bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry
Authors: Shattil SJ , Cunningham M , Hoxie JA 
Published: October 2019

Abstract
Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.
Anemia of the Belgrade rat: evidence for defective membrane transport of iron
Authors: Bowen BJ , Morgan EH 
Published: October 2019

Abstract
The mechanisms underlying the impaired utilization of transferrin-bound iron by erythroid cells in the anemia of the Belgrade laboratory rat were investigated using reticulocytes from homozygous anemic animals and transferrin labeled with 59Fe and 125I. The results were compared with those obtained using reticulocytes from phenylhydrazine-treated rats and iron-deficient rats. Each step in the iron uptake mechanism was investigated, ie, transferrin-receptor interaction, transferrin endocytosis, iron release from transferrin, and transferrin exocytosis. Although there were quantitative differences, no fundamental difference was found in any of the abovementioned aspects of cellular function when the reticulocytes from Belgrade rats were compared with those from iron-deficient animals. The basic defect in the Belgrade reticulocytes must therefore reside in subsequent steps in iron uptake, after it is released from transferrin within endocytotic vesicles, ie, in the mechanism by which it is transferred across the lining membrane of the vesicles into the cell cytosol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of reticulocyte ghosts extracts demonstrated a prominent protein band of mol wt 69,000 that was absent or present only in low concentration extracts from the other two types of reticulocytes. This may be a result of the genetic defect.
Immunological specificity and mechanism of action of IgG lupus anticoagulants
Authors: Pengo V , Thiagarajan P , Shapiro SS , Heine MJ 
Published: October 2019

Abstract
Although observations have implied that lupus anticoagulants have immunologic specificity toward anionic phospholipids, this assumption has been directly demonstrated in only one patient with a monoclonal IgM paraprotein. We tested the generality of this hypothesis directly by isolating five IgG lupus anticoagulants from patients with lupuslike syndromes and/or thrombosis. IgG lupus anticoagulant fractions were isolated free of other plasma proteins and free of contaminating phospholipid by adsorption to and elution from cardiolipin-cholesterol- dicetyl phosphate liposomes, followed by chromatography on protein A- Sepharose. Cardiolipin liposomes, but not phosphatidylcholine liposomes, were capable of removing all, or nearly all, lupus anticoagulant activity from patient plasma. The affinity-purified IgG preparations reacted with cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid, but not with phosphatidylcholine or phosphatidylethanolamine, and inhibited calcium- dependent binding of prothrombin and of factor X to phosphatidylserine- coated and to cardiolipin-coated surfaces. F(ab')2 fragments retained lupus anticoagulant activity and bound to cardiolipin in an enzyme- linked immunosorbent assay (ELISA). Anticardiolipin and lupus anticoagulant activity were both present in acidic fractions on isoelectric focusing. These data strongly suggest that most, if not all, lupus anticoagulants are antibodies that have immunologic specificity towards anionic phospholipids, thereby blocking the calcium- mediated binding of vitamin K-dependent coagulation factors to coagulation-active phospholipid surfaces.
Immunophenotyping of acute myeloid leukemia using monoclonal antibodies and the alkaline phosphatase-antialkaline phosphatase technique
Authors: Hanson CA , Gajl-Peczalska KJ , Parkin JL , Brunning RD 
Published: October 2019

Abstract
The leukemic cells from 41 cases of acute myeloid leukemia (AML) and 17 cases of acute lymphocytic leukemia (ALL) were immunophenotyped by the alkaline phosphatase-antialkaline phosphatase (APAAP) immunocytochemical technique utilizing eight monoclonal antibodies (MoAb) reactive with cells of myeloid origin and seven MoAb reactive with lymphoid antigens. Ninety percent of the cases of AML reacted with one or more of the pan-myeloid MoAb, My7, My9, or 20.3. Reactivity of the myeloid panel of MoAb showed some correlation with the French- American-British (FAB) classification of AML. Five of six cases of acute promyelocytic leukemia (APL) were HLA-DR negative; the one HLA-DR- positive APL had a minor population of HLA-DR-negative promyelocytes. OKM5 and/or My4 reacted with 16 of 16 monocytic leukemias. No specific marker of early erythroid development was identified. AP3, a MoAb reactive with platelet glycoprotein (GPIIIa), was specific for acute megakaryoblastic leukemia. Immunocytochemistry was also helpful in classifying seven cases of AML with equivocal or negative routine cytochemistry. Two cases of AML had minor populations of blasts detected by the APAAP technique that were immunologically distinct from the major blast population; these minor populations emerged as the predominant cell type at relapse. Two cases of ALL expressed multiple myeloid and lymphoid antigens. Two other cases that morphologically were ALL reacted with only myeloid MoAb; one consisted entirely of immature basophils on ultrastructural examination. Immunophenotyping results using the APAAP technique were comparable with those obtained with flow cytometry. The APAAP technique is a reliable method for immunophenotyping leukemia that complements other methods of immunologic evaluation. The primary advantages of this method include its use with routinely prepared blood and bone marrow smears and the ability to correlate immunocytochemical reactions with morphology.
Erythropoietin production by the fetal liver in an adult environment
Authors: Flake AW , Harrison MR , Adzick NS , Zanjani ED 
Published: October 2019

Abstract
To gain insight into the mammalian liver to kidney erythropoietin (Ep) switch, we heterotopically transplanted livers from preswitch, switched, and postswitch fetal and newborn lambs into normal adult sheep. Recipients' serum Ep and circulating reticulocyte levels were serially determined until rejection of the graft and compared with identical samples from sham-operated control adult ewes. Transplantation of preswitch and switched fetal livers caused an impressive rise in recipients' serum Ep activity and provoked a corresponding increase in reticulocytosis. In contrast, Ep activity and reticulocyte counts did not change from preoperative levels in adult ewes transplanted with postswitch livers or in the sham-operated controls. The production of Ep by the preswitch fetal liver in the adult environment was not dependent on the presence or absence of host kidneys and was stimulated by anemic hypoxia. These results suggest that the fetal liver is capable of producing relatively large amounts of Ep activity, and the production of Ep can be maintained in the adult environment in the presence of functional adult kidneys. This argues against suppression of liver Ep production by renal Ep, or some other factor in the postnatal environment, and suggests that the liver to kidney switch of Ep production during ontogeny may represent a genetically determined event.
Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species
Authors: Bar-Ner M , Eldor A , Wasserman L , Matzner Y , Cohen IR , Fuks Z , Vlodavsky I 
Published: October 2019

Abstract
Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.
Detection of HIV antigen and specific antibodies to HIV core and envelope proteins in sera of patients with HIV infection
Authors: Cao YZ , Valentine F , Hojvat S , Allain JP , Rubinstein P , Mirabile M , Czelusniak S , Leuther M , Baker L , Friedman-Kien AE 
Published: October 2019

Abstract
The sera of well-characterized populations were examined for three markers of human immunodeficiency virus (HIV) infection; HIV antigen (HIV Ag), and antibodies to HIV envelope (gp41) and core (p24) proteins. Of 563 serum samples tested, 251 were from HIV-infected patients diagnosed as having AIDS manifested by opportunistic infections (AIDS-OI), AIDS-associated Kaposi's sarcoma (AIDS-KS), or AIDS-related complex (ARC). One hundred seventy-six specimens tested were from asymptomatic high-risk individuals, and 136 were from heterosexual control subjects or patients with non-AIDS-related disease. None of the 136 control individuals tested had HIV Ag or HIV antibodies to either p24 or gp41. Of the 427 HIV-seropositive individuals, 99% to 100% were positive for gp41 antibodies to HIV. In contrast, the seroprevalence of p24 antibodies to HIV varied from 23% to 83% and appeared to be inversely associated with the severity of the patients' clinical symptoms. When specimens were analyzed for the presence of HIV Ag, in seropositive individuals the prevalence rate for this marker was lowest (1.4%) in asymptomatic individuals and highest (50%) in the AIDS-OI diagnosed group. Also, 240 cases with AIDS-KS, AIDS-OI, and ARC and the group of asymptomatic high-risk individuals were analyzed for T helper/T lymphocytes (T4) cell number and T4/T8 ratio; only one (2.0%) HIV Ag-positive case showed a T4 cell number greater than 400 and a normal T4/T8 ratio. These studies appear to demonstrate a direct correlation between the presence of HIV Ag and the severity of clinical complications of HIV infection.
Deposition of autofluorescent eosinophil granules in pathologic bone marrow biopsies
Authors: Samoszuk MK , Espinoza FP 
Published: October 2019

Abstract
Eosinophil granules are intensely autofluorescent when excited by green light. To determine if eosinophils degranulate in the bone marrows of patients with a variety of diseases, we used green light epifluorescence microscopy to examine deparaffinized and dezenkerized sections of 49 bone marrow core biopsies. In 14 of the biopsies, there was striking extracellular deposition of intensely autofluorescent eosinophil granules in addition to numerous intact eosinophils. Among the 14 specimens with extracellular autofluorescence were seven cases of leukemia, four cases of non-Hodgkin's lymphoma, two cases of myelofibrosis, and one case of pancytopenia with eosinophilia. In the remaining 35 specimens, only intact eosinophils were identifiable. There was no extracellular autofluorescence in three normal marrows, four marrows from AIDS patients, or three biopsies from patients with idiopathic thrombocytopenic purpura (ITP). We conclude that green light epifluorescence microscopy identifies extracellular deposits of eosinophil granules in bone marrow biopsies of some neoplastic disorders and in diseases associated with reticulin fibrosis.
Monoclonal antibody Ki-B3 detects a formalin resistant antigen on normal and neoplastic B cells
Authors: Feller AC , Wacker HH , Moldenhauer G , Radzun HJ , Parwaresch MR 
Published: October 2019

Abstract
A new monoclonal antibody Ki-B3 produced by a fusion with leukemic cells of a centroblastic/centrocytic lymphoma (m.l. follicular) is introduced. This antibody predominantly recognizes B cells of follicular mantle and germinal center cells, as well as plasma cells in normal lymphoid tissue. Furthermore, 80% of all low- and high-grade B cell lymphomas are stained, whereas among T cell lymphomas, only four of 15 T lymphoblastic lymphomas were positive to Ki-B3. All peripheral T cell lymphomas showed a negative reaction. Additionally, Ki-B3 detects a small percentage of monocytes and some myelomonocytic leukemias. All epithelial tissues as well as all sarcomas tested were invariably negative. Ki-B3 precipitates a 220 kiloDalton (kD) molecular weight antigen similar to the leukocyte common antigen. Presumably Ki- B3 detects a subtype of the leukocyte common antigen that is predominantly expressed on mature and immature B cells. As the antigen is formalin resistant, Ki-B3 can be used in routine hematology on paraffin sections for the detection and differential diagnosis of B cell lymphomas.
The transglutaminase in vascular cells and tissues could provide an alternate pathway for fibrin stabilization
Authors: Greenberg CS , Achyuthan KE , Borowitz MJ , Shuman MA 
Published: October 2019

Abstract
A thrombin-independent transglutaminase (TG) has been identified in vascular cells and tissues from human, rabbit, rat, porcine, and bovine sources. The vascular TG had several properties that were similar but not identical to guinea pig liver TG. Both enzymes had similar chromatographic and electrophoretic properties, preferentially cross- linked the alpha-chains of fibrinogen, and reacted with polyclonal and monoclonal anti-guinea-pig liver TG antibodies. However, the TG from adult bovine aortic endothelial (ABAE) cells exhibited a novel Ca2+/Mg2+ dependence for enzymatic activity that was distinct from that of purified guinea pig liver TG. The mol wt of the vascular TG (79 +/- 3 kd) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was slightly lower than the purified guinea pig liver TG (85 +/- 9 kd). The TG antigen was detected by immunohistochemical techniques in association with the endothelial and smooth muscle cells of arteries, veins, venules, and capillaries. The TG antigen also codistributed with the fibronectin antigen along the hepatic sinusoids. The ABAE cell TG cross-linked alpha 2-plasmin inhibitor to fibrinogen and caused the modified fibrinogen to be 40- fold more resistant to plasminolysis. A thrombin-independent TG in vascular cells of blood vessels could provide an alternate pathway to inhibit fibrinolysis and promote fibrin stabilization.
An alpha-globin gene initiation codon mutation in a black family with HbH disease
Authors: Olivieri NF , Chang LS , Poon AO , Michelson AM , Orkin SH 
Published: October 2019

Abstract
The molecular basis of hemoglobin H disease in a Black family of Canadian origin was investigated. Affected individuals had a combination of deletion and nondeletion alpha-thalassemia mutations on different chromosomes. Cloning and sequencing of the DNA of one member with the nondeletion form revealed a new thalassemia mutation, an A---- G substitution, in the initiation codon of the remaining alpha-globin gene of a rightward (-alpha 3.7) deletion chromosome. This mutation abolished an Ncol restriction site and therefore is detectable in genomic DNA by Southern blot analysis.
Regulated expression of amplified human beta globin genes
Authors: Rund D , Dobkin C , Bank A 
Published: October 2019

Abstract
Gene therapy for the beta thalassemias and sickle cell anemia will require high levels of expression of human beta globin genes. One method to achieve this goal is amplification of globin genes transferred into the stem cells in the bone marrow of these patients. If the amplified genes remain normally regulated, they will then further increase their expression on being induced to differentiate along an erythroid pathway. To begin this study, we constructed a plasmid containing a neomycin resistance gene, a human beta globin gene, and a wild-type DHFR cDNA, and transfected it into mouse erythroleukemia cells. All the G418-resistant clones analyzed acquired and expressed the human beta globin gene. By serial passage of the cells in increasing concentrations of methotrexate, the exogenous human beta globin genes were stably amplified in all lines, and all increased their globin mRNA expression roughly proportional to their augmented copy number. Most of the clones further increased their beta globin expression on addition of an erythroid stimulus (dimethylsulfoxide). These results indicate that globin gene amplification may be useful in increasing globin mRNA expression in further experiments whose goal is gene therapy.
ADP-induced platelet shape change and mobilization of cytoplasmic ionized calcium are mediated by distinct binding sites on platelets: 5'- p-fluorosulfonylbenzoyladenosine is a weak platelet agonist
Authors: Rao AK , Kowalska MA 
Published: October 2019

Abstract
Platelet stimulation with ADP results in several responses, including shape change, increase in cytoplasmic ionized calcium concentration [Ca2+]i, an inhibition of adenylate cyclase. 5′-p-Fluorosulphonyl benzoyladenosine (FSBA), which covalently labels an ADP binding site on platelets, blocks platelet shape change but not the inhibition of cyclic AMP levels by ADP, whereas p-chloromercuribenzenesulfonate (pCMBS), a nonpenetrating thiol reagent, has the opposite effects. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i using platelets loaded with fluorescent Ca2+ indicators quin2 and fura-2. FSBA (50 to 200 mumol/L) induced a dose-dependent rise in [Ca2+]i, indicating that it is a weak platelet agonist. Under conditions of covalent labeling of the ADP binding sites, FSBA (50 to 100 mumol/L) did not inhibit the ADP-induced increase in [Ca2+]i or its inhibition of adenylate cyclase, whereas pCMBS (up to 1 mmol/L) abolished both these responses but not shape change. These findings suggest that ADP-induced Ca2+ mobilization and inhibition of adenylate cyclase are mediated by platelet binding sites distinct from those mediating shape change.
Defensin-rich dense granules of human neutrophils
Authors: Rice WG , Ganz T , Kinkade JM Jr , Selsted ME , Lehrer RI , Parmley RT 
Published: October 2019

Abstract
Defensins are a newly recognized class of small, cationic polypeptides that have in vitro microbicidal activity toward certain bacteria, fungi, and viruses. Human neutrophil granules were separated into 13 density fractions by using a high-resolution Percoll gradient centrifugation procedure, and the distribution of the three defensin polypeptides in these fractions was determined. Levels of defensins and several granule marker proteins were estimated in each fraction from relative staining intensities of bands following acid-urea and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of total acid-extractable proteins. These results were confirmed by enzyme immunoassay measurements of defensins and quantitative determinations of the typical azurophil granule components, myeloperoxidase, beta- glucuronidase, lysozyme, and elastase. The five higher density granule fractions (H1 through H5) contained fourfold higher relative amounts of defensins as compared with the eight lower density fractions (L1 through L8), accounting for approximately 50% of the total protein. In particular, fraction H5 was especially enriched in defensins but was relatively deficient in myeloperoxidase, beta-glucuronidase, lysozyme, and elastase. Ultrastructural morphology showed that fraction H5 contained the largest granules. Seventy percent of these granules exhibited electron-dense rims and electron-lucent central regions when stained with methanolic uranyl acetate-lead citrate, and 70% showed this same characteristic rim-staining pattern after limited reaction (30 minutes) for peroxidase with diaminobenzidine. These distinctively large, rim-stained granules were identified in intact, mature peripheral blood neutrophils as well as in human bone marrow promyelocytes, indicating that their synthesis occurs during early myeloid development. This unusual granule type may play a specialized role in the microbicidal functions of the neutrophil, distinct from that of typical azurophil granules.