bluuebunny/crossref_metadata_2025 huggingface dataset:
High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions
Authors: Rice WG , Kinkade JM Jr , Parmley RT
Published: October 2019

Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA-TCH-SP staining of isolated granule fractions revealed patterns similar to those of granules in intact neutrophils at different stages of development. Granules from human acute promyelocytic leukemia cells (HL-60) exhibited a surprisingly low density compared with typical azurophil granules from normal, mature neutrophils. The data suggest that both functional and maturational differences contribute to granule heterogeneity, and provide a new practical and conceptual framework for further defining the phenomenon of neutrophil granule heterogeneity.
Quantitative analysis of platelet surface HLA by W6/32 anti-HLA monoclonal antibody
Authors: Kao KJ , Cook DJ , Scornik JC
Published: October 2019

Class I molecules of human major histocompatibility complex (HLA) are the most important antigenic system in determining the survival of transfused platelets in alloimmunized patients. Platelets with reduced expression of a specific type of HLA antigen may escape specific anti- HLA antibody-mediated destruction. By using 125I-labeled Fab fragments of W6/32 anti-HLA monoclonal antibody and competitive protein binding assays, we measured the range of total HLA concentrations on platelets. In 12 individuals examined, the mean number of HLA-A, B, and C molecules per platelet was 81,587 +/- 20,016 (mean +/- SD); its range was between 54,782 to 116,185 molecules per platelet. After treatment with chloroquine, 79.9 +/- 7.0% (mean +/- SD, n = 6) of HLA antigens were removed from platelets as determined by binding of 125I-W6/32 Fab. A similar result was obtained when HLA antigens on chloroquine-treated platelets were evaluated with immunofluorescence flow cytometry. In contrast, chloroquine treatment did not remove integral membrane protein such as P1A1 antigens on platelets. The presence of HLA antigens in the chloroquine eluate of platelets could be demonstrated to contain HLA antigens similar in mol wts to intact class I molecules by an immunoblotting technique. These data suggest that 70% to 80% of platelet HLA antigens are adsorbed and that such HLA antigens are not proteolytic products of integral membrane class I molecules. The origin of the adsorbed platelet HLA-antigens remains to be determined.
von Willebrand's disease characterized by increased ristocetin sensitivity and the presence of all von Willebrand factor multimers in plasma
Authors: Holmberg L , Berntorp E , Donner M , Nilsson IM
Published: October 2019

In eight members of one family, platelets in platelet-rich plasma aggregated at much lower ristocetin concentrations than normal. Ivy bleeding time was variously prolonged, and von Willebrand factor antigen (vWF:Ag), ristocetin cofactor activity, and factor VIII coagulant activity were decreased. Most of the affected members had had slight to rather severe bleeding symptoms. Platelet-type von Willebrand's disease (vWD) could be ruled out. All multimers of vWF:Ag were found in plasma as well as platelets. Administration of 1-desamino- 8-D-arginine vasopressin (DDAVP) to the propositus did not cause thrombocytopenia, and platelet-poor plasma obtained immediately after did not aggregate normal platelets. The molecular defect in this family, inherited as an autosomal dominant, resembles the one in type IIB because of the response to ristocetin but differs from IIB because all vWF:Ag multimers are present in plasma and the response to DDAVP is atypical. We conclude that this family has a new subtype of vWD and propose that structural as well as functional criteria should be used for a proper classification of vWD.
Effect of danazol on clotting factor levels, bleeding incidence, factor infusion requirements, and immune parameters in hemophilia
Authors: Saidi P , Lega BZ , Kim HC , Raska K Jr
Published: October 2019

Several recent studies have reported conflicting results on the effectiveness of danazol, an attenuated androgen, in raising plasma levels of clotting factors VIII and IX in patients with hemophilia. We undertook a randomized, double-blind cross-over trial using 8 weeks' administration of danazol (D), 600 mg/d, and 8 weeks' administration of placebo (P) separated by 2 weeks of rest in 12 patients with hemophilia A and four patients with hemophilia B. Plasma factor VIII and IX levels, frequency and type of bleeding episodes, amount of factor concentrate infused, fibrinogen, fibrinolysis assays, antithrombin III, liver function, and immune parameters were followed. During the danazol phase a minimal increase was noted in the average clotting factor levels, an increase that, although statistically significant, was of hemostatically marginal magnitude. Significant increases in protein C and plasminogen levels, however, were observed during the danazol period, suggestive of danazol-mediated enhanced fibrinolysis. Clinically, bleeding frequency was significantly increased, and more clotting factor was consumed during the danazol period. Furthermore, eight episodes of hematuria and oral mucosal bleeding was reported during the danazol phase in contrast to only one episode of hematuria during the placebo phase, consistent with an enhancement of fibrinolytic activity with danazol. We conclude that danazol does not have a hemostatically significant effect on plasma levels of factor VIII and IX but may be associated with enhancement of fibrinolytic activity, resulting in increased bleeding frequency and requiring more clotting factor infusions. Therefore, danazol is not a viable alternative in the treatment of hemophilia.
Activation of human factor VII in the initiation of tissue factor- dependent coagulation
Authors: Rao LV , Rapaport SI , Bajaj SP
Published: October 2019

We have used activation peptide release assays to compare factor VII and activated factor VII (VIIa) activation of factor X, normal factor IX (IXN), and a variant factor IX (IXBmLE), which, after activation, is unable to back-activate factor VII. In purified systems, factor VII and VIIa each rapidly activated factor X, but after a one minute lag for factor VII. VIIa also readily activated both IXN and IXBmLE. Factor VII initially failed to activate substantial amounts of either IXN or IXBmLE; on further incubation factor VII activated IXN but not IXBmLE. Activation of IXN began when approximately 10% of factor VII had been converted to VIIa, as measured by 125I-factor VII radioactivity profiles. Adding factor VII to VIIa slowed its activation of IXBmLE. However, in the presence of factor X, factor VII alone rapidly activated IXBmLE. Unlike purified systems, 1 nmol/L VIIa added to factor VII-deficient plasma failed to activate factor IX. Increasing factor VII to 10 nmol/L (plasma concentration) either as native VII or VIIa yielded similar activation curves for factor IX and similar activation curves for factor X. Adding 5% VIIa to factor X-deficient plasma and to factor XII-deficient plasma substantially shortened the dilute tissue factor clotting time of only the former. These data support the hypothesis that factor VII/tissue factor complex initiates tissue factor-dependent clotting through a minimal generation of Xa. This Xa then rapidly back-activates a small amount of factor VII, following which the rates of activation of both factors IX and X increase dramatically.
Autocrine growth of interleukin 2-producing leukemic cells in a patient with adult T cell leukemia
Authors: Arima N , Daitoku Y , Ohgaki S , Fukumori J , Tanaka H , Yamamoto Y , Fujimoto K , Onoue K
Published: October 2019

Leukemic cells in the peripheral blood of a patient with adult T cell leukemia (ATL), which expressed the Tac antigen/interleukin 2 (IL2) receptor, were investigated in vitro for autocrine growth by IL 2. The cells showed spontaneous proliferation in mitogen-free medium. The spontaneous proliferation of the cells was inhibited by monoclonal anti- IL 2 or anti-Tac antibody. These cells were found to produce messenger RNA for IL 2 and secrete IL 2 during short-term culture in the same medium. Recombinant IL 2 and IL 2 secreted by the cells enhanced the proliferation of the cells in a dose-dependent manner when added to the initial culture. These findings demonstrate that an autocrine mechanism by IL 2 is involved in the proliferation of ATL cells during short-term culture.
Antithrombotic effect of a monoclonal antibody to the platelet glycoprotein IIb/IIIa receptor in an experimental animal model
Authors: Coller BS , Folts JD , Scudder LE , Smith SR
Published: October 2019

A murine monoclonal antibody directed at the platelet glycoprotein IIb/IIIa complex, which blocks platelet aggregation ex vivo, was tested for its antithrombotic effects in an established animal model of acute platelet thrombus formation in partially stenosed arteries. Infusion of 0.7 to 0.8 mg/kg of the F(ab')2 fragment of the antibody completely blocked new thrombus formation despite multiple provocations, making it the most potent antithrombotic agent tested in this model.
Purification and functional evaluation of mature neutrophils from human bone marrow
Authors: Berkow RL , Dodson RW
Published: October 2019

Human myeloid maturation proceeds within the bone marrow and results in a mature neutrophil that is released into the peripheral circulation. Previous reports have indicated that neutrophils from bone marrow demonstrate decreased adherence, impaired phagocytosis, and decreased nitroblue tetrazolium dye reduction when stimulated. Due to lack of a suitable method for isolating purified bone marrow neutrophils, these studies have been performed by microscopic techniques. We now report a method for isolating 1 X 10(8) neutrophils [bands plus polymorphonuclear leukocytes (PMNs)] from 10 mL of bone marrow aspirate sample. By means of a discontinuous Percoll-gradient centrifugation through densities of 1.085, 1.095, and 1.10 g/mL a leukocyte-rich suspension of bone marrow can be separated into three leukocyte layers. By combining the lower two leukocyte layers (M2/3), a population of neutrophils consisting of 26% bands and 63% PMNs is seen. When compared with peripheral blood PMNs, these bone marrow neutrophils had a lower alkaline phosphatase activity, decreased ingestion of Oil Red O-coated particles, impaired superoxide release on stimulation with the chemotactic peptide Fmet-leu-phe (FMLP) or the tumor promotor phorbol myristate acetate (PMA), and less activity of the NADPH-dependent oxidase. These results indicate that morphologically mature neutrophilic cells within the bone marrow exist in a still functionally immature state.
Functional and immunologic protein S levels are decreased during pregnancy
Authors: Comp PC , Thurnau GR , Welsh J , Esmon CT
Published: October 2019

Protein S, is a natural anticoagulant protein which serves as a cofactor for activated protein C. During pregnancy and in the postpartum period, functional protein S levels are significantly reduced (38% +/- 17.3%, mean +/- 1 SD) when compared to nonpregnant females (97% +/- 31.6%) (P less than 0.001). In plasma an equilibrium exists between functionally active free protein S and protein S complexed with C4b-binding protein, which is functionally inactive. As a result of this equilibrium either a decreased level of total protein S antigen or an elevation of C4b-binding protein could lead to reduced protein S activity. C4b-binding protein levels measured by enzyme- linked immunoassay are not significantly different in pregnant women versus nonpregnant controls (103.5% +/- 21.2% v 100% +/- 16.9%). However, during pregnancy and in the postpartum period, total protein S levels are reduced (68% +/- 10.7%) compared to nonpregnant controls (100% +/- 17.0%). This difference is significant at P less than 0.001. These data demonstrated that the reduction in protein S activity observed during pregnancy is a result of reduced total protein S antigen.
Rearrangement of the bcr gene in Philadelphia chromosome-negative chronic myeloid leukemia
Authors: Ganesan TS , Rassool F , Guo AP , Th'ng KH , Dowding C , Hibbin JA , Young BD , White H , Kumaran TO , Galton DA
Published: October 2019

We studied the clinical, hematologic, cytogenetic, and molecular biologic features of seven patients with Philadelphia (Ph1) chromosome- negative chronic myeloid leukemia (CML). In five cases the hematologic findings were indistinguishable from those of patients with classical Ph1-positive disease. Myeloid cells were studied by chromosome-banding techniques. One patient had a masked Ph1 chromosome (with translocation t(4;9;22)), one had a deletion involving chromosome 16, and one had a small minority population of 22q- cells without 9q+ but otherwise normal metaphases; metaphases from the other four patients were entirely normal. DNA prepared from the myeloid cells was digested with the restriction enzymes EcoRI, HindIII, BamHI and BglII. Southern analysis using a 0.6-kb fragment of the breakpoint cluster region (bcr) gene showed the presence in each patient's DNA of a germline fragment together with a rearranged fragment or fragments with at least one of the restriction enzymes. We conclude that genomic changes in the bcr gene characteristic of CML can be present in the absence of a Ph1 chromosome.
A de novo intragenic deletion of the potential EGF domain of the factor IX gene in a family with severe hemophilia B
Authors: Vidaud M , Chabret C , Gazengel C , Grunebaum L , Cazenave JP , Goossens M
Published: October 2019

We have studied a family of three patients who were severely afflicted with hemophilia B without inhibitor for their factor IX genes through the use of factor IX cDNA and genomic DNA probes. The patients had detectable (30% of normal) factor IX antigen. DNA hybridization analysis demonstrated that these patients had a partial intragenic deletion in their factor IX gene. This 2.8-kb deletion included exon d and the surrounding sequences. This exon codes for the amino acid sequence from No. 47 through 84 of the factor IX protein and contains its first potential EGF domain; the de novo occurrence of the mutation in the grandfather's germ cells was established by linkage analysis. This specific gene has been named F IXStrasbourg.
The spectrum of beta-thalassemia genes in China and Southeast Asia
Authors: Kazazian HH Jr , Dowling CE , Waber PG , Huang S , Lo WH
Published: October 2019

To make possible prenatal diagnosis of beta-thalassemia in China and Southeast Asia by direct detection of mutant beta-globin genes, we have determined the spectrum of mutations producing the disorder in this region of the world. Seventy-eight beta-thalassemia genes from Chinese and Southeast Asians were randomly obtained, and the relevant mutation was characterized in 76 (98%) of them. Seven different point mutations were found among the 78 genes studied. Of these seven beta-thalassemia alleles, two constitute 62%, and two others account for 29% of the total. Since only four alleles make up 91% of the mutant genes, prenatal diagnosis of beta-thalassemia in China and Southeast Asia should be feasible by simplified techniques for direct detection of point mutations.
The 18- to 23-kb deletion of the Macedonian delta beta-thalassemia includes the entire delta and beta globin genes
Authors: Efremov GD , Nikolov N , Hattori Y , Bakioglu I , Huisman TH
Published: October 2019

Restriction endonuclease mapping analyses were made of DNA from a few members of a Macedonian family with hematological characteristics of delta beta-thalassemia, ie, microcytosis, normal HbA2 levels, and elevated levels of HbF (7% to 14%) with G gamma (average 40.5%) and A gamma T chains (average 59.5%). A large deletion of 18 to 23 kb was present with a 5′ breakpoint within a 670-bp segment of DNA between the HpaI and NcoI restriction sites 5′ to the delta globin gene, and a 3′ breakpoint between the BamHI and HpaI restriction sites located some 9 to 13 kb 3′ to the beta globin gene. This deletion is different from those present in other types of G gamma A gamma(delta beta)zero- thalassemia. The similarity of the hematological expression of these delta beta-thalassemic conditions which have somewhat comparable 5′ breakpoints supports the idea that an important fetal hemoglobin- controlling region lies between the psi beta and delta globin genes.
Bromodeoxyuridine treatment of normal adult erythroid colonies: an in vitro model for reactivation of human fetal globin genes
Authors: Comi P , Ottolenghi S , Giglioni B , Migliaccio G , Migliaccio AR , Bassano E , Amadori S , Mastroberardino G , Peschle C
Published: October 2019

We report that bromodeoxyuridine (BrdU) addition in semi-solid cultures of normal adult erythroid progenitors causes a sharp rise of gamma- globin gene expression in erythroid colonies. Control studies were carefully carried out to exclude the possibility of toxic effects exerted by the drug in these experimental conditions. In particular, BrdU addition induces a sharp increase in the level of relative gamma- globin synthesis and content in pooled BFU-E-derived colonies: this rise is clearly observed in single bursts of the mature type (largely composed of late erythroblasts) but not in immature ones (essentially comprising early erythroblasts). Furthermore, it is associated with an increase of the G gamma/G gamma + A gamma synthetic ratio from adult up to fetal like values. Reactivation of gamma-synthesis was observed even if BrdU was added to colonies composed essentially of early erythroblasts, ie, when BrdU was added to either bursts at day 10 of culture or late CFU-E-derived clones at day 1. These in vitro observations indicate modulation of gamma-synthesis at the stage of erythroblasts from normal adults. At the molecular level we suggest that BrdU, by replacing thymidine in DNA, may inhibit the switch from a fetal-like biosynthetic program expressed in early erythroblastic differentiation to the adult program expressed in later stages of maturation.
Childhood Ki-1 lymphoma presenting with skin lesions and peripheral lymphadenopathy
Authors: Kadin ME , Sako D , Berliner N , Franklin W , Woda B , Borowitz M , Ireland K , Schweid A , Herzog P , Lange B
Published: October 2019

We describe a large-cell lymphoma of activated lymphoid cells in six children and adolescents. The presenting clinical features of regressing skin lesions and peripheral lymphadenopathy, sinus infiltration of lymph nodes, and infrequent tumor cell erythrophagocytosis resulted in initial diagnoses of malignant or regressing atypical histiocytosis in five cases. Binucleate and multinucleate tumor cells, sometimes with prominent eosinophilic nucleoli, resembled Reed-Sternberg (RS) cells and occasionally were found in a cytoarchitectural milieu that was consistent with a diagnosis of Hodgkin's disease (HD). The tumor cells did in fact express the HD-associated antigen Ki-1, but unlike most types of HD, the RS-like cells expressed common leukocyte antigen and were negative for Leu-M1. A T cell origin for the malignant cells was demonstrated with monoclonal antibodies in two cases, by focal staining for nonspecific esterase in one case, and by rearrangement of the beta- chain genes for the T cell receptor in a fourth case. These studies provide further evidence that some cases previously interpreted as malignant or regressing atypical histiocytosis and some types of HD are actually T cell disorders.
Erythrocyte-endothelial cell adherence in sickle cell disorders
Authors: Smith BD , La Celle PL
Published: October 2019

Detachment of individual sickle erythrocytes from cultured endothelial cell monolayers has been evaluated by a fluid-shearing technique in an effort to quantitate adherence at shear forces that would be anticipated in the in vivo circulation. Nonirreversibly sickled cells (non-ISC) were more adherent at normal oxygen tensions than control cells. More than 1% non-ISC remained attached to the monolayer at forces greater than physiologic shear stresses in capillary and venous circulations, and many of the most avidly attached cells, once separated, immediately reattached to adjacent endothelial cells. These data suggest that hemoglobin S-containing erythrocytes may have a higher frequency of adherence in vivo in regions of low shear stress where prolonged erythrocyte-endothelial cell contact could occur. Some of these cells detached by shear force would subsequently reattach in in vivo conditions. Plasma-enhanced attachment frequency and plasma from blood in a case of sickle crisis caused further increase. These observations further support the concept that sickle erythrocyte- endothelial cell interaction may be a significant factor in initiation of vascular occlusive events in sickle cell disease.
Cyclophosphamide cardiotoxicity: an analysis of dosing as a risk factor
Authors: Goldberg MA , Antin JH , Guinan EC , Rappeport JM
Published: October 2019

Patients who undergo bone marrow transplantation are generally immunosuppressed with a dose of cyclophosphamide (CYA) which is usually calculated based on the patient's weight. At these high doses of CYA, serious cardiotoxicity may occur, but definitive risk factors for the development of such cardiotoxicity have not been described. Since chemotherapeutic agent toxicity generally correlates with dose per body surface area, we retrospectively calculated the dose of CYA in patients transplanted at our institution to determine whether the incidence of CYA cardiotoxicity correlated with the dose per body surface area. Eighty patients who were to receive CYA 50 mg/kg/d for four days as preparation for marrow grafting underwent a total of 84 transplants for aplastic anemia, Wiskott-Aldrich syndrome, or severe combined immunodeficiency syndrome. Fourteen of 84 (17%) patients had symptoms and signs consistent with CYA cardiotoxicity within ten days of receiving 1 to 4 doses of CYA. Six of the 14 patients died with congestive heart failure. The dose of CYA per body surface area was calculated for all patients and the patients were divided into two groups based on daily CYA dose: Group 1, CYA less than or equal to 1.55 g/m2/d; Group 2, CYA greater than 1.55 g/m2/d. Cardiotoxicity that was thought to be related to CYA occurred in 1/32 (3%) of patients in Group 1 and in 13/52 (25%) patients in Group 2 (P less than 0.025). Congestive heart failure caused or contributed to death in 0/32 patients in Group 1 v 6/52 (12%) of patients in Group 2 (P less than 0.25). There was no difference in the rate of engraftment of evaluable patients in the two groups (P greater than 0.5). We conclude that the CYA cardiotoxicity correlates with CYA dosage as calculated by body surface area, and that patients with aplastic anemia and immunodeficiencies can be effectively prepared for bone marrow grafting at a CYA dose of 1.55 g/m2/d for four days with a lower incidence of cardiotoxicity than patients whose CYA dosage is calculated based on weight. This study reaffirms the principle that drug toxicity correlates with dose per body surface area.
Growth and development following marrow transplantation for leukemia
Authors: Sanders JE , Pritchard S , Mahoney P , Amos D , Buckner CD , Witherspoon RP , Deeg HJ , Doney KC , Sullivan KM , Appelbaum FR
Published: October 2019

One hundred forty-two patients between the ages of 1 and 17 years who survived disease-free more than 1 year after marrow transplantation for hematologic malignancy had growth and development evaluations from one to 14 years posttransplant (median 4 years). Prior to transplant all children received multiagent chemotherapy and 55 also received central nervous system irradiation, but none had growth and development evaluations. Marrow transplant preparation included high-dose chemotherapy and total body irradiation (TBI) given as a single dose of 9.2 to 10.0 Gy (79 patients) or as fractionated doses of 2.0 to 2.25 Gy/d for six to seven days (63 patients). After transplant abnormal thyroid function was present in 39%. Stimulated 11-desoxycortisol levels were subnormal in 24% of patients evaluated. Growth hormone (GH) deficiency was present in 17 of 25 children who received previous cranial irradiation. Partial GH deficiency was present in 4 of 25 who received previous cranial irradiation and in 6 of 18 who had not received cranial irradiation. Height velocity was decreased in all patients. After transplant, height was significantly influenced by chronic graft-v-host disease and single-dose TBI. Sixty-eight percent had delayed development of secondary sexual characteristics. Gonadal failure occurred in nearly all who were postpubertal at transplant. While it is not possible to determine how many of these endocrine abnormalities occurred as a result of treatment administered prior to transplantation, these data do demonstrate that children who become long-term survivors after marrow transplantation for hematologic malignancy have endocrine abnormalities that adversely affect growth and development.
Heterogeneity of large granular lymphocyte proliferations: delineation of two major subtypes
Authors: Chan WC , Link S , Mawle A , Check I , Brynes RK , Winton EF
Published: October 2019

Two major types of lymphocytosis of large granular lymphocytes (LGLs) were observed. The proliferating LGLs in each type had distinct immunophenotypes, functional characteristics, and probably belonged to different cell lineages. The more common form (Type A) consisted of cells derived from the T cell lineage and had the T suppressor/cytotoxic phenotype (T11+, T3+, T8+). The expression of the Leu 7 and HLA-DR antigen was variable. These cells did not have natural killer (NK) function but showed a variable degree of antibody-dependent cell-mediated cytotoxic (ADCC) activity. Neutropenia was invariably present and rheumatoid arthritis and autoantibodies were frequent associations. These lymphocytes had many similarities to the major type of LGLs present in normal adult bone marrow. The other type of LGL lymphocytosis (Type B) consisted of cells lacking the antigens T3 and T8 but expressing M1 and NKH1. These cells possessed strong NK and ADCC activity but their cell lineage was not clear. Neutropenia and autoimmune phenomena were not detected. The cytochemical characteristics of the LGL granules from both types of patients were similar but differences in ultrastructure were observed. LGLs from Type B patients proliferated in the presence of Interleukin 2 (IL-2) and 12- O-tetradecanoyl-phorbol-13 acetate (TPA). Significant changes in their basic T11+, T3-, T8- phenotype were not observed. IL-2 and TPA, however, had profound influence on the NK function of the cells with enhancement in the case of IL-2 and marked suppression when stimulated by TPA.
Alpha-thalassemia in blacks: genetic and clinical aspects and interactions with the sickle hemoglobin gene
Authors: Steinberg MH , Embury SH
Published: October 2019

Recent advances in molecular genetics have permitted detailed study of the human alpha-globin genes and the causes of alpha-thalassemia. In this review, we examine the causes of alpha-thalassemia in the black population and the consequences of the interactions between alpha- thalassemia and structural hemoglobin variants.
The heterogeneity of type IIA von Willebrand's disease: studies with protease inhibitors
Authors: Batlle J , Lopez Fernandez MF , Campos M , Justica B , Berges C , Navarro JL , Diaz Cremades JM , Kasper CK , Dent JA , Ruggeri ZM
Published: October 2019

The absence of large von Willebrand factor (vWF) multimers from plasma is a characteristic of Type IIA von Willebrand's disease (vWD) and is thought to contribute to the clinical expression of this disorder. Recently, three IIA patients have been reported in whom intermediate and large multimers could be restored if blood were collected in 5 mm EDTA, 6 mmol/L N-ethylmaleimide, and 1 mmol/L leupeptin. This suggested that absence of large multimers resulted from in vitro proteolysis. We have now collected blood from ten Type IIA vWD patients in these inhibitors but were not able to detect large multimers in the plasma of any of them. In addition, intermediate-sized multimers were reduced or completely absent in all. The inclusion of inhibitors in the citrate anticoagulant, as compared to citrate alone, was found to increase the relative proportion of intermediate multimers in some patients but had no effect in others, and in none did it restore large multimers to plasma. The results with platelet vWF were more varied. Four patients showed an absence or decrease of large multimers, whereas in seven patients large multimers were present. When compared with citrate anticoagulant alone, the inclusion of inhibitors in the anticoagulant had little or no effect on the platelet multimeric pattern. 1-Deamino-8- D-Arginine Vasopressin (DDAVP) was administered to six patients from five families. Two patients from one family showed complete correction and a third patient showed almost complete correction of her bleeding time. Two patients showed minimal correction and one showed no detectable correction. An increase in multimer size after DDAVP tended to be associated with correction of the bleeding time. However, in no case did the largest multimers appear in plasma even in patients with complete bleeding time correction. The presence or absence of inhibitors in the anticoagulant had little or no effect on the multimeric pattern after DDAVP. These results indicate that Type IIA vWD is a heterogeneous disorder in which absence of largest and intermediate multimers is an in vivo phenomenon.
Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome
Authors: Budde U , Dent JA , Berkowitz SD , Ruggeri ZM , Zimmerman TS
Published: October 2019

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.
Extracellular deposition of eosinophil granule major basic protein in lymph nodes of patients with Hodgkin's disease
Authors: Butterfield JH , Kephart GM , Banks PM , Gleich GJ
Published: October 2019

Lymph nodes from each of the four histologic types of Hodgkin's disease were examined for the presence of eosinophils and for eosinophil degranulation by immunofluorescent localization of eosinophil granule major basic protein (MBP). Eosinophil degranulation shown by MBP deposition outside of eosinophils was found in six of eight nodes from patients with nodular sclerosing disease and in two of eight nodes from patients with lymphocyte depletion-type disease. Three nodes of the mixed cellularity type, one node of the lymphocyte predominance type, and one lymph node of the lymphocyte depletion type showed one or two small foci of extracellular MBP deposition. Lymph nodes from patients without Hodgkin's disease showed no extracellular deposition of MBP. Large numbers of eosinophils were found in seven of eight nodes of the nodular sclerosing variant, but were less frequently seen among the other types of Hodgkin's disease. The presence of extracellular MBP in lymph nodes of patients with Hodgkin's disease indicates that eosinophil degranulation commonly occurs and suggests that the released eosinophil granule proteins may participate in the inflammatory reaction in this disorder more extensively than is presently appreciated.
L-triiodothyronine augments erythropoietic growth factor release from peripheral blood and bone marrow leukocytes
Authors: Dainiak N , Sutter D , Kreczko S
Published: October 2019

To investigate cellular mechanisms involved in thyroid hormone stimulation of erythropoiesis, we studied the response of erythroid burst-forming unit (BFU-E) proliferation to L-triiodothyronine (L-T3) in a serum-free culture system. When added directly to culture, L-T3 stimulates erythroid burst formation by normal human bone marrow cells. In contrast, granulocyte-macrophage colony formation is unaffected. Enhancement of erythroid burst formation by L-T3 required the presence of nylon wool adherent and/or B-4 antigen-positive light-density marrow populations. Addition of other erythropoietic factors including platelet-derived growth factor and insulinlike growth factor II did not abrogate this apparent cellular requirement. Pulse exposure of marrow and peripheral blood mononuclear cells (greater than 95% lymphocytes) to L-T3 accelerates the release of a soluble factor that augments BFU-E proliferation into serum-free liquid culture medium. Time-course studies show that this factor appears in conditioned medium (CM) coincidentally with erythroid burst-promoting activity (BPA). Furthermore, incubation of CM with an antibody known to react with and adsorb BPA from solution removes the inducible mitogen. Biochemical analysis of CM prepared from unexposed and L-T3 pulse-exposed cells indicates that the rate of protein appearance is accelerated by L-T3 in a fashion that immediately precedes growth factor release and that several polypeptides are quantitatively increased. We conclude that unlike erythropoietin, which is mitogenic for progenitor cells directly, L-T3 enhances BFU-E proliferation indirectly by augmenting the release of soluble BPA-like molecules from accessory cells in culture.
Morphological abnormalities in the lymphocytes of patients with the Wiskott-Aldrich syndrome
Authors: Kenney D , Cairns L , Remold-O'Donnell E , Peterson J , Rosen FS , Parkman R
Published: October 2019

Lymphocytes from 18 patients with the Wiskott-Aldrich Syndrome (WAS) were examined by scanning electron microscopy (SEM). Most peripheral blood lymphocytes from normal individuals are covered with slender microvillus projections, but a large proportion of lymphocytes from WAS patients were found to be relatively devoid of microvilli. A lymphocyte morphology scoring system was developed to quantify the density of microvilli: Grade 4 classified those lymphocytes with greater than 75% of the surface covered with microvilli with progressive decrements to grade 1, which were those without microvilli. The mean lymphocyte morphology score of eight normal individuals was 3.62 +/- .22. The mean lymphocyte score of WAS patients was substantially lower (2.89 +/- .27, P less than .001). In addition, WAS lymphocytes often were qualitatively abnormal, with short, blunted microvilli. These morphological criteria were used to diagnose WAS from the cord blood lymphocytes of one “at-risk” patient. Thus, WAS is the first primary immunodeficiency in which morphological abnormalities have been identified that can aid in diagnosis.
Classifying acute leukemia by immunophenotyping: a combined FAB- immunologic classification of AML
Authors: Neame PB , Soamboonsrup P , Browman GP , Meyer RM , Benger A , Wilson WE , Walker IR , Saeed N , McBride JA
Published: October 2019

A panel of commercially available monoclonal antibodies and five heteroantisera were used to distinguish and subtype 138 cases of acute leukemia (AL). The immunophenotype was compared with the French- American-British (FAB) classification obtained on the cases. The immunophenotype discriminated acute myelogenous leukemia (AML) from acute lymphoblastic leukemia (ALL) and recognized cases not distinguished by cytochemistry (22% of cases), mixed lineage phenotypes (13% of cases), and cases with separate populations of lymphoblasts and myeloblasts (one case). Using the immunologic panel and derived criteria to subtype AML, correspondence of the immunophenotype to the FAB subtypes M1, M2, M4, and M5 was possible in greater than 80% of cases. A combined classification of the immunophenotype and FAB morphology/cytochemistry was devised for AML subtyping. It is recommended that immunophenotyping should be done at least in all cases with negative orinconclusive cytochemistry. At present, we suggest that until a “gold standard” for identifying leukemic subtypes is developed, the best method for typing acute leukemia is by using a combination of morphology, cytochemistry and immunophenotyping.
In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes
Authors: Lichtenstein A , Ganz T , Selsted ME , Lehrer RI
Published: October 2019

We examined the activity of defensins, cysteine-rich cationic peptides that are abundant in the cytoplasmic granules of human and rabbit granulocytes, against various tumor targets. The three human defensins, HNP-1, HNP-2, and HNP-3, lysed human and murine targets in chromium release and dye exclusion assays. Defensin-mediated tumor cell lysis was concentration-dependent, inhibited by serum, and dependent on temperature-sensitive events. Lysis was first detected by three hours of incubation and it reached a plateau between eight and 14 hours. In vitro exposure of murine teratocarcinoma cells to HNP 1–3 abrogated their oncogenicity in vivo. Nonmalignant target cells were also susceptible to defensin-mediated lysis. Four rabbit granulocyte defensins exerted marked (NP-1, NP-2) or moderate (NP-3a, NP-3b) cytotoxic activity, whereas defensin NP-5 was not cytotoxic. When tumor cells were incubated with human defensins in combination with hydrogen peroxide, synergistic cytotoxicity was detected. As defensins are released from granulocytes by various stimuli, their release could contribute to extracellular cytotoxicity which is independent of reactive oxygen intermediates.
Subunit assembly of hemoglobin: an important determinant of hematologic phenotype
Authors: Bunn HF
Published: October 2019

Hemoglobin's physiologic properties depend on the orderly assembly of its subunits in erythropoietic cells. The biosynthesis of alpha- and beta-globin polypeptide chains is normally balanced. Heme rapidly binds to the globin subunit, either during translation or shortly thereafter. The formation of the alpha beta-dimer is facilitated by electrostatic attraction of a positively charged alpha-subunit to a negatively charged beta-subunit. The alpha beta-dimer dissociates extremely slowly. The difference between the rate of dissociation of alpha beta- and alpha gamma-dimers with increasing pH explains the well-known alkaline resistance of Hb F. Two dimers combine to form the functioning alpha 2 beta 2-tetramer. This model of hemoglobin assembly explains the different levels of positively charged and negatively charged mutant hemoglobins that are encountered in heterozygotes and the effect of alpha-thalassemia and heme deficiency states in modifying the level of the variant hemoglobin as well as Hb A2. Electrostatic interactions also affect the binding of hemoglobin to the cytoplasmic surface of the red cell membrane and may underlie the formation of target cells. Enhanced binding of positively charged variants such as S and C trigger a normally dormant pathway for potassium and water loss. Thus, the positive charge on beta c is responsible for the two major contributors to the pathogenesis of Hb SC disease: increased proportion of Hb S and increased intracellular hemoglobin concentration. It is likely that electrostatic interactions play an important role in the assembly of a number of other multisubunit macromolecules, including membrane receptors, cytoskeletal proteins, and DNA binding proteins.
Characterization of the inhibition of tissue factor in serum
Authors: Broze GJ Jr , Miletich JP
Published: October 2019

Tissue factor (TF) is a lipoprotein cofactor that markedly enhances the proteolytic activation of factors IX and X by factor VIIa. The functional activity of TF is inhibited by serum in a time- and temperature-dependent fashion. The inhibitory effect is also dependent on the presence of calcium ions and can be reversed by calcium chelation (EDTA) and dilution, thus excluding direct proteolytic destruction of TF as the mechanism for inhibition. Using crude TF, serum immunodepleted of factor VII, and serum depleted of the vitamin K- dependent coagulation factors by BaSO4 absorption, it is shown that TF factor inhibition requires the presence of VII(a), X(a), and an additional moiety contained in barium-absorbed serum. When each of the other required components were at saturating concentrations, half- maximal inhibition of TF occurred in reaction mixtures containing 2% (vol/vol) of TF at a factor VII(a) concentration of 4 ng/mL (80 pmol/L), a factor X concentration of 50 ng/mL (850 pmol/L), and a concentration of barium-absorbed serum of 2.5% (vol/vol). Catalytically active factor Xa appeared to be required for the generation of optimal TF inhibition. The results are consistent with the conclusions of Hjort that barium-absorbed serum contains a moiety that inhibits the VIIa- Ca2+-TF complex. The role of factor X(a) in the generation of the inhibitory phenomenon remains to be elucidated. The inhibitor present in serum (plasma) may in part be produced by the liver in vivo since cultured human hepatoma cells (HepG2) secrete this inhibitory activity in vitro.
Selective expression of fos proto-oncogene in human acute myelomonocytic and monocytic leukemias: a molecular marker of terminal differentiation
Authors: Mavilio F , Testa U , Sposi NM , Petrini M , Pelosi E , Bordignon C , Amadori S , Mandelli F , Peschle C
Published: October 2019

Expression of human fos proto-oncogene (c-fos) was analyzed in primary cells from 50 untreated acute lymphocytic (ALL) and myeloblastic (AML) leukemias. c-fos RNA, analyzed by blot hybridization, was detected virtually only in myelomonocytic (M4) and monocytic (M5) AML. Both M4 and M5 samples show a strong positive correlation between the amount of c-fos transcripts and the percentage of leukemic cells expressing surface antigens specific for mature monocytes and macrophages. Normal mature monocytes exhibit a detectable level of c-fos RNA, which is virtually unaltered on activation to macrophage differentiation, but is always below that observed in M4 through M5 monocyticlike cells. These data provide evidence that c-fos expression is linked to terminal monocyte and macrophage differentiation in normal and leukemic hemopoiesis.
Is heparin administration necessary during induction chemotherapy for patients with acute promyelocytic leukemia?
Authors: Goldberg MA , Ginsburg D , Mayer RJ , Stone RM , Maguire M , Rosenthal DS , Antin JH
Published: October 2019

The role of heparin in the treatment of the disseminated intravascular coagulation (DIC) associated with acute promyelocytic leukemia (APL) remains unclear. Between 1974 and 1985, we treated 27 patients with APL using four different chemotherapeutic regimens; 23/27 (85%) had evidence of DIC either at presentation or following the initiation of induction chemotherapy. The coagulopathy was treated primarily with fresh frozen plasma and platelet transfusions; only 2/27 (7%) patients received heparin. Twenty of 27 patients (74%) entered complete remission. Major bleeding or thrombotic complications occurred in 5/27 patients (19%), but 2 of these 5 patients presented after hemorrhage had already occurred. None of the 5 patients with bleeding or thrombosis entered complete remission. All of the hemorrhagic complications due to DIC in our study occurred before 1979, which may reflect changes in the management of leukemic patients. This observation emphasizes the risks inherent in the use of historical controls in this population. In conclusion the DIC associated with APL can be successfully treated with intensive blood product support without the use of heparin.
Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets
Authors: D'Angelo A , Lockhart MS , D'Angelo SV , Taylor FB Jr
Published: October 2019

Platelets stimulated with thrombin release an inhibitor of plasminogen activator (PAI), which has been shown previously to be neutralized by activated protein C (APC). The requirements for optimal neutralization of PAI activity were investigated. The releasate of gel-filtered human platelets stimulated with thrombin served as a source of PAI. When 6 X 10(8) platelets/mL were incubated with thrombin (1 IU/mL), the releasate contained 18 to 26 ng/mL PAI as determined by incubation of the releasate with urokinase and measurement of residual urokinase activity on plasminogen (S2251). Preincubation of PAI with up to 4 micrograms/mL APC for two hours yielded less than 20% neutralization of PAI activity. In the presence of protein S, phospholipid, and Ca2+, neutralization of PAI activity was time-dependent with 50% neutralization occurring in two hours with 1 microgram/mL APC. The cofactor effects of protein S and phospholipid were concentration- dependent with half-maximal acceleration at approximately 3 micrograms/mL protein S and 10 micrograms/mL phospholipid when the experiments were performed at 1 microgram/mL APC. Diisopropylfluorophosphate-inactivated APC, gla-domainless APC, and thrombin-cleaved protein S had no effect on PAI activity, indicating requirement for preservation of the APC active site and of the Ca2+ binding ability of both APC and protein S. These results suggest coordinate binding of APC and protein S onto phospholipid membrane as a prerequisite for optimal expression of PAI neutralized by APC.