bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemokine and Chemokine Receptor Gene Variants and Risk of Non-Hodgkin’s Lymphoma in Human Immunodeficiency Virus-1–Infected Individuals
Authors: Rabkin Charles S. , Yang Quan-en , Goedert James J. , Nguyen Giao , Mitsuya Hiroaki , Sei Shizuko
Published: October 2019

Normal B-lymphocyte maturation and proliferation are regulated by chemotactic cytokines (chemokines), and genetic polymorphisms in chemokines and chemokine receptors modify progression of human immunodeficiency virus-1 (HIV-1) infection. Therefore, 746 HIV-1–infected persons were examined for associations of previously described stromal cell-derived factor 1 (SDF-1) chemokine and CCR5 and CCR2 chemokine receptor gene variants with the risk of B-cell non-Hodgkin’s lymphoma (NHL). The SDF1-3′A chemokine variant, which is carried by 37% of whites and 11% of blacks, was associated with approximate doubling of the NHL risk in heterozygotes and roughly a fourfold increase in homozygotes. After a median follow-up of 11.7 years, NHL developed in 6 (19%) of 30 SDF1-3′A/3′A homozygotes and 22 (10%) of 202 SDF1-+/3′A heterozygotes, compared with 24 (5%) of 514 wild-type subjects. The acquired immunodeficiency syndrome (AIDS)-protective chemokine receptor variant CCR5-▵32 was highly protective against NHL, whereas the AIDS-protective variant CCR2-64I had no significant effect. Racial differences in SDF1-3′A frequency may contribute to the lower risk of HIV-1–associated NHL in blacks compared with whites. SDF-1 genotyping of HIV-1–infected patients may identify subgroups warranting enhanced monitoring and targeted interventions to reduce the risk of NHL.
The Rhesus Macaque as an Animal Model for Hemophilia B Gene Therapy
Authors: Lozier Jay N. , Metzger Mark E. , Donahue Robert E. , Morgan Richard A.
Published: October 2019

We have determined the 2905 nucleotide sequence of the rhesus macaque factor IX complementary DNA (cDNA) and found it to be greater than 95% identical to that of the human factor IX cDNA. The cDNA has a large 3′ untranslated region like the human cDNA, but unlike the human cDNA has two polyadenylation sites 224 nucleotides apart that are used for transcription of the messenger RNA. The deduced amino acid sequence is greater than 97% identical to that of human factor IX, differing in only 11 of 461 amino acids in the complete precursor protein. We found a single silent polymorphism in the nucleotide sequence at the third position of the codon for asparagine at position 167 in the secreted protein (AAC/AAT). All residues subject to posttranslational modifications in the human protein are also found in the rhesus factor IX sequence. The high degree of homology between the rhesus and human factor IX proteins suggested the possibility that the human factor IX protein might be nonimmunogenic in the rhesus. We tested the immunogenicity of human factor IX in three rhesus macaques by repeated intravenous injections of monoclonal antibody–purified, plasma-derived human factor IX over the course of more than a year and assessed the recovery and half-life of the infused protein, as well as in vitro indicators of antihuman factor IX antibodies. Human factor IX recovery and half-life remained unchanged over the course of a year in the three animals studied, and aPTT mixing studies showed no evidence for neutralizing antihuman factor IX antibodies. An outbred, nonhuman primate model that permits assessment of the level and duration of factor IX expression as well as vector safety would complement the use of other (mouse and canine) hemophilia B animal models in current use for the development of gene therapy for hemophilia B.
Deficient Transcription of Mouse Mast Cell Protease 4 Gene in Mutant Mice of mi/mi Genotype
Authors: Jippo Tomoko , Lee Young-Mi , Katsu Yee , Tsujino Kumiko , Morii Eiichi , Kim Dae-Ki , Kim Hyung-Min , Kitamura Yukihiko
Published: October 2019

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). We reported that expression of the mouse mast cell protease 5 (MMCP-5) and MMCP-6 genes were deficient in cultured mast cells (CMC) derived from mutant mice ofmi/mi genotype. Despite the reduced expression of both MMCP-5 and MMCP-6, their regulation mechanisms were different. Because MMCP-5 is a chymase and MMCP-6 a tryptase, there was a possibility that the difference in regulation mechanisms was associated with their different characteristics as proteases. We compared the regulation mechanisms of another chymase, MMCP-4, with those of MMCP-5 and MMCP-6. The expression of the MMCP-4 gene was also deficient in mi/mi CMC. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-4 gene in mi/mi CMC, indicating the involvement of +-MITF in transactivation of the MMCP-4 gene. Although MMCP-4 is chymase as MMCP-5, the regulation of MMCP-4 expression was more similar to MMCP-6 than to MMCP-5. We also showed the deficient expression of granzyme B and cathepsin G genes inmi/mi CMC. Genes encoding granzyme B, cathepsin G, MMCP-4, and MMCP-5 are located on chromosome 14. Because all these genes showed deficient expression in mi/mi CMC, there is a possibility that MITF might regulate the expression of these genes through a locus control region.
Influence of Monoclonal Antiplatelet Glycoprotein Antibodies on In Vitro Human Megakaryocyte Colony Formation and Proplatelet Formation
Authors: Takahashi Ryo , Sekine Nariko , Nakatake Toshihiko
Published: October 2019

The influence of antiplatelet glycoprotein (GP) antibodies on megakaryocytopoiesis in patients with idiopathic or immune thrombocytopenic purpura (ITP) has been well studied. However, the influence of GP antibodies on proplatelet formation is poorly understood. Here we investigated whether in vitro human megakaryocyte colony formation and proplatelet formation are affected by various monoclonal antiplatelet GP antibodies (MoAb). The megakaryocyte colony formation inhibition assay was performed by methylcellulose culture with modifications, using peripheral blood nonadherent mononuclear cells. The proplatelet formation inhibition assay was performed by megakaryocytes derived from CD34+ cells, stimulated with thrombopoietin + stem cell factor, which were then incubated with antiplatelet GP MoAb for 24 or 48 hours. Anti-GP-Ib MoAb (CD42b; HIP1) slightly inhibited megakaryocyte colony formation (P < .05). and strongly inhibited proplatelet formation (after 24 hours incubation, P < .0002; after 48 hours incubation, P < .0007). Anti-GP-IIb MoAb (CD41; 5B12) inhibited only proplatelet formation (only after 24 hours incubation,P < . 03). Anti-integrin vβ3MoAb (CD51/CD61; 23C6) only slightly inhibited colony size (P < .05). However, anti-GP-IIIa MoAb (CD61; Y2/51) did not inhibit either colony formation or proplatelet formation. These results suggest that antiplatelet GP MoAbs have differing effects on in vitro megakaryocyte colony formation and proplatelet formation.
Mutant N-ras Induces Myeloproliferative Disorders and Apoptosis in Bone Marrow Repopulated Mice
Authors: MacKenzie K.L. , Dolnikov A. , Millington M. , Shounan Y. , Symonds G.
Published: October 2019

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-rasreconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-rasmutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.
Missense Mutations in the gp91-phox Gene Encoding Cytochromeb558 in Patients With Cytochrome b Positive and Negative X-Linked Chronic Granulomatous Disease
Authors: Kaneda Mizuho , Sakuraba Hitoshi , Ohtake Akira , Nishida Akira , Kiryu Chika , Kakinuma Katsuko
Published: October 2019

Chronic granulomatous disease (CGD) is a disorder of host defense due to genetic defects of the superoxide (O2-) generating NADPH oxidase in phagocytes. A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the oxidase. The X-linked form of the disease is due to defects in the gp91-phox gene. We report here biochemical and genetic analyses of patients with typical and atypical X-linked CGD. Immunoblots showed that neutrophils from one patient had small amounts of p22-phoxand gp91-phox and a low level of O2-forming oxidase activity, in contrast to the complete absence of both subunits in two patients with typical CGD. Using polymerase chain reactions (PCR) on cDNA and genomic DNA, we found novel missense mutations of gp91-phox in the two typical patients and a point mutation in the variant CGD, a characteristic common to two other patients with similar variant CGD reported previously. Spectrophotometric analysis of the neutrophils from the variant patient provided evidence for the presence of heme of cytochromeb558. Recently, we reported another variant CGD with similar amounts of both subunits, but without oxidase activity or the heme spectrum. A predicted mutation at amino acid 101 in gp91-phox was also confirmed in this variant CGD by PCR of the genomic DNA. These results on four patients, including those with two variant CGD, are discussed with respect to the missense mutated sites and the heme binding ligands in gp91-phox.
Structural and Functional Consequences of Antigenic Modulation of Red Blood Cells With Methoxypoly(Ethylene Glycol)
Authors: Murad Kari L. , Mahany Kathleen L. , Brugnara Carlo , Kuypers Frans A. , Eaton John W. , Scott Mark D.
Published: October 2019

We previously showed that the covalent modification of the red blood cell (RBC) surface with methoxypoly(ethylene glycol) [mPEG; MW ∼5 kD] could significantly attenuate the immunologic recognition of surface antigens. However, to make these antigenically silent RBC a clinically viable option, the mPEG-modified RBC must maintain normal cellular structure and functions. To this end, mPEG-derivatization was found to have no significant detrimental effects on RBC structure or function at concentrations that effectively blocked antigenic recognition of a variety of RBC antigens. Importantly, RBC lysis, morphology, and hemoglobin oxidation state were unaffected by mPEG-modification. Furthermore, as shown by functional studies of Band 3, a major site of modification, PEG-binding does not affect protein function, as evidenced by normal SO4− flux. Similarly, Na+ and K+ homeostasis were unaffected. The functional aspects of the mPEG-modified RBC were also maintained, as evidenced by normal oxygen binding and cellular deformability. Perhaps most importantly, mPEG-derivatized mouse RBC showed normal in vivo survival (∼50 days) with no sensitization after repeated transfusions. These data further support the hypothesis that the covalent attachment of nonimmunogenic materials (eg, mPEG) to intact RBC may have significant application in transfusion medicine, especially for the chronically transfused and/or allosensitized patient.
Heparan Sulfate Proteoglycan Expression Is Induced During Early Erythroid Differentiation of Multipotent Hematopoietic Stem Cells
Authors: Drzeniek Zofia , Stöcker Georg , Siebertz Barbara , Just Ursula , Schroeder Timm , Ostertag Wolfram , Haubeck Hans-Dieter
Published: October 2019

Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells and their extracellular matrix are important components of the microenvironment of hematopoietic tissues and are involved in the interaction of hematopoietic stem and stromal cells. Although previous studies have emphasized the role of HS proteoglycan synthesis by BM stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan. Immunochemical, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis of this HS proteoglycan showed that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS proteoglycans is associated with the differentiation state of hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Proteoglycans were isolated from metabolically labeled cells and purified by several chromatographic steps. Isolation and characterization of proteoglycans from the cell lines HEL and ELM-D, which like TF-1 cells have an immature erythroid phenotype, showed that these cells synthesize the same HS proteoglycan, previously detected in TF-1 cells, as a major proteoglycan. In contrast, cell lines of the myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and B2, do not express HS proteoglycans. Taken together, our data strongly suggest that expression of this HS proteoglycan in hematopoietic progenitor cell lines is associated with the erythroid lineage. To prove this association we have analyzed the proteoglycan expression in the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of erythroid or granulocytic differentiation. Our data show that HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells. In contrast, during granulocytic differentiation, no expression of HS proteoglycans was observed.
Subcellular and Cell-Cycle Expression Profiles of CDK-Inhibitors in Normal Differentiating Myeloid Cells
Authors: Yaroslavskiy B. , Watkins S. , Donnenberg A.D. , Patton T.J. , Steinman R.A.
Published: October 2019

A central question in hematopoiesis is how cell-cycling behavior changes during the emergence of the differentiated state. To further understand what genetic regulators might couple proliferation status to differentiation, we studied the expression of the cell-cycle inhibitors p21 and p27 during the in vitro differentiation of normal CD34+ blast cells along the myeloid lineage. We find p27 but not p21 to be expressed in freshly harvested resting CD34+ cells. Thereafter, p21 levels peak concurrent with cellular proliferation and then decline in expression as cells undergo terminal differentiation. In contrast, p27 levels are fairly constant but the subcellular localization of p27 changes from nuclear expression to predominantly cytoplasmic expression and finally to perinuclear localization at progressive stages of differentiation. This report discusses the implications of these findings.
Anticancer Drugs Induce Caspase-8/FLICE Activation and Apoptosis in the Absence of CD95 Receptor/Ligand Interaction
Authors: Wesselborg Sebastian , Engels Ingo H. , Rossmann Evi , Los Marek , Schulze-Osthoff Klaus
Published: October 2019

Proteases of the caspase family are the critical executioners of apoptosis. Their activation has been mainly studied upon triggering of death receptors, such as CD95 (Fas/APO-1) and tumor necrosis factor-R1, which recruit caspase-8/FLICE as the most proximal effector to the receptor complex. Because apoptosis induced by anticancer drugs has been proposed to involve CD95/CD95 ligand interaction, we investigated the mechanism of caspase activation by daunorubicin, doxorubicin, etoposide, and mitomycin C. In Jurkat leukemic T cells, all drugs induced apoptosis and the cleavage of procaspase-8 to its active p18 subunit. However, cells resistant to CD95 were equally susceptible to anticancer drugs and activated caspase-8 with a similar kinetic and dose response as CD95-sensitive cells. The broad caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone prevented apoptosis and caspase-8 activation in response to CD95 and drug treatment, whereas a neutralizing CD95 decoy as well as a dominant-negative FADD construct selectively abrogated CD95, but not drug-induced effects. A potent activation of caspase-8 was also induced by cycloheximide, indicating that it was independent of protein synthesis. Our data, therefore, show that (1) anticancer drug-induced apoptosis does not require de novo synthesis of death ligands or CD95 interaction, and (2) that caspase-8 can be activated in the absence of a death receptor signaling.
A Requirement for K+-Channel Activity in Growth Factor–Mediated Extracellular Signal-Regulated Kinase Activation in Human Myeloblastic Leukemia ML-1 Cells
Authors: Xu Dazhong , Wang Ling , Dai Wei , Lu Luo
Published: October 2019

Voltage-gated K+ channels have been shown to be required for proliferation of various types of cells. Much evidence indicates that K+-channel activity is required for G1 progression of the cell cycle in different cell backgrounds, suggesting that K+-channel activity is required for early-stage cell proliferation in these cells. However, little is known about the molecular mechanisms that underlie this phenomenon. We have shown in human myeloblastic leukemia ML-1 cells that K+ channels are activated by epidermal growth factor (EGF), whereas serum starvation deprivation suppressed their activity. In addition, voltage-gated K+ channels are required for G1/S-phase transition of the cell cycle. We report here that suppression of K+ channels prevented the activation of extracellular signal-regulated protein kinase 2 (ERK-2) in response to EGF and serum. However, blockade of K+ channels did not prevent ERK-2 activation induced by 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Elimination of extracellular Ca2+ did not alter either ERK-2 activation or the effect of K+-channel blockade on ERK-2 activation. Our data demonstrate that the K+ channel is a part of the EGF-mediated mitogenic signal-transduction process and is required for initiation of the EGF-mediated mitogen-activated protein kinase (MAPK) pathways. Our findings may thus explain why an increase in K+-channel activity is associated with cell proliferation in many types of cells, including ML-1 cells.
Characterization of Wild-Type and Mutant 2-Antiplasmins: Fibrinolysis Enhancement by Reactive Site Mutant
Authors: Lee Kyung N. , Tae Weon-Chan , Jackson Kenneth W. , Kwon Soon H. , McKee Patrick A.
Published: October 2019

During human blood clotting, 2-antiplasmin (2AP) becomes covalently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in 2AP and a specific ɛ-lysyl group in each of the -chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. We found that chemically Arg-modified 2AP, which lacked plasmin-inhibitory activity, competed effectively with native 2AP for becoming cross-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombinant 2AP reported to date by other groups either lacked or possessed a low level of FXIIIa substrate activity. As a first step in the development of an engineered protein that might have potential as a localized fibrin-specific fibrinolytic enhancer, we expressed recombinant 2AP in Pichia pastoris yeast. Two forms of nonglycosylated recombinant 2AP were expressed, isolated and characterized: (1) wild-type, which was analogous to native 2AP, and (2) a mutant form, which had Ala substituted for the reactive-site Arg364. Both the wild-type and mutant forms of 2AP functioned as FXIIIa substrates with affinities and kinetic efficiencies comparable to those of native 2AP, despite each having an additional acetylated Met blocking group at their respective amino-termini. Wild-type recombinant 2AP displayed full plasmin inhibitory activity, while mutant 2AP had none. Neither the absence of glycosylation nor blockage of the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activities of wild-type 2AP. When our mutant 2AP, which lacked plasmin-inhibitory function, was added to human plasma or whole blood clots, urokinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, indicating that mutant 2AP augmented lysis by competing with native 2AP for FXIIIa-catalyzed incorporation into fibrin.
Growth Inhibition of a Human Myeloma Cell Line by All-transRetinoic Acid Is Not Mediated Through Downregulation of Interleukin-6 Receptors but Through Upregulation of p21WAF1
Authors: Chen Yi-Hsiang , Lavelle Donald , DeSimone Joseph , Uddin Shahab , Platanias Leonidas C. , Hankewych Maria
Published: October 2019

All-trans retinoic acid (ATRA) has previously been shown to inhibit the growth of OPM-2 human myeloma cells. The growth inhibition was postulated to result from a transcriptional downregulation of interleukin-6 receptor  (IL-6R) with IL-6Rβ (gp130) unaffected. To formally test this hypothesis, an expression vector designed for constitutive IL-6R expression was constructed and used for transfection of OPM-2 cells. Six stable transfectants were cloned. The expression of IL-6R was shown by immunofluorescence with anti–IL-6R antibody and 125I-IL-6 binding. In five of six transfectant clones, cellular IL-6R was 1.5- to 6-fold higher than the parental cells, with the ligand binding affinity unchanged. While ATRA reduced IL-6R expression in the parental OPM-2 cells, it enhanced its expression in these five transfectants. The clonogenic growth of these transfectants, however, remained strongly inhibited by ATRA. Further analysis, comparing the parental OPM-2 cells and a representative transfectant, clone C5, showed that IL-6 caused rapid tyrosine phosphorylation of gp130 in both OPM-2 and C5 clones. Pretreatment with ATRA greatly reduced IL-6–induced gp130 phosphorylation in OPM-2 cells, reflecting a reduction in cellular IL-6R. In contrast, IL-6–induced gp130 phosphorylation was not reduced by ATRA pretreatment in C5 cells, indicating that the expressed IL-6R was functional. Similar to OPM-2 cells, C5 cells were sensitive to growth inhibition by dexamethasone, which was entirely reversed by exogenous IL-6, suggesting that the IL-6 postreceptor signal transduction remained intact. ATRA was further shown to upregulate p21WAF1 expression and cause dephosphorylation of the retinoblastoma protein (pRB) in both OPM-2 and C5 cells. Exogenous IL-6 also failed to reverse these effects of ATRA. Thus, the growth inhibitory activity of ATRA is not mediated through cellular IL-6R downregulation and is likely to result from a direct upregulation of p21WAF1 and consequent dephosphorylation of pRB.
The Carboxy-Terminal Cell-Binding Domain of Thrombospondin Is Essential for Sickle Red Blood Cell Adhesion
Authors: Hillery Cheryl A. , Scott J. Paul , Du Ming C.
Published: October 2019

Sickle red blood cells (SS-RBCs) have enhanced adhesion to the plasma and subendothelial matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. TSP has at least four domains that mediate cell adhesion. The goal of this study was to map the site(s) on TSP that binds SS-RBCs. Purified TSP proteolytic fragments containing either the N-terminal heparin-binding domain, or the type 1, 2, or 3 repeats, failed to sustain SS-RBC adhesion (<10% adhesion). However, a 140-kD thermolysin TSP fragment, containing the carboxy-terminal cell-binding domain in addition to the type 1, 2, and 3 repeats fully supported the adhesion of SS-RBCs (126% ± 25% adhesion). Two cell-binding domain adhesive peptides, 4N1K (KRFYVVMWKK) and 7N3 (FIRVVMYEGKK), failed to either inhibit or support SS-RBC adhesion to TSP. In addition, monoclonal antibody C6.7, which blocks platelet and melanoma cell adhesion to the cell-binding domain, did not inhibit SS-RBC adhesion to TSP. These data suggest that a novel adhesive site within the cell binding domain of TSP promotes the adhesion of sickle RBCs to TSP. Furthermore, soluble TSP did not bind SS-RBCs as detected by flow cytometry, nor inhibit SS-RBC adhesion to immobilized TSP under conditions of flow, indicating that the adhesive site on TSP that recognizes SS-RBCs is exposed only after TSP binds to a matrix. We conclude that the intact carboxy-terminal cell-binding domain of TSP is essential for the adhesion of sickle RBCs under flow conditions. This study also provides evidence for a unique adhesive site within the cell-binding domain that is exposed after TSP binds to a matrix.
Incomplete T-Cell Immune Reconstitution in Two Major Histocompatibility Complex Class II–Deficiency/Bare Lymphocyte Syndrome Patients After HLA-Identical Sibling Bone Marrow Transplantation
Authors: Godthelp Barbara C. , van Eggermond Marja C.J.A. , Peijnenburg Ad , Tezcan Ilhan , van Lierde Stefaan , van Tol Maarten J.D. , Vossen Jaak M. , van den Elsen Peter J.
Published: October 2019

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II–deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8+ T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II–expressing CD4+ and in the CD8+ T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.
Deletion of the Extracellular Membrane-Distal Cytokine Receptor Homology Module of Mpl Results in Constitutive Cell Growth and Loss of Thrombopoietin Binding
Authors: Sabath Diana F. , Kaushansky Kenneth , Broudy Virginia C.
Published: October 2019

The thrombopoietin receptor, Mpl, is a member of the cytokine receptor superfamily. The extracellular domain of Mpl contains two copies of the cytokine receptor homology module (CRM). Mpl is encoded by c-mpl, the cellular homologue of the oncogene v-mpl.The oncogenic potential of v-mpl may arise from deletion of all but the 43 most membrane-proximal amino acids of the extracellular domain of the wild-type receptor. To test the hypothesis that the extracellular domain of Mpl plays a role in controlling receptor activity, we created mutants of murine Mpl in which the membrane-distal CRM was either deleted or replaced by the membrane-proximal CRM. Introduction of these mutant receptors into factor-dependent BaF3 cells led to constitutive cell growth in the absence of growth factor. Both mutant receptors failed to bind 125I-Tpo. These results suggest that the membrane-distal CRM of Mpl acts as a brake on cell proliferation and that this region is required for ligand binding.
Correction of X-Linked Immunodeficient Mice by Competitive Reconstitution With Limiting Numbers of Normal Bone Marrow Cells
Authors: Rohrer Jurg , Conley Mary Ellen
Published: October 2019

Gene therapy for inherited disorders is more likely to succeed if gene-corrected cells have a proliferative or survival advantage compared with mutant cells. We used a competitive reconstitution model to evaluate the strength of the selective advantage that Btk normal cells have in Btk-deficient xid mice. Whereas 2,500 normal bone marrow cells when mixed with 497,500 xid cells restored serum IgM and IgG3 levels to near normal concentrations in 3 of 5 lethally irradiated mice, 25,000 normal cells mixed with 475,000 xidcells reliably restored serum IgM and IgG3 concentrations and the thymus-independent antibody response in all transplanted mice. Reconstitution was not dependent on lethal irradiation, because sublethally irradiated mice all had elevated serum IgM and IgG3 by 30 weeks postreconstitution when receiving 25,000 normal cells. Furthermore, the xid defect was corrected with as few as 10% of the splenic B cells expressing a normal Btk. When normal donor cells were sorted into B220+/CD19+ committed B cells and B220−/CD19− cell populations, only the B220−/CD19− cells provided long-term B-cell reconstitution in sublethally irradiated mice. These findings suggest that even inefficient gene therapy may provide clinical benefit for patients with XLA.
Lupus Anticoagulants Form Immune Complexes With Prothrombin and Phospholipid That Can Augment Thrombin Production in Flow
Authors: Field Susan L. , Hogg Philip J. , Daly Elise B. , Dai Yan-Ping , Murray Barbara , Owens Daniel , Chesterman Colin N.
Published: October 2019

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid binding proteins, including prothrombin. We found that a murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested enhanced binding of prothrombin to 25:75 phosphatidyl serine:phosphatidyl choline vesicles in a concentration-dependent manner. We hypothesized that enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production when reactions are performed in flow. Thrombin production by purified prothrombinase components was measured in a phospholipid-coated flow reactor. The flow reactor was incubated with prothrombin, calcium ions, and the IgGs and then perfused with prothrombin, calcium ions, the IgGs, factor Va, and factor Xa. A murine monoclonal antiprothrombin antibody and 4 of 6 LA IgGs from patients with a history of thrombosis increased thrombin production up to 100% over control in the first 15 minutes. In summary, LA IgGs concentrate prothrombin on a phospholipid surface that can augment thrombin production by prothrombinase in flow. These observations suggest that LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall.
Coexistence of Two Functioning T-Cell Repertoires in Healthy Ex-Thalassemics Bearing a Persistent Mixed Chimerism Years After Bone Marrow Transplantation
Authors: Battaglia Manuela , Andreani Marco , Manna Marisa , Nesci Sonia , Tonucci Paola , Persini Barbara , della Cuna Gioacchino Robustelli , Nocera Arcangelo , Gorski Jack , Lucarelli Guido , De Palma Raffaele
Published: October 2019

Bone marrow transplantation (BMT) from an HLA-identical donor is an established therapy to cure homozygous β-thalassemia. Approximately 10% of thalassemic patients developed a persistent mixed chimerism (PMC) after BMT characterized by stable coexistence of host and donor cells in all hematopoietic compartments. Interestingly, in the erythrocytic lineage, close to normal levels of hemoglobin can be observed in the absence of complete donor engraftment. In the lymphocytic lineage, the striking feature is the coexistence of immune cells. This implies a state of tolerance or anergy, raising the issue of immunocompetence of the host. To understand the state of the T cells in PMC, repertoire analysis and functional studies were performed on cells from 3 ex-thalassemics. Repertoire analysis showed a profound skewing. This was due to an expansion of some T cells and not to a collapse of the repertoire, because phytohemagglutinin stimulation showed the presence of a complex repertoire. The immunocompetence of the chimeric immune systems was further established by showing responses to alloantigens and recall antigens in vitro. Both host and donor lymphocytes were observed in the cultures. These data suggest that the expanded T cells play a role in specific tolerance while allowing a normal immune status in these patients.
Defective Apoptotic Signal Transduction Pathway Downstream of Caspase-3 in Human B-Lymphoma Cells: A Novel Mechanism of Nuclear Apoptosis Resistance
Authors: Kawabata Yoshinari , Hirokawa Makoto , Kitabayashi Atsushi , Horiuchi Takahiro , Kuroki Jun , Miura Akira B.
Published: October 2019

Mitochondria play a central role in controlling apoptosis, and activation of the caspase cascade appears to be crucial event during the apoptotic process. Human B lymphoma Raji cells are resistant to nuclear apoptosis induced by various stimuli. Using this cell line, we have asked whether reduction of the mitochondrial transmembrane potential and activation of caspase-3 are sufficient to induce DNA fragmentation during the apoptotic process. After stimulation with cell-permeable C2-ceramide or mitochondrial permeability transition (PT) inducers, not only apoptosis-sensitive cell lines (HL-60, Jurkat, and Daudi cells), but also Raji cells showed reduction of the mitochondrial transmembrane potential (▵ψm), activation of caspase-3, and loss of clonogenic potential. However, Raji cells did not show detectable levels of nuclear apoptosis (DNA degradation). In a cell-free system, cell lysates from tetra-butylhydroperoxide (t-BHP)–treated HL-60 cells induced DNA degradation of Raji nuclei, whereas cell lysates from t-BHP–treated Raji cells failed to induce DNA degradation in either apoptosis-sensitive cell lines or apoptosis-resistant Raji cells. Cleavage of DFF-45, which is a downstream target molecule for caspase-3, was observed in Raji cells as well as in apoptosis-sensitive Daudi cells. These results indicate that there is a defective apoptotic pathway in the cytoplasm downstream of caspase-3 in Raji cells.
Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies
Authors: Cardoso Angelo A. , Veiga J. Pedro , Ghia Paolo , Afonso Hernani M. , Haining W. Nicholas , Sallan Stephen E. , Nadler Lee M.
Published: October 2019

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.
Surface Molecule Loss and Bleb Formation by Human Germinal Center B Cells Undergoing Apoptosis: Role of Apoptotic Blebs in Monocyte Chemotaxis
Authors: Segundo Carmen , Medina Francisco , Rodrı́guez Carmen , Martı́nez-Palencia Rosalı́a , Leyva-Cobián Francisco , Brieva José A.
Published: October 2019

Human tonsil germinal center (GC) B cells rapidly undergo apoptosis in culture. Annexin-V binding shows an early event in this process. In the present study, this method has been used to label apoptotic GC B cells and to analyze additional surface molecules. The expression of all of the molecules studied was reduced in apoptotic (annexin-V+) GC B cells, and the reduction was more marked for CD11a, CD21, CD22, CD49d, and CD54, molecules that participate in survival interaction for GC B cells. The analysis of CD54, one of the molecules that was more drastically reduced, showed that GC, but not mantle zone, B cells actively secrete CD54 to the culture supernatant (SN). The secreted CD54 was partly released from the GC B cells in a particulate form as demonstrated by centrifugation. Further experiments using filtration, fluorescence microscopy, electron microscopy, and flow cytometry analysis showed that GC B cells released to the culture SN a population of spherical membranous vesicles of about 0.18 μm in size, similar to the blebs described in other apoptosis systems. Bleb formation depended on active metabolism, Ca2+, and, in part, on microfilament integrity. GC B-cell–derived blebs were clearly associated with apoptosis, as antiapoptotic stimuli prevented their formation. In addition, GC B-cell–derived blebs contained the adhesion molecules previously studied. Consequently, bleb formation might contribute to the surface molecule loss occurring in apoptotic GC B cells. Finally, a chemotaxis assay showed that GC B-cell blebs were chemotactic for human monocytes, suggesting that this mechanism might operate in vivo.
A BCR-ABL Oncoprotein p210b2a2 Fusion Region Sequence Is Recognized by HLA-DR2a Restricted Cytotoxic T Lymphocytes and Presented by HLA-DR Matched Cells Transfected With an Iib2a2 Construct
Authors: ten Bosch George J.A. , Kessler Jan H. , Joosten Antonia M. , Bres-Vloemans Alexandra A. , Geluk Annemieke , Godthelp Barbara C. , van Bergen Jeroen , Melief Cornelis J.M. , Leeksma Onno C.
Published: October 2019

Peptides corresponding to the fusion site in 210 kD BCR-ABL protein b3a2 (p210b3a2) were previously shown to bind to several HLA class I and II alleles. We have found that b3a2 peptide-specific CD4-positive T-helper cells were able to recognize p210b3a2-positive chronic myelogenous leukemia (CML) blasts in a DR4 restricted manner. Until now, there were no reports of b2a2 breakpoint-specific human T-cell responses. Here we show that repetitive stimulation of T lymphocytes with a 17mer peptide covering the fusion region in p210b2a2 also leads to specific T-cell responses. CD4 and CD4/CD8 double-positive clones obtained from a b2a2 peptide-specific cell line were cytotoxic and proliferative in an HLA-DR2a (DRB5*0101) restricted fashion. Autologous Epstein-Barr virus (EBV) transformed cells, expressing BCR-ABLb2a2 on transfection, and allogeneic HLA-DR matched p210b2a2-positive cells from CML patients were, however, not lysed. BCR-ABL peptide-specific T-cell clones did respond to autologous EBV cells transfected with invariant chain (li) cDNA in which the HLA class II–associated invariant chain peptide (CLIP) was replaced by a BCR-ABL b2a2 fusion oligonucleotide sequence, illustrating the potential of these T cells to recognize an endogenous BCR-ABLb2a2ligand.
Oligoclonal TCRBV Gene Usage in B-Cell Chronic Lymphocytic Leukemia: Major Perturbations Are Preferentially Seen Within the CD4 T-Cell Subset
Authors: Rezvany Mohammad-Reza , Jeddi-Tehrani Mahmood , Österborg Anders , Kimby Eva , Wigzell Hans , Mellstedt Håkan
Published: October 2019

TCRBV (T-cell receptor B variable) gene usage and CDR3 size distribution were analyzed using reverse transcription polymerase chain reaction (RT-PCR) to assess the T-cell repertoire of 10 patients with B-cell chronic lymphocytic leukemia (B-CLL) and in nine age-matched healthy control donors. When the usage of each TCRBV gene within the CD8+ T cells of the patients was compared with that of the controls, no statistically significant difference was noted except for BV 6S1-3. In contrast, within the CD4+ T cells of the CLL patients, a statistically significant overexpression for four BV families (2, 3, 5S1, 6S1-3) was seen while an underrepresentation was noted for five BV families (10, 11, 15, 16, 19). Based on the criterion that a value of any BV higher than the mean + 3 standard deviation (SD) of healthy controls indicated an overexpression, individual patients were shown to overexpress several TCRBV genes compared with the controls. Analyses of the CDR3 length polymorphism showed a significantly higher degree of restriction within CD4+ and CD8+ T cells of the patients, as compared with the corresponding control T-cell population. There was a significant difference in the CDR3 size distribution pattern with a more polymorphic CDR3 length pattern in the age-matched controls as compared with CLL patients, suggesting different mechanisms driving the T cells towards a clonal/oligoclonal TCRBV usage in patients and controls, respectively. The results show major perturbations of T cells in CLL patients, more frequently seen in the CD4+ T-cell subset, indicating that nonmalignant CD4+ T cells may be involved in the pathogenesis of CLL, but also CD8+ T cells.
VH Gene Analysis of IgM-Secreting Myeloma Indicates an Origin From a Memory Cell Undergoing Isotype Switch Events
Authors: Sahota Surinder S. , Garand Richard , Mahroof Razeen , Smith Alastair , Juge-Morineau Nadine , Stevenson Freda K. , Bataille Regis
Published: October 2019

IgM-secreting plasma cell tumors are rare variants of typical isotype-switched multiple myeloma with a similar disease outcome. To probe the origin and clonal history of these tumors, we have analyzed VH gene sequences in 6 cases. Potentially functional tumor-derived VH genes were all derived from VH3, with the V3-7 gene segment being used by 4 of 6. All were somatically mutated, with a mean deviation from germline sequence of 5.2% (range, 3.1% to 7.1%). The distribution of replacement mutations was consistent with antigen selection in 4 of 6 cases, and no intraclonal heterogeneity was observed. Clonally related switched isotype transcripts were sought in 4 cases, and Cγ transcripts with tumor-derived CDR3 sequence were identified in 2 of 4. These findings indicate that IgM-secreting myelomas are arrested at a postfollicular stage at which somatic mutation has been silenced. Isotype switch variants show the cell of origin to be at the IgM to IgG switch point. These features indicate that the final neoplastic event has occurred at a stage immediately before that of typical isotype-switched myeloma. One possibility is that IgM myeloma involves the previously identified precursor cell of typical myeloma.
Serial Analysis of Gene Expression in Human Monocytes and Macrophages
Authors: Hashimoto Shin-ichi , Suzuki Takuji , Dong Hong-Yan , Yamazaki Nobuyuki , Matsushima Kouji
Published: October 2019

Monocytes/macrophages serve as sentinels involved in chronic inflammation and the eradication of various pathogens. To define molecularly the differentiation of blood monocytes into macrophages, we conducted serial analysis of gene expression (SAGE) in human blood monocytes/macrophages induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF. SAGE analysis of 57,560, 57,463, and 55,856 tags from monocytes, GM-CSF–, and M-CSF–induced macrophages, respectively, allowed identification of 35,037 different transcripts. Interestingly, the genes with the highest expression during differentiation from monocytes into macrophages were genes involved in lipid metabolism. Both CSF-induced macrophages expressed similar sets of genes except for several genes such as monocyte-derived chemokine (MDC), legumain, prostaglandin D synthetase, and lysosomal sialoglycoprotein. The identification of specific gene expression in human monocytes, GM-CSF–, or M-CSF–induced macrophages provides novel methods to define macrophage subsets and the maturation and activation stage of cells of macrophage lineage and, possibly, to diagnose diseases in which macrophages play a major role. This study represents the first extensive serial analysis of gene expression for any type of human hematopoietic cells.
Real-Time Analysis of Mural Thrombus Formation in Various Platelet Aggregation Disorders: Distinct Shear-Dependent Roles of Platelet Receptors and Adhesive Proteins Under Flow
Authors: Tsuji Shizuko , Sugimoto Mitsuhiko , Miyata Shigeki , Kuwahara Mitsuhiro , Kinoshita Seiji , Yoshioka Akira
Published: October 2019

We evaluated real-time processes of platelet thrombus formation on a collagen surface in a flow chamber with whole blood from patients with various platelet aggregation disorders, such as Bernard-Soulier syndrome (BSS), Glanzmann’s thrombasthenia (GTA), type 3 von Willebrand disease (vWD), and congenital afibrinogenemia (Af), who lack platelet glycoprotein (GP) Ib-IX complex, GP IIb-IIIa, von Willebrand factor (vWF), and fibrinogen, respectively. Blood from GTA patients showed impaired thrombus growth but significant initial platelet-surface interaction under all shear conditions tested (50 to 1,500 s−1). By contrast, blood from patients with BSS or type 3 vWD showed no platelet-surface interaction under high shear (≥1,210 s−1) but normal thrombus formation under low shear (≤340 s−1). When shear rate was increased stepwise to 1,500 s−1 during perfusion, the thrombus growth observed in type 3 vWD or BSS under low shear was arrested, whereas that in control blood was sharply accelerated as a function of shear rate. Overall thrombus formation in Af appeared indistinguishable from that of a control under shear rates between 50 and 1,500 s−1. However, Af thrombi formed under such conditions collapsed immediately when shear rate was further increased to 4,500 s−1, whereas thrombi of type 3 vWD or BSS formed under low shear were stable even when shear rate was elevated to 9,000 s−1 during perfusion. These findings suggest that distinct molecular mechanisms underlie the pathologic bleeding in these diseases and point to the distinct roles of two major adhesive proteins, vWF and fibrinogen. In mural thrombus formation under flow conditions, vWF, perhaps mainly through its interaction with GP Ib-IX, acts as an “initiator and promoter,” whereas fibrinogen, via its binding to GP IIb-IIIa, acts as a “stabilizer” against heightened shear forces that could lead to peeling off of platelets from the surface.