bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells
Authors: Zain Jasmine , Huang Yao-Qi , Feng XueSheng , Nierodzik Mary Lynn , Li Jian-Jun , Karpatkin Simon
Published: October 2019

Because thrombin-treated tumor cell-induced metastasis increases tumor nodule volume12 greater than nodule number, we studied the effect of thrombin on tumor cell growth in vitro and in vivo (murine B16F10 melanoma, human HCT8 colon carcinoma, DU145 prostate carcinoma). Tumor cell growth was measured after 3 to 7 days in 1% fetal calf serum (FCS) + RPMI 1640. We found that, whereas relatively low concentrations of thrombin, 0.1 to 0.5 U/mL (1-5 nmol/L) enhance tumor cell growth in vitro approximately 2- to 3-fold, higher concentrations, 0.5 to 1 U/mL (5-10 nmol/L) impaired cell growth approximately 2- to 4-fold. Impaired cell growth was associated with cell cycle arrest at G2M and increased pre-GoDNA, as well as apoptosis, measured by tumor cell binding to Annexin V and propidium iodide. Apoptosis was reversed with the general caspase inhibitor, FK-011. The enhancing and inhibiting effects were specific for thrombin (reversed with inactive diisopropyl-fluorophosphate [DFP]-thrombin) and mediated via the protease-activated receptor 1 (PAR-1). PAR-1 activation was demonstrated by (1) use of a cell line, B16F10, devoid of the 3 other thrombin receptors, PAR-3, PAR-4, and GPIb; and (2) greater sensitivity of PAR-1 transfected B16F10 and HCT8 cells to impaired cell growth/apoptosis, 3- and 14-fold, respectively. Thus, thrombin has a bimodal effect on PAR-1 in tumor cells: enhanced growth at low concentration, impaired growth/apoptosis at higher concentration.
Interleukin-13 induces PSGL-1/P–selectin–dependent adhesion of eosinophils, but not neutrophils, to human umbilical vein endothelial cells under flow
Authors: Woltmann Gerrit , McNulty Claire A. , Dewson Grant , Symon Fiona A. , Wardlaw Andrew J.
Published: October 2019

Selective eosinophil accumulation is a hallmark of diseases such as asthma. In a model of chronic eosinophilic inflammation, we have previously shown that the tethering step in eosinophil adhesion is mediated by PSGL-1 binding to P-selectin. The Th2-associated cytokine IL-13 is of potential importance in allergic disease. We have therefore investigated whether IL-13 can mediate eosinophil binding to human umbilical vein endothelial cells (HUVEC) through P-selectin. IL-13 caused dose- and time-dependent increases of P-selectin expression, as assessed by flow and laser scanning cytometry. A similar degree of expression was observed with IL-4. There was no effect on E-selectin or ICAM-1 expression. Tumor necrosis factor- induced the expression of VCAM-1, E-selectin, and ICAM-1 but had no effect on P-selectin expression. IL-13 increased the production of mRNA for surface and soluble variants of P-selectin. Under flow conditions, eosinophils, but not neutrophils, showed enhanced binding to IL-13 and to IL-4–stimulated HUVEC compared to medium-cultured cells. Eosinophil adhesion was completely inhibited by a blocking monoclonal antibody against PSGL-1 and P-selectin. Anti–VLA-4 and anti–VCAM-1 antibodies inhibited binding to a lesser extent. Thus, at physiologic levels of expression induced by Th2 cytokines, P-selectin/PSGL-1 supported eosinophil but not neutrophil adhesion. This mechanism is likely to be a key event leading to the selective accumulation of eosinophils in allergic inflammation.
Alteration of tumor necrosis factor–α T-cell homeostasis following potent antiretroviral therapy: contribution to the development of human immunodeficiency virus–associated lipodystrophy syndrome
Authors: Ledru Eric , Christeff Névéna , Patey Olivier , de Truchis Pierre , Melchior Jean-Claude , Gougeon Marie-Lise
Published: October 2019

Highly-active antiretroviral therapy (HAART) has lead to a dramatic decrease in the morbidity of patients infected with the human immunodeficiency virus (HIV). However, metabolic side effects, including lipodystrophy-associated (LD-associated) dyslipidemia, have been reported in patients treated with antiretroviral therapy. This study was designed to determine whether successful HAART was responsible for a dysregulation in the homeostasis of tumor necrosis factor- (TNF-), a cytokine involved in lipid metabolism. Cytokine production was assessed at the single cell level by flow cytometry after a short-term stimulation of peripheral blood T cells from HIV-infected (HIV+) patients who were followed during 18 months of HAART. A dramatic polarization to TNF- synthesis of both CD4 and CD8 T cells was observed in all patients. Because it was previously shown that TNF- synthesis by T cells was highly controlled by apoptosis, concomitant synthesis of TNF- and priming for apoptosis were also analyzed. The accumulation of T cells primed for TNF- synthesis is related to their escape from activation-induced apoptosis, partly due to the cosynthesis of interleukin-2 (IL-2) and TNF-. Interestingly, we observed that LD is associated with a more dramatic TNF- dysregulation, and positive correlations were found between the absolute number of TNF- CD8 T-cell precursors and lipid parameters usually altered in LD including cholesterol, triglycerides, and the atherogenic ratio apolipoprotein B (apoB)/apoA1. Observations from the study indicate that HAART dysregulates homeostasis of TNF- synthesis and suggest that this proinflammatory response induced by efficient antiretroviral therapy is a risk factor of LD development in HIV+ patients.
Glyoxalase I is involved in resistance of human leukemia cells to antitumor agent-induced apoptosis
Authors: Sakamoto Hiroshi , Mashima Tetsuo , Kizaki Atsuo , Dan Shingo , Hashimoto Yuichi , Naito Mikihiko , Tsuruo Takashi
Published: October 2019

Abnormality in the machinery of apoptosis is associated with a resistant phenotype of the tumor cell to chemotherapy. To determine the molecular basis of resistance to antitumor agent-induced apoptosis, we performed a complementary DNA (cDNA) subtractive hybridization with messenger RNA (mRNA) from human monocytic leukemia U937 and its variant UK711, which is resistant to apoptosis induced by antitumor agents. We found that glyoxalase I (GLO1), an enzyme that detoxifies methylglyoxal, is selectively overexpressed in the apoptosis-resistant UK711 cells. The GLO1 enzyme activity was significantly elevated in UK711 and UK110 cells, another drug-resistant mutant, as well as in K562/ADM, adriamycin-resistant leukemia cells, compared with their parental cells. When overexpressed in human Jurkat cells, GLO1 inhibited etoposide- and adriamycin-induced caspase activation and apoptosis, indicating the involvement of GLO1 in apoptosis suppression caused by these drugs. Moreover, cotreatment withS-p-bromobenzylglutathione cyclopentyl diester (BBGC), a cell-permeable inhibitor of GLO1, enhanced etoposide-induced apoptosis in resistant UK711 cells but not in parental U937 cells. Taken together, these results indicate that GLO1 is a resistant factor to antitumor agent-induced apoptosis in human leukemia cells and that the GLO1 inhibitor could be a drug resistance-reversing agent.
The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators
Authors: Centola Michael , Wood Geryl , Frucht David M. , Galon Jerome , Aringer Martin , Farrell Christopher , Kingma Douglas W. , Horwitz Mitchell E. , Mansfield Elizabeth , Holland Steven M. , O'Shea John J. , Rosenberg Helene F. , Malech Harry L. , Kastner Daniel L.
Published: October 2019

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designatedMEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase–polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) γ, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor β inhibited such expression. Induction by IFN-γ occurred rapidly and was resistant to cycloheximide. IFN- also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-γ and the combination of IFN- and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-γ immediate early gene.
Perfusion with sickle erythrocytes up-regulates ICAM-1 andVCAM-1 gene expression in cultured human endothelial cells
Authors: Shiu Yan-Ting , Udden Mark M. , McIntire Larry V.
Published: October 2019

Sickle cell anemia is characterized by periodic vasoocclusive crises. Increased adhesion of sickle erythrocytes to vascular endothelium is a possible contributing factor to vasoocclusion. This study determined the effect of sickle erythrocyte perfusion at a venous shear stress level (1 dyne/cm2) on endothelial cell (EC) monolayers. Sickle erythrocytes up-regulated intercellular adhesion molecule-1 (ICAM-1) gene expression in cultured human endothelial cells. This was accompanied by increased cell surface expression of ICAM-1 and also elevated release of soluble ICAM-1 molecules. Expression of vascular cell adhesion molecule-1 (VCAM-1) messenger RNA (mRNA) was also strikingly elevated in cultured ECs after exposure to sickle cell perfusion, although increases in membrane-bound and soluble VCAM-1 levels were small. The presence of cytokine interleukin-1β in the perfusion system enhanced the production of ICAM-1 and VCAM-1 mRNA, cell surface expression, and the concentrations of circulating forms. This is the first demonstration that sickle erythrocytes have direct effects on gene regulation in cultured human ECs under well-defined flow environments. The results suggest that perfusion with sickle erythrocytes increases the expression of cell adhesion molecules on ECs and stimulates the release of soluble cell adhesion molecules, which may serve as indicators of injury and/or activation of endothelial cells. The interactions between sickle red blood flow, inflammatory cytokines, and vascular adhesion events may render sickle cell disease patients vulnerable to vasoocclusive crises.
Locus control region activity by 5′HS3 requires a functional interaction with β-globin gene regulatory elements: expression of novel β/γ-globin hybrid transgenes
Authors: Rubin Joel E. , Pasceri Peter , Wu Xiumei , Leboulch Philippe , Ellis James
Published: October 2019

The human β-globin locus control region (LCR) contains chromatin opening and transcriptional enhancement activities that are important to include in β-globin gene therapy vectors. We previously used single-copy transgenic mice to map chromatin opening activity to the 5′HS3 LCR element. Here, we test novel hybrid globin genes to identify β-globin gene sequences that functionally interact with 5′HS3. First, we show that an 850-base pair (bp) 5′HS3 element activates high-level β-globin gene expression in fetal livers of 17 of 17 transgenic mice, including 3 single-copy animals, but fails to reproducibly activate Aγ-globin transgenes. To identify the β-globin gene sequences required for LCR activity by 5′HS3, we linked the 815-bp β-globin promoter to Aγ-globin coding sequences (BGT34), together with either the β-globin intron 2 (BGT35), the β-globin 3′ enhancer (BGT54), or both intron 2 and the 3′ enhancer (BGT50). Of these transgenes, only BGT50 reproducibly expresses Aγ-globin RNA (including 7 of 7 single-copy animals, averaging 71% per copy). Modifications to BGT50 show that LCR activity is detected after replacing the β-globin promoter with the 700-bp Aγ-globin promoter, but is abrogated when an AT-rich region is deleted from β-globin intron 2. We conclude that LCR activity by 5′HS3 on globin promoters requires the simultaneous presence of β-globin intron 2 sequences and the 260-bp 3′ β-globin enhancer. The BGT50 construct extends the utility of the 5′HS3 element to include erythroid expression of nonadult β-globin coding sequences in transgenic animals and its ability to express antisickling γ-globin coding sequences at single copy are ideal characteristics for a gene therapy cassette.
Therapy-related acute myeloid leukemia and myelodysplasia after high-dose chemotherapy and autologous stem cell transplantation
Authors: Pedersen-Bjergaard Jens , Andersen Mette Klarskov , Christiansen Debes H.
Published: October 2019

Therapy-related myelodysplasia (t-MDS) and acute myeloid leukemia (t-AML) after high-dose chemotherapy (HD-CT) and autologous stem cell transplantation (ASCT) for malignant diseases have become an important problem. The actuarial risk has varied, but has often been high if compared to the risk after conventional therapy. Prior chemotherapy with large cumulative doses of alkylating agents is the most important risk factor. In addition, patient age and previous radiotherapy, particularly the use of total body irradiation (TBI) in the preparative regimen for ASCT, have been identified as risk factors. In 3 studies, patients transplanted with CD34+ cells from peripheral blood after chemotherapy priming showed a higher risk of t-MDS or t-AML than patients transplanted with cells isolated from the bone marrow without priming. To what extent this higher risk relates to the prior therapy with a different contamination with preleukemic, hematopoietic precursors of the CD34+ cells obtained by the 2 methods, or is a direct result of chemotherapy priming, or of an increasing awareness of these complications, remains to be determined. The latent period from ASCT to t-MDS and t-AML has often been short, 12 months or less in 27% of the patients. Bone marrow pathology of early cases of t-MDS after ASCT has often been neither diagnostic nor prognostic, but most patients presented chromosome aberrations, primarily deletions or loss of the long arms of chromosomes 5 and 7. The prognosis was in general poor, although 17% with indolent t-MDS survived more than 18 months from diagnosis, and most of these presented a normal karyotype or a single chromosome aberration.
Phase I/II trial of neutrophil transfusions from donors stimulated with G-CSF and dexamethasone for treatment of patients with infections in hematopoietic stem cell transplantation
Authors: Price Thomas H. , Bowden Raleigh A. , Boeckh Michael , Bux Juergen , Nelson Karen , Liles W. Conrad , Dale David C.
Published: October 2019

We examined the feasibility of a community blood bank granulocyte transfusion program utilizing community donors stimulated with a single-dose regimen of subcutaneous granulocyte colony-stimulating factor (G-CSF) plus oral dexamethasone. The recipients of these transfusions were neutropenic stem cell transplantation patients with severe bacterial or fungal infection. Nineteen patients received 165 transfusions (mean 8.6 transfusions/patient, range 1-25). Community donors provided 94% of the transfusions; relatives accounted for only 6% of the transfusions. Sixty percent of the community donors initially contacted agreed to participate, and 98% of these individuals indicated willingness to participate again. Transfusion of 81.9 ± 2.3 × 109 neutrophils (mean ± SD) resulted in a mean 1-hour posttransfusion neutrophil increment of 2.6 ± 2.6 × 103/μL and restored the peripheral neutrophil count to the normal range in 17 of the 19 patients. The buccal neutrophil response, a measure of the capacity of neutrophils to migrate to tissue sites in vivo, was restored to normal in most patients following the transfusion. Chills, fever, and arterial oxygen desaturation of ≥ 3% occurred in 7% of the transfusions, but these changes were not sufficient to limit therapy. Infection resolved in 8 of 11 patients with invasive bacterial infections or candidemia. These studies indicate that transfusion of neutrophils from donors stimulated with G-CSF plus dexamethasone can restore a severely neutropenic patient's blood neutrophil supply and neutrophil inflammation response. Further studies are needed to evaluate the clinical efficacy of this therapy.
Conserved amino acids in metal-binding motifs of PDE3A are involved in substrate and inhibitor binding
Authors: Zhang Wei , Colman Robert W.
Published: October 2019

The activity of phosphodiesterase (PDE)3A requires divalent cations. Putative metal-binding sites are expected at 2 highly conserved metal-binding motifs, HXXXH(X)25E. A functional truncated recombinant PDE3A containing the catalytic domain (PDE3A▵1) and mutant proteins were expressed in a baculovirus/Sf9 cell system. All the mutant proteins had decreased catalytic efficiency (kcat/Km). Mutants H752A, H756A, and E825A had kcat of less than 0.0008 s−1 to 0.0475 s−1 compared to PDE3A▵1, with 1.86 second−1, with unchanged Km. Although E866A had a kcat of 0.235 s−1, the Kmfor cyclic adenosine monophosphate (cAMP) was increased 11-fold and the Ki for cyclic guanosine monophosphate (cGMP) was 27-fold higher than PDE3A▵1. The Ki of H836A for cGMP was 177-fold higher than that of PDE3A▵1. The Km for E971A was 5-fold higher than PDE3A▵1. These results suggest that the cAMP and cGMP binding sites are overlapping, but not identical, involving both common and different amino acids. Mutants E825A, H836A, and E866A showed low activity in a metal ion-free assay; however, their enzymatic activities were increased 4- to 10-fold in buffers containing Mn2+, Mg2+, or Co2+. This observation indicates that conserved amino acids in the second metal-binding motif might not be involved in binding divalent cations but may serve other functions such as substrate or inhibitor binding in PDE3A.
Antibodies to the FVIII light chain that neutralize FVIII procoagulant activity are present in plasma of nonresponder patients with severe hemophilia A and in normal polyclonal human IgG
Authors: Moreau Alexandre , Lacroix-Desmazes Sébastien , Stieltjes Natalie , Saenko Evgueni , Kaveri Srini V. , D'Oiron Roseline , Sultan Yvette , Scandella Dorothea , Kazatchkine Michel D.
Published: October 2019

We have analyzed the properties of anti-factor VIII (FVIII) immunoglobulin (Ig) G recovered by affinity chromatography on FVIII-Sepharose from the IgG fraction of the plasma of healthy individuals and nonresponder patients with hemophilia A. Affinity-purified anti-FVIII antibodies were found to neutralize FVIII activity and to bind to FVIII with an affinity similar to that of anti-FVIII IgG that had been affinity-purified from the plasma of inhibitor-positive hemophilia patients and of patients with anti-FVIII autoimmune disease. The antibodies also exhibited patterns of reactivity with thrombin-digested FVIII similar to those of FVIII inhibitors and preferentially recognized epitopes located in the light chain of FVIII. These observations suggest that FVIII inhibitors occurring in hemophilia A and in patients with anti-FVIII autoimmune disease originate from the expansion of preexisting natural anti-FVIII clones that exhibit FVIII-neutralizing properties.
Ferriporphyrins and endothelium: a 2-edged sword—promotion of oxidation and induction of cytoprotectants
Authors: Balla József , Balla György , Jeney Viktoria , Kakuk György , Jacob Harry S. , Vercellotti Gregory M.
Published: October 2019

Heme arginate infusions blunt the symptoms of patients with acute intermittent porphyria without evidence of the vascular or thrombotic side effects reported for hematin. To provide a rationale for heme arginate's safety, the present study examined the effects of various ferriporphyrins to sensitize human endothelial cells to free radical injury and to induce heme oxygenase and ferritin expression. Heme arginate, unlike hematin, did not amplify oxidant-induced cytotoxicity mediated by hydrogen peroxide (5.3 ± 2.4 versus 62.3 ± 5.3% 51Cr release,P < .0001) or by activated neutrophils (14.4 ± 2.9 versus 41.1 ± 6.0%, P < .0001). Nevertheless, heme arginate efficiently entered endothelial cells similarly to hematin, since both markedly induced heme oxygenase mRNA (more than 20-fold increase) and enzyme activity. Even with efficient permeation, endothelial cell ferritin content was only minimally increased by heme arginate compared with a 10-fold induction by hematin; presumably less free iron was derived from heme arginate despite up-regulation of heme oxygenase. Hematin is potentially vasculopathic by its marked catalysis of oxidation of low-density lipoprotein (LDL) to endothelial-toxic moieties. Heme arginate was significantly less catalytic. Heme arginate–conditioned LDL was less than half as cytotoxic to endothelial cells as hematin-conditioned LDL (P < .004). It is concluded that heme arginate may be less vasculotoxic than hematin since it is an effective heme oxygenase gene regulator but a less efficient free-radical catalyst.
Nitric oxide induces apoptosis in megakaryocytic cell lines
Authors: Battinelli Elisabeth , Loscalzo Joseph
Published: October 2019

Cytokines that stimulate inducible nitric oxide (NO) synthase can suppress the growth and differentiation of normal human bone marrow cells, including megakaryocytes. Since NO promotes apoptosis in other cell systems, we chose to study the determinants of apoptosis in megakaryocytic cells. We show that both exogenous and endogenous sources of NO can induce apoptosis in megakaryocytoid cell lines. The megakaryocyte growth factor thrombopoietin suppresses NO-induced apoptosis, whereas treatment with peroxynitrite, a cytotoxic product formed when NO reacts with superoxide, promotes apoptosis. Superoxide inhibitors suppress NO-induced apoptosis, and pretreatment with megakaryocyte growth and maturation factors attenuates NO-induced apoptosis. These data show that NO modulates megakaryocyte apoptosis and suggest that this process may occur in the cytokine-rich marrow milieu to regulate megakaryocyte turnover.
Failure of gelsolin overexpression to regulate lymphocyte apoptosis
Authors: Posey S. Celeste , Martelli Maria Paola , Azuma Toshifumi , Kwiatkowski David J. , Bierer Barbara E.
Published: October 2019

The actin regulatory protein gelsolin cleaves actin filaments in a calcium- and polyphosphoinositide-dependent manner. Gelsolin has recently been described as a novel substrate of the cysteinyl protease caspase-3, an effector protease activated during apoptosis. Cleavage by caspase-3 generates an amino-terminal fragment of gelsolin that can sever actin filaments independently of calcium regulation. The disruption of the actin cytoskeleton by cleaved gelsolin is hypothesized to mediate many of the downstream morphological changes associated with apoptosis. In contrast, overexpression of full-length gelsolin has also been reported to inhibit apoptotic cell death upstream of the activation of caspase-3, suggesting that gelsolin may also act prior to commitment to cell death. The authors previously observed that actin stabilization by the cell permeant agent jasplakinolide enhanced cell death upon interleukin (IL)-2 or IL-3 withdrawal from growth-factor–dependent lymphocyte cell lines, and hypothesized that actin polymerization could alter the activity of gelsolin, thus enhancing apoptosis. Here the authors show that constitutive overexpression of gelsolin did not, however, inhibit or dramatically enhance apoptotic cell death upon growth-factor withdrawal, nor did it modify sensitivity to jasplakinolide. In contrast to previous reports, overexpression of gelsolin in Jurkat T cells did not prevent or delay apoptosis induced by Fas ligation or ceramide treatment. Overexpressed gelsolin protein was cleaved during apoptosis, as seen previously in this and other cell types. In these model systems, therefore, the level of gelsolin expression was not a rate-limiting determinant in commitment to or time to the morphological changes of apoptosis.
Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells
Authors: McKenna Hilary J. , Stocking Kim L. , Miller Robert E. , Brasel Kenneth , De Smedt Thibaut , Maraskovsky Eugene , Maliszewski Charles R. , Lynch David H. , Smith Jeffrey , Pulendran Bali , Roux Eileen R. , Teepe Mark , Lyman Stewart D. , Peschon Jacques J.
Published: October 2019

The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an important role in hematopoiesis. The flt3 ligand (flt3L) is a growth factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice are treated with flt3L immature B cells, natural killer (NK) cells and dendritic cells (DC) are expanded in vivo. To further elucidate the role of flt3L in hematopoiesis, mice lacking flt3L (flt3L−/−) were generated by targeted gene disruption. Leukocyte cellularity was reduced in the bone marrow, peripheral blood, lymph nodes (LN), and spleen. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B-lymphoid progenitors were noted in the BM of flt3L−/− mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, LN, and thymus. Both myeloid-related (CD11c++ CD8−) and lymphoid-related (CD11c++ CD8+) DC numbers were affected. We conclude that flt3L has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes.
The Duffy-binding-like domain 1 of Plasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) is a heparan sulfate ligand that requires 12 mers for binding
Authors: Barragan Antonio , Fernandez Victor , Chen Qijun , von Euler Anne , Wahlgren Mats , Spillmann Dorothe
Published: October 2019

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), present on the surfaces of parasitized red blood cells (pRBC), mediates rosetting, a virulent phenotype. Here, we show that pRBC specifically bind heparan sulfate (HS) and heparin onto their surfaces and that the rosetting ligand PfEMP1 specifically adheres to heparin–Sepharose when extracted from the surfaces of radioiodinated infected RBC. An analysis of the binding properties of the different regions of PfEMP1 provides evidence that the Duffy-binding–like domain-1 (DBL-1) is the predominant ligand involved in HS and heparin binding. Soluble DBL-1 requires a minimal heparin fragment size of a 12-mer (≈4 kd) for binding and is critically dependent on N-sulfation. A 12-mer is also the minimal heparin fragment that disrupts naturally formed rosettes. DBL-1 binds specifically to erythrocytes and also to HS from endothelial cells and human aorta but not to chondroitin sulfate A, suggesting that different PfEMP1s mediate adhesion to distinct glycosaminoglycans in individual malaria parasites. Present data suggest that HS on endothelial cells may also be involved in the sequestration of pRBC. Elucidation of these binding mechanisms opens up new possibilities for therapeutic strategies targeting adhesive interactions of pRBC.
Vena caval filters: a comprehensive review
Authors: Streiff Michael B.
Published: October 2019

Hematologists are often asked to treat patients with venous thromboembolic disease. Although anticoagulation remains the primary therapy for venous thromboembolism, vena caval filters are an important alternative when anticoagulants are contraindicated. To assess the evidence supporting the utility of these devices, a comprehensive review of the English language literature was performed. Except for one randomized trial, the vena caval filter literature consists of case series or consecutive case series. The mean duration of follow-up for each of the 5 filter types varies from 6 to 18 months. All are about equally effective in the prevention of pulmonary embolism (2.6%-3.8%). Deep venous thrombosis (6%-32%) and inferior vena cava thrombosis (3.6%-11.2%) after filter placement vary widely among different filter types primarily because of differences in outcome assessment. Thrombosis at the insertion site is a common complication of filter placement (23%-36%). In view of the absence of randomized comparisons, no filter can be designated as superior in safety or efficacy. Vena caval filters represent a potentially important but poorly evaluated therapeutic modality in the prevention of pulmonary emboli. Randomized trials are necessary to establish the appropriate place for vena caval filters in the treatment of venous thromboembolic disease.
Sp1 and C/EBP are necessary to activate the lactoferrin gene promoter during myeloid differentiation
Authors: Khanna-Gupta Arati , Zibello Theresa , Simkevich Carl , Rosmarin Alan G. , Berliner Nancy
Published: October 2019

In this study, we sought to identify factors responsible for the positive modulation of lactoferrin (LF), a neutrophil-specific, secondary-granule protein gene. Initial reporter gene transfection assays indicated that the first 89 base pairs of the LF promoter are capable of directing myeloid-specific LF gene expression. The presence of a C/EBP site flanked by 2 Sp1 sites within this segment of the LF promoter prompted us to investigate the possible role of these sites in LF expression. Cotransfection studies of LF-89luc plasmid with increasing concentrations of a C/EBP expression vector in myeloid cells resulted in a linear transactivation of luciferase reporter activity. Electrophoretic mobility shift assays found that the C/EBP site is recognized by C/EBP and that both LF Sp1 binding sites bind the Sp1 transcription factor specifically in myeloid cells. Mutation of either Sp1 site markedly reduced activity of the LF-89luc plasmid in myeloid cells, and neither Sp1 mutant plasmid was transactivated by a C/EBP expression plasmid to the same extent as wild-type LF-89luc. We also transfected LF-89luc into Drosophila Schneider cells, which do not express endogenous Sp1, and demonstrated up-regulation of luciferase activity in response to a cotransfected Sp1 expression plasmid, as well as to a C/EBP expression plasmid. Furthermore, cotransfection of LF-89luc plasmid simultaneously with C/EBP and Sp1 expression plasmids resulted in an increase in luciferase activity greater than that induced by either factor alone. Taken together, these observations indicate a functional interaction between C/EBP and Sp1 in mediating LF expression.
Interferon gamma delays apoptosis of mature erythroid progenitor cells in the absence of erythropoietin
Authors: Choi Ilseung , Muta Koichiro , Wickrema Amittha , Krantz Sanford B. , Nishimura Junji , Nawata Hajime
Published: October 2019

Based on the hypothesis that interferon gamma (IFN-γ) may have stimulating effects on survival of hematopoietic progenitor cells, we examined the effect of IFN-γ on apoptosis of mature erythroid colony-forming cells (ECFCs) derived from human peripheral blood obtained from normal, healthy volunteers. When the cells were cultured in the presence of IFN-γ, even without erythropoietin (EPO), the viability of the cells was maintained for at least 36 hours. When apoptosis of ECFCs was assessed by flow cytometric analysis', using annexin V, IFN-γ reduced the extent of apoptosis of the cells, as well as EPO. DNA fragmentation of ECFCs was also reduced by IFN-γ. In cells cultured with IFN-γ alone, expression of Bcl-x was detected but the level of expression decreased gradually during incubation for 36 hours, and the expression level was lower than incubation with EPO. Fas expression and activation of downstream caspases were assessed by flow cytometric analysis or fluorometric protease assay. IFN-γ induced Fas expression of the cells without the activation of caspase8 or caspase3 during 16 hours of incubation, while deprivation of EPO induced expression of Fas and the activation of both caspase8 and caspase3. We propose that IFN-γ produces a stimulating signal for the survival of mature erythroid progenitor cells by reducing apoptosis through a mechanism other than modulating Fas and one related to the expression of Bcl-x.
Secretion of bioactive interleukin-1β by dendritic cells is modulated by interaction with antigen specific T cells
Authors: Gardella Stefania , Andrei Cristina , Costigliolo Sara , Olcese Lucia , Zocchi M. Raffaella , Rubartelli Anna
Published: October 2019

The role of interleukin-1β (IL-1β) as a regulator of the immune response, although extensively investigated, is still debated. We then studied the expression of IL-1β by human dendritic cells (DCs), the professional antigen presenting cells, and its modulation during immune reactions in vitro. Our results show that, on maturation or tetanus toxoid presentation to specific CD4+ CD40L+T lymphocytes, DCs begin to accumulate IL-1β precursor (pro–IL-1β) but do not secrete bioactive IL-1β. In contrast, interaction with alloreactive T cells results in both stimulation of pro–IL-1β synthesis and secretion of processed isoforms of the cytokine, that display biologic activity. Both CD4+ and CD8+ subsets of allospecific T lymphocytes are required: CD4+ T cells drive the synthesis of pro–IL-1β through CD40 engagement but have no effects on pro–IL-1β processing; CD8+ T cells, unable to induce synthesis of pro–IL-1β per se, are responsible for the generation of mature IL-1β by pro–IL-1β–producing DCs. Interleukin-1β–converting enzyme (ICE) inhibitors do not prevent the recovery of IL-1β bioactivity after allorecognition, indicating that allospecific CD8+ T cells may induce the release of bioactive IL-1β via mechanism(s) other than ICE activation. Altogether, these findings suggest that CD4+ and CD8+ T-lymphocyte subsets have distinct roles in the induction of IL-1β secretion by DCs and support the hypothesis that IL-1β plays a role in cell-mediated immune responses.
Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor
Authors: Stefano George B. , Cadet Patrick , Breton Christophe , Goumon Yannick , Prevot Vincent , Dessaint Jean Paul , Beauvillain Jean-Claude , Roumier Ann S. , Welters Ingeborg , Salzet Michel
Published: October 2019

We tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17β-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17β-estradiol conjugated to bovine serum albumin (E2-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17β-estradiol and E2-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17β-estradiol–stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca2+]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N′,N′-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca2+]i chelator, reduced [Ca2+]i in response to E2-BSA, and depleting [Ca2+]i stores abolished the effect of 17β-estradiol on NO release. Confocal photomicrographs using E2-BSA–FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase–polymerase chain reaction, human neutrophil granulocytes expressed ER but not ERβ, suggesting that ER may be the membrane receptor for 17β-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca2+]i.
Constitutive activation of the MAPK pathway mediates v-fes–induced mitogenesis in murine macrophages
Authors: Rovida Elisabetta , Marra Fabio , Baccarini Manuela , Sbarba Persio Dello
Published: October 2019

Fes is a nonreceptor tyrosine kinase expressed at the highest level in macrophages. We previously showed that the overexpression of c-fes in murine macrophages of the BAC-1.2F5 cell line renders these cells independent of macrophage colony-stimulating factor (MCSF) for survival and proliferation, although no direct relationship could be established between tyrosine-phosphorylated substrates of Fes- and MCSF receptor–dependent signaling and mitogenesis. In this study, we investigated whether the mitogen-activated protein kinase (MAPK) pathway is involved in the growth factor–independent growth of v-fes–overexpressing macrophages. We found a constitutively increased phosphorylation of extracellularly regulated kinase (ERK) in v-fes–overexpressing macrophages as compared with mock-infected cells. This finding was associated with activation of mitogen/extracellular signal–regulated kinase (MEK) and with constitutive localization of ERK in the nucleus. Treatment of v-fes–overexpressing cells with the MEK-specific inhibitor PD98059 markedly reduced cell growth, hyperphosphorylation, and nuclear localization of ERK, indicating that the MAPK pathway mediates the mitogenic effect of v-fes.
Mild spherocytic hereditary elliptocytosis and altered levels of α- and γ-adducins in β-adducin-deficient mice
Authors: Muro Andrés F. , Marro Martı́n L. , Gajović Srećko , Porro Fabiola , Luzzatto Lucio , Baralle Francisco E.
Published: October 2019

The membrane skeleton, a dynamic network of proteins associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Deficiencies or defects in membrane skeletal proteins are associated with inherited disorders of erythrocyte morphology and function. Adducin is one of the proteins localized at the spectrin-actin junction of the membrane skeleton. In this work we show that deficiency of β-adducin produces an 80% decrease of -adducin and a fourfold up-regulation of γ-adducin in erythrocytes. β-Adducin or any other isoform generated by translation of abnormally spliced messenger RNAs could not be detected by our antibodies either in ghosts or in cytoplasm of −/− erythrocytes. Actin levels were diminished in mutant mice, suggesting alterations in the actin-spectrin junctional complexes due to the absence of adducin. Elliptocytes, ovalocytes, and occasionally spherocytes were found in the blood film of −/− mice. Hematological values showed an increase in reticulocyte counts and mean corpuscular hemoglobin concentration, decreased mean corpuscular volume and hematocrit, and normal erythrocyte counts that, associated to splenomegaly, indicate that the mice suffer from mild anemia with compensated hemolysis. These modifications are due to a loss of membrane surface and dehydration that result in an increase in the osmotic fragility of red blood cells. The marked alteration in osmotic fragility together with the predominant presence of elliptocytes is reminiscent of the human disorder called spherocytic hereditary elliptocytosis. Our results suggest that the amount of adducin remaining in the mutant animals (presumably γ adducin) could be functional and might account for the mild phenotype.
Results of high-dose therapy for 1000 patients with multiple myeloma: durable complete remissions and superior survival in the absence of chromosome 13 abnormalities
Authors: Desikan Raman , Barlogie Bart , Sawyer Jeffrey , Ayers Dan , Tricot Guido , Badros Ashraf , Zangari Maurizio , Munshi Nikhil C. , Anaissie Elias , Spoon Dan , Siegel David , Jagannath Sundar , Vesole David , Epstein Joshua , Shaughnessy John , Fassas Athanasios , Lim Seah , Roberson Paula , Crowley John
Published: October 2019

High-dose therapy (HDT) has increased complete remission (CR) rates and survival in multiple myeloma (MM). We now report on continuous CR (CCR) and associated prognostic factors in 1000 consecutive patients receiving melphalan-based tandem HDT. Five-year CCR was 52% among 112 CR patients without chromosome 13 (▵13) abnormalities and with beta-2-microglobulin ≤ 2.5 mg/L, C-reactive protein ≤ 4 mg/L, and pre-HDT standard chemotherapy ≤ 12 months. Of all 390 CR patients without ▵13 abnormalities, 35% enjoyed 5-year CCR but none of 54 with ▵13 abnormalities. ▵13 abnormalities, present in overall 16%, reduced 5-year event-free survival from 20% to 0% and overall survival from 44% to 16% (both P < .0001). CR and a second HDT cycle applied within 6 months both extended event-free and overall survival significantly, justifying further pursuit of HDT, especially toward curing non-▵13 MM.
Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1
Authors: McRedmond James P. , Harriott Patrick , Walker Brian , Fitzgerald Desmond J.
Published: October 2019

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A2production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S741A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1.
Missense mutations in the human β fibrinogen gene cause congenital afibrinogenemia by impairing fibrinogen secretion
Authors: Duga Stefano , Asselta Rosanna , Santagostino Elena , Zeinali Sirous , Simonic Tatjana , Malcovati Massimo , Mannucci Pier Mannuccio , Tenchini Maria Luisa
Published: October 2019

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by bleeding that varies from mild to severe and by complete absence or extremely low levels of plasma and platelet fibrinogen. Although several mutations in the fibrinogen genes associated with dysfibrinogenemia and hypofibrinogenemia have been described, the genetic defects of congenital afibrinogenemia are largely unknown, except for a recently reported 11-kb deletion of the fibrinogen A-chain gene. Nevertheless, mutation mechanisms other than the deletion of a fibrinogen gene are likely to exist because patients with afibrinogenemia showing no gross alteration within the fibrinogen cluster have been reported. We tested this hypothesis by studying the affected members of two families, one Italian and one Iranian, who had no evidence of large deletions in the fibrinogen genes. Sequencing of the fibrinogen genes in the 2 probands detected 2 different homozygous missense mutations in exons 7 and 8 of the Bβ-chain gene, leading to amino acid substitutions Leu353Arg and Gly400Asp, respectively. Transient transfection experiments with plasmids expressing wild-type and mutant fibrinogens demonstrated that the presence of either mutation was sufficient to abolish fibrinogen secretion. These findings demonstrated that missense mutations in the Bβ fibrinogen gene could cause congenital afibrinogenemia by impairing fibrinogen secretion.
Engraftment, clinical, and molecular follow-up of patients with multiple myeloma who were reinfused with highly purified CD34+ cells to support single or tandem high-dose chemotherapy
Authors: Lemoli Roberto M. , Martinelli Giovanni , Zamagni Elena , Motta Maria Rosa , Rizzi Simonetta , Terragna Carolina , Rondelli Roberto , Ronconi Sonia , Curti Antonio , Bonifazi Francesca , Tura Sante , Cavo Michele
Published: October 2019

Eighty-two patients with advanced multiple myeloma (MM) were enrolled in 2 sequential clinical studies of 1 or 2 courses of myeloablative therapy with stem cell support. Conditioning regimens consisted of high-dose melphalan (MEL) with or without total body irradiation (TX1 = 35) and MEL as the first preparative regimen, followed within 6 months by busulfan and melphalan (TX2 = 47). On the basis of adequate stem cell harvest, 31 patients (TX1 = 13; TX2 = 18) were transplanted with highly purified CD34+ cells. Positively selected stem cells did not adversely affect hematopoietic reconstitution compared with unmanipulated peripheral blood stem cell. Overall, the complete remission (CR) rate of evaluative patients was 13.8% and 41% for single and double autotransplant, respectively (P = .04). Moreover, 3 patients undergoing TX2 achieved molecular remission and 2 remain PCR-negative after 36 and 24 months from autograft. The median event-free survival (EFS) durations for TX1 and TX2 were 17 and 35 months, respectively (P = .03). Actuarial 3-year overall survival for patients treated with 1 or 2 transplants are 76% and 92%, respectively (P = NS). On multivariate analysis, superior EFS was associated with low β2 microglobulin (β2-M) level at diagnosis and TX2, whereas overall survival was correlated with β2-M. Positive selection of CD34+ cells did not influence the achievement of clinical or molecular CR, as well as remission duration or survival of MM patients. Thus, whereas multiple cycles of high-dose therapy may be beneficial for patients with myeloma, the clinical impact of tumor cell purging remains highly questionable.