bluuebunny/crossref_metadata_2025 huggingface dataset:
Constitutive Activation of the JAK2/STAT5 Signal Transduction Pathway Correlates With Growth Factor Independence of Megakaryocytic Leukemic Cell Lines
Authors: Liu Richard Y. , Fan Chun , Garcia Roy , Jove Richard , Zuckerman Kenneth S.
Published: October 2019

The factor-independent Dami/HEL and Meg-01 and factor-dependent Mo7e leukemic cell lines were used as models to investigate JAK/STAT signal transduction pathways in leukemic cell proliferation. Although Dami/HEL and Meg-01 cell proliferation in vitro was independent of and unresponsive to exogenous cytokines including granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, thrombopoietin (TPO), and tumor necrosis factor- (TNF-), the growth of Mo7e cells was dependent on hematopoietic growth factors. When these cell lines were cultured in medium without cytokines, a constitutively activated STAT-like DNA-binding factor was detected in nuclear extracts from both Dami/HEL and Meg-01 cells. However, the STAT-like factor was not detectable in untreated Mo7e cells, but was activated transiently in Mo7e cells in response to cytokine treatments. The constitutively activated and cytokine-induced STAT-like DNA-binding factor in these three cell lines was identified as STAT5 by oligonucleotide competition gel mobility assays and by specific anti-STAT antibody gel supershift assays. Constitutive activation of JAK2 also was detected in the factor-independent cell lines, but not in Mo7e cells without cytokine exposure. Meg-01 cells express a p185 BCR/ABL oncogene, which may be responsible for the constitutive activation of STAT5. Dami/HEL cells do not express the BCR/ABL oncogene, but increased constitutive phosphorylation of Raf-1 oncoprotein was detected. In cytokine bioassays using growth factor-dependent Mo7e and TF-1 cells as targets, conditioned media from Dami/HEL and Meg-01 cells did not show stimulatory effects on cell proliferation. Our results indicate that the constitutive activation of JAK2/STAT5 correlates with the factor-independent growth of Dami/HEL and Meg-01 cells. The constitutive activation of JAK2/STAT5 in Dami/HEL cells is triggered by a mechanism other than autocrine cytokines or the BCR/ABL oncoprotein.
Idiotype Vaccination Using Dendritic Cells After Autologous Peripheral Blood Stem Cell Transplantation for Multiple Myeloma—A Feasibility Study
Authors: Reichardt Volker L. , Okada Craig Y. , Liso Arcangelo , Benike Claudia J. , Stockerl-Goldstein Keith E. , Engleman Edgar G. , Blume Karl G. , Levy Ronald
Published: October 2019

The idiotype (Id) determinant on the multiple myeloma (MM) protein can be regarded as a tumor-specific marker. Immunotherapy directed at the MM Id may stem the progression of this disease. We report here on the first 12 MM patients treated at our institution with high-dose therapy and peripheral blood stem cell transplantation (PBSCT) followed by Id immunizations. MM patients received PBSCT to eradicate the majority of the disease. PBSCT produced a complete response in 2 patients, a partial response in 9 patients and stable disease in 1 patient. Three to 7 months after high-dose therapy, patients received a series of monthly immunizations that consisted of two intravenous infusions of Id-pulsed autologous dendritic cells (DC) followed by five subcutaneous boosts of Id/keyhole limpet hemocyanin (KLH) administered with adjuvant. Between 1 and 11 × 106 DC were obtained by leukapheresis in all patients even after PBSCT. The administration of Id-pulsed DC and Id/KLH vaccines were well tolerated with patients experiencing only minor and transient side effects. Two of 12 patients developed an Id-specific, cellular proliferative immune response and one of three patients studied developed a transient but Id-specific cytotoxic T-cell (CTL) response. Eleven of the 12 patients generated strong KLH-specific cellular proliferative immune responses showing the patients’ immunocompetence at the time of vaccination. The two patients who developed a cellular Id-specific immune response remain in complete remission. Of the 12 treated patients, 9 are currently alive after autologous transplantation with a minimum follow-up of 16 months, 2 patients died because of recurrent MM and 1 patient succumbed to acute leukemia. These studies show that patients make strong anti-KLH responses despite recent high-dose therapy and that DC-based Id vaccination is feasible after PBSCT and can induce Id-specific T-cell responses. Further vaccine development is necessary to increase the proportion of patients that make Id-specific immune responses. The clinical benefits of Id vaccination in MM remain to be determined.
Filgrastim for Cladribine-Induced Neutropenic Fever in Patients With Hairy Cell Leukemia
Authors: Saven Alan , Burian Carol , Adusumalli Jasvant , Koziol James A.
Published: October 2019

Cladribine treatment of hairy cell leukemia (HCL) is complicated by neutropenic fever in 42% of patients despite documented infections being relatively uncommon. We performed a study of priming filgrastim followed by cladribine and then filgrastim again to determine if filgrastim would lead to a reduction of neutropenia and febrile episodes. Thirty-five patients received filgrastim and cladribine and were compared with 105 historic controls treated with cladribine alone. Cladribine was administered at 0.1 mg/kg/d by continuous infusion for 7 days. Filgrastim was administered at 5 μg/kg/d subcutaneously on days −3, −2, and −1 and then again after the completion of cladribine until the absolute neutrophil count (ANC) was ≥2 × 109/L on 2 consecutive days (days +8, +9, etc). After filgrastim priming, the median ANC increased from 0.9 × 109/L to 2.26 × 109/L (2.5-fold increase), and after cladribine, the median nadir ANC in the filgrastim-treated group was 0.53 × 109/L compared with 0.29 × 109/L among historic controls (P = .04). The median number of days to an ANC greater than 1.0 × 109/L was 9 days in the filgrastim-treated group versus 22 days among historic controls (P < 10−5). The percentage of febrile patients, number of febrile days, and frequency of admissions for antibiotics were not statistically different in the two groups. Filgrastim regularly increases the ANC in patients with HCL and shortens the duration of severe neutropenia after cladribine. This phase II study, with comparison to historical controls, failed to detect any clinical advantage from the use of filgrastim and cladribine in the treatment of HCL. Accordingly, the routine adjunctive use of filgrastim with cladribine in the treatment of HCL cannot be recommended.
TT Virus in Bone Marrow Transplant Recipients
Authors: Kanda Yoshinobu , Tanaka Yuji , Kami Masahiro , Saito Toshiki , Asai Takashi , Izutsu Koji , Yuji Koichiro , Ogawa Seishi , Honda Hiroaki , Mitani Kinuko , Chiba Shigeru , Yazaki Yoshio , Hirai Hisamaru
Published: October 2019

TT virus (TTV) is a newly discovered transfusion-transmissible DNA virus, which may cause posttransfusion hepatitis. The virus was detected in 12% of Japanese blood donors. The aim of the study is to investigate the prevalence and clinical influence of TTV in bone marrow transplant (BMT) recipients. Sera from 25 BMT recipients obtained 6 to 12 weeks after the transplant were examined for TTV-DNA by the seminested polymerase chain reaction. Serial samples were additionally analyzed in patients with TTV-DNA. Fifteen of 25 recipients (60%) were positive for TTV-DNA after transplant, whereas it was detected in only two of 20 BMT donors (10%). In patients positive for TTV-DNA before BMT, the amount of TTV-DNA decreased to an undetectable level during the myelosuppressed period after BMT. We also found that there was a novel group of TTV, G3, classified by the nucleotide sequences. The median peak alanine aminotransferase (ALT) levels were 135.0 IU/L and 116.5 IU/L (normal range, 4 to 36 IU/L) in TTV-positive and TTV-negative recipients, respectively. In one of the seven TTV-positive patients who developed hepatic injury (ALT > 150 IU/L), a serial change in the serum TTV titer showed a good correlation with the ALT level. We concluded that (1) the prevalence of TTV is high in BMT recipients, (2) TTV might be replicated mainly in hematopoietic cells, (3) transfusion-transmitted TTV may cause persistent infection, (4) a novel genetic group of TTV, G3, was discovered, and (5) TTV does not seem to frequently cause hepatic injury, although one patient was strongly suggested to have TTV-induced hepatitis.
Characterization of Bone Marrow Laminins and Identification of 5-Containing Laminins as Adhesive Proteins for Multipotent Hematopoietic FDCP-Mix Cells
Authors: Gu Yuchen , Sorokin Lydia , Durbeej Madeleine , Hjalt Tord , Jönsson Jan-Ingvar , Ekblom Marja
Published: October 2019

Laminins are extracellular matrix glycoproteins that influence the phenotype and functions of many types of cells. Laminins are heterotrimers composed of , β, and γ polypeptides. So far five , three β, and two γ polypeptide chains, and 11 variants of laminins have been proposed. Laminins interact in vitro with mature blood cells and malignant hematopoietic cells. Most studies have been performed with laminin-1 (1β1γ1), and its expression in bone marrow is unclear. Employing an antiserum reacting with most laminin isoforms, we found laminins widely expressed in mouse bone marrow. However, no laminin 1 chain but rather laminin 2, 4, and 5 polypeptides were found in bone marrow. Our data suggest presence of laminin-2 (2β1γ1), laminin-8 (4β1γ1), and laminin-10 (5β1γ1) in bone marrow. Northern blot analysis showed expression of laminin 1, 2, 4, and 5 chains in long-term bone marrow cultures, indicating upregulation of laminin 1 chain expression in vitro. Laminins containing 5 chain, in contrast to laminin-1, were strongly adhesive for multipotent hematopoietic FDCP-mix cells. Integrin 6 and β1 chains mediated this adhesion, as shown by antibody perturbation experiments. Our findings indicate that laminins other than laminin-1 are functional in adhesive interactions in bone marrow.
Human Erythropoietin Induces a Pro-Angiogenic Phenotype in Cultured Endothelial Cells and Stimulates Neovascularization In Vivo
Authors: Ribatti Domenico , Presta Marco , Vacca Angelo , Ria Roberto , Giuliani Roberta , Dell’Era Patrizia , Nico Beatrice , Roncali Luisa , Dammacco Franco
Published: October 2019

Hematopoietic and endothelial cell lineages share common progenitors. Accordingly, cytokines formerly thought to be specific for the hematopoietic system have been shown to affect several functions in endothelial cells, including angiogenesis. In this study, we investigated the angiogenic potential of erythropoietin (Epo), the main hormone regulating proliferation, differentiation, and survival of erythroid cells. Epo receptors (EpoRs) have been identified in the human EA.hy926 endothelial cell line by Western blot analysis. Also, recombinant human Epo (rHuEpo) stimulates Janus Kinase-2 (JAK-2) phosphorylation, cell proliferation, and matrix metalloproteinase-2 (MMP-2) production in EA.hy926 cells and significantly enhances their differentiation into vascular structures when seeded on Matrigel. In vivo, rHuEpo induces a potent angiogenic response in the chick embryo chorioallantoic membrane (CAM). Accordingly, endothelial cells of the CAM vasculature express EpoRs, as shown by immunostaining with an anti-EpoR antibody. The angiogenic response of CAM blood vessels to rHuEpo was comparable to that elicited by the prototypic angiogenic cytokine basic fibroblast growth factor (FGF2), it occurred in the absence of a significant mononuclear cell infiltrate, and it was not mimicked by endothelin-1 (ET-1) treatment. Taken together, these data demonstrate the ability of Epo to interact directly with endothelial cells and to elicit an angiogenic response in vitro and in vivo and thus act as a bona fide direct angiogenic factor.
Immunization With Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor as a Vaccine Adjuvant Elicits Both a Cellular and Humoral Response to Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor
Authors: McNeel Douglas G. , Schiffman Kathy , Disis Mary L.
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine for the generation and propagation of antigen-presenting cells and for priming a cellular immune response. We report here that use of recombinant human GM-CSF (rhGM-CSF), administered as an adjuvant in a peptide-based vaccine trial given monthly by intradermal injection, led to the development of a T-cell and antibody response to rhGM-CSF. An antibody response occurred in the majority of patients (72%). This antibody response was not found to be neutralizing. In addition, by 48-hour delayed type hypersensitivity (DTH) skin testing, 17% of patients were shown to have a cellular immune response to the adjuvant rhGM-CSF alone. Thymidine incorporation assays also showed a peripheral blood T-cell response to rhGM-CSF in at least 17% of the patients. The generation of rhGM-CSF–specific T-cell immune responses, elicited in this fashion, is an important observation because rhGM-CSF is being used as a vaccine adjuvant in various vaccine strategies. rhGM-CSF–specific immune responses may be incorrectly interpreted as antigen-specific immunity, particularly when local DTH responses to vaccination are the primary means of immunologic evaluation. We found no evidence of hematologic or infectious complications as a result of the development of rhGM-CSF–specific immune responses.
The Effect of Glucocorticoids on the Expression of L-Selectin on Polymorphonuclear Leukocyte
Authors: Nakagawa Motohito , Bondy Gregory P. , Waisman Dan , Minshall Diane , Hogg James C. , van Eeden Stephan F.
Published: October 2019

When active bone marrow release is induced by inflammatory stimuli, it is associated with an increase in L-selectin expression on circulating polymorphonuclear leukocyte (PMN). This contrasts sharply with glucocorticoid-induced granulocytosis that is associated with decreased L-selectin expression on PMN. The present study was designed to determine if the reduced L-selectin expression observed after glucocorticoid treatment is the result of suppression of L-selectin synthesis in the bone marrow. New Zealand white rabbits treated with dexamethasone (2.0 mg/kg, a single dose intravenously) were shown to have decreased L-selectin expression on circulating PMN 12 to 24 hours after treatment (P < .01) with a return to baseline levels by 48 hours. When dexamethasone was administered 48 hours after the bone marrow PMN were pulse labeled with the thymidine analogue, 5′-bromo-2′-deoxyuridine (BrdU), L-selectin expression on BrdU-labeled PMN released from the bone marrow was decreased (P< .01). Dexamethasone decreased L-selectin expression on segmented PMN in the bone marrow (P < .05) but not on PMN already in the circulation. We conclude that glucocorticoids decrease L-selectin expression on circulating PMN by downregulating L-selectin expression in the maturation pool of bone marrow and speculate that this is an important glucocorticoid effect that influences the recruitment of PMN into inflammatory sites.
BSAP/Pax5A Expression Blocks Survival and Expansion of Early Myeloid Cells Implicating Its Involvement in Maintaining Commitment to the B-Lymphocyte Lineage
Authors: Chiang Mark Y. , Monroe John G.
Published: October 2019

Early B lymphopoiesis is marked by plasticity between the myeloid and B lineages. An attractive model for B-lineage development is that commitment to this lineage is partly determined by the ordered expression of genes that prohibit switching to the myeloid lineage. In this regard, whereas the role of the B-cell–specific transcription factor BSAP/Pax5A in regulating B-lymphoid–restricted gene expression has been well-established, its role in maintaining B-lineage commitment is unclear. Thus, BSAP/Pax5A was constitutively expressed in the multipotent EML cell line, which can be directed toward the myeloid lineage by culture with interleukin-3 (IL-3) and retinoic acid. EML cells expressing BSAP/Pax5A successfully acquired the myeloid lineage markers CD11b and F4/80 in response to IL-3 and retinoic acid, indicating differentiation to the myeloid lineage. However, these early myeloid cells failed to expand in culture with granulocyte-macrophage colony-stimulating factor and were directed instead toward an apoptotic pathway. In parallel, primary bone marrow stem cells transduced with retrovirus constitutively expressing BSAP/Pax5A began myeloid cell differentiation, but like the transformed EML model failed to expand in response to myeloid growth factors. These studies identify a role for BSAP/Pax5A in suppressing the response to myeloid growth factors, which may be a component of the regulatory processes that limit plasticity of early B-lymphoid progenitors.
A Randomized, Double-Blind, Placebo-Controlled Study With Pegylated Recombinant Human Megakaryocyte Growth and Development Factor (PEG-rHuMGDF) as an Adjunct to Chemotherapy for Adults With De Novo Acute Myeloid Leukemia
Authors: Archimbaud Eric , Ottmann Oliver G. , Yin John A. Liu , Lechner Klaus , Dombret Herve , Sanz Miguel A. , Heil Gerhard , Fenaux Pierre , Brugger Wolfram , Barge Alan , O’Brien-Ewen Caroline , Matcham James , Hoelzer Dieter
Published: October 2019

To determine the safety, biologic, and clinical benefits of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF; Amgen, Thousand Oaks, CA) after myelosuppressive chemotherapy in acute myeloid leukemia (AML), 108 adult patients with de novo AML were randomized to receive either PEG-rHuMGDF (2.5 μg/kg/d or 5 μg/kg/d) for up to 21 doses (group A), a single dose of 2.5 μg/kg PEG-rHuMGDF, 7 daily doses of 2.5 μg/kg PEG-rHuMGDF (group B), or placebo. The greatest biologic activity was seen in group A with a median peak platelet count of 1,084 × 109/L, occurring at a median 9 days after the last dose of study drug, compared with 517 × 109/L and 390 × 109/L in group B and placebo group, respectively. Thrombocytosis (platelets >1,000 × 109/L) was seen at rates of 52%, 8%, and 9% in groups A, B, and placebo, respectively, but were not associated with any adverse event. There was no effect on median time to transfusion independent platelet recovery (≥20 × 109/L). The median time to neutrophil recovery (≥500/μL) and red blood cell transfusion requirements were similar in all groups, and there was no apparent stimulation of leukemia. PEG-rHuMGDF was biologically active and well tolerated. Further investigation of dose and scheduling is required, specifically earlier dosing before and during chemotherapy.
A Novel BTB/POZ Transcriptional Repressor Protein Interacts With the Fanconi Anemia Group C Protein and PLZF
Authors: Hoatlin Maureen E. , Zhi Yu , Ball Helen , Silvey Kirsten , Melnick Ari , Stone Stacie , Arai Sally , Hawe Nicola , Owen Gareth , Zelent Arthur , Licht Jonathan D.
Published: October 2019

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome. The phenotype includes developmental defects, bone marrow failure, and cell cycle abnormalities. At least eight complementation groups (A-H) exist, and although three of the corresponding complementation group genes have been cloned, they lack recognizable motifs, and their functions are unknown. We have isolated a binding partner for the Fanconi anemia group C protein (FANCC) by yeast two-hybrid screening. We show that the novel gene, FAZF, encodes a 486 amino acid protein containing a conserved amino terminal BTB/POZ protein interaction domain and three C-terminal Krüppel-like zinc fingers. FAZF is homologous to the promyelocytic leukemia zinc finger (PLZF) protein, which has been shown to act as a transcriptional repressor by recruitment of nuclear corepressors (N-CoR, Sin3, and HDAC1 complex). Consistent with a role in FA, BTB/POZ-containing proteins have been implicated in oncogenesis, limb morphogenesis, hematopoiesis, and proliferation. We show that FAZF is a transcriptional repressor that is able to bind to the same DNA target sequences as PLZF. Our data suggest that the FAZF/FANCC interaction maps to a region of FANCC deleted in FA patients with a severe disease phenotype. We also show that FAZF and wild-type FANCC can colocalize in nuclear foci, whereas a patient-derived mutant FANCC that is compromised for nuclear localization cannot. These results suggest that the function of FANCC may be linked to a transcriptional repression pathway involved in chromatin remodeling.
Altered Development and Cytokine Responses of Myeloid Progenitors in the Absence of Transcription Factor, Interferon Consensus Sequence Binding Protein
Authors: Scheller Marina , Foerster John , Heyworth Clare M. , Waring Jeffrey F. , Löhler Jürgen , Gilmore Gary L. , Shadduck Richard K. , Dexter T. Mike , Horak Ivan
Published: October 2019

Mice deficient for the transcription factor, interferon consensus sequence binding protein (ICSBP), are immunodeficient and develop disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of ICSBP−/− mice, we investigated the growth, differentiation, and leukemogenic potential of ICSBP−/−myeloid progenitor cells in vitro, as well as by cell-transfers in vivo. We report that adult bone marrow, as well as fetal liver of ICSBP-deficient mice harbor increased numbers of progenitor cells, which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP−/− colonies formed in the presence of M-CSF are mostly of granulocytic morphology. This disproportional differentiation toward cells of the granulocytic lineage in vitro parallels the expansion of granulocytes in ICSBP−/− mice and correlates with a 4-fold reduction of M-CSF receptor expressing cells in bone marrow. Cell transfer studies showed an intrinsic leukemogenic potential and long-term reconstitution capability of ICSBP−/− progenitors. Further experiments demonstrated strongly reduced adhesion of colony-forming cells from ICSBP−/− bone marrow to fibronectin. In summary, ICSBP−/− myeloid progenitor cells share several abnormal features with CML progenitors, suggesting that the distal parts of signaling pathways of these two disorders are overlapping.
Regulation of c-Jun-NH2 Terminal Kinase and Extracellular-Signal Regulated Kinase in Human Platelets
Authors: Bugaud Franck , Nadal-Wollbold Florence , Lévy-Toledano Sylviane , Rosa Jean-Philippe , Bryckaert Marijke
Published: October 2019

Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by IIbβ3integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0.02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to IIbβ3 integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab′)2 fragments of a monoclonal antibody specific for IIbβ3, demonstrating that, like ERK2, IIbβ3 integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of IIbβ3 integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet IIbβ3. The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by IIbβ3 engagement and positively by mechanical forces in platelets.
Fibrinogen Niigata With Impaired Fibrin Assembly: An Inherited Dysfibrinogen With a Bβ Asn-160 to Ser Substitution Associated With Extra Glycosylation at Bβ Asn-158
Authors: Sugo Teruko , Nakamikawa Chizuko , Takano Hiroshi , Mimuro Jun , Yamaguchi Shu-ichi , Mosesson Michael W. , Meh David A. , DiOrio James P. , Takahashi Noriko , Takahashi Hoyu , Nagai Koichi , Matsuda Michio
Published: October 2019

A novel BβAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser–type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BβAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin γ-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bβ-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.
CD38 Triggers Cytotoxic Responses in Activated Human Natural Killer Cells
Authors: Sconocchia Giuseppe , Titus Julie A. , Mazzoni Alessandra , Visintin Alberto , Pericle Federica , Hicks Stuart W. , Malavasi Fabio , Segal David M.
Published: October 2019

Receptors used by natural killer (NK) cells to mediate natural cytotoxicity are poorly defined, although it is now clear that a number of adhesion molecules can serve this function. CD38 transduces signals on T- and B-cell lines, and we asked whether it could trigger lytic and secretory responses in human NK cells. By using an anti-CD38 monoclonal antibody in reverse antibody-dependent cellular cytotoxicity experiments, it is shown that CD38 engagement triggers cytotoxic responses by activated NK cells, but not by cytotoxic T lymphocytes or fresh NK cells. Cross-linking with anti-CD38 F(ab′)2 caused activated NK cells to release granzymes and cytokines, but did not trigger an increase in intracellular Ca2+. Fresh NK cells acquired CD38-dependent lytic function during activation with interleukin-2 (IL-2), and inhibitor studies suggested that IL-2 stimulated the de novo expression of proteins that act between CD38 and the lytic machinery in NK cells. The induction of proteins that link commonly expressed adhesion molecules to effector mechanisms could provide a paradigm for pathogen recognition by the innate immune system.
Hepatocyte Growth Factor (HGF) Induces Interleukin-11 Secretion From Osteoblasts: A Possible Role for HGF in Myeloma-Associated Osteolytic Bone Disease
Authors: Hjertner Øyvind , Torgersen Maria Lyngaas , Seidel Carina , Hjorth-Hansen Henrik , Waage Anders , Børset Magne , Sundan Anders
Published: October 2019

Multiple myeloma is associated with unbalanced bone remodeling causing lytic bone lesions. Interleukin-11 (IL-11) promotes osteoclast formation and inhibits osteoblast activity and may, thus, be one factor involved in cancer-induced bone destruction. We have previously shown that myeloma cells produce hepatocyte growth factor (HGF). We now report that HGF induces IL-11 secretion from human osteoblast-like cells and from the osteosarcoma cell lines Saos-2 and HOS. In coculture experiments, both the myeloma cell line JJN-3 and primary myeloma cells from 3 patients induced IL-11 secretion from osteoblasts, whereas no induction was observed with the non-HGF producing myeloma cell line OH-2. Enhanced IL-11 induction was observed with physical contact between osteoblasts and myeloma cells as compared with experiments in which contact was prohibited by tissue inserts. Anti-HGF serum strongly reduced the myeloma cell-induced IL-11 secretion. Furthermore, we show that JJN-3 cells express HGF on the cell-surface. Removal of surface-bound HGF on JJN-3 cells reduced IL-11 production induced in cocultures. Transforming growth factor β1 and IL-1 potentiated the effect of HGF on IL-11 secretion, whereas an additive effect was observed with tumor necrosis factor. Thus, myeloma-derived HGF can influence the bone marrow environment both as a soluble and a surface-bound factor. Furthermore, HGF emerges as a possible factor involved in myeloma bone disease by its ability to induce IL-11.
Defective Recovery and Severe Renal Damage After Acute Hemolysis in Hemopexin-Deficient Mice
Authors: Tolosano Emanuela , Hirsch Emilio , Patrucco Enrico , Camaschella Clara , Navone Roberto , Silengo Lorenzo , Altruda Fiorella
Published: October 2019

Hemopexin (Hx) is a plasma glycoprotein mainly expressed in liver and, less abundantly, in the central and peripheral nervous systems. Hx has a high binding affinity with heme and is considered to be a major transport vehicle of heme into the liver, thus preventing both heme-catalyzed oxidative damage and heme-bound iron loss. To determine the physiologic relevance of heme-Hx complex formation, Hx-deficient mice were generated by homologous recombination in embryonic stem (ES) cells. The Hx-deficient mice were viable and fertile. Their plasma iron level and blood parameters were comparable to those of control mice and they showed no evidence of tissue lesions caused by oxidative damage or abnormal iron deposits. Moreover, they were sensitive to acute hemolysis, as are wild-type mice. Nevertheless, Hx-null mice recovered more slowly after hemolysis and were seen to have more severe renal damage than controls. After hemolytic stimulus, Hx-deficient mice presented prolonged hemoglobinuria with a higher kidney iron load and higher lipid peroxidation than control mice. Moreover, Hx-null mice showed altered posthemolysis haptoglobin (Hp) turnover in as much as Hp persisted in the circulation after hemolytic stimulus. These data indicate that, although Hx is not crucial either for iron metabolism or as a protection against oxidative stress under physiologic conditions, it does play an important protective role after hemolytic processes.
Human Cord Blood Progenitors Sustain Thymic T-Cell Development and a Novel Form of Angiogenesis
Authors: Crisa Laura , Cirulli Vincenzo , Smith Kent A. , Ellisman Mark H. , Torbett Bruce E. , Salomon Daniel R.
Published: October 2019

There is growing interest in using human umbilical cord blood (CB) for allogeneic bone marrow transplantation (BMT), particularly in children. Thus, CB has been identified as a rich source of hematopoietic progenitors of the erythroid, myeloid, and B-cell lineages. Whether CB blood cells engrafting in the BM space also comprise T-cell progenitors capable of trafficking to the thymus and reconstituting a functional thymopoiesis in young recipients is presently unknown. Here, we show that CB progenitors, engrafted in the BM of immunodeficient mice, sustain human thymopoiesis by generating circulating T-cell progenitors capable of homing to and developing within a human thymic graft. Surprisingly, development of CB stem cells in this in vivo model extended to elements of the endothelial cell lineage, which contributed to the revascularization of transplants and wound healing. These results demonstrate that human CB stem cell transplantation can reconstitute thymic-dependent T-cell lymphopoiesis and show a novel role of CB-derived hematopoietic stem cells in angiogenesis.
Recipient Tumor Necrosis Factor- and Interleukin-10 Gene Polymorphisms Associate With Early Mortality and Acute Graft-Versus-Host Disease Severity in HLA-Matched Sibling Bone Marrow Transplants
Authors: Cavet James , Middleton Peter G. , Segall Miriam , Noreen Harriet , Davies Stella M. , Dickinson Anne M.
Published: October 2019

The proinflammatory cytokine tumor necrosis factor- (TNF-) is strongly implicated in graft-versus-host disease (GVHD) and other acute bone marrow transplant (BMT) complications. The antiinflammatory interleukin-10 (IL-10) antagonizes TNF- and reduces GVHD. We previously showed association of recipient TNF (TNFd) and IL-10 (IL-10−1064) gene polymorphisms with acute GVHD severity in matched sibling BMT using only cyclosporin A monotherapy. The current study tested association of GVHD with TNFd and IL-10−1064/-1082 polymorphisms in a large cohort (144 matched sibling donor/recipient pairs) given both cyclosporine A (CyA) and methotrexate (MTX) prophylaxis. Genotype results were correlated with acute and chronic GVHD and mortality. Patients homozygous for the TNFd microsatellite allele 3 had higher early mortality: 23.7% of TNFd3/d3 homozygotes died before day 30, compared with 6.80% of non-d3/d3 recipients (P = .013). Recipients possessing longer IL-10−1064 microsatellite alleles developed more severe acute GVHD: 22.3% of recipients possessing alleles 12 to 15 developed grade III to IV GVHD, versus 3.92% of those with smaller alleles (P < .01). Other recipient or donor genotypes tested did not significantly affect GVHD or mortality. We conclude that recipient TNFd and IL-10−1064 polymorphisms associate with early mortality and severe acute GVHD in matched sibling BMT with dual prophyaxis. This supports the hypothesis of genetic predisposition towards GVHD and other BMT complications other than histocompatibility antigen disparity.
Characterization of the Murine Platelet IIb Gene and Encoded cDNA
Authors: Thornton Michael A. , Poncz Mortimer
Published: October 2019

The IIb/β3 receptor is central to platelet aggregation. Biological studies of this receptor have been limited by the inability to reproduce IIb/β3 function in a cell system. Increasingly, efforts are being directed at studies of this receptor in mice models. The structure of murine (m) β3 has been reported. We now have sequenced the mIIb gene and found that it has the same size and organization as the human gene. The exon/intron borders are reported here, as are the distances between exons. mIIb protein is 1,033 amino acids (aa), 7 and 5 aa shorter than human (h) and rodent (r) IIb, respectively, with 79% and 90% homology, respectively. As part of the comparative analysis of the 3 known IIb chains included in this report, we found that a particular region of the IIb N-terminal β-propeller is highly conserved and speculate that it directly participates in ligand binding.
Correction of Leukocyte Adhesion Deficiency Type II With Oral Fucose
Authors: Marquardt Thorsten , Lühn Kerstin , Srikrishna Geetha , Freeze Hudson H. , Harms Erik , Vestweber Dietmar
Published: October 2019

We describe a simple, noninvasive, and effective therapy for leukocyte adhesion deficiency type II (LAD II), a rare inherited disorder of fucose metabolism. This disorder leads to an immunodeficiency caused by the absence of carbohydrate-based selectin ligands on the surface of neutrophils as well as to severe psychomotor and mental retardation. The fucosylation defect in LAD II fibroblasts can be corrected by addition of L-fucose to the culture medium. This prompted us to initiate dietary fucose therapy on a patient with LAD II. Oral supplementation of fucose in this patient induced the expression of fucosylated selectin ligands on neutrophils and core fucosylation of serum glycoproteins. During 9 months of treatment, infections and fever disappeared, elevated neutrophil counts returned to normal, and psychomotor capabilities improved.
Molecular Configuration of Rh D Epitopes as Defined by Site-Directed Mutagenesis and Expression of Mutant Rh Constructs in K562 Erythroleukemia Cells
Authors: Liu Wendy , Avent Neil D. , Jones Jeffrey W. , Scott Marion L. , Voak Douglas
Published: October 2019

The Rh D antigen is the most clinically important protein blood group antigen of the erythrocyte. It is expressed as a collection of at least 37 different epitopes. The external domains of the Rh D protein involved in epitope presentation have been predicted based on the analysis of variant Rh D protein structures inferred from their cDNA sequences and their D epitope expression. This analysis can never be absolute because (1) most partial D phenotypes involve multiple amino acid changes in the Rh D protein and (2) deficiency for 1 or more epitopes may be due to gross structural alteration in the variant Rh D protein structure. We report here the amino acid requirements for the majority of D epitopes. They have been defined by generating a series of novel Rh mutant constructs by mutagenesis using an Rh cE cDNA as template and mutagenic oligonucleotide primers. When transfected into K562 cells, the D epitope expression of the derived mutant clones was then assessed by flow cytometry. The introduction of 9 externally predicted Rh D-specific amino acids on the Rh cE protein was sufficient to express 80% of all tested D epitopes, whereas other clones expressed none. We concluded from our data that the D epitope expression is consistent with at least 6 different epitope clusters localized on external regions of the Rh D protein, most involving overlapping regions within external loops 3, 4, and 6.
Regulation and Function of WASp in Platelets by the Collagen Receptor, Glycoprotein VI
Authors: Gross Barbara S. , Wilde Jonathan I. , Quek Lynn , Chapel Helen , Nelson David L. , Watson Steve P.
Published: October 2019

Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin 2β1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.
Variant Genotypes of the Low-Affinity Fcγ Receptors in Two Control Populations and a Review of Low-Affinity Fcγ Receptor Polymorphisms in Control and Disease Populations
Authors: Lehrnbecher Thomas , Foster Charles B. , Zhu Shaoxian , Leitman Susan F. , Goldin Lynn R. , Huppi Konrad , Chanock Stephen J.
Published: October 2019

Fcγ-receptors (FcγR) provide a critical link between humoral and cellular immunity. The genes of the low-affinity receptors for IgG and their isoforms, namely, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and SH-FcγRIIIb, are located in close proximity on chromosome 1q22. Variant alleles may differ in biologic activity and a number of studies have reported the frequencies of variant FcγR alleles in both disease and control populations. No large study has evaluated the possibility of a nonrandom distribution of variant genotypes. We analyzed 395 normal individuals (172 African Americans [AA] and 223 Caucasians [CA]) at the following loci: FcγRIIa, FcγRIIIa, and FcγRIIIb, including the SH-FcγRIIIb. The genotypic distributions of FcγRIIa, FcγRIIIa, and FcγRIIIb conform to the Hardy-Weinberg law in each group. There was no strong evidence that combinations of 2-locus genotypes of the 3 loci deviated from random distributions in these healthy control populations. The distribution of SH-FcγRIIIb is underrepresented in CA compared with AA (P < .0001) controls. A previously reported variant FcγRIIb was not detected in 70 normal individuals, indicating that this allele, if it exists, is very rare (<1%). In conclusion, we present data that should serve as the foundation for the interpretation of association studies involving multiple variant alleles of the low-affinity FcγR.
Formation of Dense Erythrocytes in SAD Mice Exposed to Chronic Hypoxia: Evaluation of Different Therapeutic Regimens and of a Combination of Oral Clotrimazole and Magnesium Therapies
Authors: De Franceschi Lucia , Brugnara Carlo , Rouyer-Fessard Philippe , Jouault Helene , Beuzard Yves
Published: October 2019

We have examined the effect of hydroxyurea (HU), clotrimazole (CLT), magnesium oxide (Mg), and combined CLT+Mg therapies on the erythrocyte characteristics and their response to chronic hypoxia in a transgenic sickle mouse (SAD) model. SAD mice were treated for 21 days with 1 of the following regimens (administered by gavage): control (n = 6), HU (200 mg/d; n = 6), CLT (80 mg/kg/d, n = 5), Mg (1,000 mg/kg/d, n = 5), and CLT+Mg (80 and 1,000 mg/kg/d, respectively, n = 6). Nine normal mice were also treated as controls (n = 3), HU (n = 3), and CLT+Mg (n = 3). Treatment with HU induced a significant increase in mean corpuscular volume and cell K content and a decrease in density in SAD mice. Treatment with the CLT and Mg, either alone or in combination, also increased cell K and reduced density in SAD mice. After 21 days of treatment, the animals were exposed to hypoxia (48 hours at 8% O2) maintaining the same treatment. In the SAD mice, hypoxia induced significant cell dehydration. These hypoxia-induced changes were blunted in either HU- or Mg-treated SAD mice and were completely abolished by either CLT or CLT+Mg treatment, suggesting a major role for the Gardos channel in hypoxia-induced dehydration in vivo.
Inverse Association Between IgG–Anti-κ and Antierythrocyte Autoantibodies in Patients With Cold Agglutination
Authors: Terness Peter , Navolan Dan , Opelz Gerhard , Roelcke Dieter
Published: October 2019

It has been known for a long time that IgG–anti-F(ab′)2 antibodies (Abs) are able to suppress the B-cell response. We showed that natural IgG-anti–F(ab′)2 autoantibodies appear in the serum of patients with cold agglutination. If the anti-F(ab′)2 Ab suppresses cold agglutinin (CA)-producing B cells, one would expect an inverse correlation between the titers of these two Abs. Our study confirmed this correlation. Subsequent experiments showed that some anti-F(ab′)2 Abs bind to the hinge region of IgG. It was difficult to explain how this Ab suppresses CA-producing B cells, which are of IgM isotype. Here we show that patients with cold agglutination have an IgG–anti-κ light chain autoantibody in their serum. This is another member of the anti-F(ab′)2 Ab group. Because the vast majority of CAs are IgM-κ Abs, the anti-κ Ab might suppress CA-producing B cells. If this is the case, there should be an inverse association between the titer of anti-κ Ab and CA. In a group of 302 patients, we found that high titers of the anti-κ Ab correlate with low titers of CA and vice versa (P= .009). Interestingly, this association is found only in patients whose disease is caused by noninfectious agents, including mainly B-cell proliferations (P = .0058). Our data show that the inverse correlation is not confined to a particular CA autoantibody specificity. The results are discussed in the light of recent findings showing that anti-IgM Abs may either inactivate or kill tumoral B cells by apoptosis.
The Tyrosine Kinase Abl-Related Gene ARG Is Fused toETV6 in an AML-M4Eo Patient With a t(1;12)(q25;p13): Molecular Cloning of Both Reciprocal Transcripts
Authors: Cazzaniga Giovanni , Tosi Sabrina , Aloisi Alessandra , Giudici Giovanni , Daniotti Maria , Pioltelli Pietro , Kearney Lyndal , Biondi Andrea
Published: October 2019

The Ets variant gene 6 (ETV6/TEL) gene is rearranged in the majority of patients with 12p13 translocations fused to a number of different partners. We present here a case of acute myeloid leukemia M4 with eosinophilia (AML-M4Eo) positive for the CBFb/MYH11 rearrangement and carrying a t(1;12)(q25;p13) that involves the ETV6 gene at 12p13. By 3′rapid amplification of cDNA ends-polymerase chain reaction (3′RACE-PCR), a novel fusion transcript was identified between the ETV6 and the Abelson-related gene (ARG) at 1q25, resulting in a chimeric protein consisting of the HLH oligomerization domain of ETV6 and the SH2, SH3, and protein tyrosine kinase (PTK) domains of ARG. The reciprocal transcript ARG-ETV6 was also detected in the patient RNA by reverse transcriptase-polymerase chain reaction (RT-PCR), although at a lower expression level. The ARG gene encodes for a nonreceptor tyrosine kinase characterized by high homology with c-Abl in the TK, SH2, and SH3 domains. This is the first report on ARGinvolvement in a human malignancy.