bluuebunny/crossref_metadata_2025 huggingface dataset:
A new abnormal variant of spectrin in black patients with hereditary elliptocytosis
Authors: Lecomte MC , Dhermy D , Solis C , Ester A , Feo C , Gautero H , Bournier O , Boivin P
Published: October 2019

Seven black patients with mild hereditary elliptocytosis (HE) from five unrelated families were studied. The erythrocytes of these patients exhibited an abnormal thermal sensitivity (between 45 degrees C and 47 degrees C instead of 49 degrees C). An important defect of spectrin dimer self-association was detected in two ways: (1) the proportions of spectrin dimer (SpD) extracted from membranes at 4 degrees C under low ionic strength conditions were increased between 25% and 56% (normal value 15% +/- 2%); (2) the spectrin dimer----tetramer conversion in solution were defective with an association constant value between 0.4 and 2.4 X 10(5) M-1 for a normal value of 6 +/- 0.4 X 10(5) M-1. Spectrin (Sp) from HE patients and normal volunteers (32 black and 22 white subjects) was submitted to limited tryptic digestion, followed by one- or two-dimensional separation of the peptides. Peptide patterns of crude Sp from all seven HE patients exhibited a marked and reproducible decrease in 80,000-dalton peptide (previously identified as the dimer- dimer interaction domain of the alpha-chain) and a concomitant appearance of a novel 65,000-dalton peptide. A minor fragment at 28,000 daltons was also decreased. Tryptic digestion of HE spectrin dimer and tetramer (SpT), isolated after the SpD self-association procedure in solution, revealed modifications (decrease in the 80,000-dalton peptide and presence of a 65,000-dalton peptide) predominantly in HE SpD when peptide patterns of HE SpT were quite similar to control SpT patterns. Immunoblots with anti-alpha-chain antibodies revealed that the 65,000- dalton peptide derived from the alpha-chain. Kinetic studies of Sp digestion showed that the 65,000-dalton peptide did not result from further digestion of a 74,000 intermediate and was not a precursor of 46,000- to 50,000-dalton peptides. These results show a new structural defect of Sp-alpha-chain, associated with a defective Sp dimer self- association in HE.
Preferentially expressed genes in chronic myelogenous leukemia
Authors: Mars WM , Florine DL , Talpaz M , Saunders GF
Published: October 2019

The predominant circulating cells in chronic myelogenous leukemia (CML) morphologically resemble normal myeloid precursors; however, certain characteristics indicate the two are not identical. Approximately 88% of the patients with clinically typical CML present with a cytogenetic abnormality known as the Philadelphia chromosome (Ph1). Additionally, the leukocyte alkaline phosphatase (LAP) value is decreased in CML. To investigate if there are selected genes expressed in the CML cell population, poly(A+)RNA from a chronic-phase, Ph1-positive CML patient was used for construction of a complementary DNA (cDNA) library. Recombinant clones representing moderately to abundantly transcribed sequences were selected by annealing [32P]-cDNA transcribed from homologous RNA to the library sequences and assessing radioactivity in the hybrids. From an initial 729 colonies, 417 (57.2%) displayed a hybridization signal more intense than controls, indicating these recombinant plasmids contained sequences homologous to moderately or highly expressed RNAs from this particular patient. Screening of the 417 clones--utilizing 32P-cDNAs derived from normal human placenta, an acute myelomonocytic leukemia (AMML), and two other CML samples--was used to select clones likely to represent sequences preferentially expressed in CML. Sixteen recombinants were initially selected that repeatedly failed to display hybridization with the placenta and AMML- derived probes. Further analysis of eight of these clones indicated that six contain sequences preferentially expressed in CML. One clone, C-A3, has been studied with 63 different RNA samples. This sequence is found to be highly expressed in peripheral blood cells from the chronic phase of both Ph1-positive and Ph1-negative CML as well as in a Ph1- positive acute myelogenous leukemia (AML). Expression is reduced in lymphoblastic crisis of CML (L BC-CML) and essentially absent in myeloblastic crisis of CML (M BC-CML). While preliminary, the results suggest that this probe may be useful as an aid in diagnosing Ph1- negative CML and in distinguishing M BC-CML from L BC-CML and Ph1- positive AML.
The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation
Authors: McNeely TB , Griffith MJ
Published: October 2019

The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.
Surface membrane expression by human blood leukocytes and platelets of decay-accelerating factor, a regulatory protein of the complement system
Authors: Nicholson-Weller A , March JP , Rosen CE , Spicer DB , Austen KF
Published: October 2019

The decay-accelerating factor (DAF), an integral membrane protein of approximately 75,000 mol wt that regulates the stability of the C3 convertases of the classical and alternative complement pathways, was initially isolated from normal erythrocyte stroma and used to prepare a polyclonal antiserum. Previously, anti-DAF antiserum has been used to immunoprecipitate DAF from surface-labeled normal erythrocytes and to document the deficiency of DAF on the surface of erythrocytes from patients with paroxysmal nocturnal hemoglobinuria, a condition in which erythrocytes express abnormal sensitivity to complement-mediated lysis. DAF has now been demonstrated by cytofluorography with anti-DAF F(ab')2 and fluoresceinated second antibody to be present on the surface of resting polymorphonuclear leukocytes (PMN), monocytes, lymphocytes, and platelets. Populations of PMN, monocytes, and platelets each exhibited a unimodal distribution of fluorescent staining, reflecting uniform cellular expression of DAF antigen, while the lymphocyte population had a skewed pattern of staining, indicating the heterogeneous expression of DAF antigen. For platelets, the shift in mean fluorescence channel observed with cytofluorographic analysis was minimal, but the presence of surface DAF on platelets was demonstrated by specific and saturable anti-DAF F(ab')2 binding. The DAF antigen, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of dithiothreitol- reduced anti-DAF immunoprecipitates prepared from surface-labeled, isolated populations of cells, presented a single polypeptide chain of approximately 84,000 mol wt for PMN and 75,000 to 80,000 mol wt for monocytes, T and B lymphocytes, and platelets. Thus, the complement regulatory protein, DAF, is expressed on the surface of all major types of circulating blood cells from normal donors.
Arachidonic acid incorporation by cytoplasmic lipid bodies of human eosinophils
Authors: Weller PF , Dvorak AM
Published: October 2019

The presence of cytoplasmic lipid bodies in human eosinophils and the participation of these lipid bodies in the metabolism of arachidonic acid by human eosinophils have been studied. Lipid bodies, structures of roughly spherical shape and variable size within the cytoplasm, were identified with transmission electron microscopy by their shape and variable osmiophilia and by their lack of a limiting membrane. While generally absent from eosinophils of normal peripheral blood, lipid bodies were found in tissue eosinophils and in blood eosinophils from patients with eosinophilia. A role for lipid bodies in arachidonic acid metabolism was determined with eosinophils obtained from two eosinophilic patients. After incubation for 30 to 60 minutes with 3H- arachidonic acid, purified eosinophils took up 50% to 98% of the tritium label. By electron microscopic autoradiography, almost all tritium label was localized to lipid bodies. Only 3.6% of the cell- incorporated tritium label was free arachidonic acid, while 5.8% was neutral lipids and 66% was phospholipid. Thus, most of the tritiated arachidonic acid incorporated by human eosinophils was present in esterified form, predominantly as phospholipids, and almost all of the tritiated lipids were localized ultrastructurally to cytoplasmic lipid bodies. These results provide evidence that lipid bodies of human eosinophils have a role in the cellular metabolism of arachidonic acid.
Identification of human megakaryocyte coagulation factor V
Authors: Nichols WL , Gastineau DA , Solberg LA , Mann KG
Published: October 2019

Specific monoclonal and polyclonal antibody reagents and a double antigen indirect immunofluorescence microscopy technique were used to visualize coagulation factor V in human bone marrow. Marrow aspirates were smeared directly on glass slides, or washed and cytospun onto glass slides, or processed and plated into a plasma/methylcellulose cell culture system. Morphologically identifiable colonies of megakaryocytes, erythrocytes, granulocytes, or monocytes/macrophages were removed from 14- to 18-day marrow culture dishes by micropipette and streaked onto glass slides. Smears of marrow cell preparations were air-dried, fixed, washed, and incubated sequentially with primary IgG antibody reagents and with secondary anti-IgG antibody reagents conjugated with either fluorescein or rhodamine. Preparations were examined and photographed through a microscope suitably equipped for two-color fluorescence and phase contrast analysis. Cells of megakaryocytic lineage were identified by their immunofluorescent reactivity with murine monoclonal antibody HP1–1D, specific for human platelet plasma membrane glycoprotein IIb/IIIa (GP IIb/IIIa), or by their immunofluorescent reactivity with monoclonal or polyclonal antibodies specific for von Willebrand factor (vWF) or for platelet factor 4 (PF4). Coagulation factor V in bone marrow was detected by simultaneous immunofluorescent staining with polyclonal burro anti- human factor V antibody or with a panel of murine monoclonal anti-human factor V antibodies. The double antigen immunofluorescence staining technique, incorporating appropriate controls, revealed that coagulation factor V was principally located in marrow cells simultaneously identified as megakaryocytes by antibodies to GP IIb/IIIa, vWF, or PF4. The specific immunofluorescence of factor V in megakaryocytes and platelets was eliminated when excess purified factor V antigen was preincubated with anti-factor V antibody. Our observations establish the presence of human megakaryocyte coagulation factor V, confirm the presence of human platelet factor V, and indicate that human megakaryocyte/platelet coagulation factor V is a lineage- associated protein.
Incidence of aplastic anemia in metropolitan Baltimore: a population- based study
Authors: Szklo M , Sensenbrenner L , Markowitz J , Weida S , Warm S , Linet M
Published: October 2019

Incidence rates for aplastic anemia (AA) so far have been unavailable for defined populations in the United States. A study was carried out in the Baltimore Standard Metropolitan Statistical Area (SMSA) to examine the incidence rates for aplastic anemia from 1970 through 1978. Cases of AA (N = 118) were identified through medical records (N = 77) in SMSA hospitals and death certificates (N = 41). All medical charts were reviewed by an oncologist-hematologist for validation purposes. Among whites, average annual age-adjusted incidence rates per million were higher in males (7.1) than in females (5.4), whereas in nonwhites, females had a higher rate (7.3) than males (4.7). None of the sex differences was statistically significant. Age-specific incidence rates were consistently low in young ages, with an exponential increase after age 40. Examination of time trends did not suggest changes in whites, although in blacks, mainly in males, there was a suggestion of a temporal increase. However, rates in blacks were based on small numbers, and trends were not statistically significant. An inconsistent sex differential, as well as the relative stability of rates over time at least in whites, suggests that although sexes may have different types of exposures, occupational exposures and changes in environmental factors over time cannot entirely explain the occurrence of AA in the population. In addition, the age pattern suggests that future studies should examine etiologic agents separately for the younger and the older subjects with AA.
The role of neutrophil membrane glycoprotein GP-150 in neutrophil adherence to endothelium in vitro
Authors: Harlan JM , Killen PD , Senecal FM , Schwartz BR , Yee EK , Taylor RF , Beatty PG , Price TH , Ochs HD
Published: October 2019

We have previously described two patients with a congenital defect in neutrophil function characterized by an inability to form pus. The patients' neutrophils lack a membrane glycoprotein of mol wt 150,000 daltons (GP-150) on analysis by SDS-PAGE. This glycoprotein is part of a membrane antigen complex recognized by the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produces defects in chemotaxis and phagocytosis in vitro similar to those observed in the patients. Since neutrophil adherence to vascular endothelium is prerequisite to neutrophil emigration in vivo, we examined the interaction of the patients' neutrophils and normal neutrophils treated with MoAb 60.3 with cultured endothelium. Adherence was determined as the percentage of 51Cr-labeled purified peripheral blood neutrophils which remained adherent to plastic wells or endothelial monolayers after a 45-minute incubation at 37 degrees C. The percentage of neutrophils from patient 1 remaining adherent to uncoated, fibronectin-coated, or laminin-coated plastic was similar to that observed in normal neutrophils (55% to 84% adherence with normal neutrophils v 73% to 78% adherence with the patient's neutrophils and 63% to 82% adherence with MoAb 60.3-treated normal neutrophils). The adherence of the neutrophils from patient 1 and MoAb 60.3-treated normal neutrophils to human or bovine endothelium in serum-free medium was also not significantly different from that observed in normal neutrophils (less than 10% adherence with normal, MoAb 60.3-treated, and patient neutrophils). In medium containing 10% autologous or heterologous human plasma, however, the adherence of neutrophils from patient 1 or MoAb 60.3-treated normal neutrophils to endothelial monolayers was significantly reduced (35% +/- 7% of normal neutrophils in seven experiments). Although phorbol myristate acetate (PMA) (10 ng/mL) and calcium ionophore A23187 (10(-5) mol/L) markedly increased the adherence of normal neutrophils to endothelial monolayers in serum- free medium (40% to 85% adherence), neither agent increased the adherence of the neutrophils from patient 1 or normal neutrophils treated with MoAb 60.3 (less than 5% adherence). The adherence of PMA- activated neutrophils from patient 2 to endothelial monolayers was also markedly decreased when compared with that of normal neutrophils. Postsecretory cell-free supernatants from PMA-activated normal neutrophils failed to augment adherence of neutrophils from patient 1 (less than 5% adherence).(ABSTRACT TRUNCATED AT 400 WORDS)
Do androgens enhance the response to antithymocyte globulin in patients with aplastic anemia? A prospective randomized trial
Authors: Champlin RE , Ho WG , Feig SA , Winston DJ , Lenarsky C , Gale RP
Published: October 2019

We analyzed the effect of antithymocyte globulin (ATG) with or without androgens in 121 patients with aplastic anemia. Fifty-three patients with moderate to severe aplastic anemia were prospectively randomized to receive ATG with or without oral androgens. Eleven of 26 patients (42%) receiving ATG plus androgen responded, including three complete and eight partial responses. Twelve of 27 patients (44%) receiving ATG plus placebo responded, including five complete and seven partial responses. The difference in response rates was not significant (P greater than .9). Survival was also comparable in the two groups; for patients with severe aplastic anemia, actuarial survival at two years was 55% +/- 24% (95% confidence interval) in patients receiving ATG plus androgen compared with 50% +/- 24% in the ATG plus placebo group (P = .65). Furthermore, results in both groups were indistinguishable from those obtained in 68 historical controls receiving ATG without androgens. These data indicate that androgens are not required in order to respond to antithymocyte globulin and the addition of androgens, as used in this trial, did not significantly improve response rates to ATG treatment.
Effect of heparin on the inactivation rate of human activated factor XII by antithrombin III
Authors: Pixley RA , Schapira M , Colman RW
Published: October 2019

Human antithrombin III (ATIII) is a plasma inhibitor of several serine proteases of the blood coagulation system. Previous investigations have reported that the presence of heparin has a multifold accelerating effect on the inhibition of factor XIIa and XIIf, the active species derived from factor XII. Recent studies from our laboratories have confirmed that ATIII inactivates factor XIIa and factor XIIf, but only contributes 2% to 3% to the inhibition of activated factor XII species in plasma. The major inhibitor is C1 inhibitor. Therefore, we have reexamined the heparin effect on the rate of inhibition of factor XIIa and factor XIIf in purified systems. We also have studied the effect of heparin on the inactivation of both factor XII-derived active species by various plasmas. Using purified factor XIIa and ATIII, we found that heparin (0.7 to 34.0 U/mL) increased the rate of inhibition of Factor XIIa. However, at heparin concentrations usually achieved during anticoagulant therapy (0.7 U/mL), the inhibition was accelerated only fourfold. This implies only a 6% contribution to the inhibitory effect of plasma. This suggestion was confirmed by the observation that heparin (1.5 U/mL) added to factor XII-deficient plasma and reconstituted with factor XIIa did not produce a detectable enhancement of the rate of inhibition of factor XIIa. Furthermore, using purified factor XIIf and antithrombin III, heparin (3.6 to 57.2 U/mL) increased the inactivation rate constant of factor XIIf by 1.6 to 14.0 times. This small effect was confirmed by the observation that heparin at a concentration greater than that sufficient for anticoagulation (1.4 U/mL) did not modify the inactivation rate of factor XIIf by prekallikrein-deficient plasma, and thus C1 inhibitor remains the major inhibitor even in the presence of heparin. From this study and our previous investigations on the effect of heparin on the inhibition of kallikrein and factor XIa, we conclude that heparin does not significantly affect the protease activity of purified contact activation factors or the activities expressed by these proteases in plasma.
High-dose methotrexate with leucovorin rescue: effectiveness in relapsed hairy cell leukemia
Authors: Joosten P , Hagenbeek A , Lowenberg B , Sizoo W
Published: October 2019

Six patients with relapsed hairy cell leukemia after splenectomy were treated with high-dose methotrexate and leucovorin rescue. Five patients had objective responses as determined by improved blood counts. In two of them, the response has continued for more than 14 and 44 months, respectively. Intensive treatment with high-dose methotrexate and leucovorin rescue was well tolerated and no significant myelosuppression was seen.
Variable breakpoints on the Philadelphia chromosome in chronic myelogenous leukemia
Authors: Leibowitz D , Schaefer-Rego K , Popenoe DW , Mears JG , Bank A
Published: October 2019

The abl oncogene is translocated from chromosome 9 to 22 in the creation of the Philadelphia (Ph1) chromosome. This article describes new translocation breakpoints identified in two patients with chronic myelogenous leukemia using Southern blotting and cloned human DNA probes from chromosome 9. The translocation breakpoints on chromosome 9 in both of these patients lie closer to the human cellular abl (c-abl) gene, and the chromosome 22 breakpoints are distributed more widely than previously reported. These data help to define more clearly the chromosomal span of the breakpoints and indicate that some translocations include very little chromosome 9 sequence located 5′ to the c-abl gene.
Serial studies of protein C and its plasma inhibitor in patients with disseminated intravascular coagulation
Authors: Marlar RA , Endres-Brooks J , Miller C
Published: October 2019

This study was undertaken to determine the levels of protein C antigen and activity and protein C inhibitor in sequential plasma samples of disseminated intravascular coagulation (DIC) patients. Our normal range for both protein C antigen and activity is 70 to 130 U/dL, and protein C inhibitor is 65 to 135 U/dL. A decreased level of protein C activity was found in 96% of the plasma samples from individuals with DIC; the protein C antigen was decreased in 73%. The inhibitor of protein C was decreased in all samples. Analysis of serial samples from patients with DIC reveals that protein C activity and antigen and protein C inhibitor decrease progressively during the initial stages of DIC and remain at a low level for 24 to 48 hours before gradually returning toward normal in nonfatal cases. The protein C activity decreases in parallel with protein C inhibitor and is lower than protein C antigen. In a fatal case of DIC, protein C activity and protein C inhibitor rapidly decreased to undetectable levels; however, protein C antigen was gradually decreasing but still detectable at time of death. In DIC, a discrepancy initially occurs between the activity and antigen of protein C, suggesting a complex with the inhibitor or other inactive forms of protein C. Protein C appears to play a major role in the control of DIC.
PAF-acether-induced release of tissue-type plasminogen activator from vessel walls
Authors: Emeis JJ , Kluft C
Published: October 2019

Platelet-activating factor (PAF-acether; 1–0-octadecyl-2-acetyl-sn- glyceryl-3-phosphorylcholine) induced the release of plasminogen activator in rat, both in vivo and in perfused hind legs. The released plasminogen activator was shown by immunologic and functional criteria to be tissue-type plasminogen activator (t-PA). Release of t-PA by PAF- acether could be inhibited by phospholipase inhibitors and by lipoxygenase inhibitors, but not by cyclooxygenase inhibitors. It is suggested that PAF-acether induces the release of t-PA from vascular endothelial cells by the (calcium-dependent) activation of a phospholipase-lipoxygenase pathway.
Duration of the effect of aspirin on the synthesis of thromboxane by density subpopulations of rabbit platelets stimulated with thrombin
Authors: Packham MA , Guccione MA , O'Brien KM
Published: October 2019

The controversy concerning the relationship between platelet buoyant density and platelet age is unresolved. Our earlier results with rabbit platelets indicate that the most-dense subpopulations are enriched in young platelets and that some platelets become less dense as they age. Other investigators have concluded that platelets either do not change in density upon aging or become more dense. In the present experiments, rabbit platelets were separated on discontinuous gradients of Stractan. Most-dense platelets synthesized significantly more thromboxane B2 (TXB2) (1.27 ng per 10(6) platelets) in response to thrombin (0.75 U/mL) than did least-dense platelets (0.70 ng per 10(6) platelets), indicating that the arachidonate pathway in most-dense platelets is more active than in least-dense platelets. After aspirin administration to rabbits, most-dense platelets recovered their ability to synthesize thromboxane B2 significantly more quickly than did least-dense platelets. Because the platelet cyclooxygenase that is responsible for TXB2 formation is permanently inhibited by aspirin, it is only the new platelets entering the circulation that will be able to form TXB2. These results indicate that, at least in rabbits, the most-dense platelets are enriched in young platelets, and that platelets decrease in density as they age in the circulation.
Drug-dependent and non-drug-dependent antiplatelet antibody in drug- induced immunologic thrombocytopenic purpura
Authors: Lerner W , Caruso R , Faig D , Karpatkin S
Published: October 2019

The mechanism of drug-dependent immunologic thrombocytopenic purpura (DITP) was investigated by studying the sera of four patients with classic DITP (two with quinidine-, one with acetaminophen-, and one with phenazopyridine-dependent antiplatelet antibody) using a solid- phase radioimmunoassay with 125I-staphylococcal protein A. Two forms of antiplatelet antibody could be demonstrated: one that required drug to bind to platelets and one that bound to platelets in the absence of drug. Drug-dependent antiplatelet antibody required the simultaneous addition of drug and the Fc domain of the drug-dependent IgG molecule for binding to platelets. It did not require serum complement or factor VIII-related antigen for binding to platelets. Drug-dependent binding of antibody to platelets was saturation-dependent. Non-drug-dependent antiplatelet antibody of two patients (one with quinidine-induced thrombocytopenia and the other with acetaminophen-induced thrombocytopenia) reacted with autologous platelets as well as with homologous platelets, indicating that they were autoantibodies. Both autoantibodies had disappeared when their sera were tested 23 and 138 days, respectively, after withdrawal of their initial positive sera. Non-drug-dependent antiplatelet antibody binding could be demonstrated with the F(ab')2 fragment of the purified IgG of the serum of the second patient with quinidine DITP, who did not have detectable alloantibodies against HLA. None of the four patients with non-drug- dependent antiplatelet antibody had a past or present history of autoimmune thrombocytopenic purpura.
Heterogeneity of human peripheral blood eosinophil-type colonies: evidence for a common basophil-eosinophil progenitor
Authors: Denburg JA , Telizyn S , Messner H , Lim B , Jamal N , Ackerman SJ , Gleich GJ , Bienenstock J
Published: October 2019

We have recently shown that a proportion of previously designated human eosinophil “(Eo)-type” colonies in methylcellulose contain basophils and histamine (Denburg et al Blood 61:775, 1983). In the present studies, individual Eo-type colonies have been analyzed by cell morphology as well as by biochemical assays for histamine, Charcot- Leyden crystal protein (CLC), and eosinophil granule major basic protein (MBP). Clonal origin of single Eo-type colonies was confirmed by G6PD isoenzyme analysis. Morphological observations of such colonies revealed the existence of two distinct colony types: (1) Eo type containing 100% basophils and (2) Eo type containing mixtures of basophils and eosinophils, including cells with mixed basophil- eosinophil granulation. Histamine was not detected in pure, mature peripheral blood eosinophils. Immunofluorescent studies demonstrated bright staining for CLC and MBP in 95% +/- 3% of cells in Eo-type colonies but only in 5% +/- 4% of cells in GM-type colonies. Radioimmunoassay for MBP was positive in 5/9 Eo-type and 0/10 neutrophil-macrophage (“GM-type”) colonies, with a mean level (nanogram/colony) of 11.6 +/- 4.2 per Eo-type colony; four of the latter colonies were doubly positive for both histamine and MBP. These and previous findings point out the morphological and biochemical heterogeneity of peripheral blood Eo-type colonies and provide direct evidence for the existence of a common, circulating basophil-eosinophil progenitor.
Constitutive production of leukemia differentiation, colony- stimulating, erythroid burst-promoting, and pluripoietic factors by a human hepatoma cell line: characterization of the leukemia differentiation factor
Authors: Gabrilove JL , Welte K , Lu L , Castro-Malaspina H , Moore MA
Published: October 2019

Conditioned medium (CM) obtained from a human hepatoma cell line, SK- HEP-1, contains colony-stimulating factors (CSFs) active on murine and human bone marrow-derived granulocyte and macrophage colony-forming units (CFU-GM) and a factor capable of inducing granulocyte-macrophage differentiation (GM-DF) of murine myelomonocytic leukemic cells WEHI- 3B(D+) and human promyelocytic leukemic cells HL-60 when assayed in semisolid agar cultures. The human active granulocyte-macrophage colony- stimulating factor (GM-CSF) for day 7 CFU-GM and the GM-DF for WEHI- 3B(D+) and for HL-60 are not separable by acrylamide agarose column chromatography, eluting at an apparent molecular weight between 20,000 and 35,000 daltons, or by isoelectric focusing (isoelectric point, pH 5.4). In addition, SK-HEP-1 CM contains erythroid burst-promoting activity (BPA) and a factor that promotes the growth of human mixed colonies. SK-HEP-1 cells, which grow as an adherent monolayer, appear not to be endothelial or monocytic in origin since by immunofluorescent staining they are negative for Ia (HLA-DR), monocyte antigen 1 and 2, lysozyme, and factor VIII-related antigen. Positive immunofluorescent staining for keratin and fibronectin suggests the possibility that SK- HEP-1 is an epithelial cell line. Constitutive production of GM-DF as well as other hematopoietic activities including GM-CSF, erythroid BPA, and an activity that promotes the growth of human mixed colony progenitors by a human epithelial tumor cell line, SK-HEP-1, suggests that this cell line is a valuable resource for both large-scale production of these factors and the cloning of the gene(s) that code for these regulators.
Evaluation of covalent antisickling compounds by PO2 scan ektacytometry
Authors: Johnson RM , Feo CJ , Nossal M , Dobo I
Published: October 2019

The ektacytometer, a device to measure erythrocyte flexibility, has been used to evaluate antisickling agents that covalently modify hemoglobin S (HbS). The instrument has been adapted to produce a continuous gradient of oxygen pressure in the measuring cuvette, which permitted the rapid determination of sickle cell rigidity over the complete oxygenation range. Inspection of curves allows classification of the compounds according to their mode of action: altering oxygen affinity or increasing deoxy-HbS solubility. Reagents that modify amino groups, thiols, and histidine, as well as a crosslinking agent, were examined. The method directly evaluates deoxygenated cell deformability rather than cell shape. Many of the compounds that are effective in preventing the morphological sickling of deoxygenated sickle cells do not necessarily restore cell deformability. The method also readily detects membrane damage brought about by covalent agents that nonspecifically derivatize membrane proteins. Cystamine and pyridoxal appear to improve deformability in deoxygenated SS cells at concentrations that do not damage the membrane. This method, which examines the intact cell, fills a gap in the available experimental techniques for drug evaluation, between studies of isolated hemoglobin and in vivo studies.
In vitro culture of leukemic cells in t(4;11) acute leukemia
Authors: Stong RC , Kersey JH
Published: October 2019

In the present study we utilized a semisolid culture system with feeder cells and enriched media to evaluate the growth of acute leukemia associated with the 4;11 chromosomal translocation. We compared growth of t(4;11) leukemia to typical acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). The two cases of t(4;11) leukemia tested exhibited the highest cloning efficiency of cells tested. The growth characteristics of t(4;11) leukemia were more similar to ANL than ALL.
Phenotypic and functional analysis of B cell lines from patients with multiple myeloma
Authors: Goldstein M , Hoxie J , Zembryki D , Matthews D , Levinson AI
Published: October 2019

We characterized phenotypic and functional properties of B cell lines obtained from patients with multiple myeloma to determine how well they conform to particular stages of B cell differentiation. This information is a prerequisite for using such lines as tools for studying B cell growth and the regulation thereof. Two lines, GM1312 and GM1500, expressed B1 and Ia, determinants on early B cells, but expressed little, if any, T10, a determinant expressed on plasma cells. By contrast, B1 and Ia were poorly expressed on two other lines, GM2132 and U266. T10 was expressed on GM2132 but not on U266. Using a reverse hemolytic plaque assay, we also assessed the numbers of cells actively secreting immunoglobulin (IgSCs) in such cultures to provide a functional marker of B cell differentiation. We observed consistently higher numbers of IgSCs in cultures of GM2132 than in GM1500 and GM1312. These phenotypic and functional markers were stable over several months. The data suggest that such cell lines represent early (GM1312, GM1500) and later stages (GM2132, U266) of B cell differentiation, although all lines were derived from patients with multiple myeloma.
Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells
Authors: Zipori D , Toledo J , von der Mark K
Published: October 2019

Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.
Growth and cytogenetic characteristics of bone marrow colonies from patients with 5q-syndrome
Authors: Carbonell F , Heimpel H , Kubanek B , Fliedner TM
Published: October 2019

Early erythroid precursor cells and myeloid progenitor cells (CFU-GM) from four patients with 5q-syndrome were cultured in order to study the in vitro growth patterns and to determine the clonal origin of this hematologic disorder. Cultures of CFU-GM exhibited normal colony growth, while erythroid progenitor cells demonstrated a marked decrease or absence of colony growth. Chromosomal studies indicate the 5q- chromosome is present in both hematopoietic progenitor cells, suggesting that the syndrome is an acquired clonal disease arising from a pluripotent hematopoietic stem cell. Follow-up cytogenetic studies reveal a decrease in the number of normal metaphases. This finding is consistent with reports that emphasize the slowly progressive nature of this malignant stem cell defect.
Establishment of two new myeloma cell lines from bilateral pleural effusions: evidence for sequential in vivo clonal change
Authors: Durie BG , Vela E , Baum V , Leibovitz A , Payne CM , Richter LC , Grogan TM , Trent JM
Published: October 2019

Two new human myeloma cell lines have been established from a 36-year- old woman with refractory IgG kappa multiple myeloma in whom bilateral malignant pleural effusions developed. The malignant plasma cells from each effusion were set up in a liquid culture using an L-15 medium containing catalase, glutathione, selenous acid, ascorbic acid, insulin, transferrin, additional glutamine hydrocortisone, and 2- mercaptoethanol and designated as M-3 medium. Two IgG kappa cell lines, LB -831 and LB-832, were established and proved to be Epstein-Barr virus negative using the internal repeat sequence DNA probe. Characteristic plasma cell morphology was evident by light and electron microscopy. Immunotyping revealed an IgG kappa , B1+, B2-, Ia (HLA- DR)+, CALLA+ phenotype for each cell line as well as for the original pleural fluid and bone marrow myeloma cells. The supernatants also contained IgG kappa, beta 2 microglobulin, and large amounts of osteoclast-activating factor (indicating bone-resorbing activity). Cytogenetic analysis of the LB-831 cell line revealed a nearly triploid highly abnormal karyotype with numerous clonal chromosomal abnormalities involving chromosomes 1, 3, 5, 7, 13, and 15; several structurally abnormal marker chromosomes; and a putative homogeneously staining region on chromosome 7p at band p22. Analysis of the LB-832 cell line revealed several additional clonal abnormalities. These additional cytogenetic changes suggest that in vivo sequential clonal evolution occurred in this patient. Therefore, two new but related cell lines have been established, which should prove useful for further biological studies.
Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation
Authors: Stong RC , Uckun F , Youle RJ , Kersey JH , Vallera DA
Published: October 2019

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.
Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme- linked differential antibody immunosorbent assay
Authors: Kaplan AP , Gruber B , Harpel PC
Published: October 2019

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF- deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF- deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time- dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.
Aggressive non-Hodgkin's lymphomas in immunocompromised homosexual males
Authors: Kalter SP , Riggs SA , Cabanillas F , Butler JJ , Hagemeister FB , Mansell PW , Newell GR , Velasquez WS , Salvador P , Barlogie B
Published: October 2019

During the period from 1981 through 1984, 14 immunocompromised homosexual males with intermediate or high-grade non-Hodgkin's lymphoma were seen at University of Texas M.D. Anderson Hospital and Tumor Institute. Six patients had diffuse large-cell lymphoma, seven had diffuse undifferentiated lymphoma, and one had unclassifiable lymphoma that suggested large-cell lymphoma. Eight patients had the acquired immunodeficiency syndrome (AIDS) and five had the AIDS-related complex. Kaposi's sarcoma was initially present in four patients and developed later in two others. The patients with diffuse large-cell lymphoma were characterized by more severely altered immune parameters, multicentric brain mass lesions, pretherapy opportunistic infections, lower performance status, poor response to therapy, and death in all within six months. The undifferentiated lymphoma group had preceding generalized reactive lymphadenopathy, less severe immune dysfunction, and excellent response to combination chemotherapy, with survival time greater than 19 months in three patients. Twelve of the patients had extranodal sites of lymphoma at presentation. There is a definite trend for the development of aggressive non-Hodgkin's lymphomas with unusual sites of extranodal involvement in immunocompromised homosexual males, with the potential for good tolerance to combination chemotherapy and improved survival in the subgroup without severe concomitant opportunistic infections.
Effects of dibutyryl adenosine 3',5'-cyclic monophosphate on erythropoietin production in human renal carcinoma cell cultures
Authors: Hagiwara M , Pincus SM , Chen IL , Beckman BS , Fisher JW
Published: October 2019

A human renal carcinoma from a patient with erythrocytosis, serially transplanted into athymic nude mice, was grown in primary monolayer cell cultures. After reaching confluency, the cultured cells formed multicellular hemicysts (domes), which became more abundant as the cultures approached saturation density. Erythropoietin (Ep) production by this renal carcinoma in culture was only slightly increased at the time of semiconfluency but showed a marked increase after the cultures reached confluency, in parallel with dome formation. Dibutyryl adenosine 3′,5′-cyclic monophosphate significantly (P less than .01) stimulated Ep production and dome formation in the semiconfluent and confluent cultures of the renal carcinoma.
Heterogeneity of type I von Willebrand disease: evidence for a subgroup with an abnormal von Willebrand factor
Authors: Mannucci PM , Lombardi R , Bader R , Vianello L , Federici AB , Solinas S , Mazzucconi MG , Mariani G
Published: October 2019

Type I von Willebrand disease (vWD) is characterized by equally low plasma concentrations of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RiCof) and by the presence of all vWF multimers in sodium dodecyl sulfate (SDS)-agarose gel electrophoresis. For 17 patients (13 kindreds) diagnosed with these criteria, we have studied the platelet contents of vWF:Ag and RiCof and the changes of these in plasma after DDAVP infusion. Platelet vWF:Ag and RiCof were normal in four kindreds (called “platelet normal” subgroup); following 1-deamino- 8-D-arginine vasopressin; plasma vWF:Ag, RiCof and the bleeding time (BT) became normal. In six kindreds, platelet vWF:Ag and RiCof were equally low (platelet low); after DDAVP, plasma vWF:Ag and RiCof remained low, and the BT was prolonged. In three additional kindreds, platelets contained normal concentrations of vWF:Ag, but RiCof was very low (platelet discordant); even though a complete set of multimers was found in plasma and platelets, there was a relatively small amount of large multimers. After DDAVP, plasma vWF:Ag became normal, but RiCof remained low and the BT was very prolonged. These findings demonstrated that there can be an abnormal vWF (RiCof less than vWF:Ag) even in type I vWD, coexisting with a complete set of vWF multimers (platelet discordant); that the abnormal vWF can be shown more clearly in platelets than in plasma or else in plasma after DDAVP infusion; and that DDAVP normalizes the BT only in those patients with normal platelet levels of both vWF:Ag and RiCof (platelet normal).