bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation stimuli induce receptors for plasma fibronectin on the human myelomonocytic cell line HL-60
Authors: Pommier CG , O'Shea J , Chused T , Takahashi T , Ochoa M , Nutman TB , Bianco C , Brown EJ
Published: October 2019

Plasma fibronectin (Fn) induces phagocytosis of C3b-opsonized sheep erythrocytes (EC3b) by human peripheral blood monocytes. However, Fn does not induce erythrophagocytosis of EC3b by human polymorphonuclear leukocytes (PMN), unless the PMN have been exposed to C5a or N-formyl- methionyl-leucyl-phenylalanine. Because of this difference, it is of great interest to examine Fn binding to cells that possess the capacity to differentiate into either granulocytes or monocytes. Hence, we have examined the consequences of Fn binding to the human myelomonocytic cell line, HL-60, both before and after in vitro differentiation of the HL-60, along a monocytoid or a granulocytoid pathway. Fn receptors were not found on undifferentiated HL-60, but several differentiating agents promoted the HL-60 binding of Fn-coated microspheres (Fn-ms). The peak of Fn-ms binding occurred four to five days after the induction of differentiation with dimethylsulfoxide (DMSO), and two days after induction by PMA. In addition, cells that differentiated along either the monocytoid or the granulocytoid pathway showed a marked increase in the phagocytosis of both IgG-coated erythrocytes (EA) and EC3b when they were exposed to Fn. Comparison of the effects of anti-Fn monoclonals on the binding of Fn-ms to the monocytes, PMN, and HL-60 showed that the same monoclonals block Fn-ms-binding and Fn-induced EC3b phagocytosis by all three cell types. Two monoclonal antibodies, M1/70 and A6F10, directed against membrane antigens on PMN and monocytes, inhibited Fn-ms binding. Both also blocked Fn-induced EC3b ingestion by these cells. However, neither antibody blocked Fn-ms binding or EC3b ingestion by differentiated HL-60. We conclude that differentiated HL-60 cells express functionally active Fn receptors, similar to monocytes and activated PMN, which, nonetheless, differ from normal cells in their association with the antigens recognized by M1/70 and A6F10.
Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts
Authors: Max-Audit I , Testa U , Kechemir D , Titeux M , Vainchenker W , Rosa R
Published: October 2019

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.
High-dose intravenous gammaglobulin in alloimmunized platelet transfusion recipients
Authors: Schiffer CA , Hogge DE , Aisner J , Dutcher JP , Lee EJ , Papenberg D
Published: October 2019

High-dose intravenous gammaglobulin (polyvalent immunoglobulin G) has been shown to be of benefit in some patients with immune thrombocytopenic purpura (ITP), possibly by producing reticuloendothelial system blockade. We studied this approach in patients refractory to random donor platelet transfusion using an IV IgG preparation manufactured by the Swiss Red Cross. Eleven adult patients with acute leukemia received either 0.4 g IgG/kg/d intravenously X five days (four patients) or 0.6 g/kg/d X five days (seven patients). All patients had high levels of lymphocytotoxic antibody and poor responses to random donor platelets. Except for mild headaches in two patients, there were no side effects related to the IgG infusions. All patients had significant elevations of serum IgG on the day after completion of treatment. Either random donor or partially HLA-matched platelet transfusions were administered the day after and, in some cases, during the IgG therapy. No patient had an improvement in one hour posttransfusion platelet count increments. Two additional patients received pooled platelet concentrates incubated for 30 minutes at 37 degrees C with IgG at a final concentration of 3 g% prior to transfusions. These results indicate that high-dose IgG, an extremely expensive treatment, cannot be recommended for alloimmunized adults with leukemia.
Growth, interleukin-2 production, and responsiveness to IL-2 in T4- positive T Lymphocyte populations from malignant cutaneous T cell lymphoma (Sezary's syndrome): the effect of cyclosporin A
Authors: Solbach W , Lange CE , Rollinghoff M , Wagner H
Published: October 2019

Functional analysis and surface phenotyping using monoclonal antibodies have revealed that malignant T lymphocyte populations in the peripheral blood of patients with Sezary's syndrome resemble the T helper cell populations from normal individuals. In this article we have studied the effects of the immunosuppressive drug cyclosporine A (CsA) on growth, interleukin-2 (IL-2) production, and the induction of IL-2 responsiveness of peripheral blood monocytes (PBMs) from five patients with Sezary's syndrome in vitro, using the lectin phytohemagglutinin (PHA) and the phorbol ester phorbol myristate acetate (PMA) as stimuli. The following results were obtained: PHA-induced cell proliferation was significantly more sensitive to inhibition by CsA than that induced by PMA or a combination of PMA and PHA (P less than .005). Sezary PBMs produced only small amounts of IL-2 in response to PHA. Stimulation with PMA, however, resulted in significant IL-2 production. PMA and PHA, when given in combination, acted synergistically. The low levels of IL-2 production induced by PHA or PMA were more sensitive to CsA- mediated suppression than those induced by a combination of PHA and PMA (75% and 55% suppression, respectively). CsA-mediated growth suppression could be overcome if the cultures were supplemented with appropriate amounts of exogenous IL-2. We conclude from our data, that CsA in Sezary PBMs inhibits T cell growth indirectly as a consequence of suppression of IL-2 growth indirectly as a consequence of suppression of IL-2 production. Moreover, like normal T lymphocytes, Sezary PBMs do not express the IL-2 receptor spontaneously, but can be induced to do so. CsA does not interfere with intracellular events leading to the expression and the biologic function of the IL-2 receptor.
Lipid analyses and fluidity studies by electron spin resonance of red cell membranes in hereditary high red cell membrane phosphatidylcholine hemolytic anemia
Authors: Yawata Y , Sugihara T , Mori M , Nakashima S , Nozawa Y
Published: October 2019

Membrane lipid analyses and electron spin resonance (ESR) studies of membrane fluidity were carried out on the red cells of a Japanese patient with hereditary high red cell membrane phosphatidylcholine hemolytic anemia (HPCHA). Increased amounts of phosphatidylcholine (PC) and cholesterol were found in the membrane lipids of the affected patient, despite normal plasma lipids. The order parameter of cholesterol-free pure phospholipid liposomes prepared from this patient's red cells was decreased, apparently because of the increased PC. In contrast, the order parameter of the total red cell lipid liposomes (containing free cholesterol) was essentially normal. The overall fluidity of the intact red cells was determined by ESR with a spin probe, 5-SAL. Again, the order parameters were normal in the intact red cells of the patient with HPCHA. This suggests that the concomitant increase of membrane cholesterol and phosphatidylcholine serves to maintain normal membrane fluidity in the HPCHA red cells.
Acquired von Willebrand's disease in the myeloproliferative syndrome
Authors: Budde U , Schaefer G , Mueller N , Egli H , Dent J , Ruggeri Z , Zimmerman T
Published: October 2019

An acquired hemorrhagic disorder developed in two patients in association with postsplenectomy thrombocytosis and leukocytosis during the course of the myeloproliferative syndrome. The presence of acquired von Willebrand's disease in these individuals was demonstrated by a decrease or absence of the larger von Willebrand factor (vWF) multimers, alteration of the repeating vWF multimeric “triplet,” decreased ristocetin cofactor activity (vWF:RCo), and prolonged bleeding time. The bleeding stopped in both patients after treatment with either 1-deamino-[8-D-arginine]-vasopressin (DDAVP) or Cohn fraction I. Treatment with thrombocytapheresis and azathioprine or busulfan resulted in reduction of the elevated platelet and white cell counts and was associated with partial correction of the vWF abnormalities and remission of the hemostatic abnormalities. In five additional patients with the myeloproliferative syndrome, but without bleeding symptoms, large multimers of plasma vWF were diminished also. These findings suggest that acquired von Willebrand's disease should be considered when a bleeding diathesis develops during the course of the myeloproliferative syndrome.
The generation of human long-term marrow cultures from marrow depleted of Ia (HLA-DR) positive cells
Authors: Keating A , Powell J , Takahashi M , Singer JW
Published: October 2019

Successful long-term cultures were generated from marrow buffy-coat cells incubated with either of two monoclonal anti-Ia antibodies, 7.2 and HBIOa, and complement. The mean CFU-GM toxicity for 7.2 and HBIOa was 81% and 94%, respectively, of the complement control. Cultures generated from marrows treated with 7.2 and complement produced between 30% and 163% of the CFU-GM (mean, 78.2 +/- 52.4%) and 33% of the BFU-E produced by the complement control cultures. Long-term cultures from marrows treated with the more cytotoxic HBIOa antibody yielded 261% of the CFU-GM present at culture initiation. Our data suggest that the progenitor cells necessary for generation of long-term cultures from human marrow are Ia-negative and may represent less mature stem cells than those measurable by semisolid colony assays. Furthermore, marrow treated with cytotoxic anti-Ia antibodies may be suitable for use in autologous transplantation.
Effects of variant gamma chains and sialic acid content of fibrinogen upon its interactions with ADP-stimulated human and rabbit platelets
Authors: Harfenist EJ , Packham MA , Mustard JF
Published: October 2019

When platelets are stimulated with adenosine diphosphate (ADP), fibrinogen binds to receptors on the platelet membrane, and the platelets aggregate. The primary platelet recognition sites of human fibrinogen are reported to be at the COOH-terminal ends of the gamma chains, with secondary sites in the A alpha chains. Normal human fibrinogen, which consists of three pairs of disulfide-bonded peptide chains, (A alpha, B beta, gamma)2, is heterogeneous with respect to sialic acid content and also contains a small proportion of molecules with a variant gamma chain (designated gamma'), elongated by a peptide extension at the COOH-terminus of the normal gamma chain. We separated fibrinogen into three fractions by chromatography on DEAE cellulose and tested the interactions of these fractions with ADP-stimulated human and rabbit platelets. Two fractions had the normal chain composition, (A alpha B beta, gamma)2, but different sialic acid contents (6.6 and 7.2 mol/mol), and the third fraction had the chain composition (A alpha, B beta)2 gamma gamma' and a sialic acid content of 7.2 mol/mol, which is similar to that of one of the normal fractions. In binding and aggregation experiments, we detected no significant differences between the reactions of the first two fractions, but ADP-stimulated platelets bound only 50% as much of 125I-fibrinogen from the fraction with the gamma' chains and also aggregated less extensively in the presence of this fraction. We conclude that the sialic acid content of fibrinogen does not significantly affect its interactions with platelets, but the elongated gamma' chains bind less effectively to ADP-stimulated platelets, and thus reduce the ability of fibrinogen to support aggregation. This may result from a conformational change caused by the gamma' extension or from the deletion of a portion of the normal gamma chain recognition site.
High incidence of monoclonal proteins in the serum and urine of chronic lymphocytic leukemia patients
Authors: Deegan MJ , Abraham JP , Sawdyk M , Van Slyck EJ
Published: October 2019

Chronic lymphocytic leukemia (CLL) is generally considered a nonsecretory B cell immunoproliferative disorder. Conventional electrophoretic and immunoelectrophoretic methods have revealed serum monoclonal proteins in less than 10% of these patients. However, there is increasing experimental evidence from in vitro studies demonstrating that CLL cells may secrete immunoglobulins, particularly free light chains. We examined the serum and urine of 36 consecutive CLL patients for monoclonal proteins using sensitive immunochemical methods (high resolution agarose gel electrophoresis combined with immunofixation). The results obtained were correlated with the Rai stage, quantitative immunoglobulin levels, and lymphocyte membrane immunoglobulin phenotype of the leukemic cells. Twenty-three monoclonal proteins were identified in the serum or urine of 22 patients, an incidence of 61%. Six patients had serum monoclonal proteins, seven had only urinary monoclonal proteins, and nine had monoclonal proteins in serum and urine. In every instance the monoclonal protein was the same light chain type as expressed on the leukemic cells. Our findings suggest that the monoclonal proteins observed in the serum or urine of CLL patients are secretory products of the tumor cells and that their discovery is a function of the sensitivity of the method used for their detection.
Coagulation Factor IX concentrate: method of preparation and assessment of potential in vivo thrombogenicity in animal models
Authors: Menache D , Behre HE , Orthner CL , Nunez H , Anderson HD , Triantaphyllopoulos DC , Kosow DP
Published: October 2019

Thrombosis and/or disseminated intravascular coagulation (DIC) are complications specifically associated with the use of factor IX complex in some patients. Assuming that these complications might result from zymogen overload, we have produced, using diethylaminoethyl (DEAE)- Sephadex (Pharmacia, Piscataway, NJ) and sulfated dextran chromatography, a factor IX concentrate (coagulation factor IX) that is essentially free of prothrombin, factor VII, and factor X. Factor IX specific activity is at least 5 U/mg protein, a 250-fold purification compared to plasma. Amounts of factors II, VII, and X are less than 5 units each per 100 units of factor IX. The concentrate is essentially free of activated clotting factors and contains no added heparin. In the rabbit stasis model, a dose of 200 factor IX U/kg was less thrombogenic than 100 factor IX U/kg of the DEAE-Sephadex eluate from which the concentrate was derived. Infusion of 200 factor IX U/kg did not induce DIC in the nonstasis rabbit model, whereas 100 factor IX U/kg of the DEAE-Sephadex eluate resulted in DIC in this model. Several factor IX lots were found to have shortened nonactivated partial thromboplastin times (PTTs), but were nonthrombogenic in both animal models. These data indicate that coagulation factor IX concentrate is less thrombogenic than factor IX complex.
Characterization of erythrocyte quality during the refrigerated storage of whole blood containing di-(2-ethylhexyl) phthalate
Authors: Estep TN , Pedersen RA , Miller TJ , Stupar KR
Published: October 2019

Di-(2-ethylhexyl) phthalate (DEHP) accumulates in blood brought into contact with materials utilizing this compound as a plasticizer. To determine whether this phthalate diester affects red blood cell integrity, we have compared cell morphology, plasma hemoglobin accumulation, micro-vesicle production, and the concentration of intracellular metabolites and electrolytes of erythrocytes from blood stored at 4 degrees C with and without DEHP. When sufficient emulsified DEHP was mixed with blood to give a final concentration of 300 micrograms/mL, plasma hemoglobin accumulation was reduced by an average of 70%, the percentage of cells exhibiting normal morphology was enhanced by at least 20-fold, and the volume of microvesicles released from red blood cells was reduced by 50% after 35 days of refrigerated storage compared to the values obtained from corresponding samples stored without added phthalate. Similar effects were observed regardless of whether blood was stored in nonplasticized polypropylene or tri-(2-ethylhexyl) trimellitate plasticized polyvinylchloride containers and with DEHP solubilized by a variety of emulsifiers. When 300 micrograms/mL DEHP was added to stored blood containing erythrocytes predominantly in the echinocyte conformation, many of the cells reverted to the normal discoid morphology. The addition of this quantity of DEHP to blood had no significant effect on the course of storage-induced changes in erythrocyte adenosine triphosphate (ATP), 2,3-diphosphoglycerate (2,3-DPG), sodium or potassium concentrations. The data are consistent with the hypothesis that DEHP inhibits the deterioration of the red blood cell membrane that results from the refrigerated storage of whole blood.
Terminal transferase positive acute promyelocytic leukemia: in vitro differentiation of a T-lymphocytic/promyelocytic hybrid phenotype
Authors: Paietta E , Dutcher JP , Wiernik PH
Published: October 2019

In a case of acute promyelocytic leukemia (APL), the expression of terminal deoxynucleotidyl transferase (TdT), an early lymphoid marker, was detected. Double-fluorescent staining for the myeloid-specific antigens VIM-2 and VIM-D5 in combination with specific antiserum for TdT suggested a mixed leukemic cell population consisting of a morphologically, cytochemically, and immunologically promyelocytic component (80%) and a lymphoid, TdT+ component (20%) that was myelomonocytic in morphology but otherwise without any evidence of nonlymphoid nature. Fluorescent-activated cell analysis revealed that a greater number of cells reacted with monoclonal anti-T antibodies (OKT3, OKT6, and OKT11) than could be identified as lymphoid by TdT expression. As confirmed by double-staining fluorescence microscopy, a large fraction of the promyelocytic leukemia cells were biphenotypic, expressing both myeloid and lymphoid markers (50% positive for VIM-D5 and OKT6, 30% positive for VIM-D5 and OKT3). Subsequently, in vitro differentiation experiments were performed. While treatment of the cells with GCT-conditioned medium favored proliferation, with only a weak and delayed promotion of the cells towards maturation as reflected by enhanced expression of the mature T-marker T3 but persistent expression of the thymocyte antigen, exposure to all-trans and 13-cis retinoic acid resulted in marked differentiation of both the myeloid and the lymphoid cell characteristics. Retinoid treatment resulted in the loss of TdT, a partial disappearance of the T6-antigen, and the expression of the late T cell antigen T3 by almost 70% of the cells. In addition, myeloid maturation was obvious from the morphologic appearance of the cells, as well as from the expression of the OKM1- associated antigen by a majority of the cells. This report concerns a unique case of APL in which, for the first time, a coexistence of promyelocytic and lymphoid elements was detected, with exposure of the cultured leukemic cells to retinoic acid inducing maturation along both the myeloid and the lymphoid lineage.
Neonatal purpura fulminans: a genetic disorder related to the absence of protein C in blood
Authors: Marciniak E , Wilson HD , Marlar RA
Published: October 2019

To confirm the pathogenesis and the genetic background of neonatal- onset purpura fulminans, two unrelated infants with this rare thrombotic syndrome and 47 of their asymptomatic relatives were studied. In both families, 27 subjects with hereditary partial deficiency of protein C, including both parents of each patient, were identified. The patient in whom it was possible to evaluate protein C directly showed no detectable levels of this plasma component. These findings confirm the linkage of neonatal purpura fulminans to a genetic trait with established mendelian transmission and strongly suggest that the syndrome is an expression of homozygosity for protein C deficiency. The dramatic clinical picture and the type of pathologic change that develops as a result of the lack of circulating protein C emphasize the vital role of this protein in protection from thrombin generation, mainly within the microvascular system. However, our data do not contribute to the evidence that partial familial protein C deficiency is associated with a major risk of venous thromboembolism.
Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes
Authors: Brittenham GM , Schechter AN , Noguchi CT
Published: October 2019

We examined the extent to which the intracellular polymerization of sickle hemoglobin (HbS) can account for the severity of anemia and of vaso-occlusive manifestations in the various sickling syndromes. Polymer formation in sickle cell disease depends principally on the intraerythrocytic hemoglobin composition and concentration. In our studies, the polymer fraction in sickle red cells was determined from reported mean values for hemoglobin composition and mean corpuscular hemoglobin concentration (MCHC) in 12 groups of patients with sickle hemoglobinopathies (homozygotes for HbS, with and without coexistent alpha-thalassemia or various forms of the hereditary persistence of fetal hemoglobin [HPFH], beta+-, beta 0-, and delta beta-thalassemia, and heterozygotes for HbS with HbA). The calculated HbS polymer fractions at full deoxygenation and at physiologic oxygen saturation values were closely correlated with mean blood hemoglobin concentrations. In addition, polymer fraction correlated with the ranking of the sickling syndromes by vaso-occlusive severity. We find that polymer fraction accounts for about 80% of the variability in hemolytic and clinical severity. The method of analysis presented here provides a quantitative and systematic means of assessing the role of polymer formation in the pathophysiologic manifestations of the sickling syndromes. Our results support the hypothesis that the intracellular polymerization of HbS is the primary determinant of the severity of both anemia and clinical symptomatology in the sickle hemoglobinopathies.
A monoclonal antibody that inhibits activation of human Hageman factor (factor XII)
Authors: Small EJ , Katzmann JA , Tracy RP , Ratnoff OD , Goldsmith GH Jr , Everson B
Published: October 2019

A monoclonal antibody to human Hageman factor (HF, factor XII) was derived from BALB/c mouse spleen cells fused with NS-1 mouse myeloma cells. This antibody, purified from ascites fluid, reacted with HF to inhibit the activation of HF, purified or in normal pooled plasma, as measured by a coagulation assay. The antibody did not inhibit the coagulant activity of activated HF. The antibody also inhibited the generation of amidolytic activity in HF-ellagic acid mixtures, but failed to inhibit the amidolytic properties of the carboxy-terminal fragment of HF (HFf). Amidolytic activity, absent in an HF-monoclonal antibody mixture, was generated upon treatment with insoluble trypsin. Monoclonal antibody, bound to CNBr Sepharose 4B gel (Pharmacia Fine Chemicals, Piscataway, NJ), reversibly bound HF in plasma or in buffer, without activating it. HF was then eluted with 4 mol/L guanidine HCI. The passage of 125I-labeled HF enzymatically cleaved by trypsin through a column of monoclonal antibody-CNBr Sepharose 4B gel resulted in flow- through of HFf with a molecular weight (mol wt) of 30,000 and HF fragments of mol wt 12,000. Elution with 4 mol/L guanidine HCI yielded several HF fragments (mol wt 80,000, 52,000, and 40,000) but not HFf. These data suggest that the single determinant recognized by the murine monoclonal antibody is not on HFf, but rather on the amino-terminal fragment thought to be involved in the binding activity of HF. The monoclonal anti-HF bound to CNBr-activated Sepharose 4B gel could be used to artificially deplete plasma samples of HF.
Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3
Authors: Newman PJ , Allen RW , Kahn RA , Kunicki TJ
Published: October 2019

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582–12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.
Independent stimulation of membrane potential changes and the oxidative metabolic burst in polymorphonuclear leukocytes
Authors: Seeds MC , Parce JW , Szejda P , Bass DA
Published: October 2019

Early events of stimulus-response coupling in polymorphonuclear leukocytes (PMNL) reportedly include membrane depolarization as a necessary antecedent to oxidative responses. However, depolarization by nonspecific means (ionophores) is insufficient to elicit an oxidative burst. This apparent conflict might be related to whether depolarization is due to membrane receptor-mediated stimulation of PMNL. We used two fluorescent probes and dual laser flow cytometry to monitor both membrane potential and oxidative product formation in individual PMNL, following stimulation by phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP). Dipentylindocarbocyanine [di-I-C5(3)] is one of a family of dyes that partition between cells and aqueous media as a function of transmembrane potential. The dye appeared stable in the presence of PMNL oxidative products. Oxidation of intracellularly trapped, nonfluorescent dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) provided a quantitative assessment of oxidative metabolism (H2O2 production) of stimulated PMNL. Incubating PMNL with both fluorochromes resulted in stable red [di-l-C5(3)] and green (DCF) fluorescence when examined on a Cytofluorograph. Upon stimulation by 0.03 to 0.1 nmol/L PMA, PMNL showed a unimodal apparent depolarization (decrease in di-l-C5(3) fluorescence). Oxidative activity (increased DCF fluorescence) was first seen at a concentration of PMA of 0.17 nmol/L, higher than that required to elicit depolarization. This oxidative burst appeared as a dose-dependent, graded response. Thus, receptor-mediated membrane depolarization, although antecedent to the onset of the oxidative burst, was not in itself sufficient to trigger the oxidative metabolic response. When PMNL were isolated by centrifugation through Ficoll-metrizoate, fMLP caused an apparent depolarization of a variable subpopulation of the cells. However, such purified PMNL appeared relatively unstable and often depolarized spontaneously. PMNL studied without centrifugation through Ficoll-metrizoate were stable. Moreover, fMLP stimulation of such cells did not cause membrane depolarization but did stimulate a two- to six-fold increase in DCFH oxidation. Apparently, membrane depolarization may antecede oxidative responses in PMNL, but appears to depend upon the method of cell preparation and the nature of the stimulus.
The heterozygous form of 4.1(-) hereditary elliptocytosis [the 4.1(-) trait]
Authors: Alloisio N , Morle L , Dorleac E , Gentilhomme O , Bachir D , Guetarni D , Colonna P , Bost M , Zouaoui Z , Roda L
Published: October 2019

Using clinical, morphological, genetic, and biochemical criteria, we studied ten white and North African families with hereditary elliptocytosis (HE). In four families, elliptocytic individuals displayed a highly significant reduction of band 4.1, which was recorded using two electrophoretic procedures. The 4.1a/4.1b ratio was also significantly reduced, as is usually observed in suspensions enriched in young red cells. This form of HE was invariably associated with the following characteristics: absence of clinical signs; numerous, smooth and well-elongated elliptocytes; dominant transmission; and, when investigated, normal osmotic fragility. Its frequency, among all forms of HE, is about one third as a first estimate, at least in whites and North Africans. In the other six families studied, elliptocytic subjects presented normal 4.1 bands. Again, the 4.1a/4.1b ratio was decreased, reflecting the red cell age- dependent changes in these two components. In three of these families, elliptocytosis was accompanied by clinical signs of variable intensity, and the mode of inheritance could not be unequivocally determined. Therefore, HE with a partially reduced band 4.1 defines a homogeneous variety of HE that can be isolated from other forms of HE. We suggest that it be termed the 4.1 (-) trait, so as to correspond with a previously proposed terminology.
Platelets contain proteins immunologically related to red cell spectrin and protein 4.1
Authors: Davies GE , Cohen CM
Published: October 2019

Human platelets were tested for the presence of proteins immunologically cross-reactive with red cell spectrin and protein 4.1. As assessed by indirect immunofluorescence microscopy, platelets were specifically reactive with affinity-purified rabbit antisera against red cell spectrin and protein 4.1. The immunoreactive platelet constituents were further analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, followed by electrophoretic transfer to nitrocellulose paper and immunoperoxidase staining. We found that whole platelets, membranes, and cytoskeletal preparations isolated by Triton X-100 extraction contain small amounts of proteins reacting with anti-spectrin or anti-protein 4.1 antiserum. The immunoreactive spectrin-like platelet protein has an apparent molecular weight of 240,000 and comigrates with the alpha-subunit of red cell spectrin. The major immunoreactive protein 4.1-like constituent has an apparent molecular weight of 78,000, which is slightly less than that of red cell protein 4.1. We conclude that platelets contain a spectrin- like protein which, by analogy with red cell spectrin, may have a role in membrane-cytoskeletal attachment. The properties and function of the platelet protein 4.1-like constituent are not yet known.
Patients with a deficiency of natural killer cell activity lack the VEP13-positive lymphocyte subpopulation
Authors: Ziegler-Heitbrock HW , Rumpold H , Kraft D , Wagenpfeil C , Munker R , Abo T , Balch CM , LeBien TW
Published: October 2019

Many patients with B-type chronic lymphocytic leukemia (CLL) exhibit a profound defect in their natural killer (NK) cell activity, the basis of which is still obscure. Hence, we analyzed the NK cells from peripheral blood samples from 11 patients with CLL for phenotype and function, after removal of the leukemic cells with a monoclonal antibody (BA-1) plus complement. Phenotypic analysis of these nonleukemic cells with monoclonal antibodies (MoAbs) against NK cells revealed that the CLL patients had higher percentages of HNK-1-positive cells (23.5% compared to controls with 14.7%). In contrast, VEP13- positive cells were absent or low in seven patients (0.8% compared to controls with 11.2%) and normal in four patients (10.5%). When testing NK cell activities against K562 or MOLT 4 target cells, patients with no or minimal numbers of VEP13-positive cells were found to be deficient, while patients with normal percentages of VEP13-positive cells had NK cell activity comparable to controls. Isolation by fluorescence-activated cell sorter of HNK-1-positive cells from patients lacking VEP13-positive cells and NK cell activity indicated that the majority of the HNK-1-positive cells in these patients had the large granular lymphocyte morphology that is characteristic of NK cells. Thus, the deficiency of NK cell activity in CLL patients appears to result from the absence of cells carrying the VEP13 marker.
Monocytes and macrophages synthesize and secrete thrombospondin
Authors: Jaffe EA , Ruggiero JT , Falcone DJ
Published: October 2019

hrombospondin, one of the major glycoproteins released from alpha- granules of thrombin-stimulated platelets, is a disulfide-linked trimer of 160,000-dalton subunits. Cultured human monocytes secreted thrombospondin (determined by an enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (1.45 micrograms/10(6) cells/24 hr); secretion was totally blocked by cycloheximide (1 microgram/mL). 35S-thrombospondin was isolated from 35S-methionine-labeled human monocyte postculture medium with rabbit polyclonal anti-thrombospondin coupled to protein A-Sepharose. The immunoisolated 35S-thrombospondin migrated in sodium dodecyl sulfate- polyacrylamide gels after reduction with a molecular weight of 159,000. Similar results were obtained using mouse resident peritoneal macrophages. Elicited peritoneal macrophages harvested from mice pretreated with endotoxin, casein, or thioglycollate secreted much less thrombospondin than did resident macrophages harvested from control mice. Thus, monocytes and macrophages from two different species synthesize and secrete thrombospondin, and the rate of synthesis of thrombospondin appears to depend on the state of activation of the cells.
Age-related changes in deformability of human erythrocytes
Authors: Sutera SP , Gardner RA , Boylan CW , Carroll GL , Chang KC , Marvel JS , Kilo C , Gonen B , Williamson JR
Published: October 2019

The present study was designed to further the characterization of age- related changes in the deformability of human erythrocytes. The top (approximately young) and bottom (approximately old) 10% fractions of density-separated red cells from ten normal donors were subjected to graded levels of shear stress in a rheoscope. Measurements were made of steady-state elongation (cells tank treading in a state of dynamic equilibrium) and the time course of shape recovery following abrupt cessation of shear. In parallel with the rheologic experiments, several physical and chemical properties were assayed to determine correlates of mechanical properties. These included mean cell volume, mean corpuscular hemoglobin concentration, type A1 hemoglobin, glucosylation of membrane proteins, and membrane phospholipid and protein concentration. The microrheologic observations revealed that only about 90% of the old cells retained their capacity to tank tread. However, the tank-treading cells elongated less than their younger counterparts at corresponding levels of shear stress, thus demonstrating a reduced level of deformability. Further analysis of the data indicates that increases in membrane viscosity and elastic modulus along with a significant loss in excess surface area contribute to the limitation of the ability of the older cells to change shape.
Multiple myeloma: significance of plasmablastic subtype in morphological classification
Authors: Greipp PR , Raymond NM , Kyle RA , O'Fallon WM
Published: October 2019

We classified 100 cases of myeloma before chemotherapy as mature (28), intermediate (38), immature (19), or plasmablastic (15). The plasmablastic group had an estimated median survival (Kaplan-Meier method) of ten months, compared to 35 months for the other types (P less than .05). Decreased survival in the plasmablastic group was due to more frequent deaths in the first six months. There were no significant differences in survival among the mature, intermediate, and immature groups or among patients with different morphological grade or asynchrony scores. The plasmablastic myeloma group had more frequent renal insufficiency and higher plasma cell labeling indices, which may have contributed to the shorter survival.
Monoclonal antibody AHN-1 inhibits phagocytosis by human neutrophils
Authors: Skubitz KM , Weisdorf DJ , Peterson PK
Published: October 2019

The granulocyte-specific monoclonal antibody, AHN-1, immunoprecipitates two major surface-iodinated proteins of 105,000 and 145,000 to 150,000 daltons from normal human neutrophils. In this study, the effect of AHN- 1 on a number of neutrophil functions was evaluated in vitro. Both complement- and antibody-mediated phagocytosis were inhibited when human neutrophils were pretreated with AHN-1 and opsonized bacteria were used as targets. The inhibition of phagocytosis was specific, in that lysosomal enzyme release and chemotaxis were not altered by treatment with AHN-1. AHN-1 did inhibit superoxide production by neutrophils in response to particulate stimuli, but not in response to the soluble stimulus, 12-O-tetradecanoylphorbol-13-acetate. The data indicate that one or both of these surface proteins may be important in the process of phagocytosis. AHN-1 should be useful in isolating and further characterizing the nature of these molecules.
Bone marrow from cadaver donors for transplantation
Authors: Mugishima H , Terasaki P , Sueyoshi A
Published: October 2019

To determine the feasibility of obtaining bone marrow cells from cadaver donors for transplantation, marrow cells were prepared from 17 cadaver donors. After surgical removal of the iliac crest, as many as 2 X 10(9) cells were isolated. Cadaver marrow had a lower percentage of T cells (mean of 10%) than did marrow from living donors. The T cells were lysed by a monoclonal antibody and human complement to a point at which no sheep red blood cell-rosetting cells were detected. Low levels of T colonies, however, grew out from the monoclonal antibody-treated cells. Although cell loss inevitably occurs from purification, antibody treatment, freezing, and thawing, sufficient numbers can be recovered for transplantation. The yield of stem cells was 84% for CFU-C, 39% for CFU-E, 81% for BFU-E, and 48% for CFU-GEMM. We suggest that T cell- depleted marrow cells from cadaver donors could be used for transplantation. Improved immunosuppressive therapy may be required, however, to prevent graft rejection of allogeneic marrow that may have minor histocompatibility differences.
Intragenic Factor IX restriction site polymorphism in hemophilia B variants
Authors: Hassan HJ , Orlando M , Leonardi A , Chelucci C , Guerriero R , Mannucci PM , Mariani G , Peschle C
Published: October 2019

This study includes 47 normal subjects and 25 hemophilia B patients without inhibitor(s), showing different factor IX coagulant activity and antigen levels. Genomic DNA, digested with various restriction endonucleases, was hybridized with two different factor IX probes, ie, the cDNA and the subgenomic probe for the intragenic TaqI polymorphic site. cDNA restriction patterns suggest absence of gross rearrangements and/or deletions in all hemophilic patients. The frequency of the X chromosome bearing the TaqI polymorphic site is 0.32 +/- 0.09 in hemophilic subjects v 0.36 +/- 0.06 in normal control subjects, the latter value being comparable to that reported for the normal British population. No association between this polymorphism and hemophilia B variants has been observed, thus indicating that a wide spectrum of mutations underlies this blood-clotting disorder and particularly each of its variants.
Decreased ecto-5'nucleotidase activity of peripheral blood lymphocytes in human monoclonal gammopathies: correlation with tumor cell kinetics
Authors: Massaia M , Ma DD , Boccadoro M , Golzio F , Gavarotti P , Dianzani U , Pileri A
Published: October 2019

Ecto-5′nucleotidase (5′NT) activity of peripheral blood (PB) lymphocytes was determined in 31 patients with serum monoclonal gammopathies (MG). Twenty-one patients had a diagnosis of multiple myeloma (MM), and ten patients had monoclonal gammopathy of undetermined significance (MGUS). The proliferative activity of the bone marrow plasma cells (LI%) was investigated in 28 of these MG patients by means of tritiated thymidine uptake evaluated by simultaneous autoradiography and cytoplasmic immunofluorescence. 5′NT activity was significantly lower in MG patients as compared with normal controls. MM patients had lower 5′NT activity than MGUS patients, but the difference was not significant. By contrast, MM had significantly higher LI% than MGUS patients. There was a linear regression of 5′NT on LI% which was statistically significant: the higher the LI%, the lower the 5′NT. Because the LI% is an accurate prognostic and monitoring factor in MG, this correlation indicates that 5′NT may be of assistance in predicting the clinical progress of MG patients. In seven MGs, the PB T and B lymphocytes were studied separately. The T cell subpopulation was 5′NT deficient compared to the normal controls, shown as a significant linear regression of T cell 5′NT on the LI%. This suggests that in MG there may be an alteration of nonneoplastic T lymphocytes correlated with tumor growth. The OKT8+ lymphocytes were mainly responsible for the 5′NT deficiency of unseparated T lymphocytes.
Catabolism of human tissue plasminogen activator in mice
Authors: Fuchs HE , Berger H Jr , Pizzo SV
Published: October 2019

The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenylmethylsulfonyl-125I-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125I-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125I-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125I-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125I-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 125I-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.
Platelet kinetics in patients with idiopathic thrombocytopenic purpura and moderate thrombocytopenia
Authors: Stoll D , Cines DB , Aster RH , Murphy S
Published: October 2019

We studied ten normal subjects and 20 patients with stable, untreated idiopathic thrombocytopenic purpura (ITP) and platelet counts in the range of 35,000 to 110,000/microL. The diagnosis was made by clinical criteria. Platelet-associated IgG was increased in all nine of the nine patients studied. Autologous platelets were labeled with chromium 51 and reinfused for measurement of mean cell life and platelet production rate. Mean cell life was calculated by two methods, weighted mean and multiple hit, with excellent agreement between the two. As expected, mean cell life was significantly reduced in ITP patients as compared to the normal subjects (2.9 days v. 8.0 days, P less than .001). However, mean platelet production rates in ITP patients and normal subjects, 3.5 and 3.8 X 10(9) platelets/k/d respectively, were not significantly different. Platelet production rate was above and below the normal range (2 to 5.6 X 10(9) platelets/k/d) in two and four patients, respectively. We conclude that the rate of platelet production is not increased in most patients with ITP who have platelet counts greater than 35,000/microL. We did find that platelet size was increased in eight of the 12 patients in whom it was measured, including two of the patients with low platelet production.
Specific identification of fibrin polymers, fibrinogen degradation products, and crosslinked fibrin degradation products in plasma and serum with a new sensitive technique
Authors: Connaghan DG , Francis CW , Lane DA , Marder VJ
Published: October 2019

A new method is described for identifying low concentrations of circulating derivatives of fibrinogen and fibrin, even when present in heterogeneous mixtures. This technique is applicable to plasma and serum and uses electrophoresis in 2% agarose in the presence of sodium dodecyl sulfate (SDS) followed by immunological identification of separated derivatives, using radiolabeled antifibrinogen antiserum and autoradiography. Unique electrophoretic patterns distinguish plasmic derivatives of crosslinked fibrin from those of fibrinogen and also identify crosslinked fibrin polymers produced by the combined action of thrombin and factor XIII on fibrinogen. The assay is sensitive to a concentration of 0.1 micrograms/mL of fibrinogen in serum or plasma. Fibrin polymers, plasmic degradation products of fibrinogen, and plasmic degradation products of crosslinked fibrin were detected in the plasma or serum of a patient with disseminated intravascular coagulation. Plasmic derivatives of both fibrinogen and crosslinked fibrin appeared in serum in the course of fibrinolytic therapy for pulmonary embolism, whereas during acute myocardial infarction a marked increase in the proportion of fibrin polymers in plasma was found in comparison with normal controls. Thus, the procedure can distinguish between the simultaneous processes of fibrin polymer formation, fibrinogenolysis, and fibrinolysis, and is sufficiently sensitive to detect relevant quantities of derivatives in pathologic conditions.