bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of Specific Cytomegalovirus Genotypes With Death From Myelosuppression After Marrow Transplantation
Authors: Torok-Storb Beverly , Boeckh Michael , Hoy Cynthia , Leisenring Wendy , Myerson David , Gooley Ted
Published: October 2019

A retrospective analysis of cytomegalovirus (CMV) genotype was conducted on 281 CMV isolates obtained from marrow transplant recipients. The genotyping was based on sequence variations in the gene encoding envelope glycoprotein B (gB) as detected by restriction analysis of polymerase chain reaction (PCR)-amplified gB DNA. Among all isolates studied, the distribution of gB types 1-4 was 48.4%, 16.4%, 24.6%, and 8.2%, respectively, with only 2.5% of all isolates containing more than one gB type. The association of gB types with acute graft-versus-host-disease (GVHD) and death related to myelosuppression was examined using appropriate multivariable regression models. Covariables in addition to gB type included underlying disease type, donor-recipient HLA matching, donor CMV serostatus, and age as a continuous variable. Death associated with myelosuppression occurred in 2.9% or 4 of 136 patients with gB1, 0% or 0 of 46 patients with gB2, 21.7% or 15 of 69 patients with gB3, and 17.4% or four of 23 patients with gB4. The significant association of CMV gB type with death due to myelosuppression was maintained in a multivariable analysis (P < .001). In addition, the data also suggested that gB types 3 and 4 may be associated with a reduced hazard of grades II to IV acute GVHD.
Diamine Oxidase-Gold Ultrastructural Localization of Histamine in Human Skin Biopsies Containing Mast Cells Stimulated to Degranulate In Vivo by Exposure to Recombinant Human Stem Cell Factor
Authors: Dvorak Ann M. , Costa John J. , Morgan Ellen S. , Monahan-Earley Rita A. , Galli Stephen J.
Published: October 2019

Stem cell factor (SCF ) has a major role in hematopoiesis and in the regulation of mast cell development and function. For example, recombinant human SCF (rhSCF ) can induce the development of human mast cells from precursor cells in vitro, stimulate mediator release from human skin mast cells in vitro, and promote both the development and functional activation of human skin mast cells in vivo. In the present study, we used a new ultrastructural enzyme-affinity method, employing diamine oxidase (DAO)-conjugated gold particles (DAO-gold), to detect histamine in skin biopsies obtained from patients with breast carcinomas who were receiving daily subcutaneous (SC) injections of rhSCF in a phase I study of this cytokine. We examined control biopsies obtained at sites remote from rhSCF injection as well as biopsies of rhSCF-injected skin that were obtained within 2 hours and 30 minutes of the SC injection of rhSCF at that site. The rhSCF-injected sites (which clinically exhibited a wheal-and-flare response), but not the control sites, contained mast cells undergoing regulated secretion by granule extrusion. The DAO-gold-affinity method detected histamine in electron-dense granules of mast cells in control and injected skin biopsies; however, the altered matrix of membrane-free, extruded mast cell granules was largely unreactive with DAO-gold. Notably, DAO-gold bound strongly to fibrin deposits and collagen fibers that were adjacent to degranulated mast cells. These findings represent the first morphologic evidence of histamine secretion by classical granule exocytosis in human mast cells in vivo.
Modulation of mRNA Expression of a Novel Human Myeloid-Selective CCAAT/Enhancer Binding Protein Gene (C/EBPε)
Authors: Chih Doris Y. , Chumakov Alexey M. , Park Dorothy J. , Silla Agnes G. , Koeffler H. Phillip
Published: October 2019

Human C/EBPε is a newly cloned gene coding for a CCAAT/enhancer binding protein that may be involved in the regulation of myeloid differentiation. Our studies showed that levels of C/EBPε mRNA were markedly increased in NB4 cells (promyelocytic leukemia line), because they were induced by 9-cis retinoic acid (9-cis RA) to differentiate towards granulocytes. Accumulation of C/EBPε mRNA occurred as early as 1 hour after exposure of NB4 cells to 9-cis RA (5 × 10−7 mol/L); and at 48 hours, levels were increased by 5.1-fold. Dose-response studies showed that 10−7 to 10−6 mol/L 9-cis RA (12 hours) resulted in peak levels of C/EBPε mRNA; but even 10−10 mol/L 9-cis RA increased levels of these transcripts. NB4 cells pulse-exposed (30 minutes) to all-trans retinoic acid (ATRA), washed, and cultured (3 days) with either dimethylsulfoxide (DMSO) or hexamethylene bisacetamide (HMBA) had a prominent increase in levels of C/EBPε mRNA and an increase in granulocytic differentiation, but exposure to either DMSO or HMBA alone had no effect on base levels of C/EBPε and did not induce differentiation. Macrophage-differentiation of NB4 reduced levels of C/EBPε mRNA. Nuclear run-off assays and half-life studies showed that accumulation of C/EBPε mRNA by 9-cis RA was due to enhanced transcription. Furthermore, this C/EBPε mRNA accumulation did not require synthesis of new protein factors because 9-cis RA induced C/EBPε mRNA accumulation in the absence of new protein synthesis. ATRA also induced expression of C/EBPε protein in NB4 cells, as shown by Western blotting. In contrast to the increase of C/EBPε in 9-cis RA–mediated granulocytic differentiation, the DMSO-induced differentiation of HL-60 cells down the granulocytic pathway was associated with an initial reduction of C/EBPε mRNA levels. In summary, we have discovered that expression of C/EBPε mRNA is markedly enhanced as the NB4 promyelocytes are induced by retinoids to differentiate towards granulocytes. This induction of C/EBPε mRNA expression is transcriptionally mediated and occurs in the absence of synthesis of additional protein factors. We suspect that the C/EBPε promoter/enhancer contains a retinoic acid-response element that is directly stimulated by retinoids.
A 2.7-kb Portion of the 5′ Flanking Region of the Murine Glycoprotein αIIb Gene Is Transcriptionally Active in Primitive Hematopoietic Progenitor Cells
Authors: Tropel Philippe , Roullot Valérie , Vernet Muriel , Poujol Christel , Pointu Hervé , Nurden Paquita , Marguerie Gérard , Tronik-Le Roux Diana
Published: October 2019

The continuous generation of mature blood cells from primitive multipotent progenitor cells requires a highly complex series of cellular events that are still largely unknown. To examine the molecular events associated with the commitment of these hematopoietic progenitor cells to the megakaryocytic lineage, the α subunit of the platelet integrin αIIbβ3 was used as marker. Despite an abundance of information regarding the role of this integrin in platelet adhesion and aggregation, the mechanisms that control the expression of the genes that code for these proteins are poorly understood and the earliest hematopoietic cell capable of expressing them has not been clearly identified. Thus, a strategy was developed to eradicate, using a conditional toxigene, all the hematopoietic cells capable of expressing the αIIb gene in mice. This was achieved by targeting the expression of the gene encoding the herpes simplex virus thymidine kinase (tk), specifically to these cell types, using a 2.7-kb fragment of the 5′-flanking region of the murine αIIb gene. Three transgenic lines having 1, 3, and 4 copies of the transgene, respectively were produced and analyzed. Administration of ganciclovir (GCV) to these mice induced a severe thrombocytopenia, which was due to the depletion of the entire megakaryocytic lineage, as shown by bone marrow (BM) culture and electron microscopy analysis. The time required to attain a severe thrombocytopenia was dependent on the level of the expression of the transgene and varied from 7 to 11 days. This condition was completely reversed when GCV treatment was discontinued. Progenitor cell assays showed that the αIIb promoter was active in primitive hematopoietic progenitor cells possessing myeloid, erythroid, and megakaryocytic potential and that the transcriptional activity of the promoter decreased progressively as differentiation proceeded towards the erythroid and myeloid lineages.
Multilineage Long-Term Engraftment Potential of Drug-Resistant Hematopoietic Progenitors
Authors: Bertolini Francesco , Battaglia Manuela , Lanza Annalisa , Gibelli Nadia , Palermo Belinda , Pavesi Lorenzo , Caprotti Marco , Robustelli della Cuna Gioacchino
Published: October 2019

Peripheral blood progenitor cells (PBPCs) are increasingly used instead of bone marrow for autologous or allogeneic transplantation. In this study PBPCs mobilized in cancer patients by chemotherapy and granulocyte-colony stimulating factor were collected by apheresis and first enriched by immunoaffinity removal of lineage positive cells. When these cells were exposed to both cyclophosphamide and taxol or cultured for 7 days in the presence of 5-fluorouracil, stem cell factor, and interleukin-3, 88% to 93% of the enriched PBPCs were killed and short-term clonogenic capacity in methylcellulose assays was lost, but week-5 cobblestone area-forming cell (CAFC) enrichment was higher than 10-fold in comparison to enriched PBPCs and higher than 700-fold in comparison to unmanipulated apheresis cells. After drug exposure, most of the progenitors displayed a CD34+, CD38−, multidrug-resistance (MDR+), Rhodamine 123 low, Hoechst 33342 low phenotype, and as few as 180 of these drug-resistant cells were able to generate a stable multilineage human hematopoiesis in sublethally irradiated immunodeficient mice. In these animals, the level of human hematopoietic engraftment was significantly increased by cotransplantation of irradiated cells from the human L87/4 stromal cell line. These observations are consistent with the functional isolation of a population of very early hematopoietic progenitors and might help to design new protocols for the removal of neoplastic cells from autografts.
Increased Phagocyte FcγRI Expression and Improved Fcγ-Receptor–Mediated Phagocytosis After In Vivo Recombinant Human Interferon-γ Treatment of Normal Human Subjects
Authors: Schiff Deborah E. , Rae Julie , Martin Thomas R. , Davis Bruce H. , Curnutte John T.
Published: October 2019

Recombinant human interferon-γ (rhIFN-γ) decreases the frequency of serious infections in patients with chronic granulomatous disease (CGD) through an unknown mechanism. To test the hypothesis that it exerts a beneficial effect by enhancing clearance of microbes from the bloodstream and tissues, normal human subjects were treated in vivo with rhIFN-γ. Phagocyte opsonic receptor expression, serum opsonin levels, and phagocytosis of bacteria were then measured. A 4.7-fold increase in neutrophil expression of the high-affinity Fcγ-receptor (FcγRI) was observed that peaked 48 hours after the initiation of rhIFN-γ treatment (P < .05). Monocyte expression of FcγRI, FcγRII, FcγRIII, CD11a, CD11b, CD18, and HLA-DR also significantly increased with peak expression at 48 hours. Phagocytosis by neutrophils of killed Staphylococcus aureus opsonized with heat-inactivated pooled human serum significantly improved after rhIFN-γ treatment (P < .05) and correlated with FcγRI expression by neutrophils (r = .8, P < .001). This increase in ingestion could be inhibited by anti-FcγRI monoclonal antibodies. Levels of the serum opsonin lipopolysaccharide-binding protein also significantly increased after in vivo rhIFN-γ (P < .05). These results suggest that the protective effect of rhIFN-γ in patients with CGD may involve improved microbial clearance. Moreover, improved phagocyte trafficking may occur secondary to increased expression of monocyte β2 -integrins. Because these IFN-γ–related improvements in host defense were seen in normal hosts, rhIFN-γ may have broader applications in the treatment of various disorders of immunity in addition to its demonstrated efficacy in CGD.
Total Body Irradiation and Acute Graft-Versus-Host Disease: The Role of Gastrointestinal Damage and Inflammatory Cytokines
Authors: Hill Geoffrey R. , Crawford James M. , Cooke Kenneth R. , Brinson Yani S. , Pan Luying , Ferrara James L.M.
Published: October 2019

The influence of bone marrow transplantation (BMT) conditioning regimens on the incidence and severity of graft-versus-host disease (GVHD) has been suggested in clinical BMT. Using murine BMT models, we show here an increase in GVHD severity in several donor-recipient strain combinations after intensification of the conditioning regimen by increasing the total body irradiation (TBI) dose from 900 cGy to 1,300 cGy. Increased GVHD was mediated by systemic increases in tumor necrosis factor α (TNFα). Histologic analysis of gastrointestinal tracts showed synergistic damage by increased TBI and allogeneic donor cells that permitted increased translocation of lipopolysacharide (LPS) into the systemic circulation. In vitro, LPS triggered excess TNFα from macrophages primed by the GVH reaction. In addition, macrophages isolated within 4 hours of conditioning were primed in proportion to the TBI dose itself to secrete TNFα. Thus, the higher TBI dose increased macrophage priming and increased gut damage after allogeneic BMT, causing higher systemic levels of inflammatory cytokines and subsequent severe GVHD. These data highlight the importance of conditioning in GVHD pathophysiology and suggest that interventions to prevent LPS stimulation of primed macrophages may limit the severity of GVHD after intensive conditioning for allogeneic BMT.
Complex Regulation of CDK2 During Phorbol Ester-Induced Hematopoietic Differentiation
Authors: Asiedu Clement , Biggs Joseph , Kraft Andrew S.
Published: October 2019

Phorbol myristate acetate (PMA) treatment of U937 human leukemic cells results in late G1 cell cycle arrest and terminal monocyte/macrophage-like differentiation. The PMA-induced G1 arrest involves a marked decrease in cdk2 activity, which correlates with total cdk2 dephosphorylation. Here, we show that the levels of cyclin A mRNA and protein markedly decrease during PMA-induced differentiation of U937 cells. In contrast, the level of cyclin E protein remains unchanged and in a complex with cdk2 during the entire course of PMA treatment. During the PMA-induced differentiation, cyclin E-associated cdk2 activity drops markedly. Furthermore, the amount of p27Kip1 protein associated with cyclin E/cdk2 greatly increases 24 to 72 hours after PMA treatment. The absence of changes in p27Kip1 mRNA levels by Northern blot suggest that the levels of this protein are controlled by posttranscriptional or posttranslational mechanism(s). These results show that the mechanisms mediating PMA-induced G1 arrest are complex. The inhibition of cdk2 activity is associated with (1) a decrease in cyclin A protein levels, (2) inactivation of cdk2 complexes, and (3) upregulation of p27Kip1 protein.
Development of Osteoclasts From Embryonic Stem Cells Through a Pathway That Is c-fms but not c-kit Dependent
Authors: Yamane Toshiyuki , Kunisada Takahiro , Yamazaki Hidetoshi , Era Takumi , Nakano Toru , Hayashi Shin-Ichi
Published: October 2019

Osteoclasts are hematopoietic cells essential for bone resorption. To study the derivation of these interesting cells, we developed a stepwise culture system where stromal cells promote embryonic stem (ES) cells to differentiate into mature osteoclasts. Three phases to this differentiation process include (1) induction of hematopoiesis, along with the generation of osteoclast precursors, (2) expansion of these precursors, and (3) terminal differentiation into mature osteoclasts in the presence of 1α,25-dihydroxyvitamine D3 . Although the transition of ES cells to the hematopoietic lineage was not blocked by an antibody to c-fms, later phases were dependent on a signaling through this transmembrane receptor as indicated by the finding that anti–c-fms treatment of cells in the second and third phases reduced the number of osteoclasts produced by 75% and more than 99%, respectively. Blockade of signaling through another tyrosine kinase–type receptor, c-kit, did not affect any stages of osteoclastogenesis, although generation of other hemopoietic lineages was reduced to less than 10% of untreated. When small numbers of ES cells were directly cultured under conditions that promote osteoclast differentiation, tartrate-resistant acid phosphatase-positive multinucleated cells were observed at the edge but not inside of colonies. This suggests that some types of cell-cell interactions may inhibit development of mature osteoclasts. The culture system developed here provides an important tool for osteoclast biology.
Modulation of the Apoptotic Response of Human Myeloid Leukemia Cells to a Diphtheria Toxin Granulocyte-Macrophage Colony-Stimulating Factor Fusion Protein
Authors: Frankel Arthur E. , Hall Philip D. , Burbage Chris , Vesely Joseph , Willingham Mark , Bhalla Kapil , Kreitman Robert J.
Published: October 2019

It has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor–bearing cells. We now report that DT388–GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF–receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL ). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I–GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388–GM-CSF at 4 × 10−9 mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 × 10−9 mol/L to 3 × 10−13 mol/L. GM-CSF–receptor occupancy at 0.7 nmol/L GM-CSF–ligand concentration correlated with the protein synthesis IC50 . Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL , in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388–GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.
Long-Term Depletion of Naive T Cells in Patients Treated for Hodgkin's Disease
Authors: Watanabe Nobukazu , De Rosa Stephen C. , Cmelak Anthony , Hoppe Richard , Herzenberg Leonore A. , Herzenberg Leonard A. , Roederer Mario
Published: October 2019

We investigated the representation of T cells in patients who had been treated for Hodgkin's disease (HD). We found a marked depletion in both CD4 and CD8 naive T-cell counts that persists up to 30 years after completion of treatment. In contrast, CD4 and CD8 memory T-cell subsets recovered to normal or above normal levels by 5 years posttreatment. Thus, the previously-reported long-term deficit in total CD4 T-cell counts after treatment for HD is due to specific depletion of naive T cells. Similarly, total CD8 T-cell counts return to normal by 5 years only because CD8 memory T cells expand to higher than normal levels. These findings suggest that the treatment (mediastinal irradiation) results in a longterm dysregulation of T-cell subset homeostasis. The profound depletion of naive T cells may explain the altered T-cell function in treated patients, including the poor response to immunization after treatment for HD. Further, in some individuals, we identified expansions of unusual subsets expressing low levels of CD8. Eight-color fluorescence-activated cell sorting analyses showed that these cells largely express CD8αα homodimers and CD57, consistent with the phenotype of potentially extrathymically derived T cells. In addition, these cells, both CD4+ and CD4−, are probably cytotoxic lymphocytes, as they express high levels of intracellular perforin. In adults treated for HD, an increased activity of extrathymic T-cell differentiation may partially compensate for the loss of thymic-derived T cells.
Overexpression of Cyclin E in Acute Myelogenous Leukemia
Authors: Iida Hiroatsu , Towatari Masayuki , Tanimoto Mitsune , Morishita Yoshihisa , Kodera Yoshihisa , Saito Hidehiko
Published: October 2019

Cyclin E is one of the G1 cyclins that play an important role in cell proliferation. Overexpression of cyclin E protein has been reported in several solid tumors, but little is known about the involvement of cyclin E in leukemia. In this study, we analyzed the expression of cyclin E gene product in 85 patients with acute myelogenous leukemia (AML) by Western blot analysis. In 23 of 85 AML samples (27%), cyclin E expression was enhanced in blasts. Among the French-American-British classification of AML, the ratio of the samples with enhanced cyclin E expression was high in M5 and low in M2 and M3. No rearrangements were observed by Southern blot analysis in these AML blasts with enhanced cyclin E expression. Flow cytometric analysis showed no correlation between overexpression of cyclin E and cell cycle distribution. Immunoblot analysis of cyclin D1 showed no correlation between overexpression of cyclin E and that of cyclin D1. Interestingly, p27 expression detected by Western blotting was apparently enhanced in 18 of 23 AML cells with enhanced cyclin E expression but none of 14 AML cells without enhanced cyclin E exhibited enhanced p27 expression. The rates of complete remission and of disease-free survival of the patients with M4 or M5 leukemia blasts with overexpressed cyclin E seemed to be low. Therefore, we suggest the necessity of a larger-scale study to elucidate the contribution of cyclin E overexpression to the phenotype and the prognosis of certain AML.
Disruption of the Multiple Tumor Suppressor Gene MTS1/p16INK4a/CDKN2 by Illegitimate V(D)J Recombinase Activity in T-Cell Acute Lymphoblastic Leukemias
Authors: Cayuela Jean-Michel , Gardie Betty , Sigaux François
Published: October 2019

We have recently shown that the multiple tumor suppressor gene 1 (MTS1 ) encoding the p16INK4a and p19ARF cell-cycle inhibitors is inactivated by deletion or disruption in most human T-cell acute lymphoblastic leukemias (T-ALLs), representing the most frequent genetic event thus far described in this disease. To analyze the mechanism of these chromosomal events, we used cloning, sequencing, and/or polymerase chain reaction mapping to study 15 rearrangements occurring in the MTS1 locus. We found that these breakpoints occur in two clusters (MTS1bcrα and MTS1bcrβ ). The three rearrangements occurring in MTS1bcrα correspond to a recurrent recombination juxtaposing 5′ MTS2 exon 1 and 5′ MTS1 exon 1α sequences. Breakpoints for 10 of 12 rearrangements within MTS1bcrβ are located at a polymorphic (CA) repeat, suggesting that this sequence might play a role in the clustering. For both MTS1bcrα and MTS1bcrβ, sequence analyses and PCR mapping experiments show that the tightly clustered breakpoints are located in the vicinity of heptamers whose sequence is similar to those involved in the V(D)J recombination. Moreover, short deletions, GC-rich random nucleotide additions, and clone-specific junctional sequences are present in all cases, further suggesting that the rearrangements are due to illegitimate V(D)J recombinase activity. These data are the first demonstration that a tumor suppressor gene can be inactivated by the V(D)J recombinational mechanism. Moreover, they reinforce the view that this process, normally required for T-cell differentiation, plays a crucial role in the pathogenesis of T-ALL.
Human Immunodeficiency Virus-1 env Impairs Fcγ Receptor-Mediated Phagocytosis Via a Cyclic Adenosine Monophosphate-Dependent Mechanism
Authors: Thomas Christian A. , Weinberger Ofra K. , Ziegler Benedikt L. , Greenberg Steven , Schieren Ira , Silverstein Samuel C. , El Khoury Joseph
Published: October 2019

Human immunodeficiency virus (HIV)-1 expression in mononuclear phagocytes is associated with multiple functional defects, including phagocytosis. To assess Fcγ receptor (FcγR) function in cells expressing HIV-1, human promonocytic cells (U937) acutely or chronically infected with HIV-1, or stably transfected with a noninfectious reverse transcriptase (RT) defective HIV-1 provirus (Δpol), were treated with phorbol 12-myristate 13-acetate for 48 hours and tested for their ability to ingest sheep erythrocytes coated with IgG (E-IgG). HIV-1–infected or transfected U937 cells ingested 50% to 65% fewer E-IgG than controls despite normal surface expression of FcγRs. HIV-1 specifically impaired FcγR-mediated phagocytosis, as ingestion of complement-coated erythrocytes was unaffected. U937 cells transfected with an env deficient mutant of HIV-1 ingested E-IgG normally, suggesting that the expression of HIV-1 env was required for HIV-1 to inhibit FcγR-mediated phagocytosis. Expression of HIV-1 in U937 cells was associated with an increased accumulation of intracellular cyclic adenosine monophosphate (cAMP); addition of the adenylate cyclase inhibitor 2′,5′-dideoxyadenosine to these cells decreased intracellular cAMP levels to that of controls and restored FcγR-mediated phagocytosis. Addition of either interferon (IFN)-γ or an inhibitor of cAMP-dependent protein kinase A (KT 5720) to HIV-1–transfected U937 cells also restored FcγR-mediated phagocytosis. Expression of HIV-1 induces a specific defect of FcγR function in mononuclear phagocytes that correlates with increased levels of cAMP, and can be corrected by pharmacologic manipulation.
Identification of a Region of PfEMP1 That Mediates Adherence of Plasmodium falciparum Infected Erythrocytes to CD36: Conserved Function With Variant Sequence
Authors: Baruch Dror I. , Ma Xin C. , Singh Hardeep B. , Bi Xiahui , Pasloske Brittan L. , Howard Russell J.
Published: October 2019

Adherence of mature parasitized erythrocytes (PE) of Plasmodium falciparum to microvascular endothelial cells contributes directly to the virulence and pathology of this human malaria. The malarial variant antigen, P falciparum erythrocyte membrane protein 1 (PfEMP1), has been implicated as the PE receptor for CD36 on endothelial cells. We identified the region of PfEMP1 that mediates adherence of PE to CD36 and showed that a recombinant protein fragment from this region blocked and reversed adherence of antigenically different parasites. Sequence variation was evident in the CD36 binding domain of different PfEMP1 genes, yet many highly conserved residues, particularly cysteine residues, are evident. This suggests a highly conserved shape that mediates adherence to CD36. Immunization with the CD36-binding domain elicited sera that are cross-reactive with the different recombinant proteins but are strain-specific for the PE surface. Novel anti-adherence therapeutics and a malaria vaccine may derived from exploitation of the structure of the CD36 binding domain of PfEMP1.
Expression of Bomapin, a Novel Human Serpin, in Normal/Malignant Hematopoiesis and in the Monocytic Cell Lines THP-1 and AML-193
Authors: Riewald Matthias , Chuang Trinette , Neubauer Andreas , Riess Hanno , Schleef Raymond R.
Published: October 2019

Our group recently cloned the cDNA-encoding bomapin, a member of the serine protease inhibitor (serpin) superfamily, from a human bone marrow cDNA library (J Biol Chem 270:2675, 1995). To understand its expression within the hematopoietic compartment, RNA extracted from bone marrow or peripheral blood from normal donors and patients with leukemia was reverse transcribed and analyzed by polymerase chain reaction (PCR). Bomapin PCR products were readily detected in normal bone marrow, which was designated as a medium mRNA level. In peripheral blood, bomapin expression was low or undetectable in normal donors (n = 6) and patients with chronic lymphocytic leukemia (n = 6). Blood from patients with chronic myeloid leukemia (n = 6), chronic myelomonocytic leukemia (n = 6), acute myeloid leukemia (n = 5), and acute lymphocytic leukemia (n = 5) exhibited low to medium levels of bomapin expression. Furthermore, a high level of bomapin expression was detected in one individual with acute monocytic leukemia. These data suggest that bomapin expression may be elevated in hematopoietic cells of monocytic lineage. Therefore, we analyzed the expression of bomapin within cell lines that exhibited characteristics of the monocytic lineage. Bomapin PCR products were detected in the monocytic THP-1 and AML-193 cell lines but not in CRL 7607, CRL 7541, KG-1, or K562 cells. Induction of bomapin transcripts was not detected in the latter series of cell lines following a 24-hour treatment with phorbol myristate acetate (PMA, 10−8mol/L) or tumor necrosis factor-α (TNF-α, 30 U/mL), whereas treatment of THP-1 or AML-193 cells with these agents reduced the intensity of the bomapin PCR products. Northern blotting confirmed these results and showed that the expression of bomapin in THP-1 cells was downregulated over a 4-day period by PMA and, to a lesser extent, TNF-α. Immunoblotting was used to show the presence of a 40-kD protein in THP-1 cytosol preparations. Bomapin antigen levels were correspondingly reduced after treatment with PMA. Because PMA and TNF-α induce monocytic differentiation in THP-1 and AML-193 cells, these data increase the possibility that bomapin may play a role in the regulation of protease activities specifically in early stages of cellular differentiation.
Screening for von Willebrand Disease With a New Analyzer Using High Shear Stress: A Study of 60 Cases
Authors: Fressinaud Edith , Veyradier Agnès , Truchaud Florence , Martin Isabelle , Boyer-Neumann Catherine , Trossaert Marc , Meyer Dominique
Published: October 2019

We have evaluated the performance of a new analyzer using high shear stress, the PFA-100 (Platelet Function Analyzer, Dade International, Massy, France), for screening of patients with von Willebrand disease (vWD). Whole citrated blood is aspirated through a capillary to the central aperture of a membrane coated with collagen and with a platelet agonist (either epinephrine or adenosine diphosphate [ADP]). The time required to obtain occlusion of the aperture by a platelet plug is defined as the closure time (CT). We studied 60 patients with different types of vWD and 96 normal subjects. Fourteen subjects with hemophilia and 15 patients with a platelet disorder were also analyzed. When omitting results from two patients with type 2N, the 58 other patients with type 1, type 2A, type 2B, type 3, or acquired vWD all exhibited an abnormal occlusion with collagen-ADP (sensitivity, 100%) and 56 of 58 had an abnormal CT with collagen-epinephrine (sensitivity, 96.5%). Only two patients with mild type 1 were not detected with collagen-epinephrine. In comparison, the bleeding time (BT) was normal in 20 patients: 17 with type 1, two with type 2A, and one with acquired vWD (sensitivity, 65.5%). The specificity of the PFA-100 was over 95% with both types of cartridges. Thus, the analyzer is well adapted to routine testing, as it has the advantages of simplicity and ease of execution, and demonstrates a high sensitivity, clearly superior to that of BT, for the screening of patients with vWD.
Altered Interleukin-12 Responsiveness in Th1 and Th2 Cells Is Associated With the Differential Activation of STAT5 and STAT1
Authors: Gollob Jared A. , Murphy Erin A. , Mahajan Sudipta , Schnipper Claudia P. , Ritz Jerome , Frank David A.
Published: October 2019

T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1α, and STAT1β, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12–induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12–induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-γ (IFN-γ) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-γ compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12–induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-γ is not a direct effect of IFN-γ on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-γ. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-γ production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells.
Temporal and Spatial Distribution of DNA Topoisomerase II Alters During Proliferation, Differentiation, and Apoptosis in HL-60 Cells
Authors: Sugimoto Koichi , Yamada Konagi , Egashira Motoki , Yazaki Yoshio , Hirai Hisamaru , Kikuchi Akihiko , Oshimi Kazuo
Published: October 2019

We related cellular content of DNA topoisomerase (topo) IIα and IIβ with the cell cycle position in proliferating, differentiated, and apoptotic HL-60 cells using two-dimensional flow cytometry. In logarithmically growing HL-60 cells, topo IIα increased especially in late S to G2/M phases, although the topo IIβ level was almost constant throughout the cell cycle. Induction of differentiation by all-trans retinoic acid dramatically reduced the topo IIα but not the topo IIβ level. A new G2/M population containing virtually no topo IIα appeared during differentiation and was supposed to be alive and noncycling. Two-dimensional flow cytometry of topo IIα or IIβ staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling assay showed that one topo IIβ epitope situated at the C-terminal end decreased specifically in apoptotic HL-60 cells treated with Ara-C, etoposide, and vincristine. The amounts of a topo IIα epitope and another topo IIβ epitope located at a more central portion were almost equal between apoptotic and nonapoptotic cells. Western blot analysis confirmed that topo IIβ protein was completely degraded into smaller fragments and lost its C-terminal end during apoptosis. On the contrary, a large portion of topo IIα remained of its original size, although both topo IIα and IIβ left from the nuclear fraction in apoptotic cells. Confocal laser microscopy showed nuclear localization of topo IIα and IIβ in growing HL-60 cells. Although topo IIα and IIβ were distributed throughout the cell during mitosis, only topo IIα was densely concentrated in the mitotic chromosomes. Both enzymes were dissociated from the genomic DNA even at an early phase of apoptosis and completely separated from the propidium iodide signal of DNA in the advanced stage. Chromatin condensation process in apoptosis is therefore completely topo II-independent and obviously differs from the mitotic one.
Antimalarial Activity of 77 Phospholipid Polar Head Analogs: Close Correlation Between Inhibition of Phospholipid Metabolism and In Vitro Plasmodium Falciparum Growth
Authors: Ancelin Marie L. , Calas Michèle , Bompart Jacques , Cordina Gérard , Martin Dominique , Ben Bari Mohammed , Jei Taı̈b , Druilhe Pierre , Vial Henri J.
Published: October 2019

Seventy-seven potential analogs of phospholipid polar heads, choline and ethanolamine, were evaluated in vitro as inhibitors ofPlasmodium falciparum growth. Their IC50 ranged from 10−3 to 10−7 mol/L. Ten compounds showed similar antimalarial activity when tested against three different parasite strains (2 chloroquine-sensitive strains and 1 chloroquine-resistant strain). Compounds showing marked antimalarial activity were assayed for their effects on phospholipid metabolism. The most active compounds (IC50 of 1 to 0.03 μmol/L) were inhibitors of de novo phosphatidylcholine (PC) biosynthesis from choline. For a series of 50 compounds, there was a close correlation between impairment of phospholipid biosynthesis and inhibition of in vitro malaria parasite growth. High choline concentrations caused a marked specific shift in the curves for PC biosynthesis inhibition. Concentrations inhibiting 50% PC metabolism from choline were in close agreement with the Ki of these compounds for the choline transporter inPlasmodium knowlesi-infected erythrocytes. By contrast, measurement of the effects of 12 of these compounds on rapidly dividing lymphoblastoid cells showed a total absence of correlation between parasite growth inhibition and human lymphoblastoid cell growth inhibition. Specific antimalarial effects of choline or ethanolamine analogs are thus likely mediated by their alteration of phospholipid metabolism. This indicates that de novo PC biosynthesis from choline is a very realistic target for new malaria chemotherapy, even against pharmacoresistant strains.
Neonatal Hemolytic Anemia Due to Inherited Harderoporphyria: Clinical Characteristics and Molecular Basis
Authors: Lamoril J. , Puy H. , Gouya L. , Rosipal R. , Da Silva V. , Grandchamp B. , Foint T. , Bader-Meunier B. , Dommergues J.P. , Deybach J.C. , Nordmann Y.
Published: October 2019

Porphyrias, a group of inborn errors of heme synthesis, are classified as hepatic or erythropoietic according to clinical data and the main site of expression of the specific enzymatic defect. Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by deficient activity of coproporphyrinogen III oxidase (COX). Typical clinical manifestations of the disease are acute attacks of neurological dysfunction; skin photosensitivity may also be present. We report a variant form of HC characterized by a unifying syndrome in which hematologic disorders predominate: harderoporphyria. Harderoporphyric patients exhibit jaundice, severe chronic hemolytic anemia of early onset associated with hepatosplenomegaly, and skin photosensitivity. Neither abdominal pain nor neuropsychiatric symptoms are observed. COX activity is markedly decreased. In a first harderoporphyric family, with three affected siblings, a homozygous K404E mutation has been previously characterized. In the present study, molecular investigations in a second family with neonatal hemolytic anemia and harderoporphyria revealed two heterozygous point mutations in the COX gene. One allele bore the missense mutation K404E previously described. The second allele bore an A→G transition at the third position of the donor splice site in intron 6. This new COX gene mutation resulted in exon 6 skipping and the absence of functional protein production. In contrast with other COX gene defects that produce the classical hepatic porphyria presentation, our data suggest that the K404E substitution (either in the homozygous or compound heterozygous state associated with a mutation leading to the absence of functional mRNA or protein) is responsible for the specific hematologic clinical manifestations of harderoporphyria.
A Novel Single Missense Mutation Identified Along the RH50 Gene in a Composite Heterozygous Rhnull Blood Donor of the Regulator Type
Authors: Hyland C.A. , Chérif-Zahar B. , Cowley N. , Raynal V. , Parkes J. , Saul A. , Cartron J.P.
Published: October 2019

Rare individuals who lack all of the Rh blood group antigens are called Rhnull and may be classified as “regulator” or “amorph” types. The suppression of Rh antigen expression for regulator types may be attributed to mutations of the RH50gene, which is independent of the RH locus. The RH50 gene encodes a glycoprotein that interacts with the Rh proteins to form a functional complex within the red blood cell membrane. This report describes an RH50 gene mutation for a previously unclassified Rhnull donor. Sequencing cDNA clones from Rh50 mRNA revealed a single base change (G836A) yielding a missense and nonconservative mutation (Gly279Glu) within a predicted hydrophobic domain for this membrane protein. Genomic DNA studies using polymerase chain reaction (PCR) restriction analysis and sequencing showed that the Rhnull propositus was a composite heterozygote for this mutation, carrying two alleles with the A and G at nucleotide 836, respectively. In contrast, cDNA studies showed that only the A836 sequence was present, suggesting that the second allele with G836 was apparently silent (no transcript detected). Family studies showed that the mutant RH50 allele (836A) was inherited maternally, whereas the silent RH50 allele (836G) was from paternal transmission. These findings provide further evidence that rare but diverse genetic alterations may occur along the RH50 gene where the Rhnull syndrome of the regulator type occurs. The single amino acid change (Gly to Glu) provides insight into the critical value of these residues for assembly of the Rh antigen complex within the membrane.
Syndecan-1 Is a Multifunctional Regulator of Myeloma Pathobiology: Control of Tumor Cell Survival, Growth, and Bone Cell Differentiation
Authors: Dhodapkar Madhav V. , Abe Etsuko , Theus Allison , Lacy Marie , Langford J. Kevin , Barlogie Bart , Sanderson Ralph D.
Published: October 2019

Multiple myeloma is characterized by an accumulation of malignant plasma cells in the bone marrow coupled with an altered balance of osteoclasts and osteoblasts, leading to lytic bone disease. Although some of the cytokines driving this process have been characterized, little is known about the negative regulators. We show that syndecan-1 (CD 138), a heparan sulfate proteoglycan, expressed on and actively shed from the surface of most myeloma cells, induces apoptosis and inhibits the growth of myeloma tumor cells and also mediates decreased osteoclast and increased osteoblast differentiation. The addition of intact purified syndecan-1 ectodomain (1 to 6 nmol/L) to myeloma cell lines in culture leads to induction of apoptosis and dose-dependent growth inhibition, with concurrent downregulation of cyclin D1. The addition of purified syndecan-1 in picomolar concentrations to bone marrow cells in culture leads to a dose-dependent decrease in osteoclastogenesis and a smaller increase in osteoblastogenesis. In contrast to the effect on myeloma cells, the effect of syndecan-1 on osteoclastogenesis only requires the syndecan-1 heparan sulfate chains and not the intact ectodomain, suggesting that syndecan's effect on myeloma and bone cells occurs through different mechanisms. When injected in severe combined immune deficient (scid) mice, control-transfected myeloma cells (ARH-77 cells) expressing little syndecan-1 readily form tumors, leading to hind limb paralysis and lytic bone disease. However, after the injection of syndecan-1–transfected ARH-77 cells, the development of disease-related morbidity and lytic bone disease is significantly inhibited. Taken together, our data demonstrate, both in vitro and in vivo, that syndecan-1 has a significant beneficial effect on the behavior of both myeloma and bone cells and therefore may represent one of the central molecules in the regulation of myeloma pathobiology.
Molecular Mechanisms of Type II Factor XIII Deficiency: Novel Gly562-Arg Mutation and C-Terminal Truncation of the A Subunit Cause Factor XIII Deficiency as Characterized in a Mammalian Expression System
Authors: Takahashi Nobumasa , Tsukamoto Hiroaki , Umeyama Hideaki , Castaman Giancarlo , Rodeghiero Francesco , Ichinose Akitada
Published: October 2019

To explore the biological and clinical implications of the structure/function relationships in factor XIII, mutations in two patients with type II deficiency were identified and characterized in a mammalian expression system. Nucleotide sequence analysis of the A subunit gene showed that case no. 1 had a deletion of 4 bp (AATT) in exon XI and that, in case no. 2, Gly562 (GGG) had been replaced by Arg(AGG). The deletion in case no. 1 leads to a premature termination at codon 464. Restriction digestion of amplified DNAs confirmed that both cases were homozygous for their respective mutations. Reverse transcription-polymerase chain reaction analysis demonstrated that the level of mRNA was greatly reduced in case no. 1, whereas the level of mutant mRNA expressed in case no. 2 was normal. Molecular modeling calculated that Arg562 changed the conformation of the A subunit, suggesting misfolding and/or destabilization of the molecule. To determine how these mutations impaired synthesis of the A subunit, recombinant A subunits bearing the mutations were expressed in mammalian cells. Pulse-chase experiments showed that the mutants were synthesized normally but disappeared rapidly, whereas the wild-type remained. These results indicate that both mutant proteins with an altered conformation become prone to rapid degradation, resulting in factor XIII deficiency in these patients.
Hematopoietic Remodeling in Interferon-γ–Deficient Mice Infected With Mycobacteria
Authors: Murray Peter J. , Young Richard A. , Daley George Q.
Published: October 2019

Control of intracellular bacterial infections requires interferon-γ (IFN-γ) both for establishing a Th1 T-cell response and for activating macrophages to kill the bacteria. Exposure of mice deficient in IFN-γ to mycobacterial infection produces an immune response characterized by a Th2 T-cell phenotype, florid bacterial growth, and death. We report here that IFN-γ–deficient mice infected with mycobacteria also undergo a dramatic remodeling of the hematopoietic system. Myeloid cell proliferation proceeds unchecked throughout the course of mycobacterial infection, resulting in a transition to extramedullary hematopoiesis. The splenic architecture of infected IFN-γ–deficient mice is completely effaced by expansion of macrophages, granulocytes, and extramedullary hematopoietic tissue. These features coincide with splenomegaly, an increase in splenic myeloid colony-forming activity, and marked granulocytosis in the peripheral blood. Systemic levels of cytokines are elevated, particularly interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). These results suggest that in addition to its central role in cellular immunity, IFN-γ may be a key cytokine in coordinate regulation of immune effector cells and myelopoiesis. This model should be valuable for deciphering the cross-talk between the immune response and hematopoiesis during bacterial infection and for improving our understanding of the mechanisms that control chronic infections.