bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel missense mutation in exon 4 of the factor VIII:C gene resulting in moderately severe hemophilia A
Authors: Chan V , Chan TK , Tong TM , Todd D
Published: October 2019

A new point mutation due to C----T transition at codon 189 (TCA) of the factor VIII:C gene was found in a Chinese patient with moderately severe hemophilia A. This mutation abolishes the EcoRI site (GAATTC) in exon 4 and can be directly detected by polyacrylamide gel electrophoresis of amplified genomic DNA.
Clot lysis induced by a monoclonal antibody against alpha 2-plasmin inhibitor
Authors: Sakata Y , Eguchi Y , Mimuro J , Matsuda M , Sumi Y
Published: October 2019

A monoclonal antibody (MoAb) to alpha 2-plasmin inhibitor designated JTPI-1 inhibited antiplasmin activity by interfering with formation of alpha 2-plasmin inhibitor (alpha 2-PI)-plasmin complex. With this MoAb, we observed plasma clot lysis in vitro and evaluated the potential of JTPI-1 to serve as a new therapeutic agent for thrombolysis. After adding 125I-labeled fibrinogen to plasma, clots were made by adding thrombin and calcium and were then resuspended in normal plasma containing various concentrations of JTPI-1. The presence of JTPI-1 enhanced release of the soluble 125I-labeled fibrin degradation fragment from the clots in a dose-dependent manner. With tissue plasminogen activator (t-PA)-depleted plasma, we showed that induction of clot lysis by JTPI-1 was dependent on fibrin-bound endogenous t-PA. Regulation of fibrinolysis initiated on the fibrin surface by fibrin- bound t-PA and plasminogen is mediated by alpha 2-PI cross-linked to fibrin by activated factor XIII. JTPI-1 bound to this cross-linked alpha 2-PI neutralized its activity and induced partial digestion of fibrin by plasmin. This resulted in additional binding of Glu- plasminogen to fibrin during the incubation. When 1.2 mumol/L JTPI-1 and 5 U/mL exogenous t-PA were present in the suspending plasma, the rate of clot lysis was essentially the same as that induced by 60 U/mL exogenous t-PA alone. These results suggest that JTPI-1 may be useful in reducing the amount of t-PA administered for thrombolytic therapy.
SUP-HD1: a new Hodgkin's disease-derived cell line with lymphoid features produces interferon-gamma [see comments]
Authors: Naumovski L , Utz PJ , Bergstrom SK , Morgan R , Molina A , Toole JJ , Glader BE , McFall P , Weiss LM , Warnke R
Published: October 2019

A new cell line, SUP-HD1, was established from the pleural effusion of a patient with nodular sclerosing Hodgkin's disease (NSHD). The SUP-HD1 cells had the characteristic morphology of Reed-Sternberg cells and contained acid phosphatase and nonspecific esterase. The cells lacked the Epstein-Barr virus (EBV) genome and reacted with monoclonal antibodies (MoAbs) against CD15 (Leu-M1), CD25 (Tac), CD71 (OKT9), Ki67, and HLA-Dr. However, the SUP-HD1 cells were nonreactive with MoAbs that specifically identify T lymphocytes, B lymphocytes, and macrophage/myeloid cells. Karyotype analysis of the cell line showed clonal abnormalities involving 1p13, 7p15, 8q22, and 11q23, chromosomal locations, at which breakpoints have been reported in HD. Southern blot analysis demonstrated rearrangement of the immunoglobulin heavy chain and kappa light chain genes as well as the gene for the beta chain of the T-cell receptor (TCR). Transcriptional analysis showed expression of RNAs for kappa light chain, interferon-gamma (IFN-gamma), and interleukin-2 receptor (IL-2R) but not IL-2. The SUP-HD1 cells lacked cytoplasmic and surface immunoglobulin heavy chain, but a small amount of cytoplasmic kappa light chain was detected. The presence of nuclear factor kappa B (NF kappa B), a B-lymphocyte-associated transcription factor, was demonstrated in stimulated and unstimulated cells. In addition, the SUP-HD1 cell line, produced IFN-gamma, a T-lymphocyte- associated lymphokine. Based on these data, the SUP-HD1 cells appear to be aberrant lymphocytes with characteristics of both activated B and T lymphocytes. Elaboration of lymphokines such as IFN-gamma by the malignant cells may represent one explanation for the unique clinical and pathologic features of HD.
Intact transferrin receptors in human plasma and their relation to erythropoiesis
Authors: Huebers HA , Beguin Y , Pootrakul P , Einspahr D , Finch CA
Published: October 2019

Intact transferrin receptor molecules complexed with transferrin were found in human plasma. The concentration of receptors was determined by an enzyme-linked immunosorbent assay that uses polyclonal antibodies. The mean concentration of 8,279 micrograms/L in 56 normal adults appears to be unrelated to age or sex. Additional receptor measurements were performed on plasmas from 260 subjects with erythropoietic disorders. Decreased concentration of plasma receptors was found in patients with erythroid hypoplasia and increased numbers in those with erythroid hyperplasia. Ferrokinetic measurements of erythropoiesis were compared with numbers of receptors in 148 subjects, and a close correlation was found (r = .86). Both sets of values, measured in different conditions and expressed in relation to normal, were consistent with expected values. Receptor values were unproportionally increased only in conditions of iron deficiency. It is concluded that plasma receptors have a constant relationship to tissue receptors, and their number in most instances reflects the rate of erythropoiesis.
Cabbage seed protease inhibitor: a slow, tight-binding inhibitor of trypsin with activity toward thrombin, activated Stuart factor (factor Xa), activated Hageman factor (factor XIIa), and plasmin
Authors: Carter TH , Everson BA , Ratnoff OD
Published: October 2019

An inhibitor of procoagulant and fibrinolytic enzymes was derived from cabbage seeds by a procedure using acetone precipitation, ion-exchange chromatography, and gel filtration. The cabbage seed inhibitor was a 10- Kd monomeric protein with intrachain disulfide bonds. This preparation prevented clot formation in whole blood and blocked the ability of thrombin to induce clot formation in plasma and to induce platelet aggregation. A number of proteases were inhibited, as demonstrated by using purified enzymes in amidolytic assays. Tight-binding inhibition was observed for activated Stuart factor (factor Xa) and plasmin. Inhibition of thrombin and activated Hageman factor (factor XIIa) was observed with a molar excess of inhibitor. No inhibition was detected for activated plasma thromboplastin antecedent (factor XIa), plasma kallikrein, or C1 esterase. Reaction progress curves for trypsin indicated slow, tight-binding inhibition, with an apparent inhibition constant in the nanomolar range or less. The electrophoretic mobility of trypsin was altered by the inhibitor in nondenaturing polyacrylamide gel electrophoresis (PAGE) but not in sodium dodecyl sulfate (SDS)- PAGE, indicating noncovalent bonding. Only partial reversal of trypsin inhibition could be demonstrated by washing the inhibitor from enzyme immobilized on solid beads. A dot-blot technique with cabbage seed inhibitor was capable of detecting 10 ng nitrocellulose-bound trypsin. The dot-blot technique also appeared capable of detecting plasmin. These findings demonstrated the potential utility of this inhibitor as a probe for detection of tightly bound proteases. In summary, cabbage seed extracts contain an inhibitor with activity toward a broad range of proteases important to hemostasis. To our knowledge, this agent represents the first inhibitor isolated from a plant source that inhibits thrombin.
Flow cytometric analysis of thiazole orange uptake by platelets: a diagnostic aid in the evaluation of thrombocytopenic disorders
Authors: Kienast J , Schmitz G
Published: October 2019

Thiazole orange (TO), a fluorescent dye originally synthesized for reticulocyte analysis, is characterized by a large fluorescence enhancement and high quantum yield on binding to nucleic acids, particularly RNA. In addition, the dye readily permeates live cell membranes. We applied TO staining, followed by fluorescence-activated flow cytometric analysis, to platelets in whole blood samples from hematologically normal subjects and patients with various quantitative platelet disorders. The percentage of TO-positive platelets in 50 control subjects was 8.6 +/- 2.8% (mean +/- SD) ranging from 2.8% to 15.8%. In 21 thrombocytopenic patients whose bone marrow contained normal to increased numbers of megakaryocytes, the percentage of fluorescently labeled platelets was significantly elevated (P less than .0001) to 26.9 +/- 10.9% (range, 13.3% to 57.1%). In contrast, the proportion of positively stained platelets in 23 patients with thrombocytopenia due to impaired platelet production (various conditions with reduced marrow megakaryocytes) did not significantly differ from the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (P less than .0001). Differences in the distributions of the percentage values as well as of the absolute counts for TO-positive platelets between the two patient groups were again highly significant (P less than .0001). Both the sensitivity and the specificity of this method in distinguishing between these categories of thrombocytopenia were greater than or equal to 95%. We conclude that flow cytometric analysis of platelets after staining with TO is a sensitive and specific test that rapidly provides information on the thrombopoietic activity in thrombocytopenic disorders. Our data further suggest that increased amounts of residual RNA characterize platelets released under conditions of “stress thrombopoiesis.”
Tissue factor and factor VII messenger RNAs in human alveolar macrophages: effects of breathing ozone
Authors: McGee MP , Devlin R , Saluta G , Koren H
Published: October 2019

This study was performed to determine if genes for tissue factor and factor VII proteins are expressed and regulated in vivo in lung macrophages during inflammation. Human alveolar macrophages and alveolar fluids were obtained 18 hours after healthy male adults were exposed, for 2 hours during intermittent exercise, to either air or air with 0.4 ppm ozone, added as a model toxic respiratory agent. Messenger RNA (mRNA) for both tissue factor and factor VII were demonstrated in macrophages isolated after subjects were exposed to unpolluted control air. With the same subjects examined after breathing ozone, the following changes were observed: tissue factor mRNA concentration in macrophages increased 2.6 +/- 0.47-fold. Factor VII mRNA concentration was reduced 0.64 +/- 0.24-fold. Total numbers of macrophages recovered did not change significantly. Ratios of nuclear:cytoplasmic areas of cytocentrifuged macrophages were augmented by 24.8% +/- 3%, giving morphometric evidence that immature cell forms increased in the population. In the lavage, tissue factor activity was increased 2.1 +/- 0.3-fold, while amounts of lipid phosphorous, which estimate total membrane lipids, and estimated volumes of alveolar fluid were not significantly changed. Factor VII activity and fibrinopeptide A levels in lavage were increased approximately twofold. These results using rapidly isolated, noncultured cells indicate that tissue factor and factor VII mRNA are synthesized in the alveolar macrophage population in vivo. In addition, evidence was found that as a result of breathing ozone, a shift in alveolar macrophage maturity occurred in association with tissue factor mRNA, tissue factor activity, and factor VII activity increases, and with formation of fibrinopeptide A in alveolar fluids.
Inhibition of functional properties of tetanus antigen-specific T-cell clones by envelope glycoprotein GP120 of human immunodeficiency virus
Authors: Chirmule N , Kalyanaraman VS , Oyaizu N , Slade HB , Pahwa S
Published: October 2019

We investigated mechanisms by which the soluble native envelope glycoprotein gp120 of the human immunodeficiency virus (HIV-1) suppresses antigen-driven T cell responses. For this study, exogenous interleukin-2 (IL-2)-independent, antigen-specific, CD4 positive, human T-cell clones were developed by cyclic restimulation with soluble tetanus toxoid antigen. In the presence of soluble antigen and antigen- presenting cells (APC), T-cell clones proliferated and secreted IL-2. Purified gp120 suppressed the proliferative responses of the T-cell clones with concomitant suppression of IL-2 secretion; proliferative responses of CD8+ T cells preincubated with gp120 were not inhibited. A short pulse of 20 minutes with gp120 was sufficient to inhibit the proliferative response of the T-cell clones. Anti-CD3 monoclonal antibody (MoAb)-driven proliferation of the T-cell clones was also suppressed by gp120, but responses elicited by mitogens, phorbol myristate acetate (PMA) plus calcium ionophore, ionomycin, anti-CD2 MoAbs, and a combination of anti-CD3 plus anti-CD28 MoAb driven responses remained unaffected. Investigation of signal transduction events showed that antigen-driven early activation signals via translocation of protein kinase C (PKC), increase in intracellular inositol phosphates, and increase in intracellular calcium were suppressed in gp120 pretreated, tetanus toxoid antigen-stimulated T- cell clones. One mechanism of immune suppression by gp120 may involve interference with the initiation of signal transduction through the T- cell receptor complex.
Prognostic factors in childhood T-cell acute lymphoblastic leukemia: a Pediatric Oncology Group study
Authors: Shuster JJ , Falletta JM , Pullen DJ , Crist WM , Humphrey GB , Dowell BL , Wharam MD , Borowitz M
Published: October 2019

Two hundred fifty-three children with newly diagnosed T-cell acute lymphoblastic leukemia (ALL), who were treated uniformly with modified LSA2L2 therapy, were evaluated using univariate and recursive partition analyses to define clinical or biologic features associated with risk of treatment failure. Overall event-free survival (EFS) at 4 years was 43% (SE = 4%). Factors examined included white blood cell (WBC) level, age, gender, race (black v other), presence of a mediastinal mass, hepatomegaly, splenomegaly, marked lymphadenopathy, hemoglobin level, platelet count, blast cell expression of antigens such as the common acute lymphoblastic leukemia antigen (CALLA, CD10), HLA-DR, and T-cell- associated antigens (CD3, CD4, CD8, CD7, CD5, and THY). Univariate analysis showed that age less than or equal to 5 or less than or equal to 7 years, WBC level less than 10, less than 25, less than 50 or less than 100 x 10(3)/microL, and blast cell expression of CD4, CD8, or CALLA were associated with significantly better EFS, while hepatomegaly and splenomegaly were associated with worse EFS. Recursive partitioning analysis showed that the most important single favorable prognostic factor was a WBC level less than 50 x 10(3)/microL and, for patients with WBC counts below this level, the most important predictor of EFS was blast cell expression of the pan-T antigen defined by the monoclonal antibody (MoAb), L17F12 (CD5). For patients with higher WBC levels, the most important predictor of EFS was blast cell expression of THY antigen. The recursive partitioning analysis defined three groups of patients with widely varied prognoses identified as follows: (1) those with a WBC count less than 50 x 10(3)/microL who lacked massive splenomegaly and had blasts expressing CD5 had the best prognosis (66%, SE = 7%, EFS 4 years, n = 84); (2) those with (b1) WBC counts less than 50 x 10(3)/microL with either massive splenomegaly or who had blasts lacking CD5 expression, or (b2) WBC counts greater than 50 x 10(3)/microL with expression of the THY antigen had an intermediate prognosis (39%, SE = 7% EFS at 4 years, n = 94); (3) those with WBC counts greater than 50 x 10(3)/microL and whose blasts lacked expression of THY antigen had the poorest outcome (EFS = 19% at 4 years, SE = 8%, n = 63). A three-way comparison of EFS according to these groupings showed significant differences among the three patient groups (P less than .001). The recursive partitioning was able to classify 241 (95%) of the patients.(ABSTRACT TRUNCATED AT 400 WORDS)
Heterogeneity of presenting features and their relation to treatment outcome in 120 children with T-cell acute lymphoblastic leukemia
Authors: Pui CH , Behm FG , Singh B , Schell MJ , Williams DL , Rivera GK , Kalwinsky DK , Sandlund JT , Crist WM , Raimondi SC
Published: October 2019

Presenting features of 120 consecutive children with T-cell acute lymphoblastic leukemia (ALL), representing 15% of all patients diagnosed as having ALL during the study period, were analyzed to determine relationships with treatment outcome. Patients' ages ranged from 1.7 to 18.8 years (median, 10.3 years) and their leukocyte counts from 1.7 to 1,070 x 10(9)/L (median, 100 x 10(9)/L). Central nervous system (CNS) leukemia was present in 12.5% of the cases, a mediastinal mass in 61%, and L2 lymphoblast morphology in 32%. A relatively high proportion of cases, 26%, had normal karyotypes at presentation. Of the cases tested, membrane CD1 expression was found in 38% of cases, CD3 in 33%, CD4 in 50%, CD5 in 94%, CD8 in 55%, and CD10 in 35%. Four presenting features were found to confer an increased risk of treatment failure: age greater than or equal to 15 years, L2 lymphoblast morphology, abnormal karyotype, and membrane CD3 expression. This study illustrates the heterogeneity of presentations of childhood T-cell ALL and suggests that the relative importance of risk factors in ALL differs according to immunophenotype and treatment strategy.
Effects of irradiation of recipient mice on the behavior and leukemogenic potential of factor-dependent hematopoietic cell lines
Authors: Duhrsen U , Metcalf D
Published: October 2019

After intravenous (IV) injection with factor-dependent FDC-P1 cells, irradiated DBA/2 and BALB/c mice developed transplantable leukemias owing to neoplastic transformation of the injected cells in vivo. Increasing the radiation dose shortened the preleukemic latent period, and in female mice the frequency of leukemia development was higher and the latent period shorter than in male mice. In the preleukemic period, the injected FDC-P1 cells rapidly increased in number in hematopoietic organs of irradiated animals, reaching peak levels 3 to 5 weeks after injection; factor-independent transformed cells were not detected before day 45. In unirradiated animals, these events were delayed by several weeks, and long-term survivors did not harbor detectable FDC-P1 cells. FDC-P1 cells sampled from preleukemic mice frequently showed atypical colony formation and reduced cloning efficiency in vitro, suggesting the occurrence of a distinct preleukemic change. U16.6 cells produced leukemia only in irradiated recipients, and the leukemic cells usually remained factor dependent. The two contrasting models should be of value in further analyzing the mechanisms underlying radiation- induced leukemias.
A new von Willebrand factor (vWF) defect in a patient with factor VIII (FVIII) deficiency but with normal levels and multimeric patterns of both plasma and platelet vWF. Characterization of abnormal vWF/FVIII interaction
Authors: Mazurier C , Dieval J , Jorieux S , Delobel J , Goudemand M
Published: October 2019

The patients with inherited bleeding diathesis related to quantitative, structural, and/or functional abnormalities of von Willebrand factor (vWF) are said to have von Willebrand's disease (vWD). We report here the clinical and laboratory features of a 50-year-old woman with a life- long history of excessive bleeding. Her particular laboratory data are factor VIII (FVIII) deficiency, subnormal bleeding time, and the presence of all plasma and platelet vWF multimers in normal amounts. Infused with FVIII/vWF concentrate, she showed a persistent increase in FVIII that led us to discard hemophilia A carrier or “acquired hemophilia” diagnoses. vWF devoid of FVIII purified from normal and patient's plasma by immunoaffinity on anti-vWF monoclonal antibody (MoAb) was immobilized onto polystyrene tubes that were further incubated with purified normal FVIII. The bound FVIII was evidenced using radiolabeled anti-FVIII MoAb. The data showed that the patient's vWF, in contrast to vWF purified from normal plasma, was unable to bind FVIII. Furthermore, no inhibitor of FVIII/vWF interaction was evidenced in incubating purified normal vWF with the patient's plasma before the addition of FVIII and anti-FVIII MoAb. These results support the concept that the bleeding diathesis of this patient appears to be due mainly to her abnormal vWF preventing FVIII/vWF interaction. This abnormality, which is not yet described in present classification of vWD, could be considered as a new variant of vWD.
Effect of venous shear stress on CD18-mediated neutrophil adhesion to cultured endothelium
Authors: Lawrence MB , Smith CW , Eskin SG , McIntire LV
Published: October 2019

The CD11/CD18 family of glycoproteins has been identified as a mediator of a number of adhesive interactions crucial to inflammatory responses. Using a monoclonal antibody (MoAb) against CD18 (TS1/18), the role of these molecules in polymorphonuclear neutrophil (PMNL) adhesion to cultured primary human umbilical vein endothelial cells (HUVEC) was examined under venous flow conditions. Incubation of PMNL with TS1/18 (anti-CD18) did not inhibit PMNL adhesion to interleukin-1 (IL-1)- treated HUVEC at 2.0 dynes/cm2 (TS1/18-treated 305 +/- 58 PMNL/mm2 v 334 +/- 63 PMNL/mm2 on control). Furthermore, incubation of HUVEC with R6.5.D6, an MoAb against intercellular adhesion molecule-1 (ICAM-1) did not significantly inhibit PMNL adhesion to IL-1-treated HUVEC at 2.0 dynes/cm2 (P greater than .3). In contrast to the lack of inhibition of adhesion under conditions of flow, incubation of PMNL with TS1/18 reduced PMNL adherence in static adhesion assays. PMNL migration beneath HUVEC monolayers has been shown to be stimulated by 4-hour IL-1 treatment. TS1/18 and R6.5.D6 significantly inhibited migration of PMNL beneath IL-1-treated HUVEC monolayers under flow conditions by slightly more than 80% (P less than .005). In flow experiments with CD18- deficient PMNL, virtually no transendothelial migration was observed. The effect of FMLP (10(-8) mol/L) on PMNL adhesion to untreated HUVEC at wall shear stresses ranging from 0.25 to 2.0 dynes/cm2 was also investigated. FMLP had little effect on PMNL adherence at shear stresses above 0.5 dynes/cm2 (P greater than .45). In response to FMLP exposure at lower wall shear stresses, PMNL adherence to untreated HUVEC increased 6.9-fold at 0.5 dynes/cm2 (P less than .001). At 0.25 dynes/cm2, FMLP stimulation increased PMNL adherence to untreated HUVEC 6.5-fold compared with controls (P less than .005), and FMLP failed to make CD18-deficient PMNL more adherent. In experiments with PMNL pretreated with TS1/18 (anti-CD18), there was a 67% inhibition of FMLP- stimulated adhesion at 0.5 dynes/cm2 (P less than .025). The upper threshold of CD18-mediated PMNL adhesion appears to be between 0.5 and 1.0 dyne/cm2. Above these wall shear stresses, the initial attachment of PMNL to cultured endothelium was mediated almost exclusively by CD18- independent mechanisms. By simulating some of the flow parameters in the microcirculation with well-characterized shear forces, PMNL adhesion by CD18-independent and dependent mechanisms can be differentiated. These data also indicate that CD18 is an important mediator of transendothelial migration by PMNL, which have attached to the endothelium by a CD18-independent mechanism.
Etoposide in acute nonlymphocytic leukemia. Australian Leukemia Study Group
Authors: Bishop JF , Lowenthal RM , Joshua D , Matthews JP , Todd D , Cobcroft R , Whiteside MG , Kronenberg H , Ma D , Dodds A
Published: October 2019

Previously untreated patients with acute nonlymphocytic leukemia (ANLL) aged 15 to 70 years were randomized to either cytosine arabinoside 100 mg/m2/d continuous intravenous (IV) infusion days 1 through 7, daunorubicin 50 mg/m2/d IV days 1 through 3 (7–3), or the same drugs intensified with etoposide 75 mg/m2/d IV days 1 through 7 (7–3–7) as induction therapy. Patients achieving complete remission (CR) received two courses of consolidation therapy (5–2 or 5–2–5) followed by maintenance therapy. Of 264 eligible patients, CR occurred in 56% of 7– 3 and 59% of 7–3–7 patients; 7–3–7 significantly improved remission duration (P = .01). The median remission duration was 12 months for 7–3 and 18 months for 7–3–7. Survival was similar when the two arms were compared overall. Subset analysis performed to identify patients with the most benefit showed that etoposide significantly prolonged remission duration in younger patients (less than 55 years) with a median of 12 months for 7–3 and 27 months for 7–3–7 (P = .01). Survival appeared to be prolonged with 7–3–7 in patients aged less than 55 years, with a median of 9 months for 7–3 as compared with 17 months for 7–3–7 (P = .03). In older patients (aged greater than or equal to 55 years), 7–3–7 was more toxic, with significantly more severe [World Health Organization (WHO) grade 3 or 4] stomatitis (P = .02) and no additional clinical benefit. Hematologic toxicity for induction courses was similar, with granulocytopenia less than 0.5 x 10(9)/L for a median of 16 days per course for 7–3 and 15 days for 7–3–7. Hematologic toxicity was more severe for 5–2–5 consolidation courses (P = .003). Induction and consolidation therapy intensified with etoposide resulted in significantly improved remission duration but not survival.
Magnetic resonance imaging of bone marrow in sickle cell disease: clinical, hematologic, and pathologic correlations
Authors: Mankad VN , Williams JP , Harpen MD , Manci E , Longenecker G , Moore RB , Shah A , Yang YM , Brogdon BG
Published: October 2019

A longitudinal, prospective, controlled evaluation of magnetic resonance images (MRI) of long bones in sickle cell patients was undertaken simultaneously with assessment of clinical status and hematologic parameters, including dense erythrocytes. MRI of bone marrow in sickle cell patients during steady states appeared patchy and were markedly different from those in matched controls (P approximately 0). Patients with severe patchiness were older than those with mild or moderate patchiness (P less than .03). Sixty-nine MRI were performed during 28 painful episodes occurring in 14 subjects with sickle cell disease (SCD). Increased signals on intermediate and T2-weighted images were detected in 35.7% of painful episodes. These abnormalities were distinct and not observed to occur spontaneously during the steady- state examinations (P approximately 0). Bone marrow infarcts were confirmed by biopsy in two instances and autopsy in one instance. Dense red cells decreased by 40.81% of baseline during pain crises (P = .00005), more remarkably in those who had pain in the lower extremities (P = .0145). Patients with change in MRI during pain crises had a greater percentage change in the dense cells than those without the change in MRI (69.7% v 31.3%, P = .0120).
Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell- associated protein
Authors: Holguin MH , Wilcox LA , Bernshaw NJ , Rosse WF , Parker CJ
Published: October 2019

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol- specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.
Correlations and interactions in the production of interleukin-6 (IL- 6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells: IL-6 suppresses IL-1 and TNF
Authors: Schindler R , Mancilla J , Endres S , Ghorbani R , Clark SC , Dinarello CA
Published: October 2019

Interleukin-6 (IL-6) shares several biologic properties with IL-1, including hematopoietin-1 activity and stimulation of T cells. Because many of their biologic activities overlap, we developed and used a specific radioimmunoassay (RIA) for IL-6 to compare production of this cytokine on a molar basis with that of IL-1 alpha, IL-1 beta, and tumor necrosis factor (TNF)alpha. The RIA correlated well with the hybridoma bioassay for IL-6 (r = .87, P less than .001). Freshly isolated human peripheral blood mononuclear cells (PBMC) cultured in the absence of stimuli did not produce IL-6 in most cases. Kinetics of secretion and cell-association of IL-6 were studied. In contrast to IL-1 alpha but similar to TNF, IL-6 was almost entirely secreted into the extracellular fluid. Incubation with different stimuli (lipopolysaccharide [LPS], phytohemagglutinin [PHA], Staphylococcus epidermidis, or IL-1 alpha) resulted in production of IL-6. However, on a molar basis PBMC produced approximately two to three times less IL-6 than IL-1 alpha, IL-1 beta, or TNF, regardless of the stimulus. The amount of IL-6 produced from PBMC was consistent when measured in the same subjects six time during a 12-week period. In a cohort of 38 donors, the coefficient of variation for IL-6 production was .32, compared with .92 for IL-1 beta and .96 for TNF. Comparing cytokine production by PBMC, there was a significant correlation between IL-6 and IL-1 beta (r = .72) and between IL-6 and TNF (r = .66). IL-6 did not stimulate IL-1 beta or TNF production, but suppressed IL-1 beta and TNF production induced by LPS or PHA by 30% (P less than .01). This suppression of IL-1 beta and TNF by IL-6 appears to be on the level of transcription.
Characterization of granulocyte-macrophage colony-stimulating factor receptor on the blast cells of acute myeloblastic leukemia
Authors: Onetto-Pothier N , Aumont N , Haman A , Bigras C , Wong GG , Clark SC , De Lean A , Hoang T
Published: October 2019

Iodinated granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to document the specific binding of GM-CSF to all acute myeloblastic leukemia (AML) samples examined in the present study. There was some heterogeneity in the number of GM-CSF binding sites per cell. To determine whether the low level of binding to some patient samples may be attributed to receptor occupancy by an endogenous source of GM-CSF, we devised an acid wash procedure that could remove surface- bound GM-CSF without affecting receptor properties. We thus document that GM-CSF specific binding to AML blasts before or after acid wash was the same, indicating that the observed heterogeneity in binding is not the result of receptor occupancy by an endogeneous source of GM- CSF. Saturation analyses are in favor of the presence of two classes of binding sites on AML blasts: a high-affinity receptor that binds GM-CSF with a dissociation constant (kd) of 3 to 73 pmol/L and a second class of low-affinity receptor that binds GM-CSF with a kd of 1 to 10 nmol/L. Binding studies with two established cell lines KG-1, and IRCM-8 also showed the presence of two classes of binding sites with high and low affinities. Analysis of GM-CSF titration curves in culture indicate that the median effective concentration required for stimulation of blast colony formation (EC50 = 5–36 pmol/L) were in the range of the kd of the high-affinity binding site, suggesting that this high-affinity binding site mediates the proliferative response.
Regulation of megakaryocyte ploidy in vivo in the rat
Authors: Kuter DJ , Rosenberg RD
Published: October 2019

The relationship between the bone marrow (BM) megakaryocyte and the circulating platelet was explored. Incremental changes in platelet count were made in rats by infusion of antiplatelet antibody or by platelet transfusion, and the response of megakaryocytes was measured by flow cytometry. Proportional changes in megakaryocyte ploidy were demonstrated: As the platelet count declined, ploidy increased; as the platelet count increased, ploidy decreased. Even moderate degrees of thrombocytopenia and thrombocytosis (48% and 177% of the normal platelet count) were associated with changes in ploidy. These changes were not the results of the technique used to alter the platelet count because reinfusion of platelets after 3 hours of thrombocytopenia prevented any ploidy change. These studies proved that the circulating platelet and the megakaryocyte constitute a classic feedback loop whose activity can be measured by the degree of ploidization of the megakaryocyte. The minimal duration of thrombocytopenia necessary to promote megakaryocyte ploidy changes was approximately 10 hours. Using a BM culture assay, we identified a plasma factor which induced alterations in megakaryocyte ploidy and whose level is inversely proportional to the platelet count.
Molecular anatomy of a 5q interstitial deletion
Authors: Nagarajan L , Lange B , Cannizzaro L , Finan J , Nowell PC , Huebner K
Published: October 2019

A truncated granulocyte-macrophage colony-stimulating factor (GM-CSF) allele on a putative 5q- chromosome of HL-60 cells was cloned and, by comparison with counterpart normal sequences, analyzed for clues to molecular mechanisms facilitating rearrangement and deletion. Within the 17-kilobase (kb) pair locus surrounding the truncated GM-CSF gene remnant, there are no fewer than four rearranged genomic fragments that seemingly derive from chromosome 5 region q21----23. Two of the fragments, which flank the truncated GM-CSF locus on the 5q-, are contiguous on the normal chromosome 5, centrometric to the normal GM- CSF allele, indicating at least one intrachromosomal insertion event, either preceded or followed by further deletion. Insertion and/or deletion was accompanied by juxtaposition of LINE sequences to the 5′ side of the truncated GM-CSF locus within the inserted fragment. The entire rearranged locus is embedded in repetitive sequences, which may have mediated successive insertions or deletions. The extent of such stepwise deletions, resulting in loss of genes such as interleukin-3 (IL-3), IL-4, IL-5, and GM-CSF, whose gene products are critical to differentiation within the lineage of the affected hematopoietic stem cell, may be mirrored in the heterogeneity of symptoms and 5q- deletion sizes observed in myelodysplasias and acute leukemias carrying a 5q- chromosome. Perhaps most significantly, the sequences surrounding the insertion/deletion region are suggestive of recombination signals, including direct repeats and mirrored repeats. The site of insertion of the GM-CSF 3′ region into an upstream (centromeric) locus is flanked by direct repeats; the upstream site into which it is inserted is also flanked by 12 base pair (bp) direct repeats. After insertion, one member of each pair of repeats is lost. The organization of this rearranged locus implies that direct repeats had a role in the intrachromosomal recombination/deletion event.
Binding of G-CSF, GM-CSF, tumor necrosis factor-alpha, and gamma- interferon to cell surface receptors on human myeloid leukemia cells triggers rapid tyrosine and serine phosphorylation of a 75-Kd protein
Authors: Evans JP , Mire-Sluis AR , Hoffbrand AV , Wickremasinghe RG
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma-interferon (gamma-IFN), or tumor necrosis factor-alpha (TNF-alpha) triggered the rapid, stable phosphorylation of a 75-Kd protein (p75) when incubated with permeabilized HL60 human myeloid leukemia cells in the presence of [gamma-32P] ATP. Among several chemical inducers of HL60 cell differentiation, dimethyl sulfoxide also triggered p75 labeling, but retinoic acid or 12-O-tetradecanoylphorbol-13-acetate did not elicit this response. Pretreatment of cells with G-CSF or GM-CSF for more than 30 seconds before permeabilization rendered the p75 labeling undetectable, suggesting that ligand-stimulated labeling was rapidly completed within this time in intact cells. Phosphorylation of p75 occurred on serine and tyrosine residues. This conclusion was confirmed by direct phosphoamino acid analysis. Immunoblot analysis of lysates of intact HL60 cells that had been incubated with G-CSF, GM-CSF, IFN, or TNF confirmed that tyrosine phosphorylation of a p75 also occurred in response to these cytokines in intact cells. Pretreatment of intact HL60 cells with one biologic agent or dimethyl sulfoxide abolished p75 labeling in response to incubation of permeabilized cells with a second agent, strongly suggesting that the same protein was phosphorylated in response to these treatments. p75 labeling was strictly dependent on expression of the appropriate ligand receptor. Data suggest that activation of a tyrosine kinase system is an early response to the binding of G-CSF, GM-CSF, TNF, or IFN to their respective cell surface receptors, or to the addition of dimethyl sulfoxide, and that the resulting phosphorylation event(s) may play a role in securing common elements in the biologic responses to these agents.
Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-beta
Authors: Cashman JD , Eaves AC , Raines EW , Ross R , Eaves CJ
Published: October 2019

Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive, high- proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum, which is absent from fetal calf serum. However, this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules, ie, platelet- derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor alpha (TGF-alpha), and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays, although all have known actions on mesenchymal cells including, in some cases, the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly, a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta, a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1, blocked their stimulating activity. These results suggest a model in which indirect, local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo.
Internalization of human immunodeficiency virus type I and other retroviruses by megakaryocytes and platelets
Authors: Zucker-Franklin D , Seremetis S , Zheng ZY
Published: October 2019

Direct infection of megakaryocytes and platelets by human immunodeficiency virus type I (HIV-I) or other retroviruses has not been demonstrated. To determine whether this could occur, murine bone marrow was co-cultivated with the amphotropic retrovirus-producing cell line PA317-N2, and freshly isolated normal human bone marrow and platelets were co-cultivated with HIV-infected H9 cells. In each case, ultrastructural analyses showed viruses within megakaryocytes and platelets. In murine specimens, the uptake of retrovirus was avid at all stages of differentiation. In human specimens, viral uptake was less frequent. These results suggest that direct infection of megakaryocytes could play a role in the pathophysiology of HIV- associated disease. In addition, these observations suggest that cells of the megakaryocyte lineage could serve as target cells in gene transfer experiments using retroviral-based vectors.
Fludarabine therapy in macroglobulinemic lymphoma
Authors: Kantarjian HM , Alexanian R , Koller CA , Kurzrock R , Keating MJ
Published: October 2019

Fludarabine, a fluorinated analogue of adenine, was given to 11 patients with macroglobulinemic lymphoma, all but one having failed prior standard chemotherapy. Five patients (45%) responded with more than a 50% reduction of immunoglobulin M (IgM) tumor mass for a projected median duration of longer than 1 year. The onset of remission was usually slow, with a median tumor halving time of 5.2 months in responding patients, emphasizing the importance of repeated courses of treatment. Fludarabine is an important new agent effective against macroglobulinemic lymphoma, and should be evaluated further in combination with other active modalities.
Direct demonstration of elevated aldehyde dehydrogenase in human hematopoietic progenitor cells
Authors: Kastan MB , Schlaffer E , Russo JE , Colvin OM , Civin CI , Hilton J
Published: October 2019

Relative levels of cytoplasmic aldehyde dehydrogenase (ALDH) were determined in selected subpopulations of normal human bone marrow cells using a flow cytometric assay that simultaneously detects a cell surface antigen (as a marker of cell lineage and developmental stage) and the level of ALDH. The intracellular level of this enzyme has been shown to be directly related to cellular resistance to activated cyclophosphamide and is believed to be important in the survival of cells capable of repopulating marrow in autologous bone marrow transplant procedures. Western blot analysis and flow cytometric analysis of four murine cell lines with known ALDH levels were used to establish the relation between ALDH content and fluorescence with an affinity-purified anti-mouse ALDH antibody. An affinity purified anti- human ALDH antibody, characterized by immunoblotting of cytosolic extracts of cell lines with known ALDH content, was used to determine relative ALDH levels in the marrow subpopulations. We found that hematopoietic progenitor cells express the highest level of ALDH, while lymphocytes express the lowest level. Immature erythroid cells express ALDH at a level intermediate between progenitor cells and lymphocytes.
Hematopoietic progenitors in cyclic neutropenia: effect of granulocyte colony-stimulating factor in vivo
Authors: Migliaccio AR , Migliaccio G , Dale DC , Hammond WP
Published: October 2019

The number and growth factor requirements of committed progenitor cells (colony-forming units-granulocyte/macrophage and burst-forming units- erythroid) in three patients with cyclic neutropenia (two congenital, one acquired) were studied before and during therapy with recombinant human granulocyte colony-stimulating factor (G-CSF; 3 to 10 micrograms/kg/d). When the patients with congenital disease were treated with G-CSF, the cycling of blood cells persisted, but the cycle length was shortened from 21 days to 14 days, and the amplitude of variations in blood counts increased. There was a parallel shortening of the cycle and increase of the amplitude of variations (from two- to three-fold to 10- to 100-fold) in the number of both types of circulating progenitor cells in these two patients. In the patient with acquired cyclic neutropenia, cycling of both blood cells and progenitors could not be seen. In cultures deprived of fetal bovine serum, erythroid and myeloid bone marrow progenitor cells from untreated patients and from normals differed in growth factor responsiveness. As examples, maximal growth of granulocyte/macrophage (GM) colonies was induced by granulocyte/macrophage (GM)-CSF plus G-CSF in the patients, whereas a combination of GM-CSF, G-CSF and interleukin- 3 (IL-3) was required in the normals, and erythropoietin alone induced fourfold more erythroid bursts from cyclic neutropenic patients than from normal donors (46% versus 11% of the maximal colony number, respectively). The growth factor responsiveness of marrow progenitor cells slightly changed during the treatment toward the values observed with normal progenitors. These results indicate that treatment with G- CSF not only ameliorated the neutropenia, but also increased the amplitude and the frequency of oscillation of circulating progenitor cell numbers. These data are consistent with the hypothesis that G-CSF therapy affects the proliferation of the hematopoietic stem cell.
Tumor necrosis factor induces the production of urokinase-type plasminogen activator by human endothelial cells
Authors: van Hinsbergh VW , van den Berg EA , Fiers W , Dooijewaard G
Published: October 2019

Endothelial cells play an important role in the regulation of fibrinolysis by the production of several key regulatory proteins. The cytokines tumor necrosis factor (TNF), lymphotoxin, and interleukin-1 (IL-1), but not interleukin-6, increase the production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells, whereas they have no stimulatory effect on the production of tissue-type plasminogen activator (t-PA). Primary cultures of human endothelial cells release very little urokinase-type plasminogen activator (u-PA). We report here that TNF and lymphotoxin induce, in a concentration-dependent way, the production of both cellular and secreted u-PA antigen in primary and subcultured human endothelial cells. The TNF-induced increase was accompanied by a more than 10-fold increase in u-PA mRNA. Upon stimulation of early passage umbilical vein endothelial cells by TNF, u- PA was predominantly secreted at the basolateral side, whereas PAI activity and t-PA were found in more equal amounts at the apical and basolateral sides of the cell monolayers. TNF-stimulated u-PA secretion by subcultured human aorta endothelial cells showed only a marginal polarity. The u-PA antigen was present in a plasmin-activatable form (single chain u-PA) and in a nonactivatable form (probably u-PA: PAI-1 complex). During the induction of u-PA by TNF, the ratio between plasmin-activatable u-PA and total u-PA decreased markedly. This may indicate that TNF also increases the degree of u-PA activation. The parallel induction of the synthesis and secretion of both u-PA and PAI- 1 by endothelial cells adds a new aspect to the alterations of the fibrinolytic system caused by inflammatory mediators. This aspect may be significant for the regulation of cell-associated and interstitial plasminogen activator activity.