bluuebunny/crossref_metadata_2025 huggingface dataset:
Deregulated E2F-1 blocks terminal differentiation and loss of leukemogenicity of M1 myeloblastic leukemia cells without abrogating induction of p15INK4B and p16INK4A
Authors: Amanullah Arshad , Hoffman Barbara , Liebermann Dan A.
Published: October 2019

The transcription factor E2F-1 has been postulated to play a crucial role in the control of cell cycle progression because of its ability to be bound and regulated by the retinoblastoma gene product (pRb). Exogenous expression of E2F-1, under growth restrictive conditions, was shown to result in p53-dependent programmed cell death. The consequences of deregulated expression of E2F-1 on terminal differentiation of hematopoietic cells in the absence of E2F-1–mediated apoptosis, as well as mechanistic insights into how deregulated E2F-1 may affect terminal differentiation, have not been established. The autonomously proliferating M1 myeloblastic leukemia cell line, which is null for p53 expression and can be induced by interleukin-6 (IL-6) to undergo terminal macrophage differentiation with concomitant loss of leukemogenicity, provides a particularly attractive model system to address these issues. Deregulated and continued expression of E2F-1 blocked the IL-6–induced terminal differentiation program at an early blast stage, giving rise to immature cells, which continued to proliferate without undergoing apoptosis and retained their leukemogenic phenotype. Although E2F-1 blocked IL-6–mediated terminal differentiation and its associated growth arrest, it did not prevent the rapid induction of both p15INK4B and p16INK4A, inhibition of cdk4 kinase activity, and subsequent hypophosphorylation of pRb. The results obtained imply that genetic alterations that both impair p53 function and deregulate E2F-1 expression may render hematopoietic cells refractory to the induction of differentiation and are, thereby, likely to play a major role in the progression of leukemias.
Neuroserpin reduces cerebral infarct volume and protects neurons from ischemia-induced apoptosis
Authors: Yepes Manuel , Sandkvist Maria , Wong Mike K. K. , Coleman Timothy A. , Smith Elizabeth , Cohan Stanley L. , Lawrence Daniel A.
Published: October 2019

Neuroserpin, a recently identified inhibitor of tissue-type plasminogen activator (tPA), is primarily localized to neurons within the central nervous system, where it is thought to regulate tPA activity. In the present study neuroserpin expression and its potential therapeutic benefits were examined in a rat model of stroke. Neuroserpin expression increased in neurons surrounding the ischemic core (ischemic penumbra) within 6 hours of occlusion of the middle cerebral artery and remained elevated during the first week after the ischemic insult. Injection of neuroserpin directly into the brain immediately after infarct reduced stroke volume by 64% at 72 hours compared with control animals. In untreated animals both tPA and urokinase-type plasminogen activator (uPA) activity was significantly increased within the region of infarct by 6 hours after reperfusion. Activity of tPA then decreased to control levels by 72 hours, whereas uPA activity continued to rise and was dramatically increased by 72 hours. Both tPA and uPA activity were significantly reduced in neuroserpin-treated animals. Immunohistochemical staining of basement membrane laminin with a monoclonal antibody directed toward a cryptic epitope suggested that proteolysis of the basement membrane occurred as early as 10 minutes after reperfusion and that intracerebral administration of neuroserpin significantly reduced this proteolysis. Neuroserpin also decreased apoptotic cell counts in the ischemic penumbra by more than 50%. Thus, neuroserpin may be a naturally occurring neuroprotective proteinase inhibitor, whose therapeutic administration decreases stroke volume most likely by inhibiting proteinase activity and subsequent apoptosis associated with focal cerebral ischemia/reperfusion.
New induction of leukotriene A4 hydrolase by interleukin-4 and interleukin-13 in human polymorphonuclear leukocytes
Authors: Zaitsu Masafumi , Hamasaki Yuhei , Matsuo Muneaki , Kukita Akiko , Tsuji Kosuke , Miyazaki Michiko , Hayasaki Rika , Muro Eriko , Yamamoto Shuichi , Kobayashi Ikuko , Ichimaru Tomohiro , Kohashi Osamu , Miyazaki Sumio
Published: October 2019

Interleukin (IL)-4, IL-10, and IL-13, Th2 cell–derived cytokines, play major roles in the pathophysiology of allergic diseases. These cytokines up-regulate or down-regulate the production of arachidonic acid metabolites. In this study, we have investigated the effect of IL-4, IL-10, IL-13, and other cytokines on A23187-stimulated synthesis of leukotriene (LT) B4 in human polymorphonuclear leukocytes (PMNs). Production of LTB4 was measured by specific radioimmunoassay and high performance liquid chromatography. Messenger RNA (mRNA) expression of cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and LTA4 hydrolase, which were involved in the synthesis of LTB4, was determined by reverse transcription–polymerase chain reaction and Northern blot analysis. Protein synthesis of their enzymes was determined by Western blot analysis. IL-4 and IL-13 enhanced A23187-stimulated LTB4 synthesis and increased mRNA expression and protein synthesis of LTA4hydrolase, but not those of cPLA2 or 5-LO. These results indicate that IL-4 and IL-13 transcriptionally or post-transcriptionally up-regulate the synthesis of LTB4, a potent chemotactic factor to PMNs, at the enzyme level of LTA4 hydrolase, and this up-regulation mechanism may participate in the development of allergic inflammation.
Higher-grade transformation of follicle center lymphoma is associated with somatic mutation of the 5′ noncoding regulatory region of the BCL-6 gene
Authors: Lossos Izidore S. , Levy Ronald
Published: October 2019

Follicle center lymphoma (FCL) is an indolent low-grade B-cell non-Hodgkin's lymphoma (NHL) that frequently transforms to aggressive diffuse large B-cell lymphoma (DLBCL). Histologic transformation of FCL is commonly associated with accumulation of secondary genetic alterations. The BCL-6 gene is altered by chromosomal rearrangements and mutations clustering in its 5′ noncoding regulatory region in up to 70% of primary DLBCL, but in a significantly smaller subset of FCL. Previous studies have shown that both chromosomal rearrangements and mutations could deregulateBCL-6 expression. To evaluate the association between progressive accumulation of BCL-6 regulatory region mutations and the histologic transformation of FCL, we analyzed by extensive cloning and sequencing paired biopsy specimens obtained at the time of FCL diagnosis and transformation (6 patients) or FCL relapse (3 patients). In an additional patient, biopsy specimens obtained at the time of diagnosis, FCL relapse, and subsequent transformation to DLBCL were evaluated. The presence of identical mutations in the paired diagnosis and posttransformation DLBCL specimens confirmed the common clonal origin of both the pretransformation and the posttransformation lymphomas. No new mutations in the 5′ noncoding regulatory region of the BCL-6 gene were detected in any of the specimens evaluated at the time of FCL relapse. In contrast, 5 of the 7 transformed specimens contained new mutations not found in the paired original biopsy specimens obtained at the time of FCL diagnosis or relapse. The number of these new mutations ranged from 1 to 6 per specimen. Some of the new mutations tended to cluster in certain areas of the 5′ noncoding regulatory region of the BCL-6 gene. Our results show that transformation of FCL to DLBCL is associated with accumulation of new mutations in the 5′ noncoding regulatory region of the BCL-6 gene, that by deregulation of theBCL-6 gene expression may play a role in lymphoma transformation.
Macrophage inflammatory protein 1-alpha is a potential osteoclast stimulatory factor in multiple myeloma
Authors: Choi Sun Jin , Cruz Jose C. , Craig Fiona , Chung Hoyeon , Devlin Rowena D. , Roodman G. David , Alsina Melissa
Published: October 2019

This study was designed to determine if macrophage inhibitory protein-1 (MIP-1), a recently described osteoclast (OCL) stimulatory factor,1 was present in marrow from patients with multiple myeloma (MM) and possibly involved in the bone destructive process. MIP-1, but not interleukin-1β (IL-1β), tumor necrosis factor-β (TNF-β), or interleukin-6 (IL-6), messenger RNA was elevated in freshly isolated bone marrow from 3 of 4 patients with MM compared to normal controls. Furthermore, enzyme-linked immunosorbent assays of freshly isolated bone marrow plasma detected increased concentrations of hMIP-1 (range, 75-7784 pg/mL) in 8 of 13 patients (62%) with active myeloma, in 3 of 18 patients (17%) with stable myeloma (range, 75-190.3), as well as in conditioned media from 4 of 5 lymphoblastoid cell lines (LCLs) derived from patients with MM. Mildly elevated levels of MIP-1 were detected in 3 of 14 patients (21%) with other hematologic diagnoses (range, 80.2-118.3, median value of 96 pg/mL) but not in normal controls (0 of 7). MIP-1 was not detected in the peripheral blood of any patients with MM. In addition, recombinant hMIP-1 induced OCL formation in human bone marrow cultures. Importantly, addition of a neutralizing antibody to MIP-1 to human bone marrow cultures treated with freshly isolated marrow plasma from patients with MM blocked the increased OCL formation induced by these marrow samples but had no effect on control levels of OCL formation. Thus, high levels of MIP-1 are expressed in marrow samples from patients with MM, but not in marrow from patients with other hematologic disorders or controls, and support an important role for MIP-1 as one of the major factors responsible for the increased OCL stimulatory activity in patients with active MM.
Glucocorticoids promote the proliferation and antagonize the retinoic acid–mediated growth suppression of Epstein-Barr virus–immortalized B lymphocytes
Authors: Quaia Michele , Zancai Paola , Cariati Roberta , Rizzo Silvana , Boiocchi Mauro , Dolcetti Riccardo
Published: October 2019

Glucocorticoids are able to release Epstein-Barr virus–immortalized (EBV-immortalized) lymphoblastoid B cell lines (LCLs) from the persistent growth arrest induced in these cells by retinoic acid (RA). Moreover, physiologic concentrations of glucocorticoids efficiently antagonized LCL growth inhibition induced by 13-cis-RA; 9-cis-RA; all-trans-RA; and Ro 40-6055, an RA  receptor (RAR) selective agonist. RAR expression levels, however, were not affected by glucocorticoids. Glucocorticoids, but not other steroid hormones, directly promote LCL proliferation, a phenomenon that was mainly mediated by down-regulation of the cyclin-dependent kinase (CDK) inhibitor p27Kip-1. Moreover, glucocorticoids contrasted the up-regulation of p27Kip-1, which was underlying the RA-induced LCL growth arrest, thereby indicating that glucocorticoids and RA signalings probably converge on p27Kip-1. Both antagonism of RA-mediated growth inhibition and promotion of LCL proliferation were efficiently reversed by the glucocorticoid receptor (GR) antagonist RU486, indicating that all of these effects were mediated by GR. Of note, RU486 also proved to be effective in vivo and, in mice, was able to significantly inhibit the growth of untreated LCLs as well as LCLs growth-arrested by RA in vitro. These findings provide a rational background to further evaluate the possible role of glucocorticoids in the pathogenesis of EBV-related lymphoproliferations of immunosuppressed patients. Moreover, GR antagonists deserve further consideration for their possible efficacy in the management of these disorders, and the use of schedules, including both RA and a GR antagonist, may allow a more thorough evaluation of the therapeutic potential of RA in this setting.
Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages
Authors: Faust Nicole , Varas Florencio , Kelly Louise M. , Heck Susanne , Graf Thomas
Published: October 2019

Pluripotent hematopoietic stem cells have been studied extensively, but the events that occur during their differentiation remain largely uncharted. To develop a system that allows the differentiation of cultured multipotent progenitors by time-lapse fluorescence microscopy, myelomonocytic cells were labeled with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene into the murine lysozyme M (lys) locus and using a targeting vector, which contains a neomycin resistant (neo) gene flanked by LoxP sites and “splinked” ends, to increase the frequency of homologous recombination. Analysis of the blood and bone marrow of thelys-EGFP mice revealed that most myelomonocytic cells, especially mature neutrophil granulocytes, were fluorescence-positive, while cells from other lineages were not. Removal of the neogene through breeding of the mice with the Cre-deleter strain led to an increased fluorescence intensity. Mice with an inactivation of both copies of the lys gene developed normally and were fertile.
CXC chemokine receptor 3 expression on CD34+hematopoietic progenitors from human cord blood induced by granulocyte-macrophage colony-stimulating factor: chemotaxis and adhesion induced by its ligands, interferon γ–inducible protein 10 and monokine induced by interferon γ
Authors: Jinquan Tan , Quan Sha , Jacobi Henrik H. , Jing Chen , Millner Anders , Jensen Bettina , Madsen Hans O. , Ryder Lars P. , Svejgaard Arne , Malling Hans-Jørgen , Skov Per S. , Poulsen Lars K.
Published: October 2019

CXC chemokine receptor 3 (CXCR3), which is known to be expressed predominately on memory and activated T lymphocytes, is a receptor for both interferon γ (IFN-γ)–inducible protein 10 (γIP-10) and monokine induced by IFN-γ (Mig). We report the novel finding that CXCR3 is also expressed on CD34+ hematopoietic progenitors from human cord blood stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) but not on freshly isolated CD34+ progenitors. Freshly isolated CD34+progenitors expressed low levels of CXCR3 messenger RNA, but this expression was highly up-regulated by GM-CSF, as indicated by a real-time quantitative reverse transcriptase–polymerase chain reaction technique. γIP-10 and Mig induced chemotaxis of GM-CSF–stimulated CD34+ progenitors by means of CXCR3, since an anti-CXCR3 monoclonal antibody (mAb) was found to block γIP-10–induced and Mig-induced CD34+ progenitor chemotaxis. These chemotactic attracted CD34+ progenitors are colony-forming units—granulocyte-macrophage. γIP-10 and Mig also induced GM-CSF–stimulated CD34+ progenitor adhesion and aggregation by means of CXCR3, a finding confirmed by the observation that anti-CXCR3 mAb blocked these functions of γIP-10 and Mig but not of chemokine stromal cell–derived factor 1α. γIP-10–induced and Mig-induced up-regulation of integrins (CD49a and CD49b) was found to play a crucial role in adhesion of GM-CSF–stimulated CD34+progenitors. Moreover, γIP-10 and Mig stimulated CXCR3 redistribution and cellular polarization in GM-CSF–stimulated CD34+progenitors. These results indicate that CXCR3–γIP-10 and CXCR3–Mig receptor-ligand pairs, as well as the effects of GM-CSF on them, may be especially important in the cytokine/chemokine environment for the physiologic and pathophysiologic events of differentiation of CD34+ hematopoietic progenitors into lymphoid and myeloid stem cells, subsequently immune and inflammatory cells. These processes include transmigration, relocation, differentiation, and maturation of CD34+ hematopoietic progenitors.
Nonmyeloablative stem cell transplantation for congenital immunodeficiencies
Authors: Amrolia Persis , Gaspar Hubert B. , Hassan Amel , Webb David , Jones Alison , Sturt Natalie , Mieli-Vergani Giorgina , Pagliuca Antonio , Mufti Ghulam , Hadzic Nedim , Davies Graham , Veys Paul
Published: October 2019

The optimal approach for stem cell transplantation in children with immunodeficiency has yet to be determined. Conditioning therapy is necessary for reliable engraftment and full immune reconstitution; however, the beneficial effect of cytoreductive conditioning is counterbalanced by increased short- and long-term treatment-related toxicity. Whether bone marrow transplantation with a nonmyeloablative preparative regimen was sufficient for the establishment of donor immune reconstitution, with the resultant correction of disease phenotype, was investigated. Eight patients with severe immunodeficiency states underwent T-cell replete bone marrow transplantation from a human leukocyte antigen-matched unrelated (n = 6) or sibling (n = 2) donor with nonmyeloablative conditioning using a fludarabine–melphalan–anti-lymphocyte globulin-based regimen. All patients had severe organ dysfunction that precluded transplantation with conventional conditioning. All patients were engrafted with predominantly donor hematopoiesis, and the duration of neutropenia was brief. Significant acute graft-versus-host disease (GVHD) did not develop, but one patient had limited chronic GVHD. One patient died of disease recurrence, and 3 have stable, mixed chimerism. At a median follow-up of 1 year, all patients have had good recovery of CD3+ T-cell numbers, and 6 of 7 evaluable patients have normal phytohemagglutinin stimulation indices. The rate of immune reconstitution is comparable with that of historical controls undergoing standard myeloablative protocols. Two patients with CD40 ligand deficiency now show significant expression, and a patient with adenosine deaminase deficiency has improved deoxy adenosine triphosphate metabolites. In summary, it has been demonstrated that nonmyeloablative stem cell transplantation permits rapid engraftment from both sibling and unrelated donors with minimal toxicity even in the presence of severe organ dysfunction. If long-term immune reconstitution of patients treated with this protocol is demonstrated, it is believed this approach might offer significant advantages compared with standard protocols by combining adequate immune reconstitution with reduced short- and long-term toxicity.
Prognostic factors and treatment outcome in primary progressive Hodgkin lymphoma: a report from the German Hodgkin Lymphoma Study Group
Authors: Josting Andreas , Rueffer Ulrich , Franklin Jeremy , Sieber Markus , Diehl Volker , Engert Andreas
Published: October 2019

To determine prognostic factors and treatment outcome, patients with primary progressive Hodgkin lymphoma (HD) registered in the database of the German Hodgkin Lymphoma Study Group (GHSG) were analyzed retrospectively. Detailed records from randomized prospective multicenter trials performed between 1988 and 1998 of 3807 patients recruited in these trials were reviewed. The median age of the 206 patients available was 34 years (range, 16-71). Fifty-seven patients (28%) in intermediate stage and 149 patients (72%) in advanced stage developed progressive disease (PD). One hundred and fifty-three patients (74%) were treated with salvage chemotherapy, 47 patients (23%) with salvage radiotherapy, and 6 patients (3%) did not receive any therapy due to rapid PD. Seventy patients (34%) were treated with high-dose chemotherapy (HDCT) and autologous stem cell transplantation. The 5-year freedom from second failure (FF2F) and overall survival (OS) for all patients were 17% and 26%, respectively. The 5-year FF2F and OS for patients treated with HDCT were 31% and 43%, respectively. In multivariate analysis low Karnofsky performance score at the time of progression (P < .0001), age above 50 years (P = .019), and failure to attain a temporary remission on first-line treatment (P = .0003) were significant adverse prognostic factors for OS. Patients with none of these risk factors had a 5-year OS of 55% compared with 0% for patients with all 3 of these unfavorable prognostic factors. Although HDCT is a reasonable option for selected patients with primary progressive HD, the majority did not receive HDCT. Interestingly, salvage radiotherapy gave promising results in patients with localized PD.
Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro
Authors: Gruber Andreas , Kan-Mitchell June , Kuhen Kelli L. , Mukai Tetsu , Wong-Staal Flossie
Published: October 2019

Dendritic cells (DCs) genetically modified to continually express and present antigens may be potent physiologic adjuvants for induction of prophylactic or therapeutic immunity. We have previously shown that an env and nef deleted HIV-1 vector (HIV-1ΔEN) pseudotyped with VSV-G transduced monocyte-derived macrophages as well as CD34+ precursors of DCs. Here we extended these findings with HIV-1ΔEN to highly differentiated human DCs derived in culture from circulating monocytes (DCs). In addition, a new vector derived from HIV-1ΔEN but further deleted in its remaining accessory genes vif, vpr, and vpu(HIV-1ΔEN V3) was also tested. Both vectors efficiently transduced DCs. Transduction of DCs did not significantly alter their viability or their immunophenotype when compared with untransduced DCs. Furthermore, the phagocytic potential of immature DCs, as well as their ability to differentiate into mature DCs capable of stimulating T-cell proliferation, was not affected. Finally, DCs transduced by the HIV-1ΔEN vector were able to elicit a primary antiviral cytotoxic T-cell response in autologous CD8 T cells. These results suggest that HIV-1–based vectors expressing viral antigens may be useful for in vivo active immunization as well as ex vivo priming of cytotoxic T cells for adoptive T-cell therapy.
Pathologic interaction between megakaryocytes and polymorphonuclear leukocytes in myelofibrosis
Authors: Schmitt Alain , Jouault Hélène , Guichard Josette , Wendling Françoise , Drouin Arnaud , Cramer Elisabeth M.
Published: October 2019

Idiopathic myelofibrosis (MF) is a myeloproliferative syndrome characterized by an increase in bone marrow collagen. Megakaryocytes (Mks), which store growth factors in their α granules, are known to be involved in the pathogenesis of MF. Previously, mice given bone marrow grafts infected with a retrovirus carrying murine thrombopoietin (TPO) complementary DNA developed a disease resembling human idiopathic MF. In this study, we used this murine model (TPO mice) to determine whether release of α granules is responsible for fibroblast activation and development of fibrosis. The intracellular trafficking of several α-granule proteins (von Willebrand factor, fibrinogen, and transforming growth factor β (TGFβ), which are stored in the granule matrix; and αIIbβ3 integrin and P-selectin (CD62p), which are located in the α-granule membrane) was studied with immune electron microscopy in bone marrow Mks from TPO mice. P-selectin immunolabeling increased consistently and was occasionally found lining the demarcation membrane system. Evidence of extensive emperipolesis was also found in TPO mouse Mks, involving almost exclusively neutrophil and eosinophil polymorphonuclear (PMN) cells with altered morphologic features. In parallel, the host Mks had myeloperoxidase-positive granules scattered in their cytoplasm, associated with marked ultrastructural cytoplasmic alterations and ruptured α-granule membranes. Similar observations were made in bone marrow biopsy specimens from 12 patients with idiopathic MF; indeed, there was an increased rate of emperipolesis involving mostly PMN cells, abnormal P-selectin expression, and mutual subcellular PMN and Mk alterations. This study indicates that in idiopathic MF, abnormal P-selectin distribution in Mks induces selective sequestration of PMN cells. This results in a release of α-granular proteins and growth factors, which in turn induces fibroblast activation and fibrosis deposition.
Thrombasthenic mice generated by replacement of the integrin αIIb gene: demonstration that transcriptional activation of this megakaryocytic locus precedes lineage commitment
Authors: Roux Diana Tronik-Le , Roullot Valérie , Poujol Christel , Kortulewski Thierry , Nurden Paquita , Marguerie Gérard
Published: October 2019

To analyze the transcriptional activity of the gene encoding the α subunit of the platelet integrin αIIbβ3during the hematopoietic differentiation, mice were produced in which the herpes virus thymidine kinase (tk) was introduced in this megakaryocytic specific locus using homologous recombination technology. This provided a convenient manner in which to induce the eradication of particular hematopoietic cells expressing the targeted gene. Results of progenitor cell cultures and long-term bone marrow (BM) assays showed that the growth of a subset of stem cells was reduced in the presence of the antiherpetic drug ganciclovir, demonstrating that the activation of the toxic gene occurs before the commitment to the megakaryocytic lineage. Furthermore theknock-in of the tk gene into the αIIb locus resulted in the knock-out of the αIIb gene in homozygous mice. Cultures of BM cells of these animals, combined with ultrastructural analysis, established that the αIIbglycoprotein is dispensable for lineage commitment and megakaryocytic maturation. Platelets collected from αIIb-deficient mice failed to bind fibrinogen, to aggregate, and to retract a fibrin clot. Moreover, platelet α-granules did not contain fibrinogen. Consistent with these characteristics, the mice displayed bleeding disorders similar to those in humans with Glanzmann thrombasthenia.
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a target glycoprotein in drug-induced thrombocytopenia
Authors: Kroll Hartmut , Sun Qi-Hong , Santoso Sentot
Published: October 2019

Drug-induced immune thrombocytopenia (DITP) is a serious complication of drug treatment. Previous studies demonstrated that most drug-dependent antibodies (DDAbs) react with the platelet membrane glycoprotein (GP) complexes IIb/IIIa and Ib/IX/V. We analyzed the sera from 5 patients who presented with DITP after intake of carbimazole. Notably, thrombocytopenia induced by carbimazole was relatively mild in comparison to patients with DITP induced by quinidine. The sera reacted with platelets in an immunoassay on addition of the drug. In immunoprecipitation experiments with biotin-labeled platelets and endothelial cells, reactivity with the platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) could be demonstrated, whereas neither GPIIb/IIIa nor GPIb/IX was precipitated in the presence of the drug. These results could be confirmed by GP-specific immunoassay (MAIPA) using monoclonal antibodies (mabs) against PECAM-1. In addition, the binding of DDAbs could be abolished by preincubation with soluble recombinant PECAM-1. Carbimazole-dependent antibodies showed similar reactivity with platelets carrying the Leu125 and Val125 PECAM-1 isoforms, indicating that this polymorphic structure, which is located in the first extracellular domain, is not responsible for the epitope formation. Binding studies with biotin-labeled mutants of PECAM-1 and analysis of sera with mabs against different epitopes on PECAM-1 in MAIPA assay suggested that carbimazole-dependent antibodies prominently bound to the second immunoglobulin homology domain of the molecule. Analysis of 20 sera from patients with quinidine-induced thrombocytopenia by MAIPA assay revealed evidence that DDAbs against PECAM-1 are involved in addition to anti-GPIb/IX and anti-GPIIb/IIIa. We conclude that PECAM-1 is an important target GP in DITP.
Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferon-γ production and STAT-mediated signal transduction
Authors: Carson William E. , Dierksheide Julie E. , Jabbour Saad , Anghelina Mirela , Bouchard Page , Ku George , Yu Haixin , Baumann Heinz , Shah Manisha H. , Cooper Megan A. , Durbin Joan , Caligiuri Michael A.
Published: October 2019

The administration of therapeutic doses of recombinant cytokines to patients with malignant disease can be complicated by systemic toxicities, which in their most severe form may present as a systemic inflammatory response. The combination of interleukin (IL)–18 and IL-12 has synergistic antitumor activity in vivo yet has been associated with significant toxicity. The effects of IL-18 plus IL-12 were examined in a murine model, and it was found that the daily, simultaneous administration of IL-18 and IL-12 resulted in systemic inflammation and 100% mortality within 4 to 8 days depending on the strain employed. Mice treated with IL-18 plus IL-12 exhibited unique pathologic findings as well as elevated serum levels of proinflammatory cytokines and acute-phase reactants. The actions of tumor necrosis factor–α did not contribute to the observed toxicity, nor did T or B cells. However, toxicity and death from treatment with IL-18 plus IL-12 could be completely abrogated by elimination of natural killer (NK) cells or macrophages. Subsequent studies in genetically altered mice revealed that NK-cell interferon–γ mediated the fatal toxicity via the signal transducer and activator of transcription pathway of signal transduction. These data may provide insights into methods of ameliorating cytokine-induced shock in humans.
Immunity to WT1 in the animal model and in patients with acute myeloid leukemia
Authors: Gaiger Alexander , Reese Valerie , Disis Mary L. , Cheever Martin A.
Published: October 2019

The Wilms' tumor (WT1) gene participates in leukemogenesis and is overexpressed in most types of leukemia in humans. WT1 is also detectable in many types of lung, thyroid, breast, testicular, and ovarian cancers and melanoma in humans. Initial studies evaluated whether immune responses to murine WT1 can be elicited in mice. Murine and human WT1 are similar. Thus, mouse models might lead to resolution of many of the critical issues for developing WT1 vaccines. C57/BL6 (B6) mice were injected with synthetic peptides from the natural sequence of WT1 containing motifs for binding to major histocompatibility (MHC) class II molecules. Immunization induced helper T-cell responses specific for the immunizing WT1 peptides and antibody responses specific for WT1 protein. Screening of multiple murine cancer cell lines identified 2 murine cancers, TRAMP-C and BLKSV40, that “naturally” overexpress WT1. Immunization with MHC class I binding peptides induced WT1 peptide-specific cytotoxic T-lymphocyte (CTL) that specifically lysed TRAMP-C and BLKSV40. WT1 specificity of lysis was confirmed by cold target inhibition. No toxicity was noted by histopathologic evaluation in the WT1 peptide-immunized animals. WT1 peptide immunization did not show any effect on TRAMP-C tumor growth in vivo. Immunization of B6 mice to syngeneic TRAMP-C elicited WT1-specific antibody, demonstrating that WT1 can be immunogenic in the context of cancer cells. To evaluate whether WT1 might be similarly immunogenic in humans, serum from patients with leukemia was evaluated for pre-existing antibody responses. Western blot analyses showed WT1-specific antibodies directed against the N-terminus portion of the WT1 protein in the sera of 3 of 18 patients with acute myeloid leukemia (AML).
Epolones induce erythropoietin expression via hypoxia-inducible factor-1α activation
Authors: Wanner Roger M. , Spielmann Patrick , Stroka Deborah M. , Camenisch Gieri , Camenisch Isabelle , Scheid Annette , Houck David R. , Bauer Christian , Gassmann Max , Wenger Roland H.
Published: October 2019

Induction of erythropoietin (Epo) expression under hypoxic conditions is mediated by the heterodimeric hypoxia-inducible factor (HIF)-1. Following binding to the 3′ hypoxia-response element (HRE) of the Epo gene, HIF-1 markedly enhances Epo transcription. To facilitate the search for HIF-1 (ant)agonists, a hypoxia-reporter cell line (termed HRCHO5) was constructed containing a stably integrated luciferase gene under the control of triplicated heterologous HREs. Among various agents tested, we identified a class of substances called epolones, which induced HRE-dependent reporter gene activity in HRCHO5 cells. Epolones are fungal products known to induce Epo expression in hepatoma cells. We found that epolones (optimal concentration 4-8 μmol/L) potently induce HIF-1α protein accumulation and nuclear translocation as well as HIF-1 DNA binding and reporter gene transactivation. Interestingly, the activity of a compound related to the fungal epolones, ciclopirox olamine (CPX), was blocked after addition of ferrous iron. This suggests that CPX might interfere with the putative heme oxygen sensor, as has been proposed for the iron chelator deferoxamine mesylate (DFX). However, about 10-fold higher concentrations of DFX (50-100 μmol/L) than CPX were required to maximally induce reporter gene activity in HRCHO5 cells. Moreover, structural, functional, and spectrophotometric data imply a chelator:iron stoichiometry of 1:1 for DFX but 3:1 for CPX. Because the iron concentration in the cell culture medium was determined to be 16 μmol/L, DFX but not CPX function can be explained by complete chelation of medium iron. These results suggest that the lipophilic epolones might induce HIF-1α by intracellular iron chelation.
Molecular heterogeneity of the Jknull phenotype: expression analysis of the Jk(S291P) mutation found in Finns
Authors: Sidoux-Walter Frederic , Lucien Nicole , Nissinen Riikka , Sistonen Pertti , Henry Stephen , Moulds Joann , Cartron Jean-Pierre , Bailly Pascal
Published: October 2019

Polymerase chain reaction genotyping of 32 unrelated Jknull individuals originating predominantly from Polynesia and Finland indicated that all were homozygous for the JK*Bpolymorphism and that 17 of 32, including the 14 Polynesians, carried a 3′-acceptor splice site mutation of intron 5 that resulted in the skipping of exon 6 (called mutation JkΔ6). The remaining 15 Jknull donors from Finland were homozygous for a new T871C transition resulting in a S291P amino acid substitution at a consensusN-glycosylation site of the Jk polypeptide. Transcription-translation assays revealed that the Jk(S291P) mutant was translated into a glycosylated component as efficiently as the wild-type Jk polypeptide (wt Jk)] in the presence of microsomes, thus indicating that the S291P mutation has no effect on theN-glycosylation pattern of the Jk protein. Expression studies in Xenopus oocytes revealed that the Jk(S291P) polypeptide functions as a urea transporter, but the transport activity and the membrane expression level of the mutant protein was reduced to a similar extent. A substantial fraction of the mutant protein was retained intracellularly suggesting that the transit to the plasma membrane was reduced, presumably because of the S→P mutation. After transfection in erythroleukemia K562 cells the wild-type, but not the mutant, protein was efficiently expressed at the cell surface. Because the Jk(S291P) mutant polypeptide was not present in human red cells from Jknull individuals, expression data in the erythroid context clearly indicates that the S→P mutation is the molecular basis of the Finnish Jknull phenotype.
Blood group A and B antigens are strongly expressed on platelets of some individuals
Authors: Curtis Brian R. , Edwards Jennifer T. , Hessner Martin J. , Klein John P. , Aster Richard H.
Published: October 2019

It is widely thought that expression of ABH antigens on platelets is insufficient to materially affect the survival of ABH-incompatible platelets in transfusion recipients, but anecdotal reports of poor survival of A and B mismatched platelets suggest that this is not always the case. The A and B antigen expression on platelets of 100 group A1 and group B blood donors was measured, and 7% and 4%, respectively, had platelets whose A and B antigen levels consistently exceeded the mean plus 2 SD. On the basis of flow cytometric and statistical analysis, donors whose platelets contained higher than normal levels of A antigen were subdivided into 2 groups, designated Type I and Type II (“high expressers”). Serum A1- and B-glycosyltransferase levels of A and B high expressers were significantly higher than those of group A1 and B individuals with normal expression. H antigen levels were low on the red cells of high expressers, indicating that the anomaly affects other cell lineages. Immunochemical studies demonstrated high levels of A antigen on various glycoproteins (GPs) from high-expresser platelets, especially GPIIb and PECAM (CD31). The A1 Type II high-expresser phenotype was inherited as an autosomal dominant trait in one family. The sequences of exons 5, 6, and 7 of the A1-transferase gene of one Type II A1 high expresser and exon 7 from 3 other genes were identical to the reported normal sequences. Further studies are needed to define the molecular basis for the high-expresser trait and to characterize its clinical implications.
Somatic mosaicism and compound heterozygosity in female hemophilia B
Authors: Costa Jean-Marc , Vidaud Dominique , Laurendeau Ingrid , Vidaud Michel , Fressinaud Edith , Moisan Jean-Pierre , David Albert , Meyer Dominique , Lavergne Jean-Maurice
Published: October 2019

Sequencing the complete factor IX gene of 2 sisters with hemophilia B with different phenotypes and no family history of hemorrhagic diathesis revealed a common 5′ splice site mutation in intron 3 (T6704C) in both and an additional missense mutation (I344T) in one. The presence of dysfunctional antigen in the latter strongly suggested that these mutations are in trans. Neither mutation was found in leukocyte DNA from the asymptomatic parents, but the mother was in somatic mosaicism for the shared splice site mutation. This case illustrates the importance of defining the phenotype and considering somatic mosaicism in sporadic cases. It underlines the limitations of complete gene sequencing for the detection of mosaicism and has implication for genetic counseling.
Viral and cellular cytokines in AIDS-related malignant lymphomatous effusions
Authors: Aoki Yoshiyasu , Yarchoan Robert , Braun James , Iwamoto Aikichi , Tosato Giovanna
Published: October 2019

Kaposi sarcoma-associated herpesvirus encodes viral IL-6 (vIL-6). To investigate the potential role of vIL-6 in the pathogenesis of human immunodeficiency virus (HIV)- related primary effusion lymphomas (PEL), a sensitive enzyme-linked immunosorbent assay was developed for vIL-6 and applied to the study of PEL. Whereas vIL-6 was detectable in 6 of 8 PEL effusions (range, 1390-66 630 pg/mL), it was not detectable in any of the control effusions. As expected, all PEL effusions contained human IL-6 (range, 957-37 494 pg/mL), and 7 of 8 contained detectable human IL-10 (range, 66-2,521,297 pg/mL). Human and vIL-6 have previously been shown to induce vascular endothelial growth factor, which in turn can increase vascular permeability. The results of the current study suggest that these cytokines play a central role in the pathogenesis and manifestations of PEL.
Multiple nonfunctional alleles of CCR5 are frequent in various human populations
Authors: Blanpain Cédric , Lee Benhur , Tackoen Marie , Puffer Bridget , Boom Alain , Libert Frédérick , Sharron Mathew , Wittamer Valérie , Vassart Gilbert , Doms Robert W. , Parmentier Marc
Published: October 2019

CCR5 is the major coreceptor for macrophage-tropic strains of the human immunodeficiency virus type I (HIV-1). Homozygotes for a 32-base pair (bp) deletion in the coding sequence of the receptor (CCR5Δ32) were found to be highly resistant to viral infection, and CCR5 became, therefore, one of the paradigms illustrating the influence of genetic variability onto individual susceptibility to infectious and other diseases. We investigated the functional consequences of 16 other natural CCR5 mutations described in various human populations. We found that 10 of these variants are efficiently expressed at the cell surface, bind [125I]-MIP-1β with affinities similar to wtCCR5, respond functionally to chemokines, and act as HIV-1 coreceptors. In addition to Δ32, six mutations were characterized by major alterations in their functional response to chemokines, as a consequence of intracellular trapping and poor expression at the cell surface (C101X, FS299), general or specific alteration of ligand binding affinities (C20S, C178R, A29S), or relative inability to mediate receptor activation (L55Q). A29S displayed an unusual pharmacological profile, binding and responding to MCP-2 similarly to wtCCR5, but exhibiting severely impaired binding and functional responses to MIP-1α, MIP-1β, and RANTES. In addition to Δ32, only C101X was totally unable to mediate entry of HIV-1. The fact that nonfunctional CCR5 alleles are relatively frequent in various human populations reinforces the hypothesis of a selective pressure favoring these alleles.
Targeting the Ras signaling pathway: a rational, mechanism-based treatment for hematologic malignancies?
Authors: Reuter Christoph W. M. , Morgan Michael A. , Bergmann Lothar
Published: October 2019

A series of alterations in the cellular genome affecting the expression or function of genes controlling cell growth and differentiation is considered to be the main cause of cancer. These mutational events include activation of oncogenes and inactivation of tumor suppressor genes. The elucidation of human cancer at the molecular level allows the design of rational, mechanism-based therapeutic agents that antagonize the specific activity of biochemical processes that are essential to the malignant phenotype of cancer cells. Because the frequency of RAS mutations is among the highest for any gene in human cancers, development of inhibitors of the Ras–mitogen-activated protein kinase pathway as potential anticancer agents is a very promising pharmacologic strategy. Inhibitors of Ras signaling have been shown to revert Ras-dependent transformation and cause regression of Ras-dependent tumors in animal models. The most promising new class of these potential cancer therapeutics are the farnesyltransferase inhibitors. The development of these compounds has been driven by the observation that oncogenic Ras function is dependent upon posttranslational modification, which enables membrane binding. In contrast to many conventional chemotherapeutics, farnesyltransferase inhibitors are remarkably specific and have been demonstrated to cause no gross systemic toxicity in animals. Some orally bioavailable inhibitors are presently being evaluated in phase II clinical trials. This review presents an overview on some inhibitors of the Ras signaling pathway, including their specificity and effectiveness in vivo. Because Ras signaling plays a crucial role in the pathogenesis of some hematologic malignancies, the potential therapeutic usefulness of these inhibitors is discussed.
Effect of active prenatal management on pregnancy outcome in sickle cell disease in an African setting
Authors: Rahimy Mohamed C. , Gangbo Annick , Adjou Roslyn , Deguenon Chantal , Goussanou Stephanie , Alihonou Eusebe
Published: October 2019

Sickle cell disease (SCD) is associated with an increased risk of medical complications during pregnancy. In sub-Saharan Africa, fetal and maternal mortality rates are particularly high. This study evaluated the effect of an active prenatal management program on pregnancy outcome in patients with SCD in an African setting. Pregnant women with SCD attending the National Teaching Hospital in Cotonou (The Republic of Benin, West Africa) were recruited before the 28th week of gestation. Management was based on providing information and education about SCD and improving nutritional status, malaria prevention, early detection of bacterial infections, and restricted use of blood transfusion. Maternal and fetal mortality rates and SCD-related morbidity were the principal variables assessed. One hundred and eight patients (42 SS and 66 SC) with 111 fetuses were included in the study. Thirteen fetal deaths (from 9 SS and 4 SC mothers) were recorded and 2 deaths of SC mothers. The maternal mortality rate of 1.8% was comparable with the overall maternal mortality rate for this maternity unit (1.2%). Few SCD-related events were recorded. Plasmodium falciparum malaria infection was the major cause of morbidity. Sixty-three patients (19 SS and 44 SC) successfully completed their pregnancy (58.3%) without requiring transfusion. Providing pregnant SCD patients with relevant medical care based on simple cost-effective approaches can have a positive impact on SCD-associated morbidity and mortality in an otherwise difficult setting in Africa.
Weekly polyethylene glycol conjugated L-asparaginase compared with biweekly dosing produces superior induction remission rates in childhood relapsed acute lymphoblastic leukemia: a pediatric oncology group study
Authors: Abshire Thomas C. , Pollock Brad H. , Billett Amy L. , Bradley Patricia , Buchanan George R.
Published: October 2019

The relapse rate in childhood acute lymphoblastic leukemia (ALL) is approximately 30% but few reinduction regimens have investigated the intensive use of polyethylene glycol Escherichia coliasparaginase (PEG-Asp). Therefore, we assessed the pharmocokinetics and efficacy of PEG-Asp in this setting. Children with B-precursor ALL, in first marrow and/or extramedullary relapse were eligible. Reinduction included doxorubicin on day 1, prednisone for 28 days, vincristine weekly for 4 weeks, and PEG-Asp either weekly or biweekly by randomization. Asparaginase levels and antibody to both E coli asparaginase and PEG-asp were measured weekly just before each PEG-asp dose. Overall, 129 of 144 patients (pts) (90%) achieved a complete remission (CR). There was a highly significant difference in CR rates between weekly (69 of 71; 97%) and biweekly (60 of 73; 82%) PEG-Asp dosing (P = .003). Grade 3 or 4 infectious toxicity was common (50%), but only 4 pts died of sepsis during induction. Other toxicities were infrequent and hypersensitivity was rare (6 of 144; 4%). Low asparaginase levels were associated with high antibody titers to either native (P = .024) or PEG asp (P = .0013). The CR rate was significantly associated with higher levels of asparaginase (P = .012). Patients with ALL in first relapse receiving weekly PEG-Asp had a higher rate of second remission compared with biweekly dosing. Low levels of asparaginase were associated with high antibody titers. Increased asparaginase levels may correlate with an improved CR rate. The use of intensive PEG-Asp should be explored further in the treatment of ALL.
Adiponectin, a new member of the family of soluble defense collagens, negatively regulates the growth of myelomonocytic progenitors and the functions of macrophages
Authors: Yokota Takafumi , Oritani Kenji , Takahashi Isao , Ishikawa Jun , Matsuyama Akifumi , Ouchi Noriyuki , Kihara Shinji , Funahashi Tohru , Tenner Andrea J. , Tomiyama Yoshiaki , Matsuzawa Yuji
Published: October 2019

We investigated the functions of adiponectin, an adipocyte-specific secretory protein and a new member of the family of soluble defense collagens, in hematopoiesis and immune responses. Adiponectin suppressed colony formation from colony-forming units (CFU)—granulocyte-macrophage, CFU-macrophage, and CFU-granulocyte, whereas it had no effect on that of burst-forming units—erythroid or mixed erythroid-myeloid CFU. In addition, adiponectin inhibited proliferation of 4 of 9 myeloid cell lines but did not suppress proliferation of erythroid or lymphoid cell lines except for one cell line. These results suggest that adiponectin predominantly inhibits proliferation of myelomonocytic lineage cells. At least one mechanism of the growth inhibition is induction of apoptosis because treatment of acute myelomonocytic leukemia lines with adiponectin induced the appearance of subdiploid peaks and oligonucleosomal DNA fragmentation. Aside from inhibiting growth of myelomonocytic progenitors, adiponectin suppressed mature macrophage functions. Treatment of cultured macrophages with adiponectin significantly inhibited their phagocytic activity and their lipopolysaccharide-induced production of tumor necrosis factor α. Suppression of phagocytosis by adiponectin is mediated by one of the complement C1q receptors, C1qRp, because this function was completely abrogated by the addition of an anti-C1qRp monoclonal antibody. These observations suggest that adiponectin is an important negative regulator in hematopoiesis and immune systems and raise the possibility that it may be involved in ending inflammatory responses through its inhibitory functions.