bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin
Authors: Austin RC , Rachubinski RA , Fernandez-Rachubinski F , Blajchman MA
Published: October 2019

Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.
Comparison of intravenous bolus injection or continuous infusion of recombinant single chain urokinase-type plasminogen activator (saruplase) for thrombolysis. A canine model of combined coronary arterial and femoral venous thrombosis
Authors: Rapold HJ , Wu ZM , Stassen T , Van de Werf F , Collen D
Published: October 2019

The thrombolytic efficacy of recombinant unglycosylated full length single chain urokinase-type plasminogen activator (rscu-PA, saruplase), applied either as single intravenous bolus or as a continuous infusion over 60 minutes, was studied in 5 randomized blinded groups of 5 dogs with combined copper coil induced coronary artery thrombosis and 125I- fibrin labeled femoral vein clots. Infusion of 1 mg/kg recu-PA (group I) induced coronary recanalization in 4 of 5 dogs and 98 +/- 1% (mean +/- SEM) venous clot lysis. Bolus injection of 1 mg/kg recu-PA (group II) caused reflow in 3 of 5 dogs and 88 +/- 5 percent venous clot lysis. Infusion of 0.5 mg/kg rescu-PA (group III) achieved reflow in 3 of 5 dogs and 52 +/- 6% venous clot lysis. Bolus injection of 0.5 mg/kg rscu-PA (group IV) induced reflow in 4 of 5 dogs and 48 +/- 12% venous clot lysis. Placebo infusion (group V) was associated with late recanalization in 1 of 5 dogs and 18 +/- 8% venous clot lysis. Coronary artery reocclusion after reflow was not observed in groups I and II, but occurred in 2 of 3 animals in group III and in 3 of 4 animals in group IV (P = .02). The time to reflow in responsive animals was 22 +/- 5 minutes with infusion of 0.5 or 1 mg/kg rscu-PA and 14 +/- 1 minute with bolus injection of 0.5 or 1 mg/kg (P = .14). Depletion of fibrinogen and alpha 2-antiplasmin to less than 25% of baseline levels was observed in the 5 dogs given 1 mg/kg rscu-PA by bolus and in 3 of the 5 dogs given 1 mg/kg rscu-PA via infusion, but in none of the dogs that received 0.5 mg/kg rscu-PA (P less than .001). Plasma clearance rates were 170 +/- 44 and 230 +/- 30 mL/minute after bolus injection and 190 +/- 47 and 310 +/- 56 mL/minute during infusion of rscu-PA for the 1 mg/kg and 0.5 mg/kg doses respectively. Thus, intravenous bolus injection of rscu-PA (saruplase) appears to be equipotent to an infusion over 60 minutes for both coronary and venous thrombolysis. This animal model of combined arterial and venous thrombolysis may be useful for the evaluation of new thrombolytic strategies.
Transmission of HTLV-I by blood transfusion and its prevention by passive immunization in rabbits
Authors: Kataoka R , Takehara N , Iwahara Y , Sawada T , Ohtsuki Y , Dawei Y , Hoshino H , Miyoshi I
Published: October 2019

To determine the minimum volume of blood required to transmit human T- cell leukemia virus type I (HTLV-I), heparinized blood was collected from a virus-infected female rabbit and aliquots of 10, 5, 1, 0.5, 0.1, and 0.01 mL were transfused into groups of two male rabbits each. All 10 rabbits transfused with 10 to 0.1 mL and 1 of 2 rabbits transfused with 0.01 mL seroconverted for HTLV-I after 2 to 4 weeks. HTLV-I- producing lymphoid cell lines of recipient origin were established from one seroconverted rabbit of each aliquot group. To determine the ability of passive immunization to protect against HTLV-I infection, two groups of three rabbits were first transfused with 5 mL of blood from the same virus-infected rabbit and then infused after 24 or 48 hours with 10 mL of HTLV-I immune globulin (77 mg/mL of IgG) prepared from seropositive healthy persons. None of the 24-hour immunization group seroconverted for HTLV-I during the observation period of six months; however, all of the 48-hour immunization group became seropositive after 2 to 4 weeks. These results indicate that HTLV-I can be transmitted with as little as 0.01 mL of virus-infected blood, and that passive immunization is effective in preventing cell-to-cell infection of HTLV-I when given within 24 hours of transfusion of virus- infected blood.
Bleeding complications associated with cardiopulmonary bypass
Authors: Woodman RC , Harker LA
Published: October 2019

Bleeding after CPB has been difficult to characterize and its treatment equally difficult to standardize. The complexity of this problem is related to the hemostatic process, the technical variations in the operative procedures, and the many uncontrolled variables associated with CPB, including the effects of anesthetic or pharmacologic agents, the nature of the priming solution, hemodilution, hypothermia, the type of oxygenator, and the use of transfused blood products. Although there are multiple and generally predictable complex changes in the hemostatic mechanism during CPB, the temporary loss of platelet function is the most common and clinically relevant. This transient platelet dysfunction occurs in all patients undergoing CPB; however, it only causes excessive bleeding in a small percentage of patients. Unfortunately, it has not yet been possible to predict which patients will develop hemorrhagic complications, although prolonged pump times are a contributing risk factor. Over the past decade there has been extensive investigation into the management of bleeding associated with CPB, provoked primarily by the increased awareness of transfusion- transmitted viral diseases and the inappropriately excessive use of homologous blood products. Several approaches to autotransfusion of shed blood and autologus blood donation have been developed to minimize perioperative homologous blood transfusion. Pharmacologic agents such as desmopressin, aprotinin, and topical fibrin glues have also been introduced to improve hemostasis during CPB. The protease inhibitor aprotinin is particularly promising in the reduction of bleeding associated with CPB when given prophylactically. Aprotinin may provide new insights into the mechanism of CPB-induced platelet dysfunction. Desmopressin is indicated only for the treatment of bleeding after CPB. The management of bleeding associated with CPB will undoubtedly
All-trans retinoic acid in acute promyelocytic leukemias. II. In vitro studies: structure-function relationship
Authors: Chomienne C , Ballerini P , Balitrand N , Daniel MT , Fenaux P , Castaigne S , Degos L
Published: October 2019

All-trans retinoic acid induces leukemic cells from patients with acute promyelocytic leukemia (M3) to differentiate in vitro to mature granulocytes which express the CD15 antigen and are capable of respiratory burst function. Of 35 M3 samples, only one failed to respond. In eight cases, we compared the efficacy of two naturally occurring isomers of retinoic acid, all-trans RA and 13-cis RA. Both isomers induce maximal differentiation at 10(-6) mol/L. The maximal response was maintained at 10(-7) mol/L for the all-trans but not for the 13-cis RA. We also observed that the metabolites 4-oxo-all-trans and 4-oxo-13-cis were effective at 10(-6) mol/L. This 1 order of magnitude difference in the in vitro differentiating potencies of all- trans RA and 13-cis RA in the blasts of promyelocytic leukemias predicts a difference in the clinical efficacy of the two drugs.
Identification and characterization of plasma cells in normal human bone marrow by high-resolution flow cytometry
Authors: Terstappen LW , Johnsen S , Segers-Nolten IM , Loken MR
Published: October 2019

The low frequency of plasma cells and the lack of specific cell surface markers has been a major obstacle for a detailed characterization of plasma cells in normal human bone marrow. Multiparameter flow cytometry enabled the identification of plasma cells in normal bone marrow aspirates. The plasma cells were located in a unique position in the correlation of forward light scattering, orthogonal light scattering, and immunofluorescent-labeled CD38. The identity of the sorted cell populations was confirmed by microscopic examination of Wright's stained slides and slides stained for cytoplasmic immunoglobulin using polyclonal antibodies reactive with light chains; ie, anti-kappa fluorescein isothiocyanate and anti lambda phycoerythrin (PE). The purity of the sorted plasma cells was greater than 97% (n = 4). The average frequency of plasma cells in normal bone marrow aspirates was low--0.25% of the nucleated cells (n = 7)--but surprisingly consistent between individuals (SD = .05; range 0.14% to 0.30%). A detailed analysis showed two distinct populations of plasma cells: (1) A population relatively smaller by forward light scattering expressed CD22, CD35, and sigE and was identified as early plasma cells (ie, lymphoplasmacytoid), and (2) a population larger by forward light scattering lacked these markers and was identified as mature plasma cells. The antigenic profile of the normal plasma cells was determined in two-color immunofluorescence studies. The expression of cell surface immunoglobulin G (IgG), IgA, IgE, IgD, IgM, and the cell surface antigens CD10, CD11b, CD13, CD11c, CD14, CD15, CD16, CD19, CD22, CD20, CD33, CD35, CD45, and HLA-DR was determined on the plasma cells. A significant heterogeneity in cell surface antigen expression was observed within the plasma cell population. Unexpectedly, myeloid- specific cell surface antigens such as CD33 and CD13 and the early B- cell antigen identified by CD10 were expressed on a proportion of plasma cells. These observations imply that the association of myeloid and early B-cell markers described in multiple myeloma may not be associated with the neoplasia but is a normal phenomenon.
Developmental regulation of granulocytic cell binding to hemonectin
Authors: Campbell AD , Long MW , Wicha MS
Published: October 2019

Hemonectin (HN), a component of the bone marrow (BM) extracellular matrix which promotes adhesion of cells in the granulocytic lineage, was purified to near homogeneity and tested for its ability to mediate attachment of normal and leukemic cells of granulocytic lineage. Purified HN immobilized on plastic substrates promoted serum-free attachment of normal granulocyte/macrophage progenitor cells (CFC-GM), using an in situ attachment assay in which cell attachment is inhibited by specific polyclonal antisera. When unfractionated BM cells were allowed to attach to purified HN and stained in situ, HN preferentially bound cells at earlier stages of granulocytic differentiation. These observations were confirmed using cells of the HL-60 progranulocytic cell line which mirrored this differentiation-stage specific binding to HN. HN promoted attachment of 60% of uninduced HL-60 cells which were arrested at the progranulocyte stage, whereas only 15% of uninduced HL- 60 cells attached to uncoated plastic and 4% to attached plastic coated with equal microgram quantities of bovine serum albumin (BSA). When HL- 60 cells were induced to differentiate along the granulocytic pathway by incubation with dimethylsulfoxide (DMSO), attachment to hemonectin was reduced. Thus, both primary BM granulocytic cells and a granulocytic cell line show preferential attachment of those cells at earlier stages of differentiation. This developmentally regulated binding suggests a mechanism for release of maturing BM into the peripheral circulation.
Enhanced endogenous leukotriene biosynthesis in patients treated with granulocyte-macrophage colony-stimulating factor
Authors: Denzlinger C , Kapp A , Grimberg M , Gerhartz HH , Wilmanns W
Published: October 2019

The hematopoietic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is being used in clinical trials for its potential in the treatment of hematopoietic insufficiency due to various causes. Involvement of leukotrienes in the effects of GM-CSF is suggested by analytical and pharmacologic evidence obtained in vitro. However, until now no data in support of a role of leukotrienes in GM-CSF action in vivo have been presented. In the present investigation this question was approached by measurement of endogenous cysteinyl leukotriene formation in patients treated with the cytokine for cytopenia induced by cytostatic drugs or for refractory anemia with excess of blasts (RAEB). Endogenous cysteinyl leukotriene formation was assessed by determination of urinary leukotriene metabolites using combined high- performance liquid chromatography and radioimmunoassay analysis. After GM-CSF administration a distinct increase in urinary cysteinyl leukotrienes was found in the cytopenic and the RAEB patients that ranged from 2.3- to 57-fold and 2.4- to 333-fold, respectively. In the cytopenic patients the increase in leukotriene production was correlated to an expansion of peripheral blood leukocytes; RAEB patients responded to GM-CSF with enhanced leukotriene biosynthesis even if the peripheral leukocytes decreased, possibly due to an abnormal number and/or irritability of leukotriene-producing cells. The increase in endogenous leukotriene production during therapy with GM- CSF may indicate that leukotrienes play a role in GM-CSF action in vivo.
Immunophenotypic characteristics of cerebrospinal fluid cells in children with acute lymphoblastic leukemia at diagnosis
Authors: Homans AC , Barker BE , Forman EN , Cornell CJ Jr , Dickerman JD , Truman JT
Published: October 2019

The presence of meningeal involvement in children with acute lymphoblastic leukemia (ALL) may have important prognostic and therapeutic implications. Conventional methods of diagnosing central nervous system (CNS) leukemia rely on the interpretation of cerebrospinal fluid (CSF) cell morphology, which may produce ambiguous results in the presence of minimal leukemic involvement. A methodology has been developed for immunophenotyping small numbers of CSF cells while preserving cell morphology. CSF samples from 33 children with CD10 (common ALL antigen [CALLA]) positive ALL were examined at initial presentation using both conventional morphology and this combined immunohistopathologic technique. Six (18%) of the samples contained lymphoblasts or cells considered morphologically suspicious for leukemic involvement. Nine additional samples (27% of the total) had normal CSF morphology, but contained increased numbers of CALLA positive cells. Twelve of the 33 samples were also examined for the simultaneous presence of nuclear terminal deoxynucleotidyl transferase (TdT) and demonstrated increased numbers of cells positive for both TdT and CD10. These data suggest that a large proportion of children with ALL may have abnormalities of CSF cells at initial diagnosis consistent with the presence of occult leukemic involvement.
Amplified expression of three jun family members inhibits erythroleukemia differentiation
Authors: Prochownik EV , Smith MJ , Snyder K , Emeagwali D
Published: October 2019

Several different proto-oncogenes have been shown to influence cellular differentiation. One of the most widely studied model systems has been the Friend murine erythroleukemia cell (F-MELC) line, which can be induced to undergo erythroid differentiation by a variety of chemical agents. Constitutive overexpression of either the c-myc or c-myb proto- oncogenes has been previously shown to inhibit F-MELC differentiation, whereas c-myc antisense sequences accelerate the process. To investigate the potential involvement of other proto-oncogenes and immediate early response genes in F-MELC differentiation, we studied the expression of the three known members of the jun family as well as another gene, egr-1, which, like the jun family members, is expressed as an immediate early response gene in growth factor-stimulated quiescent cells. All four genes were expressed in F-MELC, although the levels of expression and modes of regulation differed. Transfection with amplifiable c-jun, junB, or junD expression plasmids inhibited differentiation, whereas transfection with an egr-1 expression plasmid was without effect. These results indicate that jun family members play a role in mediating F-MELC differentiation. The known inhibitory effect of phorbol ester tumor promoters on F-MELC differentiation may be the result of their known stimulation of jun expression.
Transcellular biosynthesis of sulfidopeptide leukotrienes during receptor-mediated stimulation of human neutrophil/platelet mixtures
Authors: Maclouf J , Murphy RC , Henson PM
Published: October 2019

The ability of different cell types to cooperate in the metabolism of reactive intermediates of arachidonic acid such as leukotriene A4 (LTA4) is currently receiving considerable attention. Of critical importance is the demonstration that transfer of LTA4 could occur under conditions when relatively low amounts of LTA4 are produced such as would be expected for in vitro receptor-mediated stimulation. Stimulation of human neutrophils with a combination of chemotactic factor (formyl-methionyl-leucyl-phenylalanine, FMLP) and phagocytosable particles (opsonized zymosan) resulted in little production of LTC4 alone, but measurable quantities appeared when platelets were coincubated. When these agonists were added to platelets alone in the absence of neutrophils, no LTC4 was produced. In the presence of stimulated neutrophils, the final synthesis of LTC4 was shown to occur within the platelets (from neutrophil-derived LTA4) by experiments using platelets that had been prelabeled with 35S-cysteine to label intracellular platelet glutathione. Other 35S-labeled sulfidopeptide leukotriene metabolites were also produced in this coincubation of neutrophils and platelets. The observed synergy between FMLP and opsonized zymosan in the production of LTC4 when neutrophils and platelets were coincubated may involve priming the neutrophil for LTA4 production. Activation of platelets or endothelial cells with thrombin did not alter the capacity of either cell to convert exogenously added LTA4 into LTC4. This would support the suggestion that even when platelets are activated they retain their capacity to metabolize LTA4 into LTC4. Finally, previous exposure of the platelets to LTA4 did not affect subsequent metabolism of arachidonic acid by the cyclooxygenase pathway to thromboxane A2 (TXA2) except at very high concentration of LTA4. These results suggest that cell-cell interactions may be critical determinants of the profile of eicosanoids produced in physiologic and pathophysiologic circumstances. In particular, we believe that both endothelial cells and platelets can, together with neutrophils, contribute relatively large amounts of sulfidopeptide leukotrienes to inflammatory and thrombotic events. These cell-cell interactions are aspirin-insensitive; therefore, aspirin-treated platelets are capable of synthesizing the vasoconstrictor LTC4 from neutrophil LTA4 at a time when they can no longer produce thromboxane.
In vitro rosetting, cytoadherence, and microagglutination properties of Plasmodium falciparum-infected erythrocytes from Gambian and Tanzanian patients
Authors: Hasler T , Handunnetti SM , Aguiar JC , van Schravendijk MR , Greenwood BM , Lallinger G , Cegielski P , Howard RJ
Published: October 2019

To understand the molecular mechanisms that lead to sequestration of red blood cells infected with mature stages of Plasmodium falciparum and to examine the relevance of earlier studies on adherence properties of laboratory-derived P falciparum parasites to the natural parasite population, we analyzed Gambian and Tanzanian isolates for in vitro cytoadherence and antibody-mediated microagglutination. Eighteen cryopreserved isolates of ring-stage parasites were cultured for 20 to 30 hours in vitro, in the patients original erythrocytes, to the trophozoite and schizont stage. All parasites were positive in the microagglutination assay with at least one of four African hyperimmune sera. In a rosetting assay, only 2 of the 18 isolates were strongly positive (35% and 41% of parasitized erythrocytes with more than two uninfected cells bound). Thirteen isolates showed either intermediate (5% to 18%) or low (less than 5%) rosetting while three isolates did not form rosettes. Infected cell-binding of the different isolates to immobilized CD36 or thrombospondin, or C32 melanoma cells correlated with the percentage of mature parasites in the blood samples (r = .932 for CD36, r = .946 for thrombospondin, and r = .881 for C32 melanoma cells). There was a high correlation between binding to CD36 and thrombospondin (r = .982). The extent of infected cell rosetting with uninfected cells in these blood samples was not correlated with these other receptor properties. We also observed coexpression of rosetting and cytoadherence receptors on the same parasitized erythrocytes.
Deficiency of glycosyl-phosphatidylinositol-linked membrane glycoproteins of leukocytes in paroxysmal nocturnal hemoglobinuria, description of a new diagnostic cytofluorometric assay
Authors: van der Schoot CE , Huizinga TW , van 't Veer-Korthof ET , Wijmans R , Pinkster J , von dem Borne AE
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease that affects not only red cells, but other blood cells as well. The common defect is supposed to be an acquired deficiency of glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins, which may be present already at the hematopoietic stem cell level. Recently, a panel of monoclonal antibodies (MoAbs) has become available directed against various GPI- linked membrane proteins. This makes it possible to study various cell lineages for the deficiency of such proteins in PNH in more detail. Using cytofluorography, we could show that the granulocytes of 20 different PNH patients miss not only GPI-linked FcRIII (CD16 antigen), but also three other GPI-linked proteins, ie, CD24 antigen, CD67 antigen and a granulocyte-specific 50 to 80 Kd antigen. The affected granulocytes were not only neutrophils but also eosinophils, as was found in a more detailed analysis of three patients. Moreover, in all 10 PNH patients tested, the monocytes were found to be deficient for the GPI-linked CD14 antigen, and we found with CD24 and CD55 (DAF) antibodies that lymphocytes may be involved as well. However, abnormal B and T lymphocytes were detected only in a subset of patients (2 of 10 tested). The uniform deficiency of GPI-linked proteins of granulocytes allows the introduction of a new diagnostic cytofluorometric assay for PNH with MoAbs against GPI-linked granulocytic antigens. This test was positive in all PNH patients studied and not in a group of 40 control patients or 50 normal donors, with the exception of three of 16 aplastic anemia (AA) patients. In the three AA patients, subpopulations (10% to 20%) of PNH granulocytes could be detected, whereas these patients had a negative acidified serum (Ham) test. This indicates that the new test is more sensitive than the Ham test and allows the early diagnosis of PNH in AA. An advantage of the neutrophil assay is that, in contrast to the Ham test, it is not influenced by recent red-cell transfusions. Moreover, it is possible to quantify the number of affected cells by single cell analysis.
Allogeneic marrow transplantation in patients with acute myeloid leukemia in first remission: a randomized trial of two irradiation regimens [see comments]
Authors: Clift RA , Buckner CD , Appelbaum FR , Bearman SI , Petersen FB , Fisher LD , Anasetti C , Beatty P , Bensinger WI , Doney K
Published: October 2019

A randomized trial of 12.0 Gy versus 15.75 Gy of total body irradiation (TBI) was performed in patients with acute myeloid leukemia undergoing allogeneic marrow transplantation while in first complete remission. All patients received 120 mg/kg cyclophosphamide followed by TBI and marrow from HLA-identical siblings. Cyclosporine and methotrexate were used for prophylaxis against acute graft-versus-host disease (GVHD). Thirty-four patients received 2.0-Gy fractions of irradiation daily for 6 days and 37 received 2.25-Gy fractions daily for 7 days. The 3-year actuarial probabilities for relapse-free survival were 0.58 for the patients who received 12.0 Gy and 0.59 for those who received 15.75 Gy. The 3-year probabilities of relapse were 0.35 for the 12.0 Gy group and 0.12 for the 15.75 Gy group (P = .06). The 3-year probabilities of transplant-related mortality were 0.12 and 0.32, respectively (P = .04). The probability of moderate to severe acute GVHD was 0.21 for the 12.0 Gy group and 0.48 for the 15.75 Gy group (P = .02). Patients exposed to the higher irradiation dose received less immunoprophylaxis against, and had a higher incidence of, acute GVHD. The increased dose of TBI significantly reduced the probability of relapse but did not improve survival because of increased mortality from causes other than relapse.
Contamination of peripheral blood stem cell harvests by circulating neuroblastoma cells
Authors: Moss TJ , Sanders DG , Lasky LC , Bostrom B
Published: October 2019

Peripheral blood stem cells (PBSC) are being used as one alternative to autologous marrow rescue for patients with neuroblastoma and other solid malignancies. Some physicians prefer use of PBSC because less risk of tumor contamination is believed to exist. This hypothesis was evaluated by immunocytologic analysis of blood samples and concurrently drawn bone marrow (BM) samples and of PBSC harvests obtained from 31 patients with disseminated neuroblastoma. We found circulating neoplastic cells in 75% of specimens analyzed at diagnosis, in 36% during therapy, and in 14% of PBSC harvests. Tumor cells in blood obtained during therapy did not appear until 3 months after the time of diagnosis. Clearance of circulating neuroblastoma cells was documented after two courses of induction chemotherapy. Six of 13 patients with minimal or no BM disease had positive blood specimens. We conclude that substantial risk of tumor contamination of PB harvests exists and recommend that induction chemotherapy be administered before hematopoietic progenitor cells are collected from blood.
Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib
Authors: Fujimura Y , Usami Y , Titani K , Niinomi K , Nishio K , Takase T , Yoshioka A , Fukui H
Published: October 2019

Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.
Hemoglobin Montreal: a new variant with an extended beta chain due to a deletion of Asp, Gly, Leu at positions 73, 74, and 75, and an insertion of Ala, Arg, Cys, Gln at the same location
Authors: Plaseska D , Dimovski AJ , Wilson JB , Webber BB , Hume HA , Huisman TH
Published: October 2019

The unstable hemoglobin Montreal with a deletion of three amino acid residues (Asp, Gly, Leu) at positions 73, 74, and 75 of the beta chain and an insertion of four residues (Ala, Arg, Cys, Gln) at the same location was observed in a 7-year-old Canadian boy suffering from a moderate hemolytic anemia. The introduction of an extra amino acid residue and of other changes in the crevice where the heme group is located is the likely cause of the instability of this hemoglobin variant. The above listed changes were detected through analyses of tryptic peptides of the beta-Montreal chain, sequencing of amplified DNA, and hybridization of amplified DNA with appropriate, 32P-labeled, oligonucleotide probes. It is suggested that a mispairing involving the AGTG sequences at codons 66 and 67 and at codons 72 and 73 of the normal beta gene caused a repetition of a 16-bp segment, while a deletion of 10 nucleotides due to recombination or slippage followed by a second short deletion during DNA repair resulted in the modified sequence of the beta-Montreal gene.
Use of leukocyte-depleted platelet concentrates for the prevention of refractoriness and primary HLA alloimmunization: a prospective, randomized trial
Authors: van Marwijk Kooy M , van Prooijen HC , Moes M , Bosma-Stants I , Akkerman JW
Published: October 2019

Compared with conventional transfusion regimes a strong reduction in HLA alloimmunization and refractoriness to platelet transfusions is obtained when both red blood cell concentrates (RBCs) and platelet concentrates (PCs) are depleted of leukocytes by filtration. Because most of the leukocyte contamination is introduced by transfusion of RBCs, filtration of RBCs appears rational, but uncertainty exists regarding the degree of leukocyte-depletion of PCs needed for the prevention of HLA alloimmunization and refractoriness. We conducted a prospective trial and randomized patients with acute leukemia to receive leukocyte-depleted PCs prepared either by centrifugation (mean leukocyte count 35 x 10(6)/PC of 6 U) or by filtration (mean leukocyte count less than 5 x 10(6)/PC of 6 U). Both groups received RBCs that were filtered after prior removal of the buffy coat. Clinical refractoriness occurred in 46% (12 of 26) of the evaluable patients that were transfused with centrifuged PCs and only in 11% (3 of 27) in the filtered group (P less than .005). De novo anti-HLA antibodies were detected in 42% (11 of 26) patients in the centrifuged group and only in 7% (2 of 27) of the patients receiving filtered PCs (P less than .004). In 8 of 11 alloimmunized patients in the centrifuged group antibodies were detected in the first 4 weeks of transfusion therapy while none of the patients in the filtered group became immunized against HLA antigens during that period. We conclude that for the prevention of HLA alloimmunization and refractoriness to platelet transfusions from random donors, both RBCs and PCs have to be leukocyte- depleted by filtration.
Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors
Authors: Kodama H , Iizuka M , Tomiyama T , Yoshida K , Seki M , Suda T , Nishikawa S
Published: October 2019

Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.
Platelet vitronectin receptor expression differentiates Iraqi-Jewish from Arab patients with Glanzmann thrombasthenia in Israel
Authors: Coller BS , Cheresh DA , Asch E , Seligsohn U
Published: October 2019

Glanzmann thrombasthenia is a rare, inherited disorder of the platelet glycoprotein IIb/IIIa (GP IIb/IIIa) complex. We previously identified two distinct populations with this disorder in Israel, Iraqi-Jews and Arabs. The groups are indistinguishable in hemorrhagic symptoms and platelet GP IIB/IIIa receptor deficiency, but they differ in their platelet immunodetectable GP IIIa (beta 3), with the Iraqi-Jewish population expressing no detectable GP IIIa and the Arab population expressing small amounts. We have now examined the platelets of these two populations as well as normal platelets for the alpha v beta 3 vitronectin receptor. Normal platelets contained between approximately 50 to 100 alpha v beta 3 vitronectin receptors as judged by the binding of antibodies to both alpha v (LM142) and the intact alpha v beta 3 vitronectin receptor complex (LM609). In addition, normal platelets bound to immobilized vitronectin in the presence of 1 mmol/LMnCl2; the adhesion was mediated predominantly through GP IIb/IIIa, but with a distinct contribution by the alpha v beta 3 vitronectin receptor, as determined by monoclonal antibody inhibition studies. Iraqi-Jewish patients' platelets had a profound decrease in immunodetectable alpha v beta 3 vitronectin receptors, and their platelets did not adhere well to vitronectin. In contrast, Arab patients' platelets had normal or increased numbers of platelet alpha v beta 3 vitronectin receptors, and these receptors functioned well in the vitronectin adhesion assay, taking over much of the adhesion mediated by GP IIb/IIIa in normal platelets. These studies define further the heterogeneity of the molecular basis of Glanzmann thrombasthenia; they also have more widespread implications for understanding the synthesis and function of the beta 3 family of integrin receptors.
Identification of a cell-surface antigen associated with activated T lymphoblasts and activated platelets
Authors: Sutherland DR , Yeo E , Ryan A , Mills GB , Bailey D , Baker MA
Published: October 2019

We have identified and biochemically characterized an antigen, 8A3, which is expressed on activated T lymphoblasts and activated platelets. Monoclonal antibodies to 8A3 were raised against the primitive lymphoid/myeloid cell line KG1a and additionally bound to the erythroleukemia-derived cell line HEL, whilst exhibiting little or no reactivity with a panel of other hematopoietic cell lines. The 8A3 antigen was expressed on poorly differentiated T-cell leukemias and on phytohemagglutinin-activated T-cells maintained in interleukin-2 (7,000 sites/cell). This antigen, though not detected on resting platelets, was expressed on thrombin-activated platelets (2,000 sites/platelet). Antibodies to 8A3 identified polypeptides of Mr 170,000 and 150,000 in lysates of surface-iodinated KG1a cells, T lymphoblasts, and activated platelets under both reducing and nonreducing conditions. However, peptide mapping and susceptibily to glycosidases indicated that the 8A3 antigen was a monomeric glycoprotein of Mr 170,000 which contained two N-linked endoglycosidase H-sensitive glycans, and that the Mr 150,000 structure was derived from it by proteolytic degradation. The 8A3 antigen was not detectably phosphorylated in KG1a cells in vivo, nor did immune complexes containing it exhibit kinase activity in vitro. Structural and serologic characteristics of the 8A3 antigen indicate that it is different from other previously described leukocyte activation antigens including transferrin receptors, interleukin-2 receptors, members of the integrin family of adhesion molecules, or “restricted” members of the leukocyte-common antigen/CD45 cluster. Furthermore, the 8A3 antigen does not appear to be related to the other previously described activation-specific platelet molecule, GMP140/PADGEM. This antibody may be useful in monitoring T-cell activation status in some clinical situations and in characterizing clinically relevant activation-associated platelet membrane alterations.
FDP D-dimer induces the secretion of interleukin-1, urokinase-type plasminogen activator, and plasminogen activator inhibitor-2 in a human promonocytic leukemia cell line
Authors: Hamaguchi M , Morishita Y , Takahashi I , Ogura M , Takamatsu J , Saito H
Published: October 2019

We studied the effect of fibrinogen degradation products D, E, and D- dimer on a human promonocytic leukemia cell line, NOMO-1. After exposure to a 10(-5)-mol/L fragment D or D-dimer, the cells displayed macrophage-like characteristics, such as adherence to plastic surfaces, and showed approximately a twofold increase in response to the nitroblue tetrazolium reduction test. The secretion of interleukin-1 alpha (IL-1 alpha) into the medium was markedly stimulated by a 10(-5)- mol/L fragment D, E, and D-dimer, whereas a significant increase in IL- 1 beta secretion was observed only in D-dimer-stimulated cells. In addition, D-dimer induced a rapid increase in urokinase-type plasminogen activator on day 1 (0.52 +/- 0.02 ng/mL v 0.07 +/- 0.01 ng/mL in the control culture) and a slow increase in plasminogen activator inhibitor-2 on day 5 (3.9 +/- 1.6 ng/mL v 1.2 +/- 0.2 ng/mL in the control culture). An increase in tissue factor (TF) was also demonstrated on the cell surface of NOMO-1 cells exposed to fragment D or D-dimer by indirect immunofluorescence using an anti-TF monoclonal antibody. Scatchard plot analysis showed that fragment D and D-dimer bound to the NOMO-1 cells with a kd of 3.3 nmol/L and 2.7 nmol/L, respectively. These results suggest that fragment D-dimer specifically stimulates cells of monocyte-macrophage lineage to secrete key substances that regulate blood coagulation, fibrinolysis, and inflammation.
Purification and partial characterization of a human hematopoietic precursor population
Authors: Smith C , Gasparetto C , Collins N , Gillio A , Muench MO , O'Reilly RJ , Moore MA
Published: October 2019

This study reports the development of an assay, the Pre-colony-forming unit (CFU) assay, which detects human hematopoietic precursors. The Pre- CFU assay is based on the observation that precursors to CFU- granulocyte-macrophage (CFU-GM) that are undetectable in clonogenic assays differentiate into CFU-GM preferentially following treatment in suspension culture with recombinant human interleukin-1 alpha (rhIL-1 alpha) combined with rhIL-3. Using the Pre-CFU assay, hematopoietic precursors were detected in human bone marrow depleted of CFU-GM progenitors and differentiated hematopoietic elements via 4- hydroperoxycyclophosphamide treatment coupled with selection for CD34+ cells (4-HCresistant/CD34+ marrow). Additionally, the Pre-CFU assay detected recovery of hematopoiesis substantially earlier than the CFU- GM assay in primates following myeloablation with 5-fluorouracil. The Pre-CFU assay was used to asses purification of a phenotypically defined hematopoietic precursor population, the lin-CD34+ population. The lin-CD34+ population lacks detectable surface markers for T-cell, B- cell, natural killer cell, and myeloid lineage, possesses the CD34 antigen, is devoid of CFU-GM progenitors, and yields Pre-CFU assay values comparable with 4-HCresistant/CD34+ marrow. Using a combination of phenotypic analysis and Pre-CFU assay analysis, the action of rhIL-1 alpha plus rhIL-3 treatment on lin-CD34+ cells was further characterized. The data indicate that rhIL-1 alpha plus rhIL-3 treatment induces proliferation and differentiation of early hematopoietic precursors into progenitors and terminally differentiated cells, without inducing a significant expansion of the precursor population itself.
Incidence of aplastic anemia in Bangkok. The Aplastic Anemia Study Group
Authors: Issaragrisil S , Sriratanasatavorn C , Piankijagum A , Vannasaeng S , Porapakkham Y , Leaverton PE , Kaufman DW , Anderson TE , Shapiro S , Young NS
Published: October 2019

The annual incidence of aplastic anemia in metropolitan Bangkok, Thailand, and its five suburban provinces was prospectively determined. All patients first diagnosed during the period from January through December 1989 who met specific clinical and pathologic criteria were included. Thirty-two cases were identified, yielding an overall incidence of 3.7 per million. The incidence rates for the age groups 0 through 24, 25 through 59, and over 60 years were 4.3, 3.2, and 2.1 per million, respectively; the highest rate, 7.2 per million, was found for individuals aged 15 to 24 years. The male-to-female ratio was 1.9. The incidence of aplastic anemia in Bangkok is higher than that reported in recent European studies. The peak rate in young persons is almost fourfold higher than in comparable recent western studies and suggests an environmental etiology peculiar to Thailand.
Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies
Authors: Chong BH , Du XP , Berndt MC , Horn S , Chesterman CN
Published: October 2019

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease- treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex- specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.
Immunolocalization of integrin receptors in normal lymphoid tissues
Authors: Zutter MM
Published: October 2019

The integrin superfamily of cell adhesion receptors consists of heterodimeric glycoproteins composed of unique alpha and beta subunits. These receptors mediate cell-substrate and cell-cell adhesive properties for a variety of cell types. This investigation has focused on the histologic distribution of the beta 1 subfamily of integrins within lymphoid tissues including tonsil, lymph node, spleen, thymus, and appendix. The dendritic cells of both follicular center and thymic origin express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6, as well as the beta 1 integrin subunits. Most lymphoid cells in normal tissues do not express the alpha 1, alpha 2, alpha 3, alpha 5, and alpha 6 subunits, or the alpha v beta 3 integrin. The beta 1 subunit is expressed by all lymphocytes but with variable intensity. Increased levels of the alpha 5 and beta 1 subunits are observed in the follicular light zone, suggesting a role for these integrins in B-cell activation. Although the alpha 4 subunit is expressed by all lymphoid cells, an increased expression of alpha 4 and decreased expression of beta 1 by the mantle zone B-cell compartment is noted in comparison with the decreased expression of alpha 4 and increased expression of beta 1 by follicular center B-cells. These studies suggest that alpha 4 may be paired with a beta subunit other than beta 1 on the mantle zone lymphoid population. Thus, integrin expression by cells of lymphoid tissues varies with location and function and differs significantly from integrin expression observed on circulating and cultured peripheral blood lymphocytes.