bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of immature and mature myeloma cells in the bone marrow of human myelomas
Authors: Kawano MM , Huang N , Harada H , Harada Y , Sakai A , Tanaka H , Iwato K , Kuramoto A
Published: October 2019

With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two- color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA- 5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL- 6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA- 5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.
Antisense oligonucleotides to the p65 subunit of NF-kappa B block CD11b expression and alter adhesion properties of differentiated HL-60 granulocytes
Authors: Sokoloski JA , Sartorelli AC , Rosen CA , Narayanan R
Published: October 2019

NF-kappa B is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of NF-kappa B to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the p50 and p65 subunits of the NF- kappa B complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of p65 was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O- tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in CD11b integrin expression on the surface of treated cells. Furthermore, the p65 antisense oligomer effectively abolished an upregulation of CD11b that was produced by formyl-met-leu- phe and TPA. However, the p65 antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to p65 can be used to define the role of NF-kappa B in the activation pathways of neutrophils.
Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines
Authors: Ganz T , Liu L , Valore EV , Oren A
Published: October 2019

Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in multivesicular bodies in 32D cells and in cytoplasmic granules of 32D cl3 cells. Our data suggest that the enzymatic and transport machinery required to process preprodefensin to mature defensin and to store it in cytoplasmic granules is a specialized feature of cells of granulocytic lineage.
Molecular cloning of RhD cDNA derived from a gene present in RhD- positive, but not RhD-negative individuals
Authors: Arce MA , Thompson ES , Wagner S , Coyne KE , Ferdman BA , Lublin DM
Published: October 2019

The Rh blood group system plays a major role in immune and nonimmune hemolytic states. Although an Rh cDNA has been previously cloned, there is no information on which Rh antigenic protein it encodes. Using polymerase chain reaction (PCR) amplification, we have identified this original Rh clone, here designated Rh21, and an additional Rh cDNA clone, Rh13, that is 96% nucleotide- and 92% amino acid-identical to Rh21, with the substitutions scattered throughout the sequence. A molecular genetic approach was used to match this Rh clone with an Rh specificity. The mRNA transcript for Rh13 was present in reticulocytes from RhD-positive individuals, but was absent from the reticulocytes of RhD-negative individuals. Using conventional screening of genomic libraries, as well as PCR cloning, partial genomic clones for these two Rh cDNAs were obtained. Based on PCR analysis and Southern blots, the Rh21 gene was present in all individuals, but an intact Rh13 gene was only present in RhD-positive and not RhD-negative individuals. Thus, by correlating the presence of Rh mRNA and gene sequences with individual Rh phenotypes, we were able to establish that the new Rh13 cDNA clone represents the RhD protein.
Structure and expression of the RH locus in the Rh-deficiency syndrome
Authors: Cherif-Zahar B , Raynal V , Le Van Kim C , D'Ambrosio AM , Bailly P , Cartron JP , Colin Y
Published: October 2019

Red blood cell deficiency of Rh proteins is associated with morphologic and functional abnormalities of erythrocytes and with a chronic hemolytic anemia of varying severity. Rh-deficiency may be the result of homozygosity either for a silent allele at the RH locus (Rhnull amorph type) or for a recessive inhibitor gene(s) at an autosomal locus unlinked to RH locus (Rhnull regulator and Rhmod). In this report, we investigated the RH locus structure of Rh-deficient individuals by Southern analysis using cDNA and exon-specific probes deduced from the recent cloning of Rh genes (CcEe and D). As expected from family studies indicating that Rhmod and Rhnull regulator individuals are unable to express Rh antigens but are able to convey functional Rh genes from one generation to another, no alteration of the Rh genes was detected in these variants. Although Rhnull of the amorph type arose by inheritance of a pair of silent alleles at the RH locus, the general organization of the unique CcEe gene in the genome of the particular individual under examination was apparently normal and indistinguishable from a Rh-negative chromosome. More surprisingly, no mutation could be detected by sequencing the polymerase chain reaction (PCR)-amplified reticulocyte mRNAs, suggesting that the RH locus of this patient might be altered in its transcriptional activity. Through hybridization with exon-specific probes, we were also able to determine the zygosity for the D gene in DNA samples from individuals of known genotypes; using this approach, we found that Rhnull regulator variants could be either of the DD, Dd, or dd genotypes. These findings suggest that the postulated inhibitor gene(s) can negatively suppress the RH locus expression from chromosomes carrying either one or two of the Rh genes.
Evidence of hepatitis in patients receiving transfusions of blood components containing antibody to hepatitis C
Authors: Aoki SK , Holland PV , Fernando LP , Kuramoto IK , Anderson S , Sazama K , Schoening V , Johnson P , Wilber JC , Fenner L
Published: October 2019

When hepatitis C virus antibody (anti-HCV) enzyme immunoassay (EIA1) testing became available in 1990, we tested samples from previously transfused blood units, traced the recipients of reactive units, and evaluated the recipients for HCV infection during the 12 months after transfusion. Ten of 42 recipients of EIA1-reactive blood were anti-HCV reactive on follow-up by EIA1 and 12 were reactive by a second- generation assay (EIA2). Reverse transcriptase-polymerase chain reaction (RT-PCR) detected HCV RNA in 5 seronegative recipients. In all, 17 of 42 recipients (40%) of EIA1-reactive blood had evidence of HCV infection. In comparison, 54 surgery patients, who received either no transfusion or autologous EIA1-nonreactive blood, remained EIA1 nonreactive and RT-PCR negative for 1 year; 1 patient (1.8%) became EIA2 reactive (P < or = .01). Of the recipients of anti-HVC reactive blood transfusions (reactive by both EIA1 and a supplemental 4-antigen strip immunoblot assay [RIBA2]), 14 (93%) of the recipients had evidence of HCV infection compared with only 3 of 27 recipients (11%) of EIA1-reactive, RIBA2-nonreactive blood (P < or = .01). Thus, blood components reactive for anti-HCV EIA1 may have transmitted HCV up to 40% of the time, but blood components found reactive by both EIA1 and RIBA2 may transmit HCV with a frequency of greater than 90%.
Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission
Authors: Nucifora G , Larson RA , Rowley JD
Published: October 2019

The translocation between chromosomes 8 and 21, t(8;21) (q22;q22), is the most frequent abnormality in acute myeloid leukemia (AML) with French-American-British type M2 (FAB-M2) morphology. The breakpoints in this translocation have been characterized at the molecular level, and the genes involved are AML1 on chromosome 21 and ETO on chromosome 8. The rearrangement of the two chromosomes results in a fusion gene and in the production of a consistent fusion transcript on the der(8) chromosome. We have used oligonucleotide primers derived from both sides of the fusion cDNA junction and reverse transcription-polymerase chain reaction (RT-PCR) to analyze six AML-M2 patients with a t(8;21) during various stages of their disease. Two patients studied at diagnosis and one studied at first relapse are alive off therapy and in continuous complete remission for 83 to 94 months. We have detected the AML/ETO fusion transcript in recent peripheral blood samples from each of them. Three other patients also had a fusion transcript detected after 1 to 4 months in remission. Two of these patients subsequently relapsed and died whereas the third patient is alive and in continuous complete remission 70 months later. Thus, our preliminary data suggest that cells with the translocation are still circulating in t(8;21) patients in long-term remission. This finding raises serious questions regarding the interpretation of positive results obtained only with this technique that may not be suitable to decide appropriate further treatment for patients in clinical remission.
Identification of yeast artificial chromosomes containing the inversion 16 p-arm breakpoint associated with acute myelomonocytic leukemia
Authors: Liu P , Claxton DF , Marlton P , Hajra A , Siciliano J , Freedman M , Chandrasekharappa SC , Yanagisawa K , Stallings RL , Collins FS
Published: October 2019

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.
Establishment of human T-cell leukemia virus type I T-cell lymphomas in severe combined immunodeficient mice
Authors: Feuer G , Zack JA , Harrington WJ Jr , Valderama R , Rosenblatt JD , Wachsman W , Baird SM , Chen IS
Published: October 2019

Human T-cell leukemia virus type I (HTLV-I) is recognized as the etiologic agent of adult T-cell leukemia (ATL), a disease endemic in certain regions of southeastern Japan, Africa, and the Caribbean basin. Although HTLV-I can immortalize T lymphocytes in culture, factors leading to tumor progression after HTLV-I infection remain elusive. Previous attempts to propagate the ATL tumor cells in animals have been unsuccessful. Severe combined immunodeficient (SCID) mice have previously been used to support the survival of human lymphoid cell populations when inoculated with human peripheral blood lymphocytes (PBL). SCID mice were injected intraperitoneally with PBL from patients diagnosed with ATL, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or from asymptomatic HTLV-I-seropositive patients. Many of these mice become persistently infected with HTLV-I. Furthermore, after human reconstitution was established in these mice, HTLV-I-infected cells displayed a proliferative advantage over uninfected human cells. Lymphoblastic lymphomas of human origin developed in animals injected with PBL from two ATL patients. The tumor cells represented outgrowth of the original ATL leukemic clone in that they had monoclonal or oligoclonal integrations of the HTLV-I provirus identical to the leukemic clone and predominantly expressed the cell surface markers, CD4 and CD25. In contrast, cell lines derived by HTLV immortalization of T cells in vitro did not persist or form tumors when inoculated into SCID mice, indicating differences between in vitro immortalized cells and ATL leukemic cells. This system represents the first small animal model to study HTLV-I tumorigenesis in vivo.
Treatment of Diamond-Blackfan anemia with recombinant human interleukin- 3 [see comments]
Authors: Gillio AP , Faulkner LB , Alter BP , Reilly L , Klafter R , Heller G , Young DC , Lipton JM , Moore MA , O'Reilly RJ
Published: October 2019

This report describes the response of eighteen Diamond-Blackfan anemia (DBA) patients to recombinant human interleukin-3 (rhIL-3). rhIL-3 was administered subcutaneously once daily on an escalating dose schedule (0.5 to 10 micrograms/kg/d). The rhIL-3 dose was escalated every 21 days until erythroid response was attained, grade III or IV nonhematologic toxicity was observed, or the maximum rhIL-3 dose was reached. Four patients experienced clinically significant erythroid responses. Two of the responders were steroid-dependent and transfusion- independent, while two were steroid-independent and transfusion- dependent. Baseline clinical or laboratory parameters, in particular in vitro bone marrow erythroid progenitor assays, were not useful in predicting rhIL-3 response. rhIL-3 administered at 5 to 10 micrograms/kg/d was associated with an increase in total white blood cell count, secondary to increases in neutrophils, eosinophils, and lymphocytes. Patients experienced a dose-dependent elevation in absolute eosinophils across the entire dose range. Two of the responding patients remain on maintenance rhIL-3, without diminution of effect at 244 and 370 + days. rhIL-3 was discontinued in the other two responders, because of the development of deep venous thrombi.
Purification and characterization of heterogeneous pluripotent hematopoietic stem cell populations expressing high levels of c-kit receptor
Authors: Orlic D , Fischer R , Nishikawa S , Nienhuis AW , Bodine DM
Published: October 2019

Mouse pluripotent hematopoietic stem cells (PHSC) were fractionated based on size and density using counterflow centrifugal elutriation (CCE). These heterogeneous PHSC populations were further enriched by subtraction of cells with lineage-specific markers (Lin-) followed by positive sorting for c-kit expression. The cells were characterized for their functional and biochemical properties. We defined a subpopulation of c-kit-positive cells that expressed high numbers of c-kit receptors (c-kitBR). One hundred c-kitBR cells from either low- or higher-density fractions were sufficient to repopulate the lymphohematopoietic system in WBB6F1-W/Wv (W/Wv) recipients, whereas no PHSC were found in cells with low (c-kitDULL) or no (c-kitNEG) c-kit expression. Lin- c-kitBR cells were separated into RhoDULL and RhoBR subsets based on their ability to efflux rhodamine 123 (Rho). The PHSC were concentrated in Lin- c-kitBR RhoDULL cells and the number of Lin- c-kitBR RhoBR cells correlated directly with the number of day 12 colony-forming unit- spleen (CFU-S12) in each fraction. We were not able to enrich further for PHSC using monoclonal antibodies to the cell-surface markers AA4.1 or CD4, which have been used by others to isolate PHSC. The small, low- density Lin- c-kitBR subset contained PHSC and few CFU-S12. This enabled us to assay PHSC for expression of the flk-2 gene, which encodes a tyrosine kinase receptor present on fetal liver PHSC. Purified RNA from the low-density Lin- c-kitBR subset did not contain flk-2 mRNA. We suggest that AA4.1, CD4 and flk-2 are expressed as stage- specific markers on PHSC in cell cycle.
Autoantibodies against the platelet glycoproteins (GP) IIb/IIIa, Ia/IIa, and IV and partial deficiency in GPIV in a patient with a bleeding disorder and a defective platelet collagen interaction
Authors: Beer JH , Rabaglio M , Berchtold P , von Felten A , Clemetson KJ , Tsakiris DA , Kehrel B , Brandenberger S
Published: October 2019

To evaluate the physiologic importance of the different collagen receptors on platelets, we screened 806 patients admitted to the hospital because of hemorrhagic diathesis for eventual laboratory evidence of a pathologic platelet collagen interaction, and found 5 patients with an isolated deficiency in collagen-induced platelet aggregation. Four of these five patients had a partial defect, one had a complete defect. The structural and functional analysis of the platelets from the patient with a complete defect showed a deficiency in glycoprotein (GP) IV and autoantibodies against GPIIb/IIIa, GPIa/IIa, and GPIV. Patient plasma had only a minimal effect on normal control platelets and Naka-negative platelets. The analyses of the defect in the patient and of the data in the literature suggest that a single defect may not result in clinical bleeding (GPIV-deficient patients do not bleed), but may become symptomatic in combination with another defect such as the autoantibodies against GPIa/IIa, GPIV, and/or GPIIb/IIIa, all of which are involved in platelet collagen interactions (three of four of our immune thrombocytopenic purpura patients with anti-GPIV and anti-GPIIb/IIIa autoantibodies had a bleeding disorder). We hypothesize that it is the synergism of two abnormalities that results in the defective function, a mechanism that is in agreement with earlier studies on platelet collagen interaction that suggests that a double defect in platelet collagen interactions is required to become clinically apparent.
mb-1: a new marker for B-lineage lymphoblastic leukemia
Authors: Buccheri V , Mihaljevic B , Matutes E , Dyer MJ , Mason DY , Catovsky D
Published: October 2019

The expression of the Ig-linked mb-1 polypeptide was analyzed by immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase technique) using a specific monoclonal antibody in 165 cases of acute leukemia, with 88 being lymphoblastic (ALL) and 77 myeloid (AML). The purpose of the study was to investigate the specificity of this reagent for B-lineage cases and its reactivity on leukemias that coexpress myeloid and B-cell antigens (biphenotypic). The majority (89%) of 72 B- cell precursor ALL patients were positive. Of these, mb-1 was expressed in all 9 patients with early-B-ALL (CD10-, c mu-), in all 11 patients with pre-B-ALL (c mu+) and in the single case of B-ALL (smIgM+). Forty- three of 51 patients with common-ALL (CD10+, c mu+) were also positive. All 16 T-lineage ALL patients and 72 (93.5%) of the AML patients examined were mb-1 negative. Four of the 5 mb-1-positive AML patients were considered biphenotypic and expressed other B-cell antigens such as CD10, CD19, and/or cCD22 and all showed rearrangement of the Ig heavy chain genes. Within the AML cases, mb-1 and cCD22 were more useful than other B-cell antigens in detecting biphenotypic cases, and mb-1 showed the highest correlation with the clonal rearrangement of Ig heavy chain genes. These results indicate that mb-1 is a sensitive and specific reagent for B-lineage blasts that will aid in the classification of B-cell precursor ALL and in the identification of biphenotypic leukemia presenting as AML.
In situ hybridization on May-Grunwald Giemsa-stained bone marrow and blood smears of patients with hematologic disorders allows detection of cell-lineage-specific cytogenetic abnormalities
Authors: van Lom K , Hagemeijer A , Smit EM , Lowenberg B
Published: October 2019

Bone marrow and blood from patients with acute myeloid leukemia and myelodysplastic syndrome were studied by simultaneous analysis of cell morphology and karyotype. A combined technique of May-Grunwald Giemsa (MGG) for cell morphology and fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of cytogenetic aberrations allowed us to investigate cell-lineage-specific chromosomal abnormalities. We introduced video recordings to examine large numbers of cells. Briefly, evaluation was first performed on MGG slides, during which cell position and morphology were recorded on an S-VHS recorder. Subsequently, the same slides were used for FISH. This resulted in the identification of MGG-stained cells on the video screen and, at the same time, the interpretation of FISH signals in the fluorescence microscope. Specimens of bone marrow or blood samples from four patients with different hematologic malignancies were studied. One of these patients was studied before and after cytotoxic treatment. The gain or loss of chromosomes could be detected easily and morphologically assigned to the blasts in all patients and to a variable proportion of the myelomonocytic lineage in two patients, but not to the lymphocytes. Thus, this method provides new possibilities for investigating the clonality of hematologic malignancies.
Serum intercellular adhesion molecule-1 in childhood malignancy
Authors: Pui CH , Luo X , Evans W , Martin S , Rugg A , Wilimas J , Crist WM , Hudson M
Published: October 2019

Levels of soluble intercellular adhesion molecule-1 (ICAM-1) were measured in serum samples taken at diagnosis from pediatric patients with Hodgkin's disease (n = 69), acute lymphoblastic leukemia (n = 28), Wilms' tumor (n = 20), osteosarcoma (n = 17), rhabdomyosarcoma (n = 18), or Ewing's sarcoma (n = 15). Median levels of serum ICAM-1 were significantly higher in acute lymphoblastic leukemia and Hodgkin's disease than in controls and other malignancies. Levels were positively correlated with disease stage for patients with Hodgkin's disease, Ewing's sarcoma or Wilms' tumor, and with the frequency of relapse in Hodgkin's disease (P = .016). Serum levels were normal in all of 76 patients tested in remission. It remains to be determined whether increased serum ICAM-1 levels simply reflect a greater tumor burden or whether this molecule contributes directly to the progression of childhood malignancies.
Detection of monosomy 7 and trisomy 8 in myeloid neoplasia: a comparison of banding and fluorescence in situ hybridization
Authors: Kibbelaar RE , Mulder JW , Dreef EJ , van Kamp H , Fibbe WE , Wessels JW , Beverstock GC , Haak HL , Kluin PM
Published: October 2019

Fluorescence in situ hybridization (FISH) is a powerful tool for detection of numerical and structural chromosomal aberrations. We have compared conventional banding techniques and FISH for the detection of monosomy 7 (-7) and trisomy 8 (+8) in 89 patients with myeloid malignancies. Of these patients, 21 had -7, 30 had +8, four had both, and 34 had no aberrations or aberrations other than -7 or +8 as assessed by banding techniques. Sequential samples were available in 23 patients. Alphoid DNA probes specific for chromosomes no. 7 and 8 were used for FISH. As controls, 10 normal bone marrow (BM) samples were hybridized with the chromosomes no. 7 and 8 probes, and in addition all tumor samples were hybridized with a chromosome no. 1 specific probe. The cut-off value for -7 was 18% one-spot cells, and for +8 was 3% three-spot cells. FISH analysis of 44 samples with -7 or +8, and at least 10 metaphases evaluated, showed that the proportions of aberrant metaphase cells mirrored the interphase clone sizes. Most samples with nonclonal metaphase aberrations, including those with only a few metaphases, had increased numbers of aberrant interphase cells: 20% to 80% for -7, and 3% to 43% for +8. Interphase cytogenetics of the 34 samples without -7 or +8 did not show significant cell populations with -7 or +8. In four patients, -7 or +8 could not be confirmed by FISH due to additional structural aberrations, marker chromosomes, or wrongly interpreted banding results. As FISH will be used more and more in cytogenetic diagnosis, clinical follow-up, and therapy monitoring, it will be necessary to standardize FISH procedures and supplement the Standing Committee on Human Cytogenetic Nomenclature (ISCN) definitions of a clone with criteria specifically for in situ hybridization.
Abundant urokinase activity on the surface of mononuclear cells from blood and bone marrow of acute leukemia patients
Authors: Tapiovaara H , Alitalo R , Stephens R , Myohanen H , Ruutu T , Vaheri A
Published: October 2019

We have examined the mononuclear cell fraction from 35 individuals, 18 with hematologic malignancies and 17 healthy controls for the presence of cell surface-associated plasminogen activator (PA) activity. PA activity was found on the cell surface of 10 out of 12 samples from patients with acute leukemia. In addition to active urokinase (uPA) found on the cell surface in four out of five acute myeloid leukemia patients, tissue-type PA activity was detected in the same samples (3 of 5). Two out of four samples from acute lymphoid leukemia displayed only uPA activity and three out of three samples from biphenotypic leukemia were also clearly uPA-positive. Plasmin activity was not detected in any of the samples. PA activity was not found on the surface of mononuclear cells from either patients with chronic lymphoid leukemia or healthy controls and, in this respect, the cell surface- bound uPA activity behaved as a marker for acute leukemia. The finding of PA activity on the cell surface in acute leukemia suggests that there may be continuous generation of plasmin with consequent consumption of plasma plasmin inhibitors.
Effects of cyclosporin A on erythropoietin production by the human Hep3B hepatoma cell line
Authors: Vannucchi AM , Grossi A , Bosi A , Rafanelli D , Statello M , Guidi S , Saccardi R , Rossi-Ferrini P
Published: October 2019

There is evidence that the inadequate erythropoietin (Epo) production observed in patients undergoing allogeneic bone marrow transplantation (BMT) might be ascribed to an inhibitory effect caused by the immunosuppressive drug cyclosporin A (CsA). In this in vitro study, we have evaluated the effects of CsA on the release of Epo in the culture medium by the human Hep3B hepatoma cell line. In cultures incubated with both CsA and the nonimmunosuppressive CsA analog MeAla-6, but not with the CsA-unrelated immunosuppressive agent FK-506, the levels of Epo in the medium were significantly reduced in comparison with controls, at concentrations (0.01 to 1.6 mumol/L) not affecting total protein synthetic rate nor the constitutive secretion of alpha- fetoprotein. Hep3B cells were found to contain a CsA-binding molecule, with an M(r) of 18 Kd, as assessed by high performance liquid chromatography (HPLC) and ligand-blotting analysis. CsA did not affect the expression of the Epo gene, as judged by Northern blot analysis, but caused a significant amount of Epo to remain unsecreted within the cells; almost all (97% of total) of the intracellular Epo was associated with the plasma membrane subcellular fraction. We conclude that: (1) CsA is able to inhibit Epo release in vitro by Hep3B cells, further supporting the hypothesis that the drug might have a role in the inappropriately low Epo levels observed in BMT patients; (2) the inhibitory effect appears to be specific and not caused by a general impairment of protein synthesis and/or secretion; and (3) the reduced Epo levels found in the medium of CsA-treated Hep3B cultures are supposed to be the consequence of an inability of the cells to correctly process Epo molecules for the secretory pathway.
Study on the expression of ABH antigens on platelets
Authors: Ogasawara K , Ueki J , Takenaka M , Furihata K
Published: October 2019

We recently examined a case of refractoriness to HLA-matched, ABO- incompatible platelet transfusions. The transfused platelets that were rapidly cleared from the circulation of the recipient expressed an amount of B antigen more than 20 times that expressed by the blood group B platelets that were successfully transfused to the recipient. These observations led us to conduct enzyme-linked immunosorbent assay (ELISA) and immunoblotting studies of the amount of blood A and B antigens expressed on the surface of platelets from randomly selected donors. The donors were clearly classified, according to the amount of A or B antigen expressed on their platelets, into two phenotypes, the high-expression and low-expression phenotypes. By ELISA, 7% of the examined donors were determined as belonging to the high-expression phenotype of either A or B antigen. The high-expression phenotype was independent of secretor phenotype. In transferase activity assay, a donor with high expression of B had increased B transferase activity in her serum, which suggested that the high-expression phenotype might be under the control of the glycosyltransferase gene. Family studies showed a dominant inheritance pattern of the high-expression phenotype. This report provides evidence that the expression of ABH antigens on platelets is genetically determined and that the ABO group should be given some attention in platelet transfusions.
Beta 2-integrin and L-selectin are obligatory receptors in neutrophil aggregation [see comments]
Authors: Simon SI , Rochon YP , Lynam EB , Smith CW , Anderson DC , Sklar LA
Published: October 2019

We have recently found that antibodies to L-selectin, the homing receptor on neutrophils, are as effective as those to beta 2-integrin at blocking formyl peptide-stimulated aggregation. Therefore, we investigated the requirements for expression of L-selectin and beta 2- integrin on adjacent cells during aggregation. Fluorescence flow cytometry allowed characterization of aggregates on the basis of size and color, as well as antibody binding to these two adhesive molecules. Formyl peptide-stimulated aggregate formation was measured for individual populations fluorescently labeled red (LDS-751) or green (CD44-FITC), and interpopulation red-green cell conjugates. Blocking either the beta 2-integrin or L-selectin adhesive epitope with monoclonal antibody on individual cell populations resulted in an approximately 50% reduction in two-color aggregation as compared with that in unblocked samples. Shedding the L-selectin on a cell population by preincubation with complexes of lipopolysaccharide and its plasma membrane binding protein also decreased aggregation to a control population by approximately 50%. We examined the aggregation of neutrophils from patients genetically deficient in beta 2-integrin and clinically leukocyte adhesion deficient (LAD). LAD adhesion to normal neutrophils was dependent on the expression of L-selectin on LAD cells and beta 2-integrin on normal cells. Thus, the minimum requirement for adhesion between two mixed populations of neutrophils was that one population expressed the beta 2-integrin and the other expressed the L- selectin adhesive epitope.
Regulation of manganese superoxide dismutase and other antioxidant genes in normal and leukemic hematopoietic cells and their relationship to cytotoxicity by tumor necrosis factor
Authors: Kizaki M , Sakashita A , Karmakar A , Lin CW , Koeffler HP
Published: October 2019

Myeloid cells are a major source of superoxide and other oxygen metabolites. As a protective mechanism, cells express antioxidant enzymes including manganese superoxide dismutase (Mn-SOD), copper-zinc SOD (Cu/Zn-SOD), and glutathione peroxidase (GSX-PX). Even though hematopoietic cells are a major source of oxidants, little is known of their expression of antioxidants. We found that seven myeloid leukemic cell lines blocked at different stages of differentiation constitutively expressed Mn-SOD, Cu/Zn-SOD, and GSX-PX RNAs. Level of Mn-SOD activities paralleled levels of Mn-SOD RNA. Terminal differentiation of native HL-60 cells to either granulocytes or macrophages did not alter levels of Mn-SOD RNA but markedly decreased cell division. Myeloid leukemic lines sensitive to cytotoxic effects of tumor necrosis factor (TNF) as well as normal peripheral blood lymphocytes and monocytes, dramatically increased their levels of Mn- SOD RNA in the presence of TNF. In contrast, Cu/Zn-SOD and GSX-PX RNA levels did not increase in these same cells. TNF-resistant leukemic lines had higher constitutive levels of Mn-SOD RNA and activity; and these levels did not change in the presence of TNF. Antisense but not random oligonucleotides to Mn-SOD markedly increased the sensitivity to the inhibitory effects of TNF for both the native HL-60 (TNF-sensitive) and K562 (TNF-resistant) cell lines. Further studies showed that the antisense oligonucleotides entered the cells and resulted in decreased levels of Mn-SOD RNA. The data suggest that Mn-SOD may provide protection against cytotoxicity of TNF in hematopoietic cells.
Normalization of markers of coagulation activation with a purified protein C concentrate in adults with homozygous protein C deficiency
Authors: Conard J , Bauer KA , Gruber A , Griffin JH , Schwarz HP , Horellou MH , Samama MM , Rosenberg RD
Published: October 2019

Homozygous or double heterozygous protein-C deficiency can present at birth with purpura fulminans or later in life with venous thrombosis. Two homozygous patients who had previously sustained thrombotic episodes were investigated at a time when they were asymptomatic and not receiving antithrombotic therapy. The plasma levels of protein-C antigen and activity in both individuals were approximately 20% of normal. We administered a highly purified plasma-derived protein C concentrate to these individuals and monitored levels of several markers of in vivo coagulation activation. Assays for protein-C activation (activated protein C and protein C activation peptide) showed a sustained increase from reduced baseline levels, whereas thrombin generation (as measured by prothrombin fragment F1 + 2) gradually decreased over about 24 hours into the normal range. These investigations provide direct evidence that protein C is converted to activated protein C in vivo, and that the protein-C anticoagulant pathway is a tonically active mechanism in the regulation of hemostatic system activation in humans.
Acute promyelocytic leukemia: clinical relevance of two major PML-RAR alpha isoforms and detection of minimal residual disease by retrotranscriptase/polymerase chain reaction to predict relapse
Authors: Huang W , Sun GL , Li XS , Cao Q , Lu Y , Jang GS , Zhang FQ , Chai JR , Wang ZY , Waxman S
Published: October 2019

Recent data have shown that the PML-RAR alpha fusion gene resulting from translocation t(15;17) is a highly reliable molecular marker of acute promyelocytic leukemia (APL). In this study performed on 97 Chinese patients with APL, the retrotranscriptase/polymerase chain reaction (RT/PCR) was used to evaluate the clinical relevance of the long (L) or short (S) PML-RAR alpha fusion mRNA isoforms and to study minimal residual disease during clinical remission (CR). There were more early deaths during the all-trans retinoic acid (ATRA) induction treatment and more relapses within 2 years of CR in the S-type (6 of 19 cases) than in the L-type group (2 of 33 cases) (P < .025). Among 12 cases analyzed before and after the ATRA-induced CR, 9 cases (75%) showed positive RT/PCR, whereas only 3 cases showed a negative result, justifying the need for chemotherapy after ATRA-induced CR. Eleven of 62 APL patients in CR, after ATRA-induced CR and chemotherapy consolidation (follow-up, from 3 to 72 months), showed positive RT/PCR. Five of them relapsed within 1 to 6 months after the positive test; one converted to negative after further chemotherapy; and 5 remained in CR status without further PCR data. However, the latter 5 cases all received further intensive consolidation therapy after the PCR positivity. These results show that a positive RT/PCR of PML-RAR alpha is a sensitive predictor of relapse in APL.
Molecular analysis of myeloperoxidase deficiency shows heterogeneous patterns of the complete deficiency state manifested at the genomic, mRNA, and protein levels
Authors: Selsted ME , Miller CW , Novotny MJ , Morris WL , Koeffler HP
Published: October 2019

Myeloperoxidase (MPO) is a glycosylated hemoprotein contained in the azurophil granules of human polymorphonuclear leukocytes (PMNs). MPO is thought to play a role in the oxidative antimicrobial activity of neutrophils by catalyzing the formation of hypochlorous acid, a potent microbicide, from hydrogen peroxide and chloride anions. Seven unrelated individuals with complete MPO deficiency, a relatively common heritable defect of neutrophils, were identified during routine blood tests. Molecular analyses were conducted to determine the level of the abnormality in these individuals. Western blot analysis showed that 6 of the 7 donors were devoid of immunoreactive MPO protein, while neutrophils from one individual contained only the 55-Kd subunit. Northern analysis of bone marrow RNA from one MPO-deficient donor showed the presence of the normal-sized 3.3-kb transcript indicating that the defect in MPO biosynthesis in this case was posttranscriptional. Southern analysis of four MPO-deficient donors showed a normal Bgl II digestion pattern, whereas an abnormal restriction pattern was observed in a fifth individual. Although the Bgl II pattern was similar to that observed in an unrelated subject described by Nauseef (Blood 73:290, 1989), our study strongly suggests that the point mutation does not reflect a polymorphism. Taken together, these analyses show the existence of diverse abnormalities associated with MPO-deficiency that may be detected at the level of the MPO polypeptide, mRNA, and gene.
Orientation of erythrocytes in a strong static magnetic field
Authors: Higashi T , Yamagishi A , Takeuchi T , Kawaguchi N , Sagawa S , Onishi S , Date M
Published: October 2019

The frequency of exposure to strong magnetic fields has increased as the magnetic-resonance image-diagnostic technique (MRI) and passenger transport systems based on the principle of magnetic levitation have come into wider use. Accordingly, it has become necessary to more systematically assess their influence on the body and set strict guidelines on acceptable limits of magnetism exposure. Therefore, we have assessed the influence of an uniform static magnetic field (8 T in maximum) on normal erythrocytes. The erythrocytes were oriented with their disk plane parallel to the magnetic field direction. These erythrocytes were influenced even by 1 T and almost 100% of them were oriented when exposed to 4 T. Furthermore, the degree of orientation was not influenced by the state of hemoglobin (oxy: diamagnetic, deoxy and met: paramagnetic). The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result of a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of erythrocytes was found to be delta chi = 8 x 10(-22) electromagnetic units/erythrocyte. It was almost in agreement with the calculated value delta chi = 6 x 10(-22) emu/erythrocyte estimated from the diamagnetism of the membrane constituents of erythrocyte.
In vitro transcription from the human A gamma-globin gene promoter
Authors: Castle M , O'Neill D , Bank A
Published: October 2019

We report enhanced transcription from the human A gamma-globin gene promoter in nuclear extracts (NE) of erythroleukemia (K562) cells compared with that in HeLa NE. We do not observe differences in transcription levels in the two extracts with nonglobin promoter templates. Our findings, indicating preferential recognition of the globin gene promoter by nuclear factors in K562 cells, are consistent with results of studies previously reported by ourselves and others. A novel finding described here is that the addition of a double-stranded octamer motif oligonucleotide to K562 NE increases the level of transcription from the A gamma-globin gene promoter, suggesting a potential role for an octamer motif-binding factor in the repression of A gamma-globin gene transcription. A cosmid construct containing extensive human gamma- and beta-globin gene promoter and structural sequences as well as upstream control sequences also exhibits higher levels of globin gene transcription in K562 NE than in HeLa NE. Our demonstration of the feasibility of efficient, globin promoter-specific in vitro transcription of this complex template offers a novel approach for the systematic analysis of the effects of putative regulatory factors on globin gene expression in vitro in the context of a genetic environment approximating that found in vivo.
Disappearance of lysosomal storage in spleen and liver of mucopolysaccharidosis VII mice after transplantation of genetically modified bone marrow cells
Authors: Marechal V , Naffakh N , Danos O , Heard JM
Published: October 2019

Mice homozygous for the gusmps allele lack beta-glucuronidase activity and provide a useful model for human Mucopolysaccharidosis type VII (MPS VII), also known as Sly syndrome. Bone marrow (BM) transplantation was shown to correct the metabolic defect and to increase the life span of diseased animals. We have used this murine model in a preclinical study aimed at evaluating whether the techniques currently available for gene transfer into large mammalian and human BM cells will provide efficient enzyme replacement therapy in MPS patients. Autologous BM was transplanted into deficient mice after retrovirus-mediated transfer of the human beta-glucuronidase cDNA. Conditioning of recipients was performed by a single sublethal irradiation of 4.5 Gy, giving rise to low donor engraftment. In recipient mice analyzed until 145 days after gene transfer, the percentage of genetically modified hematopoietic cells was less than 5%. Nevertheless, beta-glucuronidase enzyme activity was detectable in various organs, including the brain, and disappearance of lysosomal storage was obvious in the liver and spleen. These results show that the autologous transplantation of genetically engineered BM cells could be beneficial in MPS patients.
Oligodeoxynucleotides antisense to the proto-oncogene c-mpl specifically inhibit in vitro megakaryocytopoiesis
Authors: Methia N , Louache F , Vainchenker W , Wendling F
Published: October 2019

The proto-oncogene c-mpl encodes a protein whose sequence shares striking homologies with members of the highly conserved hematopoietin receptor superfamily. This gene had been transduced in a truncated form by the acute leukemogenic murine Myeloproliferative leukemia virus, which exhibits the unique property of inducing factor-independent proliferation and terminal differentiation of a broad spectrum of hematopoietic progenitors. Presently, the ligand and the role of c-mpl in the regulation of normal hematopoiesis are unknown. To show the function of c-mpl, its expression was first examined in human purified hematopoietic cell populations and, then, an antisense strategy was used. By RNA-based polymerase chain reaction, c-mpl transcripts were detected in purified CD34+ cells, megakaryocytes, and platelets. Synthetic unmodified antisense oligodeoxynucleotides were derived from different regions of the c-mpl extracellular domain. On in vitro exposure of CD34+ cells, two antisense oligomers led to a 50% to 70% inhibition of c-mpl mRNA synthesis, whereas their respective sense had no effect. Furthermore, the decrease in c-mpl mRNA correlated with a significant inhibition (range, 54% to 81%) of in vitro megakaryocytic colony formation (CFU-MK), whereas the growth of erythroid (BFU-E) or granulomacrophage (CFU-GM) colonies was unaffected. The data provide first evidences that c-mpl is involved in megakaryocytopoiesis. In addition, the results raise the possibility that this proto-oncogene encodes the receptor for a new cytokine specifically regulating thrombocytopoiesis.
Collection of peripheral blood hematopoietic progenitors (PBHP) from patients with severe aplastic anemia (SAA) after prolonged administration of granulocyte colony-stimulating factor
Authors: Bacigalupo A , Piaggio G , Podesta M , Van Lint MT , Valbonesi M , Lercari G , Mori PG , Pasino M , Franchini E , Rivabella L
Published: October 2019

The aim of this study was to test whether prolonged administration of granulocyte colony-stimulating factor (G-CSF) would allow the collection by leukapheresis of PBHP in patients with SAA. For this purpose, nine SAA patients, 7 to 46 years old, six of whom were enrolled at diagnosis of their disease and three after previous immunosuppression had failed, were treated with antilymphocyte globulin (ALG) (day 1 to 5), cyclosporin A (5 mg/kg/d orally) (day 6 to 90) and G-CSF 5 micrograms/kg/d (day 6 to 90). A total of 40 leukaphereses were performed, (range 2 to 7 per patient), between days +10 and +168 from G- CSF treatment. White blood cell count at the time of harvest ranged from 1.2 to 18.1 x 10(9)/L. Results can be summarized as follows: the median number of cells collected per patient was 5.0 x 10(8)/kg (range 2.6 to 18.7), the median number of CD34+ cells was 1.8 x 10(6)/kg (range 0.27 to 3.8) and the median number of colony-forming units granulocyte-macrophage (CFU-GM) was 3.9 x 10(4)/kg (range 0 to 39). Twenty leukaphereses performed between days +33 and +77 of G-CSF treatment grew granulocyte macrophages and erythroid colonies in vitro. No colony growth was obtained from 20 leukaphereses performed before day +33 or after day +80. In six patients the total number of CFU-GM recovered were in the range described for autologous peripheral blood stem cell grafts. (2.6 to 39 x 10(4)/kg). In conclusion, this study suggests that circulating hematopoietic progenitors can be recovered after ALG priming and after at least 1 month of G-CSF treatment in a proportion of patients with SAA. Whether these cells will be suitable for autologous transplantation remains to be determined.