bluuebunny/crossref_metadata_2025 huggingface dataset:
A linear amino acid sequence involved in the interaction of t-PA with its endothelial cell receptor
Authors: Beebe DP , Miles LA , Plow EF
Published: October 2019

Endothelial cell receptors for tissue plasminogen activator (t-PA) have been demonstrated recently, and we have sought to identify a region of the t-PA molecule involved in its interaction with these receptors on human umbilical vein endothelial cells. Of three monoclonal antibodies against various regions of t-PA, one directed against the finger region inhibited 125I-t-PA binding to the cells. Synthetic peptides corresponding in amino acid sequences to segments from within the finger region were constructed, and one of these inhibited t-PA binding. This peptide corresponded to residues 7 through 17 of t-PA. The inhibition by this peptide was specific as other peptides from the finger region were inactive. The inhibitory peptide also did not affect the binding of another fibrinolytic ligand, urokinase, to the cells. Although a role for other regions of t-PA in binding to endothelial cells cannot be excluded, the results implicate a short span of linear amino acid sequence within the finger region in the interaction of t-PA with endothelial cells.
Nucleoside transport and proliferative rate in human thymocytes and lymphocytes
Authors: Smith CL , Pilarski LM , Egerton ML , Wiley JS
Published: October 2019

The thymus is a site of active T-lymphoid cell proliferation and DNA synthesis. In this study, the capacity of human thymocytes for nucleoside transport was assessed both by cytosine arabinoside influx and by equilibrium binding of nitrobenzylmercaptopurine riboside (NBMPR), a specific ligand for the equilibrative nucleoside transporter of leukocytes. The proportion of freshly isolated thymocytes synthesizing DNA was 8.6% +/- 2.1% (n = 12) by 3H-thymidine labeling index and 7.8% +/- 2.9% (n = 4) S-phase cells by flow cytometric analysis of DNA content. In comparison, both methods gave proliferation S-phase values less than 1% for peripheral blood lymphocytes (PBLs). Thymocytes expressed a high density of specific NBMPR binding sites (26,068 +/- 8,776 sites per cell, n = 12) as compared with PBLs (1,123 +/- 553 sites per cell, n = 8). The initial influx of cytosine arabinoside into thymocytes was 14-fold greater than into PBLs, and in both cell types the influx of nucleoside was totally inhibited by 0.5 mumol/L NBMPR, which is known to inhibit the major equilibrative nucleoside transporter in white blood cells. Depletion of mature CD3+ cells from the thymocyte preparation by anti-CD3 antibody left a residual population with both increased labeling index and up to twofold greater density of NBMPR binding sites. When PBLs were cultured for 48 hours with the T-cell mitogen phytohemagglutinin, a 40-fold increase in labeling index was observed, together with a 30-fold increase in the density of specific NBMPR binding sites. Thus, fresh thymocytes from human thymus are actively proliferating and express high densities of a functional nucleoside transporter. The more immature cells in the thymocyte population which are proliferating more actively have a greater density of nucleoside transporters than the whole population. In contrast, mitotically inactive PBLs-have few nucleoside transporters, but after mitogenic stimulation PBLs express large numbers of this transmembrane molecule.
Molecular evidence for a single clonal origin in biphenotypic concomitant chronic lymphocytic leukemia and multiple myeloma
Authors: Saltman DL , Ross JA , Banks RE , Ross FM , Ford AM , Mackie MJ
Published: October 2019

To establish the clonal origin of a case of concomitant B-cell chronic lymphocytic leukemia (IgM kappa) and multiple myeloma (IGA lambda), we analyzed the immunoglobulin (Ig) gene rearrangements in the patient's blood and bone marrow. Despite the different isotypes, pretreatment investigation of the heavy chain gene (JH) revealed a germline fragment and two identical rearrangements in the blood and marrow. Both kappa and lambda light-chain genes were rearranged in the blood, suggesting peripheral blood lymphocyte involvement in the myeloma. Analysis of the Ig genes after chemotherapy demonstrated no change in the JH or CK rearrangements; however, the lambda genes were now in a germline configuration. Our results suggest that in this patient both chronic lymphocytic leukemia and myeloma originated from the same B-cell progenitor.
Modulation of arabinosylcytosine metabolism by arabinosyl-2- fluoroadenine in lymphocytes from patients with chronic lymphocytic leukemia: implications for combination therapy
Authors: Gandhi V , Nowak B , Keating MJ , Plunkett W
Published: October 2019

Our previous studies indicated that K562 cells loaded with arabinosyl-2- fluoroadenine 5′-triphosphate (F-ara-ATP) accumulated arabinosylcytosine 5′-triphosphate (ara-CTP) at a threefold higher rate compared to the control cells. In the present study lymphocytes were obtained from patients with chronic lymphocytic leukemia before and after F-ara-A monophosphate therapy. The rate of ara-CTP accumulation after in vitro ara-C incubation was compared in lymphocytes obtained prior to therapy without any other manipulation, after ex vivo F-ara- ATP (100 mumol/L) treatment, and after in vivo F-ara-A monophosphate therapy. Lymphocytes showed a 2.2-fold (n = 23) and 1.7-fold (n = 23) median increase in the cellular concentration of ara-CTP after an ex vivo incubation with 100 mumol/L F-ara-A and 20 to 24 hours after the first dose (25 or 30 mg/m2) of F-ara-A monophosphate in vivo treatment, respectively. Although the rates of F-ara-ATP and ara-CTP accumulation varied among patients, a relationship was observed in individuals between the cellular concentration of F-ara-ATP at the beginning of the ara-C incubation and ara-CTP accumulation. These studies strongly suggest that a protocol designed to administer F-ara-A monophosphate prior to ara-C infusion will augment ara-CTP accumulation by leukemia cells.
Molecular heterogeneity in acute leukemia lineage switch
Authors: Gagnon GA , Childs CC , LeMaistre A , Keating M , Cork A , Trujillo JM , Nellis K , Freireich E , Stass SA
Published: October 2019

Six cases of acute leukemia that underwent lineage switch from acute lymphocytic leukemia to acute myelogenous leukemia are reported. The mean age of the patients was 24 years, time to conversion was 36 months, and survival after conversion was only 3 months. Of the three cases which showed abnormal metaphases at both diagnosis and conversion, two (cases 2, 5) showed related cytogenetic abnormalities, and the third showed (case 3) independent chromosomal changes. Molecular analysis for immunoglobulin heavy chain and T-cell receptor beta chain genes showed that five of the six cases had rearrangement of at least one of these lymphoid associated genes at conversion to acute myelogenous leukemia. The single case (case 3) in which there were no lymphoid gene rearrangements at conversion was also the only case in which independent karyotypic abnormalities at diagnosis and conversion were demonstrated. Our findings suggest that lineage switch can represent either relapse of the original clone with heterogeneity at the molecular level or the emergence of a second new leukemic clone without molecular heterogeneity.
Immunophenotypic analysis of clonogenic cells in acute lymphoblastic leukemia using an in vitro colony assay
Authors: Hudson AM , Makrynikola V , Kabral A , Bradstock KF
Published: October 2019

A culture system was used to analyze the expression of membrane differentiation antigens on the proliferative or clonogenic fraction of cells from cases of common (precursor B) acute lymphoblastic leukemia (c-ALL). Colonies of leukemic cells were obtained in 18 of 20 cases after 1 week in culture in a liquid layer containing recombinant interleukin-2 (IL-2), phytohemagglutinin (PHA), and B-cell growth factor over an agar feeder layer containing irradiated peripheral blood mononuclear cells plus fetal calf serum (FCS) and horse serum. Cultured cells expressed HLA-DR, CD-9, CD-10, CD-20, and CD-34 antigens, indicating conservation of precursor B phenotype. Differentiation antigen expression on the clonogenic subpopulation giving rise to leukemic colonies was assessed by treating cells prior to culture with selected cytotoxic monoclonal antibodies (MoAbs) and complement or by fluorescence-activated cell sorting. Lytic treatment with HLA-DR, CD- 10, CD-9, and CD-20 antibodies produced median reductions in colony formation of 93%, 81%, 73%, and 58% respectively. Combined treatment with CD-9 and CD-10 antibodies produced complete inhibition in 11 of 13 cases. Cell-sorting experiments after CD-10 staining indicated a good correlation with complement lysis studies and also demonstrated that the CD-19 antigen is expressed on a high proportion of clonogenic cells. Colony formation was also significantly reduced in 13 of 17 cases by lytic treatment of cells with the CD-33 antibody MY-9, suggesting expression of this “myeloid” lineage antigen on ALL clonogenic cells. The substantial case-to-case variation in expression of individual differentiation antigens indicates that leukemic cellular heterogeneity is a major consideration in ex vivo bone marrow purging using MoAbs and emphasizes the need for more critical evaluation of purging techniques.
Immunohistologic features predict clinical behavior of orbital and conjunctival lymphoid infiltrates
Authors: Medeiros LJ , Harmon DC , Linggood RM , Harris NL
Published: October 2019

The natural history of lymphoid infiltrates of the orbit and conjunctiva is poorly understood. To determine if immunohistologic features could predict clinical outcome, these features were evaluated in 61 patients with orbital and conjunctival lymphoid infiltrates, including 44 patients with lesions localized to one or both orbits or conjunctivae. Using histologic criteria, 20 infiltrates were classified as malignant lymphoma (cytologically atypical), 14 cases were classified as benign (follicular hyperplasia or inflammatory pseudotumor), and 27 infiltrates (44%) were dense infiltrates of small lymphocytes without cytologic atypia, which could not be confidently classified as benign or malignant (histologically indeterminate). Based on expression of monotypic immunoglobulin, 20 of these cases were reclassified as small lymphocytic lymphoma. For all cases, monotypic immunoglobulin expression correlated with reduced survival (P less than .05) and increased likelihood of dissemination (P less than .001). Monotypic immunoglobulin expression also correlated with increased risk of dissemination for all histologically indeterminate (small lymphocytic) infiltrates (P less than .05). Separate analysis of the localized infiltrates showed that monotypic immunoglobulin expression significantly correlated with an increased likelihood of dissemination for all 44 cases (P less than .005) and for the 22 histologically indeterminate lesions (P = .06). For the localized small lymphocytic infiltrates, monotypic immunoglobulin expression conferred a 50% risk of dissemination. In contrast, no patients with polytypic small lymphocytic infiltrates have disseminated, although one lesion locally recurred 30 months later as lymphoma. Thus, monotypic immunoglobulin expression significantly correlates with reduced patient survival and increased risk of dissemination by orbital and conjunctival lymphoid infiltrates. Monotypic small lymphocytic infiltrates without cytologic atypia behave as do low grade B-cell malignant lymphomas with a significant risk of dissemination and an indolent clinical course. The relationship of polytypic small lymphocytic infiltrates to lymphoma remains to be determined.
Prognostic implications of cytogenetic studies in an intensively treated group of children with acute lymphoblastic leukemia
Authors: Fletcher JA , Kimball VM , Lynch E , Donnelly M , Pavelka K , Gelber RD , Tantravahi R , Sallan SE
Published: October 2019

We assessed the prognostic significance of leukemia cell cytogenetics by analyzing bone marrow aspirates obtained at time of diagnosis in 165 children on a single protocol for acute lymphoblastic leukemia (ALL). These children were assigned to six mutually exclusive cytogenetic categories as follows: (1) hyperdiploid, with 50 or more chromosomes (n = 35); (2) hyperdiploid, with 47 to 49 chromosomes (n = 11); (3) diploid (n = 42); (4) pseudodiploid (n = 34); (5) hypodiploid (n = 9); and (6) insufficient data (n = 34). At a median follow-up of 5 years, there were no statistically significant differences between any of these cytogenetic categories in either event-free or overall survival. Those children with chromosomal translocations (n = 26) appeared to fare the same as those lacking translocations (n = 105). The absence of karyotypic prognostic significance was observed not only within the overall group, but also when the results were stratified by standard- risk and high-risk status. Of the specific structural chromosome changes that we studied, only the Philadelphia chromosome (Ph) appeared to confer a poor prognosis, although there were too few such cases to achieve statistical significance. Although we did not detect the event- free survival differences that have been described previously in hyperdiploid, hypodiploid, and pseudodiploid childhood ALL, our findings must be viewed as preliminary given the small number of children in some of the cytogenetic categories. We think that the prognostic implications of these cytogenetic features might have been nullified by improvements in therapy.
The breakpoint of a large deletion causing hereditary persistence of fetal hemoglobin occurs within an erythroid DNA domain remote from the beta-globin gene cluster
Authors: Feingold EA , Forget BG
Published: October 2019

The DNA juxtaposed to the gamma-globin genes as a result of a large deletion associated with hereditary persistence of fetal hemoglobin (HPFH) was studied to define the role it may play in maintaining active expression of these genes in adult erythroid cells. The DNA located immediately 3′ to the deletion breakpoint was found to function as an enhancer element in gene transfer experiments and to be specifically hypomethylated in normal erythroid cells of both fetal and adult origin. This DNA also contains a long open reading frame encoding a polypeptide chain 292 amino acids in length. Therefore, in this form of HPFH (HPFH-1), the continued expression of gamma-globin genes in adult life may result from the inclusion of these genes within a new chromosomal domain that is potentially transcriptionally active in adult erythroid cells. The 3′ breakpoint of another large deletion causing delta beta thalassemia rather than HPFH was also identified. This deletion (Spanish G gamma A gamma (delta beta) degrees thalassemia) is nearly identical in size and location to that of HPFH- 1, but extends an additional 8.5 to 9 kb in the 3′direction, and therefore results in loss of the sequences near the 3′ breakpoint of HPFH-1. Thus, the presence of these sequences appears to be important for the expression of the HPFH phenotype.
Food iron absorption in idiopathic hemochromatosis
Authors: Lynch SR , Skikne BS , Cook JD
Published: October 2019

The relationship between iron status and food iron absorption was evaluated in 75 normal volunteers, 15 patients with idiopathic hemochromatosis, and 22 heterozygotes by using double extrinsic radioiron tags to label independently the nonheme and heme iron components of a hamburger meal. In normal subjects, absorption from each of these pools was inversely correlated with storage iron, as measured by the serum ferritin concentration. In patients with hemochromatosis, absorption of both forms of iron was far greater than would be predicted from the relationship between absorption and serum ferritin observed in normal volunteers. Nevertheless, there was still a modest but statistically significant reduction in absorption of nonheme iron with increasing serum ferritin. This relationship could not be demonstrated in the case of heme iron absorption. In heterozygotes, nonheme iron absorption from a hamburger meal containing no supplementary iron did not differ significantly from that observed in normal volunteers. However, when this meal was both modified to promote bioavailability and supplemented with iron, absorption of nonheme iron was significantly elevated. These studies confirm the presence of excessive nonheme iron absorption even from unfortified meals in patients with idiopathic hemochromatosis and suggest in addition that they are particularly susceptible to iron loading from diets containing a high proportion of heme iron. Impaired regulation of nonheme iron absorption was also observed in heterozygous individuals, but a statistically significant abnormality was demonstrable only when the test meal contained a large highly bioavailable iron supplement.
Development of immunologic functions after bone marrow transplantation in 33 patients with severe combined immunodeficiency
Authors: Wijnaendts L , Le Deist F , Griscelli C , Fischer A
Published: October 2019

We retrospectively analyzed the development of lymphocytes and of the main immunological functions in 33 patients with severe combined immunodeficiency who survived at least 6 months after bone marrow transplantation (BMT). Eighteen patients received HLA-identical BM and 15 received HLA-nonidentical BM. Development of immune functions occurred faster after HLA-identical BMT as full T- and B-lymphocyte- mediated responses were present at day 186 versus 505, respectively (P = .05). In addition, antibody responses remain completely or partially absent in 8 of 15 patients of the second group. Detection of antibody response after HLA-incompatible BMT correlated with engraftment of donor B cells in informative cases. In patients who received an HLA- nonidentical BMT after chemotherapy (6 of 15), development of immune functions occurred more rapidly and 6 of 6 had B-cell functions, including normal antibody production. Autoimmunity was not uncommon and was found after HLA-incompatible BMT (4 of 15) or after HLA-partially phenotypically identical BMT (2 of 3). Antibodies were in most cases specific for blood cells. Occurrence of autoimmunity correlates with poor B-cell functions and to a lesser extent with defective T-cell responses. This type of study may lead to definition of a more accurate strategy for performing BMT in patients with severe combined immunodeficiency.
Interleukin-1 enhances survival of lethally irradiated mice treated with allogeneic bone marrow cells
Authors: Oppenheim JJ , Neta R , Tiberghien P , Gress R , Kenny JJ , Longo DL
Published: October 2019

Interleukin-1 (IL-1) enhanced the capacity of allogeneic bone marrow (BM) cells to promote survival of mice given doses of radiation (1,200 to 1,350 cGy) that are significantly higher than those generally used for BM ablation (850 to 950 cGy). Three to five times greater numbers of lethally irradiated (1,200 to 1,350 cGy) C57B1/6 (H-2b) mice given 10(7) T-cell-depleted Balb/c (H-2d) BM cells survived over 6 weeks if also treated with a single intraperitoneal (IP) dose of 10 micrograms IL-1 20 hours before or from 1 to 3 hours after radiation. The spleens of these mice were reconstituted predominantly, but not exclusively, with donor cells (54% to 91%). Histologic examination of the epidermal and gastrointestinal tissues of mice surviving more than 6 weeks did not reveal any evidence of graft-versus-host (GVH) disease; however, since 10% to 43% of the mice died between days 30 and 46, the possibility of a GVH syndrome in these mice cannot be excluded. The spleen cells from irradiated mice given BM transplants and IL-1, which consisted of greater than or equal to 85% donor cells, were able to generate specific T-cell cytotoxic killing of unrelated allogeneic donor cells but were unreactive to target cells bearing either host or donor major histocompatibility complex (MHC) class I antigens. Thus, long-term mixed chimeric survivors were tolerant to recipient and donor alloantigens but exhibited immunologic competence. These results show that IL-1 promotes survival of lethally irradiated mice and that allogeneic hematopoietic cells in such animals develop tolerance to host MHC antigens. Although there are many unanswered questions, these data suggest that IL-1 may prove clinically useful in promoting BM engraftment.
Platelet-derived growth factor concentrations in platelet-poor plasma and urine from patients with myeloproliferative disorders
Authors: Gersuk GM , Carmel R , Pattengale PK
Published: October 2019

Our enzyme-linked immunosorbent assay (ELISA) for measuring human platelet-derived growth factor (PDGF) detects nanogram quantities (ranging from 0.007 to 16 ng/100 microL) in purified PDGF standards. This assay is sensitive enough for studying plasma and urine. The range in normal volunteers was 0.6 to 2.3 micrograms/L for platelet-poor plasma and 1.4 to 3.3 micrograms/L for urine. We determined PDGF levels in the circulation (outside platelets) in patients with myeloproliferative diseases. Platelet-poor plasma and urine PDGF were significantly elevated in patients with myelofibrosis (6.2 +/- 2.0 micrograms/L for plasma; 7.8 +/- 2.4 micrograms/L for urine) and essential thrombocythemia (5.5 +/- 1.5 micrograms/L for plasma; 11.4 +/- 2.2 micrograms/L for urine), but not in patients with chronic myelogenous leukemia (2.1 +/- 0.4 micrograms/L for plasma; 2.8 +/- 1.2 micrograms/L for urine). Polycythemia vera produced an intermediate pattern: although plasma PDGF was within the normal range (2.1 +/- 0.2 micrograms/L), urine levels were increased (3.7 +/- 0.6 micrograms/L). These results show that PDGF is increased in the circulation in some but not all myeloproliferative diseases, and suggest that this is due to abnormal in vivo release from either megakaryocytes in the bone marrow or circulating platelets.
Human granulocyte colony-stimulating factor specifically binds to murine myeloblastic NFS-60 cells and activates their guanosine triphosphate binding proteins/adenylate cyclase system
Authors: Matsuda S , Shirafuji N , Asano S
Published: October 2019

The specific binding of human granulocyte colony-stimulating factor (G- CSF) to its receptors on NFS-60 cells acts as a primer for cellular proliferation. There are approximately 400 binding sites per cell, with a binding constant of about 100 pmol/L. Before the proliferative response, the affinity constant of the membrane particulate fraction to 35S-labeled guanosine triphosphate-gamma-S (35S-GTP gamma S) and the intracellular cyclic adenylate monophosphate (cAMP) level increased in the presence of G-CSF to about 2.5-fold and about fivefold higher, respectively, than the levels seen in the absence of G-CSF. The increases were time-dependent, with a peak occurring 15 minutes after the addition of G-CSF at 37 degrees C. These findings suggest that, following the binding of the G-CSF to its surface receptors, the activation of the guanosine triphosphate (GTP)-binding protein/adenylate cyclase system may be involved in the proliferation of immature myeloid cells.
Inhibition of autoantibody binding to platelet glycoprotein IIb/IIIa by anti-idiotypic antibodies in intravenous gammaglobulin
Authors: Berchtold P , Dale GL , Tani P , McMillan R
Published: October 2019

Intravenous immunoglobulin (IVIgG) causes an acute rise in the platelet count in the majority of patients with chronic immune thrombocytopenic purpura (ITP) but the mechanism(s) of action is still unknown. We evaluated the ability of three different IVIgG preparations to inhibit the in vitro binding of autoantibody to platelet glycoprotein (GP) IIb/IIIa. ITP plasma, known to contain anti-GPIIb/IIIa antibodies, was incubated overnight with either IVIgG or bovine serum albumin (BSA) followed by measurement of the autoantibody titer. Binding of autoantibody from eight ITP patients was inhibited by IVIgG in proportion to the IVIgG concentration. Using 3.2% IVIgG, compatible with therapeutic concentrations expected in vivo, mean inhibition of autoantibody binding ranged from 20.2% to 41.3%. No inhibition by IVIgG of alloantibody binding to the same or different molecules was detected (five patients with anti-GPIIb/IIIa and two with anti-HLA alloantibodies). F(ab')2 fragments of IVIgG also inhibited the binding of both plasma autoantibodies and purified anti-GPIIb/IIIA autoantibodies prepared by elution from antigen affinity columns. A portion of the anti-idiotypic antibodies could be adsorbed from IVIgG using insolubilized, purified anti-GPIIb/IIIa autoantibody. These results show that IVIgG preparations from normal donors contain anti- idiotypic antibodies directed against idiotypes located on GPIIb/IIIa autoantibodies but do not have anti-idiotypes to platelet alloantibodies against the same or different molecules. The importance of these anti-idiotypic antibodies in the therapeutic response to IVIgG remains to be established.
Modulation, shedding, and serum titers of the chronic lymphatic leukemia-associated antigen: characterization and clinical correlations
Authors: Faguet GB , Agee JF
Published: October 2019

The fate of the common chronic lymphatic leukemia antigen (cCLLa), a leukemia-associated antigen selectively expressed by clonal cells in chronic lymphatic leukemia (CLL) was examined in 31 patients. cCLLa was detected by immune precipitation assay in extracts of metabolically labeled CLL culture cells, in CLL cell culture supernatants, and in patients' sera. In vitro shed membrane cCLLa was comparable in all patients (n = 15) on a per cCLLa-positive cell basis but was independent of cCLLa density (r = .46), absolute lymphocyte count ([ALC] r = .46) and stage (r = .44). In contrast, serum cCLLa (n = 31) correlated with absolute cCLLa-positive cell count (r = .92) and to a lesser extent with stage (r = .67), but was independent of cCLLa density (r = .47). cCLLa modulation was assessed from changes in membrane density estimated by radioreceptor assay before and after in vitro exposure to anti-cCLLa monoclonal antibody (MoAb) CLL2. Immune precipitation studies of metabolically labeled CLL cells showed that modulated cCLLa was internalized as judged by its detection within modulated cells but not in their supernatants. Intact cCLLa-CLL2 complexes were not detected within the modulated cells nor in their supernatants. Regeneration of modulated cCLLa was rapid with return to baseline density levels within 24 hours of antibody removal. Modulation was specific and depended on exposure time, medium temperature, and on antibody titer; correlated with extent of disease (versus absolute lymphocyte count, r = .79; versus stage, r = .66; n = 22); was independent of cCLLa density or affinity (r = .44 to r = .57; n = 11); and was unaffected by T cells or by monocytes (n = 14).
Priming of human neutrophils with N-formyl-methionyl-leucyl- phenylalanine by a calcium-independent, pertussis toxin-insensitive pathway
Authors: Karnad AB , Hartshorn KL , Wright J , Myers JB , Schwartz JH , Tauber AI
Published: October 2019

Resting neutrophils may be “primed” to augmented effector function, eg, superoxide (O2-) production in the respiratory burst, upon a second stimulation with a variety of soluble agonists including formylated methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). At priming concentrations of FMLP (5 x 10(-9) mol/L) that did not initiate O2- generation, two metabolic activities were noted: (1) approximately a threefold increase in the baseline intracellular calcium (Ca++i) level, that was not dependent on extracellular Ca++, and (2) a rapid rise in intracellular pH that was blocked by 5-(N,N- dimethyl) amiloride (DA), that had no effect on the Ca++i response to priming. Furthermore, there were no significant increases in inositol metabolites in cells primed and stimulated with FMLP compared with cells receiving the stimulating dose of FMLP alone and pretreatment with pertussis toxin (PT) (before the addition of the priming -5 x 10(- 9) mol/L dose of FMLP), whereas abolishing the response to FMLP during the second stage of stimulation, had (1) no effect on FMLP-primed cells subsequently stimulated with PMA, and (2) only partially ablated the rise in Ca++i initiated with FMLP. That FMLP priming involved distinctive processes to those of the well characterized FMLP-coupled Ca++-dependent activation cascade was shown by the full priming effect attained in a Ca++-free buffer, which did not sustain an O2- response to a second-stage FMLP stimulation, but sustained a primed response to PMA. These data demonstrate that FMLP primes human neutrophils by a Ca++-independent and PT-insensitive pathway, offering a functional model for studying heterogeneous FMLP receptor-coupled reactions.
Enhancement of erythrocyte superoxide dismutase activity: effects on cellular oxidant defense
Authors: Scott MD , Eaton JW , Kuypers FA , Chiu DT , Lubin BH
Published: October 2019

To delineate further the role of superoxide dismutase (SOD) in red blood cell (RBC) oxidant defense, normal human erythrocytes were osmotically lysed and resealed in the presence of varying concentrations of exogenous SOD. This resulted in a dose-dependent increase in SOD activity in the resealed erythrocytes while maintaining nearly normal RBC hemoglobin concentration (less than 10% decrease from the control value), cell volume, and cellular deformability. Surprisingly, a five- or ninefold increase in SOD activity yielded no additional protection against superoxide-generating drugs (phenazine methosulfate or menadione sodium bisulfite). No significant differences were observed between the control and SOD-loaded RBCs in O2-driven methemoglobin formation or generation of thiobarbituric acid-reactive substances. In contrast, RBCs with elevated SOD activity pretreated with sodium azide (to block catalase activity) or 1-chloro-2,4- dinitrobenzene (to deplete reduced glutathione, GSH) showed significantly enhanced methemoglobin generation in response to superoxide generating drugs. No differential response was noted between the control, control-resealed, and SOD-loaded RBCs to oxidants other than superoxide. Based on our results and other data, we conclude that elevated SOD activity may imbalance cellular oxidant defense, resulting in enhanced oxidation due to the accelerated generation of H2O2, the product of O2- dismutation. This effect is significantly exacerbated under conditions in which H2O2 catabolism is altered.
Serological confirmation of human T-lymphotropic virus type I infection in healthy blood and plasma donors
Authors: Anderson DW , Epstein JS , Lee TH , Lairmore MD , Saxinger C , Kalyanaraman VS , Slamon D , Parks W , Poiesz BJ , Pierik LT
Published: October 2019

We wished to develop criteria for serological confirmation of human T- lymphotropic virus type I (HTLV-I) infection in healthy donors. Selected serum or plasma samples reactive by HTLV-I enzyme immunosorbent assay or gel-agglutination assays with at least one viral- specific band on Western immunoblot (WIB) were tested in six laboratories by four WIBs and four radioimmunoprecipitation assays (RIPAs) for antibodies to HTLV-I proteins encoded by gag (p19 and p24), env (gp46 and/or gp61), and tax (p40x) genes. One hundred forty-two donor sera were obtained from 38 Japanese, 69 American, and 35 Caribbean blood or plasma donors. Among these samples, WIB assays appeared more sensitive to p24 antibodies, whereas RIPAs were significantly more sensitive to gp61 antibodies. All sera (137) with gp61 antibodies had p24 antibodies. Of the 137 sera positive for p24 and gp61 antibodies, p19 antibodies were detected in 129 sera, and p40x antibodies were detected in 108. In sera with p19 antibodies and antibodies to env- or tax-encoded proteins, p24 antibodies were always present. Antibodies to p40x were not found in the absence of gp61 antibodies. Virological evidence of infection was found in seven American donors by lymphocyte coculture (one HTLV-I, one HTLV-II) or by polymerase chain reaction (three HTLV-I, two HTLV-II). Sera from all seven donors showed p24 and gp46 and/or gp61 antibodies. We suggest that seroreactivity to both p24 and gp46 and/or gp61 by WIB or RIPA or both are suitable criteria to confirm but not to distinguish HTLV-I and HTLV-II infections.
Prevention of transfusion-induced graft-versus-host disease in dogs by ultraviolet irradiation
Authors: Deeg HJ , Graham TC , Gerhard-Miller L , Appelbaum FR , Schuening F , Storb R
Published: October 2019

Ten dogs were given 9.2 Gy of total body irradiation and autologous bone marrow infusion followed by ten daily transfusions of leukocytes for a total of 11.5 to 36.2 (median, 18.8) x 10(8)/kg obtained via leukapheresis from histoincompatible unrelated donors. Four dogs were given unirradiated leukocytes, and all developed graft-versus-host disease (GVHD). In contrast, only two of three dogs given leukocytes irradiated with 20 mJ/cm2 of ultraviolet (UV) light (200 to 300 nm), and none of three dogs given leukocytes irradiated with 1,000 mJ/cm2 developed GVHD. These data indicate that UV irradiation abrogates the alloreactive potential of transfused leukocytes, and suggest that UV irradiation can be used to prevent the development of transfusion- induced GVHD.
Interleukin-6 is not involved in the interleukin-1-induced production of colony-stimulating factors by human bone marrow stromal cells and fibroblasts
Authors: Schaafsma MR , Fibbe WE , Van Damme J , Duinkerken N , Ralph P , Kaushansky K , Altrock BW , Willemze R , Falkenburg JH
Published: October 2019

Interleukin-6 (IL-6) is a multifunctional cytokine that plays a role in regulation of hematopoiesis. Because IL-6 is coinduced with colony- stimulating factors (CSFs) by various cell types in response to stimulation with IL-1, we investigated whether IL-6 is involved in the IL-1-induced production of CSF by human bone marrow (BM) cells in long- term culture or human fibroblasts. We showed that IL-6 does not induce CSF production by these cells. Neither addition of exogenous IL-6 nor neutralization of endogenous production of IL-6 by an anti-IL-6 monoclonal antibody (MoAb) diminished the IL-1-induced colony- stimulating activity (CSA), indicating that IL-6 did not act synergistically with IL-1. Finally, IL-6 did not influence the kinetics of IL-1-induced CSA production by human fibroblasts. We conclude that IL-6, either alone or in combination with IL-1, does not induce CSF production by human BM stromal cells or fibroblasts.
Immunophenotypes of Reed-Sternberg cells: a study of 19 cases of Hodgkin's disease in plastic-embedded sections
Authors: Casey TT , Olson SJ , Cousar JB , Collins RD
Published: October 2019

The immunophenotype of Reed-Sternberg (RS) cells in Hodgkin's disease (HD) has not been clearly defined, partly owing to difficulties in studying RS cells in cell suspensions or identifying them with certainty in frozen sections. We studied the immunophenotype of RS cells with a recently developed plastic section immunohistochemical technique on acetone-fixed tissues that affords superior morphological detail while preserving a wide variety of lymphoid differentiation antigens. Nineteen cases of HD [16 nodular sclerosing (NS), 2 mixed cellularity (MC), and 1 lymphocyte depleted (LD)] were embedded in plastic and stained for pan-B, pan-T, and various T-subset markers, as well as leukocyte common antigen (CD45), interleukin-2 (IL-2) receptor (CD25), and RS cell markers CD15 and CD30. RS cells were positive for CD45, CD15, CD30, and CD25, except for 3 cases (2 NS, 1 MC) that were CD15 negative and 2 cases (NS) that were CD45 negative. In 10 cases (NS), RS cells were positive for at least two pan-T-cell markers and CD4; pan-B cell markers were uniformly negative. RS cells in 6 cases (3 NS, 2 MC, 1 LD) were positive for at least one T-cell marker (CD2) and one B-cell marker (CD22). Two cases of NSHD showed no T- or B-cell marking. These data provide further evidence that RS cells in some cases of NSHD have T-cell phenotypes and that RS cells are not homogeneous in their immunoreactivity.
Hematologic effects of recombinant human granulocyte colony-stimulating factor in patients with malignancy
Authors: Lindemann A , Herrmann F , Oster W , Haffner G , Meyenburg W , Souza LM , Mertelsmann R
Published: October 2019

The effect of recombinant human granulocyte colony-stimulating factor (G-CSF) on hematologic parameters was evaluated in a phase I clinical study in 18 patients with advanced malignancy. G-CSF was administered once daily as a 30-minute infusion for 14 days; three patients each were treated at increasing dose levels of 1, 3, 10, 30, and 60 micrograms kg-1 day-1. A transient decrease in neutrophil and monocyte counts was observed immediately after the G-CSF infusion, followed by a dose-dependent increase of up to 15-fold. G-CSF-induced neutrophils exhibited an increased O2- radical production, and serum levels of enzymes related to granulocyte turnover, including lysozyme and elastase, were markedly elevated during therapy. A dose-dependent depression of platelet counts occurred in the second third of the treatment course, followed by a spontaneous recovery despite continuing therapy. G-CSF was well-tolerated; minor to moderate bone pain was the most common side effect. The primary course of the malignant diseases studied was not significantly altered. G-CSF appears to be an appropriate means to selectively increase the number of functionally competent polymorphonuclear phagocytes.
Granulocyte colony-stimulating factor receptors in human acute myelocytic leukemia
Authors: Budel LM , Touw IP , Delwel R , Lowenberg B
Published: October 2019

The binding of granulocyte colony-stimulating factor (G-CSF) to normal and human acute myeloid leukemia (AML) cells was investigated with radiolabeled recombinant human G-CSF (rhG-CSF). In all 14 cases of primary AML specific receptors for G-CSF were demonstrated on purified blast cells. The average numbers of G-CSF receptors ranged from very low to 428 receptors per cell (mean). Normal granulocytes showed G-CSF binding sites on their surface at higher densities (703 to 1,296 sites per cell). G-CSF receptors appeared to be of a single affinity type with a dissociation constant (kd) ranging between 214 and 378 pmol/L for AML blasts and 405 to 648 pmol/L for granulocytes. In 12 of 14 cases, including those with relatively low specific binding, G-CSF was a potent inducer of DNA synthesis of blasts in vitro; therefore, apparently relatively few receptors are required to permit activation of AML cell growth. However, in two cases cell cycling was not activated in response to G-CSF despite G-CSF receptor availability. The results show that G-CSF receptors of high affinity are frequently expressed on the blasts of human AML, but their presence may not be a strict indicator of the proliferative responsiveness of the cells to G- CSF.
Calcium mobilization in human platelets using indo-1 and flow cytometry
Authors: Jennings LK , Dockter ME , Wall CD , Fox CF , Kennedy DM
Published: October 2019

Regulation of cytoplasmic free calcium concentration is believed to be important in the response of platelets to external stimuli. A relatively new fluorescent calcium indicator, indo-1, has properties by which alterations of cytoplasmic calcium can be evaluated in single platelets by flow cytometry. Activation of platelets at a temperature lower than 37 degrees C allows examination of the heterogeneity of intracellular free calcium levels and can distinguish variations among platelets in the initiation, duration, and magnitude of calcium fluxes. The clear advantage of flow cytometric analysis of platelet cytosolic calcium is that stimulus-response coupling can now be studied on a single cell basis. Platelets were activated by addition of human alpha- thrombin or ADP at 37 degrees C or at room temperature (22 degrees C). Activation at 37 degrees C approaches more closely an in vivo response and, as expected, increases in cytosolic calcium occurred within seconds of agonist addition. Transient increases in cytoplasmic calcium levels occurred when platelets were challenged with a low concentration of agonist. Heterogeneity in cytoplasmic calcium levels was also observed at 10(-5) mol/L ADP and 0.1 U/mL alpha-thrombin. Some of this heterogeneity was no longer observed at higher concentrations of agonist (10(-4) mol/L ADP and 0.5 U/mL thrombin), suggesting that a sufficient magnitude of signal is required to induce changes in platelet cytosolic calcium. Light-scatter properties of the activated platelets were also monitored simultaneously and showed changes in response to both agonists. The ability to measure changes in cytoplasmic free calcium by ratio flow cytofluorimetry provides a new approach to study of the role of alterations in intracellular calcium in response to agonists acting through different membrane receptors as well as providing a sensitive technique to detect functional subpopulations of platelets.
Lymphokine overproduction in severe aplastic anemia is not related to blood transfusions
Authors: Hinterberger W , Adolf G , Bettelheim P , Geissler K , Huber C , Irschick E , Kalhs P , Koller U , Lechner K , Meister B
Published: October 2019

The production of interferons (IFNs), IFN-gamma, tumor necrosis factors (TNFs) and TNF-alpha (TNF-alpha) by peripheral blood mononuclear cells (PBMNCs) of untransfused and transfused, but otherwise untreated patients with severe aplastic anemia (SAA) was determined using bioassays and immunoassays. In untransfused and pretransfused SAA patients, spontaneous and lectin-induced production of these cytokines by PBMNCs was strongly enhanced. Cytokine production in untransfused SAA patients did not differ from that in pretransfused patients. Similar relative frequencies of activated (HLA-DR+) lymphocyte subpopulations present in the PBMNCs demonstrated cytokine overproduction per cells. Cytokine production was studied in three SAA patients before and after blood cell transfusions. Spontaneous and lectin-induced production of these cytokines was abnormally high and unaffected by blood transfusions. In another patient exhibiting abnormal cytokine production, the hematopoietic response to cyclosporin- A in vivo was accompanied by normalization of cytokine production in vitro. We conclude that overproduction of IFN-gamma and TNF-alpha by lectin-stimulated PBMNCs is an intrinsic abnormality of SAA unrelated to blood transfusions. Normalization of production of IFN-gamma and TNF- alpha accompanying a clinical response to cyclosporin-A may cautiously be taken as further evidence suggesting a pathogenetic role of cytokine overproduction in SAA.
Trans-activation of human globin genes by HTLV-I tax1
Authors: Fox HB , Gutman PD , Dave HP , Cao SX , Mittelman M , Berg PE , Schechter AN
Published: October 2019

We studied the effects of a known retroviral trans-activating factor, HTLV-I tax1, on transcription of human globin genes. Transfection of HeLa cells by the cloned tax1 gene stimulated activity of both the beta- and epsilon-globin promoters approximately 20-fold, as measured by chloramphenicol acetyl transferase (CAT) assays. Studies of promoter 5′- deletion mutants revealed that the trans-activation response required only 185 base pairs (bp) of beta-globin 5′-flanking sequence or 177 bp of epsilon-globin 5′ flanking sequence. These promoter regions contain either two (for beta) or three (for epsilon) copies of the pentanucleotide sequence CTGAC, which is characteristic of previously described tax1-responsive promoters. We also stably transfected tax1 into the erythroid cell line K562. Transfectants expressing tax1 showed increased transcription of epsilon-, gamma-, zeta-, and alpha-globins. This indicates that tax1 can stimulate transcription of globin genes in their native chromosomal location. This was confirmed by measurements of increases in intracellular hemoglobin as determined by an increased percentage of cells staining with benzidine and by spectrophotometric measurements of hemoglobin. The observed trans-activation of globin genes by tax1 may provide insight into normal regulation of globin genes by clarifying cis regulatory sequences. Furthermore, it suggests that the trans-acting effects of tax1 on heterologous genes are more widespread than was previously appreciated.
Intracellular localization and de novo synthesis of FcRIII in human neutrophil granulocytes
Authors: Jost CR , Huizinga TW , de Goede R , Fransen JA , Tetteroo PA , Daha MR , Ginsel LA
Published: October 2019

Immunoelectron microscopic studies in human neutrophils showed that FcRIII was present on the plasma membrane, in the Golgi complex, and in many small vesicles (120 to 180 nm). FcRIII was not found in specific or azurophilic granules as shown by immunogold double-labeling experiments, visualizing both FcRIII and either lactoferrin (a marker of specific granules) or myeloperoxidase (a marker for azurophilic granules). Because the occurrence of FcRIII in the Golgi complex suggested that biosynthesis of this receptor occurs in these cells, metabolic labeling experiments were performed. Immunoprecipitation of FcRIII from NP-40 lysates of cells labeled with 35S-methionine showed a diffuse 50- to 70-Kd band corresponding with the band noted after immunoprecipitation of FcRIII from surface iodinated cells. These findings show that de novo synthesis of FcRIII occurs in neutrophils and suggest that at least some of the small vesicles containing FcRIII derive from the Golgi complex and thus are involved in transport of newly synthesized FcRIII to the plasma membrane.
Assay of lymphokine-activated killer activity generated from bone marrow cells of children with acute lymphoblastic leukemia
Authors: Zhou MX , Findley HW Jr , Davis R , Ragab AH
Published: October 2019

We recently reported that low molecular weight B-cell growth factor (LMW-BCGF) plus recombinant interleukin-2 (rIL-2) synergistically induced lymphokine-activated killer (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). The kinetics of cell growth, antigenic phenotype, and lytic activity of the generated effector cells were further analyzed in this study. BM cells from ALL patients with active disease and in complete remission (CR) were cultured with a combination of LMW-BCGF and rIL-2. Monoclonal antibodies (anti-CD3 and anti-Leu 19) and immunomagnetic beads were used to separate LAK cells into three subsets: CD3+/Leu 19-, CD3+/Leu 19+, and CD3-/Leu 19+. Cytotoxicity assays with different subsets were performed versus K562, Raji, and autologous leukemic cells, using a 3- hour 51Cr release test. There was a significant cell expansion of 54- fold (mean value) for CD3+ cells and 15-fold for Leu 19+ cells in culture with LMW-BCGF plus rIL-2 for 7 to 14 days, whereas no cell expansion was observed in culture with rIL-2 alone. Although NK activity (K562) was generated from leukemic BM cells in culture with rIL-2 alone, it is only about one third of that generated in culture with rIL-2 plus LMW-BCGF. Analysis of lytic activity of cells generated in the latter cultures demonstrated that CD3-/Leu 19+ cells expressed highest lytic activity against NK-sensitive K562 cells as well as against NK-resistant Raji cells. CD3+/Leu 19+ cells showed median cytotoxicity, and CD3+Leu 19- cells mediated only minimal cytotoxic activity. Also, lytic activity of CD3-/Leu 19+ cells against autologous leukemic blasts was noted in patients with active disease. Our results demonstrate that LAK activity generated from BM cells by LMW-BCGF and r- IL2 is mediated mainly by two types of Leu 19+ cells: CD3-/Leu 19+ NK cells and CD3-/Leu 19+ T cells. Although CD3+ T cells (both Leu 19+ and Leu 19-) mediated less antitumor cytotoxicity than CD3-/Leu 19+ cells, the former cells were the major expanding cell population in culture with LMW-BCGF and rIL-2. The new culture system may be effective in generation of cells with LAK activity for therapeutic use.