bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential Dissemination of B-Cell Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma to the Splenic Marginal Zone
Authors: Du Ming-Qing , Peng Huai-Zheng , Dogan Ahmet , Diss Tim C. , Liu Haiqun , Pan Lang-Xing , Moseley Robin P. , Briskin Michael J. , Chan John K.C. , Isaacson Peter G.
Published: October 2019

The tendency for gastric mucosa-associated lymphoid tissue (MALT) lymphoma cells preferentially to localize around reactive B-cell follicles, both in the mucosa and regional lymph nodes, coupled with their immunophenotype, has led to the proposal that the normal cell counterpart of this lymphoma is the marginal zone B cell. In keeping with this proposition, lymphocytes expressing the lymphoma idiotype have been detected in the splenic marginal zone in a single case of gastric MALT lymphoma. To confirm that this truly represented preferential homing of MALT lymphoma to the splenic marginal zone, we have now re-examined this case, together with 17 other cases, using both immunohistochemical and molecular methods in an attempt to establish clonal identity between the gastric lymphoma and cells in the splenic marginal zone. In three cases, the spleen was characterized by marked expansion of marginal zones by cells showing the same pattern of Ig light chain restriction as the gastric lymphoma. None of the remaining 15 cases showed histologic evidence of lymphomatous infiltration. Analysis of the Ig genes by polymerase chain reaction (PCR), cloning, and sequencing confirmed clonal identity between the splenic marginal zone infiltrates and the gastric lymphoma in the histologically involved cases. Amplifiable DNA could be extracted from only 5 of the remaining 15 cases. In 3 of these cases, including the case previously studied using an anti-idiotype, involvement of the splenic marginal zone could be confirmed using microdissection and clone-specific PCR. No involvement could be detected in the remaining 2 cases. In addition, we have shown that mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the primary homing receptor of gut-mucosa for lymphocytes, was strongly expressed by the sinus lining cells of the splenic marginal zone. These results provide strong evidence for preferential involvement of the marginal zone when gastric MALT lymphomas disseminate to the spleen, which is in keeping with the notion that the marginal zone B cells are the normal counterparts of MALT lymphoma cells.
Defects in Yolk Sac Hematopoiesis in Mll-Null Embryos
Authors: Hess Jay L. , Yu Benjamin D. , Li Bin , Hanson Rob , Korsmeyer Stanley J.
Published: October 2019

Translocations involving the mixed lineage leukemia gene (MLL ), the human homolog of the Drosophila gene trithorax, are one of the most common genetic alterations in human acute leukemias. Each translocation involving MLL results in loss of one functional copy of MLL and the generation of a chimeric fusion protein with potential dominant negative or neomorphic activity. Mll is a positive regulator of Hox genes, which have been implicated in both axial skeleton patterning and hematopoietic development. Previous studies indicated that Hox gene expression is altered in Mll heterozygous (+/−) and homozygous (−/−) deficient mice. To study the role of Mll in hematopoiesis and to obtain insights into leukemogenesis, we have examined the effects of haplo-insufficiency or absence of Mll by in vitro differentiation of Mll +/+, +/−, and −/− yolk sac progenitor cells. Mll −/− colonies were fewer in number, took longer to develop, and contained fewer cells than their wild-type and heterozygous counterparts. Formation of colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), colony-forming unit-macrophage (CFU-M), and burst-forming unit-erythroid (BFU-E) was markedly decreased in Mll −/− cultures, while numbers of colony-forming unit-erythroid (CFU-E), colony-forming unit-granulocyte (CFU-G), and colony-forming unit-granulocyte macrophage (CFU-GM) were essentially unaffected. Despite the decreased numbers of colonies present, Mll −/− cultures showed all cell types without morphologic evidence of maturation arrest. These studies indicate that Mll is required for normal numbers of hematopoietic progenitors and their proper differentiation, especially along the myeloid and macrophage pathways.
Regulation of the Serum Concentration of Thrombopoietin in Thrombocytopenic NF-E2 Knockout Mice
Authors: Shivdasani Ramesh A. , Fielder Paul , Keller Gilbert-Andre , Orkin Stuart H. , de Sauvage Frederic J.
Published: October 2019

The mechanisms that regulate circulating levels of thrombopoietin (Tpo) are incompletely understood. According to one favored model, the rate of Tpo synthesis is constant, whereas the serum concentration of free Tpo is modulated through binding to c-Mpl receptor expressed on blood platelets. Additionally, a role for c-Mpl expressed on megakaryocytes is suggested, particularly by the observation that serum Tpo levels are not elevated in human immune thrombocytopenic purpura. Whereas direct binding of Tpo to platelets has been demonstrated in vitro and in vivo, the role of megakaryocytes in modulating serum Tpo levels has not been addressed experimentally. The profoundly thrombocytopenic mice lacking transcription factor p45 NF-E2 do not show the predicted increase in serum Tpo concentration. To evaluate the fate of the ligand in these animals, we injected 125I-Tpo intravenously into mutant and control mice. In contrast to normal littermates, NF-E2 knockout mice show negligible association of radioactivity with blood cellular components, consistent with an absence of platelets. There is no corresponding increase in plasma-associated radioactivity to suggest persistence in the circulation. However, a greater fraction of the radioligand is bound to hematopoietic tissues. In the bone marrow this is detected virtually exclusively in association with megakaryocytes, whereas in the spleen it is associated with megakaryocytes and small, abnormal, platelet-like particles or megakaryocyte fragments that are found within or in close contact with macrophages. These findings implicate the combination of megakaryocytes and the latter particles as a sink for circulating Tpo in NF-E2 knockout mice, and provide an explanation for the lack of elevated serum Tpo levels in this unique animal model of thrombocytopenia.
Recombinant RFB4 Immunotoxins Exhibit Potent Cytotoxic Activity for CD22-Bearing Cells and Tumors
Authors: Mansfield Elizabeth , Amlot Peter , Pastan Ira , FitzGerald David J.
Published: October 2019

Many B-cell malignancies express the CD22 antigen on their cell surface. To kill cells expressing this antigen, the RFB4 monoclonal antibody (MoAb) has been linked chemically with either deglycosylated ricin A chain or truncated versions of Pseudomonas exotoxin. These immunotoxins exhibited selective cytotoxic activity for CD22+ cells and antitumor activity in nude mouse models bearing human B-cell lymphomas. To construct a recombinant immunotoxin targeted to CD22, we first cloned the variable portions of the heavy and light chains of RFB4. The cloned Fv fragments were joined by a newly created disulfide bond to form a disulfide stabilized (ds) construct. The RFB4 construct was combined by gene fusion with PE38, a truncated version of PE. The recombinant immunotoxin was then expressed in Escherichia coli, purified by column chromatography and tested for cytotoxicity activity. RFB4(dsFv)PE38 retained its binding activity for CD22, was very stable at 37°C and exhibited selective cytotoxic activity for CD22+-cultured cell lines. Because of its favorable binding characteristics and potency for CD22-positive cell lines, RFB4(dsFv)PE38 was tested for antitumor activity in a nude mouse model of human lymphoma. CA46 cells were injected subcutaneously and then treated with the RFB4(dsFv)PE38 immunotoxin. Antitumor activity was dose responsive and was not evident when an irrelevant immunotoxin was administered on the same schedule.
Interleukin-1β Secretion Is Impaired by Inhibitors of the Atp Binding Cassette Transporter, ABC1
Authors: Hamon Yannick , Luciani Marie-Françoise , Becq Frederic , Verrier Bernard , Rubartelli Anna , Chimini Giovanna
Published: October 2019

The production of interleukin-1β (IL-1β), a powerful mediator of inflammation, is tightly regulated at several levels. However, in some pathologic conditions, a pharmacologic treatment is required to control the toxicity of excessive extracellular IL-1β. Because of the heavy side effects of most therapies used in IL-1β–mediated pathologies, a goal of pharmacologic research is the development of selective anti–IL-1β drugs. We show here that the sulfonylurea glyburide, currently used in the oral therapy of noninsulin dependent diabetes, is an inhibitor of IL-1β secretion from human monocytes and mouse macrophages. Glyburide reduces dramatically the recovery of extracellular 17-kD IL-1β in the absence of toxic effects on the cells and without affecting the synthesis or processing of the IL-1β precursor. IL-1β belongs to the family of leaderless secretory proteins released from the cell by a nonclassical secretory route. In bacteria and yeast Atp binding cassette (ABC) transporters are involved in the secretion of leaderless secretory proteins. Interestingly, glyburide blocks the anion exchanger function of ABC1, a mammalian member of the family of ABC transporters. We thus investigated the involvement of ABC1 in IL-1β secretion, through the analysis of the effects of drugs known to inhibit IL-1β secretion, on the activity of ABC1 and in turn the ability of known inhibitors of ABC1 of blocking IL-1β secretion. Our data show that IL-1β secretion and the function of ABC1 as an anion exchanger are sensitive to the same drugs, therefore suggesting an involvement of the ABC1 transporter in the secretion of leaderless proteins in mammals.
Bcl-2 Targeted Overexpression Into the Erythroid Lineage of Transgenic Mice Delays But Does Not Prevent the Apoptosis of Erythropoietin-Deprived Erythroid Progenitors
Authors: Lacronique V. , Varlet P. , Mayeux P. , Porteu A. , Gisselbrecht S. , Kahn A. , Lacombe C.
Published: October 2019

Erythropoietin (Epo) is known to control the erythroid developmental program through various biologic activities including maintenance of viability, cell proliferation, and/or cell maturation. In vitro experiments showed massive apoptosis in cultures of Epo-deprived colony-forming unit-erythroid (CFU-E) progenitors, demonstrating the Epo requirement of late-stage erythroid progenitors for survival. Based on these data, a model has been proposed whereby from CFU-E to proerythroblast stages, Epo acts rather as a survival factor than a proliferating factor. To investigate the relationship between Epo dependence and apoptotic mechanisms, we generated transgenic mice expressing the antiapoptotic human bcl-2 gene product in erythroid progenitors. Transgenic animals developed without any evidence of erythropoietic disorders. In vitro studies showed that overexpression of bcl-2 in erythroid progenitors delayed, but did not prevent the loss of CFU-E from Epo-deprivation. By measuring burst-forming unit-erythroid (BFU-E) and CFU-E–derived colonies, an enhanced sensitivity to low levels of Epo was demonstrated in adult bone marrow of transgenic mice with respect to nontransgenic animals. No spontaneous erythroid colonies were, however, observed in vitro in the absence of the cytokine, indicating that overexpression of bcl-2 is not sufficient to induce by itself a complete erythroid differentiation. Taken together, our data indicate that targeted erythroid overexpression of bcl-2 fails to alter the normal erythropoietic development in vivo and that erythroid progenitors remain strictly dependent on Epo for their survival.
Fibronectins Are Essential for Heart and Blood Vessel Morphogenesis But Are Dispensable for Initial Specification of Precursor Cells
Authors: George Elizabeth L. , Baldwin H. Scott , Hynes Richard O.
Published: October 2019

The underlying mechanisms of lethal cardiovascular defects associated with the fibronectin-null (FN.null) mutation in mouse embryos were investigated by lineage analysis of myocardial, endocardial, and endothelial cells. A wide variation in phenotype was observed on two genetic backgrounds. In the less severe class (C57/BL6 background), FN.null embryos display a defective heart. Myocardial cells express the specific marker MF-20 and are correctly localized in the anterior trunk region, but myocardial organization is disrupted, resulting in a bulbous heart tube. Endocardial cells express the specific marker platelet-endothelial cell adhesion molecule-1 (PECAM-1) and are localized within the myocardium, but the endocardium appears collapsed. Endothelial cells of two vascular beds are specified, but the aortae are distended and lack contact with the surrounding mesenchyme, while no vessels form in the yolk sac. Defects in the more severe class suggest that FNs are essential earlier in development on the 129/Sv background. Myocardial and endocardial cells are specified, but morphogenesis of the myocardium and endocardium does not occur. Aortic endothelial cells are specified and localized normally, but remain scattered. Yolk sac endothelial cells resemble those of the less severe class. We conclude that FNs are essential for organization of heart and blood vessels, but are dispensable for cellular specification in the appropriate regions within the embryo.
Cross-Resistance of CD95- and Drug-Induced Apoptosis as a Consequence of Deficient Activation of Caspases (ICE/Ced-3 Proteases)
Authors: Los Marek , Herr Ingrid , Friesen Claudia , Fulda Simone , Schulze-Osthoff Klaus , Debatin Klaus-Michael
Published: October 2019

The cytotoxic effect of anticancer drugs has been shown to involve induction of apoptosis. We report here that tumor cells resistant to CD95 (APO-1/Fas) -mediated apoptosis were cross-resistant to apoptosis-induced by anticancer drugs. Apoptosis induced in tumor cells by cytarabine, doxorubicin, and methotrexate required the activation of ICE/Ced-3 proteases (caspases), similarly to the CD95 system. After drug treatment, a strong increase of caspase activity was found that preceded cell death. Drug-induced activation of caspases was also found in ex vivo-derived T-cell leukemia cells. Resistance to cell death was conferred by a peptide caspase inhibitor and CrmA, a poxvirus-derived serpin. The peptide inhibitor was effective even if added several hours after drug treatment, indicating a direct involvement of caspases in the execution and not in the trigger phase of drug action. Drug-induced apoptosis was also strongly inhibited by antisense approaches targeting caspase-1 and -3, indicating that several members of this protease family were involved. CD95-resistant cell lines that failed to activate caspases upon CD95 triggering were cross-resistant to drug-mediated apoptosis. Our data strongly support the concept that sensitivity for drug-induced cell death depends on intact apoptosis pathways leading to activation of caspases. The identification of defects in caspase activation may provide molecular targets to overcome drug resistance in tumor cells.
Human T-Cell Leukemia Virus Type I Tax Transactivates the Promoter of Human Prointerleukin-1β Gene Through Association With Two Transcription Factors, Nuclear Factor–Interleukin-6 and Spi-1
Authors: Tsukada Junichi , Misago Masahiro , Serino Yoko , Ogawa Ryosuke , Murakami Syuichi , Nakanishi Masatsugu , Tonai Shinichi , Kominato Yoshihiko , Morimoto Isao , Auron Philip E. , Eto Sumiya
Published: October 2019

The human T-cell leukemia virus type I (HTLV-I), which infects a wide variety of mammalian cells including monocytes and macrophages, encodes a transactivating protein designated as Tax. We now report that Tax induces the human prointerleukin-1β (IL1B) gene promoter in monocytic cells. In our transient transfection assays using human THP-1 monocytic cells, a chloramphenicol acetyltransferase (CAT) construct containing the IL1B promoter sequence between positions −131 and +12 showed an approximately 90-fold increase in activity following cotransfection of a Tax expression vector. Moreover, Tax synergized with lipopolysaccharide (LPS) to induce the IL1B promoter activity. Analyses of specific nucleotide substitutions further indicated that the Tax-induced transcriptional activation requires two transcription factor binding motifs within the IL1B promoter; one is a binding site for nuclear factor (NF)-IL6 (CCAAT/enhancer binding protein β, C/EBPβ), which belongs to the basic region-leucine zipper (bZIP) family and the other for Spi-1 (PU.1), which is an Ets family protein found principally in monocytes, macrophages, and B lymphocytes. In electrophoretic mobility shift assays (EMSA) using in vivo THP-1 nuclear extracts, Tax expression in THP-1 monocytic cells significantly increased binding of the two factors to their target IL1B promoter sequences. However, in contrast to NF-IL6 and Spi-1, DNA binding activity of Oct-1, an ubiquitously expressed octamer-binding protein was not affected by Tax. Additional EMSA using in vitro translated proteins also showed that recombinant Tax enhances DNA binding of both of recombinant NF-IL6 and Spi-1 proteins. These data were supported by our glutathione S-transferase (GST)-pulldown data, which indicated that Tax physically interacts with the two proteins. Based on the results obtained from the present study, we conclude that the IL1B promoter is a Tax-responsive sequence as a result of ability of Tax to induce binding of NF-IL6 and Spi-1 to the IL1B promoter sequence through protein-protein interaction.
Stimulation of Hematopoiesis by Amifostine in Patients With Myelodysplastic Syndrome
Authors: List Alan F. , Brasfield Farah , Heaton Ruth , Glinsmann-Gibson Betty , Crook Linda , Taetle Raymond , Capizzi Robert
Published: October 2019

The aminothiol, amifostine (Ethyol; U.S. Bioscience, West Conshohocken, PA), is a cytoprotective agent that ameliorates the toxicities of anticancer therapy. In vitro, amifostine promotes the formation and survival of primitive hematopoietic progenitors derived from myelodysplastic bone marrow (BM) specimens. To evaluate the hematological effects of amifostine, 18 patients with myelodysplastic syndrome (MDS) and one or more refractory cytopenias received treatment with amifostine in a Phase I/II study. Four cohorts received intravenous treatment with 100, 200, or 400 mg/m2 amifostine three times a week, or 740 mg/m2 weekly for three consecutive weeks followed by 2 weeks observation. Nonresponding patients received a second course of therapy at the next higher dose level depending upon drug tolerance. Bone marrow (BM) progenitor growth was assessed before treatment and after day 21. Diagnoses included refractory anemia (7), refractory anemia with ringed sideroblasts (5), refractory anemia with excess blasts (RAEB) (4), and RAEB-in transformation (RAEB-t) (2). Single- or multi-lineage hematologic responses occurred in 15 patients (83%) treated with the three-times-a-week dose schedule. Fourteen patients had a 50% or greater increase in absolute neutrophil count with amifostine treatment (range, 426 to 11,348/μL). Platelet count increased in 6 (43%) of 14 patients with thrombocytopenia (absolute increase, 16,000 to 110,000/μL), and 5 of 15 red blood cell transfusion-dependent patients had a 50% of greater reduction in transfusion needs. Assayable hematopoietic progenitors increased in 13 of 15 evaluable patients; including CFU-GEMM (12), BFU-E (8), and CFU-GM (6). Amifostine doses less than or equal to 200 mg/m2 were well tolerated, whereas grade II nausea, vomiting, and fatigue was limiting at higher doses. Three patients with excess blasts before enrollment experienced an increase in BM blast percentage and two patients had evolution to acute leukemia that persisted after treatment withdrawal. We conclude that amifostine administered at doses ≤200 mg/m2 three times a week is well tolerated and has hematologic activity in patients with MDS.
Matrix Glycoprotein SC1/ECM2 Augments B Lymphopoiesis
Authors: Oritani Kenji , Kanakura Yuzuru , Aoyama Keisuke , Yokota Takafumi , Copeland Neal G. , Gilbert Debra J. , Jenkins Nancy A. , Tomiyama Yoshiaki , Matsuzawa Yuji , Kincade Paul W.
Published: October 2019

The extracellular matrix produced by stromal cells plays a critical role in lympho-hematopoiesis. It was recently discovered that matrix glycoprotein SC1/ECM2 is a component of that matrix and preliminary evidence suggested that it could contribute to the nurturing environment for B-lymphocyte precursors. A fusion protein prepared from the amino terminal portion of SC1/ECM2 and the constant region of human Ig preferentially bound to pre-B cells. Furthermore, the cloning efficiency of interleukin-7–dependent B-cell precursors was increased in a dose-dependent manner by addition of this fusion protein. We now report the complete cDNA sequence for murine SC1/ECM2 and its localization to the central region of chromosome 5. A fusion protein prepared from the full length of SC1/ECM2 and Ig was found to recognize pre-B cells in a divalent cation-dependent manner, and to augment mitogen-dependent proliferation of mature B cells, as well as the cloning of pre-B cells, but to have no influence on myeloid progenitor cells. Although SC1/ECM2 is normally a secreted protein, we show that it is also capable of augmenting lymphopoiesis when expressed as a transmembrane protein on fibroblasts. Although the C-terminal portion of SC1/ECM2 has sequence homology to osteonectin/SPARC, the unique N-terminal one fifth of the protein was sufficient to augment lymphocyte growth.
Characterization of Stromal Progenitor Cells Enriched by Flow Cytometry
Authors: Zohar Ron , Sodek Jaro , McCulloch Christopher A.G.
Published: October 2019

The progenitors for cells of bone, cartilage, fat, and muscle are thought to be derived from mesenchymal stem cells but despite extensive study of stromal cell differentiation, neither mesenchymal stem cells or the more committed, tissue-specific progenitors have been well-characterized. In this study we used flow cytometry to isolate from fetal rat periosteum a population of small, slowly cycling cells with low cytoplasmic granularity (S cells) that display stem cell characteristics. On plating, S cells exhibited a 90% higher labeling index with [3H]-thymidine compared to unsorted cells and when grown in culture generated cartilage, adipocyte, and smooth muscle phenotypes, in addition to bone. Only the S-cell population showed extensive self-renewal of cells with osteogenic potential. Electron microscopy showed that S cells have high nuclear:cytoplasmic ratios with large condensed nuclei and a paucity of cytoplasmic organelles. Freshly sorted suspensions of immunocytochemically stained S cells did not express differentiation-associated markers such as type I, II, and III collagens, alkaline phosphatase, or osteopontin. However, after attachment, S cells became immunopositive for collagens I, II, III, osteopontin, and also for the cell surface receptor CD44, which mediates cell attachment to hyaluronan and osteopontin. These studies show that viable osteogenic precursor cells with the stem cell characteristics of self-renewal, high proliferative capacity, and multipotentiality can be enriched from heterogeneous stromal cell populations with simple flow cytometric methods. These cells may be useful for regeneration of stromal tissues.
Molecular Mimicry Between the Rabies Virus Glycoprotein and Human Immunodeficiency Virus-1 GP120: Cross-Reacting Antibodies Induced by Rabies Vaccination
Authors: Bracci Luisa , Ballas Samir K. , Spreafico Adriano , Neri Paolo
Published: October 2019

The 160-170 sequence of human immunodeficiency virus (HIV)-1 gp120 mimics a nicotinic receptor-binding motif of rabies virus glycoprotein and snake neurotoxins. This sequence has been proposed to be involved in the binding of HIV-1 gp120 to the acetylcholine binding sites of nicotinic receptors. By using biomolecular interaction analysis (BIA) technology we have found that HIV-1 gp120 can bind to detergent-extracted nicotinic receptor from fetal calf muscle. The binding is inhibited by nicotine and by a synthetic peptide reproducing the gp120 160-170 sequence. The molecular mimicry between gp120 and rabies virus glycoprotein is confirmed by cross-reacting antibodies. We have found that vaccination against rabies can induce the production of anti–HIV-1 gp120 antibodies in humans. The cross-reacting antibodies are directed to the gp120 sequence involved in the mimicry with the rabies virus glycoprotein. The cross-reactivity between the rabies virus and HIV-1 has important implications in transfusion medicine. Moreover, the presence of cross-reacting antibodies between the nicotinic receptor binding site of rabies virus glycoprotein and a fragment of HIV-1 gp120 strengthens the hypothesis about the possible role of nicotinic receptors as potential receptors for HIV-1 in the central nervous system.
Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo
Authors: Fehniger Todd A. , Carson William E. , Mrózek Ewa , Caligiuri Michael A.
Published: October 2019

The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.
Several Cytogenetic Subclones May Be Identified Within Plasma Cells From Patients With Monoclonal Gammopathy of Undetermined Significance, Both at Diagnosis and During the Indolent Course of This Condition
Authors: Zandecki Marc , Laı̈ Jean-Luc , Geneviève Franck , Bernardi Franck , Volle-Rémy Hélène , Blanchet Odile , François Michel , Cosson Alain , Bauters Francis , Facon Thierry
Published: October 2019

Monoclonal gammopathy of undetermined significance (MGUS) is a frequent condition in patients over 50 years old, that ultimately leads to multiple myeloma (MM) in 20% of patients after 20 to 35 years of follow-up. Little is known about cytogenetic changes associated with this condition. We studied 19 MGUS patients both at diagnosis and after 12 to 35 months of follow-up (mean = 26), using DNA content measurement of bone marrow plasma cells (BMPC), and a new interphase fluorescence in situ hybridization technique (FISH) allowing the simultaneous identification of monotypic BMPC (fluorescent anti light-chain antibodies) and the determination of the number of copies for two different chromosomes within the same PC nucleus (one biotin-labeled probe coupled next to texas red avidin and one FITC-labeled probe). At diagnosis of the MGUS, single interphase FISH showed at least one numeric chromosome change in 13 of 19 patients, after the use of centromeric probes directed against chromosomes no. 3, no. 7, no. 9, and no. 11. At follow-up, abnormalities found at diagnosis in 13 patients were still shown. Moreover, abnormalities occurred in three of the last six patients (trisomy for one to three different chromosomes), although no patient evolved into MM. Dual interphase FISH showed that some BMPC bore numeric changes with both probes tested whereas other BMPC bore abnormality with only one of the probes tested. In patients who showed trisomy for at least three different chromosomes, distribution of numeric changes within BMPC defined significant numbers of up to seven different BMPC clones. All these various clones were shown both at diagnosis and at follow-up. In every patient, these various clones differed only for the number of abnormalities they exhibited, and could be related to each other in a model of gradual acquisition of chromosome changes. Eventually, data reported here show that MGUS patients acquire slowly, gradually, but ineluctably chromosome changes, distributed within several related subclones. However, these changes are not related to transformation into MM: among the various clones coexisting within the same patient, a peculiar change, still to demonstrate, might develop and lead to overt MM.
AF6q21, a Novel Partner of the MLL Gene in t(6; 11)(q21; q23), Defines a Forkhead Transcriptional Factor Subfamily
Authors: Hillion Josette , Le Coniat Maryvonne , Jonveaux Philippe , Berger Roland , Bernard O.A.
Published: October 2019

Fusion genes implicating the MLL gene have been recently demonstrated in various 11q23 chromosomal abnormalities in human hematopoietic malignancies. We analyzed a t(6; 11)(q21; q23) translocation detected in a secondary acute myeloblastic leukemia. This translocation results in fusion of the MLL gene on 11q23 to a previously unknown gene on chromosome 6 that differs from the previously reported MLL partner gene AF6q. The novel gene, named AF6q21, encodes a forkhead (FH) protein with strong similarities to the two FH family members whose genes are already known to be involved in chromosomal translocations of human malignancies, AFX and FKHR. Strikingly, in these translocations the breakpoints are located at the same position within the FH domains. Therefore, AF6q21, AFX, and FKHR could define a new FH subfamily particularly involved in human malignancies.
Diphtheria Toxin Fused to Granulocyte-Macrophage Colony-Stimulating Factor Eliminates Acute Myeloid Leukemia Cells With the Potential to Initiate Leukemia in Immunodeficient Mice, But Spares Normal Hemopoietic Stem Cells
Authors: Terpstra Wim , Rozemuller Henk , Breems Dimitri A. , Rombouts Elwin J.C. , Prins Arie , FitzGerald David J.P. , Kreitman Robert J. , Wielenga Jenne J. , Ploemacher Rob E. , Löwenberg Bob , Hagenbeek Anton , Martens Anton C.M.
Published: October 2019

We studied the cell kill induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) fused to Diphtheria Toxin (DT-GM-CSF ) in acute myeloid leukemia (AML) samples and in populations of normal primitive hemopoietic progenitor cells. AML samples from three patients were incubated in vitro with 100 ng/mL DT-GM-CSF for 48 hours, and AML cell kill was determined in a proliferation assay, a clonogenic assay colony-forming unit-AML (CFU-AML) and a quantitative long-term bone marrow (BM) culture ie, the leukemic-cobblestone area forming cell assay (L-CAFC). To measure an effect on cells with in vivo leukemia initiating potential DT-GM-CSF exposed AML cells were transplanted into immunodeficient mice. In two out of three samples it was shown that all AML subsets, including those with long-term abilities in vivo (severe combined immunodeficient mice) and in vitro (L-CAFC assay) were reduced in number by DT-GM-CSF. Cell kill induced by DT-GM-CSF could be prevented by coincubation with an excess of GM-CSF, demonstrating that sensitivity to DT-GM-CSF is specifically mediated by the GM-CSF receptor. Therefore, binding and internalization of GM-CSF probably occur in immature AML precursors of these two cases of AML. The third AML sample was not responsive to either GM-CSF or DT-GM-CSF. The number of committed progenitors of normal bone marrow (burst-forming unit-erythroid, colony-forming unit granulocyte- macrophage, and cobble stone area forming cell [CAFC] week 2) and also the number of cells with long-term repopulating ability, assayed as week 6 CAFC, were unchanged after exposure to DT-GM-CSF (100 ng/mL, 48 hours). These studies show that DT-GM-CSF may be used to eliminate myeloid leukemic cells with long-term potential in vitro and in immunodeficient mice, whereas normal hemopoietic stem cells are spared.
Constraints on CD4 Recovery Postchemotherapy in Adults: Thymic Insufficiency and Apoptotic Decline of Expanded Peripheral CD4 Cells
Authors: Hakim Frances T. , Cepeda Rosemarie , Kaimei Song , Mackall Crystal L. , McAtee Nanette , Zujewski JoAnne , Cowan Kenneth , Gress Ronald E.
Published: October 2019

To examine the mechanisms of CD4 reconstitution in an adult population, lymphocyte repopulation was assessed following dose-intense chemotherapy in 25 breast cancer patients, ages 33 to 69 years. Chemotherapy resulted in a greater than 60% reduction in total CD4 T cells and, in particular, a greater than 90% loss of the CD45RA+ CD4 cells. CD4 recovery was protracted, achieving less than 50% of pretreatment levels after 12 to 14 months. Two facets of the CD4 recovery were notable. First, generation of CD45RA+ CD4 cells played only a minor role in the first year, suggesting that thymic production was not the main route of CD4 regeneration. Indeed, recovery of CD45RA+ CD4 cell levels remained limited in half of the patients even after 2 years. Second, expansion of the mature peripheral CD4 cells (CD45RO+) remaining after chemotherapy was the main source of early CD4 repopulation, peaking at 3 to 6 months postchemotherapy. This expansion was limited in duration, however, and was followed by a secondary decline, such that the total CD45RO+ CD4 levels at 9 to 12 months were lower than at 6 months. When stimulated by mitogens, an increased susceptibility to apoptosis was observed in postchemotherapy CD4 cells as compared with those from normal donors. The elevated expression of markers such as HLA-DR during chemotherapy and for several months postchemotherapy is consistent with the presence of an activated T-cell population. CD4 apoptotic frequency correlated with the frequency of HLA-DR expression on T cells. Thus, CD4 recovery is constrained in adults by a limited thymic regenerative capacity and by an increased susceptibility to apoptosis within the expanding peripheral CD4 population.
Factor V Cambridge: A New Mutation (Arg306→Thr) Associated With Resistance to Activated Protein C
Authors: Williamson David , Brown Karen , Luddington Roger , Baglin Caroline , Baglin Trevor
Published: October 2019

A new factor V mutation associated with resistance to activated protein C and thrombosis (factor V Cambridge, Arg306→Thr) was found in one patient from a carefully selected group of 17 patients with venous thrombosis and confirmed APC resistance in the absence of the common Gln506 mutation. The Arg306 mutation was also present in a first degree relative who also had APC resistance. Other potential causes of APC resistance, such as a mutation at the Arg679 site and the factor V HR2 haplotype, were excluded. Subsequent screening of 585 patients with venous thromboembolism and 226 blood donors did not show any other individual with this mutation. Factor VThr306 is the first description of a mutation affecting the Arg306 APC cleavage site and is the only mutation, other than factor V Leiden (Arg506→Gln), that has been found in association with APC resistance. This finding confirms the physiologic importance of the Arg306 APC-cleavage site in the regulation of the prothrombinase complex. It also supports the concept that APC resistance and venous thrombosis can result from a variety of genetic mutations affecting critical sites in the factor V cofactor.
The Anemic Friend Virus gp55 Envelope Protein Induces Erythroid Differentiation in Fetal Liver Colony-Forming Units-Erythroid
Authors: Constantinescu Stefan N. , Wu Hong , Liu Xuedong , Beyer Wendy , Fallon Amy , Lodish Harvey F.
Published: October 2019

The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR−/− fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.
Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner
Authors: Kambe Taiho , Tada Junko , Chikuma Mariko , Masuda Seiji , Nagao Masaya , Tsuchiya Terumasa , Ratcliffe Peter J. , Sasaki Ryuzo
Published: October 2019

Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.
Effects of Increased Anionic Charge in the β-Globin Chain on Assembly of Hemoglobin In Vitro
Authors: Adachi Kazuhiko , Yamaguchi Takamasa , Pang Jian , Surrey Saul
Published: October 2019

Studies on assembly in vitro of α-globin chains with recombinant β16 Gly→Asp, β95 Lys→Glu, β120 Lys→Glu and β16 Gly→Asp, 120 Lys→Glu human β-globin chain variants in addition to human βA- and βS-globin chains were performed to evaluate effects of increased anionic charge in the β chain on hemoglobin assembly using soluble recombinant β-globin chains expressed in bacteria. A β112 Cys→Asp change was also engineered to monitor effects on assembly of increased negative charge at α1β1 interaction sites. Order of tetramer formation in vitro under limiting α-globin chain conditions showed Hb βG16D, K120E = Hb βK120E = Hb βK95E > Hb βG16D > Hb A > Hb S >>> Hb βC112D. In addition, β112 Cys→Asp chains exist as monomers rather than β4tetramers in the absence of α chains, and the β chain in Hb βC112D tetramers was readily exchanged by addition of βs. These results suggest that affinity between α and β chains is promoted by negatively-charged β chains up to a maximum of two additional net negative charges and is independent of location on the surface except at the α1β1 interaction site. In addition, our findings show that β112 Cys on the G helix is critical for facilitating formation of stable αβ dimers, which then form functional hemoglobin tetramers, and that β112 Cys→Asp inhibits formation of stable α1β1 and β1β2 interactions in α2β2 and β4 tetramers, respectively.
Soluble Fas Levels in Sera of Bone Marrow Transplantation Recipients Are Increased During Acute Graft-Versus-Host Disease But Not During Infections
Authors: Liem Linda M. , van Lopik Thea , van Nieuwenhuijze Annemarie E.M. , van Houwelingen Hans C. , Aarden Lucien , Goulmy Els
Published: October 2019

Graft-versus-host disease (GVHD) and infections are two major complications of allogeneic bone marrow transplantation (BMT). In the course of GVHD, one of the pathways that activated cytotoxic T cells use to execute their killing mechanisms is the Fas/Fas ligand pathway. This killing mechanism might be accompanied by the release of soluble Fas (sFas) in the circulation. To examine the association of serum sFas levels and post-BMT complications, we have analyzed sFas levels in sera of bone marrow recipients with and without GVHD. Postallogeneic BMT sFas levels were significantly increased during clinically relevant acute GVHD (aGVHD; P = .002). However, during infections sFas levels tended to decrease (P = .088). Yet, the simultaneous occurrence of GVHD and infections resulted in extreme high sFas levels. These results suggested that sFas release may be correlated with the amount of tissue damage, because aGVHD induces more damage than infections. The presence of significantly increased sFas levels during aGVHD provides new insights into the GVHD pathogenesis.
Reduced Retinoic Acid-Sensitivities of Nuclear Receptor Corepressor Binding to PML- and PLZF-RARα Underlie Molecular Pathogenesis and Treatment of Acute Promyelocytic Leukemia
Authors: Guidez Fabien , Ivins Sarah , Zhu Jun , Söderström Mats , Waxman Samuel , Zelent Arthur
Published: October 2019

Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RARα fusion protein and responsiveness to treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RARα fusion protein. Both PML- and PLZF-RARα possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that the above-mentioned oncogenic fusion proteins interact with the nuclear receptor corepressor N-CoR and, in comparison with the wild-type RARα protein, their interactions display reduced sensitivities to ATRA. Although pharmacologic concentration of ATRA could still induce dissociation of N-CoR from PML-RARα, it had a very little effect on its association with the PLZF-RARα fusion protein. This ATRA-insensitive interaction between N-CoR and PLZF-RARα was mediated by the N-terminal PLZF moiety of the chimera. It appears that N-CoR/histone deacetylase corepressor complex interacts directly in an ATRA-insensitive manner with the BTB/POZ-domain of the wild-type PLZF protein and is required, at least in part, for its function as a transcriptional repressor. As the above-noted results predict, histone deacetylase inhibitors antagonize oncogenic activities of the PML-RARα fusion protein and partially relieve transcriptional repression by PLZF as well as inhibitory effect of PLZF-RARα on ATRA response. Taken together, our results demonstrate involvement of nuclear receptor corepressor/histone deacetylase complex in the molecular pathogenesis of APL and provide an explanation for differential sensitivities of PML- and PLZF-RARα–associated leukemias to ATRA.
Human Immunodeficiency Virus-1 Infection Interrupts Thymopoiesis and Multilineage Hematopoiesis In Vivo
Authors: Jenkins Morgan , Hanley Mary Beth , Moreno Mary Beth , Wieder Eric , McCune Joseph M.
Published: October 2019

It is still uncertain whether multilineage hematopoietic progenitor cells are affected by human immunodeficiency virus-1 (HIV-1) infection in vivo. The SCID-hu Thy/Liv model is permissive of long-term multilineage human hematopoiesis, including T lymphopoiesis. This model was used to investigate the effects of HIV-1 infection on early hematopoietic progenitor function. We found that both lineage-restricted and multilineage hematopoietic progenitors were depleted from grafts infected with either a molecular clone or a primary isolate of HIV-1. Depletion of hematopoietic progenitors (including CD34+ cells, colony-forming units in methylcellulose, and long-term culture-initiating cells) occurred several days before the onset of thymocyte depletion, indicating that the subsequent rapid decline in thymocyte numbers was due at least in part to loss of thymocyte progenitors. HIV-1 proviral genomes were not detected at high frequency in hematopoietic cells earlier than the intrathymic T-progenitor cell stage, despite the depletion of such cells in infected grafts. Proviral genomes were also not detected in colonies derived from progenitor cells from infected grafts. These data demonstrate that HIV-1 infection interrupts both lineage-restricted and multilineage hematopoiesis in vivo and suggest that depletion of early hematopoietic progenitor cells occurs in the absence of direct viral infection.
Cytokine Production and Function in c-mpl–Deficient Mice: No Physiologic Role for Interleukin-3 in Residual Megakaryocyte and Platelet Production
Authors: Gainsford Timothy , Roberts Andrew W. , Kimura Shinya , Metcalf Donald , Dranoff Glenn , Mulligan Richard C. , Begley C. Glenn , Robb Lorraine , Alexander Warren S.
Published: October 2019

Mice lacking thrombopoietin (TPO), or its receptor c-Mpl, display defective megakaryocyte and platelet development and deficiencies in progenitor cells of multiple hematopoietic lineages. The contribution of alternative cytokines to thrombopoiesis in the absence of TPO signalling was examined in mpl−/− mice. Analysis of serum and organ-conditioned media showed no evidence of a compensatory overproduction of megakaryocytopoietic cytokines. However, consistent with a potential role in vivo, when injected intompl−/− mice, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) retained the capacity to elevate megakaryocytes and their progenitors in hematopoietic tissues and increase circulating platelet numbers. However, double mutant mice bred to carry genetic defects both in c-Mpl and IL-3 or the alpha chain of the IL-3 receptor, displayed no greater deficiencies in megakaryocytes or platelets than mpl-deficient animals, suggesting absence of a physiologic role for IL-3 in the residual megakaryocytopoiesis and platelet production in these mice.