bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Selection of Wild-Type Hematopoietic Stem Cells in a Murine Model of Fanconi Anemia
Authors: Battaile Kevin P. , Bateman Raynard L. , Mortimer Derik , Mulcahy Jean , Rathbun R. Keaney , Bagby Grover , Fleming William H. , Grompe Markus 
Published: October 2019

AbstractFanconi anemia (FA) is an autosomal recessive disorder characterized by birth defects, increased incidence of malignancy, and progressive bone marrow failure. Bone marrow transplantation is therapeutic and, therefore, FA is a candidate disease for hematopoietic gene therapy. The frequent finding of somatic mosaicism in blood of FA patients has raised the question of whether wild-type bone marrow may have a selective growth advantage. To test this hypothesis, a cohort radio-ablated wild-type mice were transplanted with a 1:1 mixture of FA group C knockout (FACKO) and wild-type bone marrow. Analysis of peripheral blood at 1 month posttransplantation showed only a moderate advantage for wild-type cells, but upon serial transplantation, clear selection was observed. Next, a cohort of FACKO mice received a transplant of wild-type marrow cells without prior radio-ablation. No wild-type cells were detected in peripheral blood after transplantation, but a single injection of mitomycin C (MMC) resulted in an increase to greater than 25% of wild-type DNA. Serial transplantation showed that the selection occurred at the level of hematopoietic stem cells. No systemic side effects were observed. Our results show that in vivo selection for wild-type hematopoietic stem cells occurs in FA and that it is enhanced by MMC administration.
Increased Sensitivity to Complement and a Decreased Red Blood Cell Life Span in Mice Mosaic for a Nonfunctional Piga Gene
Authors: Tremml G. , Dominguez C. , Rosti V. , Zhang Z. , Pandolfi P.P. , Keller P. , Bessler M. 
Published: October 2019

AbstractThe gene PIGA encodes one of the protein subunits of the 1-6-N acetylglucosaminyltransferase complex, which catalyses an early step in the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. PIGA is somatically mutated in blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH), leading to deficiency of GPI-linked proteins on the cell surface. To investigate in detail how inactivating mutations of the PIGA gene affect hematopoiesis, we generated a mouse line, in whichloxP-mediated excision of part of exon 2 occurs on the expression of Cre. After crossbreeding with EIIa-cre transgenic mice, recombination occurs early in embryonic life. Mice that are mosaics for the recombined Piga gene are viable and lack GPI-linked proteins on a proportion of circulating blood cells. This resembles the coexistence of normal cells and PNH cells in patients with an established PNH clone. PIGA(−) blood cells in mosaic mice have biologic features characteristic of those classically seen in patients with PNH, including an increased sensitivity toward complement mediated lysis and a decreased life span in circulation. However, during the 12-month follow-up, the PIGA(−) cell population did not increase, clearly showing that a Piga gene mutation is not sufficient to cause the human disease, PNH.
Transgenic Analysis of a 100-kb Human β-Globin Cluster–Containing DNA Fragment Propagated as a Bacterial Artificial Chromosome
Authors: Kaufman Richard M. , Pham Christine T.N. , Ley Timothy J. 
Published: October 2019

AbstractTo date, the normal transcriptional regulation of the human β-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human β-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human β-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F1 mice showed that 2 lines express human β globin at levels approximately equivalent to the endogenous mouse β-major genes. One line expresses no human β globin, while the remaining 6 lines show intermediate expression levels. Complete γ→β-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic→adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.
A Kinetic Model for the Homing and Migration of Prenatally Transplanted Marrow
Authors: Shaaban Aimen F. , Kim Heung Bae , Milner Ross , Flake Alan W. 
Published: October 2019

Abstract
Currently little is known about the mechanisms regulating the homing and the early engraftment of prenatally transplanted hematopoietic cells due to the lack of a relevant functional assay. In this study, we have defined a reproducible kinetic profile of the homing and the early engraftment events in a murine model of prenatal stem cell transplantation. Light density mononuclear cells (LDMCs) from adult C57Pep3b and SJL/J marrow were transplanted by intraperitoneal (IP) injection into C57BL/6 fetuses (106 LDMCs/fetus) at 14 days of gestation. The fetuses were sacrificed at early time points (1.5 to 96 hours) after transplantation. Recipient fetal liver and cord blood were analyzed for donor cell frequency and donor cell phenotype by dual color flow cytometry. Pertinent findings included the following: (1) a triphasic kinetic profile exists after in utero hematopoietic stem cell (HSC) transplantation (homing of circulating donor cells, rapid reduction of donor cell frequency, and donor cell competitive equilibration); (2) homing to the fetal liver is nonselective and reflects the phenotypic profile of the donor population; and (3) the kinetics after the prenatal transplantation of congenic or fully allogeneic cells are identical. This model will facilitate a systematic analysis of the mechanisms that regulate the homing of prenatally transplanted hematopoietic cells.
Human LTC-IC can be maintained for at least 5 weeks in vitro when interleukin-3 and a single chemokine are combined with O-sulfated heparan sulfates: requirement for optimal binding interactions of heparan sulfate with early-acting cytokines and matrix proteins
Authors: Gupta Pankaj , Oegema Theodore R. , Brazil Joseph J. , Dudek Arkaduisz Z. , Slungaard Arne , Verfaillie Catherine M. 
Published: October 2019

AbstractWe have shown that stromal O-sulfated heparan sulfate glycosaminoglycans (O-S-GAGs) regulate primitive human hematopoietic progenitor cell (HPC) growth and differentiation by colocalizing heparin-binding cytokines and matrix proteins with HPC in stem cell “niches” in the marrow microenvironment. We now show that long-term culture-initiating cells (LTC-IC) are maintained for 5 weeks in the absence of stroma when O-S-GAGs are added to IL-3 and either MIP-1 or PF4 (LTC-IC maintenance without GAGs, 32 ± 2%; with GAGs, 95 ± 7%; P &lt; .001). When cultured with 5 additional cytokines, O-S-GAGs, IL-3, and MIP-1, LTC-IC expanded 2- to 4-fold at 2 weeks, and 92 ± 8% LTC-IC were maintained at 5 weeks. Similar results were seen when PF4 replaced MIP-1. Although O-S-GAG omission did not affect 2-week expansion, only 20% LTC-IC were maintained for 5 weeks. When O-S-heparin was replaced by completely desulfated-, N-sulfated (O-desulfated), or unmodified heparins, LTC-IC maintenance at week 5 was not better than with cytokines alone. Unmodified- and O-S-heparin, but not desulfated- or N-sulfated heparin, bound to MIP-1, IL-3, PF4, VEGF, thrombospondin, and fibronectin. However, the affinity of heparin for thrombospondin and PF4, and the association and dissociation rates of heparin for PF4, were higher than those of O-S-heparin. We conclude that (i) although cytokines may suffice to induce early expansion, adult human LTC-IC maintenance for longer than 1 month requires O-S-GAGs, and (ii) HPC support may depend not only on the ability of GAGs to bind proteins, but also on optimal affinity and kinetics of interactions that affect presentation of proteins in a biologically active manner to progenitors. (Blood. 2000;95:147-155)
Fibrinogen is a component of a novel lipoprotein particle: Factor H–related protein (FHRP)–associated lipoprotein particle (FALP)
Authors: Park C. Thomas , Wright Samuel D. 
Published: October 2019

AbstractWe have previously described a novel lipoprotein particle consisting of phospholipids, apolipoprotein A-I (apoAI), lipopolysaccharide binding protein (LBP) and Factor H-related proteins (FHRP), and we termed these particles FALP (FHRP-associated lipoprotein particles). Highly purified preparations of FALP contain variable amounts of an unidentified polypeptide triplet of Mr ∼85 000 (tp85). Here we report that tp85 represents fragment D of fibrinogen, as confirmed by N-terminal amino acid sequencing and Western blot analysis with an antifibrinogen antibody. The physical association of fibrinogen with other components of FALP in plasma was further confirmed by sandwich ELISA by using monoclonal antibodies against apoAI, FHRP or LBP to capture the particles and polyclonal antifibrinogen as the detecting antibody. Furthermore, affinity chromatography with anti-FHRP-1–specific IgG showed that fibrinogen is co-immunodepleted with FALP and approximately 17% of total plasma fibrinogen are bound to FALP. LBP is a lipid transfer protein that moves lipopolysaccharide (LPS) to a binding site on CD14 or high-density lipoprotein (HDL). To determine whether fibrinogen affects the lipid transfer activity of LBP on FALP, this activity was measured in FALP prepared with and without fibrinogen. Neither activity of LBP was affected by fibrinogen. The abundance of FALP suggests, instead, an effect of FALP on the function or clearance of fibrinogen or fragment D. (Blood. 2000;95:198-204)
Threonine-145/Methionine-145 variants of baculovirus produced recombinant ligand binding domain of GPIbα express HPA-2 epitopes and show equal binding of von Willebrand factor
Authors: Li Chester Q. , Garner Stephen F. , Davies Julian , Smethurst Peter A. , Wardell Mark R. , Ouwehand Willem H. 
Published: October 2019

AbstractGlycoprotein (GP) Ib is the functionally dominant subunit of the platelet GPIb-IX-V receptor complex, with the von Willebrand factor (vWF) binding site residing on the amino-terminus. A threonine for methionine-145 replacement of GPIb is associated with the human platelet antigen (HPA)-2 system. To study the structural and functional consequences of this mutation, both forms of GPIb were expressed as calmodulin fusion proteins in insect cells. Both recombinant proteins were recognized by their respective alloantibodies, independent of glycosylation or intactness of disulfide bonds, and gave similar results to platelet-derived GPIb in antibody detection assays. Resonant mirror studies showed that vWF binding was not affected by the HPA-2 mutation; however, vWF binding was partially inhibited by IgG HPA-2 antibodies. Our data are compatible with an involvement of the leucine-rich repeat domain of GPIb in vWF binding and indicate that recombinant GPIb may be used to detect HPA-2 antibodies. (Blood. 2000;95:205-211)
Viral latent membrane protein 1 (LMP-1)–induced CD99 down-regulation in B cells leads to the generation of cells with Hodgkin's and Reed-Sternberg phenotype
Authors: Kim Soon Ha , Shin Young Kee , Lee Im-soon , Bae Young Mee , Sohn Hae Won , Suh Young Ho , Ree Howe J. , Rowe Martin , Park Seong Hoe 
Published: October 2019

AbstractRecently we reported that the down-regulation of CD99 (Mic2) is a primary requirement for the generation of Hodgkin's and Reed-Sternberg (H-RS) cells seen in Hodgkin's disease. In this study, we provide evidence that the down-regulation of CD99 is induced by high expression of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1), which is highly expressed in H-RS cells of EBV-associated Hodgkin's disease. To investigate the effect of LMP-1 on the expression of CD99 in vitro, we established a stable cell line by transfecting an SV40-early promoter driven-LMP-1 expression construct into a neoplastic lymphoblastoid B cell line, IM9, in which the level of endogenous LMP-1 expression is almost negligible. In this cell line, the overexpression of LMP-1 led to the down-regulation of CD99 and the acquisition of morphological and functional characteristics of H-RS cells indistinguishable from those in lymph nodes of Hodgkin's disease patients and in CD99-deficient B cells. In addition, induced LMP-1 expression in an EBV-negative B cell clone, BJAB, directly caused the down-regulation of surface CD99 expression. Northern and Western analysis data, showing that overexpression of LMP-1 negatively influenced the expression of CD99, were supported by experiments in which a CD99 promoter-driven luciferase promoter reporter construct transfected into 293T cells was down-regulated when LMP-1 was coexpressed. Therefore, our data strongly suggest that the EBV LMP-1 protein plays a pivotal role in the down-regulation of CD99 via transcriptional regulation, which leads to the generation of the H-RS cells. (Blood. 2000;95:294-300)
Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells
Authors: Fulda Simone , Strauss Gudrun , Meyer Eric , Debatin Klaus-Michael 
Published: October 2019

AbstractActivation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)
Activation of C-C β-chemokines in human peripheral blood γδ T cells by isopentenyl pyrophosphate and regulation by cytokines
Authors: Cipriani Barbara , Borsellino Giovanna , Poccia Fabrizio , Placido Roberta , Tramonti Daniela , Bach Simona , Battistini Luca , Brosnan Celia F. 
Published: October 2019

AbstractHuman γδ T lymphocytes respond to viral, bacterial, protozoal, and tumoral antigens, but their precise function remains unknown. In adults the major circulating γδ T-cell subset expresses the Vγ9Vδ2 T-cell receptor and responds to protease-resistant phosphorylated derivatives found in many pathogens. In this study we show that activation of Vδ2+ cells with the nonpeptidic antigen isopentenyl pyrophosphate (IPP) rapidly induces (within 4-12 hours) the C-C chemokines MIP-1, MIP-1β, and lymphotactin but not MCP-1. The most robust response was obtained for MIP-1β. IPP induction of MIP-1 and MIP-1β was not affected by costimulation with interleukin-4 (IL-4), IL-10, TGF-β, or interferon-γ (INF-γ). However, IL-12 significantly enhanced IPP-induced expression and release of MIP-1 that was down-regulated by TGF-β whereas the induction of MIP-1β by IPP+IL-12 was refractory to cotreatment with TGFβ indicating that these chemokines are differentially regulated by these cytokines. Vδ2+ T cells also expressed a wide range of C-C chemokine receptors including CCR1, CCR5, and CCR8, all of which were down-regulated following activation. We conclude that Vδ2+ cells can be rapidly induced by components of bacterial cell walls to express high levels of proinflammatory chemokines, supporting an important role for these cells in the early stages of the inflammatory responses to many common pathogens. (Blood. 2000, 95:39-47)
Limiting numbers of G156A O6-methylguanine–DNA methyltransferase-transduced marrow progenitors repopulate nonmyeloablated mice after drug selection
Authors: Davis Brian M. , Koç Omer N. , Gerson Stanton L. 
Published: October 2019

Abstract
The limited efficacy of hematopoietic gene therapy can be improved by in vivo selection for transduced long-term repopulating cells (LTRC). We selected for G156A MGMT (▵MGMT) transduced LTRC present in 5 × 104 to 100 × 104 marrow cells infused into nonmyeloablated mice by the administration of O6-benzylguanine (BG) and BCNU every 3 to 4 weeks. To facilitate engraftment, mice were given a nonablative dose of BG and BCNU before infusion. Without selection, ▵MGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, ▵MGMT+ CFU were frequently detected, and their proportions increased with each treatment cycle. After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted with 75% to 100% ▵MGMT+ CFU for at least 6 months, representing up to 940-fold enrichment. Thus, BG and BCNU stem cell toxicity allows ▵MGMT-transduced LTRC to repopulate the bone marrow. This degree of selection pressure in nonmyeloablated mice is far greater than that observed in previous drug-resistance gene transfer studies. These data support our approved clinical trial to select for drug-resistant, transduced hematopoietic cells, potentially decreasing cumulative drug-induced myelosuppression in patients with cancer. These data also suggest that ▵MGMT may be a potent, dominant, selectable marker for use in dual gene therapy.
Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells
Authors: Zain Jasmine , Huang Yao-Qi , Feng XueSheng , Nierodzik Mary Lynn , Li Jian-Jun , Karpatkin Simon 
Published: October 2019

AbstractBecause thrombin-treated tumor cell-induced metastasis increases tumor nodule volume12 greater than nodule number, we studied the effect of thrombin on tumor cell growth in vitro and in vivo (murine B16F10 melanoma, human HCT8 colon carcinoma, DU145 prostate carcinoma). Tumor cell growth was measured after 3 to 7 days in 1% fetal calf serum (FCS) + RPMI 1640. We found that, whereas relatively low concentrations of thrombin, 0.1 to 0.5 U/mL (1-5 nmol/L) enhance tumor cell growth in vitro approximately 2- to 3-fold, higher concentrations, 0.5 to 1 U/mL (5-10 nmol/L) impaired cell growth approximately 2- to 4-fold. Impaired cell growth was associated with cell cycle arrest at G2M and increased pre-GoDNA, as well as apoptosis, measured by tumor cell binding to Annexin V and propidium iodide. Apoptosis was reversed with the general caspase inhibitor, FK-011. The enhancing and inhibiting effects were specific for thrombin (reversed with inactive diisopropyl-fluorophosphate [DFP]-thrombin) and mediated via the protease-activated receptor 1 (PAR-1). PAR-1 activation was demonstrated by (1) use of a cell line, B16F10, devoid of the 3 other thrombin receptors, PAR-3, PAR-4, and GPIb; and (2) greater sensitivity of PAR-1 transfected B16F10 and HCT8 cells to impaired cell growth/apoptosis, 3- and 14-fold, respectively. Thus, thrombin has a bimodal effect on PAR-1 in tumor cells: enhanced growth at low concentration, impaired growth/apoptosis at higher concentration.
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice
Authors: Peled Amnon , Kollet Orit , Ponomaryov Tanya , Petit Isabelle , Franitza Suzanna , Grabovsky Valentin , Slav Michal Magid , Nagler Arnon , Lider Ofer , Alon Ronen , Zipori Dov , Lapidot Tsvee 
Published: October 2019

Abstract
Hematopoietic stem cell homing and engraftment require several adhesion interactions, which are not fully understood. Engraftment of nonobese/severe combined immunodeficiency (NOD/SCID) mice by human stem cells is dependent on the major integrins very late activation antigen–4 (VLA-4); VLA-5; and to a lesser degree, lymphocyte function associated antigen–1 (LFA-1). Treatment of human CD34+cells with antibodies to either VLA-4 or VLA-5 prevented engraftment, and treatment with anti–LFA-1 antibodies significantly reduced the levels of engraftment. Activation of CD34+ cells, which bear the chemokine receptor CXCR4, with stromal derived factor 1 (SDF-1) led to firm adhesion and transendothelial migration, which was dependent on LFA-1/ICAM-1 (intracellular adhesion molecule–1) and VLA-4/VCAM-1 (vascular adhesion molecule–1). Furthermore, SDF-1–induced polarization and extravasation of CD34+/CXCR4+ cells through the extracellular matrix underlining the endothelium was dependent on both VLA-4 and VLA-5. Our results demonstrate that repopulating human stem cells functionally express LFA-1, VLA-4, and VLA-5. Furthermore, this study implies a novel approach to further advance clinical transplantation.
Glycoprotein Ia gene C807T polymorphism and risk for major adverse cardiac events within the first 30 days after coronary artery stenting
Authors: von Beckerath Nicolas , Koch Werner , Mehilli Julinda , Böttiger Corinna , Schömig Albert , Kastrati Adnan 
Published: October 2019

Abstract
The glycoprotein complex Ia/IIa (GP Ia/IIa) is a major collagen receptor on platelets and other cell types. Recently, linked polymorphisms within the coding region of the GP Ia gene (C807T and G873A) were identified that are related to GP Ia/IIa surface expression. The T807/A873 allele is associated with high expression, whereas the C807/G873 allele is associated with low surface expression of GP Ia/IIa. Subsequently, the T807 allele was found to be associated with coronary and cerebral infarction in younger patients. Platelet adhesion to the vessel wall plays a pivotal role in thrombosis after coronary artery stent placement. The goal of this study was to test whether C807T polymorphism is associated with a higher incidence of thrombotic events following coronary stenting. Consecutive patients treated with coronary stent placement (n = 1797) were genotyped for C807T polymorphism with polymerase chain reaction and allele-specific fluorogenic probes. The composite end point was defined as death, myocardial infarction, or urgent target vessel revascularization within 30 days of stent implantation. The genotype distribution of the study population was CC in 36.5%, CT in 46.7%, and TT in 16.8% of the patients. The incidence of the composite end point was 6.5% in T allele carriers and 5.3% in noncarriers (odds ratio for T allele carriage 1.23 [95% confidence interval, 0.81-1.86],P = .33). After adjusting for other baseline characteristics, the odds ratio for the composite end point was 1.15 (0.76-1.75). Therefore, C807T genotype has no significant influence on the major adverse events occurring after coronary artery stenting.