bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet size does not correlate with platelet age
Authors: Thompson CB , Love DG , Quinn PG , Valeri CR
Published: October 2019

The relationship between platelet size and in vivo aging was investigated in the baboon using size-dependent platelet subpopulations separated by counterflow centrifugation. The separation characteristics, size, lactate dehydrogenase (LDH) activity, and dense- body content of the baboon platelet subpopulations were similar to those previously observed in studies of human platelets. Three independent labeling techniques were used: (1) in vivo labeling with 75Se-methionine, (2) in vitro labeling with 51Cr, and (3) in vivo labeling with 14C-serotonin. Maximal incorporation of all three labels showed a close correlation between the mean platelet volume (MPV) of each fraction and the platelet radioactivity. The onset of incorporation and rate of accumulation of 75Se-methionine were comparable in all fractions when corrected for differences in volume, suggesting that platelet size heterogeneity was present from the time of release of the platelets from the bone marrow. Survival studies using 51Cr and 14C-serotonin showed no translocation of the label from one fraction to another in the circulation over time. In vivo survival values for the three radionuclides showed a slight but significant correlation between the lifespan and the MPV of the fractions. The data suggest that large platelets were not younger platelets, but rather platelets with a longer life-span. Platelet size heterogeneity is the result of production factors in the bone marrow and not maturation in the circulation.
Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro
Authors: Laposata M , Dovnarsky DK , Shin HS
Published: October 2019

When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.
Effect of mouse interferon on growth and differentiation of mouse bone marrow cells stimulated by two different types of colony-stimulating factor
Authors: Yamamoto-Yamaguchi Y , Tomida M , Hozumi M
Published: October 2019

The effects of mouse L-cell interferon (IFN) on growth of mouse bone marrow cells and their differentiation into macrophages and granulocytes were investigated in a liquid suspension culture system with two different types of colony-stimulating factor (CSF). Within 7 days, most bone marrow cells differentiated into macrophages in the presence of macrophage colony-stimulating factor (M-CSF) derived from mouse fibroblast L929 cells, but into both granulocytes (40%) and macrophages (23%) in the presence of a granulocyte-macrophage colony- stimulating factor (GM-CSF) from mouse lung tissue. IFN inhibited growth of bone marrow cells with both M-CSF and GM-CSF, but had 20 times more effect on bone marrow cells stimulated with M-CSF than on those stimulated with GM-CSF. A low concentration of IFN (50 IU/ml) stimulated production of macrophages by GM-CSF in liquid culture medium, whereas it selectively inhibited colony formation of macrophages in semisolid agar culture. IFN caused no detectable block of late stages of differentiation; mature macrophages and granulocytes were produced even when cell proliferation was inhibited by IFN. These results indicate that IFN preferentially affects growth and differentiation of the cell lineage of macrophages among mouse bone marrow cells.
Natural killer and suppressor T-cell chronic lymphocytic leukemia
Authors: Palutke M , Eisenberg L , Kaplan J , Hussain M , Kithier K , Tabaczka P , Mirchandani I , Tenenbaum D
Published: October 2019

A patient with low-grade lymphocytosis, splenomegaly, and neutropenia, but adequate myeloid leukogenesis, was found to have chronic lymphocytic leukemia, which represented a clonal proliferation of a distinct T-lymphocyte subset. The lymphocytes did not form E rosettes but had an OKT3+, OKT4+, OKT6+, OKT8+, OKT11+, HNK-1+, HNK-36+, OKIa1+, OKM1+ phenotype and functionally had suppressor and natural killer activity. Morphologically, they were large granular lymphocytes, which were strongly acid phosphatase positive and nonspecific esterase negative. They did not respond to mitogens, or to allogeneic cells. Initially, the spleen appeared to be the most involved organ and, judging from the high proportion of leukemic splenic lymphocytes in the S and G2/M phases of the cell cycle, was also the organ of origin of the leukemic cells. Only a few leukemic cells in the blood and bone marrow were in S and G2/M phases. After splenectomy, the lymphocyte count rose considerably and the bone marrow became progressively more infiltrated by tumor nodules. One year after diagnosis, the patient was started on chemotherapy because of progressive anemia. He responded to the chemotherapy by normalization of the hemoglobin and neutrophil count and had a moderate decrease in the bone marrow involvement and peripheral lymphocytosis.
Enzyme analysis of lymphoproliferative diseases: a useful addition to cell surface phenotyping
Authors: van de Griend RJ , van der Reijden HJ , Bolhuis RL , Melief CJ , von dem Borne AE , Roos D
Published: October 2019

Lymphocytes from patients with acute and chronic T-cell malignancy or chronic T gamma lymphocytosis were characterized by studying the activity of three enzymes involved in purine metabolism and by determining the isoenzyme pattern of lactate dehydrogenase (LDH) in addition to analysis of surface marker expression with monoclonal antibodies. Four clinically different types of disease were distinguished on the basis of the enzyme parameters. Lymphocytes from patients with acute lymphocytic leukemia (T-ALL) showed an enzyme profile similar to that of normal thymocytes, i.e., an elevated level of adenosine deaminase (ADA) activity as compared with normal T lymphocytes, reduced activities of purine 5′nucleotidase (5′NT) and purine nucleoside phosphorylase (PNP), and a binomial distribution of the LDH isoenzyme pattern. Cells from “null”-ALL patients had an ADA/PNP ratio that was intermediate between that of normal T cells and that of T-ALL cells or thymocytes, but their 5′NT activity and LDH isoenzyme pattern were thymocyte-like. Patients with chronic T-cell proliferation were subdivided into those with chronic T gamma lymphocytosis and those with proven chronic T malignancy. The lymphocytes from these patients had ADA and PNP activities within the ranges of those of normal T lymphocytes. However, the ADA activity and/or the ADA/PNP ratio were consistently higher in the cells from the patients with chronic T gamma lymphocytosis than in those with chronic T malignancy. The enzyme profile of the cells from the T gamma patients was similar to that of T gamma cells of normal individuals. The cells from patients with chronic T malignancies showed a heterogeneous enzyme pattern as compared with that of normal T lymphocytes. Analysis with monoclonal antibodies enabled us to distinguish null-ALL patients from the other leukemias studied, but a distinction between chronic and acute T-cell proliferation disease, for instance, was not possible with monoclonal antibodies alone. Our data demonstrate that the enzyme profiles studied provide supplementary information for classification and diagnosis of lymphoproliferative diseases to that obtained with cell surface markers alone.
Specific toxicity of 2-chlorodeoxyadenosine toward resting and proliferating human lymphocytes
Authors: Carson DA , Wasson DB , Taetle R , Yu A
Published: October 2019

2-Chlorodeoxyadenosine (CdA), an adenosine-deaminase-resistant purine deoxynucleoside, is markedly toxic toward human T-lymphoblastoid cell lines in vitro and is an effective agent against L1210 leukemia in vivo. The present studies have examined the toxicity, and in some cases, metabolism, of CdA in (1) multiple established human cell lines of varying phenotype, (2) leukemia and lymphoma cells taken directly from patients, (3) normal bone marrow cells, and (4) normal peripheral blood lymphocytes. Nanomolar concentrations of CdA blocked the proliferation of lymphoblastoid cell lines with a high ratio of deoxycytidine kinase to deoxynucleotidase. The drug had virtually no effect on the growth of cell lines derived from solid tissues. The CdA inhibited the spontaneous uptake of tritiated thymidine by many T and non-T, non-B acute lymphoblastic leukemia cell specimens at concentrations less than or equal to 5 nM. The same concentrations did not impair either thymidine uptake or granulocyte-monocyte colony formation by normal bone marrow cells. In common with deoxyadenosine, but unlike several other agents affecting purine and purine metabolism, CdA was lethal to resting normal T lymphocytes and to slowly dividing malignant T cells. In both resting and proliferating lymphocytes, the CdA was phosphorylated by deoxycytidine kinase and entered a rapidly turning over nucleotide pool. Dividing lymphocytes also incorporated abundant CdA into DNA. The selective toxicity of CdA toward both dividing and resting lymphocytes may render the drug useful as an immunosuppressive or antileukemic agent.
Analysis of B-cell antigens in normal reactive lymphoid tissue using four B-cell monoclonal antibodies
Authors: Hofman FM , Yanagihara E , Byrne B , Billing R , Baird S , Frisman D , Taylor CR
Published: October 2019

Lymph nodes from 13 cases of reactive hyperplasia were examined with four different monoclonal antibodies to B cells. B-1 recognizes an antigen of 30,000 daltons on B cells. CB-2 was prepared with normal spleen and binds to a glycolipid. BA-1 labels surface immunoglobulin- positive cells, but not T lymphocytes or monocytes. B-532 recognizes an antigenic determinant of 45,000 daltons. Using the immunoperoxidase method on frozen sections of reactive lymph nodes, the staining patterns of these four unique antibodies showed dramatic differences. B- 1 labeled 80%-90% of the germinal center cells and 10%-50% of the mantle region. Few interfollicular cells were positive. CB-2 stained predominantly in the mantle area (50%-90% positive cells), with moderate staining in the germinal center as well and less than 1% positive cells in the diffuse cortex. BA-1 exhibited predominant labeling in the mantle (50%-90%), with little staining in the germinal center. A large number of cells in the interfollicular, subcapsular, and medullary regions expressed the BA-1 antigen. The B-5 antibody demonstrated intense staining in the follicle (50%-95%). This staining often appeared to be polarized within the germinal center. The mantle zone demonstrated staining of 30%-50% of the cells. The different staining patterns of the B-cell antibodies, as demonstrated by the in situ distribution of positive cells within the lymph node, may reflect stages of development or activation of the B-cell population.
Increased respiratory burst in myeloperoxidase-deficient monocytes
Authors: Locksley RM , Wilson CB , Klebanoff SJ
Published: October 2019

Studies of the respiratory burst in myeloperoxidase (MPO) deficient monocytes were undertaken to assess the physiologic consequence of the absence of MPO in these cells. As previously demonstrated with neutrophils, MPO-deficient monocytes had a greater initial rate, duration, and total superoxide production in response to phagocytosis of zymosan than did normal monocytes. Introduction of purified eosinophil peroxidase (EPO) into the phagosome by binding the enzyme to the surface of the zymosan particles changed the hypermetabolic characteristics of superoxide production in MPO-deficient cells to more closely resemble normal cells, but had no effect on superoxide generation by the normal monocytes. Further, inactivation of the bound EPO before ingestion restored the supranormal respiratory burst by the MPO-deficient cells. Iodination by MPO-deficient monocytes was significantly depressed as compared to normal monocytes following the ingestion of zymosan (1.9 versus 10.1 nmole I-/10(7) monocytes/30 min; p less than 0.01). In contrast, iodination was markedly augmented in MPO-deficient cells compared to normal cells after ingestion of zymosan coated with EPO (208 versus 70 nmole I-/10(7) monocytes/30 min; p less than 0.005), presumably reflecting the greater amounts of hydrogen peroxide formed by MPO-deficient cells. There were no differences in the levels of endogenous scavengers of reactive oxygen products (catalase, superoxide dismutase, glutathione peroxidase and reductase, and total glutathione) in MPO-deficient and normal monocytes that would account for the enhanced respiratory burst of MPO-deficient cells. These findings support a role for peroxidase in the termination of the respiratory burst of monocytes.
N-formylmethionyl-leucyl-[3H]phenylalanine binding, superoxide release, and chemotactic responses of human blood monocytes that repopulate the circulation during leukapheresis
Authors: Alteri E , Leonard EJ
Published: October 2019

Human blood monocytes comprise two subpopulations: one migrates to the chemoattractant, N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe), and has saturable binding sites for this peptide; the other does not migrate and exhibits little peptide binding. To determine if expression of binding sites was a function of monocyte maturation, we depleted human subjects of blood monocytes by leukapheresis so that the circulation was repopulated by monocytes released from the bone marrow. Pre- and postleukapheresis monocytes were then compared for fMet-Leu- [3H]Phe binding, superoxide generation, and chemotactic responses. No significant differences in peptide binding curves were found, suggesting that receptor expression was stable over the maturational span represented by these two groups of cells. This supports the hypothesis that there are two distinct lineages of monocytes with respect to expression of receptors for fMet-Leu-Phe. An additional finding of interest was that the number of chemotactically responsive cells immediately postleukapheresis was half the control. This was a transient state; monocyte responses were normal 3 hr after termination of leukapheresis, suggesting that they rapidly become functionally mature.
The amount of platelet-bound albumin parallels the amount of IgG on washed platelets from patients with immune thrombocytopenia
Authors: Kelton JG , Steeves K
Published: October 2019

The biologic relevance of the increased platelet-associated IgG (PAIgG) on platelets from patients with idiopathic thrombocytopenic purpura (ITP) is unclear. Platelets from ITP patients are often larger than normal, and it is possible that the increased IgG is not specific but passively related to platelet size. The measurement of platelet-bound albumin could provide information concerning the specificity of the platelet-bound IgG, since albumin, like IgG, is a plasma protein, but unlike IgG, is not an active participant in immunologic reactions. Albumin is also a normal constituent of platelet membrane, and increased platelet albumin could indicate an increased platelet mass. Platelet-bound albumin, IgG, and total platelet protein were measured on both intact and disrupted platelets from healthy individuals (n = 25) and patients with ITP (n = 21). Platelet IgG and albumin were measured in an immunoradiometric assay using intact antisera and F(ab')2 fragments prepared from the same antisera. There was no relationship between platelet-bound IgG or albumin, and platelet size measured by either platelet protein or platelet volume, (r less than 0.3 for all interactions). In contrast, there was a significant correlation between platelet-bound albumin and platelet-bound IgG (r = 0.7, n = 21, p less than 0.001). Those patients with elevated platelet PAIgG also had elevated platelet albumin, and this relationship was irrespective of the total platelet protein content or mean platelet volume. It is possible that the increased platelet-bound IgG in ITP reflects an increase in platelet surface area or contaminating platelet fragments that are not manifested as an increase in platelet volume or total platelet protein. Alternatively, a platelet membrane abnormality may occur in ITP that results in the uptake of significant amounts of plasma proteins. Either possibility implies that not all of the IgG on platelets from patients with ITP is pathologic IgG.
The kinetic properties of the ecto-ATPase of human peripheral blood lymphocytes and of chronic lymphatic leukemia cells
Authors: Gutmann HR , Chow YM , Vessella RL , Schuetzle B , Kaplan ME
Published: October 2019

This study examines whether the activity of the Mg2+-dependent ecto- ATPase of the surface membrane of the human lymphocyte is changed in chronic lymphocytic B-cell leukemia (CLL-B) and may be an indicator of malignant transformation. The ecto-ATPase activities of preparations consisting predominantly of T or B cells were compared to each other and to the ecto-ATPase of the CLL peripheral blood lymphocytes (PBL). The specific activities and kinetic constants of the ecto-ATPase of the cell preparations were determined with [gamma-32P] adenosine triphosphate (ATP) as substrate. B-enriched lymphocytes had nearly fourfold greater specific activity and apparent Vmax than T-enriched lymphocytes, while the Km values of both cell types showed no significant difference. The specific activities and kinetic constants of the ecto-ATPase of the CLL PBL were significantly higher than the corresponding values of PBL or of B-enriched lymphocytes. Judging from the kinetic constants the ecto-ATPase of the CLL-B lymphocyte appears to be an enzyme that is distinctly different from that of the normal B cell. On the basis of the kinetic properties, the ecto-ATPase of the B cell appears to be identical with that of the T cell. The differences in the maximal velocities of the hydrolysis of ATP by B and T cells are likely due to a greater number of enzymatic sites on the B cell.
Antithymocyte globulin reacts with many normal human cell types
Authors: Greco B , Bielory L , Stephany D , Hsu SM , Gascon P , Nienhuis A , Young N
Published: October 2019

Antithymocyte globulin (ATG) is frequently effective therapy for aplastic anemia. Its mechanism of action is often assumed to be upon a lymphocyte inhibitor of hematopoiesis. However, specificity for T lymphocytes would not be anticipated from consideration of the method of preparing ATG. In fact, using flow microfluorometry and fluorescence immunohistochemistry, we have found that ATG binds to virtually all circulating lymphocytes, granulocytes, and platelets, as well as to bone marrow cells. Extensive absorption of ATG with either granulocytes or lymphocytes does not eliminate reactivity with the opposite cells, indicating that ATG recognizes some distinct antigens on each cell type. Treatment of cells with ATG blocks the binding of monoclonal antibodies directed against either lymphocyte differentiation or histocompatibility antigens. ATG also binds to visceral tissues, including thymus and testis cell membranes and the nuclear and cytoplasmic components of tonsil, kidney, liver, breast, lung, and intestine. In vitro cytotoxicity of ATG was demonstrated for both T and non- T lymphocytes and platelets. Despite its name, ATG is not specific for a particular cell type, and it would be premature to conclude that its effect is mediated through a specific lymphocyte population.
Role of ADP-ribosyl transferase in differentiation of human granulocyte- macrophage progenitors to the macrophage lineage
Authors: Francis GE , Gray DA , Berney JJ , Wing MA , Guimaraes JE , Hoffbrand AV
Published: October 2019

Nuclear adenosine diphosphate-ribosyl (ADP-ribosyl) transferase is a chromatin-bound enzyme catalyzing the transfer of ADP-ribose from NAD+ to chromatin proteins. The physiologic function of this covalent modification of chromatin has not been fully established, but roles in both DNA repair and in differentiation have been proposed. We demonstrate that three specific inhibitors of ADP-ribosyl transferase (5-methylnicotinamide, 3-methoxybenzamide, 3-aminobenzamide) inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage. Differentiation to the neutrophil-granulocyte lineage is much less affected. The inhibition of macrophage differentiation seems to relate to the ability of these compounds to inhibit ADP-ribosyl transferase. A structural analogue (3- methoxybenzoic acid), which is not inhibitory for the enzyme, did not inhibit macrophage differentiation. Additional evidence for a role of ADP-ribosyl transferase in the differentiation of granulocyte- macrophage progenitors was obtained from experiments in which enzyme activity levels were measured in permeabilized marrow cells. Marrow cell ADP-ribosyl transferase activity increased after 3-hr stimulation by the differentiation/proliferation stimulus--granulocyte-macrophage colony-stimulating activity (GM-CSA). Unstimulated marrow cells showed low or undetectable levels of enzyme activity.
The mechanism of action of the antiinflammatory agents dexamethasone and Auranofin in human polymorphonuclear leukocytes
Authors: Coates TD , Wolach B , Tzeng DY , Higgins C , Baehner RL , Boxer LA
Published: October 2019

Human polymorphonuclear neutrophils (PMN) were treated with the antiinflammatory agents dexamethasone or Auranofin. PMN treated with dexamethasone in a dose range of 0.25–1 microM or Auranofin, 5–15 mM, were stimulated with 10(-7)M N-formyl-methionyl-leucyl-phenylalanine (FMLP). These agents were shown to inhibit the functional responses of degranulation and superoxide production in a dose-dependent manner. Similarly, the change in electrophoretic mobility, reflecting cell surface charge, was blocked. While both agents inhibited change in the fluorescence of the calcium chelate probe chlorotetracycline (CTC), the pattern of inhibition was significantly different. Dexamethasone appeared to inhibit the CTC response during its latter phases, while Auranofin inhibited all aspects of the CTC response. Auranofin was additionally shown to significantly decrease specific binding of FMLP, as well as the number of FMLP receptors. The two agents thus appear to act by different mechanisms. Dexamethasone is shown to have an effect on membrane-bound calcium release as measured by CTC, while Auranofin interferes with receptor binding.
Evidence for the separate human T-lymphocyte subpopulations that collaborate with autologous monocyte/macrophages in the elaboration of colony-stimulating activity and those that suppress this collaboration
Authors: Verma DS , Johnston DA , McCredie KB
Published: October 2019

We investigated the interaction of monocyte/macrophages and autologous T lymphocytes in the methanol extraction residue (MER) of BCG-induced production of granulocyte-macrophage colony-stimulating activity (CSA). Coincubation of monocyte/macrophages and T lymphocytes at a 1:3 ratio produces an optimum collaboration; a change to a 1:9 ratio diminished this collaboration. Coincubation of monocyte/macrophages and T lymphocytes primed with lithium carbonate (2 meq/liter) for 40 hr synergistically increased CSA elaboration and prevented the decline in CSA noted for the 1:9 monocyte/macrophage: T lymphocyte ratio. In contrast, concanavalin-A-primed T lymphocytes did not enhance CSA elaboration at any monocyte/macrophage:T lymphocyte ratio except, occasionally, at 1:9. However, this was overcome if the T lymphocytes were primed with both concanavalin-A and lithium carbonate before their coincubation with monocyte/macrophages. Further cell-mixing experiments revealed that concanavalin-A-primed T lymphocytes contained a subpopulation that suppressed monocyte/macrophage and T-lymphocyte collaboration. Activation of suppressor T lymphocytes could be effectively prevented by lithium carbonate and, in a dose-dependent manner, by irradiation. Also, suppressor T lymphocytes not only diminished the elaboration of colony-stimulating factor(s), but also elaborated an inhibitor of granulocyte-macrophage colony-forming cells. We further demonstrated that the respective hemopoietic helper and suppressor T-lymphocyte activities could be enriched with OKT8- (or OKT4+) and OKT8+ subpopulations.
In vivo modulation of murine myelopoiesis following intravenous administration of prostaglandin E2
Authors: Gentile P , Byer D , Pelus LM
Published: October 2019

The effects of in vivo administration of prostaglandin E2 (PGE2) on several hematologic parameters were investigated in intact mice under both steady-state conditions and in mice hematopoietically rebounding following a sublethal injection of cyclophosphamide. Intravenous injection of native PGE2, or 16,16 dimethyl-PGE2, an enzymatically stable analog of PGE2, resulted in the significant suppression of nucleated bone marrow and splenic cellularity, total resident nucleated peritoneal cells, and the absolute number of detectable granulocyte- macrophage progenitor cells (CFU-GM) per femur or spleen when administered for 3 or 7 consecutive days. The in vivo effects of 16,16 dimethyl-PGE2 were more pronounced on the cyclophosphamide-treated mice. Dose titration analysis of the effects of 16,16 dimethyl-PGE2 revealed significant suppression of hematologic parameters over a concentration range of 10 micrograms-10(-5) micrograms/mouse/day (10(- 5) M-10(-11) M). The reduction in total nucleated marrow, splenic, and peritoneal cellularity observed following PGE2 administration resulted from a selective effect on nonspecific esterase-positive cells. In situ morphological analysis of the progeny of CFU-GM proliferating in cultures established from mice treated with PGE2 in vivo indicated that the reduction in absolute CFU-GM observed resulted from a preferential effect on those colony-forming cells restricted to monocyte-macrophage differentiation. Prostaglandin F2 alpha was without stimulatory or inhibitory effects in vivo on the hematopoietic parameters investigated.
A monoclonal antibody (WT1) for detecting leukemias of T-cell precursors (T-ALL)
Authors: Vodinelich L , Tax W , Bai Y , Pegram S , Capel P , Greaves MF
Published: October 2019

The selectivity of a monoclonal anti-T antibody, designated WT1, has been assessed in a series of 906 leukemias and lymphomas. In acute lymphoblastic leukemias, WT1 reacts comprehensively and selectively with thymic acute lymphoblastic leukemia (ALL) cells in untreated or relapsed patients, thus overriding the extensive antigenic diversity of this cancer and the immaturity of the cell type involved. All 80 cases of thymic ALL examined were WT1-positive. In addition, 18 cases of presumptive prethymic ALL were also WT1-positive, but were unreactive with other maturation-linked T-cell markers. The phenotype WT1+ HLA-DR TdT+ appears to be unique to T-ALL and can therefore be used systematically for the differential diagnosis of this poor prognosis subtype of ALL. Virtually all ALL cases can now be placed into one of two major subgroups representing transformed precursors of either the T- or B-cell lineage. WT1 identifies a single polypeptide of approximately 40,000 mol wt and is similar to two previously described monoclonal antibodies.
Apparent molecular weight of purified human factor VIII procoagulant protein compared with purified and plasma factor VIII procoagulant protein antigen
Authors: Weinstein MJ , Fulcher CA , Chute LE , Zimmerman TS
Published: October 2019

We have compared apparent molecular weights of purified factor VIII procoagulant protein (VIII:C) and VIII:C antigen (VIII:CAg) by two different NaDodSO4 gel electrophoretic techniques. In a discontinuous NaDodSO4–7.5% polyacrylamide system, reduced and unreduced VIII:C, purified from commercial factor VIII concentrates by a monoclonal antibody immunoadsorption technique, showed a major doublet at mol wt 0.79 and 0.8 X 10(5) and less intense bands extending up to 1.9 X 10(5). In NaDodSO4–4% polyacrylamide/0.5% agarose gels (NaDodSO4–4% PAAGE), purified VIII:C had a major band of mol wt 1.0 X 10(5), with minor bands of mol wt 0.96, 1.1, 1.4, 1.6, 1.8, 2.2, and 2.4 X 10(5). In NaDodSO4–4% PAAGE of 125I-anti-VIII:C-Fab-VIII:CAg complexes, the major and minor forms of VIII:CAg in purified VIII:C had the same molecular weight as above when calculated by subtracting the molecular weight of 125I-Fab from 125I-Fab-VIII:CAg. In both plasma and factor VIII concentrate, a band of mol wt 2.4 X 10(5) predominated, and minor VIII:CAg forms of mol wt 2.6, 1.8, 1.2 and 1.0 X 10(5) were also visible. We conclude that the molecular weight of plasma VIII:CAg forms agree with those derived from protein stains of purified VIII:C in the NaDodSO4–4% PAAGE system, but that consistently lower molecular weight values are obtained for purified VIII:C in the discontinuous system. Both native and either disaggregated or proteolyzed VIII:C species are present in the purified VIII:C preparation.
Common ALL with pre-B-cell features showing (8;14) and (14;18) chromosome translocations
Authors: Mufti GJ , Hamblin TJ , Oscier DG , Johnson S
Published: October 2019

A 21-yr-old man presented with inguinal lymphadenopathy and splenomegaly. The peripheral blood showed a high blast cell count. The morphological and immunologic features of the blast cells were consistent with the diagnosis of common acute lymphoblastic leukemia (ALL) with 15% pre-B-cells. Banded karyotype analysis of the blood and the marrow cells, using the technique of methotrexate synchronization, revealed the presence of (8;14) and (14;18) chromosome translocations, a finding that has not been previously documented. The significance of these findings is discussed.
Pyruvate kinase isozyme (PK-Greenville) with defective allosteric activation by fructose-1,6-diphosphate: the role of F-1,6-P modulation in normal erythrocyte metabolism
Authors: Paglia DE , Valentine WN , Holbrook CT , Brockway R
Published: October 2019

A child with chronic hemolytic anemia since birth was found to have erythrocyte pyruvate kinase (PK) in a highly unusual form relative to other mutant isozymes when characterized by International Committee for Standardization in Hematology criteria. Most properties of the partially purified isozyme (designated PK-Greenville) were altered minimally, if at all, except for nearly total insensitivity to allosteric activation by fructose-1,6-diphosphate (F-1,6-P). One parent appeared to be heterozygous for a null gene and the other for an allele governing production of the mutant isozyme. Apparent restriction of the molecular defect to ineffective activation kinetics suggests that the F- 1,6-P binding site on erythrocyte PK is functionally as well as physically allosteric. The magnitude of the metabolic block at the PK step and the clinical severity indicate that allosteric modulation by F- 1,6-P is a crucial property of PK in normal erythrocyte metabolism.
Identification of hemopoietic cells responsive to colony-stimulating factor by autoradiography
Authors: Shadduck RK , Pigoli G , Caramatti C , Degliantoni G , Rizzoli V , Porcellini A , Waheed A , Shiffer L
Published: October 2019

Binding of radiolabeled L-cell colony-stimulating factor (CSF) was studied using murine bone marrow and fetal liver cells. With 10(7) cells, saturation of binding was seen with approximately 500,000 cpm of 125I-CSF. Minimal binding was detected after one hour incubation with tracer at 37 degrees C; however, marked cellular uptake of radioactivity was noted after 24-hr exposure to CSF. As judged by autoradiographs, small numbers of myeloblasts, promyelocytes, and large mononuclear cells were labeled with 1-hr exposure to tracer. By 6 hr of incubation, 50%-70% of myeloblasts and promyelocytes and small numbers of late granulocytic cells were labeled. Virtually all myeloblasts and promyelocytes and approximately 50% of myelocytes, metamyelocytes, polymorphonuclear granulocytes, and monocytes were labeled after 24-hr exposure to the radioiodinated CSF. Label was not detected on erythroblasts, eosinophils, or megakaryocytes. Suspensions of fetal liver cells had lower uptake of radioactivity than bone marrow cells. This appeared to result from a lesser concentration of granulocytic cells in fetal liver, as labeling of individual cells was similar with both tissues. In additional experiments, CSF binding to marrow cells was assessed after 30-min exposure to tracer at 0 degrees C. Uptake of 125I-CSF exceeded that observed after 24-hr incubation at 37 degrees C. With this technique, cellular label was also confined to granulocytic and monocytic cells. These findings suggest that purified CSF reacts with and may stimulate immature and mature cells of the granulocytic and monocytic lineages.
Retinoic acid treatment of acute promyelocytic leukemia: in vitro and in vivo observations
Authors: Flynn PJ , Miller WJ , Weisdorf DJ , Arthur DC , Brunning R , Branda RF
Published: October 2019

We describe in vitro studies and a therapeutic trial of retinoic acid (RA) in a patient with acute promyelocytic leukemia (APL) refractory to chemotherapy. Bone marrow promyelocytes from the patient, prior to RA, matured morphologically in liquid culture with RA (97% maturing myeloid cells compared with 26% in control cultures at 7 days). RA-cultured cells displayed leukocyte alkaline phosphatase activity and cytoplasmic maturation (by electron microscopy). Retinoic-acid-treated cells, compared to controls, demonstrated increased functional maturation, with phagocytosis of opsonized zymosan (90% versus 10%) and production of superoxide (measured by nitroblue tetrazolium reduction) in response to phorbol ester, opsonized zymosan, or the chemotaxin F-met-leu-phe. There was no evidence of active proliferation in the cultures. RA- treated cells continued to show 15;17 chromosomal translocation after 7 days in culture. The patient was treated with oral 13-cis-retinoic acid (100 mg/sq m/day) for 13 days. During that time, the peripheral white blood count rose from 300 cu mm to 6,700 cu mm, and the maturing myeloid cell count rose from 54 cu mm to 3,800 cu mm. Bone marrow maturing cells increased from 1.8% to 8.0%. Despite the increasing number of maturing myeloid cells, the patient died on day 13 from disseminated candidiasis. These data confirm that RA induces maturation of leukemic promyelocytes in vitro and suggest that similar maturation is achievable in vivo. We suggest that oral retinoic acid may be a useful adjunct in the treatment of APL.
Characterization of a monoclonal antibody that reacts with an activation antigen on human B cells: reactions on mitogen-stimulated blood lymphocytes and cells of normal lymph nodes
Authors: Frisman D , Slovin S , Royston I , Baird S
Published: October 2019

We describe a monoclonal antibody that reacts with human B-lymphoid cells. We have characterized the reactions of this antibody on normal blood lymphocytes, with and without pokeweed mitogen stimulation, bone marrow lymphocytes, and on frozen sections of normal lymph nodes. The antibody, B532, appears to recognize an activation antigen on human B cells. This activation antigen can apparently be induced both by infection with Epstein-Barr virus and by stimulation with antigens and mitogens, but it is lost on plasma cells. In normal lymph nodes, the antigen is confined to germinal center cells and some of the cells of the “mantle” that surrounds germinal centers. The antigen is not present on T cells.
Characterization of human mast cells in long-term culture
Authors: Horton MA , O'Brien HA
Published: October 2019

Recent studies in rodents have demonstrated that mast cells derived from lymphoid tissues can be grown in longterm culture, provided that supportive growth factors or stromal fibroblasts are added; such findings have not been reported in man. Furthermore, although a hemopoietic origin for mast cells is supported by transplantation studies in mice, the exact origin of the human mast cell or its relationship to the circulating basophil and other hemopoietic cell lineages is unknown. We have investigated the requirements for in vitro growth of human mast cells derived from the infiltrated bone marrow of a patient with systemic mastocytosis, and have characterized both the mast cells proliferating in these cultures and those obtained from splenic infiltrates. Our data approached two questions: (1) Is there any evidence for the origin of mast cells from a bone-marrow-derived stem cell, and, if so, (2) what lineage relationship is there between mast cells and granulopoietic cells, including basophils? First, we have shown the expression of hemopoietic tissue-specific antigens by mast cells, strongly supporting a bone marrow origin for the mast cell in man (at least for those mast cells analyzed here). Second, the complete lack of granulocyte-monocyte markers contrasts with the phenotype of the basophil and suggests that mast cells diverge considerably from other granulopoietic cells during the acquisition of their differentiated specialized functions.
An immunologic study of spouses and siblings of asymptomatic hemophiliacs
Authors: Ragni MV , Bontempo FA , Lewis JH , Spero JA , Rabin BS
Published: October 2019

A transmissible agent may be responsible for the recent occurrence of the acquired immune deficiency syndrome (AIDS) in female heterosexual partners and infant contacts of AIDS victims or of persons at increased risk for AIDS. Therefore, we evaluated the clinical and immunologic status of 18 hemophiliac-spouse pairs and 19 hemophiliac-sibling pairs. Using surface marker assays of lymphocyte subpopulations, we found a mean T helper cell/T suppressor cell (H/S) ratio +/- SEM in 18 hemophiliac adults of 1.11 +/- 0.15 (11 less than 1.00), in their 18 spouses of 1.88 +/- 0.13 (none less than 1.00), in 19 hemophiliac children of 1.54 +/- 0.11 (3 less than 1.00), and in their 19 siblings of 1.87 +/- 0.11 (none less than 1.00). Both hemophiliac adult and hemophiliac children mean H/S ratios differed significantly (p less than 0.05) from the control mean ratio of 2.22 +/- 0.16 and from the mean of their respective nonhemophiliac spouse or sibling group (p less than 0.05). The mean ratio of hemophiliac adults differed significantly from that of hemophiliac children (p less than 0.05). These findings suggest that lymphocyte alterations in hemophiliacs are secondary to their therapy, are not influenced by genetic factors, and are not related to an infectious agent that is easily transmissible by close contact.
Lithium stimulation of murine hematopoiesis in liquid culture: an effect mediated by marrow stromal cells
Authors: Quesenberry PJ , Coppola MA , Gualtieri RJ , Wade PM , Song Z , Doukas MA , Shideler CE , Baker DG , McGrath EH
Published: October 2019

Lithium has previously been observed to stimulate in vitro Dexter culture hemopoiesis with increases in granulocytes, megakaryocytes, and pluripotent stem cells (CFU-S). In the present study, a two-phase murine Dexter culture system was established to study the mechanism of lithium-mediated stem cell stimulation. Different lots of horse sera or fetal calf sera were found to have markedly different effects on Dexter culture growth; given the appropriate sera supplementation, supernatant cells from Dexter cultures established from C57BL/6J mice 3 wk previously were free of stromal-forming capacity, but had stem cells and could grow on 900–950 R irradiated stroma. Conversely, in vitro irradiation (900–950 R) of 3-wk cultures resulted in a stem-cell-free adherent monolayer that could support growth for up to 9 wk in culture. The stroma from Dexter cultures preexposed to lithium chloride (1.0 mmole/liter) for 3 wk, irradiated (900 R), and then refed with 3-wk Dexter supernatant cells has an enhanced capacity to support cell production, CFU-S, and probably granulocyte-macrophage colony-forming cell (GM-CFU-C) production, as compared to stroma not preexposed to lithium. Lithium carryover was ruled out in these experiments. These data indicate that lithium stimulates CFU-S and in vitro granulopoiesis by an indirect effect on a radioresistant adherent stromal cell.
Transient versus permanent expression of cancer-related glycopeptides on normal versus leukemic myeloid cells coinciding with marrow egress
Authors: van Beek W , Tulp A , Bolscher J , Blanken G , Roozendaal K , Egbers M
Published: October 2019

Release of mature cells from the bone marrow (BM) into the peripheral blood (PB) compartment is supposed to be triggered by changes in cell surface constituents, most probably in glycoproteins. The supposed importance of glycoproteins in marrow exit prompted us to investigate glycopeptides, i.e., the carbohydrate part of the cell-surface-located glycoproteins of isolated human bone marrow cells of the myeloid series at different stages of maturation. Fractionation of cells was performed by a four-step procedure, comprised of density gradient centrifugation and velocity sedimentation at unit gravity in specially designed separation chambers. With this method, promyelocytes/myeloblasts, granulocytes from bone marrow, and granulocytes from peripheral blood were isolated in high quantity with purities up to 90%, 90%, and 100%, respectively. Surface glycopeptides of the various myeloid cells were investigated by gel filtration analysis after metabolic labeling with radioactive fucose or after external labeling with periodate- borotritide under mild conditions. Within the normal myeloid maturation sequence, mature granulocytes within the bone marrow were found to transiently express altered surface glycopeptides, which disappeared after release into the peripheral blood. These oligosaccharide structures appeared similar to those encountered on leukemic blast cells, known as “cancer-related glycopeptides.” In contrast to normal granulocytes from BM, leukemic blast cells retained these aberrant carbohydrate structures on their surface after marrow release. A possible role for cancer-related glycopeptides in the process of marrow cell exit might be hypothesized.
Perturbations in the erythroid marrow progenitor cell pools may play a role in the augmentation of HbF by 5-azacytidine
Authors: Torrealba-de Ron AT , Papayannopoulou T , Knapp MS , Fu MF , Knitter G , Stamatoyannopoulos G
Published: October 2019

In vivo observations on the kinetics of F cells and of fetal hemoglobin (HbF) synthesis and in vitro studies of erythroid progenitors, their number, and the gamma-gene expression in their progeny were carried out in baboons (Papio cynocephalus) treated with 5-azacytidine. Maximum effect on the increase of HbF production in vivo was observed only when an expanded erythroid marrow population was present. In these animals, as well as in normal animals, treatment resulted in a significant reduction of the late erythroid progenitor cell pools (erythroid clusters and erythroid colony-forming units, CFU-E) in the marrow. This reduction was more pronounced among those progenitors grown in the absence of added erythropoietin, and it was followed by a rebound a few days after treatment cessation, reflecting the accumulation of regenerating progenitors. An early increase in the in vitro synthesis of HbF in erythroid clusters and CFU-E colonies was observed. This increase was further documented at the cellular level, with immunofluorescent labeling of colonies with monoclonal anti-gamma- globin chain antibodies. In contrast to the findings in late progenitors, the number of erythroid burst-forming unit (BFU-E) colonies and the synthesis of HbF in these colonies was not influenced significantly by 5-azacytidine treatment. It is proposed that the toxic effects of 5-azacytidine on late progenitors, leading to faster mobilization of earlier progenitors to the next more mature compartment, play a role in the in vivo augmentation of HbF synthesis by this drug. This perturbation in the progenitor cell population kinetics and the presumed hypomethylation of the surviving differentiating cells may act synergistically to produce a maximum HbF response after 5-azacytidine treatment.
Primary therapy of acute promyelocytic leukemia: results of amsacrine- and daunorubicin-based therapy
Authors: Arlin Z , Kempin S , Mertelsmann R , Gee T , Higgins C , Jhanwar S , Chaganti RS , Clarkson B
Published: October 2019

Remission rates for patients with acute promyelocytic leukemia (APL) have improved with the use of anthracyclines and proper management of disseminated intravascular coagulopathy. In a prospective randomized trial of chemotherapy in patients with acute nonlymphoblastic leukemia, there were 16 patients with APL. All 7 of the patients receiving the amsacrine-containing regimen and 5 of 9 receiving the daunorubicin- containing regimen achieved a remission. All patients, except 2 of the 3 who underwent bone marrow transplantation, remain alive and in remission from 1+ to 25+ mo. Amsacrine is an effective replacement for daunorubicin in the treatment of APL, and its use does not compromise the favorable remission duration characteristic of APL.
Mechanical properties of oxygenated red blood cells in sickle cell (HbSS) disease
Authors: Nash GB , Johnson CS , Meiselman HJ
Published: October 2019

Little data exist for the mechanical properties of individual irreversible or reversible sickle cells (ISC and RSC, respectively), nor is the process of ISC formation well understood. For oxygenated ISC and density-fractionated RSC, we have used micropipette techniques to measure cell surface area (SA) and volume (V), membrane shear elastic modulus (mu), time constant for viscoelastic shape recovery (tc), and hence to calculate membrane surface viscosity (eta = mu X tc). Volume loss associated with increasing cell density was accompanied by a proportionately smaller surface area decrease; SA/V ratio thus increased for denser cells, with ISC having the highest values. Membrane area loss by fragmentation must thus be accompanied by an accelerated decrease in cell volume. ISC had relatively rigid membranes (mu 130% above normal controls) and tc close to normal values, so that their effective membrane viscosity was more than double control. RSC had viscoelastic properties close to control, but showed wider variation between sickle cell donors and within samples. Measurements on density-separated RSC showed that, on average, mu was nearly constant, but that tc was longer for the densest cells, with their eta approaching ISC levels. A small subpopulation of RSC were found that had mu close to ISC values. Hypotonically swollen ISC (with internal hemoglobin concentration decreased to normal levels) retained their increased membrane stiffness but had markedly decreased tc, so that their eta approached normal values. The results show that elevated hemoglobin concentration influences the viscoelastic behavior of ISC and RSC, but that an irreversible change in membrane elasticity also occurs for ISC. These data suggest that ISC formation occurs via a two- stage process: (1) accelerated volume loss leading to increased cytoplasmic and effective membrane viscosity; (2) a sharp rise in membrane rigidity, presumably linked to membrane structural alteration.