bluuebunny/crossref_metadata_2025 huggingface dataset:
Leukocyte migration inhibition of buffy coats from patients with autoimmune thrombocytopenic purpura when exposed to normal platelets: modulation by transfer factor
Authors: Borkowsky W , Karpatkin S
Published: October 2019

Cellular-mediated immunity was studied in autoimmune thrombocytopenic purpura (ATP) patients by investigating leukocyte migration inhibition (LMI) following the interaction of normal platelets with patients' lymphocytes. When normal platelets were incubated with leukocyte buffy coats of ATP patients, the migration index (MI) was significantly impaired compared to buffy coats from normal subjects, employing 4 different concentrations of platelets. At the highest platelet concentration (10(9)/ml), MI was 0.87 +/- 0.04 (SEM) for ATP lymphocytes compared to 1.05 +/- 0.05 (p less than 0.01) for normal lymphocytes. Nine of 21 patients had an MI less than 0.80, whereas all control subjects had MIs greater than 0.85. Similar results were obtained at 2 different platelet membrane concentrations. At 500 micrograms/ml, the MI for ATP lymphocytes was 0.74 +/- 0.04, compared to 0.98 +/- 0.08 (p less than 0.01) for normal lymphocytes (12 experiments). An inverse relationship was noted between platelet count and lymphokine production in ATP patients (r = 0.815, p less than 0.001, 10 experiments). Transfer factor from an ATP patient in remission converted an abnormal LMI response of 0.68 +/- 0.04 from a patient with severe thrombocytopenia to 0.84 +/- 0.07 (p less than 0.005, 8 experiments). Similar results were obtained with transfer factor from 2 other patients in remission. Transfer factor from a patient with severe thrombocytopenia converted a normal response of 1.04 +/- 0.05 of normal subjects to a lower response of 0.88 +/- 0.04 (p less than 0.03, 12 experiments). Thus, lymphocytes of ATP patients are primed to recognize and be perturbed by normal platelets, whereas normal lymphocytes are not. This indicates specificity of the antigen- lymphocyte reaction in ATP patients. Transfer factor is capable of modulating this response in vitro.
Phagolysosomal pH of human neutrophils
Authors: Cech P , Lehrer RI
Published: October 2019

Human neutrophils sequester yeast cells or zymosan particles in two classes of phagocytic vacuoles: sealed and unsealed. The pH of sealed vacuoles was measured by a newly devised fluorometric procedure that used fluoresceinated zymosan particles, derived from Saccharomyces cerevisiae, as a pH probe. Five minutes after initiation of phagocytosis, sealed vacuoles were alkaline, exhibiting a pH of 7.80 +/- 0.19 (mean +/- SEM). Their acidity subsequently increased, so that the pH fell to 7.38 +/- 0.25 after 15 min. 6.35 +/- 0.35 after 30 min, and 5.68 +/- 0.26 after 60 min. The implications of these findings for neutrophil microbicidal mechanisms are discussed.
Establishment of a new peroxidase-positive human myeloid cell line, PL- 21
Authors: Kubonishi I , Machida K , Niiya K , Sonobe H , Ohtsuki Y , Iwata K , Miyoshi I
Published: October 2019

A myeloid cell line, designated PL-21, was established from the peripheral blood of a patient with acute promyelocytic leukemia. The PL- 21 cell line grew in single-cell suspension, with a doubling time of 48– 64 hr, and consisted of promyelocytes with fine immature nuclei and prominent azurophilic granules in the cytoplasm. PL-21 cells were positive for peroxidase, naphthol AS-D chloroacetate esterase, and Sudan Black B staining. Under the usual culture conditions, a small proportion of these cells differentiated into mature granulocytes, and this differentiation was enhanced by the addition of dimethyl sulfoxide in the culture medium. PL-21 cells had receptors for the Fc portion of IgG and complement, intracytoplasmic lysozyme and phagocytic activity, but lacked Epstein-Barr virus-associated nuclear antigen. Chromosome analysis of this cell line revealed a human male polyploid karyotype with 13q+ and double minute chromosomes. This new myeloid cell line may provide useful material for the study of proliferation and differentiation of human leukemia cells.
Specific receptors for phorbol diesters on freshly isolated human myeloid and lymphoid leukemia cells: comparable binding characteristics despite different cellular responses
Authors: Goodwin BJ , Moore JO , Weinberg JB
Published: October 2019

Freshly isolated human leukemia cells have been shown in the past to display varying in vitro responses to phorbol diesters, depending on their cell type. Specific receptors for the phorbol diesters have been demonstrated on numerous different cells. This study was designed to characterize the receptors for phorbol diesters on leukemia cells freshly isolated from patients with different kinds of leukemia and to determine if differences in binding characteristics for tritium-labeled phorbol 12,13-dibutyrate (3H-PDBu) accounted for the different cellular responses elicited in vitro by phorbol diesters. Cells from 26 patients with different kinds of leukemia were studied. PDBu or phorbol 12- myristate 13-acetate (PMA) caused cells from patients with acute myeloblastic leukemia (AML), acute promyelocytic (APML), acute myelomonocytic (AMML), acute monocytic (AMoL), acute erythroleukemia (AEL), chronic myelocytic leukemia (CML) in blast crisis (myeloid), acute undifferentiated leukemia (AUL), and hairy cell leukemia (HCL) (n = 15) to adhere to plastic and spread. However, they caused no adherence or spreading and only slight aggregation of cells from patients with acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), or CML-blast crisis (lymphoid) (n = 11). All leukemia cells studied, irrespective of cellular type, displayed specific receptors for 3H-PDBu. The time courses for binding by all leukemia types were similar, with peak binding at 5–10 min at 37 degrees C and 120 min at 4 degrees C. The binding affinities were similar for patients with ALL (96 +/- 32 nM, n = 4), CLL (126 +/- 32 nM, n = 6), and acute nonlymphoid leukemia (73 +/- 14 nM, n = 11). Likewise, the numbers of specific binding sites/cell were comparable for the patients with ALL (6.2 +/- 1.3 X 10(5) sites/cell, n = 4), CLL (5.0 +/- 2.0 X 10(5) sites/cell, n = 6), and acute nonlymphoid leukemia (4.4 +/- 1.9 X 10(5) sites/cell, n = 11). Thus, the differing responses to phorbol diesters of various types of freshly isolated leukemia cells appear to be due to differences other than initial ligand-receptor binding.
Impaired natural killer activity in patients with chronic lymphocytic leukemia is associated with a deficiency of azurophilic cytoplasmic granules in putative NK cells
Authors: Kay NE , Zarling JM
Published: October 2019

This study was undertaken to gain further insight into the severely impaired natural killer (NK) activity we and others have previously observed in patients with chronic lymphocytic leukemia (CLL). Normal individuals' NK cells are large granular lymphocytes (LGL) that (A) bind to and lyse NK-sensitive cells, including K562, (B) express receptors for the Fc portion of IgG (FcR+ cells), and (C) express cell surface antigens reactive with monoclonal antibodies OKM1, 9.6, and OKT11A. We thus examined lymphocytes depleted of monocytes and B cells, from 6 CLL patients and 6 normal individuals, that were identified on the basis of binding to K562, expressing OKM1, or expressing receptors for the Fc portion of IgG. In the CLL patients studied, lymphocytes that bind to K562 cells, as well as OKM1+ cells isolated by fluorescence activated cell sorting, were morphologically similar to LGL of normal individuals, with the exception that more than 75% of the patientsx' cells were deficient in azurophilic cytoplasmic granules, which typify normal individuals LGL. Furthermore, although the percentages of the patients' FcR+ cells reactive with OKT11A, 9.6 and OKM1 were very similar to those of normals, the majority of the patients' FcR+ cells were deficient in azurophilic granules and lacked NK activity. These findings indicate that the impaired NK activity in CLL patients is associated with cells that are phenotypically and morphologically NK cells, but which lack azurophilic granules that are thought to play a role in NK-mediated lysis.
A cytotoxic monoclonal antibody detecting a novel B cell membrane antigen expressed predominantly on cells bearing surface membrane immunoglobulin
Authors: Jephthah J , Terasaki PI , Hofman F , Wolde-Marian W , Peter JB , Billing R
Published: October 2019

A new human B lymphocyte membrane antigen, CB2, has been detected by a mouse monoclonal IgM antibody. CB2 appears to be predominantly expressed on normal and malignant cells expressing surface membrane immunoglobulin (SmIg). By indirect immunofluorescence, the number of CB2-positive cells in normal peripheral blood correlated well with the number of SmIg-positive cells. Cytotoxicity studies on isolated cell populations showed that CB2 was present on normal B cells isolated from the spleens of 52 donors and on peripheral blood B cells from 8 donors. Monocytes, T cells, granulocytes, platelets, and red cells were CB2 negative. Only malignant cells expressing SmIg were positive. These included B-CLL, B lymphoma, prolymphocytic leukemia, and B lymphoma cell lines Daudi, Raji, and Conception. SmIg-negative leukemia cells, such as common acute lymphoblastic leukemia, acute and chronic myelogenous leukemia, and T cell leukemias, were negative. Blocking studies with human immunoglobulin suggests that the CB2 antigen is not directed against immunoglobulin determinants. Immunoperoxidase studies on normal lymph node sections show that CB2-positive cells are predominantly present in the mantle region of the follicle, whereas B1- positive cells are mainly in the germinal center.
Anti-HEL cell monoclonal antibodies recognize determinants that are also present in hemopoietic progenitors
Authors: Papayannopoulou T , Brice M , Yokochi T , Rabinovitch PS , Lindsley D , Stamatoyannopoulos G
Published: October 2019

The characteristics of nine monoclonal antibodies (MoAbs) produced using the uninduced cells of a human erythroleukemia line (HEL) as immunogen are described. These antibodies were grouped into four categories by their differences in recognition of normal cells and cells of hemopoietic cell lines. The four MoAbs of group A recognize determinants that are expressed in a large proportion of normal bone marrow cells and other mature cells. The two MoAbs of group B (53/5, 53/6) and the two MoAbs of group C (54/23, 54/39) recognize small proportions of bone marrow cells, whereas the single MoAb of group D (53/10) essentially recognizes only HEL cells. Competition experiments revealed two pairs of competing Abs (53/5 and 53/6; 54/23 and 54/39). In complement-dependent cytotoxicity of progenitors, 53/6 produced 90%- 100% inhibition of CFU-E, BFU-E, and CFU-C growth; 54/39 30%–60% inhibition of BFU-E and CFU-C growth; 53/10 produced a variable degree of inhibition of CFU-E and BFU-E. Cell sorting using 53/6 resulted in approximately a 10–12-fold enrichment of CFU-E, BFU-E, and CFU-C among the positive cells. Cell sorting with 54/23 resulted in recovery of over 90% of BFU-E and 100% of CFU-C among the 23.5% of sorted cells showing strong or intermediate positivity. These findings suggest that HEL cells possess surface characteristics that are expressed in several classes of hemopoietic progenitors.
Correlation of two in vitro tests with clinical response to immunosuppressive therapy in 54 patients with severe aplastic anemia
Authors: Torok-Storb B , Doney K , Brown SL , Prentice RL
Published: October 2019

Two in vitro tests were applied to 54 consecutive patients with severe aplastic anemia who were treated in Seattle with antithymocyte globulin. In the first test, peripheral blood mononuclear cells were collected from each patient before antithymocyte globulin therapy and then treated with a panel of monoclonal antibodies and complement. The treated peripheral blood mononuclear cells were assayed for erythroid burst-forming units (BFU-E). This test was designed to determine whether removing various subpopulations of peripheral blood mononuclear cells would increase the number of detectable BFU-E. In the second test, peripheral blood was collected within 48 hr after completion of antithymocyte globulin therapy, and cells were immediately assayed for BFU-E without any further treatment. Data from both tests were analyzed to determine whether the in vitro results correlated with patient response to therapy. Binary logistic regression analyses indicate that a modest correlation (p = 0.04) exists between test 1 in vitro results and patient response to therapy. However, the strength of this association appears to decrease as the interval between diagnosis and treatment increases. In contrast, test 2 had a very significant correlation (p = 0.001) with response to therapy among patients diagnosed more than 1 mo prior to treatment, whereas such an association was not apparent among patients treated within 1 mo of diagnosis.
Inhibition of CFU-NM and CFU-EOS by mature granulocytes
Authors: Bjornson BH , Pincus SH , DiNapoli AM , Desforges JF
Published: October 2019

Approximately half of the colony-forming units-culture (CFU-C) from normal peripheral blood are eosinophilic. The purpose of our study was to determine: (1) whether progenitor cells committed to eosinophil or neutrophil maturation would be differentially affected by feedback inhibition, and (2) whether mature eosinophils added to the feeder layers of the culture would inhibit the proliferation of CFU-C in a manner similar to that described for neutrophils. Concentrated eosinophils and neutrophils, obtained by separation on a metrizamide gradient, were added to feeder layers containing either 10(6) autologous whole mononuclear cells (WMNC) or 0.1 ml of leukocyte conditioned media (LCM). The average number of colonies was 123/10(6) nonadherent cells (NAC) cultured. When neutrophils or eosinophils were added to the WMNC feeder layer, the percent inhibition of growth was 40.2% +/- 1.6% (mean +/- SEM) and 42.3% +/- 5.4%, respectively, but the ratio of neutrophil to eosinophil colonies remained constant. No effect was seen when neutrophils or eosinophils were added to an LCM feeder layer. Thus, it appears that the differential control of neutrophil versus eosinophil production in vitro is not regulated through feedback inhibition by mature granulocytes. In addition, these studies suggest that eosinophils, as well as neutrophils, cause inhibition of CFU-C growth when intact cells are the source of colony-stimulating factor (CSF).
Electron microscopic observations on platelet aggregation induced by cationized ferritin
Authors: Yamazaki H , Suzuki H , Yamamoto N , Tanoue K
Published: October 2019

Washed and gel-filtered human platelets were dose-dependently aggregated by the addition of cationized ferritin (CF). Ca++ and plasma factors were not necessary to induce the aggregation. Immediately after the addition of CF, CF particles were attached to the surface of platelets that showed discoid form, as observed electron microscopically. Some platelets were connected to each other through the CF particles located on their membranes. After the addition of CF, the following was observed: at 15 sec after, platelets showed a round form and were aggregated to each other; at 3 min after, centralization of granules was clearly seen and the aggregates increased their size during the time course; at 3–5 min after, the CF-connected aggregates were found locally. Around the aggregates, other platelets were aggregated, though not through the membrane-located CF. Observing with a lumiaggregometer, the aggregation showed a biphasic curve associated with adenosine triphosphate (ATP) release. The second part of aggregation curve was inhibited by PGI2, PGE1, aspirin, N- ethylmaleimide, and apyrase. The first part of the aggregation curve was inhibited only by heparin. Neuraminidase treatment also inhibited the aggregation dose-dependently. These findings suggest that neutralization of the platelet surface negative charge by a positively charged macromolecule can trigger platelet aggregation, which is followed by the release reaction.
Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V
Authors: Fulcher CA , Gardiner JE , Griffin JH , Zimmerman TS
Published: October 2019

Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79–80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79–80,000 (or 71–72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.
Platelet-collagen interactions: increase in rate of adhesion of fixed washed platelets by factor VIII-related antigen
Authors: Aihara M , Cooper HA , Wagner RH
Published: October 2019

A simple technique using an aggregometer and fixed washed human platelets (FWP) and fibrillar collagen has been used to evaluate the contribution of the two components of the factor VIII (FVIII) complex to platelet-collagen interactions. FWP bound individually to collagen fibrils in suspension, and both the total number of FWP bound and the rate of adhesion increased with increasing collagen concentration. Von Willebrand's disease (vWD) type I or normal plasma immunoadsorbed with anti-factor VIII-related antigen (anti-FVIIIR:Ag) antiserum gave 20% and vWD type IIa gave 50% of the rate of adhesion obtained with normal, hemophilia A, or hemophilia A with inhibitor plasma, but the same percent adhesion was found with all plasmas. The rate of adhesion of both vWD type I and type IIa was corrected by the addition of purified FVIII complex. These results indicated that the FVIIIR:Ag and not the factor VIII coagulant activity (FVIII:C) in normal plasma or purified FVIII complex caused an accelerating effect on the rate at which FWP bound to collagen. Collagen fibrils not only bound FWP, but also adsorbed the FVIII complex with preferential adsorption of the forms of FVIIIR:Ag with the greatest ristocetin cofactor (FVIIIR:RCoF) activity. Saturation of collagen with FWP did not change the adsorption pattern of the FVIII complex. Also anti-FVIIIR:Ag blocked the accelerating effect of the FVIII complex but not the adhesion of FWP. Thus, FWP and FVIIIR:Ag appeared to bind to separate sites on collagen.
Effect of age on hematopoiesis in man
Authors: Lipschitz DA , Udupa KB , Milton KY , Thompson CO
Published: October 2019

We have shown previously that the cause of anemia in healthy elderly subjects can usually not be identified. In this study, hematopoiesis was examined in 18 healthy elderly subjects with unexplained anemia and in 15 young and 15 healthy elderly individuals without anemia. No reduction in circulating testosterone was noted, making decreased androgen levels as a cause for the anemia unlikely. The 2,3 diphospho- glycerate (2,3DPG) levels in the anemic subjects were significantly higher than their corresponding controls, suggesting that the anemia was pathologic, as no increase would be expected if the low hemoglobin was a physiologic adjustment to age. The anemia was associated with a reduction in marrow normoblast and CFU-E number, but no decrease in BFU- E levels was seen. This suggests that the mechanism of the anemia is a decrease in stem cell proliferation. This could be caused by a reduction in circulating erythropoietin or a defect in end organ response. A second possibility is that a basic cellular abnormality exists. The presence of an overall reduction in hematopoiesis in anemic elderly (decreased peripheral blood counts, reduced marrow myeloid precursors, and CFU-C levels) makes this especially likely. The abnormality may be caused by a mechanism unrelated to the aging process. The fact that nonanemic elderly also have reductions in hematopoiesis suggests that age contributes to the defect.
Role of endothelial cells in human hematopoiesis: modulation of mixed colony growth in vitro
Authors: Ascensao JL , Vercellotti GM , Jacob HS , Zanjani ED
Published: October 2019

The identification of clonal human multipotent hematopoietic progenitors has permitted an analysis of the growth factor requirements for these cells. Human endothelial cell cultures were used to examine the effects of media conditioned by the endothelial cells on human multipotent (CFU-mix) and committed erythroid (BFU-E, CFU-E) and myeloid (CFU-GM) precursors. These studies demonstrate that endothelial cells produce proteins of approximately 30,000 daltons, with isoelectric focusing points of 4.5 and 7.2, which stimulate the growth of human BFU-E and CFU-mix. A heat-labile protein(s) of 30,000 and 15,000 daltons stimulated the proliferation and differentiation of granulocyte-macrophage (CFU-GM) colonies. No erythropoietin was detected in endothelial cell supernatants. This suggests that endothelial cells, a normal component of marrow stroma, play an active role in the modulation of human hematopoietic stem cell growth.
Characterization of an oligopeptide chemoattractant receptor on human blood monocytes using a new radioligand
Authors: Benyunes MC , Snyderman R
Published: October 2019

The study of chemoattractant receptors on human monocytes had been limited by the lack of a radioligand suitable for use with the small numbers of cells routinely available from human donors. A new synthetic oligopeptide radioligand f[35S]met-leu-phe, with a higher specific radioactivity than was available with the tritiated compound, was used to characterize a chemoattractant receptor on freshly isolated human blood monocytes. These cells bind f[35S]met-leu-phe with a dissociation constant (KD) of 30.2 +/- 5.6 nM and contain 84,000 +/- 11,300 receptors per cell. f[35S]met-leu-phe does not bind specifically to blood lymphocytes. The specificity of the oligopeptide receptor on monocytes is indistinguishable from the oligopeptide chemoattractant receptor on human polymorphonuclear leukocytes. Using f[35S]met-leu- phe, it will now be feasible to study the chemotactic peptide receptor on small numbers of partially purified peripheral blood monocytes from patients with defects of immune function.
Congenital transcobalamin II deficiency presenting atypically with a low serum cobalamin level: studies demonstrating the coexistence of a circulating transcobalamin I (R binder) complex
Authors: Carmel R , Ravindranath Y
Published: October 2019

A case of transcobalamin II deficiency with several unique features is described. The clinical presentation was typical, except for a slightly delayed age at presentation and the occurrence of apparent neurologic dysfunction from the beginning. The unusual biochemical feature was a low serum cobalamin level (97 pg/ml). Several cobalamin-binding protein abnormalities coexisted and antedated cobalamin therapy. Chief among these was the complexing of all serum R binder (transcobalamin I), leaving the patient with no detectable R binder. This defect appeared to be transient. Noteworthy, too, was a prominent binder of 70,000 mol wt that also carried the bulk of his serum cobalamin after therapy; it was prominent in his presumably heterozygous relatives too. The interrelationship between all these abnormalities is intriguing but unclear. The abnormality in transcobalamin II deficiency is clearly not limited solely to deficiency of transcobalamin II. It is also evident that this entity must now be considered in the differential diagnosis of low serum cobalamin levels in infancy.
Estrogen stimulates von Willebrand factor production by cultured endothelial cells
Authors: Harrison RL , McKee PA
Published: October 2019

Monolayers of cultured human umbilical vein endothelial cells were exposed to 17 beta-estradiol and compared to control cultures with respect to levels of von Willebrand factor (vWF) released into the media after 3–5 days of incubation. The amount of functional vWF activity was assessed by ristocetin-induced platelet aggregation and by a radioreceptor platelet assay. vWF antigen was quantitated by immunoassay. The DNA content of each monolayer was determined fluorometrically and used as a measure of cell number. By all assays, vWF levels in the media from the estradiol-treated endothelial cells were reproducibly and significantly higher when compared with control values. The amount of vWF produced by the cultured endothelial cells showed a dose-response effect to the estradiol added to the media. The estradiol-treated cells produced approximately 1.3 +/- 0.30 micrograms vWF/ml/micrograms DNA at 2 ng estradiol/ml, compared with control cultures that produced 0.75 +/- 0.16 microgram vWF/ml/micrograms DNA (p less than 0.001). The estradiol-treated monolayers consistently contained slightly greater amounts of DNA than control cultures: 2.0 +/- 0.10 micrograms versus 1.7 +/- 0.12 micrograms DNA (p less than 0.001). By multivariant analysis, however, the differences in cell number could only account for less than or equal to 10% of the elevation in the level of vWF that occurred in response to estradiol. By SDS-agarose electrophoresis and radioimmunoblotting, the vWF within the cytosol of the endothelial cells was found to possess a multimeric pattern similar to that found for either purified plasma vWF or vWF released into media overlying endothelial cell cultures. Our studies indicate that estrogen directly stimulates endothelial cells to increase their rate of production of vWF and, in addition, causes a slight increase in endothelial cell replication. These data may bear on the observation that administration of estrogen to some women with von Willebrand's disease causes an increase in their functional levels of vWF.
Identification of cytidine diphosphodiesters in erythrocytes from a patient with pyrimidine nucleotidase deficiency
Authors: Swanson MS , Markin RS , Stohs SJ , Angle CR
Published: October 2019

Pyrimidine 5′-nucleotidase deficient (PND) erythrocytes contain elevated levels of pyrimidine nucleotides and relatively normal purine nucleotide levels. The composition of this nucleotide pool has been examined by others, but not all of the abnormal red cell metabolites in this disorder were identified. We have isolated and positively confirmed the identity of cytidine diphosphate (CDP)-choline and CDP- ethanolamine from PND red cells using methods including proton FT-NMR, spectroscopy, and comparative mass spectrometry. The concentrations of these and other pyrimidine nucleotidase-deficient erythrocyte nucleotides were determined using anion-exchange high performance liquid chromatography and ultraviolet (u.v.) detection. The pyrimidine diphosphodiesters appear to be the most prominent abnormal pyrimidine nucleotides in PND red cells, accounting for 55% of the total red cell pyrimidine nucleotides in this disorder. It is proposed that these abnormal phosphodiesters may be related to the accelerated hemolysis in PND.
On the maturation order of AML cells: a distinction on the basis of self-renewal properties and immunologic phenotypes
Authors: Wouters R , Lowenberg B
Published: October 2019

To investigate the heterogeneous cellular structure of human acute myeloid leukemia (AML), subpopulations of cells were distinguished by two combined criteria: proliferation and differentiation. Purified blast cells were fractionated from blood or bone marrow of patients with newly diagnosed AML and colonies and clusters grown in phytohemagglutinin (PHA)-leukocyte feeder cultures. Large colonies, small colonies, macroclusters, and microclusters were recloned separately to assess the replicative capacities as a function of clone size. Large colonies showed higher proliferative capacities than did small ones, etc. Anti-Ia and an antigranulocyte (B4.3) monoclonal antibody (MoAb) were then employed to evaluate the stage of differentiation of AML cells in two patients before and following colony culture. Alterations of the immunologic phenotypes appeared during colony formation. This suggested differentiation of cells to more mature B4.3 granulocyte antigen-positive stages. MoAb-dependent cell lysis with the two antibodies was subsequently performed to assess the phenotypes of the precursors of the colonies and clusters. Leukemic colony-forming cells were Ia-positive and B4.3-negative and different from cluster-forming cells, which were largely Ia-negative and B4.3- negative. These data suggest that the cell organization of AML fits a maturation scheme containing immature cells with relatively high proliferative capacities, intermediate cells with low proliferative capacities, and end cells that are nonreplicative, and each with specific phenotypes.
Nonhistone protein antigen profiles of five leukemic cell lines reflect the extent of myeloid differentiation
Authors: Goldberger A , Brewer G , Hnilica LS , Briggs RC
Published: October 2019

The human leukemic cell lines, K562, KG-1, and HL-60, and the blast subclones, KG-1a and HL-60 blast, were utilized to relate differences in nonhistone protein antigens to stages of myeloid cell differentiation. Chromatin proteins were separated on SDS- polyacrylamide gels, transferred electrophoretically to nitrocellulose sheets, and visualized by the peroxidase-antiperoxidase method of Sternberger. Screening with antisera raised against total and dehistonized chromatin and a nuclear extract from these cells revealed quantitative as well as qualitative differences between the cell lines. A decrease in antigen content seemed to parallel progressive stages of myeloid cell development. The results indicate that a number of chromosomal protein antigens are lost or modified during differentiation. An antigen(s) of approximately 55,000 molecular weight was found in HL-60 chromatin, but was not present in its less differentiated subclone or in the other lines representative of earlier stage cells. Upon the induction of HL-60 cells to mature to end stages with 4 microM retinoic acid, a significant increase in the mol wt 55,000 activity was seen. This antigen was detected only with antisera against HL-60 total chromatin and granulocyte nuclei, and it was found only in normal mature granulocytes and in the later stage cells of the HL-60 culture. Thus, the antigen appears to be associated with a differentiated myeloid function.
Pre-B cell leukemia associated with chromosome translocation 1;19
Authors: Carroll AJ , Crist WM , Parmley RT , Roper M , Cooper MD , Finley WH
Published: October 2019

Chromosome banding studies on 60 children with acute lymphocytic leukemia (ALL), including “null,” pre-B, B, and T cell phenotypes, were performed. In 4 of 17 patients with pre-B cell ALL, we noted a previously undescribed chromosome translocation, t(1;19)(q23;q13). This translocation was not found in patients with “null” cell, B cell, or T cell ALL. Since each patient with the 1;19 translocation experienced early treatment failure, t(1;19)(q23;q13) may mark a subgroup of patients with pre-B cell ALL who have an especially poor prognosis.
Two-color flow cytometric measurement of DNA distributions of rat megakaryocytes in unfixed, unfractionated marrow cell suspensions
Authors: Jackson CW , Brown LK , Somerville BC , Lyles SA , Look AT
Published: October 2019

The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.
Inhibition of platelet function by cis-unsaturated fatty acids
Authors: MacIntyre DE , Hoover RL , Smith M , Steer M , Lynch C , Karnovsky MJ , Salzman EW
Published: October 2019

The uptake of free fatty acids has previously been shown to affect the capping of lymphocytes, and there is evidence that different types of fatty acids may partition into separate lipid domains in cell surface membranes. In studies of gel-filtered human platelets, we found that cis-unsaturated fatty acids (1–35 microM) inhibited platelet shape change, aggregation, and secretion of 5-hydroxytryptamine induced by thrombin, adenosine diphosphate (ADP), collagen, U46619 (a thromboxane A2 analog), or plant lectins, but not that induced by A23187, a calcium ionophore. Trans-unsaturated and saturated fatty acids had little or no inhibitory effect. The inhibitory effects of cis-unsaturated fatty acids were not affected by inhibition of adenylate cyclase or cyclooxygenase. 14C-labeled fatty acids were taken up into platelet lipids. The maximum platelet-inhibitory effect of cis-unsaturated fatty acids was seen when over 90% of the platelet label was still in the form of free fatty acids. Platelet inhibition could be reversed by washing the platelets by gel filtration. Binding of platelet agonists to the platelet was not inhibited by the fatty acids. Cis-unsaturated fatty acids, but not trans-unsaturated or saturated fatty acids, decreased fluorescence polarization of platelets or isolated platelet membranes monitored with 1,6-diphenyl- 1,3,5-hexatriene. The potency of the fatty acids as inhibitors of platelet aggregation was inversely correlated with their melting points. These data suggest that inhibition of receptor-mediated platelet responses by cis-unsaturated fatty acids results from perturbation of the platelet membrane in specific lipid domains.
Is there a threshold level of fetal hemoglobin that ameliorates morbidity in sickle cell anemia?
Authors: Powars DR , Weiss JN , Chan LS , Schroeder WA
Published: October 2019

When the clinical manifestations of 272 patients with sickle cell anemia are compared with their level of fetal hemoglobin (HbF), the results suggest that there may be a threshold above which HbF is effective in ameliorating the morbidity of this disease. The age of entry of these SS patients into the study ranged from birth to 56 yr; the average length of follow-up was 11 yr for a total of 3,011 patient- years of clinic observation. HbF was determined quantitatively by microchromatographic procedures; the mean for HbF was 10% +/- 6% with a range from 2% to 32%. For major organ failure, analyzed as termination events of morbidity, such as stroke or aseptic necrosis, the threshold appears to be 10%, whereas for recurrent clinical events, such as crisis or pulmonary disorders, it is 20%. No linear trend was found between HbF levels and morbidity. If a threshold exists, it is important to recognize this fact when attempts are made to raise the level of HbF in patients with sickle cell disease.
Thrombin Metz: characterization of the dysfunctional thrombin derived from a variant of human prothrombin
Authors: Rabiet MJ , Jandrot-Perrus M , Boissel JP , Elion J , Josso F
Published: October 2019

Thrombin Metz and normal thrombin, resulting from activation of the respective prothrombins by factor Xa in the presence of calcium, phospholipid, and factor Va, were purified by chromatography on sulfopropyl Sephadex. By physicochemical criteria, thrombin Metz is identical to normal thrombin. Its functional properties were investigated in some reactions in which thrombin is classically involved. Thrombin Metz exhibits less than 4% of fibrinogen clotting activity. Both Km and Kcat, determined on S2238, are abnormal. Titration with the high-affinity competitive inhibitor of thrombin, DAPA, shows that fluorescence enhancement of the probe is only 34% in binding to thrombin Metz when compared to that observed in binding to normal thrombin. High-performance liquid chromatography has been used to measure the simultaneous rate of release of fibrinopeptides A and B. A decreased release rate for both fibrinopeptides, more marked for fibrinopeptide B, results in a slow fibrin polymerization, as followed by absorbance at 450 nm. Thrombin Metz is less than 5% as effective as normal thrombin in inducing platelet aggregation. Interaction with antithrombin III is slower than normal when followed by SDS gel electrophoresis and inhibition of the amidolytic activity of thrombin on S2238. This abnormality is not observed in the presence of heparin. However, thrombin Metz binds less tightly to a heparin-Sepharose column, and the direct inhibition of heparin on its activity on S2238 is weaker. From these results, we can predict that the defect in thrombin Metz affects the catalytic site or its vicinity and, jointly or consequently, the region of interaction of thrombin with antithrombin III and heparin.
Red cell membrane and cation deficiency in Rh null syndrome
Authors: Ballas SK , Clark MR , Mohandas N , Colfer HF , Caswell MS , Bergren MO , Perkins HA , Shohet SB
Published: October 2019

A 52-yr-old multiparous white female was found to have Rh null blood type. She had macrocytic anemia, with reticulocytosis (15%-20%), of long duration. Although stomatocytes in peripheral blood were numerous and osmotic fragility was increased, suggesting increased cell water, the RBC cation content, and thus cell water, was decreased. Cell dehydration was confirmed by an increased proportion of high density RBC on Stractan density gradients. The deformability of RBC from four gradient subpopulations was measured in the ektacytometer as a function of suspending medium osmolality. Analysis of these measurements showed an abnormal reduction in cell surface area with increasing cell density, thus explaining the increased osmotic fragility of whole blood. This was confirmed by a density-dependent reduction in cell cholesterol content, suggesting membrane instability in vivo. Rh null subpopulations showed a twofold increase in both ouabain-sensitive and - insensitive Na-K ATPase activity and 86Rb transport, even in the dense fraction with the fewest reticulocytes. No membrane protein or glycoprotein abnormality was detected by SDS-PAGE. The associated deficiencies of both membrane surface area and cation content in Rh null cells, as well as increased Na-K pump activity, suggest a pleiotropic functional interrelationship among Rh antigen, membrane stability, and cation regulation.
Increases in circulating megakaryocyte growth-promoting activity in the plasma of rats following whole body irradiation
Authors: Miura M , Jackson CW , Lyles SA
Published: October 2019

To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.
Phenotypically and functionally distinct subpopulations of human lymphocytes with T cell markers also exhibit different cytochemical patterns of staining for lysosomal enzymes
Authors: Landay A , Clement LT , Grossi CE
Published: October 2019

Human peripheral blood lymphocytes that express T cell markers, when reacted for the cytochemical localization of lysosomal acid hydrolases, display two major patterns of staining, i.e., dot-like and scattered granular. Previous attempts to fractionate T cells according to surface markers have yielded populations of cells with heterogeneous patterns of cytochemical staining. In this study, peripheral blood cells forming rosettes with sheep erythrocytes have been fractionated by sequential staining with two monoclonal antibodies, D12 and 2D2, followed by fluorescence-activated cell sorting. These reagents have been shown previously to recognize a subpopulation of cells capable of suppressing T cell proliferation. All of the cells positive for D12 and 2D2 stained for acid hydrolases with the scattered granular pattern, whereas the large majority of the cells negative for both markers stained with the dot-like pattern. It is concluded that suppressor cells within the E+ cell fraction have the cytochemical characteristics of large granular lymphocytes.
Cytofluorometric detection of B cell clonal excess: a new approach to the diagnosis of B cell lymphoma
Authors: Weinberg DS , Pinkus GS , Ault KA
Published: October 2019

A sensitive cytofluorometric technique, the “kappa-lambda test,” permits detection of small numbers of monoclonal B lymphocytes (clonal excess). Such a method might represent a new diagnostic tool for diagnosis of non-Hodgkin’s lymphomas, potentially providing definitive evidence of lymphomatous involvement in cases equivocal by standard immunologic methods. To determine the significance of detecting B cell clonal excess in lymphoid tissues, we applied the kappa-lambda test to cell suspensions from 60 consecutive specimens suspected to involvement by malignant lymphoma. Results were correlated with the pathologic diagnosis and with standard cell marker studies in each case. B cell clonal excess was observed in 24 of the 25 cases of non-Hodgkin’s lymphoma of B cell origin, including a single case involving early detection of recurrence. None of the remaining cases, including benign reactive hyperplasia, T cell lymphoma, and Hodgkin’s disease, showed evidence of B cell clonal excess. Selective examination of cell subpopulations was also achieved using this cytofluorometric method. We conclude that the detection of B cell clonal excess by the kappa-lambda test represents a new approach to the diagnosis of B cell lymphoma, which provides certain advantages over more standard methods of cell marker analysis.
In vitro cytodestruction of human leukemic cells using murine monoclonal antibodies and human complement
Authors: Stepan DE , Bartholomew RM , LeBien TW
Published: October 2019

We have investigated the ability of murine monoclonal antibodies (MoAb) to lyse human leukemic cells in vitro using human serum as a source of complement (C'). The human C'-fixing ability of five of seven MoAb is documented. Studies with two of these MoAb (BA-1 and BA-2) indicated that their human C'-fixing ability and subsequent lysis of leukemic cells was through activation of the classical pathway of C', was independent of donor serum source, and occurred with a number of different target cells. BA-1 and BA-2 could effectively lyse fresh leukemic cells in the presence of 100% human serum, and BA-1 plus human serum could effectively lyse leukemic cells in the presence of a 20- fold excess of normal human bone marrow cells. Our results have potential implications for immunotherapy trials utilizing murine MoAb.
The production of granulocyte-monocyte colony-stimulating activity by isolated human T lymphocyte subpopulations
Authors: Hesketh PJ , Sullivan R , Valeri CR , McCarroll LA
Published: October 2019

Isolated human T lymphocyte subpopulations were obtained by fluorescence-activated cell sorting using the murine monoclonal antibodies, OKT4 and OKT8. The capabilities of the isolated lymphocytes to produce granulocyte-monocyte colony-stimulating activity (CSA) in response to mitogen challenge were assessed by in vitro assays employing light density nonadherent bone marrow cells. Essentially, no CSA production was noted by any isolated T lymphocyte population [OKT4 positive (+) or OKT8 positive (+)] cultured alone or following the addition of 10(4) autologous monocytes/ml. When phytohemagglutinin (PHA) alone was added, OKT4+ lymphocytes elaborated small amounts of CSA. With the addition of concanavalin A (Con-A) alone, both OKT4+ and OKT8+ cells were able to produce modest amounts of CSA. Significantly enhanced CSA production was observed when either OKT4+ or OKT8+ lymphocytes were coincubated with autologous monocytes in the presence of mitogen. We conclude that highly purified T lymphocyte subpopulations, free of monocytes as assessed by nonspecific esterase staining, can elaborate small amounts of CSA in response to PHA or Con- A challenge. A synergistic augmentation of CSA production was noted with coincubation of sorted lymphocytes and autologous monocytes in the presence of mitogen. Finally, our results suggest that the ability of T lymphocytes to make CSA is not exclusively limited to either the OKT4+ or OKT8+ defined subsets.