bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasmin effect on platelet glycoprotein Ib-von Willebrand factor interactions
Authors: Adelman B , Michelson AD , Loscalzo J , Greenberg J , Handin RI
Published: October 2019

We have studied the effect of streptokinase on platelets in platelet- rich plasma (PRP) and of plasmin on washed platelets. By three and one- half minutes after the addition of 50,000 IU/mL streptokinase to PRP, the maximum rate of ristocetin-induced platelet agglutination declined 40%, and by 60 minutes, it declined 70%. During the same time interval, the thrombin time increased from 20 seconds to over 120 seconds. At a concentration as low as 50 IU/mL, streptokinase reduced the maximum rate of ristocetin-induced platelet agglutination by 50% and prolonged the thrombin time to 1.5 times control value. Streptokinase added to PRP also caused inhibition of platelet aggregation following stimulation by 2.9 mumol/L adenosine diphosphate, 0.25 U/mL thrombin, and 0.025 mg/mL collagen. Plasmin, 0.05 to 1.0 CU/mL, reduced ristocetin-mediated agglutination of washed platelets in the presence of von Willebrand factor (vWF) from 66% of control to 2% of control, following a one-hour incubation. Autoradiograms produced following sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) of plasmin- treated 125I-surface-labeled platelets demonstrated progressive loss of a protein with a molecular weight (mol wt) of 180,000; simultaneously, a protein with mol wt 135,000 appeared on autoradiograms produced following SDS-PAGE of the surrounding platelet medium. These proteins are similar in molecular weight to glycoprotein (gp) Ib, a platelet surface receptor for vWF, and glycocalicin, a proteolytic fragment of gpIb. By use of an enzyme-linked immunosorbent assay (ELISA) based immunoinhibition assay for glycocalicin, we were able to demonstrate that plasmin treatment of washed platelets released a glycocalicin- related antigen into the surrounding medium and that appearance of this material corresponding to loss of vWF-dependent, ristocetin-induced agglutination.
Killer cell activity of human monoblastic leukemia cells as detected with a monocyte-specific target cell
Authors: Ziegler-Heitbrock HW , Munker R , Thiel E , Krebs I , Riethmuller G
Published: October 2019

Peripheral blood leukemia cells from patients with acute monoblastic leukemia (AMoL) were tested for killer cell activity against target cells that detected natural killer cell-mediated or monocyte-mediated spontaneous cytotoxicity. The fibrosarcoma cell line Wehi 164, pretreated with actinomycin D to induce susceptibility to lysis, specifically detects the activity of unstimulated human monocytes. In four of six cases of AMoL, high killer cell activity could be measured against this target. In three of these four cases, the killer cell activity could be assigned exclusively to the leukemic clone, based on the high leukocyte counts and the resultant dilution of normal cells, as evidenced by marker and by functional analysis. While leukemic cells with killer cell activity against Wehi 164 contained 34% to 45% cells that were positive for binding of the 63D3 monoclonal antibody, the two leukemic samples without killer cell activity contained only 1% and 12% 63D3-positive cells. Cell sorting of 63D3-positive and -negative cells from two leukemias with killer cell activity demonstrated that the killer cell activity was restricted to the 63D3-positive fraction of AMoL cells. These data demonstrate that monoblastic leukemia cells can be potent killer cells and that killing activity is linked to the 63D3- defined cell surface molecule.
Von Willebrand factor in the vessel wall mediates platelet adherence
Authors: Stel HV , Sakariassen KS , de Groot PG , van Mourik JA , Sixma JJ
Published: October 2019

A monoclonal antibody directed against the von Willebrand factor moiety (vWF) of factor VIII-von Willebrand factor (FVIII-vWF), which blocks ristocetin-induced platelet aggregation as well as the binding of FVIII- vWF to platelets in the presence of ristocetin, inhibited platelet adherence to human artery subendothelium when present in normal flowing blood. This monoclonal antibody, CLB-RAg 35, inhibited platelet adherence as a function of the shear rate. At wall shear rates below 500 s-1, platelet adherence was not affected, but at higher shear rates platelet adherence was gradually inhibited, reaching an average of 11% of the normal value at 2,500 s-1. Indirect immunofluorescence established the reactivity of CLB-RAg 35 with vWF present in artery subendothelium. Pretreatment of normal vessel walls with this antibody inhibited adherence of platelets in blood from a patient with severe homozygous von Willebrand's disease and in blood from normal individuals. The inhibition was shear-rate dependent and significant at high shear rates (2,500 s-1). By adding increasing amounts of purified FVIII-vWF to normal blood, the inhibition was gradually overcome. These data indicate that vWF present in the vessel wall contributes appreciably to platelet adherence. At high wall shear rates, platelet adherence is mediated virtually completely by both plasma FVIII-vWF and vWF in the vessel wall. At low wall shear rates (below 500 s-1), platelet adherence occurs independent of FVIII-vWF in plasma and vWF in the vessel wall.
The role of activated human platelets in prothrombin and factor X activation
Authors: Rosing J , van Rijn JL , Bevers EM , van Dieijen G , Comfurius P , Zwaal RF
Published: October 2019

The effect of activated human platelets in intrinsic factor X activation was compared with their effect in prothrombin activation. Compared with unstimulated platelets, platelets triggered by the combined action of collagen plus thrombin showed a tenfold activity increase in prothrombin activation, and a 20-fold rate enhancement in factor X activation. Treatment of collagen plus thrombin-stimulated platelets with N.naja phospholipase A2 almost completely abolished their activity in prothrombin and factor X activation. Since no significant cell lysis occurs during phospholipase treatment, this indicates that platelet phospholipids, exposed at the membrane exterior, play an essential role in the interaction of platelets with the proteins of the prothrombin and factor X-activating complexes. The time course of generation of the procoagulant platelet surface was different when the amount of coagulation factors present in the assay systems was varied. At suboptimal concentrations of coagulation factors, maximum platelet activity was reached after a shorter time period than at saturating concentrations. When measured at suboptimal amounts of coagulation factors, the platelet activity in prothrombin and factor X activation is also more sensitive to phospholipase treatment. Experiments with synthetic phospholipid mixtures show that prothrombin and factor X activation are optimal at low mol% phosphatidylserine when high concentrations of factor Va and factor VIIIa are employed. The optimal mol% phosphatidylserine increases when the concentrations of nonenzymatic protein cofactors are lowered. These findings are discussed in relation to a model in which phosphatidylserine, exposed at the outer surface of activated platelets, plays an essential role in prothrombin and factor X activation. It is proposed that this phosphatidylserine is not homogeneously distributed in the platelet outer membrane, but that areas with different phosphatidylserine density participate in coagulation factor activation.
Inherited phosphofructokinase deficiency in dogs with hyperventilation- induced hemolysis: increased in vitro and in vivo alkaline fragility of erythrocytes
Authors: Giger U , Harvey JW , Yamaguchi RA , McNulty PK , Chiapella A , Beutler E
Published: October 2019

Two male English springer spaniel dogs with a chronic hemolytic anemia and sporadic hemolytic crises, historically related to “stress” situations, were studied. Although canine erythrocytes are in general known to be more alkaline fragile, erythrocytes from both patients began to lyse earlier, at significantly lower pH values (near pH 7.4 at 37 degrees C), than erythrocytes from control dogs. Hyperventilation induced by 30 minutes of exercise, placement in a 39 degrees C water bath, or intravenous doxapram increased venous blood pH in dog 1 and control dogs, but transient hemoglobinemia, hemoglobinuria, and severe bilirubinuria occurred only in the studied patient. The erythrocyte phosphofructokinase (PFK) activity was severely decreased in both dogs (10% of controls). The erythrocyte 2,3-diphosphoglycerate content was markedly reduced and the cell chloride content was consequently increased. This change in cell chloride content is related to an increase in the erythrocyte pH, which may partially explain the pathogenesis of hemolysis in canine PFK deficiency. Thus, these studies demonstrate a presumably inherited erythrocyte PFK deficiency in English springer spaniels, which causes an increased in vitro and in vivo erythrocyte alkaline fragility. Dogs with PFK deficiency and inducible hemolytic crises may become a valuable genetic animal model in which to study the pathophysiology of hemolysis.
The E rosette-associated antigen of T cells can be identified on blasts from patients with acute myeloblastic leukemia
Authors: Mirro J , Antoun GR , Zipf TF , Melvin S , Stass S
Published: October 2019

Monoclonal antibody T-11, considered specific for the sheep erythrocyte rosette-associated antigen of T cells, reacted with leukemic blasts from four of 23 patients with morphologic and cytochemical criteria for acute myeloblastic leukemia (AML). Although 83%, 87%, 88%, and 96% of the blasts from these patients reacted with T-11, only one patient demonstrated a small percentage of heat-stable E rosettes (5%). Antibody 9.6, which also reacts with the E rosette-associated antigen, was tested on blasts from two of the T-11-positive patients and was also strongly reactive (96% and 98%). Dual staining of blasts from these two patients demonstrated a small number of cells that simultaneously expressed the E rosette-associated antigen and myeloid- associated cytochemistries (myeloperoxidase [MPO] and Sudan black B). Additionally, leukemic blasts were identified that simultaneously expressed the E rosette-associated antigen and contained Auer rods. Antibody OKT-11 immunoprecipitated a 48,100-dalton glycoprotein from these leukemic blasts that is similar in molecular weight to that previously determined for the T cell surface protein (Tp50), thus providing strong evidence that this molecule can be found in some cases of AML. Because cells simultaneously expressing both the E rosette- associated antigen and MPO were identified, it would appear likely that leukemic blasts with only the E rosette-associated antigen or only MPO arose from the same progenitor. Our findings further demonstrate that the epitopes identified by antibodies OKT-11, T-11, and 9.6 are not always associated with, or sufficient for, 37 degrees C E rosette formation and can be found on blasts from patients with AML.
Neutrophils autoinactivate secretory products by myeloperoxidase- catalyzed oxidation
Authors: Clark RA , Borregaard N
Published: October 2019

The neutrophil response to inflammatory stimuli involves the formation of reactive oxygen species and secretion of granule enzymes. In studying secretion of vitamin B12 binding protein by human neutrophils, we noted a major decrease in total recoverable activity from the extracellular fluid plus the stimulated cells (54% of resting cells). Recovery of B12 binding protein from neutrophils exposed to phorbol myristate acetate or opsonized zymosan was significantly enhanced on addition of heme enzyme inhibitors (azide, cyanide) or catalase or when halide-free medium was used. The changes in B12 binding protein recovery were attributable entirely to increases in extracellular fluid levels, and cell pellet content was unaffected. These data indicate extracellular destruction of functional B12 binding protein by the halide-dependent heme enzyme myeloperoxidase and H2O2. Kinetic studies demonstrated rapid secretion of B12 binding protein in the first two to five minutes, followed by its inactivation over the next 20 to 30 minutes. A cell-free extract of vitamin B12 binding protein was readily inactivated on exposure to purified myeloperoxidase, H2O2, and a halide. These findings document a functional interaction among products of the neutrophil specific granules (B12 binding protein), azurophil granules (myeloperoxidase), and metabolic burst (H2O2). They provide an interesting model for the modulation of the inflammatory response by oxidation of secretory products of neutrophils.
Fechtner syndrome--a variant of Alport's syndrome with leukocyte inclusions and macrothrombocytopenia
Authors: Peterson LC , Rao KV , Crosson JT , White JG
Published: October 2019

This study reports a family comprising four generations in whom nephritis, deafness, congenital cataracts, macrothrombocytopenia, and leukocyte inclusions were observed in varying combinations in eight of 17 members. The family differs from others reported in that their hematologic abnormalities include not only macrothrombocytopenia, but also small, pale blue cytoplasmic inclusions in the neutrophils and eosinophils. Light microscopic appearance of the inclusions resembled that of toxic Dohle bodies and inclusions of May-Hegglin anomaly, but their ultrastructural appearance was unique. The inclusions consisted of clusters of ribosomes and small segments of rough endoplasmic reticulum (RER). They lacked the parallel 10-nm filaments characteristic of May-Hegglin anomaly and the parallel strands of RER seen in toxic Dohle bodies. Platelets were large, but their light and ultrastructural appearance was not significantly different from normal platelets. Platelet aggregation in response to epinephrine, arachidonate, thrombin, adenosine diphosphate, collagen, and ristocetin was normal. Levels of nucleotides and serotonin were elevated in proportion to cell volume. The concentration of adenosine triphosphate secreted and the percentage of arachidonic acid converted to thromboxane B2 were proportional to cell number. Deafness was high-tone sensorineural. Renal disease ranged from microscopic hematuria to end- stage renal failure necessitating dialysis and kidney transplantation. All affected adults had cataracts. This family represents a variant of Alport's syndrome with cataracts and leukocyte inclusions that, because of the associated macrothrombocytopenia, may be confused with May- Hegglin anomaly.
Rapid method for isolation of normal human peripheral blood eosinophils on discontinuous Percoll gradients and comparison with neutrophils
Authors: Roberts RL , Gallin JI
Published: October 2019

Previous studies on human eosinophils often have used cells from patients with hypereosinophilia syndrome or parasitosis owing to the difficulty in isolating pure populations of eosinophils from normal individuals. In the present study, human eosinophils were isolated with a purity of 97%, with 70% recovery from normal individuals with blood eosinophil counts of less than 3%. Human eosinophils are denser than neutrophils, but the range of densities of the two cell types overlap, making purification of eosinophils by density-gradient centrifugation difficult. However, if neutrophils were exposed to the chemotactic peptide (f-Met-Leu-Phe), which did not stimulate eosinophils, the neutrophils' density decreased, shifting them away from the density of eosinophils. Whole normal blood anticoagulated with EDTA was incubated at 37 degrees C for 15 minutes with 10(-6) mol/L f-Met-Leu-Phe and then layered over a discontinuous Percoll gradient (65% and 75% in diluted phosphate-buffered saline) and centrifuged at 400 g for 25 minutes at 22 degrees C. The cell layer between the 65% and 75% Percoll was collected and washed, and hypotonic lysis was used to remove erythrocytes. This cell layer contained 97.3 +/- 0.7% eosinophils (N = 8) with a yield of 4.9 X 10(4) eosinophils per milliliter of whole blood, or 70% of the total eosinophil count. The isolated eosinophils were in a quiescent state but responded to Escherichia coli endotoxin- activated serum with shape change and chemotaxis, membrane depolarization, and reduced nitroblue tetrazolium (96.0 +/- 1.0%), when stimulated with phorbol myristate acetate. In phagocytic assays, 89.3 +/- 1.3% of the eosinophils ingested Candida albicans v 96.0% +/- 1.0% of neutrophils. In contrast, the eosinophils did not respond chemotactically, alter membrane potential, or reduce nitroblue tetrazolium when treated with f-Met-Leu-Phe, and studies with f-Met-Leu- [3H]Phe showed that normal eosinophils lacked expression of receptors for f-Met-Leu-Phe. In control studies, normal eosinophils that were not exposed to f-Met-Leu-Phe during purification also failed to respond to f-Met-Leu-Phe, indicating intrinsic differences between normal eosinophils and neutrophils. Thus, exposure of whole blood to f-Met-Leu- Phe, followed by separation on Percoll is a simple method for rapid isolation of normal human eosinophils.
Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium
Authors: van Hinsbergh VW , Bertina RM , van Wijngaarden A , van Tilburg NH , Emeis JJ , Haverkate F
Published: October 2019

Confluent cultures of endothelial cells from human umbilical cord were used to study the effect of activated human protein C (APC) on the production of plasminogen activators, plasminogen activator-inhibitor, and factor VIII-related antigen. Addition of APC to the cells in a serum-free medium did not affect the production of tissue-type plasminogen activator (t-PA) or factor VIII-related antigen; under all measured conditions, no urokinase activity was found. However, less plasminogen activator-inhibitor activity accumulated in the conditioned medium in the presence of APC. This decrease was dose dependent and could be prevented by specific anti-protein C antibodies. No decrease was observed with the zymogen protein C or with diisopropylfluorophosphate-inactivated APC. APC also decreased the t-PA inhibitor activity in endothelial cell-conditioned medium in the absence of cells, which suggests that the effect of APC is at least partly due to a direct effect of APC on the plasminogen activator- inhibitor. High concentrations of thrombin-but not of factor Xa or IXa-- had a similar effect on the t-PA inhibitor activity. The effect of APC on the plasminogen activator-inhibitor provides a new mechanism by which APC may enhance fibrinolysis. The data suggest that activation of the coagulation system may lead to a secondary increase of the fibrinolytic activity by changing the balance between plasminogen activator(s) and its (their) fast-acting inhibitor.
Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III
Authors: Wolf M , Boyer C , Tripodi A , Meyer D , Larrieu MJ , Mannucci PM
Published: October 2019

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.
Increased expression of a novel c-abl-related RNA in K562 cells
Authors: Leibowitz D , Cubbon R , Bank A
Published: October 2019

The c-abl locus is translocated from chromosome 9 to chromosome 22 in chronic myelogenous leukemia (CML), creating the Philadelphia chromosome (22q-, Ph1), one of the most consistent chromosomal abnormalities found in human hematologic malignancy. The K562 cell line is a human cell line originally derived from a patient with CML. We have isolated cloned human c-abl probes to analyze the organization and expression of abl genes in patients with CML and in K562 cells. With these probes, we confirm the amplification of abl genes in K562 cells. In addition, we demonstrate the presence of increased amounts of a novel RNA species hybridizing to a c-abl probe in K562 cells. This same large RNA species is present in addition to two normal transcripts in the leukemic cells of patients with CML. These results provide evidence that the c-abl locus is abnormally expressed in CML.
Studies of a familial platelet disorder
Authors: Dowton SB , Beardsley D , Jamison D , Blattner S , Li FP
Published: October 2019

At least 22 members of a large kindred have a bleeding tendency resulting from an autosomal dominant disorder of platelet production and function. Phenotypic manifestations include mild to moderate thrombocytopenia, bleeding time prolongation, and abnormal platelet aggregation. Platelet survival time is normal. The platelet disorder in this family appears to differ from known hereditary thrombocytopenic or thrombocytopathic syndromes and may represent a new genetic disease. Six family members reportedly developed hematologic neoplasms: acute monocytic leukemia nine years after treatment for congenital neuroblastoma; lymphosarcoma at age 10 years; myeloid leukemia at age 23 years; acute myelocytic leukemia at age 62 years; leukemia of unknown type at age 48 years; and lymphocytic lymphoma at age 52 years.
Human bone marrow and peripheral blood T lymphocyte depletion: efficacy and effects of both T cells and monocytes on growth of hematopoietic progenitors
Authors: Levitt L , Kipps TJ , Engleman EG , Greenberg PL
Published: October 2019

The efficacy of four separate methods of human bone marrow T lymphocyte depletion was assessed, and the effect of T cells and monocytes on in vitro growth of marrow (CFU-GEMM, BFU-E, and CFU-GM) and peripheral blood (BFU-E) hematopoietic progenitors was determined. Extent of T cell depletion was assessed by multiparameter fluorescent cell sorter (FACS) analysis and by functional studies. Cells staining positively by FACS analysis for one or more of three separate fluorescent pan-T cell monoclonal antibodies (MCAbs) comprised 8.4% to 9.5% of control marrow mononuclear cells (MNCs). T cells constituted 3.2% to 5.1% of marrow following single, sequential, or combination treatment with two different pan-T cell MCAbs (Leu 1 and TM1) plus complement, 1.5% to 2.2% of marrow following solid-phase immunoabsorption (“panning”), 0.2% of marrow after sheep cell rosetting, and only 0.05% of marrow after FACS selective cell sorting and gated separation. T cells made up 59% to 73% of control peripheral blood MNCs and 0.8% to 2.8% of peripheral MNCs following sheep cell rosetting plus treatment with Leu 1 MCAb and complement. Mitogen (PHA, Con A) and allogeneic MLC-induced blastogenic responses (stimulation indices, experimental/control or E/C) revealed a concordant decrement in marrow T cell function after MCAb plus complement (E/C of 3.9 to 9.0), after panning (E/C of 1.6 to 3.5) and after sheep cell rosetting (E/C of 0.7 to 1.3), compared with control marrow (E/C of 5.3 to 15.7). After T cell depletion, marrow BFU-E growth was 95% to 120% of control, CFU-GM growth was 90% to 108% of control, and CFU-GEMM growth was 89% to 111% of control. Marrow T cell and/or monocyte depletion did not alter erythropoietin-dependent BFU-E growth in the absence of Mo-conditioned medium (81% to 95% of control), and the addition of as many as 50 to 100 X 10(3) purified marrow monocytes or T cells to 10(5) autologous nonadherent T cell-depleted marrow target cells had a negligible (P greater than .1) effect on marrow BFU-E growth in vitro. Peripheral blood (PB) BFU-E/10(5) T- depleted target cells were 106% +/- 19% of expected; PB BFU-E growth was significantly diminished after monocyte depletion alone (7% +/- 6% of expected) or after monocyte plus T cell depletion (8% +/- 4% of expected).(ABSTRACT TRUNCATED AT 400 WORDS)
Glycoprotein-180 deficiency: genetics and abnormal neutrophil activation
Authors: Weisman SJ , Berkow RL , Plautz G , Torres M , McGuire WA , Coates TD , Haak RA , Floyd A , Jersild R , Baehner RL
Published: October 2019

Neutrophil function was studied in a patient with polymorphonuclear leukocyte (PMN) glycoprotein-180 deficiency and in her parents. PMNs of the patient had abnormal chemotaxis, phagocytosis, adherence, surface charge, and membrane-associated events of activation. Selective defects to C3b, immunoglobulin G (IgG), phorbol myristate acetate (PMA) and N- formyl-methionyl-leucyl-phenylalanine (FMLP) are described, although C3b receptor density was normal. The parents were found to have abnormal adherence to nylon-wool fibers, abnormal transmembrane potential depolarization with PMA, and reduced amounts of glycoprotein- 180 in their PMNs. These studies provide further evidence that the oxidative burst has several different pathways for activation. They demonstrate that the absence of a single PMN surface glycoprotein is associated with a broad spectrum of PMN functional abnormalities. Finally, the observations made in the parents support an autosomal recessive mode of inheritance.
Regulatory factors specific for adult and embryonic globin genes may govern their expression in erythroleukemia cells
Authors: Anagnou NP , Yuan TY , Lim E , Helder J , Wieder S , Glaister D , Marks B , Wang A , Colbert D , Deisseroth A
Published: October 2019

In order to test if trans-acting regulatory factors specific for globin genes of the adult and embryonic stages of development exist in erythroid cells, transcriptionally active embryonic and adult globin genes on the same chromosome were transferred by cell fusion from the human leukemia cell K562 into phenotypically adult mouse erythroleukemia cells. Restriction-fragment-length polymorphisms of the K562 zeta (embryonic) globin genes were used to establish that all three copies of human chromosome 16 present in the K562 cell showed the same pattern of human globin gene expression after transfer to the mouse erythroleukemia cell. Adult (alpha) but not embryonic (zeta) human globin mRNA was detected in all nine of the independently derived mouse erythroleukemia hybrid cells, each of which contained human chromosome 16. Restriction endonuclease studies of the K562 alpha- and zeta-globin genes after transfer into the mouse erythroleukemia cell showed no evidence of rearrangements or deletions that could explain this loss of zeta-globin gene expression. These data suggest that regulation of globin gene expression in these erythroleukemia cells involves trans-acting regulatory factors specific for the adult and embryonic stages of development.
Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds
Authors: Ragni MV , Sinha D , Seaman F , Lewis JH , Spero JA , Walsh PN
Published: October 2019

The relationship of clinical bleeding tendency and factor XI antigen (XI:Ag) in factor XI deficiency was studied in 78 members of 25 factor XI-deficient kindreds. Factor XI:Ag was measured in a competitive radioimmunoassay, using monospecific, heterologous anti-factor XI antibody. 125I-labeled factor XI, and staphylococcal protein A as the precipitating agent. Deficiency of factor XI clotting activity (XI:C), less than 0.62 U/mL, occurred in 48 individuals, 22 of whom experienced postoperative or posttraumatic bleeding: Their mean factor XI:C was 0.21 +/- 0.04 U/mL (SEM), and factor XI:Ag was 0.23 +/- 0.04 U/mL. The remaining 26 had no clinical bleeding, many despite surgical challenge: Their mean factor XI:C was 0.30 +/- 0.04 U/mL, and factor XI:Ag was 0.34 +/- 0.05 U/mL. In all, 13 kindreds had between 1 and 11 members with bleeding; the other 12 had none with deficient hemostasis. Two heterozygous factor XI-deficient individuals appeared to be positive for cross-reacting material (CRM+). The slope of the regression line for factor XI:C and factor XI:Ag data points in the 78 individuals tested did not differ from control, and all points fell within 95% confidence limits derived from control. In conclusion, bleeding tendency appears to be consistent within a given kindred and is not determined exclusively by factor XI:C or factor XI:Ag levels.
Adult acute lymphoblastic leukemia phenotypes defined by monoclonal antibodies
Authors: Sobol RE , Royston I , LeBien TW , Minowada J , Anderson K , Davey FR , Cuttner J , Schiffer C , Ellison RR , Bloomfield CD
Published: October 2019

Pretreatment peripheral blood and/or bone marrow blasts from 90 adults with acute lymphoblastic leukemia (ALL) were analyzed as part of a prospective treatment protocol study. Specimens were tested by immunofluorescence cytofluorometry for reactivity with the following monoclonal antibodies (MoAbs): BA-1 (B cell antigen); T101, OKT11 (pan- T cell antigens [T]); 3A1 (T cell antigen); MCS-2 (myeloid antigen); J5 common ALL antigen (CALLA); BA4 (Ia antigen [Ia]); BA-2 (lymphohematopoietic antigen). Four major phenotypic groups were identified: B lineage ALL (BA-1+T-) (64%), T lineage ALL (T+BA-1-MCS-2- ) (13%), unclassified ALL (BA-1-MCS-2-CALLA-T-) (9%) and myeloid antigen ALL (MCS-2+CALLA-T-) (7%). An additional group of patients, miscellaneous ALL (7%), was comprised of cases with unusual marker profiles. In B lineage ALL, all cases tested were Ia+MCS-2-, and the vast majority were CALLA+ (84%). In T lineage ALL, 42% expressed CALLA or Ia positivity. In unclassified ALL, the predominant phenotype was Ia+BA-2+. In myeloid antigen ALL, two of four tested were 3A1+ and all cases evaluated were BA-1-. Patients with myeloid antigen ALL were older (median age, 66 years) than patients in the other groups. The T lineage ALL group had higher leukocyte counts (median WBCs, 183,000/microL) and an increased incidence of anterior mediastinal mass at presentation. All patients received identical induction therapy. In CALLA+B lineage ALL, 30 of 46 (65%) achieved a complete remission. While the number of patients evaluated was small, 9 of 9 CALLA-B- lineage ALL and only two of six myeloid antigen ALL cases responded with a complete remission. The data suggest that these MoAbs are useful in the characterization of adult ALL.
Regulation of erythropoiesis in long-term hamster marrow cultures: role of bone marrow-adherent cells
Authors: Eastment CE , Ruscetti FW
Published: October 2019

The initial establishment of hamster long-term bone marrow (LTBM) cultures requires formation of an adherent stromal layer, but continued long-term proliferation of these cultures is best accomplished by removal of the suspension cells from the adherent layer and subsequent incubation in liquid suspension culture. Continued maintenance of bone marrow cells in the presence of the adherent layer for more than four to six weeks leads to a decline and eventual disappearance of erythroid and multipotent colony-forming cells. Addition of erythropoietin to LTBM suspension cultures produces mature, hemoglobinized erythrocytes. Incubation of the same cells plus erythropoietin in the presence of autologous parental adherent layers markedly inhibits both terminal erythroid differentiation and the number of detectable erythroid burst- forming units (BFU-Es). This erythroid inhibition occurs primarily within the first 24 hours with little or no effect on CFU-GEMMs or granulocyte-macrophage colony-forming units (GM-CFUs). However, continued incubation for seven days produces a reduction in all parameters. Removal of suspension cells from the adherent layer and restimulation with erythropoietin allows regeneration of erythropoiesis. Pretreatment of suspension cells with erythropoietin for 96 hours before exposure to the adherent culture only slightly inhibits erythropoiesis, suggesting that more mature erythroid progenitors are unaffected. Conditioned medium from the marrow adherent layer (ALCM) produces similar erythroid inhibitory effects in LTBM cultures with as little as two hours of incubation. The inhibition is actively produced by the adherent cells, since cycloheximide abolishes its production, while indomethacin has no apparent effect. Adherent marrow stromal cells may regulate hemopoiesis through negative as well as positive humoral signals, and they are particularly effective in erythroid regulation.
Small cell lung cancer, endocrine cells of the fetal bronchus, and other neuroendocrine cells express the Leu-7 antigenic determinant present on natural killer cells
Authors: Bunn PA Jr , Linnoila I , Minna JD , Carney D , Gazdar AF
Published: October 2019

Small cell lung cancer is distinguished from other lung cancer histologic types by possessing a variety of neuroendocrine properties. Anti-Leu-7 is a monoclonal antibody that recognizes a 110,000-dalton molecular weight glycoprotein initially described on natural killer cells and subsequently reported on a variety of normal and malignant neural and neuroendocrine cell types. We have found intense anti-Leu-7 binding to a large number of small cell lung cancers, while other lung cancer types were negative or showed only weak and focal binding. Other antigens expressed by natural killer cells, lymphocytes, and monocytes were never or less often expressed on small cell lung cancer cells. In addition, we report for the first time anti-Leu-7 binding by carcinoids, carotid body tumors, pheochromocytomas, endocrine cells of the fetal bronchus and the adult intestine, and select pancreatic islet cells. Anti-Leu-7 binding by small cell lung cancer is consistent with a derivation from pulmonary precursor cells, and anti-Leu-7 staining is clinically useful for the identification of human neuroendocrine tumors of the amine precursor uptake and decarboxylation (“APUD”) type.
The leftward deletion alpha-thal-2 haplotype in a black subject with hemoglobin SS
Authors: Embury SH , Gholson MA , Gillette P , Rieder RF
Published: October 2019

We have identified a black individual with homozygous sickle cell anemia who is the silent carrier of alpha-thalassemia (genotype - alpha/alpha alpha) due to heterozygosity for the leftward deletion alpha-thal-2 haplotype. This deletion has not been described previously in a black subject and is the only leftward deletion that we have found among 255 alpha-thal-2 chromosomes from sickle cell subjects. Its effects on the clinical, hematologic, biosynthetic, and cellular pathology of sickle cell anemia resemble those reported for the common alpha-thalassemia genotypes of the black population.
Isolation of microtubule coils from normal human platelets
Authors: White JG , Krumwiede M
Published: October 2019

The blood platelet is the only human cell known to have a circumferential band of microtubules. However, the mechanisms involved in assembly of the multi-looped coil, its interaction with the cell membrane to support discoid shape, and constriction into tight rings around centrally concentrated organelles in activated platelets are unknown. Separation of the microtubule rings from intact platelets would permit new approaches to solution of these questions. The present study has used simultaneous detergent extraction and fixation to isolate intact microtubule coils in significant numbers from suspended platelets for the first time. Isolated coils closely resembled the circumferential band observed in thin sections of plastic embedded platelets and in platelets prepared by the negative-stain whole-mount method. Enough microtubule coils could be recovered from suspensions of concentrated platelets to permit counting and quantitation on microscope grids. Results of this study will permit new approaches to clarification of the structural physiology of platelet microtubule coils.
Diphenylhydantoin-induced pure red cell aplasia
Authors: Dessypris EN , Redline S , Harris JW , Krantz SB
Published: October 2019

The pathogenesis of diphenylhydantoin-induced pure red cell aplasia was investigated in the case of a 32-year-old man who developed pure red cell aplasia while he was under treatment with diphenylhydantoin. The patient's serum IgG purified from serum drawn at the time of diagnosis suppressed normal allogeneic marrow colony-forming (CFU-E) and burst- forming (BFU-E) and autologous blood BFU-E growth in vitro only in the presence of diphenylhydantoin. This IgG-diphenylhydantoin complex had no effect on CFU-GM growth in vitro. Normal IgG or patient's IgG purified from serum drawn after the remission of red cell aplasia had no effect on erythroid colony formation in vitro in the presence of diphenylhydantoin. The IgG-diphenylhydantoin complex exerted no direct cytotoxic effect on normal marrow erythroblasts, CFU-E, and BFU-E, nor did it interfere with the action of erythropoietin on marrow erythroblasts. These studies suggest that diphenylhydantoin-induced red cell aplasia is immunologically mediated through an IgG inhibitor, which requires the presence of the drug to suppress erythroid colony formation in vitro. This inhibitor seems to exert its effect on erythroid progenitors at or beyond the stage of differentiation of CFU- E, but not on erythroblasts.
Regulation of heme synthesis in erythroid cells: hemin inhibits transferrin iron utilization but not protoporphyrin synthesis
Authors: Ponka P , Schulman HM
Published: October 2019

The inhibition of delta-aminolevulinic acid (ALA) synthase activity by heme is commonly thought to regulate the overall rate of heme synthesis in erythroid cells. However, since heme inhibits erythroid cell uptake of iron from transferrin, we have tested the hypothesis that in reticulocytes heme regulates its own synthesis by controlling the cellular acquisition of iron from transferrin rather than by controlling the synthesis of ALA. We found that hemin added to reticulocytes in vitro inhibits not only the total cell incorporation of 59Fe from transferrin but also the incorporation of [2–14C]-glycine and transferrin-bound 59Fe into heme. However, hemin did not inhibit [2 –14C]-glycine incorporation into protoporphyrin. Furthermore, cycloheximide, which increases the level of non-hemoglobin heme in reticulocytes, also inhibited [2–14C]-glycine into heme but not into protoporphyrin. With high concentrations of ferric pyridoxal benzoylhydrazone (Fe-PBH), which, independent of transferrin and transferrin receptors, can be used as a source of iron for heme synthesis in reticulocytes, significantly more iron is incorporated into heme than from saturating concentrations of Fe-transferrin. This suggests that some step (or steps) in the pathway of iron from extracellular transferrin to protoporphyrin limits the overall rate of heme synthesis in reticulocytes. In addition, hemin in concentrations that inhibit the utilization of transferrin-bound iron for heme synthesis has no effect on the incorporation of iron from Fe-PBH into heme. Our results indicate that in reticulocytes heme inhibits and controls the utilization of iron from transferrin but has no effect on the enzymes of porphyrin biosynthesis and ferrochelatase. This mode of regulation of heme synthesis may be a specific characteristic of the hemoglobin biosynthetic pathway.
The effect of 5-azacytidine and its analogues on blast cell renewal in acute myeloblastic leukemia
Authors: Motoji T , Hoang T , Tritchler D , McCulloch EA
Published: October 2019

Blast cells from patients with acute myeloblastic leukemia were exposed to 5-azacytidine (5-aza) and its analogues 5-aza 2′-deoxycytidine (5- aza-dr) and 6-azacytidine (6-aza). Simple negative exponential survival curves were obtained for the three drugs, but the sensitivity varied; 5- aza-dr was most toxic, 6-aza was least toxic, and 5-aza was intermediate. Colonies surviving drug exposure were replated; 5-aza and 5-aza-dr were found to increase secondary plating efficiency, whereas 6- aza was inactive. The findings provide indirect evidence for a role for DNA methylation in the regulation of blast cell self-renewal.
The monoclonal antibodies alpha S-HCL 1 (alpha Leu-14) and alpha S-HCL 3 (alpha Leu-M5) allow the diagnosis of hairy cell leukemia
Authors: Schwarting R , Stein H , Wang CY
Published: October 2019

To define cell surface antigens associated with hairy cell leukemia (HCL), and to gain better insight into the origin of this disease, we developed monoclonal antibodies against spleen cells of a patient with this disease. Although none of these antibodies alone proved specific for the leukemic cells, two of them, designated alpha S-HCL 1 (alpha Leu-14) and alpha S-HCL 3 (alpha Leu-M5) were found to be valuable in the diagnosis of HCL when used in combination. alpha S-HCL 1 recognizes an antigen associated with greater than 95% of B cells in the peripheral blood. Biochemical analysis identified this antigen as a single polypeptide chain with a molecular weight of 150,000 daltons (150 kilodaltons). alpha S-HCL 1 expression on hairy cells is markedly increased when compared with normal B lymphocytes isolated from peripheral blood, tonsils, and spleens. alpha S-HCL 3 reacts with an antigen present on hairy cells but also on monocytes, macrophages, in a lower density on neutrophils, and a small percentage (less than 2%) of lymphocytes. The antigen recognized by alpha S-HCL 3 is composed of a non-covalently linked biomolecular complex of 90 and 150 kilodaltons. Since the HCL 3 antigen was not detectable on other lymphomas of either T or B cell type, the co-expression of S-HCL 1 and S-HCL 3 on hairy cells is a unique marker for this disease.
Drug-induced endocytosis of neonatal erythrocytes
Authors: Matovcik LM , Junga IG , Schrier SL
Published: October 2019

The erythrocytes of the newborn infant have many properties that distinguish them from those of adults, and their membranes are also different from those of adult erythrocytes. We compared the ability of adult and neonatal RBCs to undergo endocytosis on exposure to drugs. Using a quantitative method, we showed that neonatal erythrocytes undergo a greater degree of endocytosis than do adult RBCs in response to primaquine, vinblastine, and chlorpromazine, and are sensitive to lower concentrations of the drugs. Some forms of drug-induced endocytosis are red cell age-dependent; when RBCs were separated by density gradient centrifugation, the membranes of the younger, less dense populations of both the neonatal and adult RBCs were capable of more extensive internalization than those of the denser, older RBCs. Neonatal RBCs of a given density undergo more endocytosis than do adult RBCs of the same density, suggesting that the membrane of the neonatal RBC is less stable and capable of more of the reorganization reflected in endocytosis than is the adult RBC membrane.
Cell-bound autologous immunoglobulin in erythrocyte subpopulations from patients with sickle cell disease
Authors: Green GA , Rehn MM , Kalra VK
Published: October 2019

Previously, we have demonstrated a parallel between most-dense (bouyant density) sickle erythrocyte subpopulations and most-dense aged normal red cells in the organization of membrane components in the intact cell. The present study has addressed the possibility that a corresponding similarity may exist between most-dense sickled red cell subpopulations and aged normal erythrocytes in the development of membrane protein components that function as receptors for autologous immunoglobulin (Ig). Autologous IgG retained by density-fractionated erythrocytes has been estimated by a nonequilibrium 125I-protein A (Staphylococcus aureus) binding assay. Results show that most-dense sickle cell fractions contain more (2.7-fold and 1.8-fold, P less than .005) cell-bound IgG in comparison to younger sickle erythrocyte fractions sedimenting at low density. Parallel findings were obtained after similar analyses of normal (homozygous-A) erythrocyte fractions. Detection of the presence of specific IgG was also carried out by direct binding of fluorescein isothiocyanate-conjugated anti-human IgG to density-separated red cell fractions followed by analyses of the fluorescent cell populations by flow cytometry. Results showed significantly higher levels of IgG bound to most-dense (12.1% +/- 2.5% and 8.8% +/- 0.5%-) sickle red cell subpopulations (P less than .005) in comparison to younger sickle erythrocyte fractions sedimenting at low densities (3.8% +/- 0.32% and 4.7% +/- 1.6% IgG-positive red cell subpopulation). These results indicate that some of the same membrane changes that occur at about 120 days in normal red cells are also apparent in the chronologically younger (life span in vivo, ten to 40 days) sickle erythrocyte. The increased retention of IgG by most-dense irreversibly sickled cell-enriched fractions in comparison to least- dense reversibly sickled cells or pre-irreversibly sickled erythrocyte fractions, suggests that alterations in the topography of the sickle cell membrane during the transformation in vivo to the most-dense irreversibly sickled cell morphology may produce the unmasking of cryptic antigenic sites. In addition, these findings may indicate that opsonization of specific erythrocyte subpopulations may play a role in the pathophysiology of sickle cell disease.
Bone marrow transplantation for acute nonlymphocytic leukemia in first remission: analysis of prognostic factors
Authors: Bostrom B , Brunning RD , McGlave P , Ramsay N , Nesbit M Jr , Woods WG , Hurd D , Krivit W , Kim T , Goldman A
Published: October 2019

Prognostic factors were reviewed retrospectively for 39 children and adults aged 1 to 40 years (median 14 years) with acute nonlymphocytic leukemia (ANLL) who attained a first remission and underwent bone marrow transplantation from November 1976 to July 1983. The preparation regimen for transplantation was cyclophosphamide (60 mg/kg/d for two days) followed by total body irradiation (either 750 cGy single dose at 26 cGy/min, n = 37, or 1,320 cGy fractionated at 10 cGy/min, n = 2). Twenty-three patients are surviving disease free with a median followup of three years. The three-year estimated disease-free survival is 55% +/- 17% (+/- 2 SE). Five patients have relapsed from 92 to 756 days after transplantation for an estimated relapse rate of 21% +/- 18%. Two factors, the white blood cell (WBC) count and the French-American- British (FAB) classification at leukemia diagnosis were found to be of prognostic importance. Patients with a WBC of less than 20,000/microL at diagnosis had a three-year estimated disease-free survival of 74% +/- 18% v 26% +/- 24% for those with a WBC of greater than or equal to 20,000 (P = .008). The estimated relapse rate was 6% +/- 12% for patients with a WBC at diagnosis less than 20,000 v 53% +/- 38% for patients with a WBC at diagnosis of greater than or equal to 20,000 (P = .01). Patients with myeloid morphology at diagnosis (FAB M1,2,3) had an estimated relapse rate of 9% +/- 12% v patients with monocytoid morphology (FAB M4,5a) whose estimated relapse rate was 58% +/- 44% (P = .05). Our data suggest that a high WBC count at poor prognostic factors for patients with ANLL who undergo bone marrow transplantation in first remission after conditioning with cyclophosphamide plus total body irradiation.