sera of 25 of 50 patients with rheumatoid arthritis and 36 of 50 with autoimmune chronic active hepatitis but in none of 160 controls comprising 24 patients with alcoholic cirrhosis, 36 with multiple myeloma, and 100 healthy subjects. Through the use of enriched populations of hemopoietic cells, this antibody was shown to be cell- specific, reacting only with the nucleus of the mature neutrophil. It was unreactive with nuclei of progenitor cells in the myeloid series and with nuclei of eosinophils, monocytes, lymphocytes, and thymocytes. It reacted with a determinant that appeared to be a differentiation antigen. This cell-specific autoantibody may prove to be of value in analytical studies of granulocyte maturation.
Histochemical demonstration of terminal deoxynucleotidyl transferase in leukemia
Authors: Hecht T , Forman SJ , Winkler US , Santos S , Winkler KJ , Carlson F , Maslow WC , Borer WZ , Blume KG
Published: October 2019

White blood cells from 22 patients with leukemia and lymphoma were studied for the presence of terminal deoxynucleotidyl transferase with a peroxidase-antiperoxidase method. The enzyme was detected in leukemic cells of 5 patients with acute lymphoblastic leukemia and 1 patient with chronic myelogenous leukemia, whereas 16 patients with different forms of leukemia or lymphoma were negative for this enzyme. Comparative studies using a biochemical and an indirect immunofluorescence assay revealed complete concordance between these three methods.
A clinicopathologic correlation of the idiopathic hypereosinophilic syndrome. II. Clinical manifestations
Authors: Schooley RT , Flaum MA , Gralnick HR , Fauci AS
Published: October 2019

The idiopathic hypereosinophilic syndrome, a disorder characterized by peripheral blood and bone marrow eosinophilia associated with single or multiple organ system dysfunction attributable to tissue invasion by eosinophils has, in the past, been associated with an extremely poor prognosis. Recently, we reported the favorable impact of a therapeutic protocol consisting of prednisone and/or hydroxyurea on the morbidity and mortality of this syndrome. We have reviewed the clinical and hematologic features upon admission and the subsequent clinical courses of 32 patients with this disease referred to the NIH between 1965 and 1979 in an effort to determine which features suggest a more rapidly progressive course. A grading system based on 22 clinical features involving the 8 organ systems commonly affected by the illness was devised. The disease followed a more aggressive course in patients with evidence of cardiac or neurologic dysfunction at the time of initial NIH evaluation. Although splenomegaly, in and of itself, caused little morbidity, splenic enlargement at presentation appeared to be a predictor of a more aggressive course. The clinical grading system accurately predicted which patients would require no specific antihypereosinophilic therapy, which patients would respond adequately to corticosteroids, and which patients would require therapy with cytotoxic agents. It is proposed that this clinical grading system, and the hematologic grading system outlined in the accompanying report be used as aids in the selection of initial therapy in this group of patients.
Hemophilic dog model for evaluating therapeutic effectiveness of plasma protein fractions
Authors: Kingdon HS , Hassell TM
Published: October 2019

Therapeutic effectiveness of factor VIII inhibitor bypassing materials has been evaluated in dogs with hemophilia A. A standardized template gingival biopsy was performed using local anesthesia. Hemophilic dogs bled extensively from the biopsy site, whereas in normal dogs the wound was sealed within 5 + /- 2 min. If untreated, the hemophilic dogs frequently bled for several days. Factor VIII infusion stopped the bleeding promptly. Some experimental preparations of factor VIII inhibitor bypassing materials were shown to be therapeutically effective, whereas others were not. Intravascular thrombi could not be demonstrated histologically. The model should prove useful for evaluating factor VIII inhibitor bypassing materials and also for evaluating their mechanism of action.
Variant von Willebrand's disease and pregnancy
Authors: Hanna W , McCarroll D , McDonald T , Painter P , Tuller J , Chen J , Lange R
Published: October 2019

The clinical course and coagulation profile of a pregnant patient with variant von Willebrand's disease were followed from the second trimester through puerperium. The clinical course was characterized by a normal delivery and absence of abnormal bleeding or need for replacement therapy. The coagulation profile demonstrated an increase in factor VIII procoagulant activity, factor-VIII-related antigen, and platelet aggregation activity in response to ristocetin prior to delivery. Postpartum, these factors decreased to prepregnancy values with distinctly different patterns. Factor VIII procoagulant activity continued to rise for 5 days after delivery and then decreased with a half-life of approximately 6 days. Factor-VIII-related antigen began to decrease just prior to delivery, displaying a half-life or approximately 6 days. Ristocetin cofactor activity, however, dropped immediately postpartum and displayed a half-life of approximately 6 hr. The ristocetin cofactor activity was associated with factor-VIII- related antigen, which displayed a significantly smaller molecular weight than does normal factor-VIII-related antigen. Larger aggregates of factor-VIII-related antigen. Larger aggregates of factor-VIII- related antigen did not appear during the pregnancy, and ristocetin cofactor activity could not be demonstrated in fragments of less than 0,8 x 10(6).
Heterogeneity in human prothrombin: analysis of cause
Authors: Bajaj SP , Rapaport SI , Prodonos C , Russell WA
Published: October 2019

Two fractions of human prothrombin can be isolated from single donor plasma by the technique of heparin-agarose chromatography in (sodium) citrate buffer, pH 7.5, as previously reported for pooled plasma. The two fractions, designated H-II1 and H-II2, are found in a ratio of approximately 4:1. Both forms comigrate in sodium dodecyl sulfate gel electrophoresis; however, under nondenaturing electrophoretic conditions, each fraction migrates as a discrete entity with a different mobility. The larger fraction (H-II1) has a faster mobility towards the anode. Isoelectric focusing in urea of H-II1 reveals that it has two components, a minor component with a pl of 5.25 (H-II1a) and a major component with a pl of 5.40 (H-II1b). H-II2 has a pl of 5.6 H- II1 and H-II2 possess the same amino terminal residue (alanine, 0.87- 0.92 mole/mole) and the same number of gamma -carboxyglutamic acid residues (9.8–10.5). Their amino acid composition is indistinguishable. However, the two fractions of prothrombin differ in their content of neutral sugar and of sialic acid residues. Removal of sialic acid with neuraminidase abolishes the electrophoretic heterogeneity. Thus, the charge heterogneity of the three variants of prothrombin found in normal human plasma appears to result exclusively from differences in the number of sialic acid residues attached to the protein moiety of the molecule.
Immunologic abnormalities in myelofibrosis with activation of the complement system
Authors: Gordon BR , Coleman M , Kohen P , Day NK
Published: October 2019

Eighteen patients with agnogenic myeloid metaplasia with myelofibrosis were studied for clinical and laboratory evidence of immunologic dysfunction. Clinical findings included the presence of arthritis, vasculitis, and erythema nodosum. Laboratory abnormalities included the presence of circulating immune complexes, antinuclear antibodies, positive direct Coombs tests, elevated latex fixations, and a circulating lupus type anticoagulant. Total hemolytic complement was markedly depressed in four patients. Analysis of complement (C) components C1-C9 and factor B demonstrated significant reduction of only C3 and factor B. By crossed-immunoelectrophoresis, both C3 and factor B, but not C4, were cleaved, indicating that C activation was occurring predominantly via the alternative pathway. The control proteins beta 1H and C3b inactivator were decreased in three of four patients with hypocomplementemia. These data suggest that immunologic mechanisms associated with activation of the complement system play an important role in the disease process of some patients with agnogenic myeloid metaplasia with myelofibrosis.
Globin synthesis in erythroid bursts that mature sequentially in culture. I. Studies in cultures of adult peripheral blood BFU-Es
Authors: Papayannopoulou T , Nakamoto B , Kurachi S , Stamatoyannopoulos G
Published: October 2019

To study whether the culture time at which the burst populations mature influences the expression of fetal hemoglobin in bursts, we measured hemoglobin synthesis in cohorts of fully hemoglobinized erythroid bursts maturing sequentially in cultures of adult peripheral blood BFU- Es. In 13 of 15 experiments, a decline in gamma/gamma + beta ratio was noted as the culture time advanced. On the average, erythroid bursts that mature during the third culture week showed lower levels of fetal Hb synthesis compared to bursts that are already mature in the second culture week. The decline of gamma/gamma + beta ratio with culture time was also noted in erythroid bursts composed of immature erythroblasts. The enhanced HbF formation in peripheral blood BFU-E cultures is thus most pronounced among the bursts that become hemoglobinized early, and there is a tendency for normalization of HbF synthesis in bursts that mature in late culture days. These results can be interpreted by several alternatives, including the possibility that the expression of high HbF levels in the early days of adult BFU-E cultures is a reflection of premature commitment to terminal differentiation of progenitors that possess an active HbF program. The present data indicate that the variation of HbF synthesis with culture time should be taken into consideration when the influence of various culture conditions of HbF synthesis is studied in BFU-E cultures.
Modification of normal human myelopoiesis by 12-0 tetradecanoylphorbol- 13-acetate (TPA)
Authors: Abrahm JL , Smiley R
Published: October 2019

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), induces macrophage characteristics in the HL-60 promyelocytic leukemia cell line. These same cells can be induced to develop mature myeloid characteristics with a variety of other stimuli. Since normal colony- forming units-culture (CFU C) also have the dual capability of developing colonies with myeloid or monocyte characteristics, the effect of TPA on normal human CFU-C development was studied. To carry out these studies, a method was developed to identify cells histochemically within agar cultures as containing either the myeloid marker, chloroacetate esterase (CAE), or the monocyte marker, nonspecific esterase (NSE). Cells from normal donors were placed into agar cultures with placenta conditioned medium (PCM), TPA in various concentrations, or combinations of PCM and TPA as stimulating materials, and examined after 7–14 days of incubation. TPA alone at concentrations of 5 x 10(-7) M to 10(-9) M stimulated cluster formation. With increasing concentrations of TPA, the percentage of clusters positive for NSE progressively increased, while CAE-positive clusters decreased. I contrast, when TPA at concentrations greater than 10(-9) M was added to PCM, the total number of clusters and colonies decreased. This resulted from a decrease in the number of clusters and colonies that contained the myeloid marker CAE, whereas the number positive for the monocyte marker NSE remained unchanged. These studies demonstrate two effects of TPA on normal CFU-C. In the absence of other sources of colony stimulating activity (CSA), TPA induces clusters. In the presence of PCM, it inhibits the production of myeloid colonies and clusters. Under both conditions, it favors the development of colonies and/or clusters containing predominantly monocytes.
Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis
Authors: Kunicki TJ , Nurden AT , Pidard D , Russell NR , Caen JP
Published: October 2019

Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.
The effects of glucocorticoid therapy on glucocorticoid receptors in leukemia and lymphoma
Authors: Shipman GF , Bloomfield CD , Smith KA , Peterson BA , Munck A
Published: October 2019

Measurement of glucocorticoid receptors appears to be useful for selecting which patients with leukemia and lymphoma should receive glucocorticoid therapy. To determine the effect of recent or concurrent glucocorticoid therapy on the number of measured tumor glucocorticoid receptor sites, 18 patients with leukemia and lymphoma were studied. Baseline determinations of numbers of glucocorticoid receptors were performed on the malignant cells circulating in the patients' peripheral blood. Glucocorticoid therapy was then instituted consisting of dexamethasone 4 mg p.o. every 6 hr. Repeat determinations of the number of glucocorticoid receptor sites were performed within 24 hr and at various subsequent times from the start of therapy. When compared to baseline receptor numbers, 16 of 18 patients demonstrated a decrease in receptor number (median decrease 1651 sites/cell) after the start of glucocorticoid therapy. The magnitude of the change in receptor number was independent on the initial number of receptors. Our results suggest that in order accurately interpret glucocorticoid receptor numbers in patients with leukemia and lymphoma, glucocorticoid should not be administered for 3 wk prior to determinations of receptor levels.
Survival of patients with localized diffuse histiocytic lymphoma
Authors: Sweet DL , Kinzie J , Gaeke ME , Golomb HM , Ferguson DL , Ultmann JE
Published: October 2019

Twenty-eight patients with previously untreated diffuse histiocytic lymphoma (DHL) were identified to be in pathologic stage (PS) I (11), IE (3), II (8), or IIE (6) by exploratory laparotomy and splenectomy. Six patients were treated with total nodal radiotherapy; 14 with an extended mantle; 5 with an inverted Y or whole abdomen; and 3 with an involved field. Twenty-six patients achieved a complete remission (93%) and 2 patients had persistent local disease. The median survival and disease-free survival and for the complete response group are 56 and 51.5 mo, respectively. Ten of the 11 stage I or IE patients had supradiaphragmatic lymph node disease. Patients with stage I or IE disease (n = 14) demonstrated a median survival of 72.5 mo and a median disease-free survival of 69.5 mo; there was 1 disease-related death. Patients with stage II or IIE disease (n = 14) demonstrated a median survival of 33 mo and median disease-free survival of 29.5 mo; there were 10 relapses or deaths. Patients in stages I, IE, II, or IIE with infradiaphragmatic disease (n = 7) had a median survival of 36 mo, while patients with supradiaphragmatic presentation (n = 21) demonstrated median survival of 68 mo (p = 0.37). The data indicate that patients with diffuse histiocytic lymphoma with stage I supradiaphragmatic lymph node disease are curable using radiotherapy alone, achieving a 93% 11-yr actuarial disease-free survival. Patients with stage II or IIE diseases are not readily curable with radiation therapy alone, achieving a 33% 11-yr actuarial disease-free survival; radiotherapy with adjuvant chemotherapy or chemotherapy alone should be considered for this group.
Immunochemical studies on Tn erythrocyte glycoprotein
Authors: Lee LT , Frank S , de Jongh DS , Howe C
Published: October 2019

Glycoproteins were extracted from membranes of erythrocytes that displayed Tn polyagglutination and were compared chemically and immunologically with glycoproteins of group O, MN cells. Tn glycoprotein had lower than normal NANA : protein and sugar : protein ratios, as revealed by direct analysis and polyacrylamide gel electrophoresis, and displayed slower immunoelectrophoretic mobility than glycoproteins of group O, MN cells. Agglutination of Tn cells by Salvia sclarea lectin was inhibited by Tn glycoprotein but not by O, MN glycoprotein. Tn and MN glycoproteins were equally potent inhibitors of influenza virus HA. Our findings indicate than Tn-specific determinants are part of the glycophorin molecule.
The effect of malonyldialdehyde on erythrocyte deformability
Authors: Pfafferott C , Meiselman HJ , Hochstein P
Published: October 2019

The addition of malonyldialdehyde (5–20 micro M) to human erythrocytes results in a marked decrease in cellular deformability as measured with a counter-rotating, cone plate Rheoscope when low shear forces (2.5–25 dynes/sq cm) are applied. At high shear forces (125–500 dynes/sq cm), malonyldialdehyde at 5 micro M had no effect on deformability, although at concentrations of 10 and 20 micro M a small but statistically significant decrease was evident. These effects of this crosslinking agent are observed in the absence of alterations in cell volume and intracellular viscosity. The results obtained are in accord with the view that the polymerization of membrane constituents may contribute to the events that lead to the removal from the circulation of either aging cells or cells exposed to peroxidation-initiating agents.
Antibody-dependent cytolysis of the human leukemia cell line K-562 in the absence of effector cells or complement
Authors: Wust CJ , Green M , Lozzio CB , Lozzio BB
Published: October 2019

K-562 human leukemia cells grown in the presence of specific goat anti- K-562 gamma globulin, F(ab)'2, Fab', or Fab showed a decrease in DNA and protein syntheses and a loss of cell viability within several hours. Eventually all cells died and lysed within 2–5 days. These events occurred in the absence of added effector cells or complement. The effect on the cell was not due to proteolytic or other external lytic activity generated at the cell surface, since 51Cr-labeled bystander cells or antibody-sensitized bystander cells (chicken erythrocytes or K-562 cells) were not lysed when added to cultures of K- 562 cells incubated with immune globulin. The anti-K-562 gamma globulin was not observed to patch or cap on K-562 cells, but the ability of bound immunoglobulin to fix complement decreased markedly (50% in 60 min), so that no complement-mediated cytolysis could be observed at 2 hr. However, the immunoglobulin, at least portions of it, reacted with fluorescein--labeled rabbit anti-goat gamma globulin for about 16 hr. 125I-labeled immune globulin appeared to be internalized and released into the medium as small degradation products. 125I-labeled Fab did not appear to be internalized, since it was released intact and more rapidly from cells than the intact globulin.
Cyclic hematopoiesis: effects of lithium on colony-forming cells and colony-stimulating activity in grey collie dogs
Authors: Hammond WP , Dale DC
Published: October 2019

The cycling of blood cell counts in grey collie dogs with cyclic hematopoiesis can be eliminated by treatment with oral lithium carbonate. To explore the mechanism by which lithium alters this stem cell disorder, studies of bone marrow granulocyte-macrophage progenitor cells (CFU-C), neutrophil colony-forming cells (neutrophilic CFU-C), and colony-stimulating activity (CSA) were performed. In untreated dogs, the proportions of CFU-C were found to fluctuate cyclically, but the cyclic fluctuations in neutrophil colony-forming cells were even more marked, with numbers decreasing to undetectable levels during each period of neutrophilia. Dogs on lithium, however, did not cycle the numbers of total or neutrophilic CFU-C. Tritiated thymidine suicide rates were not altered by treatment with lithium. Serum CSA levels and bone marrow cell elaboration of CSA were not increased by lithium. These studies suggest that lithium corrects cyclic neutropenia by a direct effect on the differentiation and proliferation of CFU-C; normalization of the proportion of CFU-C that enter neutrophilopoiesis appears to be an important effect of the lithium therapy.
Cold-induced contact surface activation of the prothrombin time in whole blood
Authors: Palmer RN , Gralnick HR
Published: October 2019

Studies of the prothrombin time (PT) have revealed that contact with borosilicate or commercial siliconized borosilicate markedly shortens the PT. This shortening is related to the activation of the contact phase of blood coagulation. This shortening of the PT occurs in both normal whole blood and plasma when stored in borosilicate or siliconized borosilicate tubes at 4 degree C and to a lesser degree at room temperature. Studies indicated the importance of several coagulation factors in decreasing the PT. The PT did not change in blood deficient in factor XII or in plasma deficient in Fletcher factor or high molecular weight kininogen, while blood deficient in CI esterase inhibitor (CI INH) had the most profound shortening. Shortening of the PT correlated directly with increased levels of factor VII. When purified CI INH was added to normal blood, it markedly reduced the activation of factor VII and the shortening of the PT in a dose-related manner. These studies indicate the pivotal roles of the contact phase of coagulation in initiating activation of the PT and of CI INH in inhibiting the activation of the coagulation factor(s) responsible for the cold-promoted activation of factor VII.
Ploidy and proliferative characteristics in monoclonal gammopathies
Authors: Latreille J , Barlogie B , Johnston D , Drewinko B , Alexanian R
Published: October 2019

Measurements were performed in 143 patients with monoclonal gammopathy of the cellular substrate in the bone marrow, including analysis of DNA and RNA content and tritiated thymidine labeling index. Aneuploidy by DNA content was present in 80% of 115 patients with active multiple myeloma and in 4 of 9 patients with benign monoclonal gammopathy, but was absent in all 12 patients with myeloma in remission and in 7 individuals with Waldenstrom's macroglobulinemia. With regard to prognosis in multiple myeloma, a low pretreatment plasma cell labeling index of less than or equal to 1% heralded longer survival than that observed in patients with a labeling index less than 1%, independent of myeloma tumor burden and ploidy pattern, except for a subset of 17 patients with a low-degree hyperdiploid abnormality whose survival was not affected by the magnitude of the pretreatment labeling index. Thus, besides tumor burden, tumor proliferative activity and ploidy both appear to have additional prognostic importance for patients with multiple myeloma.
Bone marrow cell count per cubic millimeter bone marrow: a new parameter for quantitating therapy-induced cytoreduction in acute leukemia
Authors: Hiddemann W , Clarkson BD , Buchner T , Melamed MR , Andreeff M
Published: October 2019

A new technique is introduced for determining the number of bone marrow cells per cubic millimeter marrow, providing an accurate and objective means for quantitating therapy-induced cytoreduction. The method requires a correction for admixed peripheral blood in bone marrow aspirates to measure the fraction of remaining pure marrow. While cell kinetic differences between blood, aspirates, and biopsies identify the proportion of contaminating blood cells, the ratio of red cell hematocrits in blood and aspirate gives the volume of trapped blood. By combining both procedures, bone marrow cell counts per unit volume pure marrow result (BMC/cu mm BM), which were found highly reproducible. Blast cell counts (BMBC/cu mm BM) were obtained by additional morphological differentiation. BMC and BMBC/cu mm BM were monitored in 16 patients with acute nonlymphoblastic leukemia treated with daunorubicin, cytosine arabinoside, and 6-thioguanine in combination and in 4 patients with end-stage acute leukemias and non-Hodgkin's lymphomas during high-dose thymidine therapy. Total and daily therapy- induced cytoreduction rates were significantly greater (P less than 0.01) in responders than nonresponders to either regimen. Changes in BMC/cu mm BM were also found representative for changes in BMBC/cu mm BM, since the majority of bone marrow cells were blasts. In acute leukemia. BMC/cu mm BM thus provides accurate and objective measurements of treatment efficacy in vivo and after short periods of drug exposure. Differences in cytoreduction rates within the group of responders also suggest possible prognostic implications.