bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lymphoid Reconstitution After Autologous PBSC Transplantation With FACS-Sorted CD34+ Hematopoietic Progenitors
Authors: Bomberger Catherine , Singh-Jairam Meeta , Rodey Glenn , Guerriero Anastasia , Yeager Andrew M. , Fleming William H. , Holland H. Kent , Waller Edmund K. 
Published: October 2019

AbstractT-cell and B-cell reconstitution was studied in nine patients who received fluorescence activated cell sorter (FACS)-sorted autologous CD34+ hematopoietic progenitor cells (HPC). The mean numbers of T cells (CD3+), B cells (CD19+) and CD34+ HPC administered to each patient were .004, .002, and 1.8 × 106 cells/kg, respectively. After high-dose myeloablative chemotherapy (busulfan, cyclophosphamide, etoposide) CD34+ HPC were infused and lymphoid reconstitution was monitored using flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VDJ T-cell receptor (TcR) sequences. Restoration of normal numbers of peripheral blood T cells and B cells among recipients of FACS-sorted CD34+ HPC was delayed compared to recipients of non-T-cell–depleted PBSC autografts. In both patient groups, the circulating T cells were primarily CD4−, CD8+, αβ TcR+, and CD45RO+, CD45RA− during the first 2 months after transplant. Subsequent increases in the frequency of CD45RA+ CD45RO− T cells occurred at 2 to 3 months after transplant, suggesting maturation of CD34+hematopoietic progenitors to “naive” T cells. Analysis of the TcR repertoire after hematopoietic reconstitution demonstrated decreased diversity of Vβ TcR expression associated with global decreases in the absolute number of total peripheral blood T cells and most Vβ TcR+ subsets. Three of nine recipients of FACS-sorted CD34+ HPC demonstrated significant increases in the percentage of γδ+ peripheral T cells and CD5+ B cells at 3 to 9 weeks after transplantation, and all patients had transient oligoclonal expansions of T cells expressing specific Vβ TcR. Transplantation with highly purified CD34+ HPC results in reduced diversity of the peripheral T-cell repertoire during the early post-transplant period compared with patients receiving unmanipulated or MoAb-depleted transplants.
Expression of a Knocked-In AML1-ETO Leukemia Gene Inhibits the Establishment of Normal Definitive Hematopoiesis and Directly Generates Dysplastic Hematopoietic Progenitors
Authors: Okuda Tsukasa , Cai Zhongling , Yang Shouli , Lenny Noel , Lyu Chuhl-joo , van Deursen Jan M.A. , Harada Hironori , Downing James R. 
Published: October 2019

AbstractThe t(8;21)-encoded AML1-ETO chimeric product is believed to be causally involved in up to 15% of acute myelogenous leukemias through an as yet unknown mechanism. To directly investigate the role of AML1-ETO in leukemogenesis, we used gene targeting to create anAML1-ETO “knock-in” allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO(AML1-ETO/+) died around E13.5 from a complete absence of normal fetal liver–derived definitive hematopoiesis and lethal hemorrhages. This phenotype was similar to that seen following homozygous disruption of either AML1 orCBFβ. However, in contrast to AML1- or CBFβ-deficient embryos, fetal livers from AML1-ETO/+ embryos contained dysplastic multilineage hematopoietic progenitors that had an abnormally high self-renewal capacity in vitro. To further document the role of AML1-ETO in these growth abnormalities, we used retroviral transduction to express AML1-ETO in murine adult bone marrow–derived hematopoietic progenitors. AML1-ETO–expressing cells were again found to have an increased self-renewal capacity and could be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation.
Interactions of Erythropoietin, Granulocyte Colony-Stimulating Factor, Stem Cell Factor, and Interleukin-11 on Murine Hematopoiesis During Simultaneous Administration
Authors: Roeder I. , de Haan G. , Engel C. , Nijhof W. , Dontje B. , Loeffler M. 
Published: October 2019

Abstract
We investigated how in vivo effects of single hematopoietic cytokines change if given in combination for a prolonged time. Mice were treated with every combination of recombinant human (rh) erythropoietin (EPO), rh granulocyte colony-stimulating factor (G-CSF), recombinant rat (rr) stem cell factor (SCF), and rh interleukin (IL)-11 by continuous infusion over 7 days (full factorial design with three dose levels for each cytokine). Burst-forming unit–erythroid (BFU-E), colony-forming unit–erythroid (CFU-E), and colony-forming unit–granulocyte-macrophage (CFU-GM) were determined in bone marrow and spleen, reticulocytes, hematocrit, granulocytes, and thrombocytes in the peripheral blood. An analysis of variance (ANOVA) and multiple comparison of means was used to evaluate the data. For several cell types, cytokine effects superimposed in an additive way if combined. However, in a large number of circumstances, nonadditive pairwise interactions were found. They differed in type and magnitude involving high-dose saturation, high-dose antagonistic effects, and even effect reversals (qualitative interactions). Hence, in general, it was not possible to foresee the combination effects on the basis of existing knowledge of single effects. On the other hand, the cytokine network was robust and no system hazards were observed under multiple cytokine combinations. The results illustrate that the cytokine network has nonlinear dynamic properties in vivo with dose-response characteristics of one cytokine being continuously modified by other cytokines.
6q Deletions in Acute Lymphoblastic Leukemia and Non-Hodgkin's Lymphomas
Authors: Merup Mats , Moreno Teresa Calero , Heyman Mats , Rönnberg Kristina , Grandér Dan , Detlofsson Rut , Rasool Omid , Liu Yie , Söderhäll Stefan , Juliusson Gunnar , Gahrton Gösta , Einhorn Stefan 
Published: October 2019

Abstract
Deletions on the long arm of chromosome 6 are frequently found in acute lymphoblastic leukemia (ALL) and non-Hodgkin's lymphomas (NHL). We have used polymerase chain reaction analysis to study loss of heterozygosity of 16 microsatellite markers on chromosome 6 in 74 ALL and 54 NHL patients. Our results show that deletions of 6q in ALL are more frequent than what has been reported in previous studies, occurring in at least 32% of the patients. The corresponding figure for NHL patients is 7%. Our results define a region of minimal deletion in ALL of less than 500 kb between markers D6S1709 and D6S434. The common region of deletion in NHL is located telomeric of this region. Thus, two different tumor suppressor genes on chromosome 6q seem to be relevant for the development of lymphoid malignancies.
Erythroid Maturation and Globin Gene Expression in Mice With Combined Deficiency of NF-E2 and Nrf-2
Authors: Martin Florence , van Deursen Jan M. , Shivdasani Ramesh A. , Jackson Carl W. , Troutman Amber G. , Ney Paul A. 
Published: October 2019

AbstractNF-E2 binding sites, located in distant regulatory sequences, may be important for high level α- and β-globin gene expression. Surprisingly, targeted disruption of each subunit of NF-E2 has either little or no effect on erythroid maturation in mice. For p18 NF-E2, this lack of effect is due, at least in part, to the presence of redundant proteins. For p45 NF-E2, one possibility is that NF-E2–related factors, Nrf-1 or Nrf-2, activate globin gene expression in the absence of NF-E2. To test this hypothesis for Nrf-2, we disrupted the Nrf-2 gene by homologous recombination. Nrf-2–deficient mice had no detectable hematopoietic defect. In addition, no evidence was found for reciprocal upregulation of NF-E2 or Nrf-2 protein in fetal liver cells deficient for either factor. Fetal liver cells deficient for both NF-E2 and Nrf-2 expressed normal levels of α- and β-globin. Mature mice with combined deficiency of NF-E2 and Nrf-2 did not exhibit a defect in erythroid maturation beyond that seen with loss of NF-E2 alone. Thus, the presence of a mild erythroid defect in NF-E2–deficient mice is not the result of compensation by Nrf-2.
The Fetal Origin of B-Precursor Leukemia in the Eμ-ret Mouse
Authors: Zeng Xiang-Xing , Zhang Haige , Hardy Richard R. , Wasserman Robert 
Published: October 2019

AbstractBefore the clinical onset of B-precursor lymphoblastic leukemia, Eμ-ret mice have an expansion of late pro-B cells (CD45R+CD43+CD24+BP-1+) within the bone marrow. To characterize the early effects of the transgene product on lymphopoiesis, we initially sequenced the Ig heavy chain (IgH) rearrangements within the late pro-B cells in 24-day-old Eμ-ret and transgene negative mice. In both mouse populations, the IgH rearrangements were polyclonal, predominately nonproductive, and exhibited similar V, D, and J gene usage. However, the frequency of N regions, a marker of postnatal lymphopoiesis, was notably different. At the VD junction, N regions were found in 25 of 25 (100.0%) rearrangements from transgene-negative mice compared with 12 of 36 (33.3%) rearrangements from Eμ-ret mice. At the DJ junction, N regions were found in 21 of 25 (84.0%) rearrangements from transgene negative mice compared with 4 of 36 (11.1%) rearrangements from Eμ-ret mice. Subsequently, we sequenced the clonal IgH rearrangements from 9 leukemias that developed in 10-to 38-week-old mice and found that 7 leukemias had a least 1 rearrangement that lacked N regions at the DJ junction. In addition, V replacement events were observed in the 1 leukemia studied in detail. Terminal deoxynucleotidyl transferase, the enzyme responsible for N region addition, was expressed at markedly lower levels in late pro-B cells from 7- to 10-day-old Eμ-ret mice compared with transgene-negative mice. Examination of fetal lymphopoiesis in Eμ-ret mice identified a relative increase in early (CD45R+CD43+CD24+BP-1−) and late pro-B cells and a decrease in more differentiated CD43− B-lineage cells. Fetal early pro-B cells from Eμ-ret mice proliferated threefold to fivefold greater but differentiated to a lesser extent than those from transgene negative mice when cultured in vitro with interleukin-7. These data suggest that the B precursor leukemias in adult Eμ-ret mice arise from the progeny of pro-B cells generated in utero.
Two New Pseudopod Morphologies Displayed by the Human Hematopoietic KG1a Progenitor Cell Line and by Primary Human CD34+Cells
Authors: Francis Karl , Ramakrishna Ramprasad , Holloway William , Palsson Bernhard O. 
Published: October 2019

AbstractA primitive human hematopoietic myeloid progenitor cell line, KG1a, characterized by high expression of the CD34 surface antigen has been observed to extend long, thin pseudopodia. Once extended, these pseudopods may take on one of two newly described morphologies, tenupodia or magnupodia. Tenupodia are very thin and form in linear segments. They adhere to the substrate, can bifurcate multiple times, and often appear to connect the membranes of cells more than 300 μm apart. Magnupodia are much thicker and have been observed to extend more than 330 μm away from the cell. Magnupods are flexible and can exhibit rapid dynamic motion, extending or retracting in a few seconds. During retraction, the extended material often pools into a bulb located on the pod. Both morphologies can adhere to substrates coated with fibronectin, collagen IV, and laminin as well as plastic. The CD34 and CD44 antigens are also present on the surface of these podia. Primary human CD34+ cells from fetal liver, umbilical cord blood, adult bone marrow, and mobilized peripheral blood extend these podia as well. The morphology that these pseudopods exhibit suggest that they may play both sensory and mechanical roles during cell migration and homing after bone marrow transplantation.
Flt3 Ligand Promotes the Generation of a Distinct CD34+Human Natural Killer Cell Progenitor That Responds to Interleukin-15
Authors: Yu Haixin , Fehniger Todd A. , Fuchshuber Pascal , Thiel Karl S. , Vivier Eric , Carson William E. , Caligiuri Michael A. 
Published: October 2019

AbstractInterleukin-15 (IL-15) is produced by human bone marrow (BM) stromal cells and can induce CD34+ hematopoietic progenitor cells (HPCs) to differentiate into CD56+CD3−natural killer (NK) cells in the absence of stromal cells. IL-15 mediates its effects by signaling through the β and γcchains of the IL-2/15 receptor (R). The c-kit ligand (KL), also produced by stromal cells, enhances the expansion of NK cells from CD34+ HPCs in the presence of IL-15, but alone has no ability to differentiate NK cells. Mice deficient in KL do not appear to have a quantitative deficiency in NK cells, suggesting that other stromal cell factors may contribute to NK cell expansion. Flt3 ligand (FL) is also produced by BM stromal cells and has homology with KL. Furthermore, mice with a targeted disruption of the FL gene have reduced numbers of NK cells. We evaluated here the effects of FL on human NK cell development and expansion from CD34+ HPCs. Like KL, FL significantly enhanced the expansion of NK cells from CD34+ HPCs in the presence of IL-15, compared with IL-15 alone. However, FL alone had no effect on NK cell differentiation. We therefore explored the mechanism by which FL promotes IL-15–mediated NK cell development. FL was found to induce IL-2/15Rβ (CD122) expression on CD34bright HPCs. The CD34brightCD122+ cell coexpressed CD38, but lacked expression of CD7, CD56, NK cell receptors (NKRs), or cytotoxic activity in the absence of IL-15. Using limiting dilution analysis in the presence of IL-15 alone, we demonstrated that the FL-induced CD34brightCD122+ HPCs had an NK cell precursor frequency 20- to 60-fold higher than the CD34dim/negCD122− HPCs and 65- to 235-fold higher than fresh CD34+ HPCs. KL had similar effects as FL, but induced a significantly lower percentage of CD34brightCD122+ cells (P ≤ .01). Both FL and KL also increased IL-15R transcript in CD34+ HPCs. Culture of CD34+ HPCs in FL or KL, followed by culture in IL-15 alone, induced expression of both C-type lectin and Ig-superfamily NKRs on CD56+ cells. These data collectively support a role for FL in early human NK cell development. FL or KL generate a unique CD34brightCD122+CD38+ human NK cell intermediate from CD34+ HPCs that lacks NK features yet is IL-15–responsive. IL-15 is then required for the induction of CD56 and NKRs, LGL morphology, cytotoxic activity, and the ability to produce abundant cytokines and chemokines.
Eosinophil Tethering to Interleukin-4–Activated Endothelial Cells Requires Both P-Selectin and Vascular Cell Adhesion Molecule-1
Authors: Patel Kamala D. 
Published: October 2019

AbstractWe examined the mechanisms used by eosinophils to tether and accumulate on interleukin-4 (IL-4)–stimulated human umbilical vein endothelial cells (HUVECs) under flow conditions. As previously reported, HUVECs treated for 24 hours with 20 ng/mL IL-4 had increased expression of P-selectin and vascular cell adhesion molecule-1 (VCAM-1) but not E-selectin. We found that eosinophils tethered and rolled on IL-4–stimulated HUVECs at physiologic shear stresses. Eosinophil rolling was quickly followed by firm adhesion. Treatment with either an anti–P-selectin monoclonal antibody (MoAb) or an anti–VCAM-1 MoAb decreased both eosinophil tethering and accumulation at 2 dyn/cm2. VCAM-1 interacts with 4-integrins expressed on eosinophils. We found that an anti–4-integrin MoAb also blocked eosinophil tethering and accumulation at 2 dyn/cm2. None of these MoAbs alone had an impact on eosinophil accumulation at lower shear stresses, but when either an anti–VCAM-1 or an anti–4-integrin MoAb was used in combination with an anti–P-selectin MoAb, all eosinophil tethering and accumulation on IL-4–stimulated HUVECs were blocked. This was true at both high and low shear stresses. These data show that both P-selectin and VCAM-1 are required to tether eosinophils at high shear stresses, but at low shear stresses these adhesion proteins can act independently to recruit eosinophils to IL-4–stimulated HUVECs.
Enhanced Retroviral Transduction of 5-Fluorouracil–Resistant Human Bone Marrow (Stem) Cells Using a Genetically Modified Packaging Cell Line
Authors: Povey Joanna , Weeratunge Nishanthi , Marden Chloe , Sehgal Amita , Thrasher Adrian , Casimir Colin 
Published: October 2019

AbstractPluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.
Interleukin-10 Abrogates the Inhibition of Epstein-Barr Virus–Induced B-Cell Transformation by Memory T-Cell Responses
Authors: Bejarano M.T. , Masucci M.G. 
Published: October 2019

AbstractIn vitro infection of human B lymphocytes by Epstein-Barr virus (EBV) results in their growth transformation and establishment of immortalized lymphoblastoid cell lines. The virus was found to encode a homologue of the pleiotropic cytokine interleukin-10 (IL-10), which has wide-ranging effects on the immune system. We investigated the effect of human IL-10 (hIL-10) and viral IL-10 (vIL-10) on EBV-specific immunological memory, as assessed by the inhibition of EBV-induced B-cell transformation by the autologous T cells. We found that IL-10 abrogates the inhibitory capacity of T cells. This IL-10 effect is mediated through suppression of T-cell activation-induced IL-2 and interferon-γ production and through a direct enhancement of EBV-infected B-cell growth.
Truncation of Glycoprotein (GP) IIIa (▵ 616-762) Prevents Complex Formation With GPIIb: Novel Mutation in Exon 11 of GPIIIa Associated With Thrombasthenia
Authors: Ferrer Milagros , Tao Jianming , Iruı́n Gema , Sánchez-Ayuso Matilde , González-Rodrı́guez José , Parrilla Roberto , González-Manchón Consuelo 
Published: October 2019

AbstractThis work reports the molecular genetic study of a patient who suffered from Glanzmann thrombasthenia (GT). Structural analysis of the glycoprotein (GP) IIb and GPIIIa genes showed the presence of a homozygous G1846→T transversion in exon 11 of GPIIIa that changes Glu616→Stop. Cytometric and immunochemical analysis indicated that platelet GPIIb-IIIa was absent in the proband but present at normal levels in the heterozygous relatives. The following observations indicate that this mutation is responsible for the thrombasthenic phenotype of the proband. (1) We failed to detect mutations other than [T1846]GPIIIa in the coding region of both GPIIb and GPIIIa genes. (2) The G1846→T mutation was observed in either parent and a brother of the proband, but none of 100 unrelated individuals carried this defect. (3) Pulse-chase and immunoprecipitation analysis of GPIIb-IIIa complexes in cells transiently cotransfected with cDNAs encoding normal GPIIb and [T1846]GPIIIa showed neither maturation of GPIIb nor complex formation and surface exposure of GPIIb-▵GPIIIa. These observations indicate that the sequence from Glu616 to Thr762 in GPIIIa is essential for heterodimerization with GPIIb. Polymerase chain reaction-based analysis demonstrated the presence of normal levels of full-length GPIIIa-mRNA in the proband and in heterozygous relatives. In addition, a shortened transcript, with a 324-nucleotide deletion, resulting from in-frame skipping of exons 10 and 11, was detectable upon reamplification of the DNA. Thus, unlike other nonsense mutations, [T1846]GPIIIa does not lead to abnormal processing or reduction in the number of transcripts with the termination codon.
Structural Studies of Fibrinolysis by Electron Microscopy
Authors: Veklich Yuri , Francis Charles W. , White Janice , Weisel John W. 
Published: October 2019

AbstractFibrin is degraded by the fibrinolytic system in which a plasminogen activator converts plasminogen to plasmin, a serine protease that cleaves specific bonds in fibrin leading to solubilization. To elucidate further the biophysical processes involved in conversion of insoluble fibers to soluble fragments, fibrin was treated with either plasmin or the combination of plasminogen and plasminogen activator, and morphologic changes were observed using scanning electron microscopy. These changes were correlated with biochemical analysis and with characterization of released, soluble fragments by transmission electron microscopy. Initial changes in the fibrin matrix included creation of many free fiber ends and gaps in the continuity of fibers. With more extensive digestion, free fiber segments associated laterally, resulting in formation of thick fiber bundles. Supernatants of digesting clots, containing soluble derivatives, were negatively contrasted and examined by transmission electron microscopy. Large, complex fragments containing portions of multiple fibers were observed, as were pieces of individual fibers and smaller fragments previously identified. Some large fragments had sharply defined ends, indicating that they had been cleaved perpendicularly to the fiber direction. Other fibers showed splayed ends or a lacy meshwork of surrounding protofibrils. Longer times generated more small fragments whose molecular composition could be inferred from their appearance. These results indicate that fibrinolytic degradation results in larger pieces than previously identified and that plasmin digestion proceeds locally by transverse cutting across fibers rather than by progressive cleavage uniformly around the fiber.