bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective Ablation of Human T-Cell Lymphotropic Virus Type 1 p12I Reduces Viral Infectivity In Vivo
Authors: Collins Nathaniel D. , Newbound Garret C. , Albrecht Björn , Beard Jennifer L. , Ratner Lee , Lairmore Michael D.
Published: October 2019

Human T-cell lymphotropic virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia and HTLV-1-associated myelopathy. Novel, yet conserved RNA transcripts encoded from open reading frames (ORFs) I and II of the viral pX region are expressed both in vitro and in infected individuals. The ORF I mRNA encodes the protein p12I, which has been shown to localize to cellular endomembranes, cooperate with bovine papillomavirus E5 in transformation, as well as bind to the IL-2 receptor β and γ chains and the H+ vacuolar ATPase. It is unknown what role p12I plays in the viral life cycle. Using an infectious molecular clone of HTLV-1 (ACH) and a derivative clone, ACH.p12I, which fails to produce the p12Imessage, we investigated the importance of p12I in infected primary cells and in a rabbit model of the infection. ACH.p12I was infectious in vitro as shown by viral passage in culture and no qualitative or quantitative differences were noted between ACH and ACH.p12I in posttransfection viral antigen production. However, in contrast to ACH, ACH.p12I failed to establish persistent infection in vivo as indicated by reduced anti-HTLV-1 antibody responses, failure to demonstrate viral p19 antigen production in peripheral blood mononuclear cell (PBMC) cultures, and only transient detection of provirus by polymerase chain reaction in PBMC from ACH.p12I-inoculated rabbits. These results are the first to show the essential role of HTLV-1 p12I in the establishment of persistent viral infection in vivo and suggest potential new targets in antiviral strategies to prevent HTLV-1 infection.
Induction of Differentiation in Acute Promyelocytic Leukemia Cells by 9-cis Retinoic Acid α-Tocopherol Ester (9-cisTretinoin Tocoferil)
Authors: Makishima Makoto , Umesono Kazuhiko , Shudo Koichi , Naoe Tomoki , Kishi Kenji , Honma Yoshio
Published: October 2019

Acute promyelocytic leukemia (APL) has a specific genetic rearrangement between the retinoic acid receptor (RAR)-α gene and the pml nuclear protein gene. All-trans retinoic acid (ATRA) induces granulocytic differentiation of APL-derived cells and is used to treat APL patients. However, ATRA interacts with normal cells with RAR throughout the entire body, and when used at high doses or over a long duration, it induces several adverse effects. The development of drugs that selectively act on APL cells may contribute to increasing the therapeutic efficacy of APL treatment as well as elucidating the mechanisms of response to ATRA. In this study, 9-cis retinoic acid α-tocopherol ester (9CTT) inhibited the proliferation of APL-derived NB4 and HT93 cells and induced differentiation markers, such as granulocytic maturation, nitroblue tetrazolium reduction, and CD11b expression, in these cells. The effects of 9CTT on non-APL cells, including HL-60 and U937 cells, were much weaker than those on APL cells, and tretinoin tocoferil (TT), which is an α-tocopherol ester of ATRA, did not induce the differentiation of APL cells as effectively as 9CTT. The differentiation-inducing effects of 9CTT were inhibited by RAR antagonists. 9CTT and TT similarly induced the transactivating activity of RARs, but were not effective on RXRs. 9CTT downregulated the expression of PML/RAR-α protein more effectively than TT, which suggests that it may be involved in the selectivity of 9CTT against APL cells. Interestingly, 9CTT enhanced the differentiation of APL cells induced by ATRA, 9-cis retinoic acid, and synthetic retinobenzoic acids. Combined with 1α,25-dihydroxyvitamin D3 (VD3), 9CTT also more than additively induced the differentiation of APL cells. Thus, 9CTT, alone or in combination with other retinoids or VD3, may be useful for the treatment of APL.
Individual Cell Analysis of the Cytokine Repertoire in Human Immunodeficiency Virus-1–Infected Monocytes/Macrophages by a Combination of Immunocytochemistry and In Situ Hybridization
Authors: Esser Ruth , Glienke Wolfgang , Andreesen Reinhard , Unger Ronald E. , Kreutz Marina , Rübsamen-Waigmann Helga , von Briesen Hagen
Published: October 2019

The expression of many cytokines is dysregulated in individuals infected with the human immunodeficiency virus-1 (HIV-1). To determine the effects of HIV-1 infection on cytokine expression in individual cells (at the single cell level), we investigated the intracellular levels of proinflammatory cytokines (tumor necrosis factor [TNF]-α, interleukin [IL]-1β, IL-6, and IL-8) and hematopoietic growth factors (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) in monocyte-derived macrophages, mock-infected, or infected with HIV-1 by immunocytochemical staining for cytokine protein and compared this with secreted cytokine levels as determined by specific enzyme-linked immunosorbent assay (ELISA). No difference in the frequency or intensity of cell-associated immunocytochemical cytokine staining could be observed between HIV-1 and mock-infected cells even though the level of secreted proinflammatory cytokines increased and the hematopoietic growth factors decreased in HIV-1–infected cultures. Furthermore, equal expression of cytokine mRNA was observed in all cells in the culture regardless of whether the cells were productively infected with HIV-1 as determined by double-labelling immunocytochemical staining for HIV-1 p24 antigen and in situ hybridization for cytokine mRNA expression. These results indicate that HIV-1 infection results in dysregulation of intracellular cytokine mRNA expression and cytokine secretion not only in HIV-1–infected cells, but also through an indirect way(s) affecting cells not producing virus.
The Bactericidal/Permeability-Increasing Protein (BPI) Is Present in Specific Granules of Human Eosinophils
Authors: Calafat Jero , Janssen Hans , Tool Anton , Dentener Mieke A. , Knol Edward F. , Rosenberg Helene F. , Egesten Arne
Published: October 2019

Eosinophils participate in the inflammatory response seen in allergy and parasitic infestation, but a role in host defense against bacterial infection is not settled. The bactericidal/permeability-increasing protein (BPI) has been demonstrated in neutrophils and it exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species. Using the Western blot technique, a 55-kD band, corresponding to BPI, was detected in lysates from both neutrophils and eosinophils. The localization of BPI in immature and mature eosinophils was investigated using immunoelectron microscopy. BPI was found in immature and mature specific granules of eosinophils and was detected in phagosomes as well, indicating release of the protein from the granules into the phagosomes. Using a specific enzyme-linked immunosorbent assay, eosinophils were shown to contain 179 ng of BPI/5 × 106 eosinophils compared with 710 ng BPI/5 × 106 neutrophils. The presence of BPI in eosinophils suggests a role for these cells in host defense against Gram-negative bacterial invasion or may suggest a role for BPI against parasitic infestation.
Platelet/Endothelial Cell Adhesion Molecule-1 Serves as a Costimulatory Agonist Receptor That Modulates Integrin-Dependent Adhesion and Aggregation of Human Platelets
Authors: Varon David , Jackson Denise E. , Shenkman Boris , Dardik Rima , Tamarin Ilya , Savion Naphtali , Newman Peter J.
Published: October 2019

Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kD member of the Ig gene superfamily that is expressed on the surface of circulating platelets, monocytes, neutrophils, and selective T-cell subsets. It is also a major component of the endothelial cell intercellular junction. Previous studies have shown that cross-linking PECAM-1 on the surface of leukocytes results in the activation of adhesion molecules of both the β1 and β2integrin family. In addition, the process of leukocyte transendothelial migration appears to be mediated, at least in part, by homophilic adhesive interactions that take place between leukocyte and endothelial cell junctional PECAM-1 molecules. However, little is known about the functional role of this membrane glycoprotein in human platelets. In the present study, we examined the effects of PECAM-1 engagement on integrin-mediated platelet-extracellular matrix or platelet-platelet interactions. Bivalent, but not monovalent, anti–PECAM-1 monoclonal antibodies (MoAbs) specific for membrane-proximal Ig-homology domain 6 significantly augmented platelet deposition (increased surface coverage) and aggregation (increased average size) onto extracellular matrix, under both oscillatory or defined low shear flow conditions (200 s−1) in a modified cone and plate viscometer. Moreover, bivalent anti-domain 6 MoAbs were capable of serving as costimulatory agonists to markedly enhance both adenosine diphosphate (ADP)- and platelet activating factor (PAF)-induced platelet aggregation responses. These antibodies appeared to act via outside-in signal transduction through PECAM-1, as evidenced by the fact that their binding (1) led to conformational changes in the αIIbβ3 integrin complex, (2) induced surface expression of P-selectin, and (3) resulted in the tyrosine phosphorylation of PECAM-1. Together, these data support a role for PECAM-1 in cellular activation and suggest that PECAM-1 may serve as a costimulatory agonist receptor capable of modulating integrin function in human platelets during adhesion and aggregation.
Molecular Cloning and Characterization of Decay-Accelerating Factor Deficiency in Cromer Blood Group Inab Phenotype
Authors: Wang Li , Uchikawa Makoto , Tsuneyama Hatsue , Tokunaga Katsushi , Tadokoro Kenji , Juji Takeo
Published: October 2019

An additional decay-accelerating factor (DAF) mutation, designated as Inab phenotype in the Cromer blood group system, was recently identified in a 28-year-old Japanese woman (H.A.). The red blood cells of H.A., like those of other Inab phenotype individuals, were negative for Cromer system antigens, Cra, Tca, Dra, UMC, and IFC. The deficiency of DAF on the red blood cells of H.A. has been shown by immunoblotting with a murine monoclonal antibody to DAF. Molecular analysis has shown that H.A. is homozygous for a single nucleotide substitution, C1579→A, at the position 24 bp upstream of the 3′-end of exon 2 of the DAF gene. This substitution causes the activation of a novel cryptic splice site and results in the production of mRNA with a 26 bp deletion. The deletion introduces a reading frame shift and creates a stop codon immediately downstream of the deletion. Translation of mRNA would be terminated at the first amino acid residue of the second short consensus repeat (SCR2) domain (exon 3) of DAF. The functional domains of DAF's complement regulatory activity and the carboxy-terminal signal domains for glycosylphosphatidylinositol (GPI) anchoring are predicted to be lacking in H.A. Thus, there would be no DAF present on the cell surface.
Effects of Cytokines on Platelet Production From Blood and Marrow CD34+ Cells
Authors: Norol Françoise , Vitrat Natacha , Cramer Elisabeth , Guichard Josette , Burstein Samuel A. , Vainchenker William , Debili Najet
Published: October 2019

The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF– or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.
Antibodies From Patients With Heparin-Induced Thrombocytopenia/Thrombosis Recognize Different Epitopes on Heparin: Platelet Factor 4
Authors: Suh Jang-Soo , Aster Richard H. , Visentin Gian P.
Published: October 2019

Antibodies associated with heparin-induced thrombocytopenia/thrombosis (HITT) are now thought to be specific for complexes formed between heparin and platelet factor 4 (PF4), a basic protein found normally in platelet alpha granules. How these antibodies cause thrombocytopenia and, in some patients, thrombosis, is not fully understood, in part because purified antibodies that could be labeled and used as probes to characterize target epitopes have not been available. We developed a novel method for antibody purification involving binding to and elution from PF4 complexed to heparin immobilized by end-linkage (EL) to a solid phase. Isolated antibodies were functional and after biotinylation, reacted with heparin: PF4 complexes in the same manner as unlabeled antibodies. Using these probes, we found that antibodies from 11 patients with HITT recognized two, and probably three, distinct sites on heparin: PF4 complexes. The antibodies did not bind to PF4 complexed with heparin immobilized by multiple chemical cross-linkages, suggesting that the heparin molecule must be in a flexible, relatively unconstrained state to react with PF4 in such a way as to create sites for HITT antibody binding.
Expression and Activation of the Nonreceptor Tyrosine Kinase Tec in Human B Cells
Authors: Kitanaka Akira , Mano Hiroyuki , Ellen Conley Mary , Campana Dario
Published: October 2019

The tyrosine kinase Tec belongs to a new group of structurally related nonreceptor tyrosine kinases that also includes Btk and Itk. Previous studies have suggested that these kinases have lineage-specific roles, with Tec being involved mainly in the regulation of cytokine-mediated myeloid cell growth and differentiation. In this study, we investigated expression and activation of Tec in human B-lymphoid cell lines representing different stages of B-cell maturation, including pro-B (RS4;11, 380, REH), pre-B (NALM6), and mature B (Ramos, and one Epstein-Barr virus [EBV]-transformed lymphoblastoid line) cells. Like Btk, Tec protein was expressed in all B-cell lines tested. Tec was also highly expressed in two EBV-transformed lymphoblastoid lines derived from patients with X-linked agammaglobulinemia (XLA) lacking Btk expression, as well as in tonsillar lymphoid cells. In surface immunoglobulin-positive B cells (Ramos), ligation of the B-cell antigen receptor (BCR) with anti-IgM antibodies caused marked tyrosine phosphorylation of Tec and increased Tec tyrosine kinase activity. Likewise, cross-linking of CD19 with a monoclonal antibody in BCR-negative pro-B (RS4;11, 380) and pre-B (NALM6) cells induced Tec tyrosine phosphorylation and increased Tec autophosphorylation, as well as Btk activation. Tyrosine phosphorylation of Tec, but not of Btk, was detectable in RS4;11 cells after CD38 ligation, suggesting that these kinases are regulated differently. We conclude that Tec is expressed and can be stimulated throughout human B-cell differentiation, implying that this tyrosine kinase plays a role in B-cell development and activation.
Application of Interphase Fluorescence In Situ Hybridization for the Detection of the Burkitt Translocation t(8;14)(q24;q32) in B-Cell Lymphomas
Authors: Siebert Reiner , Matthiesen Peter , Harder Svetlana , Zhang Yanming , Borowski Annekathrin , Zühlke-Jenisch Reina , Metzke Simone , Joos Stefan , Weber-Matthiesen Klaus , Grote Werner , Schlegelberger Brigitte
Published: October 2019

The translocation t(8;14)(q24;q32) is the characteristic chromosomal aberration of Burkitt's-type lymphomas and leukemias (BLs). On the molecular level, the t(8;14) juxtaposes the c-myc gene in 8q24 next to the IgH locus in 14q32, resulting in overexpression of the transcription factor c-Myc. The detection of a t(8;14) is a major aim in the diagnostic process of all patients with high-grade B-cell lymphomas because treatment strategies differ between BL and other high-grade lymphomas. As chromosome analyses are sometimes hampered by the low yield or poor quality of metaphase spreads and as the application of molecular genetic techniques is limited by the distribution of the 8q24 breakpoints over a region of about some hundred kilobases, we set out to establish an interphase fluorescence in situ hybridization (FISH) assay for the detection of the t(8;14). A cosmid probe hybridizing to the IgH constant region in 14q32 was combined with a differently labeled probe of pooled cosmid clones spanning the c-myc locus in 8q24. Interphase nuclei lacking a t(8;14) show two separated signals corresponding to each probe, whereas interphase nuclei carrying a t(8;14) display a split of the c-myc probe and a colocalization of at least one of the splitted signals with the IgH probe. Based on the results of extensive control studies, the cutoff level for this stringent (type I) criteria was set at 2%. Additionally, colocalization of at least one c-myc signal with one IgH signal alone (without signal split for the c-myc probe) was used as a less stringent (type II) criteria with a cutoff limit of 11%. Nine BLs and one Burkitt-like lymphoma were investigated by this approach. Cytogenetically, all tumors contained a translocation t(8;14)(q24;q32) except for one BL, in which cytogenetic analysis had failed. In interphase FISH, all lymphomas and leukemias met the less stringent criteria for the diagnosis of the t(8;14). Additionally, in all tumors but the Burkitt-like lymphoma, a t(8;14) could be diagnosed according to the stringent criteria. The percentage of cells found to harbor the t(8;14) by FISH ranged from 4.3% to 100%. Comparison of cytogenetic and FISH results revealed a significantly lower percentage of t(8;14)+ interphase nuclei than metaphase cells (P =  .004). In conclusion, the described FISH assay provides a feasible and sensitive tool for the routine detection of the translocation t(8;14) in interphase cells which might also offer new insights into the biology of high-grade B-cell lymphomas.
Protein C Inhibitor Acts as a Procoagulant by Inhibiting the Thrombomodulin-Induced Activation of Protein C in Human Plasma
Authors: Elisen Marc G.L.M. , von dem Borne Peter A.Kr. , Bouma Bonno N. , Meijers Joost C.M.
Published: October 2019

Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited by PCI. The paradoxical inhibitory effect of PCI on both coagulant and anticoagulant proteases raised questions about the role of PCI in plasma. We studied the role of thrombomodulin (TM)-dependent inhibition of thrombin by PCI in a plasma system. Clotting was induced by addition of tissue factor to recalcified plasma in the absence or presence of TM, and clot formation was monitored using turbidimetry. In the absence of TM, PCI-deficient plasma showed a slightly shorter coagulation time compared with normal plasma. Reconstitution with a physiologic amount of PCI gave normal clotting times. Addition of PCI to normal plasma and protein C–deficient plasma resulted in a minor prolongation of the clotting time. This suggested that PCI can act as a weak coagulation inhibitor in the absence of TM. TM caused a strong anticoagulant effect in normal plasma due to thrombin scavenging and activation of the protein C anticoagulant pathway. This effect was less pronounced when protein C–deficient plasma was used, but could be restored by reconstitution with protein C. When PCI was added to protein C–deficient plasma in the presence of TM, a strong anticoagulant effect of PCI was observed. This anticoagulant effect was most likely caused by the TM-dependent thrombin inhibition by PCI. However, when PCI was added to normal plasma containing TM, a strong procoagulant effect of PCI was observed, due to the inhibition of protein C activation. PCI-deficient plasma was less coagulant in the presence of TM. A concentration-dependent increase in clotting time was observed when PCI-deficient plasma was reconstituted with PCI. The combination of these results suggest that the major function of PCI in plasma during coagulation is the inhibition of thrombin. A decreased generation of activated protein C is a procoagulant consequence of the TM-dependent thrombin inhibition by PCI. We conclude that TM alters PCI from an anticoagulant into a procoagulant during tissue factor-induced coagulation.
In Vitro Regulation of FcRIα Expression on Human Basophils by IgE Antibody
Authors: MacGlashan Donald , McKenzie-White Jane , Chichester Kristine , Bochner Bruce S. , Davis Frances M. , Schroeder John T. , Lichtenstein Lawrence M.
Published: October 2019

In vivo studies suggested the possibility of an IgE-dependent regulation of high-affinity (FcRI) IgE receptor expression on basophils. The current studies extend these observations to in vitro cultures of human basophils. Incubation of basophils for 3 to 4 weeks resulted in a slow dissociation of IgE antibody, during which time FcRI expression decreased, as measured by flow cytometry using the anti-FcRIα monoclonal antibody, 22E7, or by measuring FcRIα mass by Western blotting of whole-cell lysates. Culture of basophils with IgE resulted in upregulation of FcRIα expression by both flow cytometry and Western blotting of whole-cell lysates. Upregulation followed a linear time course during 2 weeks of culture. The relative increase in FcRIα density depended on the starting density; with starting densities of FcRIα of 10,000 to 170,000 per basophil, the upregulation varied 20- to 1.1-fold, respectively. Upregulation occurred in high-purity basophils, was not influenced by IgG at concentrations up to 1 mg/mL, and was inhibited by dimeric IgE. Heat-inactivated IgE was less effective and the monoclonal antibody CGP51901 that prevents IgE binding to FcRIα blocked the ability of IgE to induce upregulation. The dose-response curve for IgE-induced upregulation had an effective concentration50 of 230 ng/mL. Although the receptor through which IgE induces this upregulation is not yet known, several characteristics suggest that the upregulation is mediated by IgE interacting through FcRIα itself.
TEL-AML1 Fusion Transcript in Relapsed Childhood Acute Lymphoblastic Leukemia
Authors: Seeger Karlheinz , Adams Hans-Peter , Buchwald Dirk , Beyermann Birgit , Kremens Bernhard , Niemeyer Charlotte , Ritter Jörg , Schwabe Dirk , Harms Dörte , Schrappe Martin , Henze Günter
Published: October 2019

The cryptic translocation t(12;21)(p13;q22) has been recently recognized as the most common genetic rearrangement in B-lineage childhood acute lymphoblastic leukemia (ALL). The resulting fusion transcript, termed TEL-AML1, has been associated with an excellent prognosis at initial ALL diagnosis. Hence, we postulated that the incidence of TEL-AML1 fusion should be lower in patients with ALL relapse. To address this assumption and to investigate the prognostic significance of TEL-AML1 expression in relapsed childhood ALL, bone marrow samples of 146 children were analyzed by reverse-transcriptase (RT)-polymerase chain reaction (PCR). All children were treated according to Berlin-Frankfurt-Münster (BFM) ALL relapse trial protocols (ALL-REZ BFM 90-96). Their clinical features and outcome were compared with those of 262 patients who could not be tested due to lack of bone marrow samples. Thirty-two of 146 children with relapsed ALL were TEL-AML1–positive. Four of the negative patients had T-lineage and nine Philadelphia chromosome (Ph1)-positive leukemia. Thus, the incidence ofTEL-AML1 in relapsed Ph1-negative, B-cell precursor ALL is 32 of 133 (24%). The 32 TEL-AML1–positive and 101 negative patients differed significantly with respect to duration of last remission (42.5 v 27 months; P = .0001) and age at initial diagnosis (53.5 v 74 months;P = .0269). At a median follow-up time of 21.5 months, children positive for TEL-AML1 had a significantly (P = .0011) higher probability of event-free survival (EFS; 0.79 v 0.33). The predominant majority of patients had been treated for initial ALL according to German multicenter BFM (108 of 133) or Cooperative ALL study group (CoALL) (19 of 133) frontline protocols. The comparison of tested and not-tested (N = 262) patients showed no significant difference.TEL-AML1 positivity predicted a favorable short-term outcome; long-term results are unknown. Screening for TEL-AML1 should become routine at relapse diagnosis and might be used for therapy stratification in future trials.
Clinical-Grade Functional Dendritic Cells From Patients With Multiple Myeloma Are Not Infected With Kaposi's Sarcoma-Associated Herpesvirus
Authors: Tarte Karin , Olsen Sonja J. , Yang Lu Zhao , Legouffe Eric , Rossi Jean-François , Chang Yuan , Klein Bernard
Published: October 2019

Bone marrow dendritic cells (DC) from patients with multiple myeloma (MM) were recently reported to be infected with Kaposi's sarcoma-associated herpesvirus (KSHV). Because immunotherapy strategies using DC are very promising in this disease, we looked for KSHV DNA in clinical-grade DC generated in vitro from MM patients. Adherent apheresis cells from MM patients were maintained for 7 days in clinical-grade X-VIVO 15 culture medium supplemented with granulocyte-macrophage colony-stimulating factor, interleukin-4, or interleukin-13. Tumor necrosis factor α was added for the last 2 days. We obtained a cell population with a DC phenotype able to endocytose fluorescein isothiocyanate (FITC)-dextran and efficiently activate resting allogenic T lymphocytes. To detect KSHV DNA, we used polymerase chain reaction (PCR) followed by Southern blotting of PCR product with a sensitivity detecting a few copies of viral DNA. All the PCR were repeated in a blinded fashion three times, on 1 μg and 0.2 μg of genomic DNA, in two different laboratories. Clinical-grade DC from 10 (91%) of 11 patients were not infected with KSHV. The apheresis cells and the purified CD34+ cells from the same patients were also negative. A very weak PCR band was detected with DC from one patient, but the initial apheresis cells were negative. The detection of KSHV infection in 1 (9%) of 11 MM patients probably represents background seroprevalence. It seems likely that functional and clinical-grade DC from MM patients can safely be used in clinical trials.
Binding of Tissue-Plasminogen Activator to Fibrin: Effect of Ultrasound
Authors: Siddiqi Farhan , Odrljin Tatjana M. , Fay Philip J. , Cox Christopher , Francis Charles W.
Published: October 2019

Ultrasound reversibly alters the structure of polymerized fibrin, an effect that could influence tissue-plasminogen activator (t-PA) binding. We have, therefore, characterized the effects of ultrasound on binding of t-PA to fibrin using a novel system in which radiolabeled, active-site blocked, single chain tissue-plasminogen activator flowed through a fibrin gel at constant rate, and specific binding was determined by monitoring incorporation of radiolabel. Results using polymerized fibrin were compared with those using a surface of fibrin immobilized on Sepharose beads in a similar system. Interaction of t-PA with surface-immobilized fibrin involved two classes of binding sites (Kd = 31 nmol/L and 244 nmol/L) and a maximum binding ratio of 3.8 mol t-PA/mol fibrin. Ultrasound increased Kd for the high affinity site to 46 nmol/L (P < .0001), but it had no significant effects on the Kd 244 nmol/L site nor on Bmax. Tissue-plasminogen activator binding to noncrosslinked fibrin involved two sites with Kds of 267 nmol/L and 952 nmol/L, while a single Kd 405 nmol/L site was identified for crosslinked fibrin. Ultrasound had no significant effect on the binding affinity for noncrosslinked fibrin, but Bmaxwas increased in the presence of ultrasound, from 31 μmol/L to 43 μmol/L (P < .0001). Ultrasound decreased the Kd for crosslinked fibrin to 343 nmol/L (P = .026) and also increased Bmax from 22 μmol/L to 25 μmol/L (P = .015). Ultrasound also affected the kinetics of t-PA binding to fibrin, significantly accelerating the rate of dissociation by 77% ± 5% for noncrosslinked fibrin and by 69% ± 3% for crosslinked fibrin (P < .001 for each). These results indicate that ultrasound exposure accelerates t-PA binding, alters binding affinity, and increases maximum binding to polymerized fibrin, effects that may result from ultrasound-induced changes in fibrin structure.
Relationship Between Major Vault Protein/Lung Resistance Protein, Multidrug Resistance-Associated Protein, P-Glycoprotein Expression, and Drug Resistance in Childhood Leukemia
Authors: den Boer M.L. , Pieters R. , Kazemier K.M. , Rottier M.M.A. , Zwaan C.M. , Kaspers G.J.L. , Janka-Schaub G. , Henze G. , Creutzig U. , Scheper R.J. , Veerman A.J.P.
Published: October 2019

Cellular drug resistance is related to a poor prognosis in childhood leukemia, but little is known about the underlying mechanisms. We studied the expression of P-glycoprotein (P-gp), multidrug resistance (MDR)-associated protein (MRP), and major vault protein/lung resistance protein (LRP) in 141 children with acute lymphoblastic leukemia (ALL) and 27 with acute myeloid leukemia (AML) by flow cytometry. The expression was compared between different types of leukemia and was studied in relation with clinical risk indicators and in vitro cytotoxicity of the MDR-related drugs daunorubicin (DNR), vincristine (VCR), and etoposide (VP16) and the non–MDR-related drugs prednisolone (PRD) and L-asparaginase (ASP). In ALL, P-gp, MRP, and LRP expression did not differ between 112 initial and 29 unrelated relapse samples nor between paired initial and relapse samples from 9 patients. In multiple relapse samples, LRP expression was 1.6-fold higher compared with both initial (P = .026) and first relapse samples (P = .050), which was not observed for P-gp and MRP. LRP expression was weakly but significantly related to in vitro resistance to DNR (Spearman's rank correlation coefficient 0.25, P = .016) but not to VCR, VP16, PRD, and ASP. No significant correlations were found between P-gp or MRP expression and in vitro drug resistance. Samples with a marked expression of two or three resistance proteins did not show increased resistance to the tested drugs compared with the remaining samples. The expression of P-gp, MRP, and LRP was not higher in initial ALL patients with prognostically unfavorable immunophenotype, white blood cell count, or age. The expression of P-gp and MRP in 20 initial AML samples did not differ or was even lower compared with 112 initial ALL samples. However, LRP expression was twofold higher in the AML samples (P < .001), which are more resistant to a variety of drugs compared with ALL samples. In conclusion, P-gp and MRP are unlikely to be involved in drug resistance in childhood leukemia. LRP might contribute to drug resistance but only in specific subsets of children with leukemia.
Complete Deficiency of Glycophorin A in Red Blood Cells From Mice With Targeted Inactivation of the Band 3 (AE1) Gene
Authors: Hassoun Hani , Hanada Toshihiko , Lutchman Mohini , Sahr Kenneth E. , Palek Jiri , Hanspal Manjit , Chishti Athar H.
Published: October 2019

Glycophorin A is the major transmembrane sialoglycoprotein of red blood cells. It has been shown to contribute to the expression of the MN and Wright blood group antigens, to act as a receptor for the malaria parasite Plasmodium falciparum and Sendai virus, and along with the anion transporter, band 3, may contribute to the mechanical properties of the red blood cell membrane. Several lines of evidence suggest a close interaction between glycophorin A and band 3 during their biosynthesis. Recently, we have generated mice where the band 3 expression was completely eliminated by selective inactivation of the AE1 anion exchanger gene, thus allowing us to study the effect of band 3 on the expression of red blood cell membrane proteins. In this report, we show that the band 3 −/− red blood cells contain protein 4.1, adducin, dematin, p55, and glycophorin C. In contrast, the band 3 −/− red blood cells are completely devoid of glycophorin A (GPA), as assessed by Western blot and immunocytochemistry techniques, whereas the polymerase chain reaction (PCR) confirmed the presence of GPA mRNA. Pulse-label and pulse-chase experiments show that GPA is not incorporated in the membrane and is rapidly degraded in the cytoplasm. Based on these findings and other published evidence, we propose that band 3 plays a chaperone-like role, which is necessary for the recruitment of GPA to the red blood cell plasma membrane.
Erythroid Krüppel-Like Factor Is Essential for β-Globin Gene Expression Even in Absence of Gene Competition, But Is Not Sufficient to Induce the Switch From γ-Globin to β-Globin Gene Expression
Authors: Guy Louis-Georges , Mei Qi , Perkins Andrew C. , Orkin Stuart H. , Wall Lee
Published: October 2019

Different genes in the β-like globin locus are expressed at specific times during development. This is controlled, in part, by competition between the genes for activation by the locus control region. In mice, gene inactivation of the erythroid Krüppel-like factor (EKLF) transcription factor results in a lethal anemia due to a specific and substantial decrease in expression of the fetal/adult-stage–specific β-globin gene. In transgenic mice carrying the complete human β-globin locus, EKLF ablation not only impairs human β-globin–gene expression but also results in increased expression of the human γ-globin genes during the fetal/adult stages. Hence, it may appear that EKLF is a determining factor for the developmental switch from γ-globin to β-globin transcription. However, we show here that the function of EKLF for β-globin–gene expression is necessary even in absence of gene competition. Moreover, EKLF is not developmental specific and is present and functional before the switch from γ-globin to β-globin–gene expression occurs. Thus, EKLF is not the primary factor that controls the switch. We suggest that autonomous repression of γ-globin transcription that occurs during late fetal development is likely to be the initiating event that induces the switch.
Induction of Fas (Apo-1, CD95)-Mediated Apoptosis of Activated Lymphocytes by Polyclonal Antithymocyte Globulins
Authors: Genestier Laurent , Fournel Sylvie , Flacher Monique , Assossou Olga , Revillard Jean-Pierre , Bonnefoy-Berard Nathalie
Published: October 2019

Polyclonal horse antilymphocyte and rabbit antithymocyte globulins (ATGs) are currently used in severe aplastic anemia and for the treatment of organ allograft acute rejection and graft-versus-host disease. ATG treatment induces a major depletion of peripheral blood lymphocytes, which contributes to its overall immunosuppressive effects. Several mechanisms that may account for lymphocyte lysis were investigated in vitro. At high concentrations (.1 to 1 mg/mL) ATGs activate the human classic complement pathway and induce lysis of both resting and phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells. At low, submitogenic, concentration ATGs induce antibody-dependent cell cytotoxicity of PHA-activated cells, but not resting cells. They also trigger surface Fas (Apo-1, CD95) expression in naive T cells and Fas-ligand gene and protein expression in both naive and primed T cells, resulting in Fas/Fas-L interaction-mediated cell death. ATG-induced apoptosis and Fas-L expression were not observed with an ATG preparation lacking CD2 and CD3 antibodies. Susceptibility to ATG-induced apoptosis was restricted to activated cells, dependent on IL-2, and prevented by Cyclosporin A, FK506, and rapamycin. The data suggest that low doses of ATGs could be clinically evaluated in treatments aiming at the selective deletion of in vivo activated T cells in order to avoid massive lymphocyte depletion and subsequent immunodeficiency.
Expression of the Epstein-Barr Virus Protein LMP1 Mediates Tumor Regression In Vivo
Authors: Cherney Barry W. , Sgadari Cecilia , Kanegane Chiharu , Wang Frederick , Tosato Giovanna
Published: October 2019

By stimulating the expression of murine IP-10 and Mig, CXC chemokines that inhibit neovascularization and cause damage to established tumor vasculature, human B cells immortalized with Epstein-Barr virus (EBV) can promote an effective antitumor response in athymic mice. In the present study, we examined the potential role of EBV in the induction of this antitumor response. Using a panel of EBV+ and EBV− Burkitt lymphoma (BL) cell lines, a significant correlation was detected between the expression of the EBV latency gene LMP1 and the occurrence of spontaneous tumor regression in athymic mice. Inoculation of LMP1+ and LMP1− BL cells in the same subcutaneous site resulted in tumors that completely regressed in a manner indistinguishable from that induced by EBV-immortalized B cells. EBV-converted BL30 and BL41 sublines infected with B95-8 virus expressed LMP1, generated tumors that frequently regressed spontaneously, and promoted an effective antitumor response against progressively growing tumors. In contrast, the EBV− BL30 and BL41 cell lines and the EBV-converted BL30 and BL41 infected with P3HR-1 virus did not express LMP1 protein, and generated progressively growing tumors in nude mice. When transfected with the LMP1 gene, BL41 cells produced tumors that regressed spontaneously in most cases, and could induce the regression of tumors derived from BL41 cells transfected with vector alone. Tumors induced by LMP1-expressing cells expressed murine IP-10 and Mig and displayed histological evidence of extensive tumor tissue necrosis and vascular damage. We conclude that the EBV protein LMP1 is likely responsible for the antitumor response elicited by EBV-immortalized cells in athymic mice.
Detection of Infectious Simian Immunodeficiency Virus in B- and T-Cell Lymphomas of Experimentally Infected Macaques
Authors: Maggiorella Maria Teresa , Monardo Francesca , Koanga-Mogtomo Martin Luther , Cioè Livia , Sernicola Leonardo , Corrias Franco , Baroni Carlo David , Verani Paola , Titti Fausto
Published: October 2019

An increasing frequency of malignant lymphomas occurs among patients infected by human immunodeficiency virus. Because of the close similarities to human malignancies, we used a nonhuman primate model to study the pathogenesis of simian immunodeficiency virus (SIV)–associated malignancies. Specifically, we investigated (1) the presence of the SIV genome in tumor cells, (2) the presence of coinfecting viruses, and (3) the presence of a rearrangement of the immunoglobulin and c-myc genes. We observed 5 cases of non-Hodgkin's lymphomas (4 of B- and 1 of T-cell origin) among 14 SIV-infected cynomolgus monkeys. No c-myc translocation was observed in the tumors, whereas B-cell lymphomas were characterized either by a monoclonal (in 2 of 4) or by an oligoclonal (in 2 of 4) VDJ rearrangements of the immunoglobulin heavy chain gene. Molecular, biological, and immunological analyses did show the presence of infectious SIV in the tumor cells of 1 T-cell and 2 oligoclonal B-cell lymphomas. Neither Simian T-lymphotropic nor Epstein-Barr viruses were detectable, whereas Simian herpes virus Macaca fascicularis-1 was detectable at a very low copy number in 3 of 4 B-cell lymphomas; however, only 1 of these also harbored the SIV genome. These results support the possibility that SIV may be directly involved in the process of B or T lymphomagenesis occurring in simian acquired immunodeficiency syndrome.
High CD34+ Cell Counts Decrease Hematologic Toxicity of Autologous Peripheral Blood Progenitor Cell Transplantation
Authors: Ketterer Nicolas , Salles Gilles , Raba Michel , Espinouse Daniel , Sonet Anne , Tremisi Pierre , Dumontet Charles , Moullet Isabelle , Eljaafari-Corbin Assia , Neidhardt-Berard Eve-Marie , Bouafia Fadhela , Coiffier Bertrand
Published: October 2019

Optimal numbers of CD34+ cells to be reinfused in patients undergoing peripheral blood progenitor cell (PBPC) transplantation after high-dose chemotherapy are still unknown. Hematologic reconstitution of 168 transplantations performed in patients with lymphoproliferative diseases was analyzed according to the number of CD34+ cells reinfused. The number of days from PBPC reinfusion until neutrophil recovery (>1.0 × 109/L) and unsustained platelet recovery (>50 × 109/L) were analyzed in three groups defined by the number of CD34+ cells reinfused: a low group with less than or equal to 2.5 × 106 CD34+ cells/kg, a high group with greater than 15 × 106 CD34+cells/kg, and an intermediate group to which the former two groups were compared. The 22 low-group patients had a significantly delayed neutrophil (P < .0001) and platelet recovery (P < .0001). The 41 high-group patients experienced significantly shorter engraftment compared with the intermediate group with a median of 11 (range, 8 to 16) versus 12 (range, 7 to 17) days for neutrophil recovery (P = .003), and a median of 11 (range, 7 to 24) versus 14 (range, 8 to 180+) days for platelet recovery (P< .0001). These patients required significantly less platelet transfusions (P = .002). In a multivariate analysis, the amount of CD34+ cells reinfused was the only variable showing significance for neutrophil and platelet recovery. High-group patients had a shorter hospital stay (P = .01) and tended to need fewer days of antibotic administration (P = .12). In conclusion, these results suggest that reinfusion of greater than 15 × 106 CD34+ cells/kg after high-dose chemotherapy for lymphoproliferative diseases further shortens hematopoietic reconstitution, reduces platelet requirements, and may improve patients' quality of life.
Growth-Supporting Activities of Fibronectin on Hematopoietic Stem/Progenitor Cells In Vitro and In Vivo: Structural Requirement for Fibronectin Activities of CS1 and Cell-Binding Domains
Authors: Yokota Takafumi , Oritani Kenji , Mitsui Hideki , Aoyama Keisuke , Ishikawa Jun , Sugahara Hiroyuki , Matsumura Itaru , Tsai Schickwann , Tomiyama Yoshiaki , Kanakura Yuzuru , Matsuzawa Yuji
Published: October 2019

Fibronectin (FN) is supposed to play important roles in various aspects of hematopoiesis through binding to very late antigen 4 (VLA4) and VLA5. However, effects of FN on hematopoietic stem cells are largely unknown. In an effort to determine if FN had a growth-supporting activity on hematopoietic stem cells, human CD34+/VLA4bright/VLA5dullhematopoietic stem cells and a murine stem cell factor (SCF)-dependent multipotent cell line, EML-C1, were treated with or without FN in a serum and growth-factor–deprived medium, and then subjected to clonogenic assay in the presence of hematopoietic growth factors. The pretreatment of the CD34+ cells with FN gave rise to significantly increased numbers of granulocyte-macrophage colony-forming units (CFU-GM), erythroid burst colony-forming units, and mixed erythroid-myeloid colony-forming units. In addition, the numbers of blast colony-forming units and CFU-GM that developed after culture of EML-C1 cells with SCF and the combination of SCF and interleukin-3, respectively, were augmented by the pretreatment with FN. The augmented colony formation by FN was completely abrogated by the addition of CS1 fragment, but not of GRGDSP peptide, suggesting an essential role of FN-VLA4 interaction in the FN effects. Furthermore, the effects of various FN fragments consisting of RGDS-containing cell-binding domain (CBD), heparin-binding domain (HBD), and/or CS1 portion were tested on clonogenic growth of CD34+ cells. Increased colony formation was induced by CBD-CS1 and CBD-HBD-CS1 fragments, but not with other fragments lacking CBD or CS1 domains, suggesting that both CS1 and CBD of FN were required for the augmentation of clonogenic growth of hematopoietic stem/progenitor cells in vitro. In addition to the in vitro effects, the in vivo administration of CBD-CS1 fragment into mice was found to increase the numbers of hematopoietic progenitor cells in bone marrow and spleen in a dose-dependent manner. Thus, FN may function on hematopoietic stem/progenitor cells as a growth-supporting factor in vitro and in vivo.
Platelet Factor 4 Modulates Fibroblast Growth Factor 2 (FGF-2) Activity and Inhibits FGF-2 Dimerization
Authors: Perollet Catherine , Han Zhong Chao , Savona Catherine , Caen Jacques Philippe , Bikfalvi Andreas
Published: October 2019

Platelet factor 4 (PF-4) inhibits angiogenesis in vitro and in vivo. The mechanism of inhibition is poorly understood. We have investigated the mechanism of inhibition by examining the interaction of PF-4 and the fibroblast growth factor-2 (FGF-2)/fibroblast growth factor receptor (FGFR) system. PF-4 inhibited the binding of FGF-2 to high-affinity and low-affinity binding sites in murine microvascular endothelial cells (LEII cells) and proliferation. Maximum inhibition of binding to endothelial FGF receptors was observed at PF-4 concentrations between 5 and 10 μg/mL (half maximum inhibition at 0.6 μg/mL), and proliferation was completely inhibited at 2 μg/mL. At this concentration, PF-4 reduced internalization of125I–FGF-2 by threefold and delayed degradation. To gain insight into the mechanism of inhibition, we have analyzed the interaction of PF-4 with FGF-2/FGFR by using mutant heparan sulfate–deficient Chinese hamster ovary (CHO) cells transfected with the FGFR-1 cDNA (CHOm–FGFR-1) and by examining the direct interaction with FGF-2. In the absence of heparin, PF-4 inhibited binding of 125I–FGF-2 to CHOm–FGFR-1 cells in a concentration-dependent manner, although not completely. In the presence of heparin, PF-4 abolished totally the stimulatory effect of heparin. Furthermore, PF-4 complexed to FGF-2 and inhibited endogenous or heparin-induced FGF-2 dimerization. These results indicate that PF-4 interacts with FGF-2 by complex formation, inhibiting FGF-2 dimerization, binding to FGF receptors, and internalization. This mechanism most likely contributes to the antiangiogenic properties of PF-4.