bluuebunny/crossref_metadata_2025 huggingface dataset:
Fibrinolysis during liver transplantation in humans: role of tissue- type plasminogen activator
Authors: Dzik WH , Arkin CF , Jenkins RL , Stump DC
Published: October 2019

Human liver transplantation is frequently associated with a coagulopathy and bleeding diathesis developing during the anhepatic phase of surgery. The hemostatic defect has been attributed in part to accelerated fibrinolysis. In this study we evaluated changes in specific blood fibrinolytic parameters occurring in eight adult patients undergoing first-time orthotopic liver transplantation. Five of the eight patients experienced moderate to severe systemic fibrinolysis as reflected by alpha 2-antiplasmin consumption and fibrinogen degradation with the concomitant appearance of fibrin(ogen) degradation products. In association with these changes, an increase in tissue-type plasminogen activator (t-PA) activity and t-PA antigen levels was also observed. Fibrinolysis was most pronounced during the anhepatic phase of surgery and decreased after revascularization of the grafted liver. Three additional patients who underwent the same procedure manifested much less evidence of systemic fibrinolytic activation and had minimal elevation of t-PA antigen levels or activity. Urokinase-type plasminogen activator levels, although elevated in three patients, were disassociated from increased t-PA levels and concomitant systemic fibrinolysis. The operative course of those patients developing t-PA-associated fibrinolysis was characterized by shock, acidosis, generalized bleeding, and a need for substantially greater blood product support during surgery. These findings suggest that the observed fibrinolytic defect is related to increased circulating plasma levels of t-PA, presumably resulting from a combination of increased intravascular release and decreased hepatic clearance of t-PA. These observations may have implications for intraoperative therapy for the transplant-related coagulopathy and its associated bleeding.
Gamma gene promoter and enhancer structure in Seattle variant of hereditary persistence of fetal hemoglobin
Authors: Gelinas RE , Rixon M , Magis W , Stamatoyannopoulos G
Published: October 2019

A variant of hereditary persistence of fetal hemoglobin (HPFH), first described in a patient from Seattle, was studied by structural analysis of the gamma-globin genes. A family study suggested that the determinant for this form of HPFH, in which the HbF contains both G gamma- and A gamma-globin chains, segregated with the beta S gene. No deletions or other abnormalities were detected in the fetal to adult globin gene region by genomic mapping studies. All four gamma-globin genes were isolated from a cosmid library, and allelic pairs of gamma- globin genes were distinguished by linkage to either the beta S- or beta A-globin gene. Nucleotide sequence analysis of the four gamma- globin gene promoters revealed a total of three discrepancies compared with a reference sequence, but these were judged unlikely to be the underlying determinants. Sequence analysis of the enhancer region located 3′ to the A gamma-globin gene from the putative HPFH chromosome revealed three base substitutions, whereas this region was normal in the A gamma-globin gene linked to the beta A gene. These data raise the possibility that an alteration of enhancer function rather than promoter function could be the basis for this condition.
Discordant effect of interferon on natural killer activity and tumor cell sensitivity to lysis in hairy cell leukemia
Authors: Lahat N , Aghai E , Kotler A , Kinarty A , Sobel E , Gruener N , Froom P
Published: October 2019

We studied the action of alpha-interferon (IFN) and interleukin-2 (IL- 2) on natural killer (NK)-rich fractions and autologous tumor cells from two patients with hairy cell leukemia (HCL). The addition of IFN or IL-2 to the NK-rich fractions resulted in a significant increase in NK activity against the autologous tumor cells. This stimulatory effect was blocked if the target hairy cells (HCs) were preincubated with either IFN or IL-2. Pretreatment of the HCs with anti-Tac antibody entirely prevented the blocking effect of IL-2 and partially the blocking effect of IFN. One patient was treated with recombinant alpha c-IFN. After 2 months there was a dramatic reduction in the number of HCs in the peripheral blood coincident with the loss of the protection effect of IFN against NK lysis of the patient's HCs. NK activity against autologous tumor cells correlated poorly with that against the K562 cell line. We conclude that there is a discordant effect of IFN and IL-2 on NK activity and HC sensitivity to lysis. The Tac receptor appears to play a role in this sensitivity. Caution should be exercised in extrapolating the effects of NK activity against K562 cells to those on HC targets.
Independent prognostic significance of a nuclear proliferation antigen in diffuse large cell lymphomas as determined by the monoclonal antibody Ki-67
Authors: Grogan TM , Lippman SM , Spier CM , Slymen DJ , Rybski JA , Rangel CS , Richter LC , Miller TP
Published: October 2019

To assess the prognostic significance of the growth fraction in diffuse large cell lymphoma (DLCL), we studied 105 DLCL patients with the monoclonal antibody Ki-67 applied to frozen tissue sections. Ki-67 detects a nuclear antigen associated with cell proliferation not found in resting cells. Ki-67 findings and other clinical prognostic factors were correlated with outcome using univariate and multivariate analyses in the proportional hazards model. High proliferative activity, defined as nuclear Ki-67 expression in greater than 60% of malignant cells (Ki- 67 greater than 60), was found to be a strong predictor of poor survival among these patients (P = .003, log-rank). The 19 patients with Ki-67 greater than 60% had a median survival of 8 months compared with a median survival of 39 months for the 86 patients with Ki-67 less than or equal to 60%. Examination of pretreatment clinical variables indicated the patient groups were similar with regard to age, sex, stage, B symptoms, tumor bulk, and lactate dehydrogenase (LDH). Both patient groups received comparable curative intent therapy and showed comparable complete response rate precluding treatment differences as modifying outcome. Multivariate analysis indicated Ki-67 is an independent predictor of survival (multivariate P = .006). Further statistical analysis using only B-cell DLCL patients treated with CHOP (63 patients) indicated that Ki-67 greater than 60 retained strong prediction of poor outcome (P = .002, log-rank) among this homogeneous group. We conclude that high proliferative activity (Ki-67 greater than 60) is an independent factor allowing laboratory prediction of probable poor outcome of DLCL.
Selective generation of erythroid burst-promoting activity by recombinant interleukin 2-stimulated human T lymphocytes and natural killer cells
Authors: Skettino S , Phillips J , Lanier L , Nagler A , Greenberg P
Published: October 2019

Because T lymphocytes and natural killer (NK) cells produce a variety of growth factors and interleukin 2 (IL2) modulates the activity of both, we assessed the ability of IL2 to stimulate human T cells and NK cells to produce hematopoietic growth factors detectable in clonogenic marrow culture. Human recombinant interleukin 2 (rIL2) added directly to cultures of human bone marrow that had been depleted of monocytes or depleted of both monocytes and T cells caused no significant alteration of myeloid (CFU-GM) or erythroid colony formation. Conditioned media harvested from rIL2-stimulated (greater than 100 U/mL) peripheral blood mononuclear cells, T cells, Leu-2 cells, and Leu-3 cells all had erythroid burst-promoting activity (BPA) but lacked myeloid colony- stimulating factor (GM-CSF) or CFU-GM-inhibitory activity. These T cells were IL2 receptor-negative, and the addition of anti-IL2 receptor monoclonal antibody (anti-Tac) to T cell cultures did not abrogate this IL2-stimulated BPA production. In addition, Percoll gradient-enriched, large granular lymphocytes (LGL) were separated by fluorescence- activated cell sorting into Leu-11+ (NK) cells and Leu-11- (low-density Leu-4+ T) cell fractions. rIL2 stimulated LGL, Leu-11+ and Leu-11- cells to produce BPA but not detectable GM-CSF or CFU-GM-inhibitory activity. Leu-11+ (NK) cells were Tac-negative from days 0 through 14 of culture. We conclude that rIL2 at high concentrations stimulated T cells, Leu-2 and Leu-3 cell subsets, LGL, and NK cells to produce BPA but not GM-CSF and that this stimulation may be mediated by an IL2 receptor distinct from Tac or by an epitope of the IL2 receptor not recognized by the anti-Tac antibody.
Slow disappearance of peripheral blast cells: an independent risk factor indicating poor prognosis in children with acute lymphoblastic leukemia
Authors: Rautonen J , Hovi L , Siimes MA
Published: October 2019

The aim of this study was to find out whether the time required for disappearance of peripheral blast cells, or blast clearance, could be used to identify patients with a slow response to treatment associated with a poor prognosis of acute lymphoblastic leukemia (ALL). Our series consisted of 158 children with newly diagnosed ALL. The mean follow-up time was 69 months (range 22 to 140 months). Blast clearance was significantly associated with length of event-free survival. Only two of nine children with blast clearance greater than or equal to 2 weeks and 4 of 11 children with blast clearance of 11 to 13 days were in remission at the time of analysis as compared with 86 of 138 of the children with more rapid blast clearance. The respective 5-year event- free survivals were 17%, 36%, and 60% (P = .003). Multivariate analysis showed that the relative risk of death or relapse in patients with blast clearance of greater than 10 days was 5.2-fold (95% confidence limits 2.1 to 13.1) as compared with the others (P less than .001). Our results indicate that patients with a slow response to treatment can be identified by simple differential peripheral cell counts during the early induction phase well before or even instead of performance of a more invasive bone marrow aspiration.
Granulocyte/macrophage colony-stimulating factor stimulates monocyte and tissue macrophage proliferation and enhances their responsiveness to macrophage colony-stimulating factor
Authors: Chen BD , Clark CR , Chou TH
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a specific humoral growth factor that stimulates both neutrophilic granulocyte and macrophage production by bone marrow hematopoietic progenitor cells. GM-CSF also stimulates the proliferation and clonal growth of both tissue macrophages and blood monocytes. Although at low concentrations GM-CSF was unable to support the long-term growth of tissue macrophages, it greatly enhanced their responsiveness to macrophage CSF (M-CSF, or CSF-1). This effect was also observed by treating macrophages with GM-CSF for a short time. GM-CSF did not compete with M-CSF for binding to M-CSF receptors nor was it inactivated by treatment with anti-M-CSF antiserum. Treatment of tissue macrophages with GM-CSF led to a rapid but transient downregulation of M-CSF receptors; prolonged incubation at 37 degrees C, however, resulted in a restoration and upregulation of M-CSF receptors. Identical effects were observed with both native or recombinant GM-CSF. This study suggests that GM-CSF regulates tissue macrophage production by two modes of action: (a) direct stimulation of macrophage proliferation, and (b) enhancement of their responsiveness to M-CSF.
Flow cytometric analysis of normal human megakaryocytes
Authors: Tomer A , Harker LA , Burstein SA
Published: October 2019

Megakaryocytes from normal routine human bone marrow aspirates were analyzed by flow cytometry for size, fine cell structure and granularity, membrane expression of glycoprotein (GP) IIb/IIIa and ploidy. Marrow cells were initially enriched for megakaryocytes by a Percoll density gradient and megakaryocytes were labeled with a fluoresceinated monoclonal antibody directed to the GPIIb/IIIa complex. The cells were fixed with paraformaldehyde and stained with propidium iodide (PI) for DNA quantitation. Using two-color flow cytometry, megakaryocytes were identified by their high membrane immunofluorescence and their ploidy was determined according to the relative fluorescence intensity of the PI. Forward light scatter (FSC), correlating with cell size, 90 degrees side light scatter (SSC), reflecting primarily cell internal fine structure and granularity, and total cell membrane fluorescence were examined. To evaluate independently the relationship between size and cell membrane fluorescence obtained by flow cytometry, megakaryocytes were sorted directly on slides and analyzed by a laser-based anchored cell analyzer (ACAS). There was a strong correlation among size, SSC, and the level of membrane fluorescence. The mean diameter of megakaryocytes was 28.1 +/- 12.3 micron. The modal ploidy distribution was 16N with approximately one-fifth of the cells less than or equal to 4N. The mean FSC and SSC levels increased with increasing ploidy. However, the marked overlap observed between the ranges of these parameters in adjacent ploidy classes suggested that size and SSC increase continuously rather than by discrete steps as is characteristic for ploidy. The total surface membrane fluorescence was correlated with cell size (r = 0.98) as measured by FSC or directly by the ACAS (r = 0.85), and with cell ploidy (r = 0.99) indicating an augmentation in total membrane GPIIb/IIIa expression with an increase in cell size and ploidy. However, estimated GPIIb/IIIa fluorescence density was inversely correlated with FSC suggesting that the GPIIb/IIIa surface epitope density is decreased with increasing cell maturity. We conclude that flow cytometry is a useful technique for the rapid analysis of human megakaryocytes obtained by marrow aspiration, and should be applicable to studies of pathologic states.
Immunocytochemical localization of fibrinogen during thrombin-induced aggregation of washed human platelets
Authors: Suzuki H , Kinlough-Rathbone RL , Packham MA , Tanoue K , Yamazaki H , Mustard JF
Published: October 2019

Because thrombin aggregates afibrinogenemic platelets and platelets from patients with the gray platelet syndrome and because antibodies to fibrinogen inhibit thrombin-induced aggregation only at low concentrations of thrombin, the role of fibrinogen in the formation of thrombin-induced aggregates was investigated further with human platelets washed and resuspended in Tyrode-albumin solution containing apyrase, either with or without added Ca2+ (2 mmol/L). Samples for immunocytochemical assessment of fibrinogen distribution were taken at several times (up to five minutes) after aggregation induced by 0.5 U/mL of thrombin. Glutaraldehyde-fixed samples were embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. By 10 seconds, small aggregates formed, and granules were centralized; alpha granules were heavily labeled with immunogold, but the platelet surface was not. As large aggregates formed, granule swelling or fusion occurred, and in some areas granule material seemed to be in contact with the exterior. In these experiments with no added fibrinogen, there were some clusters of gold particles on the platelet surfaces remote from sites of granule discharge, but there were large areas where platelets were in close contact with little or no fibrinogen detectable between them. No fibrin was visible up to five minutes after the addition of thrombin, which indicated that fibrinogen from the granules does not readily become available for fibrin formation in the ambient fluid. Similar results were obtained in media with and without added Ca2+. Thus at least some aggregation in response to thrombin can occur without the participation of released fibrinogen, and much of the granule fibrinogen appears to remain localized at sites where granules fuse with the plasma membrane or the open canalicular system. Incubation of unstirred samples with thrombin for ten minutes resulted in the formation of small aggregates, extensive gold label in regions connected to the exterior of the platelets, but very little gold labeling of the platelet membrane and no visible fibrin formation. When the platelets were aggregated in the presence of external fibrinogen, the morphological changes within the platelets were the same, but fibrinogen rapidly became associated with the entire platelet surface, and visible fibrin formed within 30 seconds in the medium containing 2 mmol/L Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS).
Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe
Authors: Wu QY , Bahnak BR , Coulombel L , Kerbiriou-Nabias D , Drouet L , Pietu G , Meulien P , Pavirani A , Caen JP , Meyer D
Published: October 2019

To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.
A simple approach to prenatal diagnosis of beta-thalassemia in a geographic area where multiple mutations occur
Authors: Cai SP , Zhang JZ , Huang DH , Wang ZX , Kan YW
Published: October 2019

We describe a simple approach for detecting beta-thalassemia mutations in geographic areas such as southern China where multiple mutations are known to occur. Segments of the beta-globin gene were amplified in vitro by using the polymerase chain reaction. Dot blot hybridization of the amplified DNA with oligonucleotide probes corresponding to the six mutations found in southern China could directly identify the mutations causing beta-thalassemia in the affected families. The increased number of target sequences after amplification allows the use of 35S-labeled probes, which are reusable for up to 3 months. The mutations can be determined in two days.
Hereditary poikilocytic anemia associated with the co-inheritance of two alpha spectrin abnormalities
Authors: Iarocci TA , Wagner GM , Mohandas N , Lane PA , Mentzer WC
Published: October 2019

This report describes a black family in which two distinct structural defects of alpha spectrin were inherited singly and in combination. The propositus, who has a poikilocytic hemolytic anemia that shares many of the features of hereditary pyropoikilocytosis (HPP) or homozygous elliptocytosis, is a compound heterozygote for both the spectrin alpha 1/65 and spectrin alpha 1/50a defects as demonstrated by electrophoretic analysis of spectrin tryptic fragments. The spectrin alpha 1/65 defect alone was found in his mother and sibling, while the spectrin alpha 1/50a defect was present in the father and another sibling. The red cell spectrin content was normal in all family members. The functional consequences of inheritance of these two spectrin defects were compared with those found in an unrelated patient with classic HPP who had the alpha 1/50a spectrin defect and was spectrin deficient as well. Prolonged incubation at 37 degrees C resulted in striking budding, fragmentation, and sphering of classic HPP red cells but only minimal changes in propositus cells. The percentage of spectrin dimers was increased tenfold in classic HPP, sevenfold in the propositus, and threefold in other family members. Mechanical stability of erythrocyte ghosts, measured by ektacytometry, was reduced severely in both classic HPP and in the propositus, but only moderately in other family members. Thus, co-inheritance of two alpha spectrin defects can result in a poikilocytic hemolytic anemia milder than that usually found in HPP. The greater clinical severity of HPP may be a consequence of the presence of spectrin deficiency, a finding absent in the propositus.
Prevention of refractoriness and HLA-alloimmunization using filtered blood products
Authors: Sniecinski I , O'Donnell MR , Nowicki B , Hill LR
Published: October 2019

Depletion of leukocytes from all blood products may decrease the incidence of alloimmunization to HLA antigens present on the white cells and thus delay the onset of refractoriness to random donor platelet support. In order to test this hypothesis, 54 patients with hematologic malignancy or marrow aplasia were entered on a prospective randomized trial using cotton-wool filtration as a method of leukocyte depletion of red cell and platelet concentrates. Forty patients were considered evaluable; 20 patients received filtered products and 20 patients in the control group received standard unfiltered products. The filter was 99% efficient in removal of leukocytes (average number of WBC/platelet product, 6 X 10(6)). Platelet loss by this technique was 8%. Alloimmunization was assessed by detection of de novo formed lymphocytotoxic and platelet specific antibodies by microcytotoxicity test, Staph A protein radioimmunoassay, and solid phase red cell adherence test. In the group receiving filtered products, three of 20 (15%) patients developed lymphocytotoxic antibodies while ten of 20 (50%) patients in the control group developed cytotoxic antibodies (P = .01 by actuarial methods). Platelet antibodies were detected in seven of ten alloimmunized patients in the control group and three of three patients in the study group. Clinical evidence of refractoriness was seen in three of 20 patients in the filtered group and ten of 20 in the control group (P = .01 by actuarial methods). The cost of filtration was a fraction of the cost of a plateletpheresis product. Filtration appears to be an effective and economical method for reducing alloimmunization and clinical refractoriness to random donor platelets in patient receiving long-term transfusion support.
The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity
Authors: Fortier N , Snyder LM , Garver F , Kiefer C , McKenney J , Mohandas N
Published: October 2019

In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta- subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.
Cellular and molecular studies on infant null acute lymphoblastic leukemia
Authors: Katz F , Malcolm S , Gibbons B , Tilly R , Lam G , Robertson ME , Czepulkowski B , Chessells J
Published: October 2019

We have studied the cellular and molecular basis of eight cases of infant null acute lymphoblastic leukemia (ALL). All eight patients were under 9 months of age and presented with leukocyte counts in excess of 60 X 10(9)/L, organomegaly, and in two cases CNS infiltration. Although seven cases were morphologically classified as ALL, one patient had both lymphoid and myeloid features. Phenotypic analysis of leukemic blasts from all patients showed a typical null ALL pattern, ie, CD10 (common ALL antigen)-negative, strongly HLA-DR-positive, and CD19 (B4)- positive. The presence of terminal deoxynucleotidyl transferase (TdT) at presentation was positive in six patients' cells and negative in two. Two patients also expressed the myeloid-associated markers CD33 (MY9) and CD15 (TG1), and coexpression of CD19 and CD33 was confirmed in these two by using dual marker flow cytometry (fluorescence- activated cell sorting). Electron microscopic examination of the same two patients' cells showed the presence of monocytoid blasts that labeled with the pan-B cell antibody B4 (CD19). Short-term culture of one of these patients cells in the presence of phorbol ester resulted in the majority of the cells exhibiting myeloid markers, strong nonspecific esterase positivity, and phagocytic properties. Cytogenetic analysis showed the common feature in 7 of 8 cases to be a break in band 11q23. Molecular analysis of DNA from the blast cells of all eight patients showed rearrangement of the immunoglobulin heavy-chain genes in all cases without, however, any evidence of kappa light-chain rearrangement. T cell receptor genes were present in the germline configuration in all cases. Rearrangements of the c-ets 1 oncogene, which maps to band 11q23, were not detected, thus providing no evidence for involvement of this oncogene in the common disease process. Our data indicate that although infant null ALL may present as a heterogeneous disease the similarity of many features between cases suggests a common derivation from a precursor cell sharing phenotypic and genotypic features of both B and myeloid progenitor cells.
Lymphocyte aggregation in response to adrenergic stimulation
Authors: Hammerschmidt DE , Jeanneret C , Husak M , Lobell M , Jacob HS
Published: October 2019

A nonanemic chronic lymphocytic leukemia patient with nearly 500,000 lymphocytes/microL underwent leukapheresis when she presented with CNS symptoms and retinal vascular engorgement. Respiratory distress developed during the cell separator run, which led us to ask whether the procedure could have changed the adhesive properties of her cells. C5a desarginine, N-f-Met-Leu-Phe, adenosine diphosphate, and collagen all failed to aggregate her lymphocytes in vitro, but arachidonic acid, excess free calcium, and 4 mumol/L epinephrine did aggregate the cells. Arachidonate-induced aggregation appeared to be a toxic phenomenon: the ED50 for aggregation was statistically indistinguishable from that for cytotoxicity, and aspirin only mildly blunted the response. In contrast, epinephrine-induced aggregation was not associated with lactic dehydrogenase release or the loss of trypan blue exclusion and was blunted by propranolol; radiopindolol-binding studies confirmed the presence of a beta-adrenergic receptor. There were approximately 3,000 receptors/cell, with no statistically significant difference between normal and chronic lymphocytic leukemia B cells or between B cells and T cells (separated by rosetting techniques). The Kd for the B cells' receptor, however, was less than that for T cells by a factor of ten (P less than .01). We conclude that B cells may aggregate when stimulated and that they--like T cells--have beta-adrenergic receptors. Adrenergically mediated changes in B cell adhesiveness may play a role in regulating lymphocyte traffic; in the rare patient with truly enormous B cell counts, we postulate that they may be an occasional cause of morbidity.
Modulation of nitrosourea resistance in myeloid leukemias
Authors: Gerson SL , Trey JE
Published: October 2019

Drug resistance in myeloid leukemias may be mediated by an increased capacity to repair chemotherapy-induced DNA damage. Some tumor cell lines that are resistant to nitrosoureas contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase). This protects cells by removing cytotoxic, nitrosourea-induced O6-alkylguanine adducts. We measured the level of alkyltransferase activity in myeloid leukemic cells freshly obtained from patients to determine whether the alkyltransferase was an important factor in nitrosourea resistance in these cells and whether inactivation of this protein could sensitize leukemic cells to nitrosoureas. Myeloid leukemic cells from patients with acute nonlymphocytic leukemia and chronic myelogenous leukemia had higher levels of alkyltransferase than did myeloid precursors from normal donors (P less than .01). This difference did not appear to be due to the state of differentiation of the leukemic or normal cells. To show that this repair protein mediated nitrosourea resistance in leukemic cells, cells were treated with the modified base O6- methylguanine to selectively and irreversibly inactivate the alkyltransferase and then exposed to 1,3-bis (2-chloroethyl)-1- nitrosourea (BCNU). An 18-hour incubation in 0.5 mmol/L O6- methylguanine caused an 87% +/- 3.6% decrease in alkyltransferase activity in leukemic cells and a 73% +/- 8.6% decrease in normal myeloid precursors. After treatment with O6-methylguanine, clonogenic leukemic cells from ten different donors became much more sensitive to BCNU, with a decrease in the dose needed to reduce colony survival by 50% (LD50) of 6.3 +/- 1.4-fold. A lesser effect was seen on CFU-GM, BFU- E, and CFU-GEM where the LD50 decreased two- to threefold. These studies show that nitrosourea resistance in myeloid leukemic cells can be abrogated by inactivation of the DNA repair protein O6-alkylguanine- DNA alkyltransferase. This method of biochemical modulation of DNA repair will sensitize leukemic cells to nitrosoureas in vitro and has the potential of increasing the therapeutic index of nitrosoureas in this disease.
Gamma-interferon modulates human monocyte/macrophage transferrin receptor expression
Authors: Taetle R , Honeysett JM
Published: October 2019

Although circulating human monocytes do not express transferrin (Tf) receptors, cultured adherent blood cells display high-affinity Tf binding sites. In the present studies, effects of various cytokines and biologically active proteins on human monocyte/macrophage Tf receptors were investigated. After culture, Tf receptor expression by adherent blood cells was time dependent and plateaued by 7 days. The addition of interleukin-1 (IL-1), alpha-interferon (alpha-IFN), granulocyte/macrophage-colony stimulating factor (GM-CSF), or human IgG to macrophages cultured for 4 days did not alter Tf receptor expression. Fe-saturated, human Tf caused a significant, dose-dependent decrease in receptor expression. At a dose of 100 U/mL, gamma- interferon (gamma-IFN) significantly increased Tf receptor expression by macrophages cultured for 4 (230% +/- 51% of control) or 7 days (150% +/- 22%). Scatchard analyses showed increased binding sites but no change in receptor affinity. Northern and slot blot analysis of cellular mRNA from macrophages cultured for 4 to 7 days and exposed to gamma-IFN showed a two- to fivefold increase in Tf receptor mRNA, but less than or equal to 30% increase in beta-actin mRNA. Ferritin content of gamma-IFN-treated macrophages was 47% to 63% of control cells. Net uptake of 59Fe from Tf by gamma-IFN-treated cells was 10% to 17% of control, but uptake of radiolabeled Tf was comparable. When macrophages were labeled with 59Fe and then exposed to gamma-IFN, cell-associated Fe was reduced by 43%, indicating that gamma-IFN caused macrophage Fe release. gamma-IFN specifically modulates Tf receptor display by inducing Fe release and reducing cellular Fe content. Regulation of Tf receptor expression in macrophages is controlled by cellular Fe content and is thus similar to regulatory mechanisms in dividing cells.
2',3'-Dideoxyadenosine is selectively toxic for TdT-positive cells
Authors: Spigelman Z , Duff R , Beardsley GP , Broder S , Cooney D , Landau NR , Mitsuya H , Ullman B , McCaffrey R
Published: October 2019

The 2′,3′-dideoxynucleosides (ddNs) are currently undergoing clinical evaluation as antiretroviral agents in HIV-infected individuals. When phosphorylated, the ddNs (ddNTPs) function as chain-terminating substrate analogues with reverse transcriptase, thereby inhibiting HIV replication. These nucleoside analogues can also inhibit, by chain- terminating additions, the primitive lymphoid DNA polymerase, terminal deoxynucleotidyl transferase (TdT). To determine the effect of possible intracellular chain-terminating additions of ddNMPs by TdT, we exposed a series of TdT-positive and TdT-negative cell lines to 2′,3′- dideoxyadenosine (ddA), a representative ddN. At ddA concentrations 25- fold higher than required for inhibition of HIV replication, progressive dose-related cytotoxicity was observed in the TdT-positive cell lines. This was accentuated by the adenosine deaminase inhibitor Coformycin (CF), presumably by enhancing the intracellular generation of ddATP from ddA. A central role of TdT in mediating the ddA/CF cytotoxicity was suggested by studies in a pre-B-cell line rendered TdT positive by infection with a TdT cDNA-containing retroviral vector. After a 48-hour continuous exposure period to 250 mumol/L ddA and 30 mumol/L CF, 30% cell death was observed in the TdT-negative parental line, whereas 90% cell death was observed in the TdT-positive daughter line. Exposure of fresh TdT-positive leukemic cells to ddA/CF for 72 hours ex vivo resulted in cytotoxicity (six cases of acute lymphocytic leukemia [ALL]) while not affecting TdT-negative acute leukemic cells (six cases). We conclude that ddA/CF selectively damages TdT-positive cells, presumably by chain-terminating additions of ddAMP, and that this may have therapeutic relevance in TdT-positive malignant disease.
Biochemical characterization and purification of HILDA, a human lymphokine active on eosinophils and bone marrow cells
Authors: Godard A , Gascan H , Naulet J , Peyrat MA , Jacques Y , Soulillou JP , Moreau JF
Published: October 2019

We previously described a lymphokine termed HILDA (for human interleukin DA) produced by T-lymphocyte alloreactive clones after antigenic stimulation. This factor sustains the growth of a murine IL3- sensitive cell line (DA2). In addition, HILDA is a potent activator of eosinophils and displays a burst-promoting activity on human bone marrow. In the present study, HILDA was purified to homogeneity from T- cell clone supernatant using successively sequential concentration, concanavalin A (ConA) affinity chromatography with differential elution (alpha-D glucopyranoside and alpha-D mannopyranoside), high-performance liquid chromatography (HPLC) gel filtration and reverse-phase HPLC. The pure material appeared as a 38-kd glycoprotein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions. Biologic activity could be recovered from SDS- PAGE gel slices corresponding to the 38-kd band. We conclude from the specificity of the DA-2 cell line and biochemical characteristics described that this lymphokine is different from other known factors produced by human T lymphocytes.
In vivo modulation of myelopoiesis by prostaglandin E2. III. Induction of suppressor cells in marrow and spleen capable of mediating inhibition of CFU-GM proliferation
Authors: Pelus LM , Gentile PS
Published: October 2019

Intravenous (IV) injection of 0.1 to 10 micrograms of authentic prostaglandin E2 (PGE2) in intact steady-state mice induces a population of bone marrow and spleen cells having the capacity to suppress CFU-GM proliferation when admixed with normal bone marrow cells. Equivalent suppression of CFU-GM committed to monocytic as well as granulocytic differentiation was observed using colony-stimulating factors (CSFs) differing in their lineage specificities and by direct morphological analysis of proliferating clones. Kinetic analysis indicates that suppressive bone marrow cells appear within 2 hours after PGE2 injection, are maximal at 6 hours, and are no longer observed by 24 hours postinjection. Positive and negative selection studies using monoclonal antibodies indicate that the PGE2-induced suppressor cells react positively with anti-GMA 1.2, MAC1, and F4/80 monoclonal antibodies, suggesting a myeloid/monocytic origin. As few as 1,000 positively selected bone marrow or spleen cells were able to inhibit maximally normal CFU-GM proliferation by 50,000 control bone marrow cells. Suppression of normal CFU-GM can be substituted for by 24- hour cell-free supernates from unseparated bone marrow cells or GMA 1.2 or F4/80 positively selected marrow or spleen cells from PGE2-treated but not control mice. These supernates also inhibited BFU-E proliferation. Injection of as few as 2 million bone marrow cells from PGE2-treated mice into steady-state mice or animals hematopoietically rebounding following a sublethal injection of cyclophosphamide significantly suppressed total CFU-GM proliferation in recipient mice within 6 hours. In summary, these studies describe the detection of a novel hematopoietic control network induced by PGE2 in intact mice.
Reduction in pO2 decreases the fibrinolytic potential of cultured bovine endothelial cells derived from pulmonary arteries and lung microvasculature
Authors: Wojta J , Jones RL , Binder BR , Hoover RL
Published: October 2019

The effect of anoxia on the fibrinolytic potential of cultured endothelial cells derived from bovine pulmonary artery and bovine lung microvasculature was studied. Both cell types reacted with an increase in plasminogen activator inhibitor (PAI) activity and a decrease in the plasminogen activator (PA) activity in the media after incubation under anoxic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin autography and reverse fibrin autography indicated that the change in fibrinolytic potential was due to an impaired release of PA and not an increase in the production of PAI. Although anoxia did not affect the viability of the cells as judged by 51Cr release, their metabolism was influenced, which is reflected by increases in the levels of lactate in cell lysates and media. Furthermore, the effect of short-term anoxia on PA and PAI could not be reversed by reoxygenation for 24 hours. The results are discussed in terms of helping to explain the tendency of reocclusion after successful thrombolytic therapy, the development of pulmonary hypertension, and the thrombotic tendency of areas with an impaired circulatory supply.
Further specificity characterization of von Willebrand factor inhibitors developed in two patients with severe von Willebrand disease
Authors: Lopez-Fernandez MF , Martin R , Lopez-Berges C , Ramos F , Bosch N , Batlle J
Published: October 2019

Circulating inhibitors against von Willebrand factor (vWF) that show the properties of heterologous IgG antibodies have been described in a few patients with severe von Willebrand disease (vWD). The present study provides further characterization of inhibitors from two patients with severe vWD. Inhibitors in both, like polyclonal rabbit antibody, detected all sizes of multimers and the complex structure of each multimer from platelets and plasma of normal individuals as well as from plasma of patients with IIA, IIB, and IIC vWD. Both inhibitors and the rabbit antibody reacted mainly with the intact 225-Kd vWF subunit and the 189-H and 140-Kd fragments in contrast to monoclonal antibodies specific for vWF fragments that detected a higher relative proportion of 176-Kd fragment. Furthermore, all these antibodies recognized fragment III, although one inhibitor and rabbit polyclonal antibody reacted poorly and the other inhibitor did not react at all with reduced fragment II of vWF digested with Staphylococcus aureus V-8 protease. These data suggest that although human inhibitors from severe vWD patients may behave, to some extent, as polyclonal heterologous antibodies against native vWF, the former show striking differences in their target specificity as well as a much broader specificity than that described for human factor VIII inhibitors.
The effect of stem cell proliferation regulators demonstrated with an in vitro assay
Authors: Pragnell IB , Wright EG , Lorimore SA , Adam J , Rosendaal M , DeLamarter JF , Freshney M , Eckmann L , Sproul A , Wilkie N
Published: October 2019

Spleen colony formation after transplantation of bone marrow cells into irradiated mice has been used as an assay for hematopoietic stem cells (CFU-S), but has serious limitations intrinsic to an in vivo assay. In this report we describe experiments using an in vitro clonogenic assay that is especially suitable for studies of stem cell regulation as defined growth factors and normal untreated bone marrow can be used. We have demonstrated that the colony-forming cells have proliferative properties in common with CFU-S and respond to specific proliferation regulators previously detected using the spleen colony assay.
Value of beta 2-microglobulin level and plasma cell labeling indices as prognostic factors in patients with newly diagnosed myeloma
Authors: Greipp PR , Katzmann JA , O'Fallon WM , Kyle RA
Published: October 2019

Beta 2-microglobulin (beta 2M) has been proposed as a prognostic factor in multiple myeloma (MM), but beta 2M levels are reported to correlate with other prognostic indicators such as stage and creatinine level. This study addressed the independent prognostic values of these and other variables, including plasma cell labeling indices (LI), in patients with newly diagnosed MM. beta 2M levels were measured with an enzyme-linked immunosorbent assay. LI were determined with a [3H]thymidine autoradiography method. By multivariate analysis and Kaplan-Meier survival analysis, the uncorrected beta 2M level remained the most significant prognostic factor after adjustment for age. Stage and creatinine level were closely related to beta 2M level and were no longer predictive of outcome after adjustment for age and beta 2M. Plasma cell LI varied independently of beta 2M level and remained predictive. A subset of patients with plasma-blastic myeloma had poor survival since beta 2M level and plasma cell LI were high. By using beta 2M level and LI, three risk groups were defined: low (beta 2M less than 4 micrograms/mL and LI less than 0.4%, median survival 48 months); intermediate (beta 2M less than 4 micrograms/mL and LI greater than or equal to 0.4%, median survival 29 months); and high (beta 2M greater than or equal to 4 micrograms/mL, median survival 12 months). Such grouping may better identify MM patients who might benefit from new treatment regimens.
Rearrangement of T-cell delta locus in lymphoproliferative disorders
Authors: Tkachuk DC , Griesser H , Takihara Y , Champagne E , Minden M , Feller AC , Lennert K , Mak TW
Published: October 2019

Studies of lymphoproliferative disorders using immunoglobulin and T- cell receptor genes have contributed to our understanding of clonality and lineages of these disorders. In this study, we examined the rearrangement of the recently discovered T-cell delta chain genes in a variety of lymphoproliferative diseases. We show here that six of 14 T- cell lymphomas and five of 23 B-cell lymphomas or B-cell leukemia cell lines have rearranged the delta loci, while two of two hyperimmune reactions retain germline configuration within these genes. Seven of ten cases of AILD were rearranged, and Lennert's lymphoma, which has been previously described as a T-cell malignancy, also contains rearrangements in the delta chain genes (three of five). Large cell anaplastic lymphomas positive for the activation antigen CD 30 also contain rearrangement in about one-half (five of 11) of the tumors examined. Two of seven of the Hodgkin's lymphomas studied contained a rearrangement for this gene. This study indicates that this newly identified T-cell delta gene is useful in evaluating clonality but is not lineage specific. However, with only one exception (in 28 rearrangements), this gene rearranges in tumors with gamma and beta chain gene rearrangements, indicating that when used in conjunction with the other TcR genes, delta rearrangement may also be useful in evaluating lineages.
Erythrocyte glutathione S-transferase deficiency and hemolytic anemia
Authors: Beutler E , Dunning D , Dabe IB , Forman L
Published: October 2019

A patient with unexplained erythrocyte glutathione-S-transferase (GST) deficiency has been detected among 513 unrelated persons with hemolytic anemia. An otherwise healthy adult male, the deficient individual had a mild hemolytic anemia with splenomegaly, indirect hyperbilirubinemia, and cholelithiasis. Because he was adopted and childless, the hereditary nature of the defect could not be established. The residual enzyme activity was only about 15% of mean normal. Depletion of glutathione (GSH) from the cells by 1-chloro-2,4-dinitrobenzene (CDNB), a substrate for GST, was somewhat decreased in the red cells from the patient, suggesting that a functional defect existed. The kinetic properties of the residual enzyme and the ratio of activity to antigenicity were normal. Modest decreases in leukocyte and platelet GST activities were documented. Although a cause-and-effect relationship between the GST deficiency and hemolysis may exist, this cannot be proven in the absence of affected family members.
Upstream promoter mutation associated with a modest elevation of fetal hemoglobin expression in human adults
Authors: Gilman JG , Mishima N , Wen XJ , Kutlar F , Huisman TH
Published: October 2019

In hereditary persistence of fetal hemoglobin, Hb F (alpha 2 gamma 2) is elevated after birth. Screening of sickle cell patients has revealed a family with elevated Hb F and high A gamma values. The propositus was a sickle cell patient with approximately 25% Hb F and 68.4% A gamma. He was heterozygous for the Benin (#19) and Mor beta S haplotypes. Five AS relatives with the Mor haplotype had 2.5% +/- 0.9% fetal hemoglobin and 92.8% +/- 2.8% A gamma, whereas two with the Benin haplotype had normal fetal hemoglobin (0.5%). The Mor haplotype is thus associated with the elevated Hb F in this family. The 13-kilobase (kb) Bg/II fragment containing the G gamma and A gamma genes of the Mor haplotype was cloned, and the G gamma and A gamma promoters sequenced from -383 to beyond the Cap sites. The Mor G gamma gene was normal, but the A gamma gene had a unique C----T mutation at -202. A different mutation at -202 of G gamma (C----G) was previously detected by other researchers in association with considerably higher Hb F in AS cases (15% to 25%). These data suggest either that -202 mutations affect the G gamma and A gamma promoters differently or that different nucleotide substitutions at -202 have divergent effects. Alternatively, additional unknown mutations could cause the differences in gene expression.