bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of human neutrophils by monoclonal antibody PMN7C3: cell movement and adhesion can be triggered independently from the respiratory burst
Authors: Melnick DA , Meshulam T , Manto A , Malech HL
Published: October 2019

Anti-neutrophil monoclonal antibody PMN7C3 (IgG3) recognizes glycoproteins bearing the oligosaccharide lacto-N-fucopentaose III, including the C3bi receptor, LFA-1, and p150,95 on the plasma membrane and a group of granule-associated glycoproteins. We have previously shown that binding of this antibody to polymorphonuclear leukocytes (PMNs) stimulates a transient rise in cytosolic free calcium concentration but does not trigger the neutrophil respiratory burst. We now demonstrate that binding of PMN7C3 (and five other monoclonal antibodies recognizing the same antigen) to human neutrophils activates several other cellular responses. Addition of PMN7C3 to monolayers of neutrophils induces a rapid change in cell shape followed by pseudopod formation and increased migration. With incubation at 37 degrees C, the neutrophils aggregate in clusters (leukoagglutination). Quantitation of cell movement in a multiwell chemotaxis assembly or by migration of PMNs under agarose revealed that PMN7C3 is both chemotactic and chemokinetic. Pretreatment with the antibody inhibits subsequent chemotactic response to other stimuli. Monoclonal antibodies binding to other neutrophil antigens do not mimic these effects. These data suggest that cell movement and adhesion can be triggered independently from the respiratory burst. PMN7C3 may be a useful probe with which to study the events that link receptor-ligand binding to cellular response.
High fetal hemoglobin production in sickle cell anemia in the eastern province of Saudi Arabia is genetically determined
Authors: Miller BA , Salameh M , Ahmed M , Wainscoat J , Antognetti G , Orkin S , Weatherall D , Nathan DG
Published: October 2019

Homozygous sickle cell disease in the eastern province of Saudi Arabia is clinically mild. Circulating fetal hemoglobin levels of 16.0 +/- 7.4% were found in these anemic patients, but only 1.09 +/- 0.97% in their sickle trait parents. To determine whether these sickle cell anemia patients inherit an increased capacity to synthesize fetal hemoglobin, a radioimmunoassay of fetal and adult hemoglobin was performed on erythroid progenitor (BFU-E)-derived erythroblasts from Saudi Arabian sickle cell patients and their parents. Mean fetal hemoglobin content per BFU-E-derived erythroblast from Saudi Arabian sickle cell patients was 6.2 +/- 2.4 pg/cell or 30.4 +/- 8.6% fetal hemoglobin (normal 1.1 +/- 0.7 pg/cell and 5.1 +/- 1.8%). Linear regression analysis of % HbF in peripheral blood versus % HbF per BFU-E- derived cell showed a positive correlation with an r of 0.65. The variance of the intrinsic capacity to produce HbF may account for almost 40% (r2) of the variance of circulating fetal hemoglobin but other factors, particularly selective survival of F cells, must also contribute significantly. Despite virtually normal HbF levels in sickle trait parents of these Saudi patients, mean fetal hemoglobin production per BFU-E-derived erythroblast in these individuals was elevated to 3.42 +/- 1.79 pg/cell or 16.1 +/- 6.4% fetal hemoglobin, and the magnitude of fetal hemoglobin production found in parents correlated with that of the patients. These data indicate that the high fetal hemoglobin in Saudi sickle cell disease is genetically determined but expressed only during accelerated erythropoiesis. Further evidence of such genetic determination was provided by analysis of DNA polymorphisms within the beta-globin gene cluster on chromosome 11. This revealed a distinctive 5′ globin haplotype (+ + - + +) on at least one chromosome 11 in all high F SS and AS tested. The precise relationship of this haplotype to HbF production in this population remains to be defined.
Establishment of eosinophilic sublines from human promyelocytic leukemia (HL-60) cells: demonstration of multipotentiality and single- lineage commitment of HL-60 stem cells
Authors: Tomonaga M , Gasson JC , Quan SG , Golde DW
Published: October 2019

Recent observations indicating that the HL-60 human acute promyelocytic leukemia cell line contains a minor eosinophil population in addition to neutrophil and mononuclear phagocyte progenitors suggest the multipotentiality of HL-60 stem cells. In order to clarify multilineage differentiation and commitment to single-lineage progenitors we analyzed HL-60 colonies formed in methylcellulose. In an HL-60 parent line with a relatively high eosinophil content (5.5%), 36% of the spontaneous colonies consisted partly or wholly of eosinophilic cells. After two rounds of subcloning in methylcellulose, two eosinophilic sublines and two neutrophilic sublines were established. These lines have been in continuous liquid culture for more than four months, and they show stable single-lineage differentiation. Purified biosynthetic GM-CSF, which stimulates normal CFU-GM and CFU-EO, induced monocytic differentiation but no eosinophilic differentiation in the neutrophilic sublines and no neutrophilic or monocytic differentiation in the eosinophilic sublines. These observations indicate that HL-60 stem cells are multipotent and capable of spontaneous commitment to single- lineage progenitors. The eosinophilic HL-60 sublines should facilitate studies on the production and function of human eosinophils and the single-lineage sublines will allow further analysis of leukemic cell differentiation and stem cell commitment.
Analysis of elevated fibrin(ogen) degradation product levels in patients with liver disease
Authors: vanDeWater L , Carr JM , Aronson D , McDonagh J
Published: October 2019

Plasma and serum from patients with liver disease and elevated fibrin(ogen) degradation product (FDP) levels as measured by latex agglutination were analyzed by immunoblotting to characterize the FDP in these patients. An antihuman fibrinogen antibody was used that recognizes fibrinogen, fibrin monomer, soluble high molecular weight fibrinogen and fibrin polymers, as well as high molecular weight cross- linked degradation fragments, and the smaller fragments X, Y, D-dimer, D, and E. The analytic procedures were validated with plasma and serum from patients known to have intravascular fibrinolysis associated either with disseminated intravascular coagulation (DIC) or with thrombolytic therapy. The samples demonstrated a spectrum of plasmin degradation fragments on the immunoblots. Twenty-eight of 35 patients with liver disease (80%) had no evidence of plasmin degradation fragments in their plasma or serum. The cause of the elevated FDP levels as measured by latex agglutination was thought to be fibrin monomer or unclottable fibrinogen that was retained in the sera of some of these patients. Seven patients (20%) were found to have circulating plasmin degradation fragments. In addition to liver disease, however, these patients all had an illness (sepsis, shock, and pancreatic carcinoma) independently associated with intravascular coagulation and fibrinolysis. Three patients who lacked plasmin fragments also had pancreatic carcinoma or sepsis. The two groups of liver disease patients could not be clearly differentiated on the basis of clinical or laboratory evidence, but the blotting procedure proved to be a useful discriminator.
Increased red blood cell deformability due to isoxsuprine administration decreases platelet adherence in a perfusion chamber: a double-blind cross-over study in patients with intermittent claudication
Authors: Aarts PA , Banga JD , van Houwelingen HC , Heethaar RM , Sixma JJ
Published: October 2019

Platelet transport towards the vessel wall is influenced by the hematocrit, red blood cell (RBC) size, and shape. Recent in vitro studies have indicated that RBC deformability may also influence platelet transport. The observation that isoxsuprine, a known vasodilating drug, caused increased RBC deformability in vitro and decreased platelet transport in vitro prompted us to study the effects of this drug in vivo. The study was performed in a double-blind cross- over study of isoxsuprine v placebo in ten patients with peripheral arterial insufficiency. RBC deformability was estimated from viscosity measurements using the blood viscosity equation of Dintenfass and expressed as T value. Platelet transport was studied in an annular perfusion chamber according to Baumgartner. Human umbilical arteries were used as blood vessels. Perfusion studies were performed with whole blood or with RBCs of the patients mixed with normal platelets and plasma at a standardized hematocrit and platelet count. An increase in RBC deformability concomitant with a decrease in platelet adherence was observed in patients on isoxsuprine with a drop in T value of approximately 0.06 (from 0.91 toward 0.86), and a concomitant decrease in platelet adherence of 20% to 40%. These observations differed significantly from the results in the placebo group and showed a significant group-period interaction at the cross-over of medication (analysis of variance). The effects on platelet adherence were observed at high vessel wall shear rate (1,800 s-1) with perfusates consisting of patients' RBCs and donor plasma and platelets at standardized hematocrit and platelet count. No differences were observed under these conditions at a shear rate of 300 s-1. When whole blood of patients was used, nonsignificant effect was observed at shear rates of 300 s-1 and 1,800 s-1. This was probably caused by the added noise due to variations in hematocrit and platelet number. These data demonstrate that isoxsuprine increases RBC deformability, and they suggest the possibility of decreasing platelet-vessel wall interaction in vivo by manipulation of RBC deformability.
Interaction of factor XIa and antithrombin in the presence and absence of heparin
Authors: Beeler DL , Marcum JA , Schiffman S , Rosenberg RD
Published: October 2019

We have studied the interaction between purified human factor XIa and antithrombin in the presence and absence of well-characterized preparations of heparin. The concentrations of hemostatic enzyme, protease inhibitor, and mucopolysaccharide were 5.76 X 10(-8) mol/L, 5.76 X 10(6) mol/L, and either 5.88 X 10(6) mol/L or 0, respectively. Kinetic investigation of this process using a tritiated factor IX substrate demonstrated that the pseudo first-order rate constants of this reaction in the presence and absence of heparin are approximately 1.0 min-1 and approximately 0.025 min-1, respectively. Thus, the rate of hemostatic enzyme-protease inhibitor complex formation is accelerated by about 40-fold in the presence of saturating levels of the mucopolysaccharide. These results were confirmed in a qualitative manner by directly monitoring the generation of factor XIa-antithrombin interaction product with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis using an antibody population specific for the protease inhibitor.
Clinical evaluation of platelet concentrates stored for one to five days
Authors: Schiffer CA , Lee EJ , Ness PM , Reilly J
Published: October 2019

There are no large-scale data available describing the increments obtained with platelet concentrates stored for varying durations. Platelet concentrates prepared by standard techniques were stored at 22 degrees C with horizontal agitation in PL-732 bags and administered to clinically stable, nonalloimmunized recipients known to respond well to random donor platelet transfusions. The platelet concentrates were stored in a mean volume of 65.0 mL (range 54–80 mL) with an average yield of .72 X 10(11) platelets per unit of platelet concentrates (N = 100 consecutive units). There was no significant deterioration of pH during storage. Mean corrected count increments ranged between 16,600 (N = 146 transfusions) after 1 day of storage to 13,300 (N = 34 transfusions) after 5 days of storage. Although these differences were statistically significant (P less than .003, analysis of variance), the overall deterioration in increments was quite modest. Platelet concentrates can be stored under standard conditions for 1 to 5 days with acceptable clinical results.
Purification and characterization of human platelet glutathione-S- transferase
Authors: Loscalzo J , Freedman J
Published: October 2019

A glutathione-S-transferase was isolated and purified to homogeneity from human platelets. With a combination of ammonium sulfate fractionation and chromatographic methods, 0.2 mg of pure enzyme was obtained from 9 X 10(11) platelets with a 12% recovery. The purified enzyme had a specific activity of 7.5 U per milligram, representing an approximately 1,100-fold purification. The enzyme was found to be anionic, with an isoelectric point of 4.6. With reduced glutathione as a co-substrate, platelet glutathione-S-transferase was most active with the synthetic substrate, 1-chloro-2,4-dinitrobenzene, less active with 1,2-dichloro-4-nitrobenzene, and essentially inactive with nitroglycerin and 1,2-epoxy-3-(p-nitrophenoxy)-propane. The pH optimum for activity with glutathione and 1-chloro-2,4-dinitrobenzene was 7.0. Indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyindole-3-acetic acid), a chlorobenzene derivative, noncompetitively inhibited human platelet glutathione-S-transferase with an apparent KI of 0.23 mmol/L. This study represents the first complete purification and characterization of a glutathione-S-transferase from platelets. The presence of this enzyme in the platelet, within which high concentrations of reduced glutathione coexist, suggests the potential importance of the platelet in detoxification reactions and in the synthesis of the glutathione adducts of leukotriene metabolism.
Biology of human megakaryocyte factor V
Authors: Gewirtz AM , Keefer M , Doshi K , Annamalai AE , Chiu HC , Colman RW
Published: October 2019

To learn more about human megakaryocyte coagulation cofactor V (FV), we studied the expression of this protein in normal bone marrow megakaryocytes and in megakaryocytes cloned from their colony-forming unit in FV-depleted plasma clot cultures. Mouse monoclonal antibodies directed against either the light chain or an activation peptide of human FV and a rabbit polyclonal, monospecific FV antiserum were used as probes for these experiments in conjunction with a variety of immunochemical detection techniques. All morphologically recognizable megakaryocytes were shown to contain FV. The origin of this protein appeared to be both from FV bound to the cell as well as from endogenous FV in the majority of cells examined. The existence of a population of small bone marrow mononuclear cells that simultaneously expressed platelet glycoproteins and FV was also noted. Such cells represented approximately 70% of all small cells positive for platelet glycoproteins. In contrast, only about 40% of megakaryocyte colonies cloned in FV-deficient medium contained cells with immunochemically detectable FV. FV expression was most clearly demonstrated in large cells in the colonies, whereas smaller, presumably less mature cells labeled weakly or not at all. Synthesis of FV by human megakaryocytes was documented using elutriation-enriched cells incubated in 35S- methionine-containing medium. Megakaryocyte lysates and medium conditioned by these cells were subjected to immunoaffinity column purification. Column eluates analyzed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and autoradiography revealed radioactive bands comigrating with the heavy and light chains of thrombin-activated FV. These studies suggest that human megakaryocytes both bind and synthesize FV. Expression of these traits appears to be related to cell maturation, with binding ability appearing earlier than the ability to synthesize this protein. Finally, although the ability to bind FV appears to be universal among megakaryocytes, our culture data suggest that synthesis may be a restricted, or constitutively expressed property of these cells.
Effects of therapy with T101 ricin A-chain immunotoxin in two leukemia patients
Authors: Laurent G , Pris J , Farcet JP , Carayon P , Blythman H , Casellas P , Poncelet P , Jansen FK
Published: October 2019

Two leukemia patients, refractory to chemotherapy, were treated with T101-ricin A-chain immunotoxin (T101 IT). Patient 1 (T-ALL) received a single 13.5 mg dose of T101 IT IV (12-hour infusion). Patient 2 (B-CLL) was treated with a daily 25 mg dose of T101 IT IV (two-hour infusion) over three consecutive days. Patient 2 also received 300 mg of chloroquine IM on days two and three as enhancer. In vivo binding of T101 IT was demonstrated by FACS analysis using either an antimouse Ig- FITC or anti-A-chain-FITC antibodies. Following IT therapy, the expression of T65 antigen on target cells dropped to 50% and 20% of pretreatment levels, respectively. In patient 1, circulating blast cells remained unsaturated during therapy while in patient 2, cells were fully saturated for four to six hours following each infusion. Pharmacokinetic studies showed a rapid clearance of T101 IT after IV administration. Antimouse and anti-A-chain antibodies could not be detected. There were no treatment-related adverse effects. In patient 1 a rapid but transient decrease of target cells was observed, possibly related to the administration of the antibody part of T101 IT. In contrast, patient 2 showed a 40% reduction of the lymphocyte count, which remained stable over a period of 2 weeks. Such a clinical benefit following IT therapy in patient 2 could be ascribed to the absence of circulating free antigen and the complete saturation of target cells.
Purified plasma factor XIIa aggregates human neutrophils and causes degranulation
Authors: Wachtfogel YT , Pixley RA , Kucich U , Abrams W , Weinbaum G , Schapira M , Colman RW
Published: October 2019

Plasma kallikrein has been shown to aggregate human neutrophils and release human neutrophil elastase. However, neutrophils resuspended in factor XII-deficient plasma released only 30% of the elastase compared with normal plasma. Isolated human neutrophils were aggregated in a concentration-dependent fashion by 0.06 to 0.6 U/mL factor XIIa (0.022 to 0.22 mumol/L). Factor XIIa (0.1 to 1.0 U/mL) also induced neutrophil degranulation as evidenced by a concentration-dependent release of the specific granule protein, lactoferrin, and azurophilic granule protease, elastase. The release of neutrophil elastase was biphasic, reaching 40% of maximum at 15 seconds with maximal release by 90 minutes. The active site of factor XIIa was required, since the synthetic inhibitor, D-Pro-Phe-Arg-CH2Cl, which reacts with an essential histidine, and the natural plasma inhibitor, Cl-inhibitor, which interacts with the critical serine, both inhibit by more than 90% the release of elastase. The heavy chain is also required, since factor XII fragments failed to aggregate neutrophils or stimulate degranulation. Factor XIIa (0.6 U/mL) can completely correct the defect in elastase release evident in factor XII-deficient plasma. These studies demonstrate that factor XIIa, at concentrations potentially obtainable in plasma in disease states, can activate neutrophils, and thus may participate in the inflammatory response.
Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization
Authors: Reber P , Furlan M , Rupp C , Kehl M , Henschen A , Mannucci PM , Beck EA
Published: October 2019

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called “fibrinogen Milano l.” The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303–356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.
Inhibition of prostaglandin E2 restores defective lymphocyte proliferation and cell-mediated lympholysis in recipients after allogeneic marrow grafting
Authors: Klingemann HG , Tsoi MS , Storb R
Published: October 2019

Prostaglandins are said to influence T and B cell function by inhibiting the generation of interleukin 2 (IL 2) and the formation of suppressor lymphocytes. After bone marrow transplantation, patients usually have a profound immunodeficiency that persists in recipients with chronic graft-v-host disease (GVHD) and generally resolves in long- term survivors without GVHD. In vitro tests of lymphocyte function such as allogeneic mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) have been shown to be impaired in many patients. We postulated that prostaglandin E2 (PGE2) plays a role in the impaired in vitro tests. To test this hypothesis, we studied in vitro tests in the presence of PGE2 antagonists, indomethacin, and anti-PGE2 antiserum with cells from 22 short-term patients (less than 100 days postgrafting) and 32 long-term survivors with or without GVHD. Results show that blockade of PGE2 release by indomethacin and anti-PGE2 significantly (P less than .01) enhanced the MLC (+67%) and the CML responses (+10.5%) of cells from long-term survivors with chronic GVHD but not from those of long-term, stable recipients. No enhancement of MLC and CML activity was observed with cells from donors of long-term recipients. In patients shortly after marrow grafting, enhancement in the MLC was not significant. However, CML activity in this patient group was significantly increased (+12.5% in recipients with no GVHD, 8.5% in those with acute GVHD, P less than .01). Indomethacin also suppressed the activity of nonspecific suppressor cells in patients with chronic GVHD. When cells from patients with chronic GVHD were treated with recombinant IL 2 and IL 2 combined with indomethacin, it was possible to get an additional augmentation of lymphocyte proliferation after the addition of indomethacin to IL 2-treated cultures. Thus it is very likely that PGE2 inhibits T lymphocyte proliferation, not exclusively by inhibition of IL2 production or activity. We conclude that PGE2, among other factors, may play a role in the pathogenesis of the immunodeficiency after transplantation. PGE2 does not act primarily by interfering with IL2 but presumably by inducing a suppressorlike activity.
A new von Willebrand variant (type I, New York): increased ristocetin- induced platelet aggregation and plasma von Willebrand factor containing the full range of multimers
Authors: Weiss HJ , Sussman II
Published: October 2019

We report three members of a family who had reduced levels of plasma von Willebrand factor (vWF) and increased ristocetin-induced platelet aggregation (RIPA) (aggregation of platelet-rich plasma with ristocetin at a concentration of 0.45 mg/mL), as previously reported in type IIB and pseudo-von Willebrand's disease (vWD). However, in contrast to the latter two disorders in which the larger vWF multimers are absent in plasma, the entire range of vWF multimers was observed in the patients' plasma after sodium dodecyl sulfate-agarose gel electrophoresis, and all vWF multimers (including the largest) were present in the same proportion as in normal plasma and type I vWD. Thus, despite increased RIPA, the levels and multimeric pattern of vWF in this family's plasma were indistinguishable from those in type I vWD in which RIPA is usually decreased. Addition of ristocetin to the patients' platelet- rich plasma resulted in the removal of vWF (and, more selectively, of the large multimers) at lower concentrations of ristocetin than normal, as in type IIB and pseudo-vWD. The defect in the patients was localized to their vWF, which had an enhanced capacity for aggregating washed normal platelets in the presence of low concentrations of ristocetin and for aggregating pseudo-vWD platelets (in the absence of ristocetin). Both glycoproteins (GP) Ib and IIb-IIIa were involved in the enhanced aggregation response. RIPA (at low ristocetin concentrations) in the patients' platelet-rich plasma was abolished by a monoclonal antibody (AP1) to GPIb and was markedly reduced by monoclonal antibodies (10E5 and LJP9) that block adenosine diphosphate and thrombin-induced binding of vWF and fibrinogen to GPIIb-IIIa but was unaffected by an antibody (LJP5) that only blocks vWF binding. Partial inhibition of the initial aggregation slope (and complete inhibition of second phase aggregation) was achieved with creatine phosphate/creatine phosphokinase. EDTA blocked second-phase aggregation but was without effect on the initial slope. The findings in this family combine some features of both type I vWD (normal pattern of vWF multimers in plasma) and type IIB vWD (increased RIPA) and further demonstrate the increasing complexity of the structure-function relationships in vWD.
Synthesis of eosinophil-associated enzymes in HL-60 promyelocytic leukemia cells
Authors: Fischkoff SA , Brown GE , Pollak A
Published: October 2019

Eosinophils derived from HL-60 cells share many of the abnormalities of granule histochemistry and morphology frequently seen in eosinophils of patients with certain malignancies, especially those seen in acute myelomonocytic leukemia with abnormal eosinophils (FAB class M4eo). In order to understand the pathogenesis of these abnormalities, four enzymes, characteristic of the eosinophil, were studied in HL-60 promyelocytic leukemia cells at various stages of eosinophilic differentiation. Using biochemical and ultrahistochemical techniques, the following differences from normal eosinophil development were demonstrated. First, both myeloperoxidase and eosinophil peroxidase coexisted in the population of maturing HL-60 eosinophils. Second, the granules formed from the condensation of material in vacuoles which were derived from dilated segments of the endoplasmic reticulum; the role of the Golgi apparatus in processing of peroxidase appeared minimal. Third, low levels of lysophospholipase and arylsulfatase were present in the cells compared to normal eosinophils. Finally, crystallizations resembling precursor structures of Auer rods appeared in the granules of about 5% of the cells. These findings suggest that several disorders of the control of protein synthesis and processing exist in HL-60 eosinophils which may be responsible for the abnormal granule morphology and histochemistry.
Polymorphonuclear leukocyte heterogeneity in neonates and adults
Authors: Krause PJ , Malech HL , Kristie J , Kosciol CM , Herson VC , Eisenfeld L , Pastuszak WT , Kraus A , Seligmann B
Published: October 2019

We have used a mouse monoclonal antibody (31D8) to determine whether differences in neutrophil (PMN) subpopulations might help explain decreased PMN chemotaxis in neonates compared with that of adults. 31D8 has been shown to bind heterogeneously to adult PMNs. Approximately 80% of the PMNs that strongly bind 31D8 (31D8 “bright”) are the same cells that depolarize and migrate chemotactically when stimulated with the chemoattractant N-formyl-methionylleucylphenylalanine, while the 20% that weakly bind 31D8 fail to similarly respond. All neonatal PMNs bound 31D8 heterogeneously. There was a smaller population of 31D8 “bright” cells in neonates at birth (76% +/- 6%, n = 45) compared with that of neonates at three to 15 days of age (82% +/- 5%, n = 10, P less than 0.002) and both were smaller than that of adults (88% +/- 4%, n = 45, P less than 0.001 and P less than 0.001). Neonatal cord PMNs, which traversed a micropore filter in a modified Boyden chemotaxis chamber in the presence of a chemoattractant, had an increased percentage of 31D8 “bright” cells (89% +/- 7%) than did PMNs which remained above the filter (82% +/- 7%, n = 10, P = 0.034). PMN chemotaxis was less in neonates at birth (32.7 +/- 4.5 micron) than at three to six days of age (36.8 +/- 11.3 micron) and both were decreased compared with that of adults (69.1 +/- 12.4 micron, P less than 0.001 and P less than 0.001). These findings indicate that decreased PMN chemotaxis in neonates may be due in part to a smaller PMN subpopulation of highly motile cells.
Ultrastructural localization of coagulation factor V in human platelets
Authors: Wencel-Drake JD , Dahlback B , White JG , Ginsberg MH
Published: October 2019

The distribution and transport in platelets of human coagulation Factor V was investigated by immunofluorescent and immunoelectron microscopy. In resting intact platelets, little surface staining was observed by immunofluorescence. In permeable resting cells, punctate staining similar to that reported for fibrinogen (Fbg), thrombospondin (TSP), fibronectin (Fn), von Willebrand factor (VWF), B-thromboglobulin (BTG), and platelet Factor 4 (PF4) was observed. Double label immunofluorescent staining for Fbg and Factor V demonstrated colocalization, suggesting their presence in the same intracellular structure. Thrombin stimulation induced the appearance of larger (approximately 0.5 mu) immunofluorescent masses of these proteins which exactly colocalized. Thus, at the light level, Factor V and Fbg are localized in the same structure in resting and thrombin-stimulated cells. On the ultrastructural level, an alpha granule localization for Fbg has previously been established. We have extended our immunofluorescent observations regarding the localization of Factor V in human platelets by use of transmission electron microscopy of antibody-stained ultrathin frozen sections. In resting cells, staining of virtually all alpha granules was observed for Factor V. In contrast, consistent staining was absent from other organelles including plasma membranes, mitochondria, and vacuolar structures which may represent the open canalicular system. These data thus establish at the ultrastructural level an alpha granule localization of human coagulation Factor V.
A new variant of type II von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor (type IIF)
Authors: Mannucci PM , Lombardi R , Federici AB , Dent JA , Zimmerman TS , Ruggeri ZM
Published: October 2019

A patient with a lifelong bleeding disorder was diagnosed as having Type II von Willebrand disease. The larger multimers of von Willebrand factor were absent from her plasma but present in platelets. A high- resolution electrophoretic technique was used to study the complex structure of individual von Willebrand factor multimers. In normal plasma, each multimer could be resolved into five bands: a more intense central one and four less intense, two moving faster and two slower than the central band. In normal platelets, each multimer could also be resolved into five bands. The central one had a mobility similar to that in plasma, whereas the four satellite bands had a mobility that differed from that of the corresponding plasma bands. In the patient, platelet von Willebrand factor antigen content and ristocetin cofactor activity were normal, and von Willebrand factor showed the same structure of individual multimers as seen in normal platelets. On the other hand, plasma von Willebrand factor antigen and ristocetin cofactor activity were decreased, and the structure of individual von Willebrand factor multimers was different from that of normal plasma and similar to that seen in normal and patient's platelets. After infusion of 1-deamino-8-D-arginine vasopressin, the largest von Willebrand factor multimers, as well as new satellite bands with a mobility similar to those in normal plasma, appeared in the patient plasma, and the levels of von Willebrand factor antigen and ristocetin cofactor activity became normal. Yet no relevant change in the prolonged bleeding time was observed. This new variant of von Willebrand disease, therefore, is characterized by the presence of a dysfunctional von Willebrand factor molecule that exhibits unique structural abnormalities in plasma but appears to be normal in platelets. The designation of Type IIF is proposed for this type of von Willebrand disease in accordance with the terminology that has been previously used.
Activation of plasminogen by tissue plasminogen activator on normal and thrombasthenic platelets: effects on surface proteins and platelet aggregation
Authors: Stricker RB , Wong D , Shiu DT , Reyes PT , Shuman MA
Published: October 2019

Tissue plasminogen activator (TPA) converts plasminogen to plasmin within the fibrin clot, thus localizing activation of fibrinolysis. To determine the extent to which platelets promote activation of plasminogen by TPA, we studied the interaction of TPA and plasminogen with unstimulated platelets. Normal washed platelets incubated in the presence of physiologic concentrations of plasminogen (180 micrograms/mL) and TPA (20 ng/mL) failed to generate plasmin activity. In contrast, incubation of platelets with TPA concentrations achieved during thrombolytic therapy (40 to 800 ng/mL) produced a tenfold to 50- fold increase in plasmin activity. After exposure to plasminogen and 200 ng/mL of TPA for one hour, platelets failed to agglutinate in the presence of ristocetin. Incubation of platelets suspended in autologous plasma with 400 ng/mL of TPA for one hour also inhibited ristocetin- induced agglutination. Exposure of platelets to plasminogen and increasing concentrations of TPA correlated with a decrease in glycoprotein Ib (GPIb) and an increase in glycocalicin, as shown by immunoblotting. The glycoprotein IIb/IIIa (GPIIb/IIIa) complex and a 250,000-dalton protein also disappeared from washed platelets after incubation with plasminogen and 200 ng/mL of TPA for one hour. These platelets failed to aggregate in the presence of adenosine diphosphate (ADP) or gamma thrombin, although aggregation in response to calcium ionophore A23187 and arachidonic acid remained intact. However, aggregation in response to all four agonists was normal when platelets were incubated with TPA in the presence of autologous plasma. Platelets from a patient with Glanzmann's thrombasthenia also generated plasmin in the presence of TPA. Hydrolysis of GPIb and inhibition of ristocetin- induced agglutination occurred to a lesser extent with these platelets than with control platelets. We conclude that platelets provide a surface for activation of plasminogen by pharmacologic amounts of TPA. Plasmin generation leads to degradation of GPIb and decreased ristocetin-induced agglutination in normal and thrombasthenic platelets, as well as degradation of GPIIb/IIIa in normal washed platelets and inhibition of ADP and gamma thrombin-induced aggregation. These findings suggest that pharmacologic concentrations of TPA may cause platelet dysfunction due to plasmin generation on the platelet surface.
Human T lymphotropic virus type III infection of human alveolar macrophages
Authors: Salahuddin SZ , Rose RM , Groopman JE , Markham PD , Gallo RC
Published: October 2019

The human T-cell lymphotropic virus type III (HTLV-III) is the etiologic agent of the acquired immunodeficiency syndrome (AIDS) and preferentially infects T4 lymphocytes. Other cell types, notably B lymphocytes and other nonlymphoid cells, also have been reported to be infected in vitro by HTLV-III. We now report on the susceptibility of human pulmonary macrophages to infection with HTLV-III in vitro. Alveolar macrophages infected with HTLV-III produced low levels of virus that could be transferred to allogeneic human peripheral blood mononuclear leukocytes as long as 2 weeks after initiation of infection. Unlike HTLV-III infection of T lymphocytes, macrophages appeared more resistant to viral-mediated cytopathic effects. Primary cultures of pulmonary macrophages from two of four patients with AIDS spontaneously produced low levels of virus detected as precipitable reverse transcriptase activity, suggesting that these cells were infected in vivo. Because tissue macrophages are long-lived cells, they may act as a reservoir of HTLV-III, capable of transmitting the virus to other susceptible cells such as T lymphocytes, causing periodic low- level viremia. Macrophage infection with HTLV-III may be one mechanism for the establishment of viral persistence in infected hosts.
Cytochemical, immunologic, chromosomal, and molecular genetic analysis of a novel cell line derived from Hodgkin's disease
Authors: Kamesaki H , Fukuhara S , Tatsumi E , Uchino H , Yamabe H , Miwa H , Shirakawa S , Hatanaka M , Honjo T
Published: October 2019

A novel cell line, KM-H2, was established from the pleural effusion of a patient with Hodgkin's disease of mixed cellular type. Multiple phenotypic studies were carried out with this cell line. Acid phosphatase and nonspecific esterase activities were detected. Rosette formation with T lymphocytes and the receptors for C3b and Fc portion of IgG were positive. Among the antigens tested with a total of 22 monoclonal antibodies defining hematopoietic cell subsets or lineages, Ki-1, Leu-M1, MCS1, HLA-DR, and OKT9 antigens were found to be positive. The other antigens reportedly specific for T cells, B cells, natural killer (NK) cells, monocytes, interdigitating reticulum (IR) cells and dendritic reticulum cells were negative. These phenotypic features were identical to those of the Sternberg-Reed (SR) and Hodgkin (H) cells in the fresh materials reported by other researchers. Moreover, the KM-H2 cells and the parental pleural effusion cells shared several structural chromosome anomalies. These findings indicated that the KM-H2 cells are derived from the SR and H cells. Molecular genetic analysis of the KM-H2 cells disclosed that the human immunoglobulin JH gene was rearranged but not the JK gene, and that the human T cell receptor beta chain gene was of the germline type. Based on these properties of the KM-H2 cells, Hodgkin's disease may be derived from a cell lineage other than T cell or B cell.
The percentage of dense red cells does not predict incidence of sickle cell painful crisis
Authors: Billett HH , Kim K , Fabry ME , Nagel RL
Published: October 2019

To test the hypothesis that the tendency of hemoglobin S (HbS) to polymerize within cells is the major determinant of the incidence of vaso-occlusive episodes, we have examined the effect of the percentage of dense cells (as measured by Percoll-Stractan continuous density gradient centrifugation) on the frequency of painful crises in a group of 36 patients with sickle cell disease. No correlation was found between the percentage of dense cells and admissions for crisis. Among the patients with known alpha-gene status (n = 25), the strong correlation between decreased dense cells and alpha-thalassemia (- alpha/alpha alpha) reported previously was confirmed (P less than .001). In addition, in this small subset, patients with alpha- thalassemia (-alpha/alpha alpha) appeared to have a marginally increased number of admissions for sickle cell crisis (t = 2.1910, P less than .05), which was independent of the percentage of dense cells. We conclude that the percentage of dense sickle cells cannot predict the incidence of painful crisis, suggesting that other factors (microcirculatory regulation or other humoral and cellular factors) are more important in the generation maintenance of painful crises than the necessary, but not sufficient, tendency of HbS-containing red cells to sickle.
Platelet membrane microparticles in blood bank fresh frozen plasma and cryoprecipitate
Authors: George JN , Pickett EB , Heinz R
Published: October 2019

Cryoprecipitate has been demonstrated to correct the bleeding abnormality of patients with some congenital (storage pool disease) and acquired (uremia) platelet abnormalities, but the reason for this effect is unknown. We found significant platelet contamination in plasma harvested to prepare fresh frozen plasma and cryoprecipitate. The platelet membrane microparticles produced by freezing and thawing of the plasma were highly concentrated in cryoprecipitate and may contribute to its therapeutic effect.
Metabolism of ara-C by blast cells from patients with ANLL
Authors: Ross DD , Thompson BW , Joneckis CC , Akman SA , Schiffer CA
Published: October 2019

The dose-response relationship between extracellular concentration of cytosine arabinoside (ara-C) and intracellular formation of the putative active metabolites of ara-C [ara-C incorporation into DNA and intracellular pools of ara-C in triphosphate form (ara-CTP)] was investigated in blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL) by exposing these cells in vitro to 10, 100, or 1,000 nmol/L of ara-C. We studied 23 untreated patients who subsequently achieved complete remission (CR) with a regimen using daunorubicin and conventional doses of ara-C (ara-C-sensitive group), and 30 patients judged to be ara-C-resistant either by failing initial induction therapy (16 patients) or by having relapsed on an ara-C- containing maintenance regimen (14 patients). In both patient groups, ara-C incorporation into DNA and intracellular ara-CTP both displayed statistically significant increases in response to increasing extracellular concentrations of ara-C (P = .0001 in both cases), with the rate of increase of ara-CTP greater than that of ara-C incorporation. Moreover, blast cells from all patients, even those who were most clinically resistant to ara-C, were able to form ara-CTP and to incorporate ara-C into DNA. Each tenfold increment in extracellular ara-C concentration caused an 8.5-fold increase in ara-CTP, but only a 3.6-fold increase in ara-C incorporation into DNA. Thus, the efficiency of incorporation of ara-C into DNA (defined as the ratio of ara-C incorporation to ara-CTP pools) decreased by 58% with each tenfold increment in the extracellular concentration of ara-C (P less than .0001), presumably as a result of the inhibitory effect of ara-CTP on DNA polymerase. Using an analysis of covariance, modest differences were found in the levels of the ara-C metabolite variables in the ara-C- sensitive group as compared with the resistant group. However, because there was considerable overlap in ara-C metabolite formation among the patient groups, it was not possible to predict clinical outcome by these in vitro assessments of ara-C metabolism.
Platelet aggregation by fibrinogen polymers crosslinked across the E domain
Authors: McManama G , Lindon JN , Kloczewiak M , Smith MA , Ware JA , Hawiger J , Merrill EW , Salzman EW
Published: October 2019

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.
HLA-DR-associated invariant chain is highly expressed in chronic lymphocytic leukemia
Authors: Narni F , Kudo J , Mars W , Calabretta B , Florine DL , Barlogie B , Saunders GF
Published: October 2019

Total RNA extracted from peripheral blood lymphocytes of a patient with B-cell chronic lymphocytic leukemia (CLL) and the poly (A+) RNA was purified. A cDNA library was constructed and approximately 4,000 clones were screened in order to identify genes preferentially expressed in CLL. A relatively low repetition frequency characterizes the majority of the abundant mRNA species present in CLL lymphocytes. One clone, corresponding to the mRNA encoding the HLA-DR-associated invariant chain, was selected and its expression was examined in different leukemic cell populations and in normal tissues. DNA-RNA hybridization studies showed that the invariant chain mRNA (In-mRNA) is detectable in RNA preparations from human blood cells and their precursors, whereas no In-mRNA is found in several other tissues examined. Among various normal and leukemic leukocyte populations, the highest levels of In- mRNA are found in CLL. Therefore, a role of In-chain mRNA as a marker of CLL is proposed. Our data support a relationship between high levels of invariant chain mRNA and the out of cycle condition of CLL peripheral blood lymphocytes.