bluuebunny/crossref_metadata_2025 huggingface dataset:
A unique precipitating autoantibody against plasma thromboplastin antecedent associated with multiple apparent plasma clotting factor deficiencies in a patient with systemic lupus erythematosus
Authors: Poon MC , Saito H , Koopman WJ
Published: October 2019

A 42-yr-old woman with systemic lupus erythematosus without bleeding diathesis developed a prolonged activated partial thromboplastin time that was not corrected by normal plasma. An inhibitor that acted rapidly and inactivated 0.5 U/ml plasma thromboplastin antecedent (PTA, factor XI) at a 1:200 plasma dilution was demonstrated. In addition to a low titer of PTA (less than 0.01 U/ml), plasma assayed at 20-fold dilution also showed low titers of Hageman (factor XII, 0.02 U/ml), Fletcher (plasma prekallikrein, 0.02 U/ml), and Fitzgerald (high molecular weight kininogen, less than 0.01 U/ml) factors. The titer of these factors, except PTA, returned to normal upon further plasma dilution or upon removal of the inhibitor by protein A adsorption. Thus, the inhibitor appeared to interfere with these clotting factor assays, possibly by inactivating PTA in the substrate plasmas in the test system. Its specificity was further confirmed. The inhibitor did not interfere with surface-induced proteolytic cleavage of Hageman factor. Surface-induced generation of plasma kallikrein activity (amidolysis of H-D-pro-phe-arg-pNa and cold-promoted factor VII activity enhancement) requires only Hageman, Fletcher, and Fitzgerald factors and was normal. Reactions requiring all 4 contact phase factors, including PTA, such as surface-induced generation of plasmin activity (amidolysis of H-D-val-leu-lys-pNa) and activated Christmas factor (factor IXa) activity, were defective. Furthermore, the inhibitor bound to agarose-protein A inactivated and removed PTA selectively from normal plasma. The inhibitor was an IgG-lambda autoantibody that precipitated PTA. The inactivated activated PTA (factor XIa) without the requirement for an additional cofactor. Furthermore, it inhibited surface-induced activation of PTA by interfering with its proteolytic cleavage upon glass surface exposure and with its binding onto the reactive surfaces.
Evidence for a multistep pathogenesis of a myelodysplastic syndrome
Authors: Raskind WH , Tirumali N , Jacobson R , Singer J , Fialkow PJ
Published: October 2019

Somatic cell genetic approaches utilizing the cellular mosaicism present in women heterozygous for glucose-6-phosphate dehydrogenase (G6PD) have provided information relevant to the pathogenesis of some neoplastic disorders. With these techniques, we studied a 61-year-old woman with a myelodysplastic syndrome. GdB/GdA heterozygosity was demonstrated in skin and cultured T lymphocytes, which exhibited both A and B type G6PD. In contrast, erythrocytes, platelets, granulocytes, and marrow nucleated cells displayed almost exclusively G6PD type B. In addition, 21 of 24 Epstein-Barr virus-transformed B lymphoblastoid lines that expressed a single immunoglobulin light chain showed only type B G6PD, suggesting that the stem cells involved by this disease were clonal and could differentiate to B lymphocytes as well as to mature granulocytes, erythrocytes , and platelets. Cultured skin fibroblasts and phytohemagglutinin-stimulated lymphocytes were karyotypically normal, but two independent abnormalities were found in marrow--47,XX, +8 and 46,XX,del(11)(q23). None of 14 type B G6PD lymphoblastoid lines analyzed in detail contained these karyotypic abnormalities, which strongly suggests that a visible chromosomal alteration is not the sole step in the development of this disease. We hypothesize that at least two events are involved in the pathogenesis of this patient's myelodysplasia: one causing proliferation of a clone of genetically unstable pluripotent stem cells and another inducing chromosomal abnormalities in its descendants.
The mechanism of in vitro clot lysis induced by vascular plasminogen activator
Authors: Tran-Thang C , Kruithof EK , Bachmann F
Published: October 2019

The contribution of vascular plasminogen activator (v-PA) to the lysis of whole blood and plasma clots was investigated. v-PA released into the circulation after infusion of deamino-D-arginine vasopressin (DDAVP) was shown to bind quantitatively to plasma clots. Its apparent molecular weight, determined by the SDS-PAGE fibrin-agarose underlay method, was approximately 68,000 daltons, and its activity was quenched by antibodies against human tissue plasminogen activator (t-PA). Clots prepared from post-DDAVP plasma or post-DDAVP whole blood, rich in v- PA, did not lyse when incubated in imidazole buffer or normal plasma, as determined by the release of 125I from radiolabeled clots. However, clots made of v-PA-poor plasma or whole blood, incubated in v-PA-rich plasma, underwent substantial lysis. The concentration of PA in clots incubated in v-PA-rich plasma progressively increased in relation to the initial concentration of v-PA in the surrounding plasma. The results suggest that, at low concentrations of circulating v-PA, a hemostatic plug will lyse at a very low rate. However, when the v-PA concentration in the clot environment is increased, v-PA will accumulate progressively onto fibrin and induce thrombolysis.
In vitro activation of the contact (Hageman factor) system of plasma by heparin and chondroitin sulfate E
Authors: Hojima Y , Cochrane CG , Wiggins RC , Austen KF , Stevens RL
Published: October 2019

A large number of negatively charged macromolecules, including DNA, glycosaminoglycans, and proteoglycans, were tested as possible activators of the contact (Hageman factor) system in vitro. Activation was assessed by conversion of prekallikrein to kallikrein, as determined by amidolytic assay and by cleavage of 125I-Hageman factor into 52,000- and 28,000-dalton fragments. Of particular interest to these studies, heparin proteoglycan and glycosaminoglycan from rat peritoneal mast cells, and squid chondroitin sulfate E, which is representative of the glycosaminoglycan from cultured mouse bone marrow derived mast cells, induced the reciprocal activation between Hageman factor and prekallikrein. In addition, naturally occurring heparin glycosaminoglycans from pig mucosa, bovine lung, and rat mast cells also induced activation. In contrast, native connective tissue matrix glycosaminoglycans and proteoglycans from several sources were inactive, although when one such chondroitin sulfate was further sulfated in vitro, it gained activity. When the negative charge of the activating agents was blocked by the addition of hexadimethrine bromide, the cleavage of 125I-Hageman factor in the presence of prekallikrein was prevented. The active negatively charged macromolecules induced cleavage of 125I-high molecular weight kininogen in normal plasma but not in Hageman factor-deficient or prekallikrein- deficient plasmas. Reconstitution of prekallikrein-deficient plasma with purified prekallikrein restored the kininogen cleavage upon addition of the active proteoglycans. These results suggest that both heparin from connective tissue mast cells and highly sulfated chondroitin sulfate E from cultured mouse bone marrow derived mast cells (which are considered synonomous with mucosal mast cells) could activate the contact system of plasma subsequent to an activation secretion response.
Bleeding and thrombosis in the myeloproliferative disorders
Authors: Schafer AI
Published: October 2019

Bleeding and thrombosis are major causes of morbidity and mortality in patients with myeloproliferative disorders. The significance of uncontrolled polycythemia as a risk factor for thrombosis in these patients has been established. However, the role of thrombocytosis in the pathogenesis of hemostatic complications remains controversial. Abnormalities of platelet function and prolongation of the bleeding time occur in a highly variable number of cases. Specific platelet defects that have been identified in the myeloproliferative defects include abnormal platelet morphology, acquired storage pool disease, platelet membrane abnormalities, and abnormal arachidonic acid metabolism. Causal relationships between any of these specific abnormalities and either bleeding or thrombosis have not been clearly established. The therapeutic efficacy of myelosuppression to reduce the platelet count in patients with thrombocytosis and the role of antiplatelet drugs in the myeloproliferative disorders are controversial issues.
Monoclonal antibodies specific for platelet glycoproteins react with human monocytes
Authors: Bai Y , Durbin H , Hogg N
Published: October 2019

Three monoclonal antibodies, P256 , P140, and P112 , react with the 135,000 mol wt IIb component of the glycoprotein IIb/IIa complex. They also react with a 200,000-mol wt protein present at low levels in the complex. Using immunofluorescence techniques, monoclonal antibodies P140 and P112 , but not P256 , can be shown to bind to 80% of human monocytes. However, P256 was able to immunoprecipitate the IIb/IIIa complex from detergent-solubilized monocytes, suggesting that the P256 epitope is less accessible on monocytes than on platelets. The monoclonal antibodies also precipitated molecules of approximately 200,000 mol wt from the monocytes. Two other monoclonal antibodies, J15 (specific for the IIb/IIIa complex) and AN51 (which reacts with the second major platelet glycoprotein complex, I), also react with monocytes. Binding of the monoclonal antibodies to the histiocytic cell line, U937, and promyelocytic cell line, HL-60, reflected the pattern of reaction with monocytes. The presence on monocytes of these glycoproteins, instrumental to the role of platelets in clotting, raises the possibility that monocytes might have similar functions in particular circumstances.
Tissue factor in microvesicles shed from U87MG human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis
Authors: Bastida E , Ordinas A , Escolar G , Jamieson GA
Published: October 2019

Microvesicles (diameter ca 200 nm) from the cell-free supernatant of U87MG human glioblastoma cell caused platelet aggregation and coagulation in a manner identical with that previously shown for the intact cells. Both activities were inhibited by dansylarginine -N-(3- ethyl-1,5-pentanediyl) amide (DAPA), confirming the thrombin-dependent nature of both activities. The specific activities per microgram of protein were 2–10 times greater in the microvesicles than in the plasma membrane fraction, suggesting localization in specific membrane domains. Sucrose density centrifugation gave a single protein peak (density 1.14) with congruent procoagulant and platelet aggregating activities. Both activities required the extrinsic pathway, as shown by studies with factor-deficient plasmas, and both were inhibited by heating (60 min/100 degrees C), by reduction and alkylation, and by incubation of the microvesicles with rabbit anti-bovine brain tissue factor antibody. These observations were confirmed using microvesicles from the HL-60 human promyelocytic leukemia cells, which are known to contain tissue factor activity. The results suggest that both procoagulant and proaggregating activities are causally related through the presence of tissue factor in the microvesicles. Studies with the Baumgartner perfusion apparatus showed that U87MG microvesicles increased the size of adherent thrombi nearly tenfold and that these thrombi were associated with nucleated cells from the blood. The increase in adherent thrombi did not occur if perfusion was carried out in the presence of DAPA, confirming the role of thrombin in their formation.
Mevalonic acid induces DNA synthesis in chronic lymphocytic leukemia cells
Authors: Larson RA , Yachnin S
Published: October 2019

Mevalonic acid can stimulate leukemic cells from some patients with B cell chronic lymphocytic leukemia (CLL) to enter the cell cycle in vitro and to synthesize DNA. Unlike normal human peripheral blood lymphocytes, which require the presence of granulocytes for a maximal DNA synthetic response to mevalonic acid, CLL cells do not require a helper cell. Only the physiologically active R(-) enantiomer of mevalonic acid is active in initiating DNA synthesis, and no stimulatory effect is noted after addition of the precursors of mevalonic acid or of its known products. The mitogenic responses to mevalonic acid measured in CLL cells from 35 patients varied widely (range of stimulation indices, 1.2–18.6), but the DNA synthetic response of CLL cells from 9 patients to mevalonic acid exceeded those seen with such common lymphocyte mitogens as concanavalin A, phytohemagglutinin, poke-weed mitogen, or a phorbol ester. The mevalonate-responsive CLL cell was enriched in the E(-), M(+) rosette subpopulation. When CLL cells were incubated in lipoprotein-depleted serum-containing medium, the presence of low density lipoprotein (LDL) cholesterol increased their sensitivity to mevalonic acid transformation, suggesting that a nonsterol product of mevalonic acid metabolism may be involved. Mevalonic acid, which is essential for the growth of cells programmed to divide or stimulated to divide by mitogens, can by itself initiate cell replication in relatively inert G0 phase normal and neoplastic lymphocyte populations.
Spontaneous iron overload in alpha-thalassemic mice
Authors: Van Wyck DB , Popp RA , Foxley J , Witte MH , Witte CL , Crosby WH
Published: October 2019

Because clinical disorders of spontaneous iron overload have no experimental counterpart, we studied iron distribution (atomic absorption analysis) and intestinal absorption (59Fe) in mice with hereditary alpha-thalassemia. Mice heterozygous for a radiation-induced alpha-Hb gene deletion exhibit a mild hemolytic anemia, like the human condition, with microcytosis, reticulocytosis, splenomegaly, and chemical evidence of defective alpha-chain synthesis. Quantitative iron determination showed that total iron content in spleen, liver, and kidney, but not heart or lung, of adult alpha-thalassemic mice was greater (P less than .05) than that in unaffected littermates. Iron concentration was also increased in liver (P less than .001), spleen (P = .025), kidney (P = .058), and heart (P = .010); in general, the greater the iron concentration in liver, the greater that in spleen (r = .39, P = .009), kidney (r = .70, P less than .001), and heart (r = .46, P less than .001). In mice examined 8 months postoperatively, splenectomy, as compared to sham operation, significantly raised iron content in extrasplenic tissues, but did not affect total body iron. At 10–11 weeks of age, but no longer at 12–14 weeks, thalassemic mice showed higher rates of iron absorption than age-matched controls. Thus, alpha-thalassemic mice display an early occurring iron absorption defect, leading to a modest, sustained, nonprogressive iron overload, and thereby represent a valuable model for exploring disorders of iron homeostasis.
Adherence of sickle erythrocytes to vascular endothelial cells: requirement for both cell membrane changes and plasma factors
Authors: Mohandas N , Evans E
Published: October 2019

Hebbel and colleagues have proposed that increased adherence of sickle red cells to vascular endothelium may initiate vasoocclusive events in sickle cell disease. We have developed a micropipette technique to obtain direct, quantitative measure of the adherence of individual red cells to vascular endothelial cells. Using this technique, we found that the vast majority of sickle cells suspended in autologous plasma were strongly adherent to endothelial cells, whereas only a small fraction of normal cells were weakly adherent. Influence of plasma factors on adherence was determined by measuring adherence of sickle cells suspended in normal plasma and normal cells suspended in sickle plasma. Although over 90% of sickle cells adhered to endothelial cells in autologous plasma, the percentage of adherent cells decreased dramatically to less than 20% when the same sickle cells were suspended in normal plasma. In contrast, adhesion of normal red cells suspended in sickle plasma was only modestly increased compared to adhesion in autologous normal plasma. Our results provide direct evidence for markedly enhanced adherence of sickle cells to endothelial cells. In addition, they suggest that both cell membrane changes and plasma factors contribute to this interaction. The requirement for sickle plasma further implies that temporal changes in plasma factors may play an important role in determining the onset of vasoocclusive crisis.
A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets
Authors: Peerschke EI , Coller BS
Published: October 2019

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.
Characterization of a novel HTLV-infected cell line
Authors: Koeffler HP , Chen IS , Golde DW
Published: October 2019

A man from Chile developed an aggressive mature T cell leukemia associated with marked eosinophilia. The neoplastic lymphocytes were of T helper surface phenotype, and they expressed the p24 and p19 antigens of human T cell leukemia virus (HTLV). A cell line (ME) was established from the patient's peripheral blood cells that was initially composed of eosinophils and T and B lymphocytes. The B lymphocytes of the cell line are polyclonal and contain Epstein-Barr virus (EBV) DNA. Many of the T lymphocytes, a few of the B lymphocytes, and none of the eosinophils express HTLV p19 and p24 antigens. By 6 months of culture, the ME line no longer contained eosinophils. A variant line of ME was established; this variant (ME-2) is notable because the cells (greater than 80%) adhere tightly to the bottom of the culture flask; they do not express T lymphocyte markers, but 30% of the cells contain cytoplasmic mu heavy immunoglobulin chains. These pre-B and null lymphocytes contain p19 and p24 antigens (80% of cells), have the HTLV- I genome, and are able to transform normal T lymphocytes in vitro. We isolated a B lymphocyte clone (11A) from ME that expresses cytoplasmic immunoglobulin (70% of cells) and p19 and p24 antigens (75% of cells), contains the EBV and HTLV genomes, and can transform T lymphocytes from normal volunteers. These data show that B lymphocytes can be infected with HTLV, although no disease of HTLV-infected B lymphocytes has been reported. The T lymphocytes from normal adult peripheral blood were easily immortalized (about 70% efficiency) by cocultivation with lethally irradiated ME cells. Twenty-five of 27 of the transformant lines were composed of T lymphocytes with helper antigens, and two of the lines were of T suppressor antigen phenotype. All the cell lines that were tested constitutively produce lymphokines, including colony- stimulating factor (CSF), erythroid-potentiating activity (EPA), macrophage migration-inhibitory factory (MIF), neutrophil-inhibitory factor (NIF), and differentiation-inducing factor (DIF).
Augmentation of neutrophilic granulocyte progenitors in the bone marrow of mice with tumor-induced neutrophilia: cytochemical study of in vitro colonies
Authors: Lee MY , Lottsfeldt JL
Published: October 2019

Transplantation of CE mammary carcinoma into mice has been shown to produce marked neutrophilia. Previous studies in vivo indicated a significant increase in marrow neutrophil production in these mice, but regulatory mechanisms of this neutrophilia have not been well understood. In order to obtain information about neutrophil production mechanisms at the progenitor cell level, the profile of marrow granulocyte-macrophage progenitors in mice with neutrophilia induced by this tumor was quantitatively analyzed by cytochemical staining of in vitro colonies to distinguish colonies of neutrophils (N-colony), macrophages (M-colony), and mixed cells (NM-colony). Cell cycle kinetics of progenitors were studied by in vivo administration of cytocidal drugs. The absolute number of N-colonies in a femur increased significantly and reached three times normal three to four weeks after tumor implantation. The number of NM-colonies also increased significantly by the fourth week, but the number of M-colonies was unchanged. The number of N-colonies in a femur related directly to the degree of neutrophilia. The increased number of N-colonies from the marrow of tumor-bearing mice was not attributed to a different time course of colony growth nor to a different sensitivity to CSA; instead, a significantly larger fraction of neutrophilic progenitors from the tumor-bearing mice were in active cell cycle than were those of normal mice. The day 14 tumor-bearing mouse serum demonstrated N-colony stimulating activity while the sera of normal mice and day 7 tumor- bearing mice were inhibitory for in vitro colony growth. These studies demonstrated an increase in the numbers and turnover rate of marrow neutrophilic progenitors in CE tumor-induced neutrophilia, suggesting that this tumor stimulates proliferation of these progenitors in vivo.
Density ultracentrifugation of sickle cells during and after pain crisis: increased dense echinocytes in crisis
Authors: Warth JA , Rucknagel DL
Published: October 2019

An increase in the number of irreversibly sickled cells (ISCs) in pain crisis has been found by some investigators but not others. We have used the technique of discontinuous arabinogalactan density-gradient ultracentrifugation of whole blood to study ISCs from patients with sickle cell anemia (SCA) during pain crisis and again when pain free (5- 331 days after crisis). Nine patients have been studied through ten episodes of pain crisis. Five layers with densities from 1.128 g/mL to 1.158 g/mL have been used. Careful classification of the cells using Nomarsky optics demonstrated highly significant changes occurring in the layers of the gradient. The changes involve the appearance of an increased percentage of echinocytic ISCs and echinocytic cells that were not ISCs, especially in the denser gradient layers, during crisis, and their replacement by normal-appearing discocytes in the pain-free state. There was no change in ISCs that were not echinocytic. Data collected previously demonstrated that reduced glutathione activity correlated with increased echinocytosis in our gradient layers. This indicates that the echinocytic change may occur as a result of oxidant stress. Hemoglobin F levels and the percentage of hemoglobin F from reticulocytes showed no consistent change to coincide with the rise in normal-appearing discocytes in the lightest layers after crisis. Our data indicate that pain crisis occurs in association with an echinocytic change, which may be induced by oxidant injury. The rise in normal-appearing cells after crisis may reflect increased hemoglobin F production in some patients but mainly relates to the disappearance of these echinocytic erythrocytes.
The effects of tumor-promoting phorbol esters on human granulopoiesis in vitro
Authors: Sullivan R , Brodie RA , Larsen NE , Gans PJ , McCarroll LA
Published: October 2019

In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro. We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA. Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA. However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA. To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short- term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium. Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others. Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.
Filterability of subpopulations of leukocytes: effect of pentoxifylline
Authors: Schmalzer EA , Chien S
Published: October 2019

Human leukocytes were separated by density into two fractions, one containing predominantly granulocytes (FI) and the other, lymphocytes and monocytes (FII). The filterability of these fractions and their mixture was determined from the pressure measured during constant flow through 5-microns Nuclepore filters. The pressure-time curve of FI indicates the behavior of a relatively homogeneous cell population. The FII pressure-time curve can be analyzed to distinguish the effect of the more numerous and more filterable lymphocytes from that of the sparser but less filterable monocytes. Pressure generated by mixed leukocytes, which had been treated with 1 and 10 mmol/L of pentoxifylline (PTX) or its metabolite I, was substantially less than untreated control; at 10 mmol/L, the pressure was reduced to about 50% of control. PTX appears to affect the filterability of monocytes and polymorphonuclear leukocytes but not lymphocytes. Scanning electron microscopy showed an inhibition of protopod formation in the treated granulocytes. The degree of cell adhesion to the filter, as measured by the number of cells remaining on the outflow side of the filter, was similar in all groups (FI and FII, treated and untreated). The results indicate that the variations in filterability between the leukocyte subpopulations and the improvement by PTX treatment reflect differences in the cells' ability to deform under the test conditions.
An objective sign in painful crisis in sickle cell anemia: the concomitant reduction of high density red cells
Authors: Fabry ME , Benjamin L , Lawrence C , Nagel RL
Published: October 2019

The etiopathologic basis of painful crisis in sickle cell anemia is largely unknown, and no objective criteria for its diagnosis and follow- up exist at present. We have studied 11 patients through 14 painful crises and observed a significant decrease of the densest fraction of red cells in 12 of the 14 crises as determined by isopycnic Percoll- Stractan continuous density gradients. If the first observation is normalized to 100%, the average decrease in dense cells was 77% with a range of 36% to 94%. The time needed for the percentage of dense cells to return to the steady-state level varied from seven to more than 30 days. These findings were in sharp contrast to the stability of the density pattern observed in another group of sickle cell patients, who were studied during crisis-free periods. The mechanism of the disappearance of dense cells could involve selective destruction by the reticuloendothelial (RE) system, selective sequestration in the areas of vasoocclusion, or a combination of both factors.
Abnormal VIII: von Willebrand factor patterns in the plasma of patients with the hemolytic-uremic syndrome
Authors: Moake JL , Byrnes JJ , Troll JH , Rudy CK , Weinstein MJ , Colannino NM , Hong SL
Published: October 2019

Plasma VIII:von Willebrand factor antigen (VIII:vWF) levels were elevated approximately two- to eightfold in seven patients (three adults and four children) during acute episodes of thrombocytopenia, renal failure, and hemolytic anemia (the hemolytic-uremic syndrome, HUS). In all seven patients, there was an alteration in plasma VIII:vWF patterns during these acute HUS episodes, so that the largest VIII:vWF forms were relatively decreased. Plasma VIII:vWF multimer patterns returned to normal, or nearly to normal, as platelet counts returned to preexisting levels, even in the patients whose recovery of renal function was incomplete and whose plasma VIII:vWF antigen level remained above normal. The sister of one of the HUS patients had a similar clinical prodrome (gastroenteritis) that was not followed by thrombocytopenia or renal failure and was not accompanied by an elevated level or abnormal forms of plasma VIII:vWF. These results suggest that an alteration in VIII:vWF metabolism, distribution, or interaction with platelets is associated with acute HUS episodes. In contrast to patients with chronic relapsing thrombotic thrombocytopenic purpura, none of the HUS patients (either during or after the acute HUS episodes) had a defect in the conversion of unusually large VIII:vWF multimers derived from endothelial cells to the VIII:vWF forms found in normal plasma.
Bone marrow histologic pattern--the best single prognostic parameter in chronic lymphocytic leukemia: a multivariate survival analysis of 329 cases
Authors: Rozman C , Montserrat E , Rodriguez-Fernandez JM , Ayats R , Vallespi T , Parody R , Rios A , Prados D , Morey M , Gomis F
Published: October 2019

In previous studies, the prognostic value of bone marrow (BM) histologic patterns in chronic lymphocytic leukemia (CLL) has been demonstrated. In order to investigate whether such a value is independent of other prognostic parameters, a multivariate survival analysis (Cox's regression model) was undertaken in a series of 329 CLL patients in whom a BM had been performed. The following binary variables were included in the analysis: age (more than 60 years), lymphadenopathy (more than two areas involved), splenomegaly, hepatomegaly, absolute lymphocyte count (more than 30,000 microL), anemia (hemoglobin less than 10 g/dL), thrombocytopenia (less than 100,000 microL), and BM pattern (diffuse v nondiffuse). Three variables entered the regression at significant level: BM pattern (P less than .001), anemia (P less than .001), and hepatomegaly (P = .03). The model was also tested by expressing the variables in a continuous way when possible. Again, BM pattern entered first in the regression (P less than .001), followed by the hepatomegaly (P = .002), hemoglobin level (P = .02), and lymphadenopathy (P = .04). When both the binary and the continuous models were tested separately in 227 patients with BM as initial staging procedure and in 102 patients in whom this was performed later during the course of the disease, in all instances, BM pattern entered first in the regression at a highly significant level. BM histologic pattern appears to be a better single prognostic parameter than any one of the variables employed in current clinical staging systems. A combined clinicopathologic system incorporating the BM pattern, together with the usual clinical variables, is presented.
Production of T lymphocyte colony-forming units from precursors in human long-term bone marrow cultures
Authors: Touw I , Lowenberg B
Published: October 2019

T cell differentiation in human marrow was studied in Dexter type long- term bone marrow cultures. In these cultures, T lymphocyte colony- forming units (TL-CFU), E rosette-forming cells (E+), and T3+, T4+, and T8+ cells (assayed by indirect immunofluorescence) were found to be present for at least 7 weeks. It was investigated whether the existence of T cells in long-term culture resulted from the persistence of inoculated T lymphocytes or from the production by immature progenitors. No significant numbers of E+, T3+, T4+, or T8+ cells were detected in cultures that were established from E+ lymphocyte-depleted bone marrow, indicating little or no production of T lymphocytes from E- negative precursors. On the other hand, bone marrow cells purged of E+ lymphocytes did not contain TL-CFU, but appeared to regain high numbers of TL-CFU during Dexter culture; this suggested that an earlier step in T cell differentiation may take place in this culture system. The generation of TL-CFU in the E-negative long-term marrow cultures only occurred when an adherent stroma layer had been established in the culture flask; it did not require added mitogens or detectable interleukin 2 in the culture medium. TL-CFU in fresh marrow (TL-CFU II) are mature (E+, T3+) T cells and are capable of producing helper (T4+) and suppressor/cytotoxic (T8+) phenotype cells in colonies. The TL-CFU newly formed in E-depleted Dexter cultures (TL-CFU I) are distinct from this population, as they are E-negative and give rise to colonies of the helper type only. T3 cell depletion of the marrow inoculum prior to culture did not prevent the appearance of TL-CFU I in long-term culture; this suggests that TL-CFU I are derived from an E- and T3- precursor (pre-TL-CFU).
Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow
Authors: Bodger MP , Hann IM , Maclean RF , Beard ME
Published: October 2019

Pluripotent hemopoietic progenitor cells (CFU-GEMM, cells forming mixed hemopoietic colonies in methylcellulose) from human bone marrow were enriched 90-fold by positive selection on the fluorescence-activated cell sorter using monoclonal antibody RFB-1. Bone marrow cells were separated by cell size, using log 90 degrees light scatter, and the cell fraction containing CFU-GEMM was further separated by relative fluorescence intensity for the RFB-1 antigen. Further enrichment, up to 150-fold, was achieved by depleting bone marrow of T cells and mature myeloid cells prior to RFB-1 selection. These procedures yield a cell fraction containing 51% blast cells, 2% promyelocytes, and 47% undifferentiated (lymphocyte-like) mononuclear cells, although only 1% of the cells formed a mixed colony. CFU-GEMM are strongly positive for the RFB-1 antigen, whereas morphologically identifiable erythroblasts, myeloblasts, and promyelocytes are weakly RFB-1+. This suggests that the relative concentration of the RFB-1 antigen on bone marrow cells is inversely related to their maturity. The greatly increased recovery of CFU-GEMM after the separation of bone marrow by log 90 degrees light scatter and the removal of T cells and mature myeloid cells suggested that accessory cells that normally regulate the cloning efficiency of CFU-GEMM were removed.
Enhancement of chemotactic factor-stimulated neutrophil oxidative metabolism by leukotriene B4
Authors: Gay JC , Beckman JK , Brash AR , Oates JA , Lukens JN
Published: October 2019

Leukotriene B4 (LTB4) is a potent primary stimulator of neutrophil chemotaxis, aggregation, and degranulation and induces superoxide production at higher concentrations. In order to determine whether LTB4 modulates neutrophil responses to oxidative stimuli, human neutrophils (PMNs) were incubated with LTB4 prior to stimulation with f-Met-Leu-Phe (fMLP, 10(-7) mol/L), opsonized zymosan (OZ, 250 micrograms/mL), or phorbol myristate acetate (PMA, 32 nmol/L). Superoxide (O2-) production by stimulated PMNs was assessed by the superoxide dismutase-inhibitable reduction of cytochrome c. LTB4 alone did not stimulate O2- production in concentrations below 10(-7) mol/L and had no effect on the O2- assay. In the concentration range of 10(-12) to 10(-8) mol/L, LTB4 did not alter O2- release induced by OZ or PMA. In contrast, LTB4-treated cells demonstrated enhanced O2- production following exposure to fMLP, and in the presence of 10 nmol/LLTB4, generated 180% +/- 41% of O-2 quantities produced by control cells (n = 23). Enhancement was LTB4 dose-dependent, was maximal in the range of 1 to 10 nmol/L LTB4, was not reversed by removal of the lipid from the medium prior to fMLP stimulation, and was not dependent on the presence of Ca++ or Mg++ in the suspending medium. Chemiluminescence of fMLP-stimulated neutrophils was increased to 323% of controls in neutrophils preincubated with 10 nmol/L LTB4. Unlike augmentation of oxidative responses to fMLP seen with other degranulating stimuli, enhancement by LTB4 was not correlated with an increase in 3H-fMLP receptor binding. These results indicate that, in addition to its primary effects on neutrophil function, LTB4 modulates PMN oxidative responses to the chemotactic peptide and, thus, may amplify the release of oxygen metabolites at inflammatory foci.
Abnormal prothrombin crossed-immunoelectrophoresis in patients with lupus inhibitors
Authors: Edson JR , Vogt JM , Hasegawa DK
Published: October 2019

Prothrombin deficiency has been known to occur in association with lupus inhibitors for over 25 years. We studied 21 patients with lupus inhibitors and found that four of five with prothrombin deficiency and ten of 16 with quantitatively normal prothrombin had abnormal prothrombin crossed-immunoelectrophoresis (CIEP) characterized by material moving slower in the first dimension of electrophoresis than normal prothrombin. In two patients with prothrombin deficiency, all prothrombin measured by quantitative assay and all slow-moving material on CIEP were removed by treatment with Staphylococcal protein A (SPA). These patients had free antibody, which bound to normal plasma prothrombin, forming larger amounts of slow-moving material on CIEP. A third patient with prothrombin deficiency had only partial removal of prothrombin after SPA treatment. Two patients with quantitatively normal prothrombin had all slow-moving material on CIEP and about one fourth of the prothrombin by quantitative assay removed by SPA treatment. There was no correlation among the strength of the inhibitor, the presence of a “cofactor effect,” and the prothrombin abnormality. These data suggest that heterogeneous antiprothrombin antibodies, with or without prothrombin deficiency, are present in the majority of patients with lupus inhibitors.
Cleavage and activation of human factor IX by serine proteases
Authors: Enfield DL , Thompson AR
Published: October 2019

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.
Acute monoblastic leukemia in infancy and early childhood: successful treatment with an epipodophyllotoxin
Authors: Odom LF , Gordon EM
Published: October 2019

Four of five infants and young children with acute monoblastic leukemia, a disease that heretofore has been highly refractory to therapy, were successfully treated with sequential infusions of a podophyllin derivative employed as a single agent over a protracted period of time. In three of the five children, monocytic leukemia cutis was present at birth. Treatment was begun in two of them when the disease had progressed to systemic involvement at a few months of age, and in the third when disease was still localized. The other two children were 11 and 18 months of age at initial presentation with widespread disease. Four children are off therapy 11 months, 26 months, 5 years, and 6 years, respectively.
Neutrophil cytoplasts: relationships of superoxide release and calcium pools
Authors: Torres M , Coates TD
Published: October 2019

Activation of human polymorphonuclear neutrophils (PMNs) by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) leads to a transient increase in intracellular level of ionized calcium and an alteration of the plasma membrane permeability. Calcium has been proposed as a second messenger for activation of the PMN. Modulation of intracellular pools of calcium is of importance in the regulation of PMN activation. We have studied the changes in membrane-bound and cytoplasmic calcium in PMN and PMN devoid of granules and nucleus by quantifying changes in chlorotetracycline (CTC) and Quin 2 fluorescence and comparing their relation to O(2) release. Similar to PMN, PMN cytoplasts (PMN-CPs) produce equivalent amounts of O(2) in response to 10(-7) mol/L fMLP. The decrease in CTC fluorescence following fMLP stimulation is not significantly different in PMN-CP (-9.9% +/- 3.7%) from that observed in PMN (-12.7% +/- 2.33%), suggesting that the trigger pool of Ca++ is present in PMN-CPs. Although PMNs show a net increase in free Ca++ as measured by Quin 2, PMN-CPs display a lower sustained rise, which is totally abolished in the absence of external Ca++. PMN-CPs release O(2) efficiently in the absence of external Ca++ when stimulated with 10(-7) mol/L fMLP, whereas PMNs release significantly less O(2) under the same conditions. Our results suggest that a rapid rise in free Ca++, as monitored by Quin 2 fluorescence, is not required for expression of full activation of the oxidase system and release of O(2) from PMN-CPs.
A study of the kinetics of ADP-triggered platelet shape change
Authors: Hantgan RR
Published: October 2019

The rapid transformation of human blood platelets from inert discoid cells to spheroechinocytes that is induced by adenosine diphosphate (ADP) has been followed by right-angle light scattering intensity measurements using a laser light source and a sensitive photomultiplier. Two steps have been observed, and their rate constants have been determined as a function of pH and [ADP] and in the presence and absence of calcium for both platelet-rich plasma and gel-filtered platelets. Both steps are significantly faster in the presence of physiologic levels of calcium. Platelets were fixed prior to and during activation, then examined by phase-contrast and scanning electron microscopy. The light scattering and morphological changes support a model in which, under physiologic conditions of pH, temperature, ionic strength, and calcium concentration, the initial rapid event in platelet shape change is the loss of discoid shape, with a decay time of two to three seconds, leading to an intermediate with short pseudopods. The slower extension of long pseudopods occurs next, with a time constant of approximately seven to eight seconds. These results may help to resolve the contradictory descriptions of the mechanism of platelet shape change that have recently appeared in the literature.
Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma
Authors: Kruithof EK , Tran-Thang C , Ransijn A , Bachmann F
Published: October 2019

This report describes a plasmatic, fast-acting, specific inhibitor (antiactivator) of tissue-type plasminogen activator (t-PA) and urokinase (UK). After addition of t-PA to human plasma, biexponential decay of activity occurred. The initial rapid inhibition of t-PA activity (half-life of approximately one minute) was correlated with the formation of a complex of a molecular weight of 110,000, suggesting a molecular weight in the order of 40,000 for the antiactivator. Diisopropylfluorophosphate (DFP)-inactivated t-PA did not form complexes with antiactivator. The second-order rate constant for the interaction of t-PA with antiactivator is in the order of 10(7) mol/L-1 sec-1. In plasma, UK added at low concentrations rapidly formed complexes of a mol wt of 95,000. Preincubation of the plasma with t-PA prevented complex formation of UK, and vice versa, suggesting that the same inhibitor inactivates both t-PA and UK. After exhaustion of the antiactivator, t-PA and UK formed complexes with alpha 2-antiplasmin and C1′-inhibitor at a low rate.