bluuebunny/crossref_metadata_2025 huggingface dataset:
Collagen Induces Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein in Human Platelets
Authors: Oda Atsushi , Ochs Hans D. , Druker Brian J. , Ozaki Katsutoshi , Watanabe Chiaki , Handa Makoto , Miyakawa Yoshitaka , Ikeda Yasuo
Published: October 2019

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by mutations of the WAS protein (WASP) gene. All hematopoietic stem cell-derived lineages, including platelets, express WASP. Platelets from WAS patients are smaller than their normal counterparts and defects in platelet aggregation and actin polymerization have been reported. To determine if WASP is important for normal platelet function, we examined its role in signal transduction. We found that collagen but not thrombopoietin or thrombin induces a rapid and robust increase in tyrosine phosphorylation of platelet-associated WASP. Collagen-induced tyrosine phosphorylation of WASP was inhibited by cytochalasin D and wortmannin, respectively, suggesting that actin polymerization and phosphatidylinositol 3-kinase (PI3-kinase) play a role in the induction of tyrosine phosphorylation of WASP. Binding of glutathion S-transferase (GST)-Grb2 to WASP was seen in the lysate of resting platelets. The binding was reduced when lysates from collagen-stimulated platelets were incubated with GST-Grb2, suggesting that tyrosine phosphorylation of WASP may directly or indirectly modulate the adapter function of WASP. Although thrombin- and thrombopoietin-induced increase in tyrosine phosphorylation of WASP is negligible or marginal, WASP from thrombin-activated platelets became incorporated into the Triton X-100–insoluble 10,000gsedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes during aggregation. Lastly, we found that WASP is cleaved in response to activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that collagen specifically induces an increase in tyrosine phosphorylation of WASP and that WASP is involved in signaling during thrombin-induced aggregation by its redistribution to the cytoskeleton and its cleavage during aggregation.

© 1998 by The American Society of Hematology.
Activation of p38 MAP Kinase and JNK But Not ERK Is Required for Erythropoietin-Induced Erythroid Differentiation
Authors: Nagata Yuka , Takahashi Noriko , Davis Roger J. , Todokoro Kazuo
Published: October 2019

p38 MAP kinase (p38) and JNK have been described as playing a critical role in the response to a variety of environmental stresses and proinflammatory cytokines. It was recently reported that hematopoietic cytokines activate not only classical MAP kinases (ERK), but also p38 and JNK. However, the physiological function of these kinases in hematopoiesis remains obscure. We found that all MAP kinases examined, ERK1, ERK2, p38, JNK1, and JNK2, were rapidly and transiently activated by erythropoietin (Epo) stimulation in SKT6 cells, which can be induced to differentiate into hemoglobinized cells in response to Epo. Furthermore, p38-specific inhibitor SB203580 but not MEK-specific inhibitor PD98059 significantly suppressed Epo-induced differentiation and antisense oligonucleotides of p38, JNK1, and JNK2, but neither ERK1 nor ERK2 clearly inhibited Epo-induced hemoglobinization. However, in Epo-dependent FD-EPO cells, inhibition of either ERKs, p38, or JNKs suppressed cell growth. Furthermore, forced expression of a gain-of-function MKK6 mutant, which specifically activated p38, induced hemoglobinization of SKT6 cells without Epo. These results indicate that activation of p38 and JNKs but not of ERKs is required for Epo-induced erythroid differentiation of SKT6 cells, whereas all of these kinases are involved in Epo-induced mitogenesis of FD-EPO cells.

© 1998 by The American Society of Hematology.
Long-Term Follow-Up of Patients With Hairy Cell Leukemia After Cladribine Treatment
Authors: Saven Alan , Burian Carol , Koziol James A. , Piro Lawrence D.
Published: October 2019

Hairy cell leukemia is a chronic B-cell disorder that follows an indolent, but progressive course. Cladribine (2-chlorodeoxyadenosine) induces complete remissions in the majority of patients after a single course. We report the long-term outcomes, including response rates and their duration; time-to-treatment failure (TTF) rates; retreatment results; toxicities; and survival rates of patients treated at Scripps Clinic (La Jolla, CA). A total of 358 patients with hairy cell leukemia were treated with cladribine at 0.087 or 0.1 mg/kg body weight per day by continuous intravenous infusion for 7 days. The expected number of second neoplasms was based on the National Cancer Institute’s Surveillance Epidemiology and End Results data. Of 349 evaluable patients, 319 (91%) achieved an initial complete response and 22 (7%) a partial response with an overall median duration of response follow-up of 52 months. Ninety patients (26%) had relapsed at a median of 29 months. The TTF rate for all 341 responders was 19% at 48 months, 16% for complete responders, and 54% for partial responders. Of 53 evaluable patients treated with second courses of cladribine at first relapse, 33 (62%) achieved complete responses and 14 (26%) partial responses. Twenty-seven patients (8%) developed second neoplasms (only 1 hematopoietic) with an observed-to-expected ratio of 1.88 (95% confidence interval, 1.24 to 2.74). The overall survival rate was 96% at 48 months. Single courses of cladribine induced long-lasting complete responses in the vast majority of patients. Relapse rates for complete responders were low. Patients who relapse can be successfully retreated with cladribine. Cladribine has high efficacy and a favorable acute and long-term toxicity profile when administered to patients with hairy cell leukemia.

© 1998 by The American Society of Hematology.
The Zinc Finger Transcription Factor Egr-1 Activates Macrophage Differentiation in M1 Myeloblastic Leukemia Cells
Authors: Krishnaraju Kandasamy , Hoffman Barbara , Liebermann Dan A.
Published: October 2019

We previously have shown that the zinc finger transcription factor Egr-1 blocked granulocytic differentiation of HL-60 cells, restricting differentiation along the monocytic lineage. Egr-1 also was observed to block granulocyte colony-stimulating factor (G-CSF)–induced differentiation of interleukin-3 (IL-3)–dependent 32Dcl3 hematopoietic precursor cells, endowing the cells with the ability to be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) for terminal differentiation along the macrophage lineage. To better understand the function of Egr-1 as a positive modulator of monocytic differentiation, in this work we have studied the effect of ectopic expression of Egr-1 on the murine myeloblastic leukemic cell line M1, which is induced for differentiation by the physiological inducer IL-6. It is shown that, unlike in HL-60 and 32Dcl3 cells, ectopic expression of Egr-1 in M1 cells resulted in activation of the macrophage differentiation program in the absence of differentiation inducer. This included the appearance of morphologically differentiated cells, decreased growth rate in mass culture, and cloning efficiency in soft agar, and expression of endogenous c-myb and c-myc mRNAs was markedly downregulated. Untreated M1Egr-1 cells also exhibited cell adherence, expression of Fc and C3 receptors, and upregulation of the myeloid differentiation primary response genes c-Jun, junD, andjunB and the late genetic markers ferritin light-chainand lysozyme. Ectopic expression of Egr-1 in M1 cells also dramatically increased the sensitivity of the cells for IL-6–induced differentiation, allowed a higher proportion of M1 cells to become terminally differentiated under conditions of optimal stimulation for differentiation, and decreased M1 leukemogenicity in vivo. These findings demonstrate that the functions of Egr-1 as a positive modulator of macrophage differentiation vary, depending on the state of lineage commitment for differentiation of the hematopoietic cell type.

© 1998 by The American Society of Hematology.
Stimulation of Mouse and Human Primitive Hematopoiesis by Murine Embryonic Aorta-Gonad-Mesonephros–Derived Stromal Cell Lines
Authors: Xu Ming-jiang , Tsuji Kohichiro , Ueda Takahiro , Mukouyama Yoh-suke , Hara Takahiko , Yang Feng-Chun , Ebihara Yasuhiro , Matsuoka Sahoko , Manabe Atsushi , Kikuchi Akira , Ito Mamoru , Miyajima Atsushi , Nakahata Tatsutoshi
Published: October 2019

We report here on a novel stromal cell line, AGM-S3, derived from the aorta-gonad-mesonephros (AGM) region of a 10.5 days postcoitum (dpc) mouse embryo. The AGM-S3 cells promoted production of hematopoietic progenitors and day-12 spleen colony-forming cells from Lin−c-Kit+Sca-1+ murine primitive hematopoietic cells. They also supported for 6 weeks generation of human multipotential progenitors from cord blood CD34+CD38− primitive hematopoietic cells. Human long-term repopulating hematopoietic stem cells (LTR-HSC) with the potential to reconstitute hematopoiesis in NOD/SCID mice were maintained on AGM-S3 cells for at least 4 weeks. Flow cytometric analysis showed that CD13, vascular cellular adhesion molecule-1, and Sca-1 were expressed on AGM-S3 cells. Because stem cell factor, interleukin-6 (IL-6), and oncostatin M, but not IL-3, IL-11, leukemia- inhibitory factor, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, thrombopoietin, and Flk2 ligand were detected in reverse transcription-polymerase chain reaction analysis of AGM-S3 cells, the cells seem to express species-cross reactive molecule(s) other than the cytokines examined and which act on primitive hematopoietic progenitor/stem cells. This cell line is expected to elucidate molecular mechanisms regulating early hematopoiesis and pave the way for developing strategies for expansion of human transplantable HSC.

© 1998 by The American Society of Hematology.
A Recombinant Soluble Form of the Integrin IIbβ3 (GPIIb-IIIa) Assumes an Active, Ligand-Binding Conformation and Is Recognized by GPIIb-IIIa–Specific Monoclonal, Allo-, Auto-, and Drug-Dependent Platelet Antibodies
Authors: Peterson Julie A. , Visentin Gian P. , Newman Peter J. , Aster Richard H.
Published: October 2019

The IIb-IIIa glycoprotein complex is a favored target for allo-, auto-, and drug-dependent antibodies associated with immune thrombocytopenia. A soluble, recombinant form of the GPIIb-IIIa heterodimer that could be produced in large quantities and maintained in solution without detergent could provide a useful experimental tool for the study of platelet-reactive antibodies, but previous attempts to produce such a construct have yielded only small quantities of the end product. Using a baculovirus expression system and the dual-promoter transfer vector P2Bac, we were able to express soluble GPIIb-IIIa complex (srGPIIb-IIIa) lacking cytoplasmic and transmembrane domains in quantities of about 1,000 μg/L, about 40 times greater than reported previously. The high yield achieved may be related to inclusion of the entire extracellular region of the GPIIb light chain in the construct. srGPIIb-IIIa reacts spontaneously with fibrinogen, and this interaction is totally inhibited by the peptide RGDS. Reactions of 24 GPIIb-IIIa–specific antibodies evaluated (12 monoclonal, 3 allo-specific, 3 auto-specific, and 6 drug-dependent) with srGPIIb-IIIa were indistinguishable from reactions with platelet GPIIb-IIIa. Thus, srGPIIb-IIIa spontaneously assumes an active, ligand-binding conformation and contains epitopes for all monoclonal and human antibodies tested to date. srGPIIb-IIIa can be produced in large quantities, can readily be modified by site-directed mutagenesis, and should facilitate identification of epitopes recognized by GPIIb-IIIa–specific antibodies, study of the mechanism(s) by which certain drugs promote antibody binding to GPIIb-IIIa in drug-induced thrombocytopenia and structure-function relationships of GPIIb-IIIa.

© 1998 by The American Society of Hematology.
Lysis of Plasma Clots by Urokinase-Soluble Urokinase Receptor Complexes
Authors: Higazi Abd Al-Roof , Bdeir Khalil , Hiss Edna , Arad Shira , Kuo Alice , Barghouti Iyad , Cines Douglas B.
Published: October 2019

Single-chain urokinase plasminogen activator (scuPA), the unique form secreted by cells, expresses little intrinsic plasminogen activator activity. scuPA can be activated by proteolytic cleavage to form a two-chain enzyme (tcuPA), which is susceptible to inhibition by plasminogen activator inhibitor type I (PAI-1). scuPA is also activated when it binds to its cellular receptor (uPAR), in which case the protein remains as a single chain molecule with less susceptibility to PAIs. Fibrin clots are invested with PAI-1 derived from plasma and from activated platelets. Therefore, we compared the fibrinolytic activity of complexes between scuPA and recombinant soluble uPAR (suPAR) to that of scuPA, tcuPA, and tcuPA/suPAR complexes. scuPA/suPAR complexes mediated the lysis of plasma-derived fibrin clots 14-fold more extensively than did equimolar concentrations of scuPA and threefold more extensively than did tcuPA or tcuPA/suPAR, respectively. The enhanced catalytic activity of scuPA/suPAR required that all three domains of the receptor be present, correlated with its PAI-1 resistance, was not dependent on fibrin alone, and required a plasma cofactor that was identified as IgG. Human IgG bound specifically to suPAR and scuPA/suPAR as determined by using affinity chromatography and immunoprecipitation. Plasma depleted of IgG lost most of its capacity to promote the fibrinolytic activity of scuPA/suPAR, and the activity of the complex was restored by adding plasma concentrations of purified IgG. These studies indicate that scuPA/suPAR can function as a plasminogen activator in a physiological milieu.

© 1998 by The American Society of Hematology.
Arg89Cys Substitution Results in Very Low Membrane Expression of the Duffy Antigen/Receptor for Chemokines in Fyx Individuals
Authors: Tournamille Christophe , Le Van Kim Caroline , Gane Pierre , Le Pennec Pierre Yves , Roubinet Francis , Babinet Jérôme , Cartron Jean Pierre , Colin Yves
Published: October 2019

The Duffy (FY) blood group antigens are carried by the DARC glycoprotein, a widely expressed chemokine receptor. The molecular basis of the Fya/Fyb and Fy(a-b-) polymorphisms has been clarified, but little is known about the Fyxantigen and the FY*X allele associated with weak expression of Fyb, Fy3, Fy5, and Fy6 antigens. We analyzed here the structure and expression of the FY gene in 4 Fy(a-bweak) individuals. As compared with Fy(a-b+) controls, the Fy(a-bweak) red blood cell membranes contained residual amount of DARC polypeptide and these cells were poorly bound by anti-Fy antibodies and chemokines. The FY gene from Fy(a-b+) and Fy(a-bweak) individuals differed by one substitution, C286T. The resulting Arg89Cys amino acid change reduced the binding of anti-Fy antibodies and chemokines to DARC transfectants. We concluded that the Fybweak donors carried theFY*X allele at the FY locus and that the Fyxantigen corresponds to highly reduced expression of a grossly normal Fyb polypeptide caused by the Arg89Cys substitution. Because FY is a single copy gene, this defect should also affect DARC expression in nonerythroid cells. Because the Fyx phenotype is not associated with apparent clinical consequences, we discussed these findings in the light of the putative roles of DARC in various tissues. Finally, we developed a Fyx DNA typing assay that should be useful for genetic studies and clinical transfusion medicine.

© 1998 by The American Society of Hematology.
The G185R Mutation Disrupts Function of the Iron Transporter Nramp2
Authors: Su Maureen A. , Trenor Cameron C. , Fleming Judith C. , Fleming Mark D. , Andrews Nancy C.
Published: October 2019

Microcytic anemia (mk) mice and Belgrade (b) rats have severe iron deficiency anemia due to defects in intestinal iron transport and erythroid iron utilization. Both animal mutants carry the same missense mutation in Nramp2, the first mammalian iron transporter to be identified. This mutation, in which glycine 185 is changed to arginine (G185R), occurs within predicted transmembrane domain 4 of the protein. We have performed site-directed mutagenesis of murine Nramp2, focusing on amino acids of transmembrane domain 4 that are highly conserved among Nramp-like proteins. We have expressed each mutant form in transfected cells and examined iron transport function, subcellular localization, and protein amounts. All tested forms of Nramp2 localize to the plasma membrane and to transferrin-containing endosomes. Most transmembrane domain 4 mutations affect the amount of protein detected and consequently show diminished iron transport. The G185R mutation, however, causes near total loss of Nramp2 function that cannot be fully explained by a decreased amount of protein, indicating that G185R disrupts iron transport through an alteration in the function of Nramp2, rather than degradation of the protein.

© 1998 by The American Society of Hematology.
Transduction of Murine Bone Marrow Cells With an MDR1 Vector Enables Ex Vivo Stem Cell Expansion, but These Expanded Grafts Cause a Myeloproliferative Syndrome in Transplanted Mice
Authors: Bunting Kevin D. , Galipeau Jacques , Topham David , Benaim Ely , Sorrentino Brian P.
Published: October 2019

Attempts to expand repopulating hematopoietic cells ex vivo have yielded only modest amplification in stem cell numbers. We now report that expression of an exogenous human multi-drug resistance 1 (MDR1) gene enables dramatic ex vivo stem cell expansion in the presence of early acting hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the MDR1 gene or a variant of human dihydrofolate reductase (DHFR), and then expanded for 12 days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor. When these cells were injected into nonirradiated mice, high levels of long-term engraftment were only seen with MDR1-transduced grafts. To verify that expansion of MDR1-transduced repopulating cells had occurred, competitive repopulation assays were performed using MDR1 expanded grafts. These experiments showed progressive expansion of MDR1-transduced repopulating cells over the expansion period, with a 13-fold overall increase in stem cells after 12 days. In all of the experiments, mice transplanted with expanded MDR1-transduced stem cells developed a myeloproliferative disorder characterized by high peripheral white blood cell counts and splenomegaly. These results show that MDR1-transduced stem cells can be expanded in vitro using hematopoietic cytokines without any drug selection, but enforced stem cell self-renewal divisions can have adverse consequences.
An Antibody From a Patient With Ranitidine-Induced Thrombocytopenia Recognizes a Site on Glycoprotein IX That Is a Favored Target for Drug-Induced Antibodies
Authors: Gentilini G. , Curtis B.R. , Aster R.H.
Published: October 2019

Although thrombocytopenia associated with the use of histamine H2 receptor (H2R) antagonists has been described, a drug-dependent, platelet-reactive antibody has not previously been identified in such cases. We studied serum from a patient who developed acute, severe thrombocytopenia after exposure to the H2 receptor antagonist, ranitidine, and identified an antibody that reacted with normal platelets in the presence of this drug at pharmacologic concentrations. In flow cytometric and immunoprecipitation studies, the antibody was shown to be specific for the glycoprotein Ib/IX complex (GPIb/IX). From the pattern of monoclonal antibody (MoAb) inhibition and the reactions of antibody with Chinese hamster ovary (CHO) cells transfected with GPIX and GPIbβ, we found that the patient's antibody is specific for an epitope on GPIX close to, or identical with a site recognized by the MoAb SZ1 that is a common target for antibodies induced by quinine and quinidine, drugs structurally unrelated to ranitidine. These findings provide evidence that immune thrombocytopenia can be caused by sensitivity to an H2 R antagonist and suggest that the SZ1 binding site on GPIX may be a common target for drug-induced antibodies. Further studies of the epitope for which SZ1 is specific may provide clues to the mechanism(s) by which drugs promote tight binding of antibody to a membrane glycoprotein and cause platelet destruction in patients with drug sensitivity.
The Contribution of the Three Hypothesized Integrin-Binding Sites in Fibrinogen to Platelet-Mediated Clot Retraction
Authors: Rooney Michael M. , Farrell David H. , van Hemel Bettien M. , de Groot Philip G. , Lord Susan T.
Published: October 2019

Fibrinogen is a plasma protein that interacts with integrin αIIbβ3 to mediate a variety of platelet responses including adhesion, aggregation, and clot retraction. Three sites on fibrinogen have been hypothesized to be critical for these interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the C-terminus of the γ chain and two Arg-Gly-Asp (RGD) sequences in the Aα chain. Recent data showed that AGDV is critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide evidence, using engineered recombinant fibrinogen, that no one of these sites is critical for clot retraction; fibrinogen lacking all three sites still sustains a relatively normal, albeit delayed, retraction response. Three fibrinogen variants with the following mutations were examined: a substitution of RGE for RGD at position Aα 95-97, a substitution of RGE for RGD at position Aα 572-574, and a triple substitution of RGE for RGD at both Aα positions and deletion of AGDV from the γ chain. Retraction rates and final clot sizes after a 20-minute incubation were indistinguishable when comparing the Aα D97E fibrinogen or Aα D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recombinant fibrinogen. Similar results were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from platelet α-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites are important for an initial step in clot retraction, but not for a subsequent step. With the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second step of clot retraction.
Role of JAK3 in CD40-Mediated Signaling
Authors: Jabara Haifa H. , Buckley Rebecca H. , Roberts Joseph L. , Lefranc Gerard , Loiselet Jacques , Khalil Georges , Geha Raif S.
Published: October 2019

CD40 is a member of the tumor necrosis factor receptor family and plays an important role in B-cell survival, growth, differentiation, and isotype switching. Recently, CD40 has been shown to associate with JAK3, a member of the family of Janus Kinases, which are nonreceptor protein kinases involved in intracellular signaling mediated by cytokines and growth factors. To investigate the role of JAK3 in CD40-mediated signaling, we studied the effect of CD40 stimulation on B-cell proliferation, IgE isotype switching, and upregulation of surface expression of CD23, ICAM-1, CD80, and LT-α in JAK3-deficient patients. Our studies show that stimulation of B cells with monoclonal antibody to CD40 in the presence of interleukin-4 (IL-4) or IL-13 resulted in similar responses in JAK3-deficient patients and normal controls. This suggests that JAK3 is not essential for CD40-mediated B-cell proliferation, isotype switching, and upregulation of CD23, ICAM-1, CD80, and LT-α surface expression.
Analysis of Major Histocompatibility Complex Class I, TAP Expression, and LMP2 Epitope Sequence in Epstein-Barr Virus–Positive Hodgkin's Disease
Authors: Murray Paul G. , Constandinou Christothea M. , Crocker John , Young Lawrence S. , Ambinder Richard F.
Published: October 2019

The Epstein-Barr virus (EBV)-encoded latent membrane proteins, LMP1 and LMP2, are consistently expressed by the malignant Hodgkin/Reed-Sternberg (HRS) cells of EBV-associated Hodgkin's disease (HD). Cytotoxic T lymphocyte (CTL) responses to both of these proteins have been shown in the blood of EBV-seropositive individuals, yet in HD the apparent failure of the CTL response to eliminate HRS cells expressing LMP1 and LMP2 in vivo has given rise to the suggestion that HD may be characterized by the presence of defects in antigen processing/presentation or in CTL function. This study has used immunohistochemistry to show high-level expression of major histocompatibility complex (MHC) class I molecules by the HRS cells of EBV-associated HD and either low level or absence of expression of MHC class I molecules on HRS cells of EBV-negative tumors. In addition, HRS cells expressed high levels of transporter-associated proteins (TAP-1, -2), irrespective of the presence of latent EBV infection. These results suggest that global downregulation of MHC class I molecules does not account for the apparent ability of EBV-infected HRS cells to evade CTL responses, but may be important in the understanding of EBV-negative disease.We have also sequenced an epitope in LMP2A (CLGGLLTMV) that is restricted through HLA A2.1, a relatively common allele in Caucasian populations, and showed that this epitope is wild type in a small group of EBV-associated HLA A2.1-positive HD tumors. This result may be relevant to proposed immunotherapeutic approaches for EBV-positive HD patients that target CTL epitopes.
Interleukin-4 and -13 Induce Upregulation of the Murine Macrophage 12/15-Lipoxygenase Activity: Evidence for the Involvement of Transcription Factor STAT6
Authors: Heydeck Dagmar , Thomas Leo , Schnurr Kerstin , Trebus Frank , Thierfelder William E. , Ihle James N. , Kühn Hartmut
Published: October 2019

When human monocytes or alveolar macrophages are cultured in the presence of interleukin (IL)-4 or IL-13, the expression of the reticulocyte-type 15-lipoxygenase is induced. In mice a 15-lipoxygenase is not expressed, but a leukocyte-type 12-lipoxygenase is present in peritoneal macrophages. To investigate whether both lipoxygenase isoforms exhibit a similar regulatory response toward cytokine stimulation, we studied the regulation of the leukocyte-type 12-lipoxygenase of murine peritoneal macrophages by interleukins and found that the activity of this enzyme is upregulated in a dose-dependent manner when the cells were cultured in the presence of the IL-4 or IL-13 but not by IL-10. When peripheral murine monocytes that do not express the lipoxygenase were treated with IL-4 expression of 12/15-lipoxygenase mRNA was induced, suggesting pretranslational control mechanisms. In contrast, no upregulation of the lipoxygenase activity was observed when the macrophages were prepared from homozygous STAT6-deficient mice. Peritoneal macrophages of transgenic mice that systemically overexpress IL-4 exhibited a threefold to fourfold higher 12-lipoxygenase activity than cells prepared from control animals. A similar upregulation of 12-lipoxygenase activity was detected in heart, spleen, and lung of the transgenic animals. Moreover, a strong induction of the enzyme was observed in red cells during experimental anemia in mice. The data presented here indicate that (1) the 12-lipoxygenase activity of murine macrophages is upregulated in vitro and in vivo by IL-4 and/or IL-13, (2) this upregulation requires expression of the transcription factor STAT6, and (3) the constitutive expression of the enzyme appears to be STAT6 independent. The cytokine-dependent upregulation of the murine macrophage 12-lipoxygenase and its induction during experimental anemia suggests its close relatedness with the human reticulocyte-type 15-lipoxygenase despite their differences in the positional specificity of arachidonic acid oxygenation.
Rh-Deficiency of the Regulator Type Caused by Splicing Mutations in the Human RH50 Gene
Authors: Chérif-Zahar Baya , Matassi Giorgio , Raynal Virginie , Gane Pierre , Delaunay Jean , Arrizabalaga Beatrix , Cartron Jean-Pierre
Published: October 2019

The Rh polypeptides and the glycoproteins Rh50, CD47, LW, and glycophorin B, which interact in the red blood cell membrane to form a multisubunit complex, are lacking or are severely reduced in the Rh-deficiency syndrome. We previously reported that in several Rhnull patients the RH50 gene was altered at the coding sequence level, resulting in either a single amino acid substitution or the synthesis of a truncated polypeptide. In the present report, we have detected two mutations in the intronic region of the RH50 gene that identify a new molecular mechanism involved in Rh-deficiency. The first mutation affected the invariant G residue of the 3′ acceptor splice-site of intron 6, causing the skipping of the downstream exon and the premature termination of translation. The second mutation occurred at the first base of the 5′ donor splice-site of intron 1. Both these mutations were found in homozygote state. RNase protection assays demonstrated that the Rh50 mRNA level was strongly reduced or undetectable in the 3′ and 5′ splice mutants, respectively. The different mutations affecting the RH50 gene are indicative of an heterogeneous mutational pattern, which further supports the hypothesis that the lack of the Rh50 protein may prevent the assembly or transport of the Rh membrane complex to the red blood cell surface.
Host Reactive Donor T Cells Are Associated With Lung Injury After Experimental Allogeneic Bone Marrow Transplantation
Authors: Cooke Kenneth R. , Krenger Werner , Hill Geoff , Martin Thomas R. , Kobzik Lester , Brewer Joanne , Simmons Raymond , Crawford James M. , van den Brink Marcel R.M. , Ferrara James L.M.
Published: October 2019

Noninfectious lung injury is common after allogeneic bone marrow transplantation (BMT), but its association with acute graft-versus-host disease (GVHD) is unclear. Using a murine BMT system where donor and host differ by multiple minor histocompatibility (H) antigens, we investigated the nature of lung injury and its relationship both to systemic GVHD and host-reactive donor T cells. Lethally irradiated CBA hosts received syngeneic BMT or allogeneic (B10.BR) T-cell–depleted (TCD) bone marrow (BM) with and without the addition of T cells. Six weeks after BMT, significant pulmonary histopathology was observed in animals receiving allogeneic BMT compared with syngeneic controls. Lung damage was greater in mice that received allogeneic T cells and developed GVHD, but it was also detectable after TCD BMT when signs of clinical and histologic acute GVHD were absent. In each setting, lung injury was associated with significant alterations in pulmonary function. Mature, donor (Vβ6+and Vβ3+) T cells were significantly increased in the broncho-alveolar lavage (BAL) fluid of all allogeneic BMT recipients compared with syngeneic controls, and these cells proliferated and produced interferon-γ (IFN-γ) to host antigens in vitro. These in vitro responses correlated with increased IFN-γ and tumor necrosis factor-α (TNF-α) in the BAL fluid. We conclude that alloreactive donor lymphocytes are associated with lung injury in this allogeneic BMT model. The expansion of these cells in the BAL fluid and their ability to respond to host antigens even when systemic tolerance has been established (ie, the absence of clinical GVHD) suggest that the lung may serve as a sanctuary site for these host reactive donor T cells. These findings may have important implications with regard to the evaluation and treatment of pulmonary dysfunction after allogeneic BMT even when clinical GVHD is absent.
Divergent Effects of Interleukin-4 and Interferon-γ on Macrophage-Derived Chemokine Production: An Amplification Circuit of Polarized T Helper 2 Responses
Authors: Bonecchi Raffaella , Sozzani Silvano , Stine Johnny T. , Luini Walter , D’Amico Giovanna , Allavena Paola , Chantry David , Mantovani Alberto
Published: October 2019

Macrophage-derived chemokine (MDC) is a CC chemokine that recognizes the CCR4 receptor and is selective for T helper 2 (Th2) versus T helper 1 (Th1) cells. The present study was designed to investigate the effect of the prototypic Th2/Th1 cytokines, interleukin-4 (IL-4) and interferon-γ (IFN-γ), on the production of MDC by human monocytes. IL-4 and IL-13 caused a time-dependent (plateau at 24 hours) and concentration-dependent (EC50 2 and 10 ng/mL, respectively) increase of MDC mRNA levels in monocytes. Increased expression of MDC mRNA was associated with protein release in the supernatant. MDC expression and production induced by IL-4 and IL-13 were inhibited by IFN-γ. IFN-γ also suppressed the constitutive expression of MDC in mature macrophages and dendritic cells. These results delineate an amplification loop of polarized Th2 responses based on differential regulation of MDC production by IL-4 and IL-13 versus IFN-γ and on the selectivity of this chemokine for polarized Th2 cells.

© 1998 by The American Society of Hematology.
Activated Endothelial Cells Induce Apoptosis in Leukemic Cells by Endothelial Interleukin-8
Authors: Terui Yasuhito , Ikeda Masayuki , Tomizuka Hiroshi , Kasahara Tadashi , Ohtsuki Tetsuya , Uwai Masaya , Mori Masaki , Itoh Takehito , Tanaka Miyuki , Yamada Muneo , Shimamura Seiichi , Ishizaka Yukihito , Ikeda Kazuma , Ozawa Keiya , Miura Yasusada , Hatake Kiyohiko
Published: October 2019

Tumor cells are eradicated by several systems, including Fas ligand-Fas and tumor necrosis factor (TNF)-tumor necrosis factor receptor (TNFR). In the previous study, we purified an apoptosis-inducing factor (AIF) to homogeneity from a medium conditioned by PDBu-treated HL-60 cells. N-terminal sequence analysis showed that AIF is identical to endothelial interleukin-8 (IL-8). A novel apoptosis system, in which endothelial cells participate via endothelial IL-8 release, is identified here. Human umbilical vein cells (VE cells) produce and secrete IL-8 by stimulation of IL-1 and TNF-. Endothelial IL-8, which is secreted from VE cells by stimulation of IL-1 and TNF- , induces apoptosis in myelogenous leukemia cell line K562 cells. Monocyte-derived IL-8 could not induce apoptosis in K562 cells. Moreover, interaction between VE cells and K562 cells induces the release of endothelial IL-8 from VE cells, and the attached K562 cells undergo apoptosis. Moreover, interactions between VE cell and other cell lines, such as HL-60, U937, Jurkat, and Daudi, induce the secretion of endothelial IL-8 and the induction of apoptosis in cell lines. Endothelial IL-8 significantly inhibits tumor growth of intraperitoneal and subcutaneous tumor mass of K562 cells and induces apoptosis in their cells in vivo. Endothelial IL-8 plays an important role in apoptosis involving endothelial cells, which may provide us with a new therapy for hematological malignancies.

© 1998 by The American Society of Hematology.
Efficacy and Costs of Granulocyte Colony-Stimulating Factor in Allogeneic T-Cell Depleted Bone Marrow Transplantation
Authors: Lee Stephanie J. , Weller Edie , Alyea Edwin P. , Ritz Jerome , Soiffer Robert J.
Published: October 2019

Hematopoietic growth factors have shown clinical benefits in patients undergoing chemotherapy and stem cell transplantation, but few studies have been performed to assess whether the benefits are worth the costs. We reviewed 196 patients undergoing T-cell depleted related donor bone marrow transplantation (BMT) between 1990 and 1996 to assess the effect of growth factor use on time to engraftment and costs of hospitalization. Beginning in 1994, based on encouraging results in autologous transplantation, patients (n = 81) were treated with granulocyte colony-stimulating factor (G-CSF) starting at day +1 after marrow infusion until engraftment. Between January 1, 1990 and January 1, 1994, patients (n = 115) did not receive growth factor. CD6 depletion of donor marrow was the only form of prophylaxis against graft-versus-host disease (GVHD). Despite receiving a lower stem cell dose (P = .004), the group receiving G-CSF had a decreased time to engraftment (20 days v 12 days, P < .0001) and time from marrow infusion to discharge (23 days v 17 days,P < .0001). In multivariate modeling, the use of G-CSF was the most significant factor predicting time to engraftment and discharge. Incidence of grades II-IV GVHD, early mortality, percentage of patients who engrafted, and relapse rates did not differ between the groups. Inpatient charges during the first 50 days after marrow infusion (including readmissions) were available on 110 patients and were converted to costs using departmental ratios of costs of charges. Median costs were significantly lower in the group receiving G-CSF ($80,600 v $84,000, P = .0373). Thus, use of G-CSF in this setting allows earlier hospital discharge with lower costs.

© 1998 by The American Society of Hematology.
Decreased Rejection and Improved Survival of First and Second Marrow Transplants for Severe Aplastic Anemia (A 26-Year Retrospective Analysis)
Authors: Stucki Anne , Leisenring Wendy , Sandmaier Brenda M. , Sanders Jean , Anasetti Claudio , Storb Rainer
Published: October 2019

Between 1970 and 1996, 333 patients with severe aplastic anemia underwent HLA-matched related marrow transplant after conditioning with cyclophosphamide (CY). Thirty-five percent of patients transplanted between 1970 and 1976 (group 1), 12% of those transplanted between 1977 and 1981 (group 2), and 9% of patients transplanted between 1982 and 1997 (group 3) had graft rejection. Graft rejection occurred later among group 3 patients (median, 180 days) than among those in groups 1 and 2 (medians, 28 and 47 days, respectively; P < .001 group 3 v 2). In group 3, 92% of rejecting patients underwent a second transplant, compared with 78% and 77% in groups 1 and 2, respectively. Group 1 patients received various conditioning regimens before second transplant, whereas most patients of groups 2 and 3 received CY combined with antithymocyte globulin (ATG). Graft-versus-host disease (GVHD) prophylaxis after second transplant consisted of methotrexate (MTX) for all group 1 and 2 patients, whereas group 3 patients received MTX combined with cyclosporine (CSP). Over the three time periods studied, first graft rejection decreased from 35% to 9%, and the proportion of rejecting patients undergoing second transplants increased from 77% to 92%. The 10-year probability of survival after second transplants increased from 5% to 83%. Multivariate analysis showed MTX/CSP GVHD prophylaxis to be a significant factor accounting for the increase in patient survival after second transplant.

© 1998 by The American Society of Hematology.
A Gln747→Pro Substitution in the IIb Subunit Is Responsible for a Moderate IIbβ3Deficiency in Glanzmann Thrombasthenia
Authors: Tadokoro Seiji , Tomiyama Yoshiaki , Honda Shigenori , Arai Morio , Yamamoto Naomasa , Shiraga Masamichi , Kosugi Satoru , Kanakura Yuzuru , Kurata Yoshiyuki , Matsuzawa Yuji
Published: October 2019

To clarify a molecular defect responsible for moderate IIbβ3 deficiency, we examined two unrelated patients, MT and MS, suffering from type II and type I Glanzmann thrombasthenia (GT), respectively. Sequence analysis of polymerase chain reaction (PCR) fragments derived from platelet mRNA showed a single A→C substitution at nucleotide (nt) 2334 leading to a Gln747→ Pro in IIb in both patients. Allele-specific restriction enzyme analysis (ASRA) of genomic DNA demonstrated that patient MT was homozygous for the Gln747→Pro substitution and patient MS was compound heterozygous for this substitution and for an RNA splice mutation at the consensus sequence of the splice acceptor site of exon 18 (AG→AA). Furthermore, ASRA showed that, among 17 unrelated Japanese GT patients, this Gln747→Pro substitution was detected in 4 patients, including MT and MS (homozygous, 2 patients; heterozygous, 2 patients). Cotransfection of Pro747IIb and β3 constructs into 293 cells resulted in moderate reduction in the amount of IIbβ3 within the transfected cells as well as on the cell surface. However, Pro747IIbβ3 bound the ligand mimetic monoclonal antibody (MoAb) PAC-1 after activation of IIbβ3 by the MoAb PT25-2, suggesting that the mutant IIbβ3 possesses the ligand-binding function. The association between the mutant proIIb and β3 was not disturbed. Surface labeling and pulse chase study showed that the Gln747→Pro substitution moderately impaired both intracellular transport of the IIbβ3 heterodimers to the Golgi apparatus and endoproteolytic cleavage of proIIb into heavy and light chains. By contrast, replacement of Gln747 with Ala by mutagenesis did not impair IIbβ3expression on the cell surface. These results suggest that the presence of Pro, rather than the absence of Gln, at amino acid residue 747 on IIb is responsible for moderate IIbβ3 deficiency.

© 1998 by The American Society of Hematology.
Lipopolysaccharide Induces the Antiapoptotic Molecules, A1 and A20, in Microvascular Endothelial Cells
Authors: Hu Xiaolong , Yee Esther , Harlan John M. , Wong Fred , Karsan Aly
Published: October 2019

The effect of lipopolysaccharide (LPS) on endothelial cells is a key component of the inflammatory response seen in Gram-negative sepsis. LPS does not cause death of cultured human endothelial cells. However, when the expression of new proteins is inhibited by cycloheximide, microvascular endothelial cells in culture undergo apoptosis. This finding suggests that LPS induces apoptotic and antiapoptotic pathways, with the antiapoptotic response being dependent on the synthesis of new proteins. Concurrent activation of apoptotic and antiapoptotic pathways has previously been documented for tumor necrosis factor (TNF). In the case of TNF, the antiapoptotic signal has been attributed to at least two cytoprotective proteins: the Bcl-2 homologue, A1, and the zinc-finger protein, A20. In this study, we demonstrate that both these molecules are induced in microvascular endothelial cells by LPS. Enforced overexpression of either A1 or A20 inhibits LPS and cycloheximide-initiated apoptosis. Induction of A1 and A20 does not require synthesis of intermediary proteins, but is dependent on the presence of soluble CD14. In addition, we show that inhibition of signaling by the transcription factor, NF-κB, blocks accumulation of A1 and A20 mRNA. Taken together, our findings suggest that LPS directly induces expression of the cytoprotective proteins, A1 and A20, via a CD14-dependent pathway requiring activation of NF-κB.

© 1998 by The American Society of Hematology.
8Cl-cAMP Cytotoxicity in Both Steroid Sensitive and Insensitive Multiple Myeloma Cell Lines Is Mediated by 8Cl-Adenosine
Authors: Halgren Robert G. , Traynor Ann E. , Pillay Shafali , Zell Joann L. , Heller Kimberly F. , Krett Nancy L. , Rosen Steven T.
Published: October 2019

We have examined the cytotoxic effects of cyclic adenosine-3′,5′-monophosphate (cAMP) derivatives on multiple myeloma cells lines and determined that the 8-Chloro substituted derivative (8Cl-cAMP) is one of the most potent. We report here that 8Cl-cAMP is cytotoxic to both steroid sensitive and insensitive myeloma cells with a half maximal concentration of approximately 3 μmol/L. 8Cl-cAMP toxicity in myeloma cells is dependent on phosphodiesterase activity in the serum of cell culture medium. A metabolite of 8Cl-cAMP, 8-Chloro-adenosine (8Cl-AD), kills myeloma cells as effectively as 8Cl-cAMP. Adenosine deaminase (ADA) converts 8Cl-AD into 8Cl-inosine and abrogates the cytotoxic effects of 8Cl-cAMP, 8Cl-AMP, and 8Cl-AD, as does 5-(p-Nitrobenzyl)-6-Thio-Inosine (NBTI), an inhibitor of nucleoside uptake. These data suggest that 8Cl-cAMP must be converted to 8Cl-AD and that 8Cl-AD is the compound that enters the cell. Contrary to glucocorticoid-mediated cell death in myeloma cells, the pathway of 8Cl-AD–mediated cell death appears to be independent of interleukin-6 (IL-6) actions. Although the exact mode of action for this agent is currently unknown, its ability to kill steroid sensitive and insensitive multiple myeloma cells in an IL-6 independent fashion may offer exciting new therapeutic options.

© 1998 by The American Society of Hematology.
Detection of Clonal Hodgkin and Reed-Sternberg Cells With Identical Somatically Mutated and Rearranged VH Genes in Different Biopsies in Relapsed Hodgkin’s Disease
Authors: Vockerodt Martina , Soares Marta , Kanzler Holger , Küppers Ralf , Kube Dieter , Hansmann Martin-Leo , Diehl Volker , Tesch Hans
Published: October 2019

Hodgkin’s disease (HD) represents a malignant lymphoma in which the putative malignant Hodgkin and Reed-Sternberg (H-RS) cells are rare and surrounded by abundant reactive cells. Single-cell analyses showed that H-RS cells regularly bear clonal Ig gene rearrangements. However, there is little information on the clinical evolution of HD in a given patient. In this study, we used the single-cell polymerase chain reaction (PCR) to identify H-RS cells with clonal Ig gene rearrangements in biopsy specimens of patients with relapsed HD. The obtained clonal variable region heavy-chain (VH) gene rearrangements were used to construct tumor-clone-specific oligonucleotides spanning the complementarity determining region (CDR) III and somatically mutated areas in the rearranged VHgene. A number of biopsies were obtained during a period of 3 years from two HD patients. H-RS cells with identical VHrearrangements were detected in two separate infiltrated lymph nodes from one patient with nodular sclerosis HD. In a second patient with mixed cellularity HD subtype, clonal VH rearrangements with identical sequences were detected in infiltrated spleen and two lymph node biopsies. Despite the high sensitivity of the PCR method used (one clonal cell in 105 mononuclear cells), residual H-RS cells were not found in peripheral blood, leukapheresis material, purified CD34+ stem cells or bone marrow. The results show that different specimens from relapsed patients suffering from classical HD carry the same clonotypic IgH rearrangements with identical somatic mutations, demonstrating the persistence and the dissemination of a clonal tumor cell population. Thus, PCR assays with CDRIII-specific probes derived from clonal H-RS cells are of clinical importance in monitoring the dissemination of HD and tumor progression and could be useful for analysis of minimal residual disease after autologous stem cell transplantation.

© 1998 by The American Society of Hematology.