bluuebunny/crossref_metadata_2025 huggingface dataset:
Mapping of a syndrome of X-linked thrombocytopenia with thalassemia to band Xp11-12: further evidence of genetic heterogeneity of X-linked thrombocytopenia
Authors: Raskind Wendy H. , Niakan Kathy K. , Wolff John , Matsushita Mark , Vaughan Ty , Stamatoyannopoulos George , Watanabe Chiaki , Rios Jacinda , Ochs Hans D.
Published: October 2019

X-linked thrombocytopenia with thalassemia (XLTT; Online Mendelian Inheritance in Man [OMIM] accession number 314050) is a rare disorder characterized by thrombocytopenia, platelet dysfunction, splenomegaly, reticulocytosis, and unbalanced hemoglobin chain synthesis. In a 4-generation family, the gene responsible for XLTT was mapped to the X chromosome, short arm, bands 11-12 (band Xp11-12). The maximum lod score possible in this family, 2.39, was obtained for markers DXS8054 and DXS1003, at a recombination fraction of 0. Recombination events observed for XLTT and markers DXS8080 and DXS8023 or DXS991 define a critical region that is less than or equal to 7.65 KcM and contains the gene responsible for the Wiskott-Aldrich syndrome (WAS; OMIM accession number 301000) and its allelic variant X-linked thrombocytopenia (XLT; OMIM accession number 313900). Manifestations of WAS include thrombocytopenia, eczema, and immunodeficiency. In WAS/XLT the platelets are usually small, and bleeding is proportional to the degree of thrombocytopenia. In contrast, in XLTT the platelet morphology is normal, and the bleeding time is disproportionately prolonged. In this study no alteration in the WAS gene was detected by Northern blot or Western blot analysis, flow cytometry, or complimentary DNA dideoxynucleotide fingerprinting or sequencing. As has been reported for WAS and some cases of XLT, almost total inactivation of the XLTTgene-bearing X chromosome was observed in granulocytes and peripheral blood mononuclear cells from 1 asymptomatic obligate carrier. The XLTT carrier previously found to have an elevated :β hemoglobin chain ratio had a skewed, but not clonal, X-inactivation pattern favoring activity of the abnormal allele. Clinical differences and results of the mutation analyses make it very unlikely that XLTT is another allelic variant of WAS/XLT and strongly suggest that X-linked thrombocytopenia mapping to band Xp11-12 is a genetically heterogeneous disorder.
Human megakaryocytes and platelets contain the estrogen receptor β and androgen receptor (AR): testosterone regulates AR expression
Authors: Khetawat Gopal , Faraday Nauder , Nealen Michele L. , Vijayan K. Vinod , Bolton Everlie , Noga Stephen J. , Bray Paul F.
Published: October 2019

Gender differences in vascular thromboses are well known, and there is evidence that platelets may be involved in these differences and that sex hormones affect platelet function. We characterized the expression of the estrogen receptor  (ER ), estrogen receptor β (ER β), progesterone receptor (PR), and androgen receptor (AR) in the megakaryocyte lineage. Megakaryocytes generated ex vivo from normal human CD34+ stem cells contained RNA for ER β and AR, which increased with cell differentiation. Platelets and human erythroleukemia (HEL) cells also contained ER β and AR transcripts. No ER  or PR messenger RNA or protein was detected in the megakaryocyte lineage. Immunofluorescence microscopy showed that ER β protein was present in glycoprotein (GP) IIb+ megakaryocytes and the HEL megakaryocytic cell line in a predominantly cytoplasmic location. AR showed a cytoplasmic and nuclear distribution in GPIIb+ and GPIIb− cells derived from CD34+ cells and in HEL cells. Western immunoblotting confirmed the presence of ER β and AR in platelets. Megakaryocyte and HEL AR expression was up-regulated by 1, 5, and 10 nmol/L testosterone, but down-regulated by 100 nmol/L testosterone. These findings indicate a regulated ability of megakaryocytes to respond to testosterone and suggest a potential mechanism through which sex hormones may mediate gender differences in platelet function and thrombotic diseases.
Granule exocytosis, and not the Fas/Fas ligand system, is the main pathway of cytotoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans
Authors: Yasukawa Masaki , Ohminami Hideki , Arai Junko , Kasahara Yoshihito , Ishida Yasushi , Fujita Shigeru
Published: October 2019

We investigated the cytotoxicity mechanisms of alloantigen-specific human CD4+ and CD8+ cytotoxic T lymphocytes (CTLs) using cells from family members with the Fas gene mutation. Alloantigen-specific CD4+ and CD8+ CTL bulk lines and clones were generated from 2 individuals by stimulation of their peripheral blood lymphocytes with allogeneic Fas−/− or Fas+/− cell lines that were established from B-lymphocytes of a patient with Fas deficiency and her mother, respectively. Both CD4+ and CD8+CTL bulk lines and clones directed against allogeneic HLA antigens exerted cytotoxicity against Fas−/− and Fas+/− cells to almost the same degree. The cytotoxicity of CD4+ and CD8+ CTLs appeared to be Ca2+-dependent and was completely inhibited by concanamycin A, an inhibitor of perforin-mediated cytotoxicity. Messenger RNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B were all detected in these CD4+CTLs with the use of the reverse transcriptase polymerase chain reaction. The majority of CD4+ CTL clones that showed Fas-independent cytotoxicity were TH0, as determined by their cytokine production profile. These data, obtained with the use of a novel experimental system, clearly show that the main pathway of cytotoxicity mediated by alloantigen-specific human CD4+as well as by CD8+ CTLs is granule exocytosis, and not the Fas/Fas ligand system.
Retroviral transfer of the hENT2 nucleoside transporter cDNA confers broad-spectrum antifolate resistance in murine bone marrow cells
Authors: Patel Divyen H. , Allay James A. , Belt Judith A. , Sorrentino Brian P.
Published: October 2019

Antifolate drugs such as methotrexate are commonly used in cancer chemotherapy. It may be possible to increase the antitumor activity of antifolates by the coadministration of drugs that inhibit nucleoside transport, thereby blocking the capacity of tumor cells to salvage nucleotide precursors. An important limitation of this approach is severe myelosuppression caused by many of these drug combinations. For this reason, we have developed a gene therapy strategy to protect bone marrow cells against combined treatment with antifolates and nitrobenzylmercaptopurine riboside (NBMPR), a potent inhibitor of thees nucleoside transporter. A retroviral vector (MeiIRG) was constructed that expressed the NBMPR-insensitive eitransporter, hypothesizing that transduced bone marrow cells would survive drug treatment because of the preservation of nucleoside salvage pathways. In vitro clonogenic assays confirmed that the MeiIRG vector did protect myeloid progenitors against the toxic effects of 3 different antifolates when each was combined with NBMPR. On testing this system in vivo, decreased myelosuppression was observed in mice transplanted with MeiIRG-transduced bone marrow cells and subsequently treated with trimetrexate and NBMPR-P. In these mice, significant increases were noted in absolute neutrophil count nadirs, reticulocyte indices, and the numbers of myeloid progenitors in the bone marrow. Furthermore, a survival advantage was associated with transfer of the MeiIRG vector, indicating that significant dose intensification was possible with this approach. In summary, the MeiIRG vector can decrease the toxicity associated with the combined use of antifolates and NBMPR-P and thereby may provide a strategy for simultaneously sensitizing tumor cells while protecting hematopoietic cells.
Coinfection of multiple strains of Epstein-Barr virus in immunocompetent normal individuals: reassessment of the viral carrier state
Authors: Srivastava Gopesh , Wong Kai Y. , Chiang Alan K. S. , Lam King Y. , Tao Qian
Published: October 2019

This study reassesses the occurrence of Epstein-Barr virus (EBV) diversity and coinfection versus dominance of a single viral strain within immunocompetent normal carriers. Polymerase chain reaction analysis of several different polymorphic loci of the EBV genome was performed on collections of peripheral blood mononuclear cells and multiple lymphoid and epithelial tissues of the same individuals. Autopsy specimens from 15 individuals who died of causes unrelated to EBV infection served as normal viral carriers. Unexpectedly, coinfection of multiple distinct strains of EBV of the same type (usually type 1) and less frequently of both types 1 and 2 was found to be very high within individual viral carriers. These data indicate that coinfection with multiple EBV strains is much more prevalent in normal carriers than previously appreciated, which in turn has direct implications on EBV persistence, host–viral interaction and pathogenesis.
Neutrophil polarity and locomotion are associated with surface redistribution of leukosialin (CD43), an antiadhesive membrane molecule
Authors: Seveau Stéphanie , Keller Hansuli , Maxfield Frederick R. , Piller Friedrich , Halbwachs-Mecarelli Lise
Published: October 2019

This study analyzed the behavior of an antiadhesive membrane molecule, CD43, in neutrophil polarization and locomotion. CD43 cross-linking by antibodies induced neutrophil locomotion, with CD43 molecules clustered at the uropod of polarized neutrophils. In contrast, CD11b/CD18 cross-linking by antibodies did not affect either cell polarization or locomotion. Stimulation of suspended or adherent neutrophils with chemotactic peptide results in cell polarization and locomotion and a concomitant redistribution of CD43 to the uropod. This process is entirely reversible. The study also investigated which actin-binding protein could be involved in CD43 lateral redistribution. -Actinin and moesin are preferentially adsorbed on Sepharose beads bearing a recombinant CD43 intracellular domain. Analysis by immunofluorescence confocal microscopy shows a codistribution of moesin during CD43 lateral redistribution. By contrast, -actinin is located at the leading edge, an area devoid of CD43. These results shed new light on the role of CD43 membrane redistribution, which appears to be directly related to neutrophil polarity and locomotion.
Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera
Authors: Temerinac Snežana , Klippel Steffen , Strunck Elisabeth , Röder Sabine , Lübbert Michael , Lange Winand , Azemar Marc , Meinhardt Gerold , Schaefer Hans-Eckart , Pahl Heike L.
Published: October 2019

Polycythemia vera (PV) is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients with PV are hypersensitive to several growth factors, the molecular pathogenesis of this disease remains unknown. To investigate the molecular defects underlying PV, we used subtractive hybridization to isolate complementary DNAs (cDNAs) differentially expressed in patients with PV versus normal controls. We isolated a novel gene, subsequently named PRV-1, which is highly expressed in granulocytes from patients with PV (n = 19), but not detectable in normal control granulocytes (n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from patients with chronic myelogenous leukemia (n = 4) or acute myelogenous leukemia (n = 5) or in granulocytes from patients with essential thrombocythemia (n = 4) or secondary erythrocytosis (n = 4). Northern blot analysis showed that PRV-1 is highly expressed in normal human bone marrow and to a much lesser degree in fetal liver. It is not expressed in a variety of other tissues tested. Although PRV-1 is not expressed in resting granulocytes from normal controls, stimulation of these cells with granulocyte colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA encodes an open reading frame of 437 amino acids, which contains a signal peptide at the N-terminus and a hydrophobic segment at the C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains homologous to those found in the uPAR/Ly6/CD59/snake toxin-receptor superfamily. We therefore propose that PRV-1 represents a novel hematopoietic receptor.
Tracing uptake of C3dg-conjugated antigen into B cells via complement receptor type 2 (CR2, CD21)
Authors: Hess Michael W. , Schwendinger Michael G. , Eskelinen Eeva-Liisa , Pfaller Kristian , Pavelka Margit , Dierich Manfred P. , Prodinger Wolfgang M.
Published: October 2019

Electron microscopy was used to study the internalization and delivery of ligands for complement receptor type 2 (CR2, CD21) to endocytic compartments of B-lymphoblastoid Raji cells. Opsonized antigen was mimicked with purified C3dg conjugated to colloidal gold. C3dg-gold bound specifically to the cell surface in a time-dependent manner, and preincubation of the cells with a monoclonal antibody blocking the CR2 ligand-binding site completely inhibited any C3dg-gold binding. Notably, the binding of C3d-gold was confined to cell surface protrusions, eg, microvilli. C3dg-gold was apparently internalized through coated pits located at the bases of microvilli and could be traced to different compartments of the endocytic pathway. The morphologic characteristics and intracellular distribution of these multivesicular or multilaminar structures were compatible with those of compartments known to harbor major histocompatibility complex (MHC) class II molecules. Immunolabeling showed that the internalized C3dg-gold colocalized with MHC class II in these structures. These data provide the first ultrastructural evidence that complement-coated antigens are endocytosed by antigen-nonspecific B cells by CR2 and are delivered to the compartments in which peptide loading for antigen presentation occurs. They support the notion that CR2 may play a role in antigen presentation by B cells regardless of B-cell receptor specificity.
Molecular analysis of lineage-specific chimerism and minimal residual disease by RT-PCR of p210BCR-ABL and p190BCR-ABL after allogeneic bone marrow transplantation for chronic myeloid leukemia: increasing mixed myeloid chimerism and p190BCR-ABL detection precede cytogenetic relapse
Authors: Serrano Josefina , Roman Jose , Sanchez Joaquin , Jimenez Antonio , Castillejo Juan A. , Herrera Concepcion , Gonzalez Maria Gracia , Reina Luisa , Rodriguez Maria del Carmen , Alvarez Miguel A. , Maldonado Juan , Torres Antonio
Published: October 2019

We studied lineage-specific chimerism and minimal residual disease (MRD) in sequential posttransplant samples from 55 patients who underwent unmanipulated (n = 44) or partially T-cell–depleted (n = 11) allogeneic bone marrow transplantation (BMT) for chronic myeloid leukemia (CML). Chimerism was assessed by polymerase chain reaction (VNTR [variable number of tandem repeats]-PCR) analysis in highly purified CD19+, CD3+, CD15+, and CD56+ cell fractions, whereas MRD was investigated in whole blood by reverse transcriptase–PCR (RT-PCR) of both p210BCR-ABL and p190BCR-ABL hybrid transcripts. Of 55 patients, 14 (including 6 T-cell–depleted patients) had cytogenetic relapse at 5-80 months and progressed to hematologic relapse, while 41 patients remained in prolonged cytogenetic remission 12-107 months post-BMT. Before leukemia recurrence, patients in the relapse group showed a consistent evolution pattern sequentially featured by persistent p210BCR-ABL positivity, increasing mixed chimerism (MC) in myeloid cells, p190BCR-ABL positivity, and, finally, cytogenetic relapse. Myeloid MC preceded cytogenetic relapse by 2-12 months, whereas p190BCR/ABL was detected 1-6 months prior to cytogenetic relapse in 11 patients and concomitant with cytogenetic relapse in 3 patients. In the remission group, all patients invariably tested negative for p190BCR-ABL; 10 patients tested positive for p210BCR-ABL at variable time-points but showed persistent full donor chimerism (DC), whereas 31 patients tested p210BCR-ABL negative and displayed full DC or transient MC due to the persistence of recipient T cells. Two patients in the relapse group were successfully reinduced into molecular remission with donor lymphocyte infusion. Sequential molecular analysis after such treatment showed the inverse pattern to that observed prior to relapse, ie, progressive disappearance of p190BCR-ABL transcripts, conversion of myeloid chimerism to donor type, and, finally, p210BCR-ABL negativity. We conclude that lineage-specific chimerism and p190BCR-ABL messenger RNA (mRNA) analyses contribute a better characterization of CML evolution after BMT and enable early identification of patients at the highest risk of relapse.
The t(5;17) acute promyelocytic leukemia fusion protein NPM-RAR interacts with co-repressor and co-activator proteins and exhibits both positive and negative transcriptional properties
Authors: Redner Robert L. , Chen J. Don , Rush Elizabeth A. , Li Hui , Pollock Sheri L.
Published: October 2019

The t(5;17) variant of acute promyelocytic leukemia (APL) fuses the genes for nucleophosmin (NPM) and the retinoic acid receptor alpha (RAR). Two NPM-RAR molecules are expressed as a result of alternative RNA splicing. Both contain RAR sequences that encode the DNA binding, heterodimerization, and ligand activation domains of RAR. This study was designed to test the ability of these fusion proteins to act as transcriptional activators of retinoic acid responsive promoters. The NPM-RAR fusion proteins bind to retinoic acid response element sequences as either homodimers or as heterodimers with RXR. Transcription of retinoic acid–inducible promoters is activated by the fusion proteins in the presence of retinoic acid. The level of transactivation induced by the NPM-RAR fusions differs from the level of transactivation induced by wild-type RAR in both a promoter and cell specific fashion, and more closely parallels the pattern of activation of the PML-RAR fusion than wild-type RAR. In addition, NPM-RAR decreases basal transcription from some promoters and acts in a dominant-negative fashion when co-transfected with wild-type RAR. Both NPM-RAR and PML-RAR interact with the co-repressor protein SMRTe in a manner that is less sensitive than RAR to dissociation by retinoic acid. Retinoic acid induces binding of the co-activator protein RAC3. These data indicate that the NPM-RAR fusion proteins can modulate expression of retinoid-responsive genes in a positive or negative manner, depending on context of the promoter, and lend support to the hypothesis that aberrant transcriptional activation underlies the APL phenotype.
Autosomal-dominant giant platelet syndromes: a hint of the same genetic defect as in Fechtner syndrome owing to a similar genetic linkage to chromosome 22q11-13
Authors: Toren Amos , Rozenfeld-Granot Galit , Rocca Bianca , Epstein Charles J. , Amariglio Ninette , Laghi Ferdinando , Landolfi Raffaele , Brok-Simoni Frida , Carlsson Lena E. , Rechavi Gideon , Greinacher Andreas
Published: October 2019

Families with 3 different syndromes characterized by autosomal dominant inheritance of low platelet count and giant platelets were studied. Fechtner syndrome is an autosomal-dominant variant of Alport syndrome manifested by nephritis, sensorineural hearing loss, and cataract formation in addition to macrothrombocytopenia and polymorphonuclear inclusion bodies. Sebastian platelet syndrome is an autosomal-dominant macrothrombocytopenia combined with neutrophil inclusions that differ from those found in May-Hegglin syndrome or Chediak-Higashi syndrome or the Dohle bodies described in patients with sepsis. These inclusions are, however, similar to those described in Fechtner syndrome. Other features of Alport syndrome, though, including deafness, cataracts, and nephritis, are absent in Sebastian platelet syndrome. Epstein syndrome is characterized by macrothrombocytopenia without neutrophil inclusions, in addition to the classical Alport manifestations—deafness, cataracts, and nephritis—and it is also inherited in an autosomal-dominant mode. We mapped the disease-causing gene to the long arm of chromosome 22 in an Italian family with Fechtner syndrome, 2 German families with the Sebastian platelet syndrome, and an American family with the Epstein syndrome. Four markers on chromosome 22q yielded an LOD score greater than 2.76. A maximal 2-point LOD score of 3.41 was obtained with the marker D22S683 at a recombination fraction of 0.00. Recombination analysis placed the disease-causing gene in a 3.37-Mb interval between the markers D22S284 and D22S693. The disease-causing gene interval in these 3 syndromes is similar to the interval described recently in an Israeli family with a slightly different Fechtner syndrome than the one described here. Recombination analysis of these 3 syndromes refines the interval containing the disease-causing gene from 5.5 Mb to 3.37 Mb. The clinical likeness and the similar interval containing the disease-causing gene suggest that the 3 different syndromes may arise from a similar genetic defect.
Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria
Authors: Genini Davide , Adachi Souichi , Chao Qi , Rose David W. , Carrera Carlos J. , Cottam Howard B. , Carson Dennis A. , Leoni Lorenzo M.
Published: October 2019

Adenine deoxynucleosides induce apoptosis in quiescent lymphocytes and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. To explain why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis, several mechanisms have been proposed, including the direct binding of dATP to the pro-apoptotic factor Apaf-1 and the activation of the caspase-9 and -3 pathways. In this study it is shown, by means of several assays on whole cells and isolated mitochondria, that 2-chloro-2′-deoxyadenosine (2CdA) and 2-choloro-2′-ara-fluorodeoxyadenosine (CaFdA) disrupt the integrity of mitochondria from primary chronic lymphocytic leukemia (B-CLL) cells. The nucleoside-induced damage leads to the release of the pro-apoptotic mitochondrial proteins cytochrome c and apoptosis-inducing factor. The other adenine deoxynucleosides tested displayed comparable DNA-damaging potency but did not affect mitochondrial function. Interference with mitochondrial integrity, thus, may be a factor in the potent cytotoxic effects of 2CdA and CaFdA toward nondividing lymphocytes.
Enhanced antitumor immunity by fusion of CTLA-4 to a self tumor antigen
Authors: Huang Tzu-Hsuan , Wu Pin-Yi , Lee Chin-Nien , Huang Hsing-I , Hsieh Shie-Liang , Kung John , Tao Mi-Hua
Published: October 2019

The idiotypic determinant (Id) of the immunoglobulin expressed by a B-cell malignancy can serve as an effective tumor-specific antigen but is only weakly immunogenic. This study demonstrates that the immunogenicity of the tumor Id protein can be dramatically increased by directing it to antigen-presenting cells (APCs). Cytotoxic T-lymphocyte antigen 4 (CTLA-4) present on activated T cells has a strong binding affinity to both B7-1 and B7-2 molecules, which are primarily expressed on APCs. After construction of a fusion protein consisting of Id and CTLA-4 (Id-CTLA4), mice immunized with the fusion protein induced high titers of Id-specific antibody and T-cell proliferative responses without adjuvants and were protected from lethal tumor challenge. The Id-CTLA4 fusion protein was so potent that even low doses (down to 0.1 μg) of the immunogen were able to elicit strong antibody responses. By using an Id-CTLA4 mutant protein, the ability to bind B7 molecules on APCs was shown to be required for the enhanced immunogenicity of Id-CTLA4. These findings demonstrate that fusing CTLA-4 to a potential tumor antigen represents an effective approach to prime antitumor immunities in vivo and may be applicable to the design of vaccines for a variety of other diseases.
Ontogenic emergence of the hematon, a morphogenetic stromal unit that supports multipotential hematopoietic progenitors in mouse bone marrow
Authors: Blazsek Istvàn , Chagraoui Jalila , Péault Bruno
Published: October 2019

Development of the full repertoire of hematopoietic-lymphopoietic cells from a single stem cell requires specific contacts with stromal cells. The spatio-temporal organization of these cell associations in the bone marrow in ontogeny is, however, not well understood. In the adult, 10% of marrow cells form a cohort of compact aggregates, the hematon. In the hematon mesenchymal cells (Stro-1+), perivascular lipocytes (desmin+), endothelial cells (CD34+, Flk-1+, Sca-1+), and macrophages amalgamate with the hematopoietic progenitors long-term culture-initiating cells (LTC-IC), cobblestone area–forming cell (CAFC), high-proliferative-potential colony-forming unit (HPP-CFU), granulocyte-macrophage (GM)–CFU, and burst-forming unit–erythroid (BFU-E). During endochondral ossification of the femur, GM-CFU and day 7 CAFC numbers increased progressively from day 17 of gestation, but primitive, day 35 LTC-IC appeared from postnatal day 2. Unexpectedly, bone marrow (BM) taken between embryonic day 17 and day 5 was unable to support myeloid cell production in long-term cultures or to support day 35 LTC-IC growth. However, a gain in stromal cell competence occurred between days 7 and 10, which was correlated with the emergence of hematon in the BM. Thus, acquisition of hematopoietic competence by BM lags behind for approximately 10 days after the initial hematopoietic cell influx. In the adult, the hematon fraction was 3.7-fold enriched in day 35 LTC-IC over the buffy coat. It produced more GM-CFU and HPP-CFU in myeloid culture and more B cells in lymphopoietic “switch” cultures. It is reported that stromal hematopoietic units named hematons are specific morphogenetic structures that emerge at a well-defined postnatal stage of development in long bones, delineate discrete territories for hematopoietic stem cell seeding and development, embody the most productive hematogenous compartment in the BM, and probably enclose a morphogenetic organizer.
End-linked homodimers in fibrinogen Osaka VI with a Bβ-chain extension lead to fragile clot structure
Authors: Sugo Teruko , Nakamikawa Chizuko , Yoshida Nobuhiko , Niwa Kazuki , Sameshima Masazumi , Mimuro Jun , Weisel John W. , Nagita Akira , Matsuda Michio
Published: October 2019

The authors have identified a 12-residue carboxyl-terminal extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A mutation that creates AAG encoding Lys at the stop (TAG) codon, thus translating 36 base pairs in the noncoding region of the Bβ gene. The extra Cys residues appear to be involved in 1 or 2 disulfide bonds between 2 adjacent abnormal fibrinogen molecules, forming a fibrinogen homodimer as indicated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Indeed, about half of the fibrinogen molecules exist as end-linked dimers oriented in parallel or with an angle, as observed by transmission electron microscopy. These end-linked dimers may well alter the conformations of D and DD regions on fibrin assembly, leading to increased fiber branching at their sites in the growing protofibrils. By scanning electron microscopy, the Osaka VI fibrin network appears to have a lacelike structure composed of highly branched, thinner fibers than the normal fibrin architecture. Such fibrin networks may be easily damaged to form large pores when fluids are allowed to pass through the gels. The fragility of Osaka VI fibrin clots, further confirmed by permeation and compaction studies, may account for the massive bleeding observed in this patient.
Up-regulation of costimulatory/adhesion molecules by histone deacetylase inhibitors in acute myeloid leukemia cells
Authors: Maeda Takahiro , Towatari Masayuki , Kosugi Hiroshi , Saito Hidehiko
Published: October 2019

Histone deacetylase inhibitors (HDACIs) have been used to focus on the effects of inducing gene expression through the acetylation of histones which results in chromatin remodeling. The study explored whether HDACIs could induce the expression of costimulatory/adhesion molecules on acute myeloid leukemia (AML) cells, thereby effectively inducing tumor immunity. The expression of CD80, CD86, human leukocyte antigen (HLA)-DR, HLA-ABC, and intracellular adhesion molecule–1 (ICAM-1) was tested in human AML cell lines after the addition of HDACI, sodium butyrate (SB). Generally, increased expression of CD86 was observed by SB treatment in a majority of cell lines, and ICAM-1 was expressed in fewer cell lines. Essentially the same results were obtained using other HDACIs such as FR901228, trichostatin A, and trapoxin A. Quantitation of transcripts of CD86 accompanied with RNA synthesis inhibition assay and nuclear run-on assay revealed that SB up-regulates the CD86 expression transcriptionally. Furthermore, chromatin immunoprecipitation experiments showed that HDACI treatment caused remarkable acetylation on histone H3 and H4 at CD86 promoter chromatin in vivo. In 30 clinical AML samples, CD86 expression was significantly increased (P < .001) by SB treatment, and the expression of HLA-DR and ICAM-1 was moderately increased (P < .05) by SB treatment. Finally, the allogeneic mixed leukocyte reaction (allo-MLR) against HL60 cells pretreated with SB was enhanced 4-fold compared with allo-MLR obtained with non-treated HL60 cells. These results suggest that the immunotherapeutic use of HDACIs may become a novel tool for treatment of AML.
Antileukemic drugs increase death receptor 5 levels and enhance Apo-2L–induced apoptosis of human acute leukemia cells
Authors: Wen Jinghai , Ramadevi Nimmanapalli , Nguyen Diep , Perkins Charles , Worthington Elizabeth , Bhalla Kapil
Published: October 2019

In present studies, treatment with tumor necrosis factor (TNF)–related apoptosis inducing ligand (TRAIL, also known as Apo-2 ligand [Apo-2L]) is shown to induce apoptosis of the human acute leukemia HL-60, U937, and Jurkat cells in a dose-dependent manner, with the maximum effect seen following treatment of Jurkat cells with 0.25 μg/mL of Apo-2L (95.0% ± 3.5% of apoptotic cells). Susceptibility of these acute leukemia cell types, which are known to lack p53wt function, did not appear to correlate with the levels of the apoptosis-signaling death receptors (DRs) of Apo-2L, ie, DR4 and DR5; decoy receptors (DcR1 and 2); FLAME-1 (cFLIP); or proteins in the inhibitors of apoptosis proteins (IAP) family. Apo-2L–induced apoptosis was associated with the processing of caspase-8, Bid, and the cytosolic accumulation of cytochrome c as well as the processing of caspase-9 and caspase-3. Apo-2L–induced apoptosis was significantly inhibited in HL-60 cells that overexpressed Bcl-2 or Bcl-xL. Cotreatment with either a caspase-8 or a caspase-9 inhibitor suppressed Apo-2L–induced apoptosis. Treatment of human leukemic cells with etoposide, Ara-C, or doxorubicin increased DR5 but not DR4, Fas, DcR1, DcR2, Fas ligand, or Apo-2L levels. Importantly, sequential treatment of HL-60 cells with etoposide, Ara-C, or doxorubicin followed by Apo-2L induced significantly more apoptosis than treatment with Apo-2L, etoposide, doxorubicin, or Ara-C alone, or cotreatment with Apo-2L and the antileukemic drugs, or treatment with the reverse sequence of Apo-2L followed by one of the antileukemic drugs. These findings indicate that treatment with etoposide, Ara-C, or doxorubicin up-regulates DR5 levels in a p53-independent manner and sensitizes human acute leukemia cells to Apo-2L–induced apoptosis.
Flt3 mutations from patients with acute myeloid leukemia induce transformation of 32D cells mediated by the Ras and STAT5 pathways
Authors: Mizuki Masao , Fenski Regina , Halfter Hartmut , Matsumura Itaru , Schmidt Rainer , Müller Carsten , Grüning Wolfram , Kratz-Albers Karsten , Serve Susanne , Steur Claudia , Büchner Thomas , Kienast Joachim , Kanakura Yuzuru , Berdel Wolfgang E. , Serve Hubert
Published: October 2019

Somatic mutations of the receptor tyrosine kinase Flt3 consisting of internal tandem duplications (ITD) occur in 20% of patients with acute myeloid leukemia. They are associated with a poor prognosis of the disease. In this study, we characterized the oncogenic potential and signaling properties of Flt3 mutations. We constructed chimeric molecules that consisted of the murine Flt3 backbone and a 510-base pair human Flt3 fragment, which contained either 4 different ITD mutants or the wild-type coding sequence. Flt3 isoforms containing ITD mutations (Flt3-ITD) induced factor-independent growth and resistance to radiation-induced apoptosis in 32D cells. Cells containing Flt3-ITD, but not those containing wild-type Flt3 (Flt3-WT), formed colonies in methylcellulose. Injection of 32D/Flt3-ITD induced rapid development of a leukemia-type disease in syngeneic mice. Flt3-ITD mutations exhibited constitutive autophosphorylation of the immature form of the Flt3 receptor. Analysis of the involved signal transduction pathways revealed that Flt3-ITD only slightly activated the MAP kinases Erk1 and 2 and the protein kinase B (Akt) in the absence of ligand and retained ligand-induced activation of these enzymes. However, Flt3-ITD led to strong factor-independent activation of STAT5. The relative importance of the STAT5 and Ras pathways for ITD-induced colony formation was assessed by transfection of dominant negative (dn) forms of these proteins: transfection of dnSTAT5 inhibited colony formation by 50%. Despite its weak constitutive activation by Flt3-ITD, dnRas also strongly inhibited Flt3-ITD–mediated colony formation. Taken together, Flt3-ITD mutations induce factor-independent growth and leukemogenesis of 32D cells that are mediated by the Ras and STAT5 pathways.
Level of minimal residual disease after consolidation therapy predicts outcome in acute myeloid leukemia
Authors: Venditti Adriano , Buccisano Francesco , Del Poeta Giovanni , Maurillo Luca , Tamburini Anna , Cox Christina , Battaglia Alessandra , Catalano Gianfranco , Del Moro Beatrice , Cudillo Laura , Postorino Massimiliano , Masi Mario , Amadori Sergio
Published: October 2019

We used flow cytometry to quantify minimal residual disease (MRD) in 56 patients with acute myeloid leukemia (AML) expressing a leukemia-associated phenotype. Thirty-four patients aged 18 to 60 years were entered into the AML-10 protocol (induction, consolidation, and autologous stem-cell transplantation [ASCT]), whereas 22 patients older than 60 years received the AML-13 protocol (induction, consolidation, and consolidation II). After induction, the level of MRD that was best associated with treatment outcome was 4.5 × 10−4 residual leukemic cells. However, the outcome in patients with at least 4.5 × 10−4 cells (n = 26) was not significantly different from that in patients with fewer leukemic cells (n = 30); there were 15 (58%) relapses in the first group and 12 (40%) relapses in the second. After consolidation, the most predictive MRD cutoff value was 3.5 × 10−4cells: 22 patients had an MRD level of 3.5 × 10−4 cells or higher and 17 (77%) of these patients had relapse, compared with 5 of 29 patients (17%) with lower MRD levels (P < .001). An MRD level of 3.5 × 10−4 cells or higher after consolidation was significantly correlated with poor or intermediate-risk cytogenetic findings, a multidrug resistance 1 (MDR1) phenotype, short duration of overall survival, and short duration of relapse-free survival (P = .014, .031, .00022, and .00014, respectively). In multivariate analysis, this MRD status was significantly associated with a high frequency of relapse (P < .001) and a short duration of overall (P = .025) and relapse-free survival (P = .007). ASCT did not alter the prognostic effect of high MRD levels after consolidation: the relapse rate after transplantation was 70%. Thus, we found that an MRD level of 3.5 × 10−4 cells or higher at the end of consolidation strongly predicts relapse and is significantly associated with an MDR1 phenotype and intermediate or unfavorable cytogenetic findings.
MHC class II and c-kit expression allows rapid enrichment of T-cell progenitors from total bone marrow cells
Authors: Ody Christiane , Corbel Catherine , Dunon Dominique , Vainio Olli , Imhof Beat A.
Published: October 2019

T-cell progenitors in the embryonic bone marrow express the tyrosine kinase receptor c-kit. RR5, an anti-MHC class II β chain monoclonal antibody, subdivides this c-kit positive population. Intrathymic transfer experiments showed that most of the T-cell progenitors belong to the MHC class II+/c-kit+ bone marrow population in the embryo and young adult. On transplantation, these bone marrow progenitors lose this expression and differentiate into CD4 CD8 T lymphocytes. In contrast, erythroid progenitors are restricted to the MHC class II−/c-kit+ population. The MHC class II+/c-kit+ pro-T cells are metabolically active, because they stain brightly with rhodamin 123. Their cyclin A and B expression level suggests that they are in the mitotic phase of the cell cycle. Thus, we define an easy sorting protocol, which allows enrichment of T-cell progenitors from total bone marrow hemopoietic cells.
Iron homeostasis: new tales from the crypt
Authors: Roy Cindy N. , Enns Caroline A.
Published: October 2019

The enterocyte is a highly specialized cell of the duodenal epithelium that coordinates iron uptake and transport into the body. Until recently, the molecular mechanisms underlying iron absorption and iron homeostasis have remained a mystery. This review focuses on the proteins and regulatory mechanisms known to be present in the enterocyte precursor cell and in the mature enterocyte. The recent cloning of a basolateral iron transporter and investigations into its regulation provide new insights into possible mechanisms for iron transport and homeostasis. The roles of proteins such as iron regulatory proteins, the hereditary hemochromatosis protein (HFE)–transferrin receptor complex, and hephaestin in regulating this transporter and in regulating iron transport across the intestinal epithelium are discussed. A speculative, but testable, model for the maintenance of iron homeostasis, which incorporates the changes in the iron-related proteins associated with the life cycle of the enterocyte as it journeys from the crypt to the tip of the villous is proposed.
Characterization of Epstein-Barr virus–infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response
Authors: Yang Jie , Tao Qian , Flinn Ian W. , Murray Paul G. , Post Linda E. , Ma Hong , Piantadosi Steven , Caligiuri Michael A. , Ambinder Richard F.
Published: October 2019

Post-transplantation lymphoproliferative disease (PTLD) is associated with Epstein-Barr virus (EBV). Quantitative and qualitative differences in EBV in peripheral blood mononuclear cells (PBMCs) of PTLD patients and healthy controls were characterized. A quantitative competitive polymerase chain reaction (QC-PCR) technique confirmed previous reports that EBV load in PBMCs is increased in patients with PTLD in comparison with healthy seropositive controls (18 539 vs 335 per 106 PBMCs, P = .0002). The average frequency of EBV-infected cells was also increased (271 vs 9 per 106 PBMCs, P = .008). The distribution in numbers of viral genome copies per cell was assessed by means of QC-PCR at dilutions of PBMCs. There was no difference between PTLD patients and healthy controls. Similarly, no differences in the patterns of viral gene expression were detected between patients and controls. Finally, the impact of therapy on viral load was analyzed. Patients with a past history of PTLD who were disease-free (after chemotherapy or withdrawal of immunosuppression) at the time of testing showed viral loads that overlapped with those of healthy seropositive controls. Patients treated with rituximab showed an almost immediate and dramatic decline in viral loads. This decline occurred even in patients whose PTLD progressed during therapy. These results suggest that the increased EBV load in PBMCs of PTLD patients can be accounted for by an increase in the number of infected B cells in the blood. However, in terms of viral copy number per cell and pattern of viral gene expression, these B cells are similar to those found in healthy controls. Disappearance of viral load with rituximab therapy confirms the localization of viral genomes in PBMCs to B cells. However, the lack of relationship between the change in viral load and clinical response highlights the difference between EBV-infected PBMCs and neoplastic cells in PTLD.
The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells
Authors: Sirven Aude , Pflumio Françoise , Zennou Véronique , Titeux Monique , Vainchenker William , Coulombel Laure , Dubart-Kupperschmitt Anne , Charneau Pierre
Published: October 2019

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34+ hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).
Zebrafish homolog of the leukemia gene CBFB: its expression during embryogenesis and its relationship to scland gata-1 in hematopoiesis
Authors: Blake Trevor , Adya Neeraj , Kim Cheol-Hee , Oates Andrew C. , Zon Leonard , Chitnis Ajay , Weinstein Brant M. , Liu P. Paul
Published: October 2019

Mammalian CBFB encodes a transcription factor (CBFβ) that in combination with CBFα2 binds to specific DNA sequences and regulates expression of a number of hematopoietic genes.CBFB is associated with human leukemias through a chromosome 16 inversion and is essential for definitive hematopoiesis during mouse embryo development. We have isolated a zebrafishcbfb complementary DNA (cDNA) clone from a zebrafish kidney cDNA library. This cbfb is highly homologous to human and mouseCBFB/Cbfb genes at both the DNA and protein level. In biochemical analyses, cbfβ binds to human CBFα2 and enhances its DNA binding. During zebrafish development, cbfb is expressed in the lateral plate mesoderm at tail bud stage and in the intermediate cell mass (ICM, the location of embryonic hematopoiesis) between the 21- to 26-somite stages. The cbfb is also expressed in Rohon-Beard cells, cranial nerve ganglia, hindbrain, retina, branchial arches, jaw, and fin buds. Expression ofcbfb is decreased or absent in the ICM and Rohon-Beard cells in some hematopoietic mutants and is unaffected in others. We have also analyzed the expression of scl andgata-1 in the same hematopoietic mutants to ascertain the relative order of these transcription factors to cbfb in zebrafish hematopoiesis. Our results indicate that cbfb is expressed in early hematopoietic progenitors and that its expression pattern in the hematopoietic mutants is similar to that ofscl.
Plasminogen activator inhibitor-1 deficiency protects against atherosclerosis progression in the mouse carotid artery
Authors: Eitzman Daniel T. , Westrick Randal J. , Xu Zuojun , Tyson Julia , Ginsburg David
Published: October 2019

Dissolution of the fibrin blood clot is regulated in large part by plasminogen activator inhibitor-1 (PAI-1). Elevated levels of plasma PAI-1 may be an important risk factor for atherosclerotic vascular disease and are associated with premature myocardial infarction. The role of the endogenous plasminogen activation system in limiting thrombus formation following atherosclerotic plaque disruption is unknown. This study found that genetic deficiency for PAI-1, the primary physiologic regulator of tissue-type plasminogen activator (tPA), prolonged the time to occlusive thrombosis following photochemical injury to carotid atherosclerotic plaque in apolipoprotein E-deficient (apoE−/−) mice. However, anatomic analysis revealed a striking difference in the extent of atherosclerosis at the carotid artery bifurcation between apoE−/− mice and mice doubly deficient for apoE and PAI-1 (PAI-1−/−/apoE−/−). Consistent with a previous report, PAI-1+/+/apoE−/−and PAI-1−/−/apoE−/− mice developed similar atherosclerosis in the aortic arch. The marked protection from atherosclerosis progression at the carotid bifurcation conferred by PAI-1 deficiency suggests a critical role for PAI-1 in the pathogenesis of atherosclerosis at sites of turbulent flow, potentially through the inhibition of fibrin clearance. Consistent with this hypothesis, intense fibrinogen/fibrin staining was observed in atherosclerotic lesions at the carotid bifurcation compared to the aortic arch. These observations identify significant differences in the pathogenesis of atherosclerosis at varying sites in the vascular tree and suggest a previously unappreciated role for the plasminogen activation system in atherosclerosis progression at sites of turbulent flow.
Spontaneous thrombosis in mice carrying the factor V Leiden mutation
Authors: Cui Jisong , Eitzman Daniel T. , Westrick Randal J. , Christie Patricia D. , Xu Zuojun J. , Yang Angela Y. , Purkayastha Anjali A. , Yang Tony L. , Metz Alan L. , Gallagher Kim P. , Tyson Julia A. , Rosenberg Robert D. , Ginsburg David
Published: October 2019

A polymorphism in coagulation factor V, factor V Leiden (FVL), is the major known genetic risk factor for thrombosis in humans. Approximately 10% of mutation carriers experience clinically significant thrombosis in their lifetime. In a small subset of patients, thrombosis is associated with coinheritance of other prothrombotic gene mutations. However, the potential contribution of additional genetic risk factors in the majority of patients remains unknown. To gain insight into the molecular basis for the variable expressivity of FVL, mice were generated carrying the homologous mutation (R504Q [single-letter amino acid codes]) inserted into the endogenous murine Fv gene. Adult heterozygous (FvQ/+) and homozygous (FvQ/Q) mice are viable and fertile and exhibit normal survival. Compared with wild-type mice, adult FvQ/Q mice demonstrate a marked increase in spontaneous tissue fibrin deposition. No differences in fetal development or survival are observed among FvQ/Q,FvQ/+ or control littermates on the C57BL/6J genetic background. In contrast, on a mixed 129Sv-C57BL/6J genetic background,FvQ/Q mice develop disseminated intravascular thrombosis in the perinatal period, resulting in significant mortality shortly after birth. These results may explain the high degree of conservation of the R504/R506 activated protein C cleavage site within FV among mammalian species and suggest an important contribution of other genetic factors to the thrombosis associated with FVL in humans.