bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil function and pyogenic infections in bone marrow transplant recipients
Authors: Zimmerli W , Zarth A , Gratwohl A , Speck B
Published: October 2019

In a consecutive entry trial, the incidence and time course of decreased neutrophil function was assessed in 20 patients treated with allogeneic bone marrow transplantation (BMT). The aim of the study was to assess the prognostic value of low neutrophil function for late pyogenic infections. Chemotaxis, superoxide production, and phagocytic- bactericidal activity were studied before and 2, 6, 9, and 12 months after BMT. Skin window migration was quantitatively assessed 2 months after BMT. Infectious complications were recorded prospectively with preset criteria during 1 year. Six of the 20 leukemic patients had defective neutrophil function before BMT. Two months after BMT all 10 patients with greater than stage II graft-versus-host disease (GVHD), and 6 of 10 patients with less than or equal to stage II GVHD had at least one decreased function. At this time, patients with subsequent pyogenic infections had lower chemotaxis (P less than .05), phagocytic- bactericidal activity (P less than .005), and superoxide production (P less than .025) than those without. Defective skin window migration and combined defects were predictive for late pyogenic infections. At 9 months all tests were normal in seven patients surviving without GVHD. In contrast, at 9 months three of three patients, and at 1 year two of three with chronic GVHD had still decreased neutrophil function. In conclusion, neutrophil function is frequently impaired during the first months after BMT. Combined neutrophil defects predispose to pyogenic infections and indicate the patient at risk.
Administration of interleukin-6 stimulates multilineage hematopoiesis and accelerates recovery from radiation-induced hematopoietic depression
Authors: Patchen ML , MacVittie TJ , Williams JL , Schwartz GN , Souza LM
Published: October 2019

Hematopoietic depression and subsequent susceptibility to potentially lethal opportunistic infections are well-documented phenomena following radiotherapy. Methods to therapeutically mitigate radiation-induced myelosuppression could offer great clinical value. In vivo studies in our laboratory have demonstrated that interleukin-6 (IL-6) stimulates pluripotent hematopoietic stem cell (CFU-s), granulocyte-macrophage progenitor cell (GM-CFC), and erythroid progenitor cell (CFU-e) proliferation in normal mice. Based on these results, the ability of IL- 6 to stimulate hematopoietic regeneration following radiation-induced hematopoietic injury was also evaluated. C3H/HeN female mice were exposed to 6.5 Gy 60Co radiation and subcutaneously administered either saline or IL-6 (1,000 micrograms/kg) on days 1 through 3 or 1 through 6 postexposure. On days 7, 10, 14, 17, and 22, femoral and splenic CFU-s, GM-CFC, and CFU-e contents and peripheral blood white cell, red cell, and platelet counts were determined. Compared with saline treatment, both 3-day and 6-day IL-6 treatments accelerated hematopoietic recovery; 6-day treatment produced the greater effects. For example, compared with normal control values (N), femoral and splenic CFU-s numbers in IL-6-treated mice 17 days postirradiation were 27% N and 136% N versus 2% N and 10% N in saline-treated mice. At the same time, bone marrow and splenic GM-CFC values were 58% N and 473% N versus 6% N and 196% N in saline-treated mice; bone marrow and splenic CFU-e numbers were 91% N and 250% N versus 31% N and 130% N in saline-treated mice; and peripheral blood white cell, red cell, and platelet values were 210% N, 60% N, and 24% N versus 18% N, 39% N, and 7% N in saline- treated mice. These studies demonstrate that therapeutically administered IL-6 can effectively accelerate multilineage hematopoietic recovery following radiation-induced hematopoietic injury.
Role of pertussis toxin-sensitive guanosine triphosphate-binding proteins in the response of erythroblasts to erythropoietin
Authors: Miller BA , Foster K , Robishaw JD , Whitfield CF , Bell L , Cheung JY
Published: October 2019

Human progenitor-derived erythroblasts have been recently shown to respond to erythropoietin (Epo) with an increase in intracellular free calcium concentration [Cac]. To explore the role of guanosine triphosphate (GTP)-binding proteins in mediating the rise in [Cac], single day 10 erythroid burst forming unit (BFU-E)-derived erythroblasts loaded with Fura-2 were pretreated with pertussis toxin (PT), stimulated with Epo, and [Cac] measured over 18 minutes with fluorescence microscopy coupled to digital video imaging. The [Cac] increase in day 10 erythroblasts stimulated with Epo was blocked by pretreatment with PT in a dose-dependent manner but not by heat- inactivated PT. These observations provided strong evidence that a PT- sensitive GTP-binding protein is involved. To further characterize the GTP-binding protein, day 10 erythroblast membrane preparations were solubilized, electrophoresed, and immunoblotted with antibodies specific for the known PT-sensitive G-protein subunits: the three subtypes of Gia (1,2, and 3) and Goa, Gia1 or Gia3 and Gia2 were identified but no Goa was found. To examine the influence of Epo on adenylate cyclase activity, day 10 erythroblasts were initially treated with Epo, isolated membrane preparations made, and cyclic adenosine monophosphate (cAMP) production by adenylate cyclase in membrane preparations in the presence of theophylline measured. Epo did not inhibit but significantly stimulated adenylate cyclase activity. However, the mechanism of increase of [Cac] appears to be independent of adenylate cyclase stimulation because treatment of erythroblasts with the cell-permeant dibutyryl cAMP failed to increase [Cac]. In summary, pertussis toxin blocks the increase in [Cac] in erythroblasts after Epo stimulation suggesting that this response is mediated through a pertussis toxin-sensitive GTP-binding protein. Candidate PT-sensitive GTP-binding proteins identified on day 10 erythroblasts were Gia 1, 2, or 3, but not Goa.
Differential and synergistic effects of human granulocyte-macrophage colony-stimulating factor and human granulocyte colony-stimulating factor on hematopoiesis in human long-term marrow cultures
Authors: Hogge DE , Cashman JD , Humphries RK , Eaves CJ
Published: October 2019

The ability of granulocyte-macrophage colony-stimulating factor (GM- CSF) and G-CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM- CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM- CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo.
Developmental expression of mRNAs encoding platelet proteins in rat megakaryocytes
Authors: Courtney MA , Stoler MH , Marder VJ , Haidaris PJ
Published: October 2019

The committed bone marrow megakaryocyte (MK) progenitor undergoes a series of highly regulated stages of development resulting in a large multi-nucleated platelet-producing cell. We studied the developmental expression of the mRNA for two alpha granule proteins, fibronectin (FN) and fibrinogen gamma chain (gamma-FIB), and a cytoskeletal protein, actin, in MKs from marrow of Sprague-Dawley rats. By the method of in situ RNA:RNA hybridization, we showed that mRNAs for the alpha granule proteins were expressed most abundantly in a population of 15-microns diameter promegakaryocytes and in cells as small as 10 microns whose identity as immature MKs was inferred by positive staining for platelet- and MK-specific markers. gamma-FIB and FN mRNAs were present in reduced abundance in a small proportion of intermediate MKs; however, little or no expression was seen in mature platelet-producing MKs. In contrast, high levels of actin mRNA were expressed predominantly in mature, multi- nucleated MKs, and less abundantly in the immature forms. These results suggest that FN and gamma-FIB are transcribed early in MK development to permit translation and packaging of the protein into alpha granules, after which transcription ceases. On the other hand, transcription of actin occurs continuously throughout development, with highest levels in mature platelet-producing MKs, in which actin is needed for shape changes and intracellular movement of organelles. Our data suggest that in situ RNA:RNA hybridization for platelet-specific markers will provide additional criteria by which to establish MK lineage in immature marrow progenitors.
T-cell translocation gene 1 (Ttg-1) encodes a nuclear protein normally expressed in neural lineage cells
Authors: McGuire EA , Davis AR , Korsmeyer SJ
Published: October 2019

We previously identified and cloned T-cell translocation gene 1 (Ttg- 1), a putative zinc finger protein, as a result of its deregulated expression in a T-cell acute lymphoblastic leukemia cell line (RPMI 8402) with a t(11;14)(p15;q11). We have now characterized its genomic organization and identified the major transcriptional start site to lie within an initiator-like motif. Ttg-1 is normally expressed in mouse brain and not in thymus. The mouse neuroblastoma cell line, N2a, also expresses Ttg-1. Antibodies raised against a TrpE-Ttg-1 fusion protein precipitate an 18-Kd nuclear protein from metabolically labeled 8402 cells. Immunofluorescence of N2a cells shows a nuclear pattern. The two potential zinc finger domains in Ttg-1 are highly homologous to similar regions in lin-11, mec-3, and lsl-1. This data suggests that Ttg-1 may be involved in gene regulation.
Activation and proliferation signals in murine macrophages. Biochemical signals controlling the regulation of macrophage urokinase-type plasminogen activator activity by colony-stimulating factors and other agents
Authors: Hamilton JA , Vairo G , Knight KR , Cocks BG
Published: October 2019

Purified hematopoietic growth factors such as colony-stimulating factor- 1 (CSF-1) or macrophage CSF, granulocyte-macrophage CSF, and interleukin-3 or multi-CSF, stimulate the urokinase-type plasminogen activator (u-PA) activity of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages. Granulocyte-CSF was inactive. The increases in BMM u-PA activity were inhibited by the glucocorticoid dexamethasone, and by agents that raise intracellular cyclic adenosine monophosphate levels, including prostaglandin E2 and cholera toxin. These changes in u-PA activity were paralleled by corresponding changes in u-PA mRNA levels. Evidence was obtained for protein kinase C and phospholipase C-mediated stimulation of BMM u-PA activity and mRNA levels; however, no evidence was found for an involvement of Na+/H+ exchange or Na+, K(+)-ATPase activity, Ca2+ fluxes, or pertussis toxin-sensitive G proteins. Several findings point to a dissociation between macrophage u-PA expression and DNA synthesis.
Evidence of a graft-versus-lymphoma effect associated with allogeneic bone marrow transplantation
Authors: Jones RJ , Ambinder RF , Piantadosi S , Santos GW
Published: October 2019

The existence of an immunologic antileukemia reaction associated with allogeneic bone marrow transplantation (BMT) is well established. However, a similar graft-versus-tumor effect against lymphomas has not been demonstrated. We analyzed the results of BMT in 118 consecutive patients with relapsed Hodgkin's disease or aggressive non-Hodgkin's lymphoma. The 38 patients less than 50 years of age with HLA-matched donors had allogenic marrow transplants, and the other 80 patients received purged autologous grafts. The median age was 26 years in both the allogeneic and the autologous graft recipients. The patient's response to conventional salvage therapy before transplant was the only factor that influenced the event-free survival after BMT (P less than .001). Both the patient's response to salvage therapy before BMT (P less than .001) and the type of graft (P = .02) significantly influenced the probability of relapse after BMT. The actuarial probability of relapse in patients who responded to conventional salvage therapy before BMT was only 18% after allogenic BMT compared with 46% after autologous BMT. However, the actuarial probability of event-free survival at 4 years was the same, 47% versus 41%, for patients with responsive lymphomas who received allogeneic and autologous transplants, respectively (P = .8). The beneficial antitumor effect of allogeneic BMT was offset by its higher transplant-related mortality (P = .01), largely resulting from graft-versus-host disease. Allogeneic BMT appears to induce a clinically significant graft-versus- lymphoma effect. The magnitude of this effect is similar to that reported against leukemias.
Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation [published erratum appears in Blood 1991 Jul 15;78(2):545]
Authors: Cramer EM , Lu H , Caen JP , Soria C , Berndt MC , Tenza D
Published: October 2019

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha- granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.
Effects of recombinant human macrophage colony-stimulating factor on plasma cholesterol levels
Authors: Stoudemire JB , Garnick MB
Published: October 2019

Recombinant human macrophage colony-stimulating factor (rhM-CSF) is a hematopoietic growth factor that stimulates the growth, differentiation, proliferation, and activation of cells of the monocyte/macrophage lineage. rhM-CSF was administered to rabbits and nonhuman primates to evaluate effects on cholesterol homeostasis. Decreases in plasma cholesterol concentrations were observed during rhM- CSF administration. The observed mean (+/- SD) decreases over a range of doses in nonhuman primates receiving rhM-CSF by continuous intravenous infusion (CIVI) or intravenous bolus (IVB) injection were approximately 16% +/- 8% and 43% +/- 10%, respectively. Low-density lipoprotein (LDL) cholesterol levels decreased 55% +/- 9% from pretreatment baseline values in the animals receiving rhM-CSF by IVB. Normocholesterolemic New Zealand white rabbits receiving rhM-CSF over a range of doses by CIVI showed a decrease from baseline in total cholesterol of approximately 28% +/- 17%, with LDL cholesterol levels decreasing by approximately 72% +/- 33%, while high-density lipoprotein levels showed variable changes, including increased values. A decrease of 36% +/- 26% in total plasma cholesterol was observed in Watanabe Heritable Hyperlipidemic rabbits receiving rhM-CSF by CIVI for 7 days. This decrease was attributable almost entirely to decreases in LDL cholesterol, which fell approximately 34% +/- 24% from baseline. Although the mechanism of this cholesterol-lowering effect is unknown, these results strongly suggest that rhM-CSF may provide a novel treatment for hypercholesterolemia and may be useful in investigations into the mechanisms of cholesterol homeostasis and atherogenesis.
Aggressive peripheral T-cell lymphomas containing Epstein-Barr viral DNA: a clinicopathologic and molecular analysis
Authors: Su IJ , Hsieh HC , Lin KH , Uen WC , Kao CL , Chen CJ , Cheng AL , Kadin ME , Chen JY
Published: October 2019

The Epstein-Barr virus (EBV) has been shown to be associated with posttransplant lymphoma, Hodgkin's disease, and T-cell lymphoma, in addition to African Burkitt's lymphoma. In a retrospective study of 56 consecutive cases of T-cell lymphoma, EBV DNA was found by Southern blot and in situ DNA hybridization in 10 (20%) of 50 peripheral T-cell lymphomas, but in none of six cases of T-lymphoblastic lymphoma. Peripheral T-cell lymphomas containing EBV DNA could be subclassified into three categories according to histology and immunophenotypic studies: (1) T-cell lymphoma of the helper phenotype, five cases. Two cases had histologic features resembling angioimmunoblastic lymphadenopathy (AILD). (2) T-cell lymphoma of the cytotoxic/suppressor phenotype, four cases. AILD-like features could also be recognized in two cases. Reed-Sternberg-like giant cells were identified in three cases designated Hodgkin-like T-cell lymphoma. (3) Angiocentric T-cell lymphoma or lymphomatoid granulomatosis in one case, initially affecting the skin and nose; no T-cell subset could be defined. Six of the eight EBV DNA-positive patients tested for serum EBV antibodies had elevated titers of IgG antiviral capsid antigen (greater than 640) and/or early antigen (greater than 10). From combined studies of Southern blot hybridization by using EBV termini fragment probe and in situ DNA hybridization, the EBV genomes appeared to be clonotypically proliferated in the neoplastic T cells. The patients in all three groups usually had prolonged fever preceding the diagnosis, hepatosplenomegaly, an aggressive clinical course, and poor response to chemotherapy; nine died with a median survival of only 8 months. We propose that these EBV-associated aggressive T-cell lymphomas, like human T-cell leukemia/lymphoma virus-positive T-cell lymphoma, have characteristic clinicopathologic features and should be treated as a separate disease entity.
Inactivation of the retinoblastoma gene in human lymphoid neoplasms
Authors: Ginsberg AM , Raffeld M , Cossman J
Published: October 2019

The absence of wild type retinoblastoma (Rb) gene expression in a wide variety of human solid tumors suggests an etiologic role for this tumor suppressor gene in human cancer. We have evaluated the involvement of Rb gene inactivation in the pathogenesis and progression of human lymphoma and leukemia. We examined the genomic configuration and transcription of the Rb gene in cultured cell lines and primary cases of T- and B-cell lymphomas and leukemias. By Southern analysis, abnormalities of the Rb locus were identified in 1 of 5 T-cell acute lymphoblastic lymphoma (T-ALL) cell lines, 1 of 26 primary cases of T- ALL, 1 of 40 primary cases of chronic lymphocytic lymphoma/well- differentiated lymphoma (CLL/WDL), and 1 of 15 primary cases of intermediately differentiated lymphoma (IDL). By Northern analysis, markedly reduced or abnormal expression of the Rb gene was identified in 2 of 5 T-ALL cell lines, 1 of 7 primary cases of T-ALL, 1 of 5 primary cases of CLL/WDL, and 1 of 6 primary cases of IDL. These findings show that Rb gene inactivation can be associated with a broad range of lymphoid neoplasms and that loss of the tumor suppressor function of Rb may influence the pathogenesis and progression of lymphoma/leukemia.
Circulating erythropoietin levels after bone marrow transplantation: inappropriate response to anemia in allogeneic transplants
Authors: Beguin Y , Clemons GK , Oris R , Fillet G
Published: October 2019

We studied 24 recipients of autologous bone marrow transplantation (ABMT) or allogeneic BMT (BMT) to determine whether impaired erythropoietin (Epo) response to anemia could delay full erythropoietic recovery. Observed Epo levels were compared with predicted levels based on the relationship between Epo and hematocrit in 125 control subjects. Circulating Epo levels were normal during conditioning and the early posttransplant period. Between days 21 and 180, Epo levels remained normal in ABMT patients but were inappropriately low for the degree of anemia in BMT patients. Median time to full erythropoietic engraftment was longer in BMT than in ABMT recipients. Circulating Epo returned to appropriate levels after day 180, except in patients with active cytomegalovirus infection. We conclude that impaired Epo response to anemia can contribute to delayed erythropoietic recovery after allogenic BMT. Renal toxicity of ciclosporin, interaction between host and donor marrow, and cytomegalovirus infection might play a role. This study could support the use of recombinant human Epo to accelerate erythropoietic engraftment after BMT.
Detection of early human T-cell lymphotropic virus type I antibody patterns during seroconversion among transfusion recipients
Authors: Manns A , Murphy EL , Wilks R , Haynes G , Figueroa JP , Hanchard B , Barnett M , Drummond J , Waters D , Cerney M
Published: October 2019

From a cohort of human T-cell lymphotropic virus type I (HTLV-I) exposed transfusion recipients (N = 71) enrolled in the Jamaican Transfusion Study, 11 were selected for detailed laboratory evaluation. All recipients were followed at monthly intervals for 6 months and then bimonthly up to 1 year for evidence of HTLV-I seroconversion. Without regard to results on screening assays, pretransfusion and posttransfusion samples were tested with two licensed HTLV-1 whole- virus screening enzyme immunoassays (EIAs), recombinant EIAs for antibody against tax (p40x) and p21e envelope, standard whole virus Western blot (WB), WB enhanced with recombinant p21e, and radioimmunoprecipitation assay (RIPA). In the early period posttransfusion, antibody to gag core protein was predominant with anti- p24 generally appearing before anti-p19. Recombinant anti-p21e envelope protein, in EIA and WB format, was frequently the earliest envelope reactivity detected, while anti-gp46 in WB and anti-gp61/68 in RIPA system appeared later. Anti-tax antibodies appeared later in the time course of seroconversion. The whole-virus EIAs were less sensitive than the confirmatory assays. The combination of WB and RIPA or WB enhanced with recombinant p21e appeared equally effective in confirming samples as positive by the Public Health Service two gene group confirmatory algorithm. However, specificity of this assay approach could not be addressed in this study.
Hirudin interruption of heparin-resistant arterial thrombus formation in baboons
Authors: Kelly AB , Marzec UM , Krupski W , Bass A , Cadroy Y , Hanson SR , Harker LA
Published: October 2019

To determine the role of thrombin in high blood flow, platelet- dependent thrombotic and hemostatic processes we measured the relative antithrombotic and antihemostatic effects in baboons of hirudin, a highly potent and specific antithrombin, and compared the effects of heparin, an antithrombin III-dependent inhibitor of thrombin. Thrombus formation was determined in vivo using three relevant models (homologous endarterectomized aorta, collagen-coated tubing, and Dacron vascular graft) by measuring: (1) platelet deposition, using gamma camera imaging of 111In-platelets; (2) fibrin deposition, as assessed by the incorporation of circulating 125I-fibrinogen; and (3) occlusion. The continuous intravenous infusion of 1, 5, and 20 nmol/kg per minute of recombinant hirudin (desulfatohirudin) maintained constant plasma levels of 0.16 +/- 0.03, 0.79 +/- 0.44, and 3.3 +/- 0.77 mumol/mL, respectively. Hirudin interrupted platelet and fibrin deposition in a dose-dependent manner that was profound at the highest dose for all three thrombogenic surfaces and significant at the lowest dose for thrombus formation on endarterectomized aorta. Thrombotic occlusion was prevented by all doses studied. In contrast, heparin did not inhibit either platelet or fibrin deposition when administered at a dose that maximally prolonged clotting times (100 U/kg) (P greater than .1), and only intermediate effects were produced at 10-fold that dose (1,000 U/kg). Moreover, heparin did not prevent occlusion of the test segments. Hirudin inhibited platelet hemostatic function in concert with its antithrombotic effects (bleeding times were prolonged by the intermediate and higher doses). By comparison, intravenous heparin failed to affect the bleeding time at the 100 U/kg dose (P greater than .5), and only minimally prolonged the bleeding time at the 1,000 U/kg dose (P less than .05). We conclude that platelet-dependent thrombotic and hemostatic processes are thrombin-mediated and that the biologic antithrombin hirudin produces a potent, dose-dependent inhibition of arterial thrombus formation that greatly exceeds the minimal antithrombotic effects produced by heparin.
Increased lysis of patient CD10-positive leukemic cells by T cells coated with anti-CD3 Fab' antibody cross-linked to anti-CD10 Fab' antibody
Authors: Oshimi K , Seto T , Oshimi Y , Masuda M , Okumura K , Mizoguchi H
Published: October 2019

An anti-CD3 Fab' x anti-CD10 Fab' bispecific hybrid F(ab')2 antibody (Ab) was generated. This bispecific Ab had a molecular mass of 100 to 110 Kd, and the capacity to react with both CD3+ T cells and CD10+ acute lymphoblastic leukemia (ALL) cells. We studied whether cytotoxic T lymphocytes (CTLs) could lyse patient CD10+ ALL cells after addition of the bispecific Ab. As effector CTLs, interleukin-2 (IL-2)-stimulated peripheral blood mononuclear cells (PBMCs) and CTL clones were used. When IL-2-stimulated PBMCs were assayed for cytotoxicity to 61Cr- labeled CD10+ ALL cells, their activity was shown to be markedly enhanced by the addition of the bispecific Ab. Most of the CTL clones established lacked cytotoxicity for CD10+ ALL cells, but addition of the bispecific Ab induced a significant level of cytotoxicity. CTLs derived from ALL patients also showed significant cytotoxicity for autologous CD10+ ALL cells after addition of the bispecific Ab. However, this Ab did not affect the cytotoxicity of CTLs when CD10- leukemic cells were used as the targets. These findings suggest that the bispecific Ab can be used for immunotherapy in patients with CD10+ ALL.
Selective hypersensitivity to granulocyte-macrophage colony-stimulating factor by juvenile chronic myeloid leukemia hematopoietic progenitors
Authors: Emanuel PD , Bates LJ , Castleberry RP , Gualtieri RJ , Zuckerman KS
Published: October 2019

Juvenile chronic myelogenous leukemia (JCML) is a good model for the study of myeloproliferation because JCML hematopoietic progenitor cells grow in vitro at very low cell densities without the addition of exogenous stimulus. Previous studies have demonstrated that this proliferation is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF), and that removal of monocytes from the cell population before culture eliminates this “spontaneous” myeloproliferation, suggesting a paracrine role of monocyte stimulation. However, subsequent studies have shown that increased GM-CSF production from the JCML monocytes is not a consistent finding and therefore not a plausible sole mechanism. In examining hematopoietic growth factor dose- response curves, both JCML GM and erythroid nonadherent progenitor cell populations displayed a marked and selective hypersensitivity to GM- CSF. Responses to interleukin-3 and G-CSF were identical to control dose-response curves. This is the first demonstration of a myeloid leukemia in which hypersensitivity to a specific growth factor appears to be involved in the pathogenesis of the disease.
Synergistic effects of nerve growth factor and granulocyte-macrophage colony-stimulating factor on human basophilic cell differentiation
Authors: Tsuda T , Wong D , Dolovich J , Bienenstock J , Marshall J , Denburg JA
Published: October 2019

We have recently shown that nerve growth factor (NGF) promotes human granulopoiesis, specifically augmenting basophilic cell differentiation observed in methylcellulose hematopoietic colony assays of human peripheral blood. Because the NGF effect was seen in the presence of conditioned medium derived from a human T-cell line (Mo-CM) containing granulocyte-macrophage colony-stimulating factor (GM-CSF), we examined interactions of purified NGF and recombinant human GM-CSF (rhGM-CSF) on granulocyte growth and differentiation. rhGM-CSF stimulated a dose- dependent increase in methylcellulose colony growth at concentrations between 0.1 U/mL and 10 U/mL, and in the presence of NGF at 500 ng/mL this effect was enhanced. The number of basophilic cell colony-forming units (CFU-Baso) and histamine-positive colonies increased synergistically when NGF was added to rhGM-CSF. Furthermore, because Mo- CM acts with sodium butyrate to promote basophilic differentiation of alkaline-passaged myeloid leukemia cells, HL-60, we also examined the interaction of NGF and Mo-CM or rhGM-CSF using this assay. In the presence of NGF, Mo-CM at concentrations of 0.5% to 20% vol/vol, and rhGM-CSF at concentrations of 0.1 U/mL to 100 U/mL synergistically increased histamine production by butyrate-induced, alkaline-passaged HL-60 cells; this was associated with the appearance of metachromatic, tryptase-negative, IgE receptor-positive cells. The effects of rhGM-CSF or Mo-CM were completely abrogated by a specific anti-rhGM-CSF neutralizing antibody in methylcellulose, with or without NGF; the NGF synergy with rhGM-CSF in the HL-60 assay was also inhibited by either anti-rhGM-CSF or anti-NGF antibody. These studies support the notion that differentiation in the basophilic lineage may be enhanced by NGF acting to increase the number of GM-CSF-responsive basophilic cell progenitors.
In vitro myelosuppressive effects of the parvovirus minute virus of mice (MVMi) on hematopoietic stem and committed progenitor cells
Authors: Segovia JC , Real A , Bueren JA , Almendral JM
Published: October 2019

The interaction of two strains of the parvovirus minute virus of mice (MVM) with the mouse hematopoietic system has been studied. The immunosuppressive strain MVMi, but not the prototype virus MVMp, inhibited hematopoiesis in vitro, as judged by colony-forming assays of the erythroid burst-forming unit and granulocyte-monocyte colony- forming unit (CFU-GM) progenitors. Interestingly, primitive hematopoietic cells of the stem compartment (CFU-S12d), were equally susceptible to the MVMi cytotoxic infection, unravelling an unprecedented feature of virus-hematopoiesis interactions. The replication of both strains of MVM virus was evaluated in primary myeloid cells of long-term bone marrow cultures. A high viral DNA synthesis and maturation was observed in MVMi-infected myeloid cells, but it was undetectable in MVMp infections; moreover, the expression of the cytotoxic nonstructural NS-1 protein, a more reliable parameter of cell permissiveness to MVM infection, was only detected in MVMi- infected cells. Correspondingly, MVMi was propagated to high titers of infectious virus and it mediated an acute myelosuppression in these cultures. We conclude that MVMi has a wider tropism than was previously suspected and it is proposed that cytotoxic infection of hematopoietic stem cells, besides that of committed progenitors, may provide an additional basis to understand the pathogenesis of certain animal and human bone marrow failures of viral etiology.
The effect of 5-fluorouracil on erythropoiesis
Authors: Rich IN
Published: October 2019

The effects of a single dose (150 mg/kg) of 5-fluorouracil on mature erythroid and erythropoietic and multipotential in vitro precursor populations in the bone marrow and spleen and circulating biologically (erythroid colony forming unit [CFU-E] assay) and immunologically active (enzyme-linked immunosorbent assay) erythropoietin (Epo) are described. All mature erythroid (reticulocytes, erythrocytes) and in vitro erythropoietic precursors (CFU-E, erythroid burst-forming unit [BFU-E]) are severely reduced, if not eradicated. Transient repopulation of the pure BFU-E and CFU-E populations on days 6 and 7, respectively, produces a marked reticulocytosis after day 9. Circulating Epo increases to above normal values by day 2. However, whereas biologically active Epo remains constant at this level until day 9, immunologically active Epo continually increases; by day 12, however, both assays detect circulating Epo levels of about 400 mU/mL. In vitro multipotential stem cells (BFU-E mix) are reduced to 32% on day 1, 7.6% on day 2, and return to normal values between days 4 and 5. The survival and repopulation kinetics of the BFU-E mix imply a stem cell population more mature than the high proliferative potential colony-forming cells. However, the BFU-E mix may be responsible for erythropoiesis repopulating ability.
Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells
Authors: Terstappen LW , Huang S , Safford M , Lansdorp PM , Loken MR
Published: October 2019

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.
2.5S nerve growth factor enhances survival, phagocytosis, and superoxide production of murine neutrophils
Authors: Kannan Y , Ushio H , Koyama H , Okada M , Oikawa M , Yoshihara T , Kaneko M , Matsuda H
Published: October 2019

The effect of 2.5S nerve growth factor (NGF) on survival, phagocytosis, and superoxide production of murine neutrophils was examined and compared with the effects of interleukin-3 (IL-3) and recombinant GM colony-stimulating factor (rGM-CSF). NGF enhanced the viability of neutrophils isolated from peripheral blood and peritoneal cavity in a dose-dependent way. IL-3 50 U/mL had no effect. rGM-CSF 50 U/mL had an effect similar to that of 50 ng/mL NGF. NGF also enhanced the phagocytosis of hydrophilic microspheres by peritoneal neutrophils, and the activity of NGF was greater than that of IL-3 or rGM-CSF. NGF enhanced the superoxide production induced by phorbol 12-myristate 13- acetate (PMA), which acts at postreceptor sites, and that induced by opsonized zymosan, a receptor-mediated ligand. The stimulating activity of NGF was largely comparable to that of rGM-CSF. The present data show that NGF bound to neutrophils enhances their survival, phagocytosis, and superoxide production. Thus, we postulate that NGF plays an important role in the inflammatory processes.
Differential tissue expression of the Lewis blood group antigens: enzymatic, immunohistologic, and immunochemical evidence for Lewis a and b antigen expression in Le(a-b-) individuals
Authors: Orntoft TF , Holmes EH , Johnson P , Hakomori S , Clausen H
Published: October 2019

The Lewis blood group system comprises two main carbohydrate antigens, Le(a) and Le(b). Lewis typing has traditionally been based on serologic determinations using erythrocytes and saliva. Several recent studies have demonstrated that erythrocyte Lewis phenotype may change during pregnancy or disease, and inappropriate Lewis antigens have been found in both normal and neoplastic tissue. To evaluate whether these observations are in conflict with the presently proposed genetic and biosynthetic basis of the Lewis blood group system, we performed a combined enzymatic, immunohistologic, and immunochemical study of Lewis antigen expression in normal and neoplastic tissues, as well as erythrocytes, plasma, and saliva of Le(a-b-)-typed individuals. Of six cancer-bearing patients typed Le(a-b-), three were identified as nongenuine owing to the presence of alpha 1----4fucosyltransferase activity (alpha 1----4FT) and Lewis antigens in saliva and three were identified as genuine (lacking alpha 1----4FT and Lewis antigens in saliva). These genuine Le(a-b-) individuals were shown to express significant alpha 1----4FT in tissues, and Lewis antigens were detected in tissues by immunohistology as well as immunochemistry. We conclude that the Lewis phenotype obtained by serologic determination of erythrocytes and saliva does not apply to all tissues. We discuss biosynthetic and genetic consequences of this finding.
High-dose cytarabine and daunorubicin induction and postremission chemotherapy for the treatment of acute myelogenous leukemia in adults
Authors: Phillips GL , Reece DE , Shepherd JD , Barnett MJ , Brown RA , Frei-Lahr DA , Klingemann HG , Bolwell BJ , Spinelli JJ , Herzig RH
Published: October 2019

Seventy consecutive adult patients with acute myelogenous leukemia (AML), median age 44 years, received high-dose cytarabine (3 g/m2 every 12 hours for 12 doses) followed by daunorubicin (45 mg/m2 daily for three doses) for remission induction. A single, identical course was planned for postremission therapy. Complete remission (CR) was achieved in 63 patients (90%, 95% confidence interval [CI] 83% to 97%), 60 after a single course. Eight patients were selected to undergo elective bone marrow transplantation (BMT) during first CR. Of the remaining 55 patients, 40 (73%) underwent planned post-CR therapy; 15 patients did not, owing to early relapse, excessive toxicity from the induction chemotherapy, or refusal. Nineteen patients, including 13 who received planned post-CR therapy, remain in continuous CR at a median follow-up of 5.2 years (range 3.0 to 7.1 years). The 5-year actuarial leukemia- free survival was 30% (95% Cl, 19% to 42%) for all patients achieving CR and 32% (95% Cl, 19% to 47%) for the 40 patients who received the planned post-CR chemotherapy. Analysis of various putative prognostic factors for CR and overall and leukemia-free survival showed significance for a previous history of myelodysplasia, higher initial leukocyte counts, certain French-American-British (FAB) types, and certain abnormal karyotypes. None of these factors was consistently significant regarding the above parameters, although small patient numbers in certain analyses may have obscured significant associations. Myelosuppression was occasionally prolonged after remission induction and especially post-CR therapy. Severe cerebellar toxicity was observed in 13 patients; in 11 cases, this toxicity was fully reversible. Other serious complications were infrequent. Intensive chemotherapy with high- dose cytarabine and daunorubicin has substantial antileukemic activity in adult AML, and may represent an improvement over conventional therapy. Relapses were common, however, even in patients who received planned therapy, and substantial toxicity was observed. The optimum use of this regimen in AML remains to be determined.
Interleukin-5 is a human basophilopoietin: induction of histamine content and basophilic differentiation of HL-60 cells and of peripheral blood basophil-eosinophil progenitors
Authors: Denburg JA , Silver JE , Abrams JS
Published: October 2019

Cytokine-induced differentiation of basophils may contribute to various inflammatory processes. We examined the effects of recombinant human interleukin-5 (IL-5) and other human cytokines in vitro on myeloid colony formation in methylcellulose and on alkaline passaged HL-60 basophilic cell differentiation. Myeloid colonies (CFU-C) at day 14, formed in the presence of either IL-3, IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or G-CSF included peripheral blood- derived progenitors of the eosinophil/basophil lineage. IL-5 stimulated a greater proportion of basophil-containing, histamine-positive, eosinophil-type colonies compared with GM-CSF, IL-3, or G-CSF. IL-5 also stimulated dose-dependent increases in histamine content of alkaline-passaged, butyrate cotreated HL-60 cells. The concentration of IL-5 required for half-maximal induction of HL-60 histamine content was similar within twofold to that needed for half-maximal stimulation of the multifactor dependent TF-1 erythroleukemic cell line. Neutralizing rat monoclonal antibodies to human IL-5 were developed and used to demonstrate that each of these IL-5 bioactivities could be specifically blocked. We conclude that in addition to its previously described eosinophil differentiation activity, IL-5 may be considered a basophilopoietin.
Accessibility of epitopes on fibrin clots and fibrinogen gels
Authors: Procyk R , Kudryk B , Callender S , Blomback B
Published: October 2019

Radiolabeled antibodies were perfused into fibrin clots and fibrinogen gels formed in vitro to assess the reactivity of selected epitopes. An antifibrinogen monoclonal antibody (MoAb) (antibody 1D4/xl-f), directed against an epitope in the A alpha-chain C-terminal region (A alpha 241– 476), bound to 35% of the epitope in crosslinked fibrin clots and 37% of the same epitope in factor XIII-induced fibrinogen gel networks. A different MoAb (4–2/xl-f, anti gamma 392–406) bound to only 7% of the epitope in both fibrin and fibrinogen gels. As expected, an antifibrin MoAb (antibody T2G1, antiB beta 15–21) did not bind to fibrinogen gels, but bound to fibrin, although to only 14% of the available T2G1- reactive epitopes. An antibody that does not recognize fibrin (antibody 1–8C6, antiB beta 1–21) predictably did not bind to fibrin clots and bound to 35% of the 1–8C6 epitopes present in fibrinogen gels, a level of binding also observed with antibody T2G1 and fibrinogen gels only after the latter were treated with thrombin. T2G1 epitope expression was affected much more than 1D4/xl-f epitope expression in clots formed in buffers of high or low ionic strength, conditions known to influence clot structure. Studies on the availability, in quantitative terms, of the T2G1-reactive epitope in fibrin clots is of particular importance because this antibody is currently being used in clinical trials as a clot imaging agent.
Stages of T-cell receptor protein expression in T-cell acute lymphoblastic leukemia
Authors: Campana D , van Dongen JJ , Mehta A , Coustan-Smith E , Wolvers-Tettero IL , Ganeshaguru K , Janossy G
Published: October 2019

In this study five monoclonal antibodies (MoAbs) to T-cell receptor (TCR) proteins (WT31, alpha F1, beta F1, TCR delta-1 and delta TCS-1) were used to identify discrete maturative stages in 40 cases of T-cell acute lymphoblastic leukemia (T-ALL). These MoAbs reacted exclusively with CD3+ T cells and did not label B-lineage and myeloid cells. In 17 of the 40 T-ALL cases studied the leukemic blasts lacked membrane and cytoplasmic TCR chains (group I). In 12 cases cells did not have membrane CD3/TCR but expressed cytoplasmic TCR proteins heterogenously: nine cases had cytoplasmic TCR beta chains (beta F1+, alpha F1-; group II), one case had cytoplasmic TCR alpha chains (alpha F1+, beta F1-; group III), and two cases were labeled by both alpha F1 and beta F1 MoAbs (group IV). The remaining 11 cases were mCD3+: nine were TCR alpha beta+ (group Va) and two exhibited TCR gamma delta (TCR delta-1+, delta TCS-1+; group Vb). The analysis of the TCR beta, -gamma, and - delta gene configurations in 23 of the 40 T-ALLs showed that: (1) the lack of TCR protein expression was due to the lack of TCR gene rearrangements only in one of nine cases; (2) five of five TCR beta+, TCR alpha- cases studied had germline TCR alpha genes (ie, no detectable TCR delta gene deletions); (3) seven of eight cases with TCR delta gene deletions expressed TCR alpha proteins, whereas in 12 of 20 of the T-ALLs with TCR beta gene rearrangements the synthesis of the corresponding protein occurred; only 2 of 16 cases with rearranged TCR delta genes expressed TCR delta chains. The T-ALL categories identified with anti-TCR MoAbs did not have additional characteristic phenotypic patterns and may correspond to the normal stages of T-cell development more precisely than those defined by other differentiation antigens.