bluuebunny/crossref_metadata_2025 huggingface dataset:
Enzymatic activities of activated and zymogen forms of human Hageman factor (factor XII)
Authors: Silverberg M , Kaplan AP
Published: October 2019

Pro-Phe-Arg chloromethylketone (PPACMK) at 5.26 microM inactivated the amidolytic activity of native human Hageman factor with an apparent first-order rate constant of 0.75 min-1. The activated forms of Hageman factor, Hfa and HFf, were also inactivated by PPACMK with rate constants 0.82 and 0.72 min-1. These numbers indicate that the activity detectable in native Hageman factor is due to contamination with activated species. Uncleaved Hageman factor reacts slowly with 40 mM diisopropyl fluorophosphate with concomitant loss of its procoagulant activity. Incubation of native Hageman factor with PPACMK does not destroy its procoagulant activity, even in the presence of the activator dextran sulphate, but PPACMK inhibits autoactivation of Hageman factor, suggesting that no active site is formed in uncleaved, surface-bound Hageman factor. The activation of prekallikrein by Hageman factor under initial-rate conditions occurs after a lag and is prevented by an inhibitor of Hageman factor from corn. The kinetics of prekallikrein activation and the effects of inhibitors provide evidence that the amidolytic and proteolytic activities of human Hageman factor reside in the activated forms derived by limited proteolysis of the native molecule.
Abnormal erythroid progenitor cells in human preleukemia
Authors: Chui DH , Clarke BJ
Published: October 2019

Ten patients with preleukemia were studied by the erythroid cell clonal culture technique. In nine of these patients, erythroid colonies derived from peripheral blood BFU-E were not observed, while the other patient had markedly decreased peripheral blood BFU-E-derived erythroid colonies in vitro. In three patients, marrow cells were also cultured and no BFU-E-derived erythroid colonies were detected. These studies indicate that immature erythroid progenitor cells, BFU-E, in patients with preleukemia are either markedly decreased in number or grossly defective in their proliferative or differentiative capacities.
Comparison of 125I-fibrinogen kinetics and fibrinopeptide A in patients with disseminated neoplasias
Authors: Mombelli G , Roux A , Haeberli A , Straub PW
Published: October 2019

To provide more information on the pathways of fibrinogen catabolism in generalized cancer, the effect of heparin on fibrinopeptide A (fpA) and on 125I-fibrinogen kinetics was studied in 15 patients with disseminated neoplasia. Three patients had evidence of venous thrombosis and in 2 additional patients a low fibrinogen level together with increased amounts of FDP/fdp and a positive ethanol test indicated disseminated intravascular coagulation (DIC). The plasma levels of fpA were grossly elevated (4.6--20, mean 11.4 ng/ml, normal values 1.01 +/- 0.45 ng/ml) in patients with thrombosis or DIC, and normal to grossly elevated (0.4--10.4, mean 6.1 ng/ml) in the other patients. Intravenous heparin bolus lowered the fpA level in 11/11 patients, and continuous heparin treatment led to an impressive suppression or complete normalization of the plasma fpA in 5/6 patients. This finding is thought to reflect heparin suppression of thrombin activity on fibrinogen. In some cases, the fpA fall after heparin bolus was slow and/or incomplete, suggesting fpA generation at sites not easily accessible to heparin or insufficient heparin dosage. The 125I- fibrinogen kinetics were characterized by a significantly shorter half- life (t1/2: 2.5 days), increased catabolic rate constant (j: 0.44 days- 1), and increased absolute turnover (68.9 mg fibrinogen/kg/day) as compared to 4 normal subjects (t1/2: 4.2 days; j: 0.26 days-1; turnover 21.7 mg fibrinogen/kg/day). As estimated from the fpA generation rates, intravascular thrombin action on fibrinogen contributed only in minor part to increase the turnover of 125I-fibrinogen. In particular, the turnover was greatly accelerated in heparin-treated patients despite impressive suppression or normalization of the fpA levels in 5/6 cases.
Allogeneic bone marrow transplantation in acute nonlymphocytic leukemia: a pilot study
Authors: Kersey JH , Ramsay NK , Kim T , McGlave P , Krivit W , Levitt S , Filipovich A , Woods W , O'Leary M , Coccia P , Nesbit ME
Published: October 2019

The objective of the current study, initiated in 1976, was to improve upon the high relapse rate and subsequent mortality in children and young adults with acute nonlymphocytic leukemia (ANLL). Seventeen patients, ages 6--28, with ANLL in first bone marrow remission, received cyclophosphamide and total body irradiation using a radiation scheme of 750 rad (7.5 Gy) total dose, delivered at a dose rate of 26 rad (26 cGy) per minute. Allogeneic marrow from HLA-matched sibling donors was followed by prophylactic therapy or graft-versus-host disease (GVHD). Median follow-up of the entire group is 20+ mo; survivors have been followed for a minimum of 14+ mo. Interstitial pneumonitis was observed in 6% of patients, and GVHD was observed in 29%. Seventy percent of patients are alive and in complete continuous remission. Two patients have relapsed (at 7 and 24 mo). Actuarial relapse-free survival is 76% at 1 yr and 64% at 5 yr. Quality of life in this disease-free survivors is excellent; all patients are free of active GVHD, receive no maintenance chemotherapy, and have high Karnofsky performances scores. High dose rate total body irradiation plus cyclophosphamide followed by allogeneic BMT may provide an opportunity for long-term complication-free survival in a substantial proportion of children and young adults with ANLL.
Fibronectin enhances in vitro monocyte-macrophage-mediated tumoricidal activity
Authors: Perri RT , Kay NE , McCarthy J , Vessella RL , Jacob HS , Furcht LT
Published: October 2019

Control of neoplastic proliferation reflects in part monocyte/macrophage destruction of target cells--destruction that evidently requires cell-cell interaction. We herein show it to involve the natural plasma opsonin, fibronectin. With two cultured human tumor lines--Malme melanoma and CAK-I renal carcinoma cells--addition of fibronectin, purified to homogeneity, enhances macrophage-mediated cytotoxicity 2--4-fold (p less than 0.01). Both fresh human monocytes or the U-937-cultured macrophage line become more lethal to tumor cells with added fibronectin. The fibronectin-enhanced monocyte and U-937 tumoricidal activity occurred in a dose-dependent fashion. Specificity of fibronectin's action was validated by use of affinity-purified rabbit antifibronectin antibody, which completely abated its enhancement of tumoricidal activity. Enhancement of tumoricidal activity did not occur when monocytes or U-937 were exposed to fibronectin-coated plates. However, the addition of soluble fibronectin to fibronectin-coated plates was then capable of enhancing cytocidal activity. These studies demonstrate that human fibronectin is capable of increasing both fresh and cultured human monocyte tumor-directed cytotoxicity. Fibronectin appears to be a potentially important circulating molecule that may favorably influence human monocyte tumor cell cytotoxicity.
Effects of a microtubule stabilizing agent on the response of platelets to vincristine
Authors: White JG , Rao GH
Published: October 2019

The discoid shape of blood platelets is supported by a circumferential bundle of microtubules. Removal of the microtubules by an antimitotic drug, vincristine, is associated with loss of lentiform appearance, formation of tubulin paracrystals, a depressed response to aggregating agents, and impaired secretory activity. Recent studies have suggested that the action of vincristine on platelet secretion and aggregation is directly related to its action on microtubules, while other work had indicated that the antimitotic drug prevents the release reaction by inhibiting prostaglandin synthesis. The present study has examined the influence of taxol, a microtubule stabilizing agent, on the response of platelets to vincristine. Taxol completely prevented vincristine- induced shape change, microtubule disassembly, and tubulin paracrystal formation, even at concentrations one-tenth that of the antimitotic drug. Pretreatment with vincristine to dissociate microtubules and convert tubulin to crystals before exposure to taxol did not affect altered shape or tubulin paracrystals, but did cause assembly of free pools of tubulin into tubular polymers. Studies of physiology confirmed that vincristine, in amounts that remove microtubules, depresses platelet aggregation and secretion, effects that could be overcome by increasing agonist concentration. Although completely preventing microtubule dissociation, taxol had no corrective influence on vincristine-induced inhibition of platelet function. Biochemical studies revealed that vincristine concentrations that disassembled microtubules and blocked secretion did not inhibit conversion of 14C- arachidonic acid to thromboxane B2. The findings suggest that vincristine inhibits platelet function through some mechanism other than disassembling microtubules, but the other mechanism does not involve inhibition of prostaglandin synthesis.
Partial purification and characterization of a growth factor for macrophage progenitor cells with high proliferative potential in mouse bone marrow
Authors: Kriegler AB , Bradley TR , Januszewicz E , Hodgson GS , Elms EF
Published: October 2019

A population of macrophage progenitor cells, with high proliferative potential, has recently been demonstrated in postfluorouracil-treated and normal mouse bone marrow (BM) in vitro, when the newly discovered growth factor (synergistic activity, SA) is combined with a macrophage colony-stimulating factor (CSF) as a proliferative stimulus. SA, shown to be present in human spleen and placental conditioned media (HSCM and HPCM, respectively) have been studied and found to be unstable to trypsin digestion and to heating at 50 degrees C or above; stable between pH 4 and 9; nonadherent to Con-A-Sepharose; and to have an isoelectric point between pH 5 and 5.8 and a molecular weight of between 14,000 and 21,000 as indicated by gel filtration chromatography. SAs from both HSCM and HPCM have been purified 89- and 122-fold, respectively, by precipitation of extraneous proteins at pH 5 followed by chromatographing twice on Sephacryl S200. Neither of these partially purified SAs contain any CSF for mouse BM. These results indicate that the SAs from HSCM and HPCM may be closely related and that they are structurally different from CSFs derived from various murine sources that have been shown to be stable to proteolytic enzymes and heat.
Clinical staging system for hairy-cell leukemia
Authors: Jansen J , Hermans J
Published: October 2019

To find a clinical staging system for patients with hairy cell leukemia, 391 patients contributed by 22 centers were analyzed using the proportional hazard survival model. Attention was paid to nonsplenectomized patients to find a staging system to predict the survival length at the time of diagnosis. On the basis of hemoglobin level and spleen size at the time of diagnosis, 3 stages could be distinguished with significantly different prognoses (stages I-III). In addition, we addressed the question of splenectomy to identify those patients who benefit from the operation. Using arbitrary, but clinically relevant, criteria to call the operation beneficial, splenectomy appears to be indicated for patients with large spleens (greater than or equal to 4 cm under costal margin) or with smaller, but palpable, spleens when anemia (Hb less than 12 g/dl) is present. The third question concerned the splenectomized patients. To indicate the patients who have a poor postsplenectomy survival, a staging system was developed on the basis of hemoglobin level and number of neutrophils at 2–3 mo after the operation (stages A-C). The validity of the two staging systems was supported by the results of an analysis of an independent test series of patients. These staging systems may be helpful for the choice of therapy and in the planning of clinical trials in patients with hairy-cell leukemia.
Adolescent and adult acute lymphoblastic leukemia: prognostic features and outcome of therapy. A study of 293 patients
Authors: Baccarani M , Corbelli G , Amadori S , Drenthe-Schonk A , Willemze R , Meloni G , Cardozo PL , Haanen C , Mandelli F , Tura S
Published: October 2019

The case histories of 293 adolescent and adult patients with acute lymphoblastic leukemia (ALL) first seen and treated between 1969 and 1979 are reviewed. A complete remission (CR) was achieved in 79% of cases. Male sex, advanced age (greater than or equal to 30 yr old), and early CNS involvement were the major determinants of remission failure. Median duration of first CR was 16 mo, with 23 patients (actuarial proportion 25%) alive and relapse-free at 5 yr. The major determinant of first CR length was white blood cell (WBC) count (best cut-off value at 35 X 10(9)/liter). First CR length was also negatively affected by early CNS involvement, morphological FAB L3 subtype, and B-cell (Smlg+) leukemia, but these features were significantly associated with a high WBC count. First CR length in patients 11–15 yr old did not differ significantly from that of patients 16–59 yr old. The negative prognostic value of T-cell (E+) leukemia was not confirmed in this adult series. CNS prophylaxis provided an effective protection against CNS relapse. Maintenance chemotherapy was apparently more effective when 4 or more than 4 drugs were employed. “Low risk” patients (WBC count less than 35 X 10(9)/liter still relapsed rather frequently (32% at 1 yr, 49% at 2 yr), with 33% of them alive and relapse-free at 5 yr. “High risk” patients (WBC count greater than or equal to 35 X 10(9)/liter +/- early CNS involvement +/- morphological L3 subtype +/- B-cell leukemia) relapsed very quickly (50% at 6 mo. 70% at 1 yr), with only 6% of them relapse-free at 5 yr.
Analysis of late infections after human bone marrow transplantation: role of genotypic nonidentity between marrow donor and recipient and of nonspecific suppressor cells in patients with chronic graft-versus-host disease
Authors: Atkinson K , Farewell V , Storb R , Tsoi MS , Sullivan KM , Witherspoon RP , Fefer A , Clift R , Goodell B , Thomas ED
Published: October 2019

Infections occurring 6 mo or later after bone marrow transplantation for severe aplastic anemia or hematologic malignancy were analyzed in 98 long-term survivors. Varicella-zoster (VZ) infections were analyzed separately from all other infections. The factor predisposing most strongly to late VZ infection was genotypic nonidentity for HLA between marrow donor and recipient. There was a suggestion that chronic graft- versus-host disease (GVHD) associated with the presence of nonspecific suppressor cells also predisposed to late VZ infection, while age less than 10 yr was protective against such infections. Chronic GVHD predisposed to late non-VZ infections, but this was not increased by the presence of nonspecific suppressor cells. HLA nonidentify between patient and marrow donor further increased the risk of late non-VZ infections over and above that due to the presence of chronic GVHD. Receipt of a syngeneic transplant appeared protective for late non-VZ infections. These findings suggest that full genotypic identity for HLA between donor and recipient may be required for optimal immune reconstitution after marrow transplantation and may denote a possible biologic role for nonspecific suppressor T cells in humans.
Immunologic and clinical effects of repeated blood exchange in familial erythrophagocytic lymphohistiocytosis
Authors: Ladisch S , Ho W , Matheson D , Pilkington R , Hartman G
Published: October 2019

Depressed cellular immune function and increased susceptibility to infection characterize familial erythrophagocytic lymphohistiocytosis (FEL), a usually fatal autosomal recessive disease. One component of the immunodeficiency is plasma-mediated inhibition of lymphocyte proliferation. We have tested whether repeated plasma or blood exchange would decrease plasma inhibitory activity and improve cellular immune function in FEL. Following this treatment, reduction in plasma inhibitory activity, reversal of depressed antigen-specific lymphocyte proliferative responses and monocyte antibody-dependent cytotoxic function in vitro, and clinical improvement were complete in two and partial in one of three patients studied. Relapse, which was ultimately fatal, was associated with recurrence of the immune defects. These findings suggest that cellular immunodeficiency in FEL is acquired and possibly related to circulating immunosuppressive activity, the removal of which is associated with transient immunologic and clinical recovery.
Karyotypic polymorphism in acute myelofibrosis
Authors: Shah I , Mayeda K , Koppitch F , Mahmood S , Nemitz B
Published: October 2019

Acute myelofibrosis (AMF) was diagnosed in a 59-yr-old black male in September 1978, on the basis of pancytopenia, lack of hepatosplenomegaly, fibrosis of the marrow, and paucity of teardrop red blood cells in the peripheral blood. Since then the patient has demonstrated an unusually long survival of 36 mo with a changing cytogenetic course. His initial 46, XY normal karyotype changed in 20 mo to trisomy 8, followed 1 yr later by 1:4 translocation in peripheral blood. Simultaneously with these changes, the fibrosis in the bone marrow progressively decreased, ultimately terminating in chronic granulocytic leukemia-like presentation with reversal to 46, XY karyotype. Fibroblast culture failed to show any evidence of cytogenetic abnormalities. The disappearance of fibrosis confirmed by trichrome and reticulin stains and lack of cytogenetic abnormalities in fibroblasts confirms the secondary role of fibrosis.
Glycogenolysis versus glucose transport in human granulocytes: differential activation in phagocytosis and chemotaxis
Authors: Weisdorf DJ , Craddock PR , Jacob HS
Published: October 2019

Granulocytes depend primarily on anaerobic glycolysis to supply the necessary energy for locomotion and chemotaxis. Either transmembrane transport of extracellular glucose or catabolism of intracellular glucose can supply glycolytic substrate. In this report, using enzymatic analysis of granulocyte glycogen, we describe conditional requirements for glycogenolysis, namely phagocytosis. With abundant extracellular glucose, granulocyte glycogen content (12.2 +/- 1.6 micrograms/10(6) cells) is not depleted whether or not incubations include various soluble chemotaxins (e.g., FMLP, C5ades arg, arachidonic acid). These chemotaxins accelerate transmembrane glucose uptake. With near complete absence (less than 6 mg/dl) of extracellular glucose, both resting and chemotaxin (FMLP, C5ades arg, arachidonic acid) stimulated granulocytes catabolize significant endogenous glycogen. Phagocytosis, however, fails to enhance glucose uptake and promotes glycogen consumption even with abundant extracellular glucose. Simple particle-phagocyte attachment without internalization (produced by cytochalasin-B) also promoted glycogen consumption, suggesting that this membrane deformation alone is a sufficient trigger for glycogenolysis. Resting or chemotactic granulocytes, therefore, can adapt their energy source pending extracellular glucose availability-- often compromised at inflammatory sites-while phagocytic cells depend primarily, if not exclusively, on endogenous glycogen stores. This differential metabolic activation defends the granulocytes energy supply and may be critical in supporting antimicrobial activity in acute inflammation.
Evidence for interaction between platelet fibrinogen receptors
Authors: Peerschke EI
Published: October 2019

Previous analysis of fibrinogen binding to human aspirin-treated gel- filtered platelets yielded upwardly concave Scatchard plots. To ascertain whether this was due to the presence of independent heterogeneous receptor populations binding fibrinogen with different affinities, the dissociation of purified 125I-fibrinogen from ADP- treated gel-filtered platelets was evaluated as a function of receptor occupancy. Dissociation of bound labeled fibrinogen was measured after 50-fold dilution with buffer containing O, 0.2, 0.8, and 2.0 mg/ml unlabeled fibrinogen. Dissociation of labeled fibrinogen increased with increasing receptor occupancy and was biphasic. With buffer alone, 76.0% +/- 5.8% (SD) of labeled fibrinogen dissociated in 30 min, with an initial rate of 0.392 +/- 0.175 min-1; with 0.2 mg/ml fibrinogen, 83.7% +/- 3.9% dissociated, with an initial rate of 0.589 +/- 0.044 min- 1; with 0.8 mg/ml, 91.8% +/- 1.3% of the labeled fibrinogen dissociated, with an initial rate of 0.910 +/- 0.028 min-1; and with 2.0 mg/ml fibrinogen, 97.3% +/- 2.3% of label dissociated, with an initial rate of 1.06 +/- 0.257 min-1 (n=5). The final rates of fibrinogen dissociation were unaffected by unlabeled fibrinogen in the dilution buffer and were not statistically different from the final dissociation rate of 0.015 +/- 0.10 min-1 observed following dilution with buffer alone. These results were neither an artifact of aspirin treatment or gel filtration, as similar observations were made using non-aspirin-treated washed platelets, nor were they an artifact of the purified fibrinogen preparations, because binding studies using whole plasma as the major source of fibrinogen also yielded upwardly concave Scatchard plots. Since the data demonstrate that the initial rate and extent of fibrinogen dissociation are dependent on fibrinogen receptor occupancy, they suggest receptor interactions possibly resulting from receptor clustering or crosslinking. Because the dissociation was biphasic, the results also suggest some heterogeneity among platelet- fibrinogen interactions.
Biochemical correlates of the differential sensitivity of subtypes of human leukemia to deoxyadenosine and deoxycoformycin
Authors: Matsumoto SS , Yu AL , Bleeker LC , Bakay B , Kung FH , Nyhan WL
Published: October 2019

Leukemic cells incubated in vitro with 2′-deoxyadenosine (dAdo) plus an inhibitor of adenosine deaminase, 2′-deoxy-coformycin (DCF), show different metabolic responses depending on the histologic and immunologic type of the leukemia. Leukemic cells were obtained from 54 patients with acute lymphoblastic leukemia (ALL), 9 with myeloid or nonlymphoblastic leukemia, 3 with chronic lymphocytic leukemia (CLL), and 3 with lymphoma. There was a wide variation in the LD50, the concentration of dAdo that caused 50% inhibition of the incorporation of 3H-thymidine into cells in the presence of 20 microM DCF. T-cell leukemia specimens were much more sensitive to dAdo than were specimens of pre-B-ALL and null-ALL. In leukemic cells that had been incubated with 14C-dAdo plus DCF, a good correlation was observed between the LD50 and the ratio of 14C-deoxyATP to ATP (correlation coefficient for the fit to a hyperbola = 0.853). The accumulation of deoxyATP by the leukemic cell specimens was correlated best with the activity of ecto- ATPase, less well with cytoplasmic 5′-nucleotidase and deoxyadenosine kinase, and poorly with adenosine deaminase and ecto-5′-nucleotidase. The clinical response to DCF therapy of a patient with T-ALL and another with pre-B-ALL was consistent with the in vitro metabolic response of their cells to DCF and dAdo.
Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets
Authors: Fernandez MF , Ginsberg MH , Ruggeri ZM , Batlle FJ , Zimmerman TS
Published: October 2019

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.
Acute leukemia associated with the t(4;11) chromosome rearrangement: ultrastructural and immunologic characteristics
Authors: Parkin JL , Arthur DC , Abramson CS , McKenna RW , Kersey JH , Heideman RL , Brunning RD
Published: October 2019

The acute leukemia associated with the t(4;11) chromosome rearrangement is characterized by relatively consistent clinical features: occurrence primarily in young individuals, hyperleukocytosis, and poor response to therapy. This study describes the morphological, ultrastructural, and immunologic characteristics of the leukemic cells from ten patients with this type of leukemia. The morphological features of the leukemic blasts vary from lymphoid-appearing to monocytic. Ultrastructurally and cytochemically, some of the lymphoid-appearing blasts possess features of myeloid origin. The immunologic phenotype is characteristically E- SIg- CALLA- BA-1- BA-2+ HLA-DR+ and TdT+. These findings suggest that the t(4;11)-associated acute leukemia represents a proliferation of an early myeloid progenitor cell.
Adipocyte development and the loss of erythropoietic capacity in the bone marrow of mice after sustained hypertransfusion
Authors: Brookoff D , Weiss L
Published: October 2019

In this electron microscopic study, erythropoiesis in mice was completely suppressed by repeated hypertransfusion for up to 6 wk. We describe a sequence of ultrastructural changes in the marrow's stromal cells that accompany the resulting shift from erythropoietic to granulopoietic tissue. These include the destruction of medullary macrophages, the accelerated development of marrow adipocytes and reticular cells, and a reduction in the amount of vascular space in the marrow. The absence of macrophages was highlighted by the complete lack of erythrophagocytosis in the marrows of hypertransfused mice that were injected with the hemolysing agent, phenylhydrazine. The changes in the marrow stroma probably underlie the shift in the marrow's hematopoietic microenvironment. Repeated phlebotomy of mice that had been hypertransfused for 2 wk evoked the appearance of unique stromal cells in the marrow, similar to cells that have been associated with accelerated erythropoiesis. The newly anemic mice were otherwise unable to mount an erythropoietic response to repeated bleeding, showing that the decline in the erythropoietic microenvironment brought on by sustained hypertransfusion was a lasting one.
A quantitative method to measure human platelet chemotaxis using indium- 111-oxine-labeled gel-filtered platelets
Authors: Lowenhaupt RW , Silberstein EB , Sperling MI , Mayfield G
Published: October 2019

Human blood platelets have been shown to migrate directionally and specifically toward collagen in plasma in vitro. We have developed a new system to monitor this behavior using a linear 7-compartment chamber with 111In-oxine-labeled gel-filtered platelets. The compartments are separated by various Nuclepore and Millipore filter membranes. Radiolabeled platelets suspended in plasma are placed in the central compartment and the other compartments are filled with platelet- free plasma. When collagen is added to an end compartment, platelets migrate toward that end. The degree of this directed movement or chemotaxis can be measured by counting the radioactivity of the contents of each compartment and then comparing the counts from radiolabeled platelets that have moved to the end that holds the chemotactic inducer with those that have randomly migrated to the opposite end, containing only plasma. This assay system allows quantitative comparisons between the chemotaxis-inducing abilities of different substances and permits the study of soluble materials. Experiments to determine the optimal conditions for the procedure are reported, and the advantages of this new method for the investigation of platelet chemotaxis and the identification of chemotaxins are discussed.
Expression of a myeloid marker on TdT-positive acute lymphocytic leukemic cells: evidence by double-fluorescence staining
Authors: Bettelheim P , Paietta E , Majdic O , Gadner H , Schwarzmeier J , Knapp W
Published: October 2019

The expression of a myeloid-specific antigen was detected on TdT- positive blast cell populations in two cases of childhood acute lymphocytic leukemia. Double-fluorescence staining by using the monoclonal antibody, VIM-D5, which is specific for cells of myeloid origin, in combination with TdT antiserum revealed that a distinct portion of the blast cells carried both markers. The finding represents the first direct demonstration of this specific biphenotype in leukemic cells and was interpreted as the abnormal expression of a myeloid antigen on lymphoid blast cells.
Childhood lymphoblastic leukemia with natural killer activity: establishment of the leukemia cell lines retaining the activity
Authors: Komiyama A , Kawai H , Miyagawa Y , Akabane T
Published: October 2019

Leukemic cells from a child with acute lymphoblastic leukemia (ALL) had high natural killer (NK) activity against K562 as determined by the 51Cr release assay at a 40:1 effector:target ratio: percent lysis was 76.8% (147.4% of normal lymphocyte value) and higher than that of control leukemic cells from 12 childhood ALL (0.1% +/- 0.3%). Two leukemic cell lines (SPI-801 and SPI-802) were established from the patient, and they were essentially the same as the freshly harvested leukemic cells in their morphology, cytochemistry, immunologic markers, and functions. The cultured cell lines as well as the fresh leukemic cells had receptors for sheep red blood cells, IgG-Fc, and C3. The cultured cells were OKM1+, Ia+ and asialo-GM1+, and were OKT-3-, OKT-4- , OKT-6-, OKT-8-, Leu-7-, human monocyte-, common ALL-, T cell-, surface Ig-, and cytoplasmic IgM- as determined by the immunofluorescence method. The two cell lines shared the same chromosome abnormalities. Their chromosomes were in hypotriploid region (63--73), and 6q-, 11p+, and several marker chromosomes were demonstrated. They had spontaneous cytotoxicity against K562 (percent lysis: up to 17.8% and 33.5% of normal lymphocyte value in SPI-801 and SPI-802, respectively) and Molt-3 (38.2% and 27.8% of normal lymphocyte value), but not against Raji and mitogen-induced normal lymphoblasts. Such phenotypic and functional characteristics of the fresh leukemic cells and cultured cells are virtually identical to those of NK cells, demonstrating a new phenotype of childhood ALL of NK cell origin.
Type IIB von Willebrand's disease: differential clearance of endogenous versus transfused large multimer von willebrand factor
Authors: Ruggeri ZM , Lombardi R , Gatti L , Bader R , Valsecchi C , Zimmerman TS
Published: October 2019

The abnormal multimeric composition of plasma von Willebrand factor in type IIB von Willebrand's disease is transiently corrected after infusion of 1-deamino-[8-D-arginine]-vasopressin. However, the larger multimers released into the circulation disappear more rapidly in these patients than in type I von Willebrand's disease or normals. We demonstrate that the larger multimers of normal von Willebrand factor transfused into a type IIB patient are cleared from the circulation more slowly than multimers of similar size endogenously released from tissue stores. The rate of disappearance of large von Willebrand factor multimers after infusion of cryoprecipitate is similar in IIB, IIA, and severe homozygous-like von Willebrand's disease. Platelets from the IIB patient exhibited normal ristocetin-induced binding of normal von Willebrand factor. However, like normal platelets, they bound IIB von Willebrand factor at lower ristocetin concentrations than required for normal von Willebrand factor. These findings provide evidence that absence of the larger multimers from IIB plasma is related to a molecular abnormality of von Willebrand factor rather than to enhanced affinity of abnormal tissue or cellular binding sites, as is the case in the recently described “pseud” von Willebrand's disease and “platelet-type” von Willebrand's disease.
Platelet-bound IgM in autoimmune thrombocytopenia
Authors: Nel JD , Stevens K , Mouton A , Pretorius FJ
Published: October 2019

Elevated levels of platelet-bound IgG (PA-IgG) are a feature of autoimmune thrombocytopenia (ATP), but it is well documented that this does not occur in all cases. This has led us to investigate the role of platelet-bound IgM (PA-IgM) in these patients using a quantitative enzyme-linked immunosorbent assay (ELISA). Forty-five determinations of PA-IgM and PA-IgG were done on 24 patients with ATP. Elevated levels of PA-IgM were found in 93.3% of the determinations, while PA-IgG was elevated in only 71.7%. In 64.4% of determinations, both were elevated. Elevated PA-IgM or PA-IgG alone occurred in 28.9% and 6.7% of determinations, respectively. These results show the hitherto unrecognized frequent involvement of PA-IgM in ATP and suggests that a complex interrelationship exists between the two immunoglobulin classes in ATP. Some of the possibilities that might explain this interrelationship are discussed.
Generalized hexokinase deficiency in the blood cells of a patient with nonspherocytic hemolytic anemia
Authors: Rijksen G , Akkerman JW , van den Wall Bake AW , Hofstede DP , Staal GE
Published: October 2019

In a patient with nonspherocytic hemolytic anemia, a hexokinase deficiency was detected in the red cells (residual activity about 25% of normal) and in blood platelets (20%-35% of normal activity). Although the total hexokinase activity in lymphocytes was normal, the amount of hexokinase type I was decreased to about 50% of normal. However, the deficiency was compensated for by the appearance of type III hexokinase. Compartmentation studies with controlled digitonin- induced cell lysis showed that this type III enzyme was localized in the cytosol, while almost all hexokinase activity in normal lymphocytes is particulate. No abnormal lymphocyte functions could be detected. The patient was homozygous for the defect. The parents and three of five sibs of the patient were apparently heterozygous with residual activities of 50%-67% of normal in their red cells, but did not show any clinical signs of hexokinase deficiency. The variant enzyme had a slightly decreased affinity for MgATP2- and a strongly increased inhibition constant for glucose-1,6-P2. Affinity for glucose, heat stability, and pH optimum were normal. In the electrophoretic pattern of red cell hexokinase, only one subtype of hexokinase I could be detected, while in normal red cells, at least three subtypes are present. In the heterozygous individuals, no enzymatic abnormalities could be detected, except for an aberration in the electropherogram of one sib.
Augmentation by platelets of granulocyte aggregation in response to chemotaxins: studies utilizing an improved cell preparation technique
Authors: Redl H , Hammerschmidt DE , Schlag G
Published: October 2019

Considerable evidence exists to suggest roles for both platelets and granulocytes (PMNs) in pulmonary injury in shock. While believing that the major contributions of the two cell types are sequential, in vitro observations suggested that direct interactions between granulocytes and platelets might also amplify tissue damage. Using isotonic Percoll density gradients to isolate PMNs, we therefore studied the effect of deliberate platelet contamination on PMN aggregation. PMN aggregation in response to N-formyl-met-leu-phe or activated complement was enhanced by the presence of 1 platelet/PMN, an effect that became maximal at 16 platelets/PMN (p less than 0.01); large mixed aggregates were formed. Lysed, aspirinated, and indomethacin-treated platelets retained their augmentative capacity, as did platelets washed by gel filtration. The effect was not mimicked by the addition of histamine or serotonin to PMN preparations. None of these platelet preparations augmented lysosomal enzyme release. We conclude that platelets augment PMN aggregation, both by forming giant mixed PMN/platelet aggregates and also by producing a labile augmentative substance, the production of which may be independent of thromboxane synthesis. We propose that direct as well as sequential platelet/PMN interactions may be important in tissue injury in shock.
Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid, and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex
Authors: Di Minno G , Thiagarajan P , Perussia B , Martinez J , Shapiro S , Trinchieri G , Murphy S
Published: October 2019

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.
Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect
Authors: Lages B , Holmsen H , Weiss HJ , Dangelmaier C
Published: October 2019

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.
Passive participation of fixed platelets in aggregation facilitated by covalently bound fibrinogen
Authors: Agam G , Livne A
Published: October 2019

The role of fibrinogen in interplatelet recognition during aggregation was examined by combining two cell types: fresh platelets (in limiting density) activated by thrombin or A23187, and formaldehyde-fixed platelets, bearing cross-linked fibrinogen. The fixed platelets did not aggregate by themselves, nor with resting platelets, but were capable of interacting with activated platelets and of participating passively in aggregation. The participation, expressed by enhanced aggregation, was assayed by the conventional turbidometric traces and by cosedimentation of fixed 3H-platelets with aggregates of fresh platelets. Platelet suspensions, prepared without special means to avert spontaneous activation, retained plasma fibrinogen to the extent of 50 micrograms/ml of a suspension containing 10(8) platelets, and the derived fixed platelets participated in aggregation, independently of added fibrinogen. The capability of such fixed platelets to participate in aggregation was sensitive to proteolytic digestion and to massive acetylation. When platelet separation was aided by apyrase or aspirin, PGE1 and gel filtration, the residual plasma fibrinogen was limited to 0.4 micrograms/ml of 10(8) platelet suspension. The derived fixed platelets were incapable of participating in aggregation unless fibrinogen was added prior to fixation. The affixed fibrinogen could not be replaced by soluble fibrinogen or affixed albumin. It is concluded that fibrinogen, which binds to platelets upon activation or is linked to them covalently, is a recognition site for platelet- platelet interaction during aggregation.
Chromosome aberrations in T lymphocytes carrying adult T-cell leukemia- associated antigens (ATLA) from healthy adults
Authors: Fukuhara S , Hinuma Y , Gotoh YI , Uchino H
Published: October 2019

Chromosomes were studied in cultured T lymphocytes carrying adult T- cell leukemia-associated antigens (ATLA) that were obtained from five Japanese anti-ATLA seropositive healthy adults. Chromosomally abnormal cells were observed in three of the five healthy adults, and these cells were clonal in two subjects. All cells examined in one subject had rearrangements of chromosome nos. 7 and 14. Clonal cells from the second had a minute chromosome of unknown origin. A few cells in the third had nonclonal rearrangements of chromosomes. Thus, ATLA-positive T lymphocytes in some anti-ATLA seropositive healthy people have chromosome aberrations.
Characterization of non-human primate antisera to acute lymphoblastic leukemia (ALL): evidence for unique antigen(s) on childhood ALL of "T" phenotype
Authors: Mohanakumar T , Coffey TW , Vaughn MP , Russell EC , Conrad D
Published: October 2019

A non-human primate antiserum was prepared to acute lymphoblastic leukemia of T-cell phenotype (T-ALL) and, after absorptions with normal blood elements, reacted by immunofluorescence and microcytotoxicity to all the T-ALL tested. In addition, the antiserum reacted with cells from about 70% of the common ALL studied and immunoprecipitated the common ALL antigen of 100,000 daltons. However, when the anti-T-ALL serum was absorbed with with lymphoblasts from common ALL, it failed to react with common ALL lymphoblasts, yet reacted significantly with cells from patients with T-ALL phenotype and defined a 100,000-dalton membrane component not found on common ALL lymphoblasts. In addition, sequential immunoprecipitation of 125I-labeled T-ALL membranes by anti- common-ALL serum followed by anti-T-ALL serum detected the T-ALL membrane component of 100,000 daltons that was not found on common ALL. Thus, our results demonstrate the presence of of a unique human T-ALL antigen present on all T-ALL distinct from the common ALL antigen.
Endogenous protein phosphorylation by resting and activated human neutrophils
Authors: Andrews PC , Babior BM
Published: October 2019

NADPH oxidase is an enzyme in the plasma membrane of the neutrophil that catalyzes the production of O2-, a species central to the oxygen- dependent killing mechanisms of this cell. The oxidase is dormant in resting cells and is activated upon the addition of a stimulus. Neutrophils of patients with chronic granulomatous disease (CGD) manifest no oxidase activity when stimulated. The possible role of protein phosphorylation in the activation of NADPH oxidase was examined in normal and CGD neutrophils by measuring the incorporation of 32Pi into proteins as determined by gel electrophoresis followed by autoradiography. Resting neutrophils from normal subjects exhibit at least 40 distinct phosphoprotein bands. The level of phosphorylation of these bands was examined after the addition of phorbol myristate acetate (PMA), opsonized zymosan, digitonin, N-formyl-methionyl- phenylalanine (FMLP), or NaF. PMA and opsonized zymosan increased the phosphorylation of a set of 6 protein bands. Digitonin and FMLP consistently caused the phosphorylation of 4 of these protein bands, while NaF failed to induce increased phosphorylation of any protein band. All activators tested caused the dephosphorylation of one specific protein band. The time course of phosphorylation (dephosphorylation) was examined using PMA as the activating agent. Increased phosphorylation of one protein band was evident by 12 sec after the addition of PMA. The most slowly phosphorylated protein band did not slow evidence of change until 5 min after the addition of PMA. Three of the phosphoproteins examined were phosphorylated either earlier than or concomitant with the activation of NADPH oxidase. CGD neutrophils were compared with normal cells for their ability to phosphorylate proteins in response to PMA. The phosphoprotein banding patterns of CGD neutrophils were identical with those of normal neutrophils in both the resting and activated states. The evidence presented shows that the phosphorylation of proteins is a prominent feature of neutrophil metabolism. The striking similarity of phosphorylation changes induced by the various activators tested suggests that protein phosphorylation may play a role in some aspects of neutrophil activation. Evidence was not obtained, however, regarding a link between protein phosphorylation and activation of NADPH oxidase.