bluuebunny/crossref_metadata_2025 huggingface dataset:
EBNA-1 Gene Sequences in Brazilian and American Patients With Hodgkin’s Disease
Authors: Chang Karen L. , Chen Yuan-Yuan , Chen Wen-Gang , Hayashi Kazuhiko , Bacchi Carlos , Bacchi Maura , Weiss Lawrence M.
Published: October 2019

We examined the types of Epstein-Barr virus–associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin’s disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P-ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P-ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis.
Inhibitory Activity of Human Lactoferrin and Its Peptide on Chondroitin Sulfate A-, CD36-, and Thrombospondin-Mediated Cytoadherence ofPlasmodium falciparum–Infected Erythrocytes
Authors: Eda Shigetoshi , Eda Keiko , Prudhomme Jacques G. , Sherman Irwin W.
Published: October 2019

Lactoferrin (LF), a human serum protein, strongly inhibited the adherence of Plasmodium falciparum–infected erythrocytes (PE) to immobilized chondroitin sulfate A (CSA)–conjugated albumin at a concentration of 100 μg/mL and blocked the PE binding to CD36-expressing Chinese hamster ovary (CHO) cells, as well as immobilized CD36 at concentrations of 5 μg/mL and 100 μg/mL, respectively. Biotinylated LF bound to CD36 in a saturable manner, and such binding was inhibited by unlabeled LF and the anti-CD36 monoclonal antibody, 8A6, suggesting specificity of binding. Additionally, LF inhibited PE binding to immobilized thrombospondin (TSP) at a concentration of 100 μg/mL, and specific binding of LF to TSP was confirmed using biotinylated LF. LF inhibited PE binding to C32 amelanotic melanoma cells in a dose-dependent manner. A peptide of LF, Arg-Asn-Met Arg-Lys-Val Arg-Gly-Pro-Pro-Val-Ser-Cys (amino acid residues 25-37 of LF), which has been suggested to contribute to LF binding to various materials, including CSA, inhibited PE binding to immobilized CSA-conjugated albumin, immobilized CD36, CD36-expressing CHO cells, immobilized TSP, and C32 amelanotic melanoma cells, as well as LF itself. These results suggest that LF peptide may provide the basis for developing agents that are able to inhibit CSA-, CD36-, and TSP-mediated cytoadherence of PE.
Human CD34+ Cells Express CXCR4 and Its Ligand Stromal Cell–Derived Factor-1. Implications for Infection by T-Cell Tropic Human Immunodeficiency Virus
Authors: Aiuti Alessandro , Turchetto Lucia , Cota Manuela , Cipponi Arcadi , Brambilla Andrea , Arcelloni Cinzia , Paroni Rita , Vicenzi Elisa , Bordignon Claudio , Poli Guido
Published: October 2019

Human CD34+ hematopoietic progenitor cells obtained from bone marrow (BM), umbilical cord blood (UCB), and mobilized peripheral blood (MPB) were purified and investigated for the expression of the chemokine receptor CXCR4 and its ligand, stromal cell–derived factor-1 (SDF-1). CXCR4 was found present on the cell surface of all CD34+ cells, although it was expressed at lower density on MPB with respect to BM CD34+ cells. Freshly isolated and in vitro–cultured CD34+ cells also coexpressed SDF-1 mRNA, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Of interest, CD34+/CD38+ committed progenitor cells, unlike primitive CD34+/CD38− cells, expressed SDF-1 mRNA. Supernatants from in vitro–cultured CD34+ cells contained substantial (3 to 8 ng/mL) amounts of SDF-1 by enzyme-linked immunosorbent assay and induced migration of CD34+ cells. Because CD34+ cells express low levels of CD4, the primary receptor of the human immunodeficiency virus (HIV), and CXCR4 is a coreceptor for T-cell tropic (X4) HIV strains, we investigated the susceptibility of CD34+cells to infection by this subset of viruses. Lack of productive infection was almost invariably observed as determined by a conventional RT activity in culture supernatants and by real-time PCR for HIV DNA in CD34+ cells exposed to both laboratory adapted (LAI) and primary (BON) X4 T-cell tropic HIV-1 strain. Soluble gp120 Env (sgp120) from X4 HIV-1 efficiently blocked binding of the anti-CD4 Leu3a monoclonal antibody (MoAb) to either human CD4+ T cells or CD34+ cells. In contrast, sgp120 interfered with an anti-CXCR4 MoAb binding to human T lymphocytes, but not to CD34+ cells. However, CXCR4 on CD34+ cells was downregulated by SDF-1. These results suggest that CXCR4 and its ligand SDF-1 expressed in CD34+ progenitors may play an important role in regulating the local and systemic trafficking of these cells. Moreover, these findings suggest multiple and potentially synergistic mechanisms at the basis of the resistance of CD34+ cells to X4 HIV infection, including their ability to produce SDF-1, and the lack of CXCR4 internalization following gp120 binding to CD4.
Characterization of the Mouse von Willebrand Factor Promoter
Authors: Guan Jiazhen , Guillot Pascale V. , Aird William C.
Published: October 2019

Expression of the von Willebrand factor (vWF) gene is restricted to the endothelial and megakaryocyte lineages. Within the endothelium, expression of vWF varies between different vascular beds. We have previously shown that the human vWF promoter spanning a region between −2182 (relative to the start site of transcription) and the end of the first intron contains information for environmentally responsive, vascular bed-specific expression in the heart, skeletal muscle, and brain. In the present study, we cloned the mouse vWF (mvWF) promoter and studied its function in cultured endothelial cells and transgenic mice. In transient transfection assays, the mvWF gene was found to be regulated by distinct mechanisms in different endothelial cell subtypes. In independent lines of transgenic mice, an mvWF promoter fragment containing DNA sequences between −2645 and the end of the first intron directed endothelial cell-specific expression in the microvascular beds of the heart, brain, and skeletal muscle as well as the endothelial lining of the aorta. In 1 line of mice, reporter gene activity was also detected in bone marrow megakaryocytes. Taken together, these findings suggest that both the mouse and human vWF promoters are regulated by vascular bed-specific mechanisms.
Enhanced Liver Uptake of Opsonized Red Blood Cells After In Vivo Transfer of FcγRIIA cDNA to the Liver
Authors: Bezdicek Petr , Worgall Stefan , Kovesdi Imre , Kim Moo-Kyung , Park Jong-Gu , Vincent Theresa , Leopold Philip L. , Schreiber Alan D. , Crystal Ronald G.
Published: October 2019

Fcγ receptors convey to phagocytic cells the ability to recognize, bind, and internalize IgG-coated cells and microorganisms. The present study demonstrates the use of adenovirus (Ad)-mediated gene transfer of human Fcγ receptor IIA cDNA to convert normally nonphagocytic cells (hepatocytes) into functional equivalents of phagocytic cells. Ad vector in vitro transfer and expression of FcγRIIA cDNA in primary rat hepatocytes was confirmed by flow cytometry anti-FcγRIIA immunodetection, and the function of the receptor was demonstrated by enhanced binding and phagocytosis of 51Cr-labeled IgG-opsonized erythrocytes. After in vivo gene transfer to rats, expression of FcγRIIA cDNA in hepatocytes was confirmed by Northern analysis and immunohistochemistry. Rats infected with the Ad vector carrying the FcγRIIA cDNA demonstrated enhanced clearance of opsonized erythrocytes, but not nonopsonized erythrocytes, from the circulation with increased sequestration within the liver. Together, these data demonstrate that Ad-mediated FcγRIIA gene transfer can convert normally IgG-nonphagocytic cells into phagocytic cells capable of recognizing, binding, and ingesting an opsonized particulate antigen, suggesting that gene transfer strategies might be used to transiently augment host defense by enhancing the clearance of immune complexes.
Effect of Costimulation and the Microenvironment on Antigen Presentation by Leukemic Cells
Authors: Buggins A.G.S. , Lea N. , Gäken J. , Darling D. , Farzaneh F. , Mufti G.J. , Hirst W.J.R.
Published: October 2019

Costimulatory signals supplied by genetically modified tumor cells can enable T-cell recognition of tumor-associated antigens that were previously silent when presented by unmodified tumor cells. Although the mechanism of the CD80/CD28 costimulation has been studied extensively in the normal T-cell/antigen-presenting cell (APC) interactions, it is unclear how expression of CD80 by tumor cells mediates its effect. We demonstrate here that optimal CD80 expression on a leukemic cell enhances T-cell recognition of alloantigen primarily by lowering the level of T-cell receptor (TCR) stimulation required for activation. CD80 expression by leukemic cells leads to increased survival of activated T cells by inducing upregulation of the antiapoptotic protein BCL-2, but not BCL-XL. The cytokine microenvironment in which T cells are activated is crucial in determining their differentiation and consequently the nature of the immune response generated. Many tumor cells produce immunosuppressive cytokines that may not favor the induction of cell-mediated immunity. In this study, the presence of CD80 on leukemic cells increased T-cell activation in vitro, but this did not result in the production of Th1 cytokines. We show that this is due to a leukemia-derived soluble factor that inhibits the production of Th1 cytokines. Optimal expression of a costimulatory molecule, therefore, enhances the ability of leukemic cells to present antigen by amplifying TCR signals, but the microenvironment generated by leukemic cells may suppress the immune response required for their eradication. Thus, strategies aimed at inducing antileukemic immunity by providing leukemic cells with costimulatory functions must ensure the presence of an appropriate microenvironment.
Prognostic Implications of the Differentiation Inhibitory Factornm23-H1 Protein in the Plasma of Aggressive Non-Hodgkin’s Lymphoma
Authors: Niitsu Nozomi , Okabe-Kado Junko , Kasukabe Takashi , Yamamoto-Yamaguchi Yuri , Umeda Masanori , Honma Yoshio
Published: October 2019

The outcome of patients with non-Hodgkin’s lymphoma has been improved by current approaches to treatment. Nevertheless, many patients either do not have a complete remission or ultimately relapse. To identify such patients, it is important to be able to predict the outcome. We previously found that the differentiation inhibitory factor/nm23 was correlated with the prognosis of acute myeloid leukemia. To examine the prognostic effect of nm23 on non-Hodgkin’s lymphoma, we established an enzyme-linked immunosorbent assay procedure to determine nm23-H1 protein levels in plasma and assessed the association of this protein level with the response to chemotherapy, overall survival, and progression-free survival in patients with aggressive non-Hodgkin’s lymphoma. The plasma concentration of nm23-H1 was significantly higher in patients with malignant lymphoma than in normal controls, especially in aggressive non-Hodgkin’s lymphoma. The complete remission rate in patients with higher nm23-H1 levels was significantly worse than that in patients with lower nm23-H1 levels. Overall survival and progression-free survival were also lower in patients with higher nm23-H1 levels than in those with lower levels. The 3-year survival rates in patients with low and high nm23-H1levels were 79.5% and 6.7% (P = .0001). A multivariate analysis of prognostic factors showed that the plasma nm23-H1level was independently associated with the survival and progression-free survival. An elevated plasma nm23-H1concentration predicts a poor outcome of advanced non-Hodgkin’s lymphoma. Therefore, nm23-H1 in plasma may be useful for identifying a distinct group of patients at very high risk.
H-Ferritin Subunit Overexpression in Erythroid Cells Reduces the Oxidative Stress Response and Induces Multidrug Resistance Properties
Authors: Epsztejn Silvina , Glickstein Hava , Picard Virginie , Slotki Itzchak N. , Breuer William , Beaumont Carole , Cabantchik Z. Ioav
Published: October 2019

The labile iron pool (LIP) of animal cells has been implicated in cell iron regulation and as a key component of the oxidative-stress response. A major mechanism commonly implied in the downregulation of LIP has been the induced expression of ferritin (FT), particularly the heavy subunits (H-FT) that display ferroxidase activity. The effects of H-FT on LIP and other physiological parameters were studied in murine erythroleukemia (MEL) cells stably transfected with H-FT subunits. Clones expressing different levels of H-FT displayed similar concentrations of total cell iron (0.3 ± 0.1 mmol/L) and of reduced/total glutathione. However, with increasing H-FT levels the cells expressed lower levels of LIP and reactive oxygen species (ROS) and ensuing cell death after iron loads and oxidative challenges. These results provide direct experimental support for the alleged roles of H-FT as a regulator of labile cell iron and as a possible attenuator of the oxidative cell response. H-FT overexpression was of no apparent consequence to the cellular proliferative capacity. However, concomitant with the acquisition of iron and redox regulatory capacities, the H-FT–transfectant cells commensurately acquired multidrug resistance (MDR) properties. These properties were identified as increased expression of MDR1 mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), P-glycoprotein (Western immunoblotting), drug transport activity (verapamil-sensitive drug efflux), and drug cytotoxicity associated with increased MDR1 or PgP. Although enhanced MDR expression per se evoked no significant changes in either LIP levels or ROS production, it might be essential for the survival of H-FT transfectants, possibly by expediting the export of cell-generated metabolites.
Genetic Evidence of an Accessory Activity Required Specifically for Cubilin Brush-Border Expression and Intrinsic Factor-Cobalamin Absorption
Authors: Xu Danbin , Kozyraki Renata , Newman Thomas C. , Fyfe John C.
Published: October 2019

Cubilin is a high molecular weight multiligand receptor that mediates intestinal absorption of intrinsic factor-cobalamin and selective protein reabsorption in renal tubules. The genetic basis of selective intestinal cobalamin malabsorption with proteinuria was investigated in a canine model closely resembling human Imerslund-Gräsbeck syndrome caused by cubilin mutations. CanineCUBN cDNA was cloned and sequenced, showing high identity with human and rat CUBN cDNAs. An intragenic CUBN marker was identified in the canine family and used to test the hypothesis of genetic linkage of the disease and CUBN loci. Linkage was rejected, indicating that the canine disorder resembling Imerslund-Gräsbeck syndrome is caused by defect of a gene product other than cubilin. These results imply that there may be locus heterogeneity among human kindreds with selective intestinal cobalamin malabsorption and proteinuria and that normal brush-border expression of cubilin requires the activity of an accessory protein.
Nitric Oxide Mediation of Active Immunosuppression Associated With Graft-Versus-Host Reaction
Authors: Bobé Pierre , Benihoud Karim , Grandjon Danièle , Opolon Paule , Pritchard Linda Louise , Huchet Roger
Published: October 2019

In the immunosuppression accompanying the lethal systemic graft-versus-host reaction (GVHR) directed against minor histocompatibility antigens in irradiated adult mice, we previously determined that non-T, non-B, L-leucine methyl ester (LME)-sensitive cells were implicated via two different mechanisms: one, which is interferon-γ (IFN-γ)–dependent and affects both T-cell proliferative responses and thymus-independent antibody production by CD5+ B cells; and a second, which is IFN-γ–independent and affects B-cell proliferative responses. Because IFN-γ induces the production of nitric oxide (NO), a potent immunosuppressive molecule, we investigated the involvement of NO in the suppression mediated by the LME-sensitive cells. Inducible NO synthase (iNOS) mRNA, iNOS protein, and the stable end products of iNOS pathway, L-citrulline and nitrite, were detected early in GVHR in LME-sensitive spleen cells taken ex vivo and could be amplified in vitro by T and B mitogens. Inhibition of NO production with arginine analogs (aminoguanidine, NG-monomethyl-L-arginine [LMMA]), like anti–IFN-γ antibodies, reversed suppression of both T-cell responses to concanavalin A and CD5+ B-cell responses, but not of B-cell response to lipopolysaccharides (LPS). The GVHR-associated, IFN-γ–dependent immunosuppression of T-cell proliferation and of antibody synthesis by CD5+ B cells is the consequence of NO production by LME-sensitive cells. Immunohistochemical analyses indicate that these cells belong to the macrophage lineage.
Basic Fibroblast Growth Factor Is Expressed by CD19/CD11c-Positive Cells in Hairy Cell Leukemia
Authors: Gruber Gerhard , Schwarzmeier Josef D. , Shehata Medhat , Hilgarth Martin , Berger Rudolf
Published: October 2019

Several features are characteristic for hairy cell leukemia (HCL). Among those are pancytopenia, bone marrow fibrosis, and the appearance of a defined tumor cell phenotype in peripheral blood (PB), bone marrow (BM), and spleen. Hairy cells (HC) coexpress antigens specific for B lymphocytes and monocytes/macrophages and thus the malignant cell does not seem to be restricted to a defined lineage. When serum or bone marrow aspirate was screened by enzyme-linked immunosorbent assay (ELISA) for basic fibroblast growth factor (bFGF), specimen derived from HCL (serum: mean value, 29 pg/mL; BM aspirate: mean value, 641 pg/mL) contained significantly higher levels than those from healthy subjects. To study whether peripheral blood mononuclear cells (PBMC) derived from patients suffering from HCL and healthy donors (HD) were capable of producing bFGF, culture supernatant (conditioned medium, [CM]) was tested for the presence of this cytokine. While bFGF was not detectable in cell cultures from HD, HCL-derived CM contained relatively high levels of bFGF. CM was successfully used for stimulation of mesenchymal cell proliferation, which could be inhibited by a neutralizing anti-bFGF antibody. Cellular activation by pokeweed mitogen (PWM) or the combination of 12-o-tetradecanoyl-phorbol-13-acetate (TPA) plus calcium ionophore (Ca-Ip) led to an enhanced mRNA expression. Results of Western blot experiments showed that HC synthesize at least three isoforms (approximately 18, 23, and 25 kD), but only the 23-kD isoform is exported. To assess the nature of the producer cell, double immunofluorescence analysis using a bFGF-specific and an anti-CD11c monoclonal antibody (MoAb) was undertaken. The majority of cells scoring positive for CD11c were also reactive with the anti-bFGF MoAb. Furthermore, enrichment of CD19/CD11c-positive cells correlated with enhanced bFGF levels, thereby supporting the argument for HC being the producer cells of bFGF. A biological function of bFGF in HCL might be mediation of chemoresistance, as 2-chlorodeoxyadenosine (2-CdA)–induced inhibition of cell proliferation can be reversed by bFGF. Endogenous bFGF production by HC is not affected by this purine analogue and 2-CdA–induced apoptosis is diminished in bFGF-producing HC as compared with normal PBMC. Therefore, bFGF expression by HC might be important for resistance to chemotherapy and survival of the malignant cells.
Differential Effects of Human Granulocyte Colony-Stimulating Factor (hG-CSF) and Thrombopoietin on Megakaryopoiesis and Platelet Function in hG-CSF Receptor-Transgenic Mice
Authors: Yang Feng-Chun , Tsuji Kohichiro , Oda Atsushi , Ebihara Yasuhiro , Xu Ming-jiang , Kaneko Azusa , Hanada Sachiyo , Mitsui Tetsuo , Kikuchi Akira , Manabe Atsushi , Watanabe Sumiko , Ikeda Yasuo , Nakahata Tatsutoshi
Published: October 2019

Granulocyte-colony stimulating factor (G-CSF) has been found to act on the neutrophilic lineage. We recently showed that human G-CSF (hG-CSF) has effects similar to early-acting cytokines such as interleukin-3 (IL-3) in the development of multipotential hematopoietic progenitors in transgenic (Tg) mice expressing receptors (R) for hG-CSF. In the present study, we examined the effects of hG-CSF on more mature hematopoietic cells committed to megakaryocytic lineage in these Tg mice. The administration of hG-CSF to the Tg mice increased the numbers of both platelets in peripheral blood and megakaryocytes in the spleen, indicating that hG-CSF stimulates megakaryopoiesis in the Tg mice in vivo. The stimulatory effect of hG-CSF was also supported by the results of studies in vitro. hG-CSF supported megakaryocyte colony formation in a dose-dependent fashion in clonal cultures of bone marrow cells derived from the Tg mice. Direct effects of hG-CSF on megakaryocytic progenitors in the Tg mice were confirmed by culture of single-cell sorted from bone marrow cells. hG-CSF showed a stronger effect on maturation of megakaryocytes in the Tg mice than that of IL-3 alone, but weaker than that of TPO alone. In addition, hG-CSF induced phosphorylation of STAT3 but not Jak2 or STAT5, while TPO induced phosphorylation of both. In contrast to TPO, hG-CSF did not enhance ADP-induced aggregation. Thus, hG-CSF has a wide variety of functions in megakaryopoiesis of hG-CSFR-Tg mice, as compared with other megakaryopoietic cytokines, but the activity of hG-CSF in megakaryocytes and platelets does not stand up to a comparison with that of TPO. Specific signals may be required for the full maturation and activation of platelets.
Demonstration That Human Mast Cells Arise From a Progenitor Cell Population That Is CD34+, c-kit+, and Expresses Aminopeptidase N (CD13)
Authors: Kirshenbaum Arnold S. , Goff Julie P. , Semere Tekli , Foster Barbara , Scott Linda M. , Metcalfe Dean D.
Published: October 2019

Human mast cells are known to arise from a CD34+/c-kit+ progenitor cell population that also gives rise to neutrophils, eosinophils, basophils, and monocytes. To further characterize cells within the CD34+/c-kit+ population that yield mast cells, this progenitor was additionally sorted for CD13, a myeloid marker known to appear early on rodent mast cells and cultured human mast cells, but not expressed or expressed at low levels on human tissue mast cells; and cultured in recombinant human (rh) stem cell factor (rhSCF), rh interleukin-3 (rhIL-3; first week only), and rhIL-6. Initial sorts revealed that although the majority of cells in culture arose from the CD34+/c-kit+/CD13−cell population, mast cells arose from a CD34+/c-kit+/CD13+progenitor cell that also gave rise to a population of monocytes. Sequential sorting confirmed that CD34+/c-kit+/CD13+cells in CD34+/c-kit+/CD13−sorts gave rise to the few mast cells observed in CD13−sorted cells. CD34+/c-kit+/CD13+cells plated as single cells in the presence of various cytokine combinations gave rise to pure mast cell, monocyte, or mixed mast cell/monocyte progeny. Addition of either rh granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or rhIL-5 to the CD34+/c-kit+/CD13+progenitor cell population cultured in rhSCF, rhIL-3, and rhIL-6 did increase the number of total cells cultured and in the case of rhIL-5, did increase total mast cell numbers. Neither rhGM-CSF or rhIL-5 led to additional cell populations, ie, even with the addition of rhGM-CSF or rhIL-5, only mast cells and monocytes grew from CD34+/c-kit+/CD13+cells. Thus, human mast cells and a population of monocytes arise from precursor cells that express CD34, c-kit, and CD13; and within which, are mast cell, monocyte, and mast/monocyte (bipotential) precursors.
Regulation of Iron Metabolism in Murine J774 Macrophages: Role of Nitric Oxide–Dependent and –Independent Pathways Following Activation With Gamma Interferon and Lipopolysaccharide
Authors: Mulero Victoriano , Brock Jeremy H.
Published: October 2019

To elucidate the pathways by which nitric oxide (NO) influences macrophage iron metabolism, the uptake, release, and intracellular distribution of iron in the murine macrophage cell line J774 has been investigated, together with transferrin receptor (TfR) expression and iron-regulatory protein (IRP1 and IRP2) activity. Stimulation of macrophages with interferon-γ (IFN-γ) and/or lipopolysaccharide (LPS) decreased Fe uptake from transferrin (Tf), and there was a concomitant downregulation of TfR expression. These effects were mediated by NO-dependent and NO-independent mechanisms. Addition of the NO synthase (NOS) inhibitor N-monomethyl arginine (NMMA) partially restored Fe uptake but either had no effect on or downregulated TfR expression, which suggests that NO by itself is able to affect iron availability. Analysis of the intracellular distribution of incorporated iron revealed that in IFN-γ/LPS-activated macrophages there was a decreased amount and proportion of ferritin-bound iron and a compensatory increase in insoluble iron, which probably consists mainly of iron bound to intracellular organelles. Finally, although NO released by IFN-γ/LPS-activated macrophages increased the iron-responsive element (IRE)-binding activity of both IRP1 and IRP2, IFN-γ treatment decreased IRP2 activity in an NO-independent manner. This study demonstrates that the effect of IFN-γ and/or LPS on macrophage iron metabolism is complex, and is not entirely due to either NO-or to IRP-mediated mechanisms. The overall effect is to decrease iron uptake, but not its utilization.
Mutator Phenotype in Human Hematopoietic Neoplasms and Its Association With Deletions Disabling DNA Repair Genes and bcl-2 Rearrangements
Authors: Indraccolo Stefano , Minuzzo Sonia , Nicoletti Laura , Cretella Elisabetta , Simon Martin , Papakonstantinou Georg , Hehlmann Rüdiger , Mion Marta , Bertorelle Roberta , Roganovic Jelena , Chieco-Bianchi Luigi
Published: October 2019

As mice carrying mutations of the DNA mismatch repair genes MSH2 and MSH6 often develop lymphoid neoplasms, we addressed the prevalence of the replication error (RER+) phenotype, a manifestation of an underlying defect of DNA mismatch repair genes, in human lymphoid tumors. We compared microsatellite instability (MSI) at 10 loci in 37 lymphoid tumors, including 16 acute lymphoid leukemias (ALL) and 21 non-Hodgkin’s lymphomas (NHL), and in 29 acute myeloid leukemias (AML). Significant differences in MSI prevalence between AMLs and ALLs emerged, and MSI occurrence was more frequent in the NHLs versus AMLs. Indeed, only 3 of 29 (10%) AMLs exhibited MSI, thus confirming its paucity in myeloid tumors, while 10 of 37 (27%) lymphoid tumors, 6 ALLs and 4 NHLs, disclosed an RER+phenotype. In 1 ALL patient, the same molecular alterations were observed in correspondence with a relapse, but were not detected during remission over a 14-month follow-up; in another ALL patient, findings correlated with impending clinical relapse. These results suggest that the study of MSI in lymphoid tumors might provide a useful molecular tool to monitor disease progression in a subset of ALLs. To correlate MSI with other known genetic abnormalities, we investigated the status of the proto-oncogene, bcl-2, in the lymphoma patients and found that 4 of 4 NHL patients with MSI carried bcl-2 rearrangements, thus linking genomic instability to enhanced cell survival in NHL; moreover, no p53 mutations were found in these patients. Finally, we addressed the putative cause of MSI in hematopoietic tumors by searching for both mutations and deletions affecting DNA repair genes. A limited genetic analysis did not show any tumor-specific mutation in MLH1 exons 9 and 16 and in MSH2 exons 5 and 13. However, loss of heterozygosity (LOH) of markers closely linked to mismatch repair genes MLH1, MSH2, and PMS2 was demonstrated in 4 of 6 ALLs and 1 of 3 AMLs with MSI. These observations indicate that chromosomal deletions might represent a mechanism of inactivation of DNA repair genes in acute leukemia.
Cloning and Characterization of EphA3 (Hek) Gene Promoter: DNA Methylation Regulates Expression in Hematopoietic Tumor Cells
Authors: Dottori Mirella , Down Michelle , Hüttmann Andreas , Fitzpatrick David R. , Boyd Andrew W.
Published: October 2019

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal and spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promoter elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the EphA3 gene promoter region. A region of 86 bp located at −348 bp to −262 bp upstream from the transcription start site was identified as the basal promoter. This region was shown to be active in both EphA3-expressing and -nonexpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive restriction enzymes and bisulfite sequencing of genomic DNA, sites of DNA methylation were identified in hematopoietic cell lines which correlated with their levels of EphA3 gene expression. We showed that EphA3 was not methylated in normal tissues but that a subset of clinical samples from leukemia patients showed extensive methylation, similar to that observed in cell lines. These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors.
Erythropoietin Receptor Mutations Associated With Familial Erythrocytosis Cause Hypersensitivity to Erythropoietin in the Heterozygous State
Authors: Watowich Stephanie S. , Xie Xiaoling , Klingmuller Ursula , Kere Juha , Lindlof Mikael , Berglund Stig , de la Chapelle Albert
Published: October 2019

Inherited mutations in the erythropoietin receptor (EPOR) causing premature termination of the receptor cytoplasmic region are associated with dominant familial erythrocytosis (FE), a benign clinical condition characterized by hypersensitivity of erythroid progenitor cells to EPO and low serum EPO (S-EPO) levels. We describe a Swedish family with dominant FE in which erythrocytosis segregates with a new truncation in the negative control domain of the EPOR. We show that cells engineered to concomitantly express the wild-type (WT) EPOR and mutant EPORs associated with FE (FE EPORs) are hypersensitive to EPO-stimulated proliferation and activation of Jak2 and Stat5. These results demonstrate that FE is caused by hyperresponsiveness of receptor-mediated signaling pathways and that this is dominant with respect to WT EPOR signaling.
Enhanced endothelialization and microvessel formation in polyester grafts seeded with CD34+ bone marrow cells
Authors: Bhattacharya Vishwanath , McSweeney Peter A. , Shi Qun , Bruno Benedetto , Ishida Atsushi , Nash Richard , Storb Rainer F. , Sauvage Lester R. , Hammond William P. , Wu Moses Hong-De
Published: October 2019

The authors have shown accelerated endothelialization on polyethylene terephthalate (PET) grafts preclotted with autologous bone marrow. Bone marrow cells have a subset of early progenitor cells that express the CD34 antigen on their surfaces. A recent in vitro study has shown that CD34+ cells can differentiate into endothelial cells. The current study was designed to determine whether CD34+ progenitor cells would enhance vascular graft healing in a canine model. The authors used composite grafts implanted in the dog's descending thoracic aorta (DTA) for 4 weeks. The 8-mm × 12-cm composite grafts had a 4-cm PET graft in the center and 4-cm standard ePTFE grafts at each end. The entire composite was coated with silicone rubber to make it impervious; thus, the PET segment was shielded from perigraft and pannus ingrowth. There were 5 study grafts and 5 control grafts. On the day before surgery, 120 mL bone marrow was aspirated, and CD34+ cells were enriched using an immunomagnetic bead technique, yielding an average of 11.4 ± 5.3 × 106. During surgery, these cells were mixed with venous blood and seeded onto the PET segment of composite study grafts; the control grafts were treated with venous blood only. Hematoxylin and eosin, immunocytochemical, and AgNO3staining demonstrated significant increases of surface endothelialization on the seeded grafts (92% ± 3.4% vs 26.6% ± 7.6%; P = .0001) with markedly increased microvessels in the neointima, graft wall, and external area compared with controls. In dogs, CD34+ cell seeding enhances vascular graft endothelialization; this suggests practical therapeutic applications.
VCAM-1 is more effective than MAdCAM-1 in supporting eosinophil rolling under conditions of shear flow
Authors: Sriramarao P. , DiScipio Richard G. , Cobb Ronald R. , Cybulsky Myron , Stachnick Greg , Castaneda Diego , Elices Mariano , Broide David H.
Published: October 2019

The ability of the 4 integrin counterligands vascular cell adhesion molecule (VCAM)-1 or mucosal addressin (MAd)CAM-1 to support eosinophil rolling or firm adhesion under conditions of physiologic flow has not been delineated. Using a parallel plate flow chamber in vitro and intravital microscopy in vivo, we demonstrate that eosinophil rolling and adhesion on VCAM-1 is mediated by both 4β1 and 4β7 integrins. Eosinophils rolled equally efficiently on both VCAM-1 2 domain and VCAM-1 7 domain, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under conditions of flow. Furthermore, activation of the eosinophil β1 integrin with monoclonal antibody (mAb) 8A2 resulted in both resistance to shear stress–induced detachment from VCAM-1 in vitro and in stable arrest of rolling eosinophils on interleukin (IL)-1β–stimulated venules in vivo. Eosinophils rolled less efficiently on MAdCAM-1– than on VCAM-1–coated coverslips under conditions of flow. However, eosinophils firmly adhered as efficiently to MAdCAM-1 as to VCAM-1. Overall, these results demonstrate that both VCAM-1 and MAdCAM-1 can support eosinophil firm adhesion under conditions of flow. In contrast, VCAM-1 is significantly more efficient than MAdCAM-1 in supporting eosinophil rolling under conditions of flow.
Soluble VCAM-1 binding to α4 integrins is cell-type specific and activation dependent and is disrupted during apoptosis in T cells
Authors: Rose David M. , Cardarelli Pina M. , Cobb Ronald R. , Ginsberg Mark H.
Published: October 2019

Soluble vascular cell adhesion molecule-1 (sVCAM-1) is generated during inflammation and can alter lymphocyte functions. The authors report that the binding of sVCAM-1 to 4 integrin-bearing cells is a dynamically regulated, active cellular process. Binding of recombinant sVCAM-1 to 4 integrins on peripheral blood mononuclear cells was cell-type specific. Circulating CD16+ NK cells constitutively bound sVCAM-1 with high affinity, whereas a subpopulation of T-lymphocytes, primarily CD45RO+ (memory), bound sVCAM-1 only after phorbol ester stimulation. sVCAM-1 binding to homogenous stable cell lines was also cell-type specific, and required active cellular processes because it was blocked by the inhibition of ATP synthesis and by Fas-induced apoptosis. Indeed, the loss of high-affinity VCAM-1 binding was an early event in apoptosis. Furthermore, an H-Ras/Raf-initiated signaling pathway also suppressed sVCAM-1 binding to 4β1 integrins. Collectively, these results showed that the capacity of 4 integrins to bind VCAM-1 is actively regulated and that this regulation may control 4 integrin-dependent cellular functions.
Prothrombin San Antonio: a single amino acid substitution at a Factor Xa activation site (Arg320 to His) results in dysprothrombinemia
Authors: Sun William Y. , Burkart Melissa C. , Holahan Joseph R. , Degen Sandra J. F.
Published: October 2019

Three members of a San Antonio, Texas, family were identified with prothrombin activity levels half the normal level but to have normal levels of antigen. All exons of the prothrombin gene from the proband were sequenced. A G-to-A mutation at nucleotide 7543 was found that resulted in the substitution of His for Arg at residue 320. The Arg320-Ile321 bond is 1 of 2 sites in prothrombin cleaved by Factor Xa in the prothrombinase complex to form thrombin. Substitution of His for Arg at this site resulted in the blockage of Factor Xa cleavage, forming a dysfunctional molecule. The proband, her mother, and her maternal aunt were found to be heterozygous for this mutation. This is the first known observation of an amino acid substitution at this site that resulted in dysprothrombinemia.
Large deletions at the t(9;22) breakpoint are common and may identify a poor-prognosis subgroup of patients with chronic myeloid leukemia
Authors: Sinclair P. B. , Nacheva E. P. , Leversha M. , Telford N. , Chang J. , Reid A. , Bench A. , Champion K. , Huntly B. , Green A. R.
Published: October 2019

The hallmark of chronic myeloid leukemia (CML) is theBCR-ABL fusion gene, which is usually formed as a result of the t(9;22) translocation. Patients with CML show considerable heterogeneity both in their presenting clinical features and in the time taken for evolution to blast crisis. In this study, metaphase fluorescence in situ hybridization showed that a substantial minority of patients with CML had large deletions adjacent to the translocation breakpoint on the derivative 9 chromosome, on the additional partner chromosome in variant translocations, or on both. The deletions spanned up to several megabases, had variable breakpoints, and could be detected by microsatellite polymerase chain reaction in unfractionated bone marrow and purified peripheral blood granulocytes. The deletions were likely to occur early and possibly at the time of the Philadelphia (Ph) chromosome translocation: deletions were detected at diagnosis in 11 patients, were found in all Ph-positive metaphases, and were more prevalent in patients with variant Ph chromosomes. Kaplan-Meier analysis showed a median survival time of 36 months in patients with a deletion; patients without a detectable deletion survived > 90 months. The survival-time difference was significant on log-rank analysis (P = .006). Multivariate analysis demonstrated that the prognostic importance of deletion status was independent of age, sex, percentage of peripheral blood blasts, and platelet count. Our data therefore suggest that an apparently simple, balanced translocation may result not only in the generation of a dominantly acting fusion oncogene but also in the loss of one or more genes that influence disease progression.
Rapid molecular response during early induction chemotherapy predicts a good outcome in childhood acute lymphoblastic leukemia
Authors: Panzer-Grümayer E. Renate , Schneider Monika , Panzer Simon , Fasching Karin , Gadner Helmut
Published: October 2019

Early response to therapy is an independent prognostic factor in childhood acute lymphoblastic leukemia. Although most patients have rapid early responses, as detected by morphology, 15% to 20% of patients have relapses. The authors evaluated residual disease by molecular methods on day 15 of minimal residual disease (MRD) therapy and compared these data with their recently established MRD-based risk stratification, defined by MRD levels 5 weeks after induction treatment and before consolidation. All 68 children treated according to current Berlin-Frankfurt-Münster (BFM) protocols went into morphologically complete remission after induction. There was a significant difference in outcome between children with rapid disease clearance and those with high levels of day-15 MRD (P = .035). Among patients with high levels of day-15 MRD, only the MRD-based risk stratification was predictive of the outcome. All patients with negative or low day-15 MRD had excellent prognoses and were in the MRD-based low-risk group. Thus, after only 2 weeks of treatment, the authors were able to identify a patient population of 20% who may benefit from the least intensive treatment.
Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase
Authors: Vanwetswinkel Sophie , Plaisance Stephane , Zhi-yong Zhang , Vanlinthout Ingrid , Brepoels Kathleen , Lasters Ignace , Collen Désiré , Jespers Laurent
Published: October 2019

Recombinant staphylokinase (SakSTAR) variants obtained by site-directed substitution with cysteine, in the core (lysine 96 [Lys96], Lys102, Lys109, and/or Lys135) or the NH2-terminal region that is released during activation of SakSTAR (serine 2 [Ser2] and/or Ser3), were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules with molecular weights of 5000 (P5), 10 000 (P10), or 20 000 (P20). The specific activities and thrombolytic potencies in human plasma were unaltered for most variants derivatized with PEG (PEGylates), but maleimide PEG derivatives had a better temperature stability profile. In hamsters, SakSTAR was cleared at 2.2 mL/min; variants with 1 P5 molecule were cleared 2-to 5-fold; variants with 2 P5 or 1 P10 molecules were cleared 10-to 30-fold; and variants with 1 P20 molecule were cleared 35-fold slower. A bolus injection induced dose-related lysis of a plasma clot, fibrin labeled with 125 iodine (125I-fibrin plasma clot), and injected into the jugular vein. A 50% clot lysis at 90 minutes required 110 μg/kg SakSTAR; 50 to 110 μg/kg of core-substitution derivatives with 1 P5; 25 μg/kg for NH2-terminal derivatives with 1 P5; 5 to 25 μg/kg with derivatives with 2 P5 or 1 P10; and 7 μg/kg with P20 derivatives. Core substitution with 1 or 2 P5 molecules did not significantly reduce the immunogenicity of SakSTAR in rabbits. Derivatization of staphylokinase with a single PEG molecule allows controllable reduction of the clearance while maintaining thrombolytic potency at a reduced dose. This indicates that mono-PEGylated staphylokinase variants may be used for single intravenous bolus injection.
Adenosine triphosphate–induced oxygen radical production and CD11b up-regulation: Ca++ mobilization and actin reorganization in human eosinophils
Authors: Dichmann Stefan , Idzko Marco , Zimpfer Ulrich , Hofmann Clemens , Ferrari Davide , Luttmann Werner , Virchow Christian , Di Virgilio Francesco , Norgauer Johannes
Published: October 2019

Eosinophils are major effector cells in cellular inflammatory conditions such as parasitic infections, atopic diseases, bullous dermatoses, and vasculitis. Biological activities of adenosine triphosphate (ATP) were characterized in human eosinophils and compared with those of other eosinophil activators such as complement fragment product C5a, platelet-activating factor (PAF), and eotaxin. ATP initiated production of reactive oxygen metabolites, as demonstrated by lucigenin-dependent chemiluminescence. Furthermore, ATP caused up-regulation of the integrin CD11b. In addition, fluorescence microscope measurements labeled with fura-2 (1-[2-(5-carboxy-oxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methyl-phenoxy)-ethane-N, N, N, N′-tetraacetic acid, pentaacetoxymethyl ester) eosinophils in the presence or absence of ethyleneglycotetraacetic acid (EGTA) indicated that there was Ca++ mobilization from intracellular stores by ATP. Flow cytometric studies showed transient actin polymerization upon stimulation with ATP and its stable analogues adenosine 5′-0-(3-thiotriphosphate) and 2-methylthioadenosine triphosphate tetrasodium (met-ATP). The reactions induced by ATP were comparable to those obtained by C5a, PAF, and eotaxin. Production of reactive oxygen metabolites and actin polymerization after stimulation with ATP was inhibited by pertussis toxin, which indicated involvement of receptor-coupled guanine nucleotide–binding proteins (Gi proteins). In addition, experiments with oxidized ATP also suggest involvement of P2X receptors in this activation process. The results show that ATP is a strong activator of eosinophils and has biological activity comparable to those of the eosinophil chemotaxins C5a, PAF, and eotaxin. The findings strongly suggest a role of ATP in the pathogenesis of eosinophilic inflammation as an activator of proinflammatory effector functions.
Effect of recombinant human erythropoietin combined with granulocyte/ macrophage colony-stimulating factor in the treatment of patients with myelodysplastic syndrome
Authors: Thompson John A. , Gilliland D. Gary , Prchal Josef T. , Bennett John M. , Larholt Kay , Nelson Richard A. , Rose Esther H. , Dugan Margaret H. ,   the GM/EPO MDS Study Group
Published: October 2019

This randomized, placebo-controlled trial was designed to assess the efficacy and safety of therapy with granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (epoetin alfa) in anemic, neutropenic patients with myelodysplastic syndrome. Sixty-six patients were enrolled according to the following French–American–British classification: refractory anemia (20), refractory anemia with excess blasts (35), refractory anemia with ringed sideroblasts (9), and refractory anemia with excess blasts in transformation (2). Patients were stratified by their serum erythropoietin levels (less than or equal to 500 mU/mL, n = 37; greater than 500 mU/mL, n = 29) and randomized, in a 2:1 ratio, to either GM-CSF (0.3-5.0 μg/kg·d) + epoetin alfa (150 IU/kg 3 times/wk) or GM-CSF (0.3-5.0 μg/kg·d) + placebo (3 times/wk). The mean neutrophil count rose from 948 to 3831 during treatment with GM-CSF ± epoetin alfa. Hemoglobin response (increase greater than or equal to 2 g/dL, unrelated to transfusion) occurred in 4 of 45 (9%) patients in the GM-CSF + epoetin alfa group compared with 1 of 21 (5%) patients with GM-CSF + placebo group (P = NS). Percentages of patients in the epoetin alfa and the placebo groups requiring transfusions of red blood cells were 60% and 92%, respectively, for the low-endogenous erythropoietin patients and 95% and 89% for the high-endogenous erythropoietin patients (P = NS). Similarly, the average numbers of units of red blood cells transfused during the 12-week study in the epoetin alfa and the placebo groups were 5.9 and 9.5, respectively, in the low-endogenous erythropoietin patients and 9.7 and 8.6 in the high-endogenous erythropoietin patients (P = NS). GM-CSF ± epoetin alfa had no effect on mean platelet count. Treatment was well tolerated in most patients, though 10 withdrew from the study for reasons related predominantly to GM-CSF toxicity.