bluuebunny/crossref_metadata_2025 huggingface dataset:
T-Cell Depletion Plus Salvage Immunotherapy With Donor Leukocyte Infusions as a Strategy to Treat Chronic-Phase Chronic Myelogenous Leukemia Patients Undergoing HLA-Identical Sibling Marrow Transplantation
Authors: Drobyski William R. , Hessner Martin J. , Klein John P. , Kabler-Babbitt Claudia , Vesole David H. , Keever-Taylor Carolyn A.
Published: October 2019

T-cell depletion (TCD) of the donor marrow graft has been shown to reduce the severity of graft-versus-host disease (GVHD) in patients with chronic-phase (CP) chronic myelogenous leukemia (CML) undergoing HLA-identical sibling allogeneic marrow transplantation. However, there has been a corresponding reduction in the graft-versus-leukemia effect so that any decrease in GVHD-related mortality has been offset by an increased rate of disease relapse. Therapy of recurrent disease with donor leukocyte infusions (DLI) has been proven to be effective salvage therapy for the majority of patients who relapse after allogeneic BMT with CP CML. However, the overall impact of salvage DLI therapy on the survival of CP CML patients initially transplanted with TCD marrow grafts is not defined. To address this question, we have evaluated a clinical strategy of TCD followed by targeted adoptive immunotherapy with DLI in 25 CP CML patients undergoing allogeneic BMT from HLA-identical siblings. All patients received a standardized preparative regimen along with ex vivo TCD and posttransplant cyclosporine as GVHD prophylaxis. Durable engraftment was observed in all 25 patients. The incidence of grade II to IV acute GVHD was 8%. The cumulative incidence of transplant-related mortality (TRM) was 4%, and the 1-year probability of overall survival was 96%. The 3-year cumulative relapse incidence was 49%. All relapsed patients received DLI to reinduce remission. The total T-cell dose administered to these patients varied from 0.1 to 5.0 × 108 T cells/kg. Complete responses were observed in 12 of 14 patients, with 1 additional patient still too early to evaluate. Three patients died of GVHD after DLI, and 1 relapsed into blast crisis after a transient cytogenetic remission. Of the remaining 10 patients, 8 are in molecular remission, 1 is alive in relapse, and 1 is receiving DLI treatment. The median follow-up after infusion of surviving DLI patients in remission is 5.3 years. The probability of overall 5-year survival for the entire population is 80%, with a median follow-up of 6.4 years. We conclude that the clinical strategy of TCD followed by targeted adoptive immunotherapy with DLI for those patients with evidence of recurrent disease is a viable transplant strategy for CP CML, resulting in 80% survival and a low risk of acute GVHD and transplant-related mortality.
Developmental Expression of Plasminogen Activator Inhibitor-1 Associated With Thrombopoietin-Dependent Megakaryocytic Differentiation
Authors: Madoiwa Seiji , Komatsu Norio , Mimuro Jun , Kimura Kouzoh , Matsuda Michio , Sakata Yoichi
Published: October 2019

Plasminogen activator inhibitor-1 (PAI-1) is present in the platelet -granule and is released on activation. However, there is some debate as to whether the megakaryocyte and platelet synthesize PAI-1, take it up from plasma, or both. We examined the expression of PAI-1 in differentiating megakaryocytic progenitor cells (UT-7) and in CD34+/CD41− cells from cord blood. UT-7 cells differentiated with thrombopoietin (TPO) resembled megakaryocytes (UT-7/TPO) with respect to morphology, ploidy, and the expression of glycoprotein IIb-IIIa. PAI-1 messenger RNA (mRNA) expression was upregulated and PAI-1 protein synthesized in the UT-7/TPO cells accumulated in the cytoplasm without being released spontaneously. In contrast, erythropoietin (EPO)-stimulated UT-7 cells (UT-7/EPO) did not express PAI-1 mRNA after stimulation with TPO because they do not have endogenous c-Mpl. After cotransfection with human wild-typec-mpl, the cells (UT-7/EPO-MPL) responded to phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor- (TNF-), and interleukin-1β (IL-1β) with enhanced PAI-1 mRNA expression within 24 to 48 hours. However, induction of PAI-1 mRNA in UT-7/EPO-MPL cells by TPO required at least 14-days stimulation. UT-7/EPO cells expressing c-Mpl changed their morphology and the other characteristics similar to the UT-7/TPO cells. TPO also differentiated human cord blood CD34+/CD41− cells to CD34−/CD41+ cells, generated morphologically mature megakaryocytes, and induced the expression of PAI-1 mRNA. These results suggest that both PAI-1 mRNA and de novo PAI-1 protein synthesis is induced after differentiation of immature progenitor cells into megakaryocytes by TPO.
All-Trans Retinoic Acid Delays the Differentiation of Primitive Hematopoietic Precursors (lin−c-kit+Sca-1+) While Enhancing the Terminal Maturation of Committed Granulocyte/Monocyte Progenitors
Authors: Purton Louise E. , Bernstein Irwin D. , Collins Steven J.
Published: October 2019

All-trans retinoic acid (ATRA) is a potent inducer of terminal differentiation of malignant promyelocytes, but its effects on more primitive hematopoietic progenitors and stem cells are less clear. In this study, we investigated the effect of ATRA on highly enriched murine hematopoietic precursor cells (lin−c-kit+Sca-1+) grown in liquid suspension culture for 28 days. ATRA initially slowed the growth of these hematopoietic precursors but prolonged and markedly enhanced their colony-forming cell production compared with the hematopoietic precursors cultured in its absence. At 7 and 14 days of culture, a substantially greater percentage of cells cultured with ATRA did not express lineage-associated antigens (55.4% at day 7 and 68.6% at day 14) and retained expression of Sca-1 (44.7% at day 7 and 79.9% at day 14) compared with cells grown in its absence (lin−cells: 31.5% at day 7 and 4% at day 14; Sca-1+: 10.4% at day 7 and 0.7% at day 14). Moreover, a marked inhibition of granulocyte production was observed in cultures continuously incubated with ATRA. Significantly, ATRA markedly prolonged and enhanced the production of transplantable colony-forming unit-spleen (CFU-S) during 14 days of liquid suspension culture. In contrast with its effects on primitive lin−c-kit+Sca-1+hematopoietic precursors, ATRA did not exert the same effects on the more committed lin−c-kit+Sca-1−progenitor cells. Moreover, the late addition of ATRA (7 days post-culture initiation) to cultures of primitive hematopoietic precursors resulted in a marked decrease in colony-forming cell production in these cultures, which was associated with enhanced granulocyte differentiation. These observations indicate that ATRA has different effects on hematopoietic cells depending on their maturational state, preventing and/or delaying the differentiation of primitive hematopoietic precursors while enhancing the terminal differentiation of committed granulocyte/monocyte progenitors.
The Thrombocytopenia of Wiskott Aldrich Syndrome Is Not Related to a Defect in Proplatelet Formation
Authors: Haddad Elie , Cramer Elisabeth , Rivière Christel , Rameau Philippe , Louache Fawzia , Guichard Josette , Nelson David L. , Fischer Alain , Vainchenker William , Debili Najet
Published: October 2019

The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34+ cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1 (SDF-1), and actin normally polymerized after SDF-1 or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.
Constitutive HOXA5 Expression Inhibits Erythropoiesis and Increases Myelopoiesis From Human Hematopoietic Progenitors
Authors: Crooks Gay M. , Fuller John , Petersen Denise , Izadi Parvin , Malik Punam , Pattengale Paul K. , Kohn Donald B. , Gasson Judith C.
Published: October 2019

The role of the homeobox gene HOXA5 in normal human hematopoiesis was studied by constitutively expressing theHOXA5 cDNA in CD34+ and CD34+CD38− cells from bone marrow and cord blood. By using retroviral vectors that contained both HOXA5and a cell surface marker gene, pure populations of progenitors that expressed the transgene were obtained for analysis of differentiation patterns. Based on both immunophenotypic and morphological analysis of cultures from transduced CD34+ cells, HOXA5expression caused a significant shift toward myeloid differentiation and away from erythroid differentiation in comparison to CD34+ cells transduced with Control vectors (P= .001, n = 15 for immunophenotypic analysis; and P < .0001, n = 19 for morphological analysis). Transduction of more primitive progenitors (CD34+CD38− cells) resulted in a significantly greater effect on differentiation than did transduction of the largely committed CD34+ population (P = .006 for difference between HOXA5 effect on CD34+v CD34+CD38−cells). Erythroid progenitors (burst-forming unit-erythroid [BFU-E]) were significantly decreased in frequency among progenitors transduced with the HOXA5 vector (P = .016, n = 7), with no reduction in total CFU numbers. Clonal analysis of single cells transduced with HOXA5 or control vectors (cultured in erythroid culture conditions) showed that HOXA5expression prevented erythroid differentiation and produced clones with a preponderance of undifferentiated blasts. These studies show that constitutive expression of HOXA5 inhibits human erythropoiesis and promotes myelopoiesis. The reciprocal inhibition of erythropoiesis and promotion of myelopoiesis in the absence of any demonstrable effect on proliferation suggests that HOXA5 diverts differentiation at a mulitpotent progenitor stage away from the erythroid toward the myeloid pathway.
Interleukin-1 (IL-1) Inhibits Growth of Cytomegalovirus in Human Marrow Stromal Cells: Inhibition Is Reversed Upon Removal of IL-1
Authors: Iwata Mineo , Vieira Jeff , Byrne Michael , Horton Heidi , Torok-Storb Beverly
Published: October 2019

A Toledo strain cytomegalovirus (CMV) containing the gene for green fluorescent protein (GFP) under the control of elongation factor-1 promoter was used to study infection of human marrow stromal cells. Two stromal cell lines were used: HS-5, which secretes copious amounts of known cytokines and interleukins; and HS-27a, which does not secrete these activities. CMV growth and spread was monitored by counting green plaques and quantitating GFP intensity. Initial studies indicated that, whereas HS-5 and 27a have similar susceptibilities to infection, as evidenced by the same number of GFP+ cells at day 2, HS-5 appears more resistant to growth and spread of CMV. Furthermore, conditioned media from HS-5 (HS-5 CM) inhibited CMV plaque formation in HS-27a, suggesting that factors secreted by HS-5 are responsible for limiting CMV growth. Neutralizing antibodies against interleukin-1 (IL-1) and IL-1β completely blocked the ability of HS-5 CM to limit viral growth, suggesting that IL-1, which is known to be present in HS-5 CM, is responsible for this effect. When exogenous IL-1β was added to CMV-infected HS-27a, both the number of plaques and the intensity of GFP was significantly reduced in IL-1–treated HS-27a compared with untreated HS-27a (the number of plaques by day 18 was 20 ± 3 v 151 ± 12/well, respectively; GFP intensity was 535 ± 165 v 6,516 ± 652/well, respectively, in 4 separate experiments). At day 21, when IL-1β–treated, CMV-infected cultures were passaged and then cultured in the absence of IL-1β, CMV growth progressed with the kinetics of the original untreated culture, indicating that the IL-1β effect is reversible. Because HS-27a expresses the type I IL-1 receptor, we speculate that the antiviral effects are mediated through IL-1–induced changes in cellular gene expression. DNA chip analysis of mRNA from IL-1β–treated and nontreated HS-27a cells has identified some candidate molecules.
Regulation of Tissue Factor Pathway Inhibitor Expression in Smooth Muscle Cells
Authors: Pendurthi Usha R. , Rao L. Vijaya Mohan , Williams J. Todd , Idell Steven
Published: October 2019

Tissue factor pathway inhibitor (TFPI) is the primary physiological inhibitor that regulates tissue factor-induced blood coagulation. TFPI is thought to be synthesized, in vivo, primarily by microvascular endothelial cells. Little is known about how TFPI is regulated under pathophysiological conditions. In this study, we determined mechanisms by which TFPI expression is regulated by human pulmonary artery smooth muscle cells (PASMC), because these cells contribute to remodeling of the pulmonary vasculature in disease. PASMC in culture constitutively synthesize and secrete TFPI. Exposure of PASMC to phorbol myristate acetate, lipopolysaccharide, tumor necrosis factor , thrombin, interleukin-1, and transforming growth factor-β had no significant effect on expression of TFPI by PASMC. By contrast, treatment of PASMC with serum and basic fibroblast growth factor (bFGF)/heparin markedly upregulated the expression of TFPI activity and antigen. On Western blot analysis, a protein consistent with full-length TFPI (42 kD) was identified in the conditioned media of PASMC, and the levels of the protein were much higher in the conditioned media of serum and bFGF/heparin-treated cells. Northern blot analysis showed that PASMC constitutively express TFPI mRNA, and treatment of cells with serum and bFGF/heparin had a minimal effect on the steady-state levels of TFPI mRNA. Nuclear run-on analysis did not show a significant increase in the transcriptional rate of TFPI gene in PASMC treated with serum or bFGF/heparin. Cycloheximide, but not actinomycin-D, treatment inhibited the serum and bFGF/heparin-induced increase in TFPI activity in PASMC. In conclusion, our data demonstrate that PASMC constitutively synthesize and secrete TFPI and serum or bFGF upregulate its expression, suggesting that growth factors that can stimulate the vessel wall in vivo might locally regulate TFPI expression. Our study also suggests that control of TFPI expression by serum or bFGF occurs via translational rather than transcriptional regulation.
Increased Fragmentation of von Willebrand Factor, Due to Abnormal Cleavage of the Subunit, Parallels Disease Activity in Recurrent Hemolytic Uremic Syndrome and Thrombotic Thrombocytopenic Purpura and Discloses Predisposition in Families
Authors: Galbusera Miriam , Noris Marina , Rossi Chiara , Orisio Silvia , Caprioli Jessica , Ruggeri Zaverio M. , Amadei Barbara , Ruggenenti Piero , Vasile Beatrice , Casari Giorgio , Remuzzi Giuseppe
Published: October 2019

We investigated here the changes in von Willebrand factor (vWF) multimers in recurrent, sporadic and familial forms of hemolytic uremic syndrome (HUS)/thrombotic thrombocytopenic purpura (TTP) to see whether they are actually proteolyzed in vivo in these patients. Molecular determinants of fragments in vWF were also characterized to identify possible sites of cleavage of the subunit. Unusually large vWF multimers were found in blood of 8 of 10 patients with recurrent HUS/TTP, both in the acute phase and in remission, but never in familial and sporadic cases. Instead, all of the groups showed evidence of enhanced fragmentation of vWF multimers during the acute phase. Increased fragmentation was also shown by decrease in native 225-kD vWF subunit. In recurrent and sporadic HUS/TTP, enhanced fragmentation normalized at remission, but the abnormality persisted in familial HUS/TTP patients. The latter findings suggest that patients with familial HUS/TTP may have a congenital abnormality in vWF processing. Analysis with specific monoclonal antibodies showed the presence of the normal vWF fragments with apparent molecular mass of 189, 176, and 140 kD in all patients; however, in 6 recurrent and in 5 familial cases, novel fragments that differed in size from normal ones were found. The size of these abnormal fragments differed from one patient to another and none of them was ever found in normal plasma. These results documented, for the first time in HUS/TTP, an abnormal cleavage of the vWF subunit that might account for the increased fragmentation observed in these patients.
Acute Systemic Reaction and Lung Alterations Induced by an Antiplatelet Integrin gpIIb/IIIa Antibody in Mice
Authors: Nieswandt Bernhard , Echtenacher Bernd , Wachs Frank-Peter , Schröder Josef , Gessner J. Engelbert , Schmidt Reinhold E. , Grau Georges E. , Männel Daniela N.
Published: October 2019

Shock is frequently accompanied by thrombocytopenia. To investigate the pathogenic role of platelets in shock, we examined the in vivo effects of monoclonal antibodies (MoAbs) against mouse platelet membrane proteins. Injection of the platelet-specific MoAb MWReg30 to the fibrinogen receptor (gpIIb/IIIa) rendered mice severely hypothermic within minutes. Isotype-matched control antibodies, even if they also recognized platelet surface antigens, did not induce comparable signs. MWReg30 induced early signs of acute lung injury with increased cellularity in the lung interstitium and rapid engorgement of alveolar septal vessels. Despite this in vivo activity, MWReg30 inhibited rather than stimulated platelet aggregation in vitro. MWReg30-binding to platelets led to phosphorylation of gpIIIa, but did not induce morphological signs of platelet activation. The MWReg30-induced reaction was abolished after treatment with MoAbs 2.4G2 to FcγRII/III and was absent in FcγRIII-deficient mice, clearly demonstrating the requirement for FcγRIII on involved leukocytes. Simultaneous administration of tumor necrosis factor exacerbated, whereas a tolerizing regimen of tumor necrosis factor or bacterial lipopolysaccharide completely prevented the reaction. These data suggest that platelet surface-deposited MWReg30-immune complexes lead to an acute Fc-mediated reaction with pulmonary congestion and life-threatening potential that could serve as an in vivo model of acute lung injury.
Reactive Plasmacytoses Are Expansions of Plasmablasts Retaining the Capacity to Differentiate Into Plasma Cells
Authors: Jego Gaëtan , Robillard Nelly , Puthier Denis , Amiot Martine , Accard Françoise , Pineau Danielle , Harousseau Jean-Luc , Bataille Régis , Pellat-Deceunynck Catherine
Published: October 2019

Circulating plasma cells in 10 cases of reactive plasmacytosis had a shared phenotype with early plasma cell (CD19+CD38+ CD138+ CD40+CD45+ CD11a+ CD49e−CD56−). In most cases, a minor subpopulation of CD28+ plasma cells was also detected. Reactive plasma cells were highly proliferative, suggesting the presence of circulating progenitors (plasmablasts). After CD138+ plasma cell removal, highly proliferative CD138− plasmablasts differentiated into CD138+ plasma cells within a few days. This differentiation, which was associated with increased CD38 and decreased HLA-DR expression, was further confirmed by a large increase in intracellular Ig content (associated with Ig secretion) and was concomitant with extensive secretion of interleukin-6 (IL-6). The addition of neutralizing anti–IL-6 and anti-CD126 (IL-6 receptor) monoclonal antibodies totally prevented Ig secretion and cell differentiation by inducing apoptosis of plasmablasts, which indicates that IL-6 is an essential survival factor for plasmablasts. This report provides the first characterization of normal plasmablasts and shows that their phenotype is not exactly that of multiple myeloma cells.
The FcγRIa (CD64) Ligand Binding Chain Triggers Major Histocompatibility Complex Class II Antigen Presentation Independently of Its Associated FcR γ-Chain
Authors: van Vugt Martine J. , Kleijmeer Monique J. , Keler Tibor , Zeelenberg Ingrid , van Dijk Marc A. , Leusen Jeanette H.W. , Geuze Hans J. , van de Winkel Jan G.J.
Published: October 2019

Within multi-subunit Ig receptors, the FcR γ-chain immunoreceptor tyrosine-based activation motif (ITAM) plays a crucial role in enabling antigen presentation. This process involves antigen-capture and targeting to specific degradation and major histocompatibility complex (MHC) class II loading compartments. Antigenic epitopes are then presented by MHC class II molecules to specific T cells. The high-affinity receptor for IgG, hFcγRIa, is exclusively expressed on myeloid lineage cells and depends on the FcR γ-chain for surface expression, efficient ligand binding, and most phagocytic effector functions. However, we show in this report, using the IIA1.6 cell model, that hFcγRIa can potentiate MHC class II antigen presentation, independently of a functional FcR γ-chain ITAM. Immunoelectron microscopic analyses documented hFcγRIa -chain/rabbit IgG-Ovalbumin complexes to be internalized and to migrate via sorting endosomes to MHC class II-containing late endosomes. Radical deletion of the hFcγRIa -chain cytoplasmic tail did not affect internalization of rabbit IgG-Ovalbumin complexes. Importantly, however, this resulted in diversion of receptor-ligand complexes to the recycling pathway and decreased antigen presentation. These results show the hFcγRIa cytoplasmic tail to contain autonomous targeting information for intracellular trafficking of receptor-antigen complexes, although deficient in canonical tyrosine- or dileucine-targeting motifs. This is the first documentation of autonomous targeting by a member of the multichain FcR family that may critically impact the immunoregulatory role proposed for hFcγRIa (CD64).
A Synthetic Peptide Derived From Human Immunodeficiency Virus Type 1 gp120 Downregulates the Expression and Function of Chemokine Receptors CCR5 and CXCR4 in Monocytes by Activating the 7-Transmembrane G-Protein–Coupled Receptor FPRL1/LXA4R
Authors: Deng Xiyun , Ueda Hirotsugu , Su Shao Bo , Gong Wanghua , Dunlop Nancy M. , Gao Ji-Liang , Murphy Philip M. , Wang Ji Ming
Published: October 2019

Because envelope gp120 of various strains of human immunodeficiency virus type 1 (HIV-1) downregulates the expression and function of a variety of chemoattractant receptors through a process of heterologous desensitization, we investigated whether epitopes derived from gp120 could mimic the effect. A synthetic peptide domain, designated F peptide, corresponding to amino acid residues 414-434 in the V4-C4 region of gp120 of the HIV-1 Bru strain, potently reduced monocyte binding and chemotaxis response to macrophage inflammatory protein 1β (MIP-1β) and stromal cell-derived factor 1 (SDF-1), chemokines that use the receptors CCR5 and CXCR4, respectively. Further study showed that F peptide by itself is an inducer of chemotaxis and calcium mobilization in human monocytes and neutrophils. In cross-desensitization experiments, among the numerous chemoattractants tested, only the bacterial chemotactic peptide fMLF, when used at high concentrations, partially attenuated calcium mobilization induced by F peptide in phagocytes, suggesting that this peptide domain might share a 7-transmembrane, G-protein–coupled receptor with fMLF. By using cells transfected with cDNAs encoding receptors that interact with fMLF, we found that F peptide uses an fMLF receptor variant, FPRL1, as a functional receptor. The activation of monocytes by F peptide resulted in downregulation of the cell surface expression of CCR5 and CXCR4 in a protein kinase C-dependent manner. These results demonstrate that activation of FPRL1 on human moncytes by a peptide domain derived from HIV-1 gp120 could lead to desensitization of cell response to other chemoattractants. This may explain, at least in part, the initial activation of innate immune responses in HIV-1–infected patients followed by immune suppression.
Low Levels of Hepatitis C Virus RNA in Serum, Plasma, and Peripheral Blood Mononuclear Cells of Injecting Drug Users During Long Antibody-Undetectable Periods Before Seroconversion
Authors: Beld Marcel , Penning Maarten , van Putten Marieke , van den Hoek Anneke , Damen Marjolein , Klein Michèl R. , Goudsmit Jaap
Published: October 2019

Screening of antibodies to hepatitis C virus (HCV) is widely used for monitoring the prevalence of HCV infections and to assess HCV infectivity. Among HCV-infected individuals in the general population, the interval between the detection of HCV RNA and the development of HCV antibodies is usually 5 to 6 weeks, but in rare cases, seroconversion may be prolonged up to 6 to 9 months. In this study, we tested for the presence of HCV RNA during the antibody-undetectable period of 19 drug-injecting HCV seroconverters to gain insight into the antibody-negative carrier status in this population. HCV seroconversion status was determined by testing the first and last serum samples obtained from each subject, using third-generation antibody screening and confirmation assays. Serial samples were tested for HCV-specific antibodies to establish the moment of seroconversion and HCV RNA by single reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA assay (bDNA) in serum. Plasma and peripheral blood mononuclear cells (PBMCs) were independently collected and tested for HCV RNA. HCV RNA-positivity was confirmed by Southern blot hybridization and sequencing of serial samples. The 19 HCV seroconverters had a mean follow-up of 5 years (range, 1 to 8 years). Of the 19, 4 were human immunodeficiency virus (HIV)-infected before HCV seroconversion. HCV RNA was detected in serum before seroconversion in 12 (63.2%) of the 19 HCV seroconverters, independent of HIV status. In 7 of these 12, the antibody-undetectable period was relatively short (2 to 10 months). The other 5, who were all HIV-negative before HCV seroconversion, had intermittent low levels of HCV RNA before seroconversion for a period of more than 12 months, with a mean of 40.8 months (range, 13 to 94 months). In all 5 individuals, independent repeats of the experiments confirmed the presence of HCV RNA in serum, and in 3 of these individuals, HCV-positivity was confirmed in independently collected plasma and PBMC samples. Low levels of HCV RNA may be present during prolonged antibody-undetectable periods before seroconversion in a number of injecting drug users. Independent of HIV status, their immune system appears to be unable to respond to these low HCV RNA levels and was sometimes only activated after reinfections with distinct HCV genotypes. These results indicate that primary HCV infection may not always elicit the rapid emergence of HCV antibodies and suggests that persistent low levels of HCV RNA (regardless of the genotype) may not elicit at all or delay antibody responses for prolonged periods of time.
A Critical Role for PU.1 in Homing and Long-Term Engraftment by Hematopoietic Stem Cells in the Bone Marrow
Authors: Fisher Robert C. , Lovelock Joshua D. , Scott Edward W.
Published: October 2019

We have previously demonstrated that PU.1 is required for the production of lymphoid and myeloid, but not of erythroid progenitors in the fetal liver. In this study, competitive reconstitution assays show that E14.5 PU.1−/− hematopoietic progenitors (HPC) fail to sustain definitive/adult erythropoiesis or to contribute to the lymphoid and myeloid lineages. PU.1−/−HPC are unable to respond synergistically to erythropoietin plus stem cell factor and have reduced expression of c-kit, which may explain the erythroid defect. Fluorescently labeled,PU.1−/−, AA4.1+, fetal liver HPC were transferred into irradiated recipients, where they demonstrated a severely impaired ability to home to and colonize the bone marrow.PU.1−/− HPC were found to lack integrins 4 (VLA-4/CD49d), 5 (VLA-5/CD49e), and CD11b (M). Collectively, this study has shown that PU.1 plays an important role in controlling migration of hematopoietic progenitors to the bone marrow and the establishment of long-term multilineage hematopoiesis.
Multimerin Processing by Cells With and Without Pathways for Regulated Protein Secretion
Authors: Hayward Catherine P.M. , Song Zhili , Zheng Shilun , Fung Roxanna , Pai Menaka , Massé Jean-Marc , Cramer Elisabeth M.
Published: October 2019

Multimerin is a massive, soluble, homomultimeric, factor V-binding protein found in platelet -granules and in vascular endothelium. Unlike platelets, endothelial cells contain multimerin within granules that lack the secretory granule membrane protein P-selectin, and in culture, they constitutively secrete most of their synthesized multimerin. To further evaluate multimerin’s posttranslational processing and storage, we expressed human endothelial cell prepromultimerin in a variety of cell lines, with and without pathways for regulated secretion. The recombinant multimerin produced by these different cells showed variations in its glycosylation, proteolytic processing, and multimer profile, and human embryonic kidney 293 cells recapitulated multimerin’s normal processing for constitutive secretion by human endothelial cells. When multimerin was expressed in a neuroendocrine cell line capable of regulated protein secretion, it was efficiently targeted for regulated secretion. However, the multimerin stored in these cells was proteolyzed more extensively than normally occurs in platelets, suggesting that endoproteases similar to those expressed by megakaryocytes are required to produce platelet-type multimerin. The impact of the tissue-specific differences in multimerin’s posttranslational processing on its functions is not yet known. Multimerin’s sorting and targeting for regulated secretion may be important for its functions and its association with factor V in secretion granules.
Depsipeptide (FR901228): A Novel Therapeutic Agent With Selective, In Vitro Activity Against Human B-Cell Chronic Lymphocytic Leukemia Cells
Authors: Byrd John C. , Shinn Charlotte , Ravi Rajani , Willis Carl R. , Waselenko Jamie K. , Flinn Ian W. , Dawson Nancy A. , Grever Michael R.
Published: October 2019

Therapy of B-cell chronic lymphocytic leukemia (CLL) has been limited by both the nonselectivity of therapeutic agents toward normal residual immune cells and inherent drug resistance. Identification of agents that spare normal immune effector cells, thus facilitating addition of immune-based therapies, and that modulate factors associated with drug resistance in CLL might represent a major therapeutic advance. Depsipeptide (FR901228) is a novel agent entering clinical trials that has selective in vitro activity against resistant leukemia cell lines. To assess its in vitro activity in CLL, we exposed peripheral mononuclear cells from CLL patients (n = 10) to varying concentrations of this agent. Viability of the CLL cells was reduced by 50% (LC50) at 4 hours, 24 hours, and 4 days at depsipeptide concentrations of 0.038, 0.024, and 0.015 μmol/L, respectively. Depsipeptide had marked selective cytotoxicity when compared with normal blood mononuclear cells, in which the LC50 was 3.44 μmol/L at 4 hours (P = .03), 0.965 μmol/L at 24 hours (P = .01), and 0.0318 μmol/L at 96 hours (P = .04). Inhibition of bone marrow progenitor cell growth was also minimal after incubation with 0.015 μmol/L (19% inhibition of colony forming unit-granulocyte-macrophage [CFU-GM]; 17% inhibition burst forming unit-erythroid [BFU-E]) and 3.44 μmol/L (24% inhibition of CFU-GM; 57% inhibition BFU-E) of depsipeptide for 4 hours, followed by a 14-day incubation period. Expression of apoptotic proteins after depsipeptide exposure (0.015 μmol/L) included no change in bcl-2, elevation of bax, and decreased expression of p27. These data demonstrate that depsipeptide has significant selective in vitro activity against human CLL cells concurrent with favorable alterations of the bcl-2:bax protein ratio and decrease in p27 expression. Such findings strongly support the early introduction of depsipeptide into clinical trials for patients with CLL.
Nested Polymerase Chain Reaction With Sequence-Specific Primers Typing for HLA-A, -B, and -C Alleles: Detection of Microchimerism in DR-Matched Individuals
Authors: Carter Anthony S. , Cerundolo Lucia , Bunce Mike , Koo Dicken D.H. , Welsh Kenneth I. , Morris Peter J. , Fuggle Susan V.
Published: October 2019

It is widely accepted that donor leukocytes survive within the recipient periphery after blood transfusion or solid organ transplantation. The significance of this microchimerism remains unclear, partially because of the insecurity of assays used to detect the donor-derived material. The techniques used to detect donor-derived DNA within recipient peripheral blood rely largely on major histocompatibility complex class II polymorphism. We and others have shown that the sensitivity of polymerase chain reaction with sequence-specific primers (PCR-SSP) typing for HLA class II alleles can be increased 100-fold by the addition of a primary amplification step (nested PCR-SSP). We have now extended this technique to encompass typing for HLA class I alleles, thereby adding flexibility to microchimerism testing by enabling testing of recipients HLA-DR matched with their donors. However, the high level of sensitivity achieved with the technique (1:100,000) leads to a concomitant decrease in the specificity that results in the amplification of unexpected products, a phenomenon we encountered in the development of our nested PCR-SSP typing system for HLA class II alleles. We describe here how it is possible to compensate for these anomalies by including multiple testing of a pretransfusion sample that acts as a specificity control, establishing a rigorous baseline for subsequent analysis.
Macrophage-Derived Chemokine Is Localized to Thymic Medullary Epithelial Cells and Is a Chemoattractant for CD3+, CD4+, CD8low Thymocytes
Authors: Chantry David , Romagnani Paola , Raport Carol J. , Wood Christi L. , Epp Angela , Romagnani Sergio , Gray Patrick W.
Published: October 2019

Macrophage-derived chemokine (MDC) is a recently identified CC chemokine that is a potent chemoattractant for dendritic cells, natural killer (NK) cells, and the Th2 subset of peripheral blood T cells. In normal tissues, MDC mRNA is expressed principally in the thymus. Immunohistochemical analysis performed on 5 human postnatal thymuses showed high MDC immunoreactivity, which was selectively localized to epithelial cells within the medulla. To examine the effects of MDC on immature T cells, we have identified cDNA clones for mouse and rat MDC. Expression of MDC in murine tissues is also highly restricted, with significant levels of mRNA found only in the thymus. Thymocytes express high-affinity binding sites for MDC (kd = 0.7 nmol/L), and, in vitro, MDC is a chemoattractant for these cells. MDC-responsive murine thymocytes express mRNA for CCR4, a recently identified receptor for MDC. Phenotypic analysis of MDC-responsive cells shows that they are enriched for a subset of double-positive cells that express high levels of CD3 and CD4 and that have reduced levels of CD8. This subset of MDC-responsive cells is consistent with the observed expression of MDC within the medulla, because more mature cells are found there. MDC may therefore play a role in the migration of T-cell subsets during development within the thymus.
Reconstitution of Early Lymphoid Proliferation and Immune Function in Jak3-Deficient Mice by Interleukin-3
Authors: Brown Michael P. , Nosaka Tetsuya , Tripp Ralph A. , Brooks James , van Deursen Jan M.A. , Brenner Malcolm K. , Doherty Peter C. , Ihle James N.
Published: October 2019

Expansion of early lymphoid progenitors requires interleukin-7 (IL-7), which functions through γc-mediated receptor activation of Jak3. Jak3 deficiency is a cause of severe combined immunodeficiency (SCID) in humans and mice. IL-3 activates many of the same signaling pathways as IL-7, such as Stat5, but achieves this effect through the activation of Jak2 rather than Jak3. We hypothesized that expansion of an IL-7–responsive precursor population through a Jak3-independent pathway using IL-3 may stimulate early lymphoid progenitors and restore lymphopoiesis in Jak3−/− mice. Newborn Jak3−/− mice that were injected with IL-3 demonstrated thymic enlargement, a 2- to 20-fold increase in thymocyte numbers, and up to a 10-fold expansion in the number of CD4+, CD8+, and B220+/IgM+ splenic lymphocytes, consistent with an effect upon an early lymphoid progenitor population. In contrast to control mice, IL-3–treated Jak3−/− mice challenged with the allogeneic major histocompatibility complex (MHC) class I-bearing tumor P815 developed a specific CD8-dependent cytotoxic T lymphocyte (CTL) response. IL-3–treated mice also mounted influenza-specific CTL responses and survival was prolonged. The beneficial effects of IL-3 are proposed to be produced by stimulation of a lymphoid precursor population of IL-7R+/IL-3R+ cells that we identified in wild-type bone marrow. In vitro, we show that an early IL-7R+ lymphoid progenitor population expresses IL-3R and proliferates in response to IL-3 and that IL-3 activates Stat5 comparably to IL-7. Clinically, IL-3 may therefore be useful treatment for X-linked and Jak3-deficient SCID patients who lack bone marrow donors.
Osteoclast-Derived Zinc Finger (OCZF) Protein With POZ Domain, a Possible Transcriptional Repressor, Is Involved in Osteoclastogenesis
Authors: Kukita Akiko , Kukita Toshio , Ouchida Mamoru , Maeda Hidefumi , Yatsuki Hitomi , Kohashi Osamu
Published: October 2019

The differentiation of osteoclasts is regulated by transcription factors expressed in cells of osteoclast lineage. We isolated here a potential transcription factor from a cDNA library of an enriched population of preosteoclasts and osteoclasts. The cDNA encodes a protein with N-terminal POZ domain and C-terminalKrüppel-like zinc fingers. We designate this protein as osteoclast-derived zinc finger (OCZF). OCZF was found to be rat homologue of mouse leukemia/lymphoma-related factor (LRF). Northern blot and in situ hybridization analysis showed OCZF mRNA at a high level in osteoclasts and kidney cells. OCZF had a nuclear targeting sequence and was localized in the nucleus of transfected cells. In addition, OCZF specifically bound to the guanine-rich consensus sequences of Egr-1 and c-Krox. Transient transfection assays indicate that OCZF can repress transcription activity like other POZ domain proteins. Furthermore, antisense but not sense phosphorothioate oligodeoxynucleotides (ODNs) for OCZF cDNA suppressed the formation of osteoclast-like multinucleated cells (MNCs) in bone marrow culture, whereas the same ODNs did not significantly affect the formation of macrophage polykaryons and mononuclear preosteoclast-like cells (POCs). These results suggest that OCZF is a unique transcription factor that plays an important role in the late stage of osteoclastogenesis.
Cultured Endothelial Cells From Human Arteriovenous Malformations Have Defective Growth Regulation
Authors: Wautier Marie-Paule , Boval Bernadette , Chappey Olivier , Enjolras Odile , Wernert Nicolas , Merland Jean-Jacques , Wautier Jean-Luc
Published: October 2019

Vascular malformations are frequent in newborns, and they persist throughout life, which differentiates them from vascular tumors (eg, hemangiomas). Arteriovenous malformations are high-flow vascular malformations. They are considered nonmalignant but can expand and become a significant clinical risk when extensive. To characterize endothelial cells from arteriovenous malformations (AMEC), we cultured cells obtained from surgical specimens and studied their properties. After selection, the cells that grew out from explants had phenotypic and antigenic features (platelet endothelial cell adhesion molecule, von Willebrand factor) of human endothelial cells. Their spontaneous proliferation rate was higher (1.8 to 6.4 times) than that of human umbilical vein, arterial, or microvascular endothelial cells. The proliferation rate of AMEC was not sensitive to the inhibitory activity of various cytokines (interleukin-1β, tumor necrosis factor-, transforming growth factor-β, Interferon-γ). In basal conditions, intercellular adhesion molecule (ICAM-1) was detected at a higher level of expression (6- to 10-fold) on AMEC, but these cells failed to express E-selectin or the vascular cell adhesion molecule (VCAM-1) after cytokine stimulation. Expression of c-ets-1 proto-oncogene was shown by in situ hybridization. The low response to cytokines, the higher propensity to proliferate, and the ets-1 expression suggest that AMEC have a defective regulation of proliferation that may be due to a reduced apoptotic process.
Hodgkin/Reed-Sternberg Cells Induce Fibroblasts to Secrete Eotaxin, a Potent Chemoattractant for T Cells and Eosinophils
Authors: Jundt Franziska , Anagnostopoulos Ioannis , Bommert Kurt , Emmerich Florian , Müller Gerd , Foss Hans-Dieter , Royer Hans-Dieter , Stein Harald , Dörken Bernd
Published: October 2019

Hodgkin’s disease is histopathologically characterized by the relative scarcity of neoplastic Hodgkin and Reed-Sternberg cells and for yet unknown reasons by an abundant reactive background of T lymphocytes and often eosinophils. Eotaxin is a CC-chemokine attracting eosinophils and T helper 2 (Th2) cells in allergic inflammation. We now report that eotaxin is strongly expressed in fibroblasts of Hodgkin’s disease tissues, whereas Hodgkin/Reed-Sternberg cells do not express this chemokine. In tissue culture, Hodgkin’s disease tumor cells induce eotaxin expression in cocultured dermal fibroblasts in a concentration leading to a specific chemotactic response of a Th2 cell clone. Production of tumor necrosis factor- (TNF-) by Hodgkin/Reed-Sternberg cells appears to be responsible for this induction, because blocking of TNF- by neutralizing antibodies prevented fibroblast eotaxin expression. Our data suggest that eotaxin is involved in the pathobiology of Hodgkin’s disease by contributing to eosinophil and T-lymphocyte recruitment.
Role of Src in the Modulation of Multiple Adaptor Proteins in FcRI Oxidant Signaling
Authors: Park Rae-Kil , Izadi Kayvon D. , Deo Yashwant M. , Durden Donald L.
Published: October 2019

Cross-linking of Fc receptors for IgA, FcR (CD89), on monocytes/macrophages is known to enhance phagocytic activity and generation of oxygen free radicals. We provide evidence here that the FcR signals through the γ subunit of FcɛRI in U937 cells differentiated with interferon γ (IFNγ). Our results provide the first evidence that FcR-mediated signals modulate a multimolecular adaptor protein complex containing Grb2, Shc, SHIP, CrkL, Cbl, and SLP-76. Cross-linking of FcRI using anti-FcRI induces the phosphorylation of the γ subunit as detected by mobility retardation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Stimulation of FcRI induced the tyrosine phosphorylation of Shc and increased the association of Grb2 with Shc and CrkL. Grb2 associates constitutively with Sos, and the latter undergoes mobility shift upon FcRI stimulation. The complex adapter proteins, Cbl and SLP-76, are physically associated in myeloid cells and both proteins undergo tyrosine phosphorylation upon FcR stimulation. These data indicate that the stimulation of FcR results in the modulation of adaptor complexes containing tyrosine-phosphorylated Cbl, Shc, SHIP, Grb2, and Crkl. Experiments performed with the Src kinase inhibitor, PP1, provide the first evidence that Src kinase activation is required for FcRI-induced production of superoxide anions and provide insight into the mechanism for FcR-mediated activation of downstream oxidant signaling in myeloid cells.
Increased Fetal and Extramedullary Hematopoiesis in Fas-Deficient C57BL/6-lpr/lpr Mice
Authors: Schneider Elke , Moreau Géraldine , Arnould Anne , Vasseur Florence , Khodabaccus Naushad , Dy Michel , Ezine Sophie
Published: October 2019

In this study, we examined the consequences of Fas deficiency on hematopoiesis in C57BL/6-lpr/lpr mice. We found a striking extramedullary increase in hematopoietic progenitor cells, comprising erythroid and nonerythroid lineages alike. These modifications preceded the lymphadenopathy, because early progenitors (colony-forming units-spleen [CFU-S] day 8) were already augmented in day-18 fetal livers of the lpr phenotype. Three weeks after birth, CFU-S increased in peripheral blood and spleen and colony-forming cells (CFU-C) began to accumulate 1 to 3 weeks later. Extramedullary myelopoiesis augmented progressively in Fas-deficient mice, reaching a maximum within 6 months. By then, mature and immature myeloid cells had infiltrated the spleen, the liver, and the peritoneal cavity. Similar changes occurred in C57BL/6-gld/gld mice, indicating that they resulted from Fas/FasL interactions. Medullary hematopoiesis was not significantly modified in adult mice of either strain. Yet, the incidence of CFU-S decreased after Fas cross-linking on normal bone marrow cells in the presence of interferon γ, consistent with a regulatory function of Fas/FasL interactions in early progenitor cell development. These data provide evidence that Fas deficiency can affect hematopoiesis both during adult and fetal life and that these modifications occur independently from other pathologies associated with the lpr phenotype.