bluuebunny/crossref_metadata_2025 huggingface dataset:
Epstein-Barr Virus (EBV) in Endemic Burkitt's Lymphoma: Molecular Analysis of Primary Tumor Tissue
Authors: Tao Qian , Robertson Keith D. , Manns Angela , Hildesheim Allan , Ambinder Richard F.
Published: October 2019

Many aspects of Epstein-Barr virus (EBV) and tumor biology have been studied in Burkitt's lymphoma (BL)-derived cell lines. However, in tissue culture, patterns of gene expression and C promoter-G (CpG) methylation often change and viral strain selection may occur. In this report, 10 cases of snap-frozen endemic BL tumors are characterized in terms of viral gene expression, promoter usage, methylation, and viral strain. EBNA1 and BamHI-A rightward transcripts (BART) were detected in 7 of 7 and LMP2A transcripts in 5 of 7 tumors with well-preserved RNA. Transcripts for the other EBNAs and for LMP1 were not detected in any tumor. These tumors differ from BL cell lines in that they lack a variety of lytic cycle transcripts. This pattern of viral gene expression in endemic BL is similar to that reported in peripheral blood mononuclear cells (PBMCs) from healthy EBV–seropositive individuals. EBNA1 transcripts originated from the Q promoter (Qp) but not C, W, or F promoters that drive transcription of EBNA1 in other circumstances. Whereas Cp has been previously shown to be entirely CpG methylated in BL, bisulfite genomic sequencing showed virtually no methylation in Qp. Type-A EBV was detected in 6 of 10 and type B in 4 of 10 cases. A previously reported 30bp deletion variant in the carboxyl terminal of LMP1 gene was detected in 5 of 10 cases. The association with both A and B strains contrasts with EBV–associated Hodgkin's disease, nasopharyngeal carcinoma, and post-transplant lymphoproliferative disease, which are much more consistently associated with A strain virus.
Fluorescence In Situ Hybridization Characterization of New Translocations Involving TEL (ETV6) in a Wide Spectrum of Hematologic Malignancies
Authors: Wlodarska Iwona , La Starza Roberta , Baens Mathijs , Dierlamm Judith , Uyttebroeck Ann , Selleslag Dominik , Francine Adrien , Mecucci Cristina , Hagemeijer Anne , Van den Berghe Herman , Marynen Peter
Published: October 2019

The ETV6 (also known as TEL) gene on chromosome 12p13 is the target of a number of translocations associated with various hematologic malignancies. The contribution of ETV6 to leukemogenesis occurs through different mechanisms that involve either its helix-loop-helix dimerization domain or its E26 transformation-specific (ETS) DNA-binding domain. Using fluorescence in situ hybridization we characterized seven newETV6 rearrangements in chronic myeloid leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma. These aberrations, not always discernible at the cytogenetic level, include a t(5;12)(q31;p13), t(6;12;17)(p21;p13;q25), t(7;12)(p15;p13), t(7;12)(p12;p13), t(7;12)(q36;p13), t(12;13)(p13;q12), and a not completely defined t(12;?)(p13;?). Loss or disruption of the secondETV6 allele by a del(12)(p12p13) or by an intragenicETV6 deletion was detected in two cases. In six cases the 12p13 breakpoint occurred in the 5′ end of ETV6, upstream to exons encoding the HLH domain, whereas the remaining case had a breakpoint between the exons coding for the HLH domain and the exons coding for the ETS domain of ETV6. These observations provide further evidence for the multiple contributions of ETV6 in the pathogenesis of a wide range of hematologic malignancies.
Wilms' Tumor (WT1) Gene Mutations Occur Mainly in Acute Myeloid Leukemia and May Confer Drug Resistance
Authors: King-Underwood L. , Pritchard-Jones K.
Published: October 2019

In a previous study of acute leukemia, we have shown thatWT1 gene mutations occur in both myeloid and biphenotypic subtypes, where they are associated with refractoriness to standard induction chemotherapy. We have now extended this study to a total of 67 cases (34 acute myeloid leukemia [AML], 23 acute lymphoblastic leukemia [ALL], 10 acute undifferentiated leukemia [AUL]/biphenotypic) and find that WT1 mutations occur in 14% of AML and 20% of biphenotypic leukemia, but are rare in ALL (one case). In contrast to the findings in Wilms' tumor, where mutations in the WT1 gene usually behave according to Knudson's two hit model for tumor suppressor genes, seven of eight leukemia-associated WT1 mutations are heterozygous, implying a dominant or dominant-negative mode of action in hematopoietic cells. In AML, the presence of a WT1 mutation is associated with failure to achieve complete remission and a lower survival rate. These data (1) confirm that WT1 mutations underlie a similar proportion of cases of AML to that seen in Wilms' tumors and (2) show for the first time that WT1 mutations can contribute to leukemogenesis of lymphoid as well as myeloid origin, suggesting that its normal role in hematopoiesis lies at a very early progenitor stage. The relationship of WT1 mutation to chemoresistance merits further investigation.
Methylation of the p15INK4b Gene in Myelodysplastic Syndromes Is Frequent and Acquired During Disease Progression
Authors: Quesnel Bruno , Guillerm Gaelle , Vereecque Rodolphe , Wattel Eric , Preudhomme Claude , Bauters Francis , Vanrumbeke Michael , Fenaux Pierre
Published: October 2019

p15INK4b gene is an inhibitor of cyclin-dependent kinase (CDK) 4 and CDK6 whose expression is induced by transforming growth factor (TGF)β. Recent reports suggest frequent methylation of the p15INK4b gene promoter in leukemias, and it has been proposed that this methylation could be necessary for leukemic cells to escape TGFβ regulation. We investigated the methylation status of p15INK4b gene in 53 myelodysplastic syndromes (MDS) cases, including nine that had progressed to acute myeloid leukemia (AML), using a recently described sensitive method where polymerase chain reaction (PCR) is preceded by bisulfite modification of DNA (methylation specific PCR). p15INK4b methylation was observed in 20 of 53 (38%) of the cases. Twenty of the 24 patients with greater than 10% bone marrow blasts had p15INK4bmethylation (including all nine patients who had progressed to AML) as compared with none of MDS patients with <10% bone marrow blasts. No correlation between karyotypic abnormalities and methylation status was found. Patients with p15INK4b methylation had a worse prognosis, but the prognostic significance of p15INK4bmethylation was no more found by multivariate analysis, due to its strong correlation to the percentage of marrow blasts. In 10 MDS cases, sequential DNA samples were available. In five of them, methylation of the p15INK4b gene was detected at leukemic transformation, but not at diagnosis. Our results showed that methylation of the p15INK4b gene in MDS is correlated with blastic bone marrow involvement and increases with disease evolution toward AML. It suggests that proliferation of leukemic cells might require an escape of regulation of the G1 phase of the cell cycle, and possibly of TGFβ inhibitory effect.
Prolonged STAT1 Activation Related to the Growth Arrest of Malignant Lymphoma Cells by Interferon-α
Authors: Grimley Philip M. , Fang Hui , Rui Hallgeir , Petricoin III Emanuel F. , Ray Subhransu , Dong Fan , Fields Karen H. , Hu Renqiu , Zoon Kathryn C. , Audet Susette , Beeler Judy
Published: October 2019

Multiple biologic effects of interferon-α (IFN-α), including cell growth inhibition and antiviral protection, are initiated by tyrosine phosphorylation of STAT proteins. Although this signal pathway has been intensively investigated, the relevance of STAT signal persistence has received scant attention. Using paired isogenic lymphoma cells (Daudi), which either are sensitive or resistant to growth inhibition by IFN-α, we found comparable initial tyrosine phosphorylation of multiple STAT proteins; however, the phosphorylation durations and associated DNA-binding activities diverged. Phosphorylation and DNA-binding capacity of STAT1 decreased after 4 to 8 hours in resistant cells, as compared with 24 to 32 hours in sensitive cells, whereas phosphorylation of STAT3 and STAT5b was briefer in both lines. Functional significance of the prolonged STAT1 signal, therefore, was explored by experimental interruption of tyrosine phosphorylation, either by premature withdrawal of the IFN-α or deferred addition of pharmacologically diverse antagonists: staurosporine (protein kinase inhibitor), phorbol 12-myristate 13-acetate (growth promoter), or aurintricarboxylic acid (ligand competitor). Results indicated that an approximately 18-hour period of continued STAT1 phosphorylation was associated with growth arrest, but that antiviral protection developed earlier. These differences provide novel evidence of a temporal dimension to IFN-α signal specificity and show that duration of STAT1 activation may be a critical variable in malignant cell responsiveness to antiproliferative therapy.
Some Human Inhibitor Antibodies Interfere With Factor VIII Binding to Factor IX
Authors: Zhong Degang , Saenko Evgueni L. , Shima Midori , Felch Matthew , Scandella Dorothea
Published: October 2019

Factor VIII (fVIII) functions as a cofactor of factor IXa in the intrinsic pathway of blood coagulation. Its absence or abnormality causes the bleeding disorder hemophilia A. About 23% of hemophiliacs who receive therapeutic fVIII infusions develop antibodies that inhibit its activity. We previously showed by inhibitor neutralization assays that the fVIII A2 and C2 domain polypeptides contain common inhibitor epitopes. Often hemophilic inhibitor plasmas were partially neutralized by C2 and more completely neutralized by fVIII light chain (A3-C1-C2), suggesting the presence of an additional major inhibitor epitope(s) within the A3-C1 domains. In immunoprecipitation assays, 17 of 18 inhibitor IgGs bound to recombinant 35S-A3-C1. Amino acids 1811-1818 of the A3 domain comprise a binding site for factors IX and IXa. Three inhibitor IgGs prevented binding of factor IXa to fVIII light chain, and the binding of each IgG to light chain was competed by A3 peptide 1804-1819. The generation of factor Xa by the fVIIIa/fIXa complex in a chromogenic assay was prevented by these inhibitors. Therefore, we propose that another important mechanism of fVIII inactivation by human inhibitors is the prevention of fVIIIa/fIXa association.
Anticoagulant Effects of 1α,25-Dihydroxyvitamin D3 on Human Myelogenous Leukemia Cells and Monocytes
Authors: Koyama Takatoshi , Shibakura Misako , Ohsawa Mai , Kamiyama Ryuichi , Hirosawa Shinsaku
Published: October 2019

The hormonally active form of vitamin D is 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is a principal regulator of calcium homeostasis. It also affects hormone secretion, cell differentiation, and proliferation by a mode of action that involves stereospecific interaction with an intracellular vitamin D receptor (VDR). We recently found that retinoids, which are vitamin A derivatives, exert anticoagulant effects by upregulating thrombomodulin (TM) and downregulating tissue factor (TF) expression in acute promyelocytic leukemia cells and monoblastic leukemia cells. Both the VDR and retinoid receptors belong to the same family of receptors. A heterodimer consisting of the retinoid X receptor and the VDR binds to vitamin D responsive elements on genes regulated by vitamin D. To determine whether 1,25(OH)2D3 would exhibit anticoagulant effects similar to retinoids, we measured the antigen level, activity, and mRNA level of TM and TF in human leukemic cells, vascular endothelial cells, and monocytes treated with 1,25(OH)2D3. We found that 1,25(OH)2D3 upregulates antigen expression, activity, and mRNA levels of TM and downregulates antigen expression, activity, and mRNA levels of TF in human monocytic leukemia cells, some acute myelogenous leukemia cells, and monocytes, but not in umbilical vein endothelial cells. Transient transfection studies with reporter plasmids in monocytic leukemia cells and mobility gel-shift assay showed interaction with 1,25(OH)2D3 and functional retinoic acid responsive elements present in the 5′-flanking region of the TM gene. However, auxiliary factors or other elements in the TM gene may contribute to VDR specificity and transactivation of the gene in specific target cells. These findings indicate that 1,25(OH)2D3 resembles the retinoids in its control of the transcription of the TM and TF genes in human monocytic cells. Analogs of 1,25(OH)2D3with anticoagulant activity may serve as adjunctive antithrombotic agents in monocytic leukemia and atherosclerotic disease.
Construction and Characterization of a Fusion Protein of Single-Chain Anti-CD20 Antibody and Human β-Glucuronidase for Antibody-Directed Enzyme Prodrug Therapy
Authors: Haisma Hidde J. , Sernee M. Fleur , Hooijberg Erik , Brakenhoff Ruud H. , Meulen-Muileman Ida H. v.d. , Pinedo Herbert M. , Boven Epie
Published: October 2019

The CD20 antigen is an attractive target for specific treatment of B-cell lymphoma. Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a nontoxic prodrug at the tumor site by an enzyme targeted by a tumor-specific antibody such as anti-CD20. We constructed a fusion protein of the single-chain Fv anti-CD20 mouse monoclonal antibody (MoAb) 1H4 and human β-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate to doxorubicin at the tumor site. The cDNAs encoding the light- and heavy-chain variable regions of 1H4 were cloned, joined by a synthetic sequence encoding a 15-amino acid linker and fused to human β-glucuronidase by a synthetic sequence encoding a 6-amino acid linker. An antibody-enzyme fusion protein-producing cell line was established by transfection of the construct into human embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody specificity, and enzyme activity were fully retained by the fusion protein. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing conditions. Gel filtration analysis indicated that the enzymatically active form of the fusion protein is a tetramer with an Mw of approximately 400 kD. The nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human β-glucuronidase. When the fusion protein was specifically bound to Daudi lymphoma cells, the prodrug induced similar antiproliferative effects as doxorubicin. Thus, it is feasible to construct a eukaryotic fusion protein consisting of a single-chain anti-CD20 antibody and human β-glucuronidase for future use in the activation of anticancer prodrugs in B-cell lymphoma.
Complement Fragment-Induced Release of Neutrophils From Bone Marrow and Sequestration Within Pulmonary Capillaries in Rabbits
Authors: Kubo Hiroshi , Graham Lori , Doyle Nicholas A. , Quinlan William M. , Hogg James C. , Doerschuk Claire M.
Published: October 2019

Infusion of complement fragments induces rapid sequestration of neutrophils within the pulmonary capillaries. This study examined the contributions of the bone marrow (BM) and the liver to the accumulation of neutrophils within the lungs. Complement fragments induced the release of neutrophils from the BM within 7 minutes of infusion, and these neutrophils sequestered in the lungs immediately upon reaching the pulmonary capillaries. Neutrophils expressing high levels of L-selectin were preferentially retained within the pulmonary microvasculature. By 30 minutes after the infusion was stopped, the circulating neutrophil counts had increased, primarily because of release from the BM. The number of neutrophils sequestered in the lung had decreased by only 27%, and the number of neutrophils in the liver increased by 223%. These studies indicate that complement fragments induce the release of neutrophils from the BM far more rapidly than previously described. These newly released neutrophils immediately sequester within the lung, increasing the number of neutrophils available to injure the lung many fold beyond the number that were circulating before infusion. The preferential retention of L-selectin–expressing neutrophils likely reflects the requirement for L-selectin–mediated adhesion in maintaining sequestered neutrophils within the pulmonary microvasculature. The number of circulating neutrophils reflects a balance between pulmonary sequestration, rapid release from the BM, and uptake by the liver and other organs.
Oxidative Modification of Low-Density Lipoprotein and Atherogenetic Risk in β-Thalassemia
Authors: Livrea M.A. , Tesoriere L. , Maggio A. , D’Arpa D. , Pintaudi A.M. , Pedone E.
Published: October 2019

We investigated the oxidative state of low-density lipoprotein (LDL) in patients with β-thalassemia to determine whether there was an association with atherogenesis. Conjugated diene lipid hydroperoxides (CD) and the level of major lipid antioxidants in LDL, as well as modified LDL protein, were evaluated in 35 β-thalassemia intermedia patients, aged 10 to 60, and compared with age-matched healthy controls. Vitamin E and β-carotene levels in LDL from patients were 45% and 24% of that observed in healthy controls, respectively. In contrast, the mean amount of LDL-CD was threefold higher and lysil residues of apo B-100 were decreased by 17%. LDL-CD in thalassemia patients showed a strong inverse correlation with LDL vitamin E (r = −0.784; P < .0001), while a negative trend was observed with LDL-β–carotene (r = −0.443; P = .149). In the plasma of thalassemia patients, malondialdehyde (MDA), a byproduct of lipid peroxidation, was increased by about twofold, while vitamin E showed a 52% decrease versus healthy controls. LDL-CD were inversely correlated with plasma vitamin E (r = −0.659; P < .0001) and correlated positively with plasma MDA (r = 0.621; P < .0001). Plasma ferritin was positively correlated with LDL-CD (r = 0.583; P =.0002). No correlation was found between the age of the patients and plasma MDA or LDL-CD. The LDL from thalassemia patients was cytotoxic to cultured human fibroblasts and cytotoxicity increased with the content of lipid peroxidation products. Clinical evidence of mild to severe vascular complications in nine of the patients was then matched with levels of LDL-CD, which were 36% to 118% higher than the mean levels of the patients. Our results could account for the incidence of atherogenic vascular diseases often reported in β-thalassemia patients. We suggest that the level of plasma MDA in β-thalassemia patients may represent a sensitive index of the oxidative status of LDL in vivo and of its potential atherogenicity.
C/EBPɛ Directly Interacts With the DNA Binding Domain of c-myb and Cooperatively Activates Transcription of Myeloid Promoters
Authors: Verbeek Walter , Gombart Adrian F. , Chumakov Alexey M. , Müller Carsten , Friedman Alan D. , Koeffler H. Phillip
Published: October 2019

C/EBPɛ is essential for granulocytic differentiation. We investigated the role of C/EBPɛ in the transcriptional activation of various myeloid-specific genes. We found that two C/EBPɛ isoforms, p32 and p30, possessing transcriptional activation domains were coexpressed in myeloid cells. Interestingly, isoform C/EBPɛ p30 but not p32 was differentially upregulated in NB-4 promyelocytic leukemia cells treated with retinoids. Both isoforms bound specifically to C/EBP sites in myeloid promoters. The kd for C/EBPɛ binding to the C/EBP site of the neutrophil elastase promoter was 4.2 nmol/L. In transfection assays using the nonhematopoietic cell line, CV-1, the p32 isoform activated promoters from the myeloid-specific mim-1, neutrophil elastase, and granulocyte colony-stimulating factor (G-CSF) receptor genes by 2.5-, 1.8-, and 1.6-fold, respectively. The p30 isoform lacked significant transcriptional activity, suggesting that other hematopoietic-specific factors were required for its function. Consistent with this prediction, transfections into the hematopoietic cell line Jurkat showed a 9.0- and 2.5-fold activation of the mim-1 promoter by the p32 and p30 isoforms, respectively. The additional 32 NH2-terminal residues made p32 a significantly more potent transcriptional activator than p30. T lymphoblasts (Jurkat cells) and immature myeloid cells (eg, Kcl22 cells) expressed high levels of the c-myb hematopoietic transcription factor. Cotransfection of c-myb with either the p32 or p30 isoform of C/EBPɛ in CV-1 cells cooperatively transactivated the mim-1 promoter by 20- and 16-fold, respectively, and the neutrophil elastase promoter by 10-and 7-fold, respectively. Pulldown assays showed that each C/EBPɛ isoform interacted directly with the DNA binding domain of the c-myb protein. Further studies showed that Kcl22 myeloid cells only contained active C/EBPɛ, but not C/EBP, C/EBPβ, or C/EBPδ. A mutation of the C/EBP site in the neutrophil elastase promoter markedly decreased the transactivation of the promoter in Kcl22 myeloblasts. These results demonstrate a role for C/EBPɛ in regulating myeloid promoters, such as neutrophil elastase, probably through a direct interaction with c-myb.
Analysis of Linked Human ɛ and γ Transgenes: Effect of Locus Control Region Hypersensitive Sites 2 and 3 or a Distal YY1 Mutation on Stage-Specific Expression Patterns
Authors: Zhu Wei , TomHon Catherine , Mason Marsha , Campbell Thomas , Shelden Eric , Richards Neil , Goodman Morris , Gumucio Deborah L.
Published: October 2019

Stage-specific expression of the human β-like globin genes is controlled by interactions between regulatory elements near the individual genes and additional elements located upstream in the Locus Control Region (LCR). Elucidation of the mechanisms that govern these interactions could suggest strategies to reactivate fetal (γ) or embryonic (ɛ) genes in individuals with severe hemoglobinopathies. This study extends an earlier analysis of a transgenic construct, HS3ɛγ, testing: (A) the effect of substitution of HS2 for HS3 on stage-specific expression of the ɛ and γ genes and, (B) the role of an evolutionarily conserved YY1 binding site in transcriptional regulation of the γ gene. The data show that both HS3ɛγ and HS2ɛγ can individually support embryonic expression of ɛ and fetal expression of Aγ. Thus, the cis regulators of distinct expression patterns for ɛ and γ are likely to reside near the genes, rather than in specific hypersensitive sites of the LCR. Alterations in Aγ expression patterns observed in transgenic lines carrying a construct with a mutation in a conserved YY1 binding site at −1086 indicate that this site might function to facilitate active transcription of the γ gene in fetal life.
Prevention of Graft-Versus-Host Disease by Induction of Immune Tolerance With Ultraviolet B-Irradiated Leukocytes in H-2 Disparate Bone Marrow Donor
Authors: del Rosario M.L.U. , Zucali James R. , Kao K.J.
Published: October 2019

Transfusions (Tx) of Ultraviolet B (UVB)-irradiated peripheral blood mononuclear leukocytes (MNL) have been shown to induce humoral immune tolerance to major histocompatability complex (MHC) antigens (Blood 88:4375, 1996). To determine whether cellular immune tolerance to MHC antigens can be induced by the same approach, transplantation of bone marrow and spleen cells from tolerant donors across the H-2 barrier was conducted to study its effect on prevention of graft-versus-host disease (GVHD). After immune tolerance induction by four weekly Tx of UVB-irradiated BALB/c (H-2d) peripheral blood MNL into CBA/HT6 (H-2k) mice, bone marrow cells (BMC) and spleen MNL from tolerant or naive CBA mice were transplanted into lethally irradiated BALB/c mice. The transplanted mice were followed by measuring body weight, peripheral leukocyte counts, GVHD, survival, and cytokine response. All BALB/c recipient mice were fully engrafted with H-2k CBA donor cells after transplantation. The severity of GVHD was significantly attenuated in BALB/c mice transplanted with BMC and spleen MNL from tolerant CBA donor mice. The recovery of peripheral leukocyte and lymphocyte counts were faster and more complete in mice transplanted with cells from the tolerant donors. The serum cytokine profile after transplantation with tolerant donor cells showed increased interleukin-4 and reduced gamma interferon that are consistent with a polarized Th2 response. The results pooled from three separate experiments showed that BALB/c mice transplanted with 5 × 106 BMC and 4 × 105spleen MNL from tolerant CBA donors had better overall survival than the control group (72% v 17%, P = .018). The findings show that transplantation with bone marrow and spleen cells from tolerant H-2 disparate donor mice is associated with significant attenuation of GVHD and better outcomes. The results also support that transfusions of UVB-irradiated leukocytes may induce cellular immune tolerance.
Expansion of genetically modified primary human hemopoietic cells using chemical inducers of dimerization
Authors: Richard Robert E. , Wood Brent , Zeng Hui , Jin Liqing , Papayannopoulou Thalia , Blau C. Anthony
Published: October 2019

The inability to deliver a therapeutic gene to a sufficient percentage of hematopoietic stem cells is the major obstacle to using gene therapy to treat blood disorders. Providing genetically corrected stem cells with a reversible growth advantage could solve this problem. To this end we have employed small synthetic molecules that can reversibly dimerize and activate fusion proteins which contain a growth factor receptor signaling domain. We have shown that the thrombopoietin receptor (mpl) signaling domain can be used in this system to expand transduced multipotential progenitor cells from mouse bone marrow. In the present study we tested a similar retroviral vector in human CD34-selected cord blood cells. Following transduction, cells cultured in the presence of the dimerizing molecule AP1903 expanded 13.8- to 186-fold relative to cells cultured in the absence of AP1903. The cell type that emerged in suspension culture was erythroid. Contrary to our results in the murine system, cell expansion was transient. Activation of mpl caused the disappearance of BFU-E followed by a transient increase in CFU-E. In contrast, mpl activation had no discernable effect on transduced myeloid progenitor cells. AP1903-mediated expansion was restricted to transduced cells, as demonstrated by immunohistochemical staining. These findings indicate that synthetic dimerizing molecules can be used to expand primary human hematopoietic cells.
Plasminogen activator inhibitor-1 and vitronectin promote vascular thrombosis in mice
Authors: Eitzman Daniel T. , Westrick Randal J. , Nabel Elizabeth G. , Ginsburg David
Published: October 2019

Occlusive thrombosis depends on the net balance between platelets, coagulation, and fibrinolytic factors. Epidemiologic information suggests that plasminogen activator inhibitor-1 (PAI-1), a central regulator of the fibrinolytic system, plays an important role in determining the overall risk for clinically significant vascular thrombosis. Vitronectin (VN), an abundant plasma and matrix glycoprotein, binds PAI-1 and stabilizes its active conformation. This study assessed the role of PAI-1 and VN expression in the formation of occlusive vascular thrombosis following arterial or venous injury. The common carotid arteries of 17 wild-type (WT) mice and 8 mice deficient in PAI-1 were injured photochemically while blood flow was continuously monitored. WT mice developed occlusive thrombi at 52.0 ± 3.8 minutes (mean ± SEM) following injury; mice deficient in PAI-1 developed occlusive thrombosis at 127 ± 15 minutes (P < .0001). Mice deficient in VN (n = 12) developed vascular occlusion 77 ± 11 minutes after injury, intermediate between the values observed for WT mice (P < .03) and mice deficient in PAI-1 (P < .01). PAI-1 and VN also affected the time to occlusion after injury to the jugular vein. Three WT mice developed occlusive venous thrombosis an average of 39.7 ± 1 minutes following the onset of injury, whereas the jugular veins of 4 mice deficient in PAI-1 and 4 deficient in VN occluded 56.7 ± 5 and 58.7 ± 2 minutes, respectively, following injury (P < .04 andP < .01 compared to WT mice). These results suggest that endogenous fibrinolysis and its regulation by PAI-1 and VN have important roles in the development of occlusive vascular thrombosis after vascular injury.
The association between the Val34Leu polymorphism in the factor XIII gene and brain infarction
Authors: Elbaz Alexis , Poirier Odette , Canaple Sandrine , Chédru François , Cambien François , Amarenco Pierre
Published: October 2019

Factor XIII catalyzes the formation of covalent bounds between fibrin monomers, thus stabilizing the fibrin clot and increasing its resistance to fibrinolysis. The frequency of a frequent Val34Leu polymorphism in the FXIII A-subunit gene has been shown to be lower in patients with myocardial infarction or venous thrombosis than in controls, whereas it was higher in patients with hemorrhagic stroke than in controls. Our aim was to study the relation between brain infarction (BI) and the FXIII Val34Leu polymorphism in 456 patients consecutively recruited with a BI confirmed by MRI, and 456 matched controls. The distribution of genotypes was different in cases (63.2% Val/Val; 30.9% Val/Leu; 5.9% Leu/Leu) compared with controls (49.8% Val/Val; 42.8% Val/Leu; 7.4% Leu/Leu; P < .001). Carrying the Leu allele was associated with an OR of 0.58 (95% CI = 0.44-0.75). A similar association was observed in cases and controls free of previous cardiovascular or cerebrovascular history (OR = 0.51; 95% CI = 0.36-0.73). No heterogeneity of this association was observed after stratification on the main BI subtypes. Adjustment for traditional vascular risk factors did not modify these findings. In addition, the effect of smoking was modified by the polymorphism (P = .05); the effect of smoking was weaker among Leu carriers than among noncarriers. In conclusion, there was a negative association of the FXIII Val34Leu polymorphism with BI, thus suggesting a protective effect of the Leu allele against thrombotic cerebral artery occlusion. In addition, our results suggest that among Leu carriers, the protective effect of the polymorphism outweighed the effect of smoking.
Multiple human serum components act as bridging molecules in rosette formation by Plasmodium falciparum-infected erythrocytes
Authors: Somner Elizabeth A. , Black Julie , Pasvol Geoffrey
Published: October 2019

Rosetting, the binding of parasitized erythrocytes to 2 or more uninfected erythrocytes, is an in vitro correlate of disease severity in Plasmodium falciparum malaria. Although cell ligands and receptors have been identified and a role for immunoglobulin M has been suggested, the molecular mechanisms of rosette formation are unknown. The authors demonstrate unequivocally that rosette formation by P falciparum-infected erythrocytes is specifically dependent on human serum, and they propose that serum components act as bridging molecules between the cell populations. Using heparin treatment and Percoll density gradient centrifugation, they have developed an assay in which parasitized erythrocytes grown in serum-containing medium and optimally forming rosettes are stripped of serum components. These infected cells were no longer able to form rosettes when mixed with erythrocytes and incubated in serum-free medium. Rosette formation was restored by the addition of serum or certain serum fractions obtained by concanavalin A (conA) affinity, anti-IgM affinity, anion exchange, and gel filtration chromatography. The authors clearly demonstrate that multiple serum components—IgM and at least 2 others—are involved in rosette formation. Those others consist of 1 or more acidic components of high-molecular mass that binds to conA (but that is not thrombospondin, fibronectin, or von Willebrand's factor) and of at least 1 more basic, smaller component that does not bind to conA. Data on the size and number of rosettes formed support the authors' hypothesis that multiple bridges are involved in this complex cellular interaction. These findings have important implications for the understanding of pathogenic adhesive interactions of P falciparum and host susceptibility to severe malaria.
Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system
Authors: Reuter Tanja , Herterich Sabine , Bernhard Oliver , Hoehn Holger , Gross Hans J.
Published: October 2019

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products.
Ectopic expression of fibroblast growth factor receptor 3 promotes myeloma cell proliferation and prevents apoptosis
Authors: Plowright Elizabeth E. , Li Zhihua , Bergsagel P. Leif , Chesi Marta , Barber Dwayne L. , Branch Donald R. , Hawley Robert G. , Stewart A. Keith
Published: October 2019

The t(4;14) translocation occurs in 25% of multiple myeloma (MM) and results in both the ectopic expression of fibroblast growth factor receptor 3 (FGFR3) from der4 and immunoglobulin heavy chain-MMSET hybrid messenger RNA transcripts from der14. The subsequent selection of activating mutations of the translocated FGFR3 by MM cells indicates an important role for this signaling pathway in tumor development and progression. To investigate the mechanism by which FGFR3 overexpression promotes MM development, interleukin-6 (IL-6)-dependent murine B9 cells were transduced with retroviruses expressing functional wild-type or constitutively activated mutant FGFR3. Overexpression of mutant FGFR3 resulted in IL-6 independence, decreased apoptosis, and an enhanced proliferative response to IL-6. In the presence of ligand, wild-type FGFR3-expressing cells also exhibited enhanced proliferation and survival in comparison to controls. B9 clones expressing either wild-type FGFR3 at high levels or mutant FGFR3 displayed increased phosphorylation of STAT3 and higher levels of bcl-xL expression than did parental B9 cells after cytokine withdrawal. The mechanism of the enhanced cell responsiveness to IL-6 is unknown at this time, but does not appear to be mediated by the mitogen-activated protein kinases SAPK, p38, or ERK. These findings provide a rational explanation for the mechanism by which FGFR3 contributes to both the viability and propagation of the myeloma clone and provide a basis for the development of therapies targeting this pathway.
Limited expression of R5-tropic HIV-1 in CCR5-positive type 1–polarized T cells explained by their ability to produce RANTES, MIP-1α, and MIP-1β
Authors: Annunziato Francesco , Galli Grazia , Nappi Filomena , Cosmi Lorenzo , Manetti Roberto , Maggi Enrico , Ensoli Barbara , Romagnani Sergio
Published: October 2019

Human T helper (Th) cells (Th1- or Th2-oriented memory T cells as well as Th1- or Th2-polarized naive T cells) were infected in vitro with an R5-tropic HIV-1 strain (BaL) and assessed for their profile of cytokine production, CCR5 receptor expression, and HIV-1 p24 antigen (p24 Ag) production. Higher p24 Ag production was found in CCR5-negative Th2-like memory T cells than in CCR5-positive Th1-like memory T cells. By contrast, p24 Ag production was higher in Th1-polarized activated naive T cells in the first 4 days after infection. However, p24 Ag production in Th1-polarized T cells became comparable or even lower than the production in Th2-polarized populations later in infection or when the cells were infected with HIV-1BaL after secondary stimulation. The higher levels of p24 Ag production by Th1-polarized naive T cells soon after infection reflected a higher virus entry, as assessed by the single round infection assay using the HIV–chloramphenicol acetyl transferase (HIV-CAT) R5-tropic virus that contains the envelope protein of HIV-1 YU2 strain. The limitation of viral spread in the Th1-polarized populations, despite the initial higher level of T-cell entry of R5-tropic strains, was due to the ability of Th1 cells to produce greater amounts of β-chemokines than Th2 cells. In fact, an inverse correlation was observed between Th1-polarized naive T cells and Th1-like memory-activated T cells in regards to p24 Ag production and the release of the following CCR5-binding chemokines: regulated on activation normal T expressed and secreted (RANTES), macrophage inflammatory protein–1 (MIP-1), and MIP-1β. Moreover, infection with the HIV-1BaL strain of Th1-polarized T cells in the presence of a mixture of anti-RANTES, anti–MIP-1, and anti–MIP-1β neutralizing antibodies resulted in a significant increase of HIV-1 expression. These findings suggest that Th1-type responses may favor CD4+ T-cell infection by R5-tropic HIV-1 strains, but HIV-1 spread in Th1 cells is limited by their ability to produce CCR5-binding chemokines.
Abundant Tax protein expression in CD4+ T cells infected with human T-cell lymphotropic virus type I (HTLV-I) is prevented by cytotoxic T lymphocytes
Authors: Hanon Emmanuel , Hall Sarah , Taylor Graham P. , Saito Mineki , Davis Ricardo , Tanaka Yuetsu , Usuku Koichiro , Osame Mitsuhiro , Weber Jonathan N. , Bangham Charles R. M.
Published: October 2019

The role of the cellular immune response in human T-cell leukemia virus type I (HTLV-I) infection is not fully understood. A persistently activated cytotoxic T lymphocyte (CTL) response to HTLV-I is found in the majority of infected individuals. However, it remains unclear whether this CTL response is protective or causes tissue damage. In addition, several observations paradoxically suggest that HTLV-I is transcriptionally silent in most infected cells and, therefore, not detectable by virus-specific CTLs. With the use of a new flow cytometric procedure, we show here that a high proportion of naturally infected CD4+ peripheral blood mononuclear cells (PBMC) (between 10% and 80%) are capable of expressing Tax, the immunodominant target antigen recognized by virus-specific CTLs. Furthermore, we provide direct evidence that autologous CD8+ T cells rapidly kill CD4+ cells naturally infected with HTLV-I and expressing Tax in vitro by a perforin-dependent mechanism. Consistent with these observations, we observed a significant negative correlation between the frequency of Tax11-19-specific CD8+ T cells and the percentage of CD4+ T cells in peripheral blood of patients infected with HTLV-I. Those results are in accordance with the view that virus-specific CTLs participate in a highly efficient immune surveillance mechanism that persistently destroys Tax-expressing HTLV-I-infected CD4+ T cells in vivo.
Evidence that immunoglobulin specificities of AIDS-related lymphoma are not directed to HIV-related antigens
Authors: Cunto-Amesty Gina , Przybylski Grzegorz , Honczarenko Marek , Monroe John G. , Silberstein Leslie E.
Published: October 2019

Chronic B-cell stimulation may be a predisposing event in the early pathogenesis of the acquired immunodeficiency syndrome (AIDS)-related lymphoma (ARL). ARL-derived immunoglobulin (Ig) genes are significantly diversified from germline, suggesting that antigenic stimulation via Ig receptors may occur prior to malignant transformation. We have evaluated 6 ARL-derived antibodies for binding to human immunodeficiency virus (HIV) and cell surface epitopes. Five cases expressed IgM, and 1 case expressed IgG. Expressed V genes were significantly diversified (3%-15%) from known germline V genes. A non-Ig producing mouse myeloma cell line was transfected with expression vectors containing the lymphoma-derived V genes. By enzyme-linked immunosorbent assay and Western blot assay, the lymphoma-derived Ig's showed no reactivity against HIV recombinant proteins. Also, no specific HIV reactivity was observed by flow cytometry with lymphoma-derived Ig's against the T-cell line infected with T-tropic HIV-1 or peripheral blood mononuclear cells infected with M-tropic HIV strains, indicating lack of binding to native HIV epitopes. However, 2 of the lymphoma-derived Ig's (ARL-7 and ARL-14) bound strongly to non–HIV-infected cells of various tissue origins. Thus, these findings suggest that the transformed B cells of AIDS-associated lymphomas may not arise from the pool of anti-HIV specific B cells but, rather, may develop from B cells responding to other antigens, including self-antigens.
Apoptosis of leukemic cells accompanies reduction in intracellular pH after targeted inhibition of the Na+/H+exchanger
Authors: Rich Ivan N. , Worthington-White Diana , Garden Oliver A. , Musk Philip
Published: October 2019

The Na+/H+ exchanger isoform 1 (NHE1) is primarily responsible for the regulation of intracellular pH (pHi). It is a ubiquitous, amiloride-sensitive, growth factor–activatable exchanger whose role has been implicated in cell-cycle regulation, apoptosis, and neoplasia. Here we demonstrate that leukemic cell lines and peripheral blood from primary patient leukemic samples exhibit a constitutively and statistically higher pHi than normal hematopoietic tissue. We then show that a direct correlation exists between pHi and cell-cycle status of normal hematopoietic and leukemic cells. Advantage was taken of this relationship by treating leukemic cells with the Na+/H+ exchanger inhibitor, 5-(N, N-hexamethylene)-amiloride (HMA), which decreases the pHiand induces apoptosis. By incubating patient leukemic cells in vitro with pharmacologic doses of HMA for up to 5 hours, we show, using flow cytometry and fluorescent ratio imaging microscopy, that when the pHi decreases, apoptosis—measured by annexin-V and TUNEL methodologies—rapidly increases so that more than 90% of the leukemic cells are killed. The differential sensitivity exhibited between normal and leukemic cells allows consideration of NHE1 inhibitors as potential antileukemic agents.
Aberrant expression of active leukotriene C4 synthase in CD16+ neutrophils from patients with chronic myeloid leukemia
Authors: Sjölinder Mikael , Stenke Leif , Näsman-Glaser Barbro , Widell Susanne , Doucet Johanne , Jakobsson Per-Johan , Lindgren Jan Åke
Published: October 2019

Elevated leukotriene (LT)C4 synthase activity was observed in peripheral blood granulocyte suspensions from patients with chronic myeloid leukemia (CML). Magnetic cell sorting (MACS) with CD16 monoclonal antibodies (mAbs), which were used to fractionate granulocytes from CML patients and healthy individuals, yielded highly purified suspensions of CD16+ neutrophils. The purity of these cell fractions was verified by extensive morphologic examination. Reverse transcriptase–polymerase chain reaction (RT-PCR) analyses, demonstrating the absence of interleukin-4 messenger RNA (IL-4 mRNA), further confirmed the negligible contamination of eosinophils in these fractions. Notably, purified CML CD16+ neutrophils from all tested patients transformed exogenous LTA4 to LTC4. These cells also produced LTC4 after activation with ionophore A23187 or the chemotactic peptide fMet-LeuPhe (N-formylmethionyl-leucyl-phenylalanine). Subcellular fractionation revealed that the enzyme activity was exclusively distributed to the microsomal fraction. Expression of LTC4 synthase mRNA in CML CD16+neutrophils was confirmed by RT-PCR. Furthermore, Western blot analyses consistently demonstrated expression of LTC4 synthase at the protein level in CML CD16+ neutrophils, whereas expression of microsomal glutathione S-transferase 2 occurred occasionally. Expectedly, LTC4 synthase activity or expression of the protein could not be demonstrated in CD16+ neutrophil suspensions from any of the healthy individuals. Instead, these cells, as well as CML CD16+neutrophils, transformed LTA4 to LTB4. The results indicate that aberrant expression of LTC4 synthase is a regular feature of morphologically mature CML CD16+neutrophils. This abnormality, possibly associated with malignant transformation, can lead to increased LTC4 synthesis in vivo. Such overproduction may be of pathophysiological relevance because LTC4 has been demonstrated to stimulate proliferation of human bone marrow–derived myeloid progenitor cells.
GCP-2–induced internalization of IL-8 receptors: hierarchical relationships between GCP-2 and other ELR+-CXC chemokines and mechanisms regulating CXCR2 internalization and recycling
Authors: Feniger-Barish Rotem , Belkin Dan , Zaslaver Alon , Gal Shira , Dori Mally , Ran Maya , Ben-Baruch Adit
Published: October 2019

The chemotactic potencies of ELR+-CXC chemokines during acute inflammation are regulated by their binding affinities and by their ability to activate, desensitize, and internalize their specific receptors, CXCR1 and CXCR2. To gain insight into the fine mechanisms that control acute inflammatory processes, we have focused in this study on the highly potent ELR+-CXC chemokine Granulocyte Chemotactic Protein 2 (GCP-2), and on its ability to control the cell surface expression of CXCR1 and CXCR2. Although GCP-2 has been considered an effective ligand for both CXCR1 and CXCR2, our findings demonstrated that it was a potent inducer of CXCR2 internalization only. A functional hierarchy was shown to exist between GCP-2 and 2 other ELR+-CXC chemokines, IL-8 and NAP-2, in their abilities to induce CXCR1 and CXCR2 internalization, according to the following: IL-8 > GCP-2 > NAP-2. By the use of pertussis toxin (PTx), it was demonstrated that the actual events of Gi-coupling to CXCR2 do not have a major role in the regulation of its internalization. Rather, CXCR2 internalization was shown to be negatively controlled by induction of signaling events, as indicated by the promotion of CXCR2 internalization following exposure to wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3 kinases and PI4 kinases. Furthermore, our results suggest that rab11+-endosomes participate in the trafficking of CXCR2 through the endocytic pathway, to eventually allow its recycling back to the plasma membrane. To conclude, our findings shed light on the interrelationships between GCP-2 and other ELR+-CXC chemokines, and determine the mechanisms involved in the regulation of GCP-2–induced internalization and recycling of CXCR2.
Spatial associations of centromeres in the nuclei of hematopoietic cells: evidence for cell-type-specific organizational patterns
Authors: Alcobia Isabel , Dilão Rui , Parreira Leonor
Published: October 2019

It is believed that the 3-dimensional organization of centromeric heterochromatin in interphase may be of functional relevance as an epigenetic mechanism for the regulation of gene expression. Accordingly, a likely possibility is that the centromeres that spatially associate into the heterochromatic structures (chromocenters) observed in the G1 phase of the cell cycle will differ in different cells. We sought to address this issue using, as a model, the chromocenters observed in quiescent normal human hematopoietic cells and primary fibroblasts. To do this, we analyzed the spatial relationships among different human centromeres in 3-D preserved cells using nonisotopic in situ hybridization and confocal microscopy. We showed quantitatively that chromocenters in all cell types do indeed represent nonrandom spatial associations of certain centromeres. Furthermore, the observed patterns of centromere association indicate that the chromocenters in these cell types are made of different combinations of specific centromeres, that hematopoietic cells are strikingly different from fibroblasts as to the composition of their chromocenters and that centromeres in peripheral blood cells appear to aggregate into distinct “myeloid” (present in monocytes and granulocytes) and “lymphoid” (present in lymphocytes) spatial patterns. These findings support the idea that the chromocenters formed in the nucleus of quiescent hematopoietic cells might represent heterochromatic nuclear compartments involved in the regulation of cell-type-specific gene expression, further suggesting that the spatial arrangement of centromeric heterochromatin in interphase is ontogenically determined during hematopoietic differentiation.