bluuebunny/crossref_metadata_2025 huggingface dataset:
FLT3 Ligand Preserves the Ability of Human CD34+ Progenitors to Sustain Long-Term Hematopoiesis in Immune-Deficient Mice After Ex Vivo Retroviral-Mediated Transduction
Authors: Dao Mo A. , Hannum Charles H. , Kohn Donald B. , Nolta Jan A.
Published: October 2019

Stromal support is required during retroviral-mediated transduction of human bone marrow-derived CD34+ cells to maintain the clonogenicity of the primitive progenitors. We hypothesized that the cytokine FLT3 ligand (FL) might be able to replace the maintenance role provided by the stroma. CD34+ progenitors from human bone marrow were transduced by the retroviral vector LN with the cytokines interleukin-3 (IL-3), IL-6, and stem cell factor (SCF ) present in all cultures. Transductions were performed with or without stromal support and with or without the inclusion of 100 U/mL FL. No significant increase in gene transfer into colony-forming cells was obtained by the addition of FL to the cultures. Transduction and survival of more primitive human hematopoietic cells was determined by growth in immune-deficient mice for 7 to 8 months. Human myeloid cells, T lymphocytes, and colony-forming progenitors were recovered from the marrow of mice that had received human cells transduced on stroma or in suspension culture with IL-3, IL-6, SCF, and FL, but not with IL-3, IL-6, and SCF alone. LN provirus was detected by polymerase chain reaction in the marrow recovered from 9 of 10 mice transplanted with human CD34+ cells transduced with stromal support, 5 of 11 mice that received human cells transduced in suspension culture with FL, but none of the 10 mice that received human cells transduced in suspension culture without FL. We conclude that FLT3 ligand, in conjunction with IL-3, IL-6, and SCF, preserves the generative capacity of primitive human hematopoietic cells during in vitro transductions in suspension culture.
Homodimerization of Erythropoietin Receptor by a Bivalent Monoclonal Antibody Triggers Cell Proliferation and Differentiation of Erythroid Precursors
Authors: Schneider Helmut , Chaovapong Warak , Matthews David J. , Karkaria Cyrus , Cass Robert T. , Zhan Hangjun , Boyle Mark , Lorenzini Tony , Elliott Steve G. , Giebel Lutz B.
Published: October 2019

Erythropoietin (EPO) stimulates proliferation and differentiation of erythroid progenitor cells. Several lines of evidence indicate that the most likely mechanism of EPO receptor (EPO-R) activation by EPO is homodimerization of the receptor on the surface of erythrocyte precursors. Therefore, we argued that it should be possible to raise EPO-R monoclonal antibodies (MoAbs) that would activate the receptor by dimerization and thus mimic EPO action. We have identified such an agonist MoAb (MoAb34) directed against the extracellular EPO binding domain of the EPO-R. This bivalent IgG antibody triggers the proliferation of EPO-dependent cell lines and induces differentiation of erythroid precursors in vitro. In contrast, the monovalent Fab fragment, which cannot dimerize the receptor, is completely inactive. The mechanism of receptor activation by homodimerization implies that at high ligand concentrations the formation of 1:1 receptor/ligand complexes is favored over 2:1 complexes, thereby turning the ligand agonist into an antagonist. Thus, EPO and MoAb34 should self-antagonize at high concentrations in both cell proliferation and differentiation assays. Our data indeed demonstrate that EPO and MoAb34 antagonize ligand-dependent cell proliferation with IC50 values of approximately 20 and 2 μmol/L, respectively. Erythroid colony formation (BFUe) is inhibited at MoAb34 concentrations above 1 μmol/L. Furthermore, we analyzed the MoAb34:EPO-R interaction using a mathematic model describing antibody-mediated receptor dimerization. The data for proliferation and differentiation activity were consistent with the receptor dimer formation on the cell surface predicted by the model.
Induction of Hypoxia-Inducible Factor-1, Erythropoietin, Vascular Endothelial Growth Factor, and Glucose Transporter-1 by Hypoxia: Evidence Against a Regulatory Role for Src Kinase
Authors: Gleadle Jonathan M. , Ratcliffe Peter J.
Published: October 2019

The induction by hypoxia of genes such as erythropoietin, vascular endothelial growth factor (VEGF ), and glucose transporter-1 (Glut-1) is mediated in part by a transcriptional complex termed hypoxia-inducible factor-1 (HIF-1). Several lines of evidence have implicated protein phosphorylation in the mechanism of activation of HIF-1 by hypoxia. Recent reports have described the activation of the tyrosine kinase src by severe hypoxia, and a role in the induction of VEGF by severe hypoxia has been proposed. This led us to examine whether src and related kinases operated more widely in the hypoxic induction of HIF-1 and HIF-1–dependent genes regulated by hypoxia. Measurements of src kinase activity in cells exposed to varying severities of hypoxia showed activation by severe hypoxia (0.1% oxygen or catalyst induced anoxia), but not 1% oxygen. This contrasted with the marked induction of HIF-1 by exposure to 1% oxygen. Manipulations of src activity were produced by transient and stable transfection of Hep3B cells. Despite substantial changes in src activity, no alteration was seen in the normoxic or hypoxic expression of erythropoietin, VEGF, or Glut-1, or in the regulation of HIF-1–dependent reporter genes inducible by hypoxia. Similarly, we found that the expression of these genes in src- or c-src kinase-deficient cells did not differ from wild-type cells at either 1% oxygen or more severe hypoxia. These results indicate that src is not critical for the hypoxic induction of HIF-1, erythropoietin, VEGF, or Glut-1.
Exocytotic Competence and Intergranular Fusion in Cord Blood-Derived Eosinophils During Differentiation
Authors: Scepek Susanne , Lindau Manfred
Published: October 2019

We studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5′-O-(3-thiotriphosphate) (GTP) γS after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from ≈700 μm2 to ≈400 μm2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.
Mcl-1, a Bcl-2 Family Member, Delays the Death of Hematopoietic Cells Under a Variety of Apoptosis-Inducing Conditions
Authors: Zhou Ping , Qian Liping , Kozopas Karen M. , Craig Ruth W.
Published: October 2019

Mcl-1 is a member of the Bcl-2 family that was identified based on increased expression in myeloblastic leukemia cells undergoing differentiation. Mcl-1 was previously found to be similar to Bcl-2 in causing a delay in apoptotic cell death in Chinese hamster ovary cells. The work described here was aimed at determining whether Mcl-1 could also exert such an effect in hematopoietic cells, because endogenous Mcl-1 expression is prominent in the hematopoietic system. A further aim was to assess the effects of Mcl-1 in cells exposed to a variety of cytotoxic stimuli, because Bcl-2 is known to have a broad spectrum of activity. To approach these aims, FDC-P1 murine myeloid progenitor cells were transfected with vectors driving either constitutive or inducible expression of Mcl-1. The introduced Mcl-1 gene was found to cause a prolongation of viability under various conditions that cause apoptotic cell death, including exposure to cytotoxic agents (the chemotherapeutic drug etoposide, calcium ionophore, or UV irradiation) and the withdrawal of required growth factors. In addition, Mcl-1 was found to interact with Bax, a member of the Bcl-2 family that promotes cell death as a homodimer but that can heterodimerize with Bcl-2 to promote cell viability. Although Mcl-1 prolonged cell viability, it did not prevent eventual cell death upon continuous exposure to a cytotoxic agent. Prolongation of viability was maximal when expression of Mcl-1 was induced before the application of the apoptotic stimulus, although some increase occurred if Mcl-1 was induced shortly thereafter and before overt apoptosis. Taken as a whole, these findings provide further parallels between Mcl-1 and Bcl-2, showing that Mcl-1 can interact with Bax in hematopoietic FDC-P1 cells and can prolong cell viability under a variety of cytotoxic conditions.
Formation of Multinucleated Giant Cells In Vitro Is Dependent on the Stage of Monocyte to Macrophage Maturation
Authors: Möst Johannes , Spötl Ludwig , Mayr Gertraud , Gasser Annette , Sarti Alessandra , Dierich Manfred P.
Published: October 2019

Multinucleated giant cells (MGC) are a common feature of granulomas that develop during various inflammatory reactions. MGC originate from fusion of monocytes or macrophages, but the exact mechanism of their generation is still unclear. In the present study, we investigated the influence of monocyte to macrophage maturation on the ability of human monocytes/macrophages to fuse with each other. MGC were generated in vitro by stimulation of human peripheral blood monocytes with cytokine containing supernatants. With freshly isolated monocytes, fusion rates of up to 90% were obtained. When monocyte to macrophage maturation was induced by culturing the cells in human serum, fusion rates gradually decreased with advancing time of the preceding culture (corresponding to the stage of differentiation) and almost no MGC formation could be obtained with 8-day-old macrophages. In contrast, fusion rates did not decrease when monocytes had been cultured under serum free conditions before stimulation. When freshly isolated monocytes were added to 1-week cultured macrophages, which had been membrane-labeled with a fluorochrome, fusion between the two populations could be induced. Because the ability for intracellular killing of certain pathogens is reduced in macrophages, fusion with monocytes (newly arriving at the site of inflammation) may represent an attempt to restore this capacity.
Protoporphyria Induced by the Orally Active Iron Chelator 1,2-diethyl-3-hydroxypyridin-4-one in C57BL/10ScSn Mice
Authors: Smith Andrew G. , Clothier Bruce , Francis Jean E. , Gibbs Anthony H. , De Matteis Francesco , Hider Robert C.
Published: October 2019

Administration in the drinking water of the orally-active iron chelator 1,2-diethyl-3-hydroxypyridin-4-one (CP94) to C57BL/10ScSn mice caused the development of hepatic protoporphyria. This was detected after 1 week and continued as long as the chelator was given (15 weeks). The more hydrophilic 1,2-dimethyl- and 1-hydroxyethyl,2-ethyl-analogues (CP20 and CP102) were also tested, but they were both inactive in inducing accumulation of protoporphyrin in the liver. Restriction of in vivo iron supply for ferrochelatase seemed a likely mode of action, but an approximately 30% decrease in activity of this enzyme was also observed when measured in vitro. Extracts of livers from mice given CP20, CP94, and CP102 showed no potential to inhibit mouse ferrochelatase, in contrast to the findings with an extract from mice treated with the known porphyrogenic chemical 4-ethyl - 3 , 5 - diethoxycarbonyl - 2 , 6 - dimethyl - 1 , 4 - dihydropyridine, -indicating that ferrochelatase inhibition did not occur by the formation of an N-ethyl-protoporphyrin derived from metabolism by cytochrome P450. CP20, CP94, CP102, and CP117 (the pivoyl ester of CP102) all caused significant depression of the levels of ferritin-iron and total nonheme iron, but only CP94 caused the significant accumulation of protoporphyrin. Protoporphyria did not occur with iron overloaded C57BL/10ScSn mice or in SWR mice that had elevated basal iron status. Although the protoporphyrin had only a small effect on the total levels of the hemoprotein cytochrome P450 in C57BL/10ScSn mice, the activity of the CYP2B isoforms of cytochrome P450 was actually induced in both strains. The results show that CP94 could cause protoporphyria in individuals of low iron status, perhaps through specifically targeting particular iron pools available to ferrochelatase and by concomitantly stimulating heme synthesis.
A Murine Model for Human Cord Blood Transplantation: Near-Term Fetal and Neonatal Peripheral Blood Cells Can Achieve Long-Term Bone Marrow Engraftment in Sublethally Irradiated Adult Recipients
Authors: Scaradavou Andromachi , Isola Luis , Rubinstein Pablo , Galperin Yelena , Najfeld Vesna , Berlin David , Gordon Jon , Weinberg Rona S.
Published: October 2019

The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo–cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.
Development of Antibodies to Fetal Calf Serum With Arthus-Like Reactions in Human Immunodeficiency Virus–Infected Patients Given Syngeneic Lymphocyte Infusions
Authors: Selvaggi Thomas A. , Walker Robert E. , Fleisher Thomas A.
Published: October 2019

In an attempt to restore immune competence to 12 human immunodeficiency virus-1 (HIV-1)–infected patients, lymphocytes from their HIV-1–uninfected identical twin siblings were cultured in medium supplemented with 5% fetal calf serum (FCS), anti-CD3 antibody, and interleukin-2 (100 IU/mL) for 10 days and then infused into the patients. After multiple infusions, at 6- to 8-week intervals, half of the patients developed arthus-like reactions within 4 to 12 hours of infusion consisting of fever <39°C, hypotension, rigors, arthralgias, myalgias, headache, and/or malaise. Preinfusion and postinfusion serum samples were evaluated for the presence of antibodies to FCS using double immunodiffusion. All preinfusion serum samples were negative by this method while 8 of the 12 patients developed antibodies to a single component of FCS after two or more infusions of lymphocytes cultured in FCS-supplemented medium. Prick skin testing to standardized beef extract was negative in all patients. There was a correlation between initial CD4 level and the development of antibodies to FCS (median initial CD4 count in FCS antibody positive patients = 362.0/μL v median initial CD4 count of nonresponders = 135.0/μL). There was no correlation with response to recall antigens in delayed-type hypersensitivity testing. We conclude that selected patients were sensitized to a single component of FCS carried on donor lymphocytes, despite thorough washing of the cells before infusion. The development of antibodies to FCS indicates that immune complex formation could have occurred after the cell infusions, resulting in the arthus-like reactions. These observations suggest that the therapeutic use of human lymphocytes cultured in FCS may expose the recipient to immunogenic substances with possible clinical sequelae.
Involvement of Fas-Mediated Apoptosis in the Inhibitory Effects of Interferon-α in Chronic Myelogenous Leukemia
Authors: Selleri Carmine , Sato Tadatsugu , Del Vecchio Luigi , Luciano Luigia , Barrett A. John , Rotoli Bruno , Young Neal S. , Maciejewski Jaroslaw P.
Published: October 2019

Interferon-α (IFN-α) is an established treatment for chronic myelogenous leukemia (CML) in chronic phase, but the mechanism of its antileukemic activity is not clear. One possible mechanism of action might include the induction of apoptosis, and especially Fas-mediated cell killing may play an important role in the elimination of malignant cells. We investigated Fas receptor (Fas-R) expression and the consequences of Fas-R triggering in CML patients. Using two-color flow cytometry, we found a significantly higher number of Fas-R–expressing CD34+ cells in the bone marrow (BM) of CML patients compared with normal subjects. We have previously shown that IFN-γ induces Fas-R expression on CD34+ cells; in this study, we investigated whether IFN-α induces Fas-R expression on CML progenitor cells. Dose-dependent induction of Fas-R expression was observed after IFN-α stimulation of CD34+ cells from CML BM. In methylcellulose culture, IFN-α alone at a therapeutic concentration showed only marginal antiproliferative effects on both normal and CML BM progenitors. In contrast, a Fas-R agonist, the anti-CD95 monoclonal antibody CH11, inhibited colony formation from normal progenitors, and the inhibition was even stronger on CML progenitors. When CML BM cells were cultured in the presence of IFN-α, Fas-R–mediated inhibition of colony growth was potentiated in a dose-dependent fashion, consistent with IFN-α induction of Fas-R expression. This functional effect did not require the presence of accessory cells, since similar results were obtained with purified CD34+ cells. In suspension cultures, we demonstrated that suppression of CML hematopoiesis by IFN-α and Fas-R agonist was exerted through Fas-R–mediated induction of apoptosis. Our findings suggest that the Fas-R/Fas-ligand system might be involved in the immunologic regulation of CML progenitor growth and that its effect can be amplified by IFN-α.
Case-Control Study Suggests a Favorable Impact of TEL Rearrangement in Patients With B-Lineage Acute Lymphoblastic Leukemia Treated With Antimetabolite-Based Therapy: A Pediatric Oncology Group Study
Authors: Rubnitz Jeffrey E. , Shuster Jonathan J. , Land Vita J. , Link Michael P. , Pullen D. Jeanette , Camitta Bruce M. , Pui Ching-Hon , Downing James R. , Behm Frederick G.
Published: October 2019

TEL gene rearrangement is the most common genetic lesion in pediatric acute lymphoblastic leukemia (ALL), occurring in about 25% of B-lineage cases. We previously showed that, among patients treated on St Jude protocols, TEL rearrangement independently conferred an excellent prognosis. To extend these results to patients treated with antimetabolite-based therapy, we performed Southern blot analysis to determine the TEL gene status of 104 cases of B-lineage ALL treated on Pediatric Oncology Group 8602, matched on age, gender, and leukocyte count. There were 52 failures among the 77 patients with germline TEL, compared with only 8 failures among 27 patients in the rearranged group. Based on a two-sided logistic regression analysis, stratified for age (subdivided at 10 years), leukocyte count (subdivided at 50,000), and gender, the estimated odds of failing by 4 years in the germline TEL group is 5.4 times that of the rearranged TEL group, with 95% confidence from 1.9 to 15.6, two-sided P = .0009. Thus, the presence of a rearranged TEL gene is also associated with an improved survival among patients treated with antimetabolite-based therapy. Our results indicate that all newly diagnosed ALL patients should be screened for TEL gene rearrangements and suggest that these patients are candidates for less intensive therapy.
Characterization of cis-Acting Sequences and trans-Acting Signals Regulating Early Growth Response 1 and c-fos Promoters Through the Granulocyte-Macrophage Colony-Stimulating Factor Receptor in BA/F3 Cells
Authors: Watanabe Sumiko , Kubota Hideya , Sakamoto Kathleen M. , Arai Ken-ichi
Published: October 2019

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF ) activates a set of genes such as c-fos, jun, myc, and early growth response gene 1 (egr-1). Studies on BA/F3 cells that express hGM-CSF receptor (hGMR) showed that two different signaling pathways controlled by distinct regions within the β subunit are involved in activation of c-fos/c-jun genes and in c-myc, respectively. However, the region(s) of the β subunit responsible for activation of the egr-1 gene and other regulatory genes has not been identified. We describe here how egr-1 promoter is activated by hGMR through two regions of the β subunit, with these regions being required for activation of the c-fos promoter. Coexpression of dominant negative (dn) Ras (N17ras) or dn JAK2 almost completely suppressed the activation of egr-1 and c-fos promoters. Deletion analysis of egr-1 promoter showed two cis-acting regions responsible for activation by hGM-CSF or mouse interleukin-3 (mIL-3), one between nucleotide positions (nt) −56 and −116, and the other between nt −235 and −480, which contains tandem repeats of the serum response element (SRE) sites. Similar experiments with the c-fos promoter showed that cis-acting regions containing the SRE/AP-1 sites is sufficient for activation by hGM-CSF. Based on these observations, we propose that signaling pathways activating egr-1 and c-fos promoters are controlled by SRE elements, either through the same or overlapping pathways that involve JAK2 and Ras.
Detection of Anaplastic Lymphoma Kinase (ALK) and Nucleolar Protein Nucleophosmin (NPM)-ALK Proteins in Normal and Neoplastic Cells With the Monoclonal Antibody ALK1
Authors: Pulford Karen , Lamant Laurence , Morris Stephan W. , Butler Lisa H. , Wood Katrina M. , Stroud David , Delsol Georges , Mason David Y.
Published: October 2019

The t(2; 5)(p23;q35) translocation, associated with anaplastic large-cell lymphoma (ALCL), results in the production of the nucleolar protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) protein. This report describes an immunocytochemical study of the distribution of ALK and NPM-ALK proteins using a new monoclonal antibody, ALK1, that recognizes a formalin resistant epitope in both the 80-kD NPM-ALK chimeric and the 200-kD normal human ALK proteins. Cytoplasmic and nuclear labeling was seen in the t(2; 5)+ SU-DHL-1 and Karpas 299 cell lines. Normal ALK protein expression was restricted to the central nervous system (in scattered neurons, glial cells, and endothelial cells). Two hundred and thirty-nine cases of lymphoma and 80 nonhematopoietic tumors were immunostained. Antibody ALK1 labeled 53.4% (39 of 73 cases) of CD30+ ALCL. A case of ALCL with a t(1; 2) translocation was ALK1+. Three cases of CD30− ALCL with prominent nucleoli showed a unique pattern of coarse granular cytoplasmic labeling. All other tumors, including Hodgkin's disease and lymphomatoid papulosis, were ALK1−. These results indicate that reliable immunostaining of routine biopsy material for NPM-ALK and ALK proteins is feasible. Such analysis is of diagnostic importance, especially because t(2; 5)+ ALCL cases have a good prognosis with appropriate treatment.
Human Platelets Display High-Affinity Receptors for Thrombopoietin
Authors: Broudy Virginia C. , Lin Nancy L. , Sabath Diana F. , Papayannopoulou Thalia , Kaushansky Kenneth
Published: October 2019

Thrombopoietin (Tpo) is a major regulator of megakaryopoiesis both in vivo and in vitro. Tpo initiates its biologic effects by binding to the Mpl receptor, which is a member of the hematopoietin receptor family. To define the Tpo binding characteristics of the Mpl receptor, we iodinated purified 70-kD recombinant human Tpo using the Bolton-Hunter reagent. Autoradiographic analysis of 125I-Tpo binding to normal human marrow mononuclear cells showed many grains specifically associated with megakaryocytes; there were no grains specifically associated with myeloblasts or erythroblasts. Equilibrium binding experiments with 125I-Tpo and normal human platelets showed a single class of high-affinity receptors (kd, 190 pmol/L) with approximately 30 Mpl receptors per platelet. Affinity cross-linking with 125I-Tpo showed that the Mpl receptor on platelets is of molecular weight ∼98 kD. Despite their sequence similarity, erythropoietin and Tpo did not cross-compete for binding to BaF3 cells engineered to coexpress Mpl receptor and erythropoietin receptor. Progeny of normal human burst-forming units-erythroid (BFU-E) contained Mpl receptor mRNA, and flow cytometric analysis showed the presence of Mpl receptor protein on the surface of these cells. These data indicate that display of the Mpl receptor is not limited to the megakaryocytic lineage, but also includes progeny of BFU-E. Like receptors for other hematopoietic cytokines, the binding affinity of the Mpl receptor for Tpo is high, with relatively few receptors displayed per cell. These results suggest that the effects of Tpo to speed red blood cell recovery after myelosuppressive therapy in vivo and to enhance colony-forming unit-erythroid generation in vitro may be mediated by direct interaction of Tpo and erythroid progenitor cells.
Efficient In Vivo Marking of Primary CD4+ T Lymphocytes in Nonhuman Primates Using a Gibbon Ape Leukemia Virus-Derived Retroviral Vector
Authors: Bunnell Bruce A. , Metzger Mark , Byrne Ellen , Morgan Richard A. , Donahue Robert E.
Published: October 2019

High efficiency retroviral-mediated gene transfer to rhesus CD4+ peripheral blood lymphocytes (PBL) was accomplished using an optimized transduction protocol using a gibbon ape leukemia virus (GaLV) envelope-containing packaging cell line PG13. Engineered CD4+ PBL were administered to three nonmyeloablated animals in three or four separate infusions over 9 months. Polymerase chain reaction (PCR) demonstrated in vivo reconstitution of the genetically engineered CD4+ PBL at levels between 1% and 10% of the circulating leukocytes. This level of gene marking indicates that up to 30% of endogenous circulating CD4+ cells can be genetically engineered. The high levels of marked lymphocytes persist for the first 3 weeks following reinfusion then decline to ≤ 0.1% over the next 21 weeks. Lymph node (LN) biopsies were performed to determine if the engineered CD4+ lymphocytes could traffic to lymphoid tissues. Marked lymphocytes were detected in LN biopsies 100 days following reinfusion of the transduced cells. Expression of retroviral vector-derived sequences was detected by reverse transcriptase (RT)-PCR analysis from CD4-enriched lymphocytes that were activated by culturing in the presence of recombinant interleukin-2 (rIL-2). A humoral immune response to fetal bovine serum (FBS) was detected in all animals following the second administration of the culture expanded CD4+ lymphocytes. No antibody response was detected to the neomycin-resistance (NeoR) transgene, the murine retroviral group-specific antigen (gag), or GaLV envelope (env) proteins.
HLA Class II–Mediated Death Is Induced Via Fas/Fas Ligand Interactions in Human Splenic B Lymphocytes
Authors: Truman Jean-Philip , Choqueux Christine , Tschopp Jürg , Vedrenne Jocelyn , Le Deist Françoise , Charron Dominique , Mooney Nuala
Published: October 2019

HLA class II molecules, expressed on the surface of antigen-presenting cells, are responsible for the presentation of antigen-derived peptides to CD4+ helper T lymphocytes. Signaling via these molecules initiates the generation of second messengers leading to programed cell death (PCD) of activated B lymphocytes. The present study examined the mechanism of HLA class II–mediated apoptosis and describes the essential role of the molecule Fas and its ligand (FasL). FasL was expressed in B lymphocytes after stimulation via HLA class II or with phorbol esters. Expression of FasL protein was significantly increased in 50% of B lymphocytes after stimulation via HLA class II, and the level of FasL mRNA was also increased either by activation with phorbol esters and ionomycin or by signaling via HLA class II. Although HLA class II signaling did not change the expression of the Fas molecule, it did lead to increased sensitivity to Fas-mediated apoptosis. The crucial role of Fas/FasL interactions was confirmed by the absence of cell death via HLA class II in B cells lacking Fas expression, and by the significant inhibition of HLA class II–mediated apoptosis in the presence of either an antagonistic anti-Fas or anti-FasL antibody. These data demonstrate FasL expression on activated human B lymphocytes and support the idea that antigen presentation could contribute to the regulation of lymphocyte populations via Fas and FasL interactions.
Polyclonal B-Cell Lymphocytosis With Features Resembling Hairy Cell Leukemia-Japanese Variant
Authors: Machii Takashi , Yamaguchi Mitsuhiro , Inoue Ryoichi , Tokumine Yukihiro , Kuratsune Hirohiko , Nagai Hiroshi , Fukuda Shunichi , Furuyama Kazumichi , Yamada Osamu , Yahata Yoshito , Kitani Teruo
Published: October 2019

Polyclonal B lymphocytosis was found in four patients having clinical and hematologic features resembling those of hairy cell leukemia (HCL). All four patients were women between 37 and 67 years of age. Three patients had splenomegaly. Lymphadenopthy was absent or slight. Persistent lymphocytosis was seen in all the patients, and anemia and/or thrombopenia was observed in three of the patients. Abnormal lymphocytes have long microvilli and prominent membranous ruffles on their surfaces. Bone marrow aspirates and biopsy specimens showed increased numbers of abnormal lymphocytes with round nuclei and abundant pale cytoplasm. Although these findings were similar to those of HCL, studies of Ig gene rearrangements and expression showed the polyclonal proliferation of B cells. We called this new disease hairy B-cell lymphoproliferative disorder (HBLD). All four patients exhibited a polyclonal increase in serum IgG. The morphology of the cells in HBLD was more similar to that of leukemia cells of a variant form of HCL (HCL-Japanese variant) than to typical HCL cells. The surface IgG+, CD5−, CD11c+, CD22+, CD24−, CD25− phenotype and the weak tartrate-resistant acid phosphatase activity in the cells were identical to those of HCL cells of the Japanese variant. Our findings suggest that the B cells in HBLD are the nonmalignant counterpart of leukemic B cells in HCL-Japanese variant.
Epstein-Barr Virus Modulates 5-Lipoxygenase Product Synthesis in Human Peripheral Blood Mononuclear Cells
Authors: Gosselin Jean , Borgeat Pierre
Published: October 2019

The effect of short-term coincubations of Epstein-Barr virus (EBV) with mononuclear cells on the synthesis of leukotrienes (LT) by monocytes was investigated. Although treatment of mononuclear cells with EBV alone had no significant effect on LT synthesis by monocytes, the preincubation of mononuclear cells with EBV before the further stimulation of the cells with either the ionophore A23187, the chemoattractant formyl-Met-Leu-Phe, or the phagocytic particles zymosan strikingly enhanced the formation of both LTB4 and LTC4 above the levels of synthesis observed with the stimuli alone. Such priming effect of EBV on LT synthesis was maximal after 15 minutes of preincubation of mononuclear cells with EBV and slowly declined at longer preincubation times; the priming effect of EBV was observed both in Hank's Balanced Salt Solution and plasma. The effect of EBV was abolished by prior treatment of viral particles by heat or by antibody raised against the glycoprotein gp350 of the viral envelope, but not by UV irradiation of the viral particles. Exposure of mononuclear cells to EBV was shown to strongly enhance the activation of the 5-lipoxygenase and the release of arachidonic acid induced upon cell stimulation with a second agonist. The release of arachidonic acid by the EBV-treated mononuclear cells was inhibitable by arachidonyl trifluoromethyl ketone, an inhibitor of the 80-kD cytosolic phospholipase A2 . Furthermore, EBV was shown to rapidly increase (maximum effect within 15 minutes) the levels of phosphorylated form of the cytosolic phospholipase A2 (as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis), a process related to the activation of this enzyme. These data show that the interaction of EBV with monocytes upregulates the formation of important lipid mediators of inflammation.
Ceramide Inhibits IgG-Dependent Phagocytosis in Human Polymorphonuclear Leukocytes
Authors: Suchard Suzanne J. , Hinkovska-Galcheva Vania , Mansfield Pamela J. , Boxer Laurence A. , Shayman James A.
Published: October 2019

Ceramide is a product of agonist-induced sphingolipid metabolism in several cell types, including polymorphonuclear leukocytes (PMNs). In adherent PMNs, the kinetics of ceramide production correspond with the termination of fMLP-stimulated H2O2 release. Furthermore, short chain ceramides inhibit fMLP-mediated H2O2 release in adherent PMNs. In the present study, we investigated the effects of short chain ceramides and sphingoid bases on phagocytosis of IgG-opsonized erythrocytes (EIgG) by suspended PMNs activated with fMLP. N-Acetylsphingosine, N-acetylphytosphingosine, phytosphingosine, sphingosine, and dihydrosphingosine, but not N-acetyldihydrosphingosine, inhibited phagocytosis of EIgG. In contrast, these same lipids did not inhibit fMLP-mediated chemotaxis. Endogenous ceramide levels increased within the first few minutes of phagocytosis, with a significant (P < .05) accumulation by 30 minutes, the time by which phagocytosis was terminated. Neutral sphingomyelinase activity paralleled the increase in ceramide, consistent with the generation of ceramide by the hydrolysis of sphingomyelin. The N-acetyl-conjugated sphingols (C2 ceramides) blocked phosphatidylethanol formation indicating that phospholipase D (PLD) is an intracellular target of ceramide action. These data suggest that ceramides, generated through activation of the sphingomyelin cycle, act as negative regulators of FcγR-mediated phagocytosis.
Traditional Beer Consumption and the Iron Status of Spouse Pairs From a Rural Community in Zimbabwe
Authors: Moyo Victor M. , Gangaidzo Innocent T. , Gomo Z.A.R. , Khumalo Hlosukwazi , Saungweme Thokozile , Kiire C.F. , Rouault Tracey , Gordeuk Victor R.
Published: October 2019

To examine the relationship between dietary iron exposure through the consumption of traditional beer and the presence of iron overload in black Africans not related by birth, we studied 28 husband and wife pairs from a rural Zimbabwean community. Lifetime traditional beer consumption was estimated by questioning subjects and iron status was assessed by repeated measurements of serum ferritin and transferrin saturation in subjects who were fasting and had received vitamin C supplementation. Each of the 56 study subjects had an estimated lifetime traditional beer consumption <1,000 L. The mean ± standard deviation (SD) concentration of iron in the supernatants of nine samples of traditional beer from the community was 46 ± 10 mg/L. Four of 28 men (14.3%) and no women had the combination of an elevated serum ferritin and a transferrin saturation <70%, suggestive of substantial iron overload. Significant correlations were not found between the iron status of the husbands and their wives or between dietary iron exposure and iron stores. Our findings suggest that dietary iron exposure may not fully explain the development of iron overload in Africans and are consistent with the hypothesis that an iron-loading gene may also be implicated in pathogenesis.
Engraftment of Embryonic Hematopoietic Cells in Conditioned Newborn Recipients
Authors: Yoder Mervin C. , Hiatt Kelly
Published: October 2019

Yolk sac hematopoiesis is characterized by restricted hematopoietic cell differentiation. Although multipotent hematopoietic progenitor cells have been identified in the early yolk sac, long-term multilineage repopulating (LTMR) hematopoietic stem cell (HSC) activity has not been demonstrable before day 11 postcoitus (PC) using standard transplantation assays. In the present study, day-10 PC yolk sac hematopoietic cells were infused into myeloablated congenic newborn pups and donor cell engraftment and multilineage reconstitution of peripheral blood cells for at least 11 months in primary recipients was observed. In contrast, transplantation of day-10 PC yolk sac cells into congenic adult recipients did not result in engraftment despite pretransplant conditioning of the recipients or use of recipients that were genetically deficient in stem cells. Although fresh yolk sac cells were incapable of reconstitution when injected into adult recipient mice, yolk sac donor-derived cells residing in the bone marrow of primary newborn transplant recipients were capable of efficient reconstitution of conditioned secondary recipient adult mice. Primary newborn and secondary adult recipient animals engrafted with yolk sac cells were observed to have normal peripheral blood white blood cell counts. Lymphocyte subsets in peripheral blood, thymus, and spleen were also similar to control animals. The distribution and frequency of lineage-restricted progenitors derived from bone marrow of secondary transplant recipients were normal. These results indicate that day-10 PC yolk sac HSCs are capable of engrafting and reconstituting the hematopoietic system of conditioned newborn but not adult recipient animals. Furthermore, the ability of the yolk sac HSCs to differentiate into all hematopoietic lineages in these recipients strongly suggests that the local cellular microenvironment plays a prominent role in regulating yolk sac HSC differentiation.
Radiotherapy-Associated Neutropenia and Thrombocytopenia: Analysis of Risk Factors and Development of a Predictive Model
Authors: Mac Manus Michael , Lamborn Kathleen , Khan Waqqar , Varghese Anna , Graef Lorin , Knox Susan
Published: October 2019

Risk factors for unscheduled interruptions in radiotherapy courses completed between June 1989 and August 1995, lasting ≥2 days, and associated with World Health Organization grade III-IV neutropenia or thrombocytopenia were studied retrospectively. A group of controls was randomly selected. Potential risk factors for myelosuppression were analyzed using univariate and multivariate analyses. The most important risk factors for treatment interruption with thrombocytopenia were concurrent chemotherapy (odds ratio [OR], 45.5; P < .001), increasing percentage of marrow irradiated (OR, 4.1 for each 20%; P < .001), and brain metastases (OR, 7.3; P = .01). Other significant (P < .05) factors were leukemia/lymphoma, bone or bone marrow metastases, and prior chemotherapy. The most important risk factors for treatment interruptions with neutropenia were concurrent chemotherapy (OR, 42.1; P < .001) and increasing percentage of marrow irradiated (OR, 3.3 for each 20%; P < .001). Similarly, the most important risk factors for treatment interruptions with both thrombocytopenia and neutropenia were concurrent chemotherapy (OR, 48.6; P < .001) and increasing percentage of marrow irradiated (OR, 3.9 for each 20%; P < .001). Other significant (P < .05) factors in these groups were bone marrow or brain metastases and previous chemotherapy. These data were used to create a model, assigning patients to groups at high, intermediate, or low risk for treatment interruption with thrombocytopenia. High-risk patients may be candidates for clinical trials of a platelet growth factor.
Host Conditioning With 5-Fluorouracil and kit-Ligand to Provide for Long-Term Bone Marrow Engraftment
Authors: van Os Ronald , Dawes Donald , Mislow John M.K. , Witsell Alice , Mauch Peter M.
Published: October 2019

Administration of kit-ligand (KL) before and after doses of 5-fluorouracil (5-FU) results in marrow failure in mice, presumably because of enhanced KL-induced cycling of stem cells, which makes them more susceptible to the effects of 5-FU. In attempt to capitalize on this effect on stem cells, we studied the ability of KL and 5-FU to allow stable donor engraftment of congenically marked marrow in a C57BL/6 (B6) mouse model. KL was administered subcutaneously at 50 μg/kg, 21 hours and 9 hours before and 3 hours after each of two doses of 5-FU (125 mg/kg) given 7 days apart to B6-recipients. Animals then received three injections of 107 congenic B6-Gpi-1a-donor bone marrow cells at 24, 48, and 72 hours after the second 5-FU dose. A separate group of animals received a single dose of either 1 × 107 or 3 × 107 donor marrow cells 24 hours after the last 5-FU dose. The level of engraftment was measured from Gpi-phenotyping at 1, 3, 6, and 8 months in red blood cells (RBCs) and at 8 months by phenotyping cells from the thymus, spleen, and marrow. Percent donor engraftment in RBCs appeared stable after 6 months. The percent donor engraftment in RBCs at 8 months was significantly higher in KL + 5-FU prepared recipients (33.0 ± 2.7), compared with 5-FU alone (18.5 ± 2.6, P < .0005), or saline controls (17.8 ± 1.7, P < .0001). In an additional experiment, granulocyte colony-stimulating factor (100 μg/dose) was added to a reduced dose of KL (12.5 μg/dose); engraftment was similar to KL alone. At 8 months after transplantation the levels of engraftment in other tissues such as bone marrow, spleen, and thymus correlated well with erythroid engraftment to suggest that multipotent long-term repopulating stem cells had engrafted in these animals. There are concerns for the toxicity of total body irradiation (TBI)- or busulfan-based regimens in young recipients of syngeneic or transduced autologous marrow who are transplanted for correction of genetic disease. In these recipients complete donor engraftment may not be needed. The results with KL and 5-FU are encouraging for the further refinement of non-TBI, nonbusulfan techniques to achieve stable mixed chimerism.
The Src Family Kinases, Fgr, Fyn, Lck, and Lyn, Colocalize With Coated Membranes in Platelets
Authors: Stenberg Paula E. , Pestina Tamara I. , Barrie Rosemary J. , Jackson Carl W.
Published: October 2019

Nonreceptor protein tyrosine kinases phosphorylate proteins, thereby activating many intracellular signaling pathways and mediating protein-protein interactions. Protein phosphorylation is regulated in large part by the subcellular localization of these kinases and their respective substrates. Src is the most studied of these kinases, although other members of the Src family have been shown to be important in the differentiation of specific cell types. Src and Src family members are reported to be membrane-associated, but detergent-extraction studies have demonstrated a major difference in the solubility of Src compared with other members of the Src family (Fgr, Fyn, Lck, Lyn, and Yes), suggesting that their subcellular distributions may be different. By immunoelectron microscopy, we demonstrate that, unlike Src, the Src-related kinases are associated with electron-dense cytoplasmic domains and plasma membrane domains that correspond in size and frequency to endocytotic vesicles and coated pits. Clusters of labeling for these kinases also were seen adjacent to granule membranes. These kinases colocalize with the coated vesicle protein, clathrin, confirming their association with this class of endocytotic vesicle. We hypothesize that this vesicular association of Src-related kinases indicates a role for them in the endocytotic vesicle–mediated uptake and trafficking of plasma proteins into platelet granules.
Upregulation of Intracellular Glutathione by Fibroblast-Derived Factor(s): Enhanced Survival of Activated T Cells in the Presence of Low Bcl-2
Authors: Hyde Helena , Borthwick Nicola J. , Janossy George , Salmon Michael , Akbar Arne N.
Published: October 2019

Activated interleukin-2 (IL-2)–dependent T cells express high levels of Bcl-2 protein. On cytokine withdrawal, Bcl-2 expression decreases and the cells die rapidly by apoptosis. We have previously shown that the survival of IL-2–deprived T cells can be promoted by factor(s) secreted by fibroblasts. Here we report that reduced glutathione (GSH), but not its oxidized counterpart GSSG, also enhances the in vitro survival of these cells. Exogenous GSH mediates its effect intracellularly, as (1) endogenous glutathione concentrations are increased up to fivefold in the presence of GSH, and (2) acivicin, an inhibitor of transmembrane GSH transport, abrogates GSH-dependent survival. The GSH-rescued T cells do not proliferate and express only low levels of Bcl-2, resembling WI38 fibroblast-rescued T cells. We, therefore, investigated a role for GSH in fibroblast-promoted T-cell survival. We show that WI38-promoted survival results in elevated GSH levels in surviving T cells and is abrogated by buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Furthermore, both WI38-promoted T-cell survival and GSH upregulation are associated with large molecular weight molecules (<30 kD). Thus, the upregulation of GSH by WI38 fibroblasts appears to be crucial in their ability to enhance the survival of cytokine-deprived activated T cells in vitro.