bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in activities and isozyme patterns of glycolytic enzymes during erythroid differentiation in vitro
Authors: Nijhof W , Wierenga PK , Staal GE , Jansen G
Published: October 2019

Late committed progenitor cells of erythropoiesis, CFU-E (colony- forming unit--erythroid), were isolated from mouse spleens to near homogeneity by a three-step enrichment procedure. The procedure included a four-day pretreatment of bled mice with the antibiotic thiamphenicol, a recovery period of 3 1/2 days, followed by centrifugal elutriation and Percoll density gradient centrifugation of the spleen cells. This practically pure CFU-E population was used to study some aspects of erythroid differentiation in vitro. Colony growth, as well as morphology and glycolytic enzyme activities of cells isolated at selected times of the 48-hour culture period, were determined. Marked declining activities of several enzymes, including hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase, and glucose-6- phosphate dehydrogenase, were observed during in vitro differentiation. The activity of diphosphoglycerate mutase was almost absent in the CFU- E, but progressively increased during differentiation. The isozyme distribution of aldolase and enolase did not change during CFU-E in vitro differentiation into the reticulocyte. Hexokinase (HK) in the CFU- E contained mainly a double-banded type I isozyme, in addition to a minor amount of HK II. During differentiation, a shift was noticed within the double-banded HK I region, whereas HK ii disappeared after one cell division. Pyruvate kinase in the CFU-E was characterized by the presence of both the K-type and the L-type isozyme and hybrids of these isozyme types. During in vitro differentiation, the production of the K-type isozyme rapidly stops in favor of the L type.
Marrow harvesting from normal donors
Authors: Buckner CD , Clift RA , Sanders JE , Stewart P , Bensinger WI , Doney KC , Sullivan KM , Witherspoon RP , Deeg HJ , Appelbaum FR
Published: October 2019

The experience at a single institution in harvesting marrow for allogeneic transplantation on 1,270 occasions from 1,160 normal donors is presented in detail, together with an analysis of all the donor complications. Four donors were less than 2 years old, and the youngest was 6 1/2 months. No special difficulties were encountered with these young donors. Hospitalization time was three days or less for 99% of the procedures. Six donors had life-threatening complications; three of a cardiopulmonary and two of an infectious nature, and one cerebrovascular embolic episode. Significant operative site morbidity, usually transient neuropathies, occurred in ten procedures. Ten percent of the donations were associated with transient postoperative fever of unknown origin. Increasing donor age was associated with a reduction of the cellularity of the marrow harvest. The use of stored autologous blood permitted the avoidance of blood bank transfusion in 81% of males, 69% of females, and 50% of children. It was concluded that the procedure was associated with a very low risk of complication, but that the involvement of normal donors in such an operation justifies stringent monitoring.
Megakaryoblastic leukemia: the characterization and identification of megakaryoblasts
Authors: Koike T
Published: October 2019

This article describes three patients with megakaryoblastic leukemia, in whom the blast cells were identified as megakaryoblasts by the platelet peroxidase (PPO) reaction. More than 70% of the blasts in these patients were positive for the PPO reaction. Ultrastructurally, acid phosphatase activity in the megakaryoblasts was detected in the nuclear envelope, the endoplasmic reticulum, and in a few granules, but not in the Golgi cisternae. Some blast cells were identified by immunofluorescence or immunoalkaline phosphatase, using monoclonal antiplatelet glycoprotein IIb/IIIa antibody. In one patient, most of the blasts were positive for anti-HLA-DR monoclonal antibody. The possible order of the appearance of markers in the maturation of the megakaryocytic cell lineage is postulated, based on the data from the present cases and those previously reported. PPO activity appears in very immature cells, which retain Ia-like antigens. Platelet-specific glycoprotein IIb/IIIa is seen in immature cells that are only recognized by PPO activity.
Lineage switch in acute leukemia
Authors: Stass S , Mirro J , Melvin S , Pui CH , Murphy SB , Williams D
Published: October 2019

Conversions of leukemic cell lineage (lymphoid or myeloid) have been reported only rarely. Our review of the cytochemical and immunophenotypic features of 89 cases of childhood leukemia in marrow relapse indicated lineage switch (lymphoid to myeloid or the reverse) in six patients (6.7%). Five patients with acute lymphoblastic leukemia (ALL) at diagnosis had converted to acute nonlymphoblastic leukemia (ANLL), and one had converted from ANLL to ALL. Each child received lineage-specific multiagent chemotherapy when initially diagnosed, and all achieved a complete remission. After conversion, four patients readily achieved second remissions with treatment for the phenotype evident at lineage switch. Two patients with ANLL at conversion failed ALL-directed reinduction, while one of the two responded to high-dose cytarabine but died during bone marrow hypoplasia, emphasizing the importance of prompt recognition of lineage switch and selection of an appropriate plan of retreatment. Cytogenetic studies disclosed evidence of clonal selection in one patient and clonal stability in two. These findings indicate an unexpectedly high frequency of lineage switch in patients who relapse in the bone marrow after intensive chemotherapy. Although specific causative factors could not be identified, our observations suggest at least two general mechanisms for lineage switch in acute leukemia. In one, chemotherapy appears to eradicate the dominant clone present at diagnosis, permitting expansion of a secondary clone with a different phenotype. In the second, drug-induced changes in the original clone may either amplify or suppress differentiation programs so that phenotypic shift is possible.
Phagocytosis of sickle erythrocytes: immunologic and oxidative determinants of hemolytic anemia
Authors: Hebbel RP , Miller WJ
Published: October 2019

Hemolytic anemia in sickle disease involves both intravascular and extravascular destruction of erythrocytes. Since the latter presumably involves the reticuloendothelial system, we have examined interactions between sickle erythrocytes and macrophages. In erythrophagocytosis assays, 18.9 +/- 7.2% of human marrow macrophages ingest sickle RBCs, while only 3.1 +/- 2.1% ingest normal RBCs. This abnormality is not explained by reticulocytosis, and it is strongly dependent upon RBC density. The interaction between sickle RBCs and macrophages appears to be partly immunologic, since it is partially blocked by Fc receptor blockade. Also, admixture of sickle RBCs (pretreated with rabbit anti- human-Ig) and Fc-receptor-bearing K562 cells results in 15.6 +/- 10.6% K562-RBC rosette formation compared with only 0.5 +/- 1.2% for normal RBCs. Regarding other factors that might promote erythrophagocytosis, sickle RBCs are found to spontaneously generate twice-normal amounts of dialdehyde byproducts of lipid peroxidation (“malondialdehyde” or MDA). Peroxide or reagent-MDA treatment of normal RBCs significantly enhances their phagocytosis, and MDA is at least 50 times more potent than other aldehydes studied here. Oxidative and immunologic effects may be related, since exposure of MDA-treated RBCs to immunoglobulin- containing human sera results in a further significant enhancement of erythrophagocytosis. For comparison of different sickle patients, an adherence assay demonstrates that sickle RBCs are 1.03 to 6.85 times more adherent to macrophages than are normal RBCs, and degree of adherence correlates significantly with irreversibly sickled cell (ISC) counts and hematologic variables reflecting hemolytic rate. We conclude that propensity for RBC interaction with macrophages is likely to be a determinant of hemolytic rate in sickle disease. Pertinent mechanisms appear to involve modification of RBC membranes by dialdehyde byproducts of excessive autoxidation and the abnormal acquisition of surface immunoglobulin on sickle RBCs, although participation of other membrane defects has not been excluded. Interestingly, the data further suggest the possibility that appearance of the “senescence antigen” in old normal RBCs represents modification of the membrane by “MDA.”
Studies of lymph nodes from patients with classical hemophilia
Authors: Andes WA , deShazo RD , Reed RJ , Harkin JC , Wang NN
Published: October 2019

Within the last 18 months, we have noted the development of unexplained lymph node enlargement in otherwise asymptomatic patients with hemophilia. Because such changes are poorly understood and, in some patient groups, may be related to the acquired immunodeficiency syndrome (AIDS), we studied the enlarged lymph nodes in four patients with severe factor VIII deficiency and abnormally low peripheral blood helper-inducer/suppressor cell (OKT4/OKT8) ratios. Surgically excised lymph nodes were studied for histopathologic, electron microscopic, and chromosomal changes. Cell suspensions from these and normal nodes were also studied using monoclonal antibodies. Excised lymph nodes showed follicular hyperplasia. Electron microscopy revealed no viral particles or vesicular rosettes. Chromosomal aberrations included an acrocentric marker chromosome in one patient and monosomy 21 in another. T lymphocyte ratios (OKT4/OKT8) in lymph node suspensions were lower than those in nodes from normal controls (1.2 v 6.1) and reflected the lymphocyte ratio in peripheral blood. Mature B cell percentages were increased in the lymph nodes from patients with hemophilia (38% v 27% in controls). Patients treated with factor VIII concentrates and male homosexuals have similarities in persistent lymph node enlargement, histologic features of follicular hyperplasia, and changes in lymph node and circulating lymphocyte subpopulations.
Clonal origin of cells restricted to monocytic differentiation in acute nonlymphocytic leukemia
Authors: Ferraris AM , Broccia G , Meloni T , Canepa L , Sessarego M , Gaetani GF
Published: October 2019

Two patients with acute monocytic leukemia and heterozygous for the Mediterranean variant of the X-linked enzyme, glucose-6-phosphate dehydrogenase (G6PD), were investigated to determine the number and type of progenitor cells involved. Mosaicism for Mediterranean G6PD was assessed by the different rate of utilization of 2-deoxy glucose-6- phosphate (2dG6P) by normal and Mediterranean variants of G6PD. The monocytoid blasts were found to express one type of G6PD only, indicating their clonal origin from a common progenitor cell, whereas all other hemopoietic cell populations tested expressed the heterozygous phenotype. The finding of a unique involvement of the monocytic line in two cases of acute nonlymphocytic leukemia (ANLL) represents further evidence of heterogeneity of stem cell involvement in ANLL.
Study of the expression of a glycoprotein of retroviral origin in the plasma of patients with hematologic disorders and in the plasma of normal individuals
Authors: Jacquemin PC , Strijckmans P , Thiry L
Published: October 2019

A 74,000 molecular weight glycoprotein was purified from the plasma of a patient with chronic myelogenous leukemia in blast crisis. Monoclonal antibodies were produced in the mouse and used to characterize this protein. It was shown to contain p15E antigenic determinants and portions of a reverse transcriptase. The level of this protein was found to be elevated in leukemic patients with high white blood cell counts and also in some patients with other hematopoietic disorders as compared to the level measured in normal individuals. The level of the protein was strongly reduced in acute leukemia patients after intense chemotherapy treatment. We tentatively conclude that this protein is of endogenous retroviral origin and perhaps regulates hematopoietic tissues.
Chromosome-mediated transfer of the malignant phenotype by human acute myelogenous leukemic cells
Authors: Minden MD , Gusella JF , Housman D
Published: October 2019

Acute myelogenous leukemia (AML) is a malignancy of the myeloid cells of the bone marrow. Recently, a number of groups have demonstrated that it is possible to study the malignant phenotype at the level of DNA through gene transfer experiments. We have used such an approach to determine whether it is possible to transfer the malignant phenotype of anchorage independence from human AML cells to anchorage-dependent rodent cells, using chromosomes as the source of genetic information. We found that chromosomes isolated from leukemic cell lines were capable of transferring the malignant phenotype of anchorage independence, whereas chromosomes derived from the lymphocytes of normal individuals were not active in this assay. Using Southern blot analysis of the DNA from transferants, we were able to show that the transfer of anchorage independence correlated with the presence of human DNA in the transferants. The pattern of human DNA in the transferants derived from different transfection experiments is compared.
Two populations of erythroid cell progenitors in paroxysmal nocturnal hemoglobinuria
Authors: Rotoli B , Robledo R , Scarpato N , Luzzatto L
Published: October 2019

We have grown erythroid cell colonies from two patients with paroxysmal nocturnal hemoglobinuria (PNH). At 11 to 13 days, individual bursts were picked and incubated for 24 hours with 3H-leucine in order to label total cell protein (mainly hemoglobin). After appropriate washing, each burst was subjected to a miniaturized acidified serum test, and lysis was measured by the release of radioactivity. In bursts from normal controls, lysis was 19% +/- 13% SD. By contrast, of 58 bursts from PNH patients, 14 had lysis similar to that of controls (mean 15.4% +/- 10.6%), while 44 had lysis ranging from 42.2% to 85.8% (mean 70.3% +/- 10.4%). Colonies sensitive to acidified serum were acetylcholinesterase (AchE) negative, whereas normal colonies were AchE- positive. Thus, based on two independent criteria, a dual population of erythroid burst-forming units (BFU-E) can be demonstrated in PNH. These data confirm directly the somatic mutation model of the pathogenesis of PNH, and by these methods the relative sizes of the normal and the PNH cell populations can be measured at the level of the erythroid cell precursors.
Factor IX and prothrombin in amniotic fluid and fetal plasma: constraints on prenatal diagnosis of hemophilia B and evidence of proteolysis
Authors: Thompson AR
Published: October 2019

Potential limitations of prenatal diagnosis of hemophilia B, as compared to hemophilia A, include (1) occurrence of far more frequent defects with abnormal circulating antigen, (2) lower levels of factor IX in fetal plasma at 16 to 20 weeks gestation, and (3) the presence of factor IX antigen in amniotic fluid. In addition, proteolysis could occur, especially with amniotic fluid contamination of fetal plasma. A sensitive polyclonal immunoradiometric assay for factor IX antigen was used to characterize the range of levels in amniotic fluids and fetal plasma samples. To assess for altered forms, factor IX species were compared to those of a homologous clotting factor, prothrombin. Fourteen postmortem abortus blood samples from fetuses of 14 to 23 weeks gestation had factor IX antigen levels that averaged 5.1 U/dL and ranged from 1.7 to 15 U/dL. Amniotic fluid factor IX antigen averaged 2.9 U/dL, with a range from 1.4 to 8.5 U/dL in 19 separate amniocentesis samples. Thus, in a male fetus at risk of hemophilia B and with a low circulating level of gene product, mixture of fetal plasma with amniotic fluid could severely limit prenatal diagnosis, assuming that the amniotic fluid factor IX is of maternal origin. Despite rapid processing of amniotic fluid samples, the prothrombin was extensively cleaved, suggesting that it had been activated in vivo. On gel electrophoresis of amniotic fluid samples, however, factor IX was only minimally cleaved. In the postmortem fetal blood specimens, prothrombin was partially cleaved. On crossed-immunoelectrophoresis, fetal plasma prothrombin showed decreased migration in calcium, compared to EDTA, indicative of mature gamma-glutamyl carboxylation. The latter presumably resulted from fetal hepatic synthesis.
Passive material behavior of granulocytes based on large deformation and recovery after deformation tests
Authors: Evans E , Kukan B
Published: October 2019

In order to better understand the in vivo rheologic behavior of white cells, we have studied the time-dependent deformability, recovery, and mechanical activation of blood granulocytes. We have used micropipette aspiration methods to measure the large deformation response and recovery after deformation characteristics of neutrophils as functions of time, temperature, and collecting media. The cell response in the pipette experiment was characterized by three time domains: the first phase was the passive deformation response to the fixed suction pressure; the second phase was an obvious transition from the passive to active motile cellular state where the cell exhibited erratic length changes in the pipette; and the third phase was the steady recovery after the suction pressure had been zeroed. Tests on white cells were carried out with three different anticoagulants to evaluate the effect of calcium on deformation and recovery behavior; also, cells were separated by centrifugation in high molecular weight dextran to determine whether or not collection and separation procedures affected the cell properties. Our results have shown that the passive deformation of granulocytes into the pipette was a continuous flow process with no approach to a static deformation limit. In addition, there was an obvious threshold pressure below which the cell would not deform and enter the micropipette. For suction pressures significantly above the threshold, granulocytes were continuously deformed with a similar functional dependence on time. The coefficient of proportionality between aspiration length and time, as well as the exponent, depended on suction pressure, pipette dimension, and temperature. It was observed that the granulocytes always recovered to the spherical state after deformation, independent of the extent of deformation or location where the cell was aspirated. Based on the recovery behavior, plus the dependence of the pressure threshold on pipette size, we propose the concept that the granulocyte membrane and cortical shell behave like a “contractile surface carpet” under tension, where the cell interior responds passively like a highly viscous liquid. The membrane cortex appears to be subject to a persistent stress (tension) of about 10(-2) dyne/cm. Our observations of passive to active transition in the pipette suction experiment indicated that granulocytes may be stimulated by deformation at room temperature. This study represents the first detailed investigation of the large deformation behavior of granulocytes, and the results indicate a simple structural model to represent the passive rheologic behavior of the granulocyte.
Dense cells in sickle cell anemia: the effects of gene interaction
Authors: Fabry ME , Mears JG , Patel P , Schaefer-Rego K , Carmichael LD , Martinez G , Nagel RL
Published: October 2019

In an attempt to uncover potential genetic sources of the clinical diversity of sickle cell anemia, we have characterized homozygous SS patients in the following ways: percentage of dense red blood cells (% F4) as determined from Percoll-Stractan continuous density gradients, alpha gene deletion, average percentage of hemoglobin F (% HbF), hemoglobin in g/dL, age, and sex. We find that alpha 4 individuals have a higher % F4 (mean 24% +/- 15%) than alpha 3 individuals (mean 12% +/- 8%) (P less than .005). Multivariate analysis demonstrated a significant correlation among % F4 levels and alpha-gene number and % HbF, and an interaction between the last two variables. The other variables considered did not significantly alter this model. As reported before, with fewer samples, we find that in the first ten years of life of SS individuals, the frequency of alpha gene deletion is 17%, which is comparable to that in the general black population, while in the group over 20 years of age, the frequency rises to 49%, implying that alpha thalassemia is associated with longer survival. These results indicate that it is necessary to consider sickle cell anemia not only as a single gene defect, but also as a disease whose clinical expression is the result of a group of genes capable of interacting at the phenotypic level.
Production of F cells in sickle cell anemia: regulation by a genetic locus or loci separate from the beta-globin gene cluster
Authors: Boyer SH , Dover GJ , Serjeant GR , Smith KD , Antonarakis SE , Embury SH , Margolet L , Noyes AN , Boyer ML , Bias WB
Published: October 2019

Levels of fetal hemoglobin (HbF) bearing reticulocytes (F reticulocytes) range from 2% to 50% in patients with sickle cell (SS) anemia. To learn whether any portion of such variation in F cell production is regulated by loci genetically separable from the beta- globin gene cluster, percentages of F reticulocytes were compared in 59 sib pairs composed solely of SS members, including 40 pairs from Jamaica and 19 from the United States. We reasoned that differences in F reticulocyte levels might arise (1) from any of several kinds of artifact, (2) via half-sib status, or (3) because one or more genes regulating F cell production segregate separately from beta S. We minimized the role of artifact by assay of fresh samples from 84 SS individuals, including both members of 38 sib pairs. In 78 of the 84 subjects, serial values for percent F reticulocytes fell within 99.9% confidence limits or were alike by t test (P greater than or equal to .05). This left 32 sib pairs for which F reticulocyte levels in each member were reproducible. When sib-sib comparisons were limited to these 32 pairs, percentages of F reticulocytes were grossly dissimilar within 12 Jamaican and 3 American sibships. Within them, the probability that sibs were alike was always less than or equal to .005 and usually less than or equal to 10(-4). We next minimized the contribution of half-sibs among Jamaicans by a combination of paternity testing and sib-sib comparison of beta-globin region DNA restriction fragment length polymorphisms, especially among discordant pairs. We thereafter concluded that at least seven to eight Jamaican pairs were composed of reproducibly discordant full sibs. There is thus little doubt that there are genes regulating between-patient differences in F cell production that are separate from the beta-globin gene cluster. Still unanswered is (1) whether or not these genes are actually linked to beta S, (2) why F reticulocyte levels in Americans tend to be lower than in Jamaicans, and (3) whether or not differences in F cell production among SS patients are regulated by several major loci or by only one.
Differentiation of a human monocytic cell line by 1,25-dihydroxyvitamin D3 (calcitriol): a morphologic, phenotypic, and functional analysis
Authors: Rigby WF , Shen L , Ball ED , Guyre PM , Fanger MW
Published: October 2019

Several recent studies have suggested a role for 1,25-dihydroxyvitamin D3 (calcitriol) in myeloid differentiation. We have examined the effects of calcitriol on the U937 monoblast cell line and found that calcitriol, at near physiologic concentrations, is a potent inhibitor of U937 growth. Moreover, calcitriol induces differentiation to a monocyte-macrophage phenotype marked by enhanced alpha-naphthyl esterase staining. Morphologic changes were attended by de novo induction of the myeloid specific antigen detected by the monoclonal antibody AML-2–23, as well as dramatic increases in Fc receptors for IgG. In addition, calcitriol induced U937 cells to perform phagocytosis and antibody-dependent cellular cytotoxicity. These results indicate the potential activity of calcitriol in myeloid differentiation and additionally suggest a role for calcitriol in monocyte-macrophage activation.
Progressive preleukemia with a chromosomally abnormal clone in a kindred with the Estren-Dameshek variant of Fanconi's anemia
Authors: Nowell P , Bergman G , Besa E , Wilmoth D , Emanuel B
Published: October 2019

One of two sisters with hypoplastic anemia and increased chromosomal fragility, but no clinical stigmata of Fanconi's anemia (FA), progressed to preleukemia (with a clone of chromosomally abnormal cells in the bone marrow), and then to acute nonlymphocytic leukemia. The findings indicate that constitutional genetic instability can lead to karyotypically aberrant neoplastic clones in the Estren-Dameshek variant of FA as well as in the more typical chromosomal fragility syndromes.
Two-cycle timed-sequential chemotherapy for adult acute nonlymphocytic leukemia
Authors: Vaughan WP , Karp JE , Burke PJ
Published: October 2019

Based on a series of clinical and laboratory studies of leukemia cell kinetics and responses to chemotherapy, we have developed an intensive timed-sequential regimen of daunorubicin and high-dose infusion 1-beta- D-arabinofuranosyl cytosine for the treatment of adult acute nonlymphocytic leukemia. Of the first 34 patients achieving complete remission (CR) with a single cycle of this therapy, four (12%) remain in complete remission without further therapy after a minimum of five years of follow-up. Treatment of relapsed patients with a second course of the same regimen at relapse and no chemotherapy in second remission increased to seven (21%) the number of patients expected to remain in remission for four years or more from their last chemotherapy. Beginning in 1980, however, we gave all consenting adults a second cycle of this chemotherapy in early first remission. Of the first 25 patients treated with a second cycle of this chemotherapy in early first remission, there was one toxic death, but 11 patients (44%) remain in CR with a median follow-up of almost three years.
A reassessment of the energy requirements for neutrophil migration: adenosine triphosphate depletion enhances chemotaxis
Authors: Lane TA , Lamkin GE
Published: October 2019

In view of previous studies demonstrating a significant correlation between adenosine triphosphate (ATP) depletion and impairment of chemotaxis (CTX) during granulocyte (PMN) storage, we sought to quantitate the relationship between CTX and PMN energy metabolism. We incubated PMNs at 37 degrees C with 2-deoxyglucose (2-dg) in the presence of 5 mmol/L glucose. As expected, ATP inhibition by 2-dg was time-dependent (T 1/2, 18 minutes) and dose-dependent, with half- maximal inhibition of ATP (ID50) with 1.3 +/- .3 mmol/L 2-dg. Similar concentrations of 2-dg inhibited lactate generation, phagocytosis, superoxide anion generation, and degranulation. The random migration of PMNs was inhibited by somewhat higher concentrations of 2-dg (ID50, 12 mmol/L). In contrast, up to 40 mmol/L 2-dg did not inhibit CTX toward synthetic peptides or activated serum. In fact, 2-dg consistently increased the CTX of PMNs toward 10(-8) mol/L f-Met-Leu-Phe (fMLP), to a maximum of 450% of control CTX using 15 mmol/L 2-dg. Half-maximal stimulation (ED50) of CTX occurred at 6.3 +/- 1.0 mmol/L 2-dg. Although maximal CTX toward optimal concentrations of fMLP was consistently increased with 2-dg, the ED50 of CTX to fMLP was unchanged (ED50 with glucose, 2.0 +/- 0.6 nmol/L fMLP; ED50 with 2-dg 2.2 +/- 0.7 nmol/L fMLP), and 2-dg did not increase fMLP receptors. In the absence of glucose, 2-dg exerted similar effects on ATP and CTX, but at doses 30- to 50-fold lower than in the presence of glucose. Other glycolytic inhibitors (iodoacetamide and sodium fluoride) exerted similar effects. Additional studies indicated that CTX enhancement by 2-dg (a) required Mg++ but not Ca++, (b) occurred with PMNs from a patient with chronic granulomatous disease, (c) was unaltered in the presence of inhibitors of proteolysis, (d) was not due to generation of a soluble agent, (e) was not due to alterations in PMN adherence, and (f) was not due to inhibition of glycosylation. We conclude that the chemotaxis, but not the random migration, of PMNs is surprisingly resistant to inhibition of energy metabolism and depletion of ATP, since concentrations of 2-dg that decreased ATP and other cell functions by more than 50% not only did not inhibit, but actually stimulated, CTX. These studies also indicate that the previously reported correlation between ATP depletion and CTX impairment observed in stored PMNs are not causally related.
Fluorescence cytophotometric analysis of megakaryocytic ploidy in culture: studies of normal and thrombocytopenic mice
Authors: Chatelain C , Burstein SA
Published: October 2019

A system for the accurate and rapid measurement of the ploidy of cultured megakaryocytes derived from megakaryocytic colony-forming cells (CFU-M) has been developed. Thirty thousand murine marrow cells per milliliter were cultured for varying time periods in agar in the presence of horse serum and pokeweed mitogen-stimulated spleen cell- conditioned medium (PWM-SCM). To ensure the inclusion of all the megakaryocytic cells in the analysis, entire agar discs were transferred onto glass slides and dried. Cells of the megakaryocytic lineage were identified by staining for acetylcholinesterase (AchE) for two hours. Subsequently, the nuclei of the cells were stained using 1.7 X 10(-5) mol/L chromomycin A3, a specific DNA-binding fluorochrome. Megakaryocytic colonies (greater than or equal to 2 AchE+ cells) were located under transmission light. The fluorescence emission of each cell of the colony was then measured by a photometer interfaced with a computer. The mean fluorescence emission of about 20 random granulocytes per slide was used as a 2N standard. There was no significant cell loss, quenching of fluorescence by AchE staining, or overlapping of colonies or cells. Approximately 100 megakaryocytes per hour could be analyzed. Modal ploidy of cultured megakaryocytes increased from 2N to 32N between days 3 and 6 in culture. Varying concentrations of PWM-SCM from 5% to 20% did not affect the ploidy distribution when examined at day 5. The heterogeneity of the ploidy of cells within colonies increased continuously with increasing cell numbers per colony. Clonal analyses of mean ploidy and ploidy heterogeneity did not show distinct types or classes of colonies; rather, the data show that megakaryocytic colonies are structured as a continuum. An inverse correlation was found between the number of cells constituting the colonies and their mean DNA content. To determine if short-term in vivo exposure of CFU-M to a thrombocytopenic environment could affect the ploidy of their progeny, mice were given rabbit antimouse-platelet serum while control animals were given normal rabbit serum. Twenty-four hours after injection, marrow derived from these animals was cultured. At day 5, the ploidy distributions and ploidy heterogeneity were identical in both treated and control groups. Thus, factor(s) that promote CFU-M proliferation do not affect megakaryocytic endoreduplication, while stimuli that acutely influence megakaryocytic ploidy in vivo do not determine the ultimate ploidy potential of megakaryocytes derived from a CFU-M.
Potentiation by red blood cells of shear-induced platelet aggregation: relative importance of chemical and physical mechanisms
Authors: Reimers RC , Sutera SP , Joist JH
Published: October 2019

Evidence has been reported to indicate that red blood cells (RBCs) may potentiate platelet adherence and platelet aggregation (PAG) in different flow systems in vitro as well as hemostatic platelet plug formation in response to vascular injury. In this study, we demonstrate that RBCs enhance PAG induced by well-defined, low-intensity, uniform, laminar shear stress. Potentiation by RBCs of shear-induced PAG was associated with appreciable loss of adenine nucleotides from 14C- adenine-labeled RBCs, the extent of which increased with increasing RBC concentration. The concentrations of RBC-derived ADP measured in the medium after shear, as determined by both high pressure liquid chromatography and the luciferin/luciferase system, were within the range of concentrations of ADP which may trigger PAG or potentiate PAG induced by low concentrations of other platelet agonists in the aggregometer. To assess the relative contribution of chemical (ADP) and physical (platelet surface transport) mechanisms in the RBC-mediated potentiation of shear-induced PAG, aliquots of citrated platelet-rich plasma (C-PRP) were exposed to shear stress in the presence of untreated RBCs or RBCs exposed to an antihemolytic concentration (5 mumol/L) of the membrane stabilizing agent, chlorpromazine (CPZ). Potentiation of shear-induced PAG in the RBC-CPZ system was significantly less than that in the untreated RBC system. However, CPZ- induced reduction of PAG potentiation was associated with an increase rather than a decrease in loss of adenine nucleotides from RBC. Furthermore, shear-induced PAG in C-PRP as well as ADP- and collagen- induced PAG in C-PRP in the aggregometer was significantly inhibited by 5 mumol/L CPZ, indicating that the observed reduced potentiation of shear-induced PAG by RBCs in the presence of CPZ was due to a direct inhibitory effect of the drug on platelets rather than a reduction of shear-induced liberation of ADP from RBCs. When aliquots of C-PRP were exposed to shear stress in the presence of RBCs completely depleted of ADP by fixation in 1% glutaraldehyde, potentiation of PAG was approximately half of that observed with intact RBCs. These findings indicate that both RBC-derived ADP and RBC-mediated platelet surface transport are involved in the potentiation by RBCs of PAG induced by laminar shear stress.
Red blood cell deformability influences platelets--vessel wall interaction in flowing blood
Authors: Aarts PA , Heethaar RM , Sixma JJ
Published: October 2019

Hematocrit and red cell size are important factors for the transport of blood platelets toward subendothelium in flowing blood. We report that red cell deformability also influences platelet transport. Red cell deformability was estimated with Couette-flow viscosimetry at a shear rate of 130 s-1 and expressed as a ‘T’ factor--a dimensionless parameter relating the relative viscosity and the hematocrit derived from the relationship: T = (1 - mu -0.4 rel)/H, where mu rel is the relative viscosity and H is the hematocrit. The normal value of T was within a narrow range (0.91 +/- 0.02). Treatment of normal red cells with isoxsuprine and chlorpromazine caused decreased rigidity and decreased T. Cholesterol loading and treatment with diamide increased rigidity and increased T. In vitro perfusion experiments in an annular perfusion system with everted human umbilical arteries were performed with perfusates to which such treated red blood cells were added to investigate their influence on platelet adherence to artery subendothelium. Platelet adherence was well correlated with red cell rigidity, with increased adherence at increased rigidity and vice versa. A change in T of 0.10 corresponded to a change in platelet adherence of approximately 50%. These effects were more pronounced at a wall shear rate of 1,800 s-1 than at 300 s-1.
Further characterization of platelet-type von Willebrand's disease in Japan
Authors: Takahashi H , Handa M , Watanabe K , Ando Y , Nagayama R , Hattori A , Shibata A , Federici AB , Ruggeri ZM , Zimmerman TS
Published: October 2019

We studied four patients who showed aggregation of platelets in platelet-rich plasma at lower concentrations of ristocetin than those required for normal platelet-rich plasma and who demonstrated an increased capacity of the platelets to bind normal von Willebrand factor. The four patients were from two Japanese families. Platelets from one family aggregated spontaneously in vitro, and platelets from both families aggregated upon the addition of normal plasma and cryoprecipitate, in the absence of ristocetin or other agonists. Analysis of the multimeric composition of von Willebrand factor by sodium dodecyl sulfate-agarose gel electrophoresis revealed a decrease in large multimers or a decrease in both large and intermediate multimers in plasma, but normal multimers in platelets. 1-Deamino-[8-D- arginine]-vasopressin caused by an immediate appearance of larger multimers in plasma, followed by the rapid disappearance of these multimers from circulating plasma. Analysis of platelet membrane glycoproteins from the patients showed that there were two distinct bands in the glycoprotein I region; one migrated in a slower region and the other in a faster region than normal glycoprotein Ib. We suggest that the platelet receptor abnormality in these patients is related to this abnormality of glycoprotein Ib.
Effect of hepatic dysfunction on oral cyclosporin pharmacokinetics in marrow transplant patients
Authors: Yee GC , Kennedy MS , Storb R , Thomas ED
Published: October 2019

The effect of hepatic dysfunction, defined as abnormal serum bilirubin level, on oral cyclosporin (CSP) pharmacokinetics was examined in 28 marrow transplant patients who received CSP for prophylaxis of graft-v- host disease. Serum CSP concentrations were measured by radioimmunoassay. Forty-one concentration-time courses were studied, divided among patients with no (less than 1.2 mg/dL), mild (1.2 to 2.0 mg/dL), and moderate (2.0 to 5.0 mg/dL) hepatic dysfunction. CSP elimination, as determined by elimination rate constant and clearance, was delayed in patients with moderate hepatic dysfunction compared to those with no hepatic dysfunction (P less than .05). The volume of distribution, lag time for absorption, maximum serum concentration, and time at which the maximum concentration was achieved was not affected by hepatic function. These data indicate that patients with moderate hepatic dysfunction have delayed CSP or CSP metabolite elimination and may be at higher risk for developing CSP-related toxicity.
Prognostic information from cytogenetic analysis in chronic B- lymphocytic leukemia and leukemic immunocytoma
Authors: Juliusson G , Robert KH , Ost A , Friberg K , Biberfeld P , Nilsson B , Zech L , Gahrton G
Published: October 2019

Fifty-five patients with a clonal expansion of B lymphocytes in the peripheral blood were studied. According to the Kiel classification, 22 patients had chronic lymphocytic leukemia (CLL), 29 had immunocytoma (IC), two had prolymphocytic leukemia, and one had centrocytic lymphoma; one was not subclassified. Cytogenetic studies after B cell mitogen stimulation showed that six patients had an extra chromosome 12 as the sole abnormality. Another ten patients had an extra chromosome 12 together with other abnormalities. One patient had dup(12). Fifteen patients showed clonal aberrations without +12. Eleven patients showed only normal metaphases, and 12 patients were not evaluated cytogenetically. The cytogenetic subgroup pattern did not distinguish between CLL and IC patients. There was no significant difference between the CLL and IC groups as regards clinical findings and prognosis. However, the cytogenetic typing proved to be of prognostic significance. Increasing numbers of chromosomal aberrations within the cell clone were significantly associated with a poorer prognosis, ie, with impairment of survival (P = .04) and therapy-free survival (P less than 10(-4]. Patients with complex karyotypes (at least clonal aberrations) showed the poorest survival (P = .007). Patients with +12 required treatment earlier than patients with a normal karyotype (P = .01) and patients with karyotypic changes other than +12 (P = .006). These latter differences were even more pronounced when only IC patients were considered (P = .005 and P = .002, respectively). A multivariate analysis revealed that +12 was as strong an indicator of poor survival as advanced Rai or Binet stages and a stronger predictor of therapy-demanding disease.
Simultaneous or sequential expression of lymphoid and myeloid phenotypes in acute leukemia
Authors: Neame PB , Soamboonsrup P , Browman G , Barr RD , Saeed N , Chan B , Pai M , Benger A , Wilson WE , Walker IR
Published: October 2019

Acute mixed myeloid-lymphoid leukemia is uncommon. We report four cases in which myeloid and lymphoid cell markers were observed simultaneously or sequentially when 94 patients with acute leukemia were phenotyped according to the French-American-British (FAB) classification system, with cytochemical stains, and with immunologically defined differentiation markers (identified by monoclonal antibodies and antiterminal deoxynucleotidyl transferase [TdT]). In one case, conversion from acute lymphoblastic leukemia to acute myeloid leukemia was noted (FAB L1, TdT+ to FAB M4, Auer rods, TdT-). In another patient, two distinct populations of myeloid and lymphoid blast cells were observed simultaneously (TdT-, LeuM1+/TdT+, LeuM1-). In two additional patients, acute leukemia was characterized by the expression of both lymphoid and myeloid markers on the same cell (TdT+/Leu M1+, B4+/Leu M1+ and greater than or equal to 70% TdT+, T11+, My9+). The Philadelphia (Ph1) chromosome was negative in all cases, though other chromosomal abnormalities were noted in three out of four cases. Malignant transformation of a pluripotential stem cell for both lymphoid and myeloid lineages, with or without the Ph1 chromosome marker, could explain the coexistence of distinct populations of lymphoblasts and myeloblasts in acute leukemia. Acute leukemia with a biphenotypic profile may reflect genome depression accompanying neoplasia.
Increased binding of fibrinogen to platelets in diabetes: the role of prostaglandins and thromboxane
Authors: DiMinno G , Silver MJ , Cerbone AM , Riccardi G , Rivellese A , Mancini M , Thiagarajan P
Published: October 2019

Previous studies suggested a role for prostaglandins or thromboxane A2, or both in the exposure of fibrinogen receptors on normal platelets in response to several aggregating agents. Platelets from diabetics are known to be more sensitive to aggregating agents and to produce more prostaglandins and thromboxane than platelets from normal subjects. We compared fibrinogen binding to platelets from diabetic subjects with binding to platelets from normal subjects and determined whether aspirin (which inhibits the formation of prostaglandins and thromboxane) would inhibit the binding of fibrinogen to platelets from diabetic subjects and whether this correlated with its effects on platelet aggregation. We found the following: Aspirin suppressed thromboxane formation and rendered the platelets less sensitive to the induction of aggregation by adenosine diphosphate (ADP) or collagen. The amount of U-46619 [( 15s]-hydroxy-11-alpha, 9-alpha [epoxy-methano]- prosta[5Z,13E]-dienoic acid, a stable analog of prostaglandin endoperoxide/thromboxane A2) necessary to induce aggregation, was similar in normal and diabetic subjects and was unchanged after ingestion of aspirin. Binding of 125I-fibrinogen following stimulation of platelets by ADP or collagen was greater in diabetic (because more binding sites were exposed) than in normal subjects. However, following stimulation by U-46619, binding was similar in diabetic and normal subjects. Aspirin caused a reduction in the exposure of binding sites on both platelets from diabetic and normal subjects, so that (in this respect) platelets from diabetic subjects became more like those from normal subjects. Effects of the monoclonal antibody B59.2, which is specific for the platelet glycoprotein IIb-IIIa complex (the presumed receptor for fibrinogen on the platelet surface) were also studied. The amount of this antibody that bound to platelets was the same for normal and diabetic subjects both before and after aspirin and with or without stimulation by ADP or collagen. In addition, B59.2 inhibited aggregation and fibrinogen binding in both platelets from diabetic and normal subjects. The combined data suggest that the glycoprotein IIb- IIIa complex of platelets from diabetic subjects is similar to that of platelets from normal subjects and that the increased fibrinogen binding and aggregation of platelets from diabetic subjects in response to ADP or collagen is mediated by increased formation of prostaglandin endoperoxide or thromboxane A2, or both.
Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics
Authors: Stong RC , Korsmeyer SJ , Parkin JL , Arthur DC , Kersey JH
Published: October 2019

A cell line, designated RS4;11, was established from the bone marrow of a patient in relapse with an acute leukemia that was characterized by the t(4;11) chromosomal abnormality. The cell line and the patient's fresh leukemic cells both had the t(4;11)(q21;q23) and an isochromosome for the long arm of No. 7. Morphologically, all cells were lymphoid in appearance. Ultrastructurally and cytochemically, approximately 30% of the cells possessed myeloid features. The cells were strongly positive for terminal deoxynucleotidyl transferase. They were HLA-DR positive and expressed surface antigens characteristic for B lineage cells, including those detected by anti-B4, BA-1, BA-2, and PI153/3. Immunoglobulin gene analysis revealed rearrangements of the heavy chain and kappa chain genes. The cells lacked the common acute lymphoblastic leukemia antigen and antigenic markers characteristic of T lineage cells. The cells reacted with the myeloid antibody 1G10 but not with other myeloid monoclonal antibodies. Treatment with 12-O-tetradecanoyl- phorbol-13-acetate induced a monocyte-like phenotype demonstrated by cytochemical, functional, immunologic, and electron microscopic studies. The expression of markers of both early lymphoid and early myeloid cells represents an unusual phenotype and suggests that RS4;11 represents a cell with dual lineage capabilities. To our knowledge, RS4;11 is the first cell line established from t(4;11)-associated acute leukemia.
Absence of a surface-connected canalicular system in bovine platelets
Authors: Zucker-Franklin D , Benson KA , Myers KM
Published: October 2019

Human platelets possess a surface-connected canalicular system (SCCS) which has been postulated to subserve endocytosis as well as secretion. Platelets of most animal species studied to date, including those of cattle, function similarly. However, ultrastructural analysis of freeze- fractured bovine platelets or thin sectioned bovine platelets treated with electron-dense tracers failed to delineate a SCCS. This observation may throw an entirely new light on the role of this organelle.