bluuebunny/crossref_metadata_2025_split huggingface dataset:
Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes
Authors: Veena Ponnazhagan , Traycoff Christie M. , Williams David A. , McMahel Jon , Rice Susan , Cornetta Ken , Srour Edward F. 
Published: October 2019

AbstractPrimitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.
The Effects of Colony-Stimulating Factor-1 on the Distribution of Mononuclear Phagocytes in the Developing Osteopetrotic Mouse
Authors: Roth Philip , Dominguez Melissa G. , Stanley E. Richard 
Published: October 2019

AbstractColony-stimulating factor-1 (CSF-1), the primary regulator of mononuclear phagocyte (M▹) production, exists as either a circulating or cell surface, membrane-spanning molecule. To establish transplacental transfer of maternal CSF-1, gestational day-17 mothers were injected intravenously with 125I-mouse CSF-1 or human rCSF-1, and the 125I-cpm or human CSF-1 concentrations were measured in fetal tissue, placenta, and fetal/maternal sera. Biologically active CSF-1 crossed the placenta and peaked in fetal tissue, placenta, and serum 10 minutes after injection. The role of CSF-1 in perinatal M▹ development was examined by studying the CSF-1–deficient osteopetrotic (csfmop/csfmop) mouse. Fetal/neonatal mice, derived from matings of either +/csfmopfemales with csfmop/csfmop males or the reciprocal pairings, were genotyped and tissue M▹ identified and quantified. In the presence of circulating maternal CSF-1 (+/csfmop mother), M▹ development incsfmop/csfmop liver was essentially complete at birth relative to +/csfmoplittermates, but significantly reduced in spleen, kidney, and lung. In the absence of circulating maternal CSF-1 (csfmop/csfmop mother), M▹ numbers at birth were reduced in csfmop/csfmopliver relative to the offspring of +/csfmopmothers, but were similar in spleen, kidney, and lung. We conclude that CSF-1 is required for the perinatal development of most M▹ in these tissues. Compensation for total absence of local CSF-1 production by circulating, maternal CSF-1 is tissue-specific and most prominent in liver, the first fetal organ perfused by placental blood. However, because some M▹ developed in the complete absence of CSF-1, other factors must also be involved in the regulation of macrophage development.
Functional Diversity of the CD8+ T-Cell Response to Epstein-Barr Virus (EBV): Implications for the Pathogenesis of EBV-Associated Lymphoproliferative Disorders
Authors: Nazaruk Rachel A. , Rochford Rosemary , Hobbs Monte V. , Cannon Martin J. 
Published: October 2019

AbstractEpstein-Barr virus (EBV)-specific CD8+ cytotoxic T cells are thought to be critical for the control of EBV, which persists in healthy individuals as a latent infection of B cells. However, recent observations have indicated that CD8+ T-cell responses are not uniformly cytotoxic and that CD8+ T cells may be subdivided into type 1 and type 2 subsets that parallel the classically described Th1 and Th2 subsets of CD4+ T cells. Using two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, we have identified two distinct but overlapping subsets of EBV-specific CD8+ T cells, the first of which expressed high levels of interferon γ (IFNγ), but little or no interleukin-4 (IL-4), whereas the second subset was IFNγ+/IL-4+ double-positive. A significant proportion of EBV-specific CD8+ T cells also expressed IL-13. Subsequent analysis of a panel of 27 EBV-specific CD8+ T-cell clones showed inverse relationships between EBV-specific cytotoxicity and secretion of IL-4, IL-10, and IFNγ, respectively. IL-10 was not secreted by the 11 most strongly cytotoxic clones, suggesting that IL-10 secretion may provide a functional definition of an EBV-specific type 2 CD8+ T-cell subset with reduced EBV-specific cytotoxicity. Finally, we have demonstrated that EBV-specific CD8+ T cells that express type 2 cytokines possess the ability to activate resting B cells. EBV-specific CD8+ T cells thus have the potential to reactivate latent EBV infection in vivo and may contribute to the development of EBV-associated lymphoproliferative disorders and lymphoma.
Non–Host-Reactive Donor CD8+ T Cells of Tc2 Phenotype Potently Inhibit Marrow Graft Rejection
Authors: Fowler Daniel H. , Whitfield Bernard , Livingston Michael , Chrobak Paul , Gress Ronald E. 
Published: October 2019

AbstractDonor CD8+ T cells capable of host reactivity inhibit marrow graft rejection, but also generate graft-versus-host disease (GVHD). To evaluate whether the Tc1- and Tc2-type subsets of CD8 cells might inhibit rejection without host reactivity, we established an F1 into-parent murine bone marrow transplant model. Donor Tc1 and Tc2 cells were generated that preferentially secreted type I or type II cytokines; both subsets possessed potent cytolytic function, and clonally deleted host-type allospecific precursor CTL in vitro. B6 hosts receiving 950 cGy irradiation did not reject the donor marrow (F1 chimerism of 78.6%; n = 10), whereas hosts receiving 650 cGy rejected the donor marrow (3.8% chimerism; n = 8). At 650 cGy irradiation, the addition of Tc2 cells to the F1 marrow resulted in extensive F1 chimerism (70.8%) in 8 of 8 recipients; in contrast, alloengraftment was not consistently observed in mice receiving Tc1 cells or unmanipulated CD8 cells. Furthermore, when the preparative regimen was further reduced to 600 cGy, only hosts receiving the Tc2-type cells did not reject the F1 marrow. We conclude that Tc2 cells potently inhibit marrow graft rejection without inducing an alloaggressive response and that non–host-reactive Tc2 cells therefore facilitate engraftment across genetic barriers with reduced GVHD.
Thrombopoietin Requires Additional Megakaryocyte-Active Cytokines for Optimal Ex Vivo Expansion of Megakaryocyte Precursor Cells
Authors: Williams J. Lynne , Pipia George G. , Datta Nabanita S. , Long Michael W. 
Published: October 2019

Abstract
Little is known concerning the interaction of thrombopoietin (TPO) with other megakaryocyte-active cytokines in directing the early events of megakaryocyte development. Culture of CD34+ cells in interleukins (IL) -1, -6, -11, plus stem cell factor (SCF; S) results in a 10- to 12-fold expansion in total cell numbers, whereas total CD41+ megakaryocytes are expanded ∼120-fold over input levels. Addition of TPO to IL-1, -6, -11, S generates a biphasic proliferation of CD41+ cells, accelerates their rate of production, and results in an ex vivo expansion of more than 200-fold. The addition of Flt-3 ligand (FL) increases CD41+ cell expansion to ∼380-fold over input levels. In the absence of TPO, ∼95% of the expanded cells show the phenotype of promegakaryoblasts; TPO and/or FL addition increases CD41 antigen density and ploidy in a subpopulation of promegakaryoblasts. A moderate (approximately sevenfold) expansion of megakaryocyte progenitor cells (colony-forming unit-megakaryocyte) occurs in the presence of IL-1, -6, -11, S, and the addition of TPO to this cocktail yields an ∼17-fold expansion. We conclude that early proliferative events in megakaryocyte development in vitro are regulated by multiple cytokines, and that TPO markedly affects these early developmental steps. However, by itself, TPO is neither necessary nor sufficient to generate a full proliferative/maturational in vitro response within the megakaryocyte compartment. TPO clearly affects terminal differentiation and the development of (some) high-ploidy human megakaryocytes. However, its limited in vitro actions on human cell polyploidization suggest that additional megakaryocyte-active cytokines or other signals are essential for the maximal development of human megakaryocytes.
Methylenetetrahydrofolate Reductase C677T Mutation, Plasma Homocysteine, and Folate in Subjects From Northern Italy With or Without Angiographically Documented Severe Coronary Atherosclerotic Disease: Evidence for an Important Genetic-Environmental Interaction
Authors: Girelli Domenico , Friso Simonetta , Trabetti Elisabetta , Olivieri Oliviero , Russo Carla , Pessotto Renzo , Faccini Giovanni , Pignatti Pier Franco , Mazzucco Alessandro , Corrocher Roberto 
Published: October 2019

Abstract
Moderate elevation of plasma total homocysteine (tHcy) is a strong and independent risk factor for coronary artery disease (CAD). It can result from genetic or nutrient-related disturbances in the transsulfuration or remethylation pathways for Hcy metabolism. A point mutation (C677T; Ala-to-Val) in the gene encoding the 5,10-methylenetetrahydrofolate reductase (MTHFR) has been recently reported to render the enzyme thermolabile and less active. Studies on the role of this mutation as a risk factor for CAD have given conflicting results. We studied a total of 415 subjects, 278 with angiographically documented multivessel CAD and 137 with angiographically documented normal coronary arteries. The overall frequency of the MTHFR V/V homozygous genotype was 15.7% (with 52.5% heterozygous and 31.8% normal). Subgroup analysis showed no significant differences between CAD and CAD-free subjects. A genotype/phenotype correlation study showed a marked effect of folate on the association between MTHFR genotypes and tHcy. Among individuals with folate levels below the median (11.5 nmol/L), fasting tHcy was significantly increased not only in V/V homozygotes (by 59%) but also, at intermediate values, in A/V heterozygotes (by 21% on average). Conversely, the mutation resulted neutral with respect to tHcy levels in subjects with adequate folate levels. We conclude that, in our population, the MTHFR C677T mutation is rather common, but it does not appear to be associated per se to CAD. A genetic-environmental interaction may contribute to the vascular risk by elevating tHcy when folate status is low.
Inhibitory Anti–Factor V Antibodies Bind to the Factor V C2 Domain and Are Associated With Hemorrhagic Manifestations
Authors: Ortel Thomas L. , Moore Karen D. , Quinn-Allen Mary Ann , Okamura Takashi , Sinclair Allen J. , Lazarchick John , Govindan Ramaswamy , Carmagnol Françoise , Kane William H. 
Published: October 2019

AbstractFactor V inhibitors may develop as spontaneous autoantibodies, as alloantibodies after exposure to bovine thrombin preparations, or in factor V–deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to life-threatening hemorrhage. We have characterized the anti–factor V antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in a prothrombinase assay. Nine IgG fractions, including the 8 patients with hemorrhage, immunoprecipitated the isolated second C-type domain (C2). The 8 IgG fractions from the symptomatic patients also immunoprecipitated recombinant chimeras containing only the N-terminal third of the factor V C2 domain, and isolated recombinant C2 domain abrogated the inhibitory effect of the antibodies. Five of the inhibitory IgG fractions blocked binding of factor V to phosphatidylserine. These results suggest that inhibitory anti–factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2 domain, whether originating spontaneously or after exposure to bovine thrombin.
Biochemical and Pharmacological Properties of SANORG 34006, a Potent and Long-Acting Synthetic Pentasaccharide
Authors: Herbert J.M. , Hérault J.P. , Bernat A. , van Amsterdam R.G.M. , Lormeau J.C. , Petitou M. , van Boeckel C. , Hoffmann P. , Meuleman D.G. 
Published: October 2019

AbstractSANORG 34006 is a new sulfated pentasaccharide obtained by chemical synthesis. It is an analog of the “synthetic pentasaccharide” (SR 90107/ ORG 31540) which represents the antithrombin (AT) binding site of heparin. SANORG 34006 showed a higher affinity to human AT than SR 90107/ORG 31540 (kd = 1.4 ± 0.3 v 48 ± 11 nmol/L), and it is a potent and selective catalyst of the inhibitory effect of AT on factor Xa (1,240 ± 15 anti–factor Xa U/mg v850 ± 27 anti-factor Xa U/mg for SR 90107/ORG 31540). In vitro, SANORG 34006 inhibited thrombin generation occurring via both the extrinsic and intrinsic pathway. After intravenous (IV) or subcutaneous (SC) administration to rabbits, SANORG 34006 displayed a long-lasting anti–factor Xa activity and inhibition of thrombin generation (TG) ex vivo. SANORG 34006 was slowly eliminated after IV or SC administration to rats, rabbits, and baboons, showed exceptionally long half-lives (between 9.2 hours in rats and 61.9 hours in baboons), and revealed an SC bioavailability near 100%. SANORG 34006 displayed antithrombotic activity by virtue of its potentiation of the anti–factor Xa activity of AT. It strongly inhibited thrombus formation in experimental models of thromboplastin/stasis-induced venous thrombosis in rats (IV) and rabbits (SC) (ED50values = 40.0 ± 3.4 and 105.0 ± 9.4 nmol/kg, respectively). The duration of its antithrombotic effects closely paralleled the ex vivo anti–factor Xa activity. SANORG 34006 enhanced rt-PA–induced thrombolysis and inhibited accretion of125I-fibrinogen onto a preformed thrombus in the rabbit jugular vein suggesting that concomitant use of SANORG 34006 during rt-PA therapy might be helpful in facilitating thrombolysis and preventing fibrin accretion onto the thrombus under lysis. Contrary to standard heparin, SANORG 34006 did not enhance bleeding in a rabbit ear incision model at a dose that equals 10 times the antithrombotic ED50 in this species and, therefore, exhibited a favorable therapeutic index. We suggest that SANORG 34006 is a promising compound in the treatment and prevention of various thrombotic diseases.
Interleukin-15 (IL-15) Can Replace the IL-2 Signal in IL-2–Dependent Adult T-Cell Leukemia (ATL) Cell Lines: Expression of IL-15 Receptor α on ATL Cells
Authors: Yamada Yasuaki , Sugawara Kazuyuki , Hata Tomoko , Tsuruta Kazuto , Moriuchi Ryozo , Maeda Takahiro , Atogami Sunao , Murata Ken , Fujimoto Kenshi , Kohno Tomoko , Tsukasaki Kunihiro , Tomonaga Masao , Hirakata Yo-ichi , Kamihira Shimeru 
Published: October 2019

AbstractInterleukin-15 receptor (IL-15R) and IL-2R have the same β and γ chains, but IL-15R has a specific α chain distinct from that of IL-2Rα, which is indispensable for the high affinity binding of IL-15. In the present study, we examined four IL-2-dependent adult T-cell leukemia (ATL) cell lines for their IL-15R expression. All cell lines bound IL-15, which was not inhibited by a 100-fold excess amount of IL-2, proliferated in response to IL-15 to the same degree as to the stimulation with IL-2, and were maintained without IL-2. The responses to 1L-15 were inhibited by the antibodies against IL-2R β or γ chains but was not by the IL-2R α chain antibody. [125I]–IL-15 exhibited a single high-affinity binding with an apparent kd of 0.17 nmol/L. Reverse transcription–coupled polymerase chain reaction (RT-PCR) showed that the cell lines had the mRNA of IL-15R α. The cell lines also had IL-15 mRNA. Despite the presence of IL-15 mRNA, the cell lines did not secrete IL-15, and the culture supernatants of fresh ATL cells and plasma from the patients did not contain a detectable amount of IL-15 with a few exceptional cases, although fresh ATL cells also responded to IL-15. These results suggest that ATL cells have the complete form of IL-15R and respond to IL-15. Such an IL-15–dependent cell proliferation mechanism might be used in the development of ATL and for the invasion and proliferation of ATL cells in the visceral organs.
Increases in Neutral, Mg2+-Dependent and Acidic, Mg2+-Independent Sphingomyelinase Activities Precede Commitment to Apoptosis and Are Not a Consequence of Caspase 3–Like Activity in Molt-4 Cells in Response to Thymidylate Synthase Inhibition by GW1843
Authors: Laethem Ronald M. , Hannun Yusuf A. , Jayadev Supriya , Sexton Connie J. , Strum Jay C. , Sundseth Rebecca , Smith Gary K. 
Published: October 2019

AbstractThymidylate synthase (TS) inhibition causes cell death, and this enzyme is the target for the important chemotherapy regime 5-fluorouracil/leucovorin. GW1843 (1843U89) is a potent and specific folate analog TS inhibitor in clinical development. Because of the importance of TS as a chemotherapy target, we are studying the mechanism of TS inhibition-induced cell death by GW1843. Ceramide is a regulatory lipid generated by the action of sphingomyelinase and is believed to signal apoptosis. The role of the ceramide in apoptotic signaling was studied in Molt-4 human T-cell leukemia cells undergoing cell death after treatment with GW1843. In response to GW1843, Molt-4 cells undergo apoptosis with both acidic pH, Mg2+-independent sphingomyelinase (ASMase) and neutral pH, Mg2+-dependent sphingomyelinase (NSMase) activities elevated as early steps in the initiation of apoptosis before Molt-4 commitment to death. These activities lead to ceramide production with kinetics consistent with a role as an effector molecule signaling the initiation of apoptosis in Molt-4 cells. These changes were found to be independent of caspase 3–like (CPP32/apopain) activity and DNA degradation, but were not separable from membrane blebbing or cell lysis in this cell line. In this report, kinetic evidence is provided for a role of ceramide in initiating GW1843-induced cell death of Molt-4 T-cell leukemia cells.
Treatment of Thrombocytopenia in Chimpanzees Infected With Human Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte Growth and Development Factor
Authors: Harker Laurence A. , Marzec Ulla M. , Novembre Francis , Sundell I. Birgitta , Waller Edmund K. , Karpatkin Simon , McClure Harold M. , Kelly Andrew B. , Stead Richard B. 
Published: October 2019

AbstractThree chimpanzees experimentally infected with human immunodeficiency virus (HIV) developed significant chronic thrombocytopenia after 5, 4, and 2 years, with peripheral platelet counts averaging 64 ± 19 × 103/μL (P = .004 compared with 228 ± 92 × 103/μL in 44 normal control animals), mean platelet volumes of 11.2 ± 1.8 fL (P &gt; .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P &lt; .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and corresponding viral loads of 534, 260, and 15 × 103 RNA viral copies/mL. Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) was administered subcutaneously (25 μg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees. PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold (from 64 ± 19 to 599 ± 260 × 103 platelets/μL;P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells); and (4) serum levels of Mpl ligand from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/μL to 9.9 ± 5.0 × 103/μL (P= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P &gt; .1). The serum levels of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal reductions during periods of thrombocytosis (P &lt; .07). PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P &gt; .5 compared with baseline values). The striking increase in peripheral platelet counts produced by PEG-rHuMGDF therapy implies that thrombocytopenia in HIV-infected chimpanzees is attributable to insufficient compensatory expansion in platelet production resulting from HIV-impaired delivery of platelets despite stimulated megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.
Interferon-γ Increases Expression of Chemokine Receptors CCR1, CCR3, and CCR5, But Not CXCR4 in Monocytoid U937 Cells
Authors: Zella Davide , Barabitskaja Oxana , Burns Jennifer M. , Romerio Fabio , Dunn Daniel E. , Revello Maria Grazia , Gerna Giuseppe , Jr Marvin S. Reitz , Gallo Robert C. , Weichold Frank F. 
Published: October 2019

AbstractChemokine receptors (CR), which can mediate migration of immune cells to the site of inflammation, also function as coreceptors for human immunodeficiency virus (HIV) entry into CD4+ T lymphocytes and antigen-presenting cells. We demonstrate here that interferon-γ (IFN-γ) increases the expression of chemokine receptors CCR1, CCR3, and CCR5 in monocytoid U937 cells as detected by cell surface molecule labeling and mRNA expression, as well as by intracellular calcium mobilization and cell migration in response to specific ligands. The increased expression of these chemokine receptors also results in an enhanced HIV-1 entry into cells. Our data provide evidence for a relationship of cellular pathways that are induced by IFN-γ with those that regulate chemokine receptor expression.