bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet-Derived Interleukin-1 Induces Cytokine Production, but not Proliferation of Human Vascular Smooth Muscle Cells
Authors: Loppnow Harald , Bil Rosita , Hirt Stephan , Schönbeck Uwe , Herzberg Mona , Werdan Karl , Theodor Rietschel Ernst , Brandt Ernst , Flad Hans-Dieter
Published: October 2019

During vascular injury, such as observed in atherosclerosis, restenosis, vasculitides, transplantation, or sepsis, vascular smooth muscle cells (SMC) can be exposed to platelets or platelet products. Under these conditions proliferation or cytokine production of SMC stimulated by platelets or platelet products may contribute to regulation of vascular pathogenesis. Thus, we investigated interleukin-6 (IL-6) and IL-8 production as well as proliferation of SMC in response to platelets or platelet lysates. Platelets not already preactivated by thrombin induced IL-6 (10- to 50-fold) or IL-8 production of unstimulated SMC in a cell number dependent fashion. Preactivation of platelets with thrombin potently increased the platelet-mediated IL-6 (50- to 1,000-fold) and IL-8 production of SMC. Hirudin specifically inhibited the activation of platelets with thrombin. Isolated platelets cultured in the absence of SMC did not contain detectable IL-6 or IL-8. Prestimulation (4 hours) of SMC with pathophysiologically relevant substances (lipopolysaccharide [LPS], tumor necrosis factor-α [TNF-α], or IL-1α) further increased the platelet-induced cytokine production. The platelet-derived SMC stimulatory activity was IL-1, since IL-1 receptor antagonist (IL-1-Ra) inhibited the platelet-induced cytokine production of SMC. Anti-platelet-derived growth factor (PDGF)-antibody did not further reduce this activity. Thrombin itself stimulated expression of IL-6 and IL-8 to some degree and induced IL-6 production of SMC synergistically with IL-1. Platelets also induced proliferation of SMC, however, anti-PDGF antibodies, rather than IL-1-Ra blocked this response. These data show that platelet-derived IL-1 stimulates cytokine production of vascular smooth muscle cells, indicating that platelet-derived IL-1 may contribute to regulation of local pathogenesis in the vessel wall by activation of the cytokine regulatory network.
Factor VII Deficiency Caused by a Structural Variant N57D of the First Epidermal Growth Factor Domain
Authors: Leonard Blair J.N. , Chen Qi , Blajchman Morris A. , Ofosu Frederick A. , Sridhara Sampath , Yang Daniel , Clarke Bryan J.
Published: October 2019

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.
Generation of Plasma Cells From Peripheral Blood Memory B Cells: Synergistic Effect of Interleukin-10 and CD27/CD70 Interaction
Authors: Agematsu Kazunaga , Nagumo Haruo , Oguchi Yumiko , Nakazawa Takayuki , Fukushima Keitaro , Yasui Kozo , Ito Susumu , Kobata Tetsuji , Morimoto Chikao , Komiyama Atsushi
Published: October 2019

B cells can differentiate into the antibody-secreting cells, plasma cells, whereas the crucial signals that positively control the entry into the pathway to plasma cells have been unclear. Triggering via CD27 by CD27 ligand (CD70) on purified peripheral blood B cells yielded an increase in the number of plasma cells in the presence of interleukin-10 (IL-10). Differentiation into plasma cells by a combination of IL-10 and CD70 transfectants occurred in CD27+ B cells but not in CD27− B cells. Moreover, addition of IL-2 to the IL-10 and CD70-transfect activation system greatly induced differentiation into plasma cells. In the presence of only IL-2, IL-4, or IL-6, CD70 transfectants did not promote differentiation into plasma cells. On the other hand, CD40 signaling increased the expansion of a B-cell pool from peripheral blood B cells primarily activated by IL-2, IL-10, and anti-CD40 monoclonal antibody (MoAb). Finally, CD27 signaling also rescued B cells from IL-10–mediated apoptosis. These data demonstrate that CD27 ligand (CD70) is a key molecule to prevent the IL-10–mediated promotion of apoptosis and to direct the differentiation of CD27+ memory B cells toward plasma cells in cooperation with IL-10.
Differential Cytotoxicity of Cord Blood and Bone Marrow–Derived Natural Killer Cells
Authors: Gardiner Clair M. , Meara Anne O' , Reen Denis J.
Published: October 2019

Allogeneic cord blood is now being widely used as a source of stem cells for hematologic reconstitution after myeloablative therapy, with reported significantly lower levels of graft-versus-host disease (GVHD) compared with the use of allogeneic bone marrow (BM). This study was undertaken to investigate biologic aspects of natural killer (NK) cell activity, as recognized effector cells of the GVHD and graft-versus-leukemia (GVL) response, from cord blood and conventional BM. NK-cell activity levels of freshly isolated cells from cord blood and BM against K562 targets were comparable. Lymphokine activated killer (LAK) cells from both hematopoietic cell sources were compared for their ability to kill target cells by necrotic or apoptotic mechanisms using specific target cell lines. Cord blood cells had significantly higher necrosis-mediated cytotoxic activity against Daudi target cells compared with BM-derived cells. Cord blood LAK cells had relatively high levels of apoptotic-mediated cytotoxicity against YAC-1 target cells, whereas BM-derived LAK cells were unable to induce apoptosis in these cells. Interleukin-2 (IL-2) induced significant granzyme B activity in cord cells in contrast to BM cells, in which very little activity was measured. Western blotting confirmed these findings, with IL-2 inducing granzyme B protein expression in cord cells but not detectable levels in BM cells. BM cells had significantly lower cell surface expression of IL-2R and prolonged culture in IL-2 was only partially able to restore their deficient apoptotic cytotoxic activity. Thus, major differences exist between cord blood-derived and BM-derived mononuclear cells with respect to their NK-cell–associated cytotoxic behavior. This could have important implications for stem cell transplantation phenomena, because it suggests that cord blood may have increased potential for a GVL effect.
VH Gene Analysis of Clonally Related IgM and IgG From Human Lymphoplasmacytoid B-Cell Tumors With Chronic Lymphocytic Leukemia Features and High Serum Monoclonal IgG
Authors: Sahota Surinder S. , Garand Richard , Bataille Regis , Smith Alastair J. , Stevenson Freda K.
Published: October 2019

An unusual group of human B-cell tumors with cellular features of chronic lymphocytic leukemia or lymphoplasmacytoid leukemia, together with high levels of a monoclonal IgG serum protein, has been investigated. Analysis of tumor-derived VH genes of neoplastic B lymphocytes was used to determine the clonal relationship between the IgM expressed or secreted by the tumor cells and the IgG serum paraprotein. In all five cases, VH gene sequences showed transcripts of IgM and IgG of common clonal origin. Sequences were derived from VH3 (4 of 5) and VH1 (1 of 5) families and were all highly somatically mutated with strong evidence for antigen selection. There was no intraclonal variation detectable in either IgM or IgG sequences. In 3 of 5 cases, in which monoclonal IgM and IgG were found in serum, the VH genes combined to Cμ or Cγ showed identical mutational patterns. However, in 2 of 5 cases, in which IgM was confined to cell expression with only monoclonal IgG in serum, sequences of the VH transcripts of IgM and IgG showed many shared mutations but also numerous differences. In these cases, the level of mutation was similar in IgM and IgG and both appeared to be antigen selected. In summary, the final neoplastic event in this group of tumors has apparently occurred at the point of isotype switch from IgM to IgG, leading to dual isotype synthesis. In the group that secreted both isotypes, the mutation pattern was identical, indicating either synthesis by a single cell, or silencing of mutational activity before switching. In the group that did not secrete IgM, cells of each isotype were distinct and reflected a divergent mutational history.
Unbalanced Expression of Bcl-2 Family Proteins in Follicular Lymphoma: Contribution of CD40 Signaling in Promoting Survival
Authors: Ghia Paolo , Boussiotis Vassiliki A. , Schultze Joachim L. , Cardoso Angelo A. , Dorfman David M. , Gribben John G. , Freedman Arnold S. , Nadler Lee M.
Published: October 2019

Although highly responsive, advanced stage follicular lymphoma (FL) is not curable with conventional treatment. This relative resistance is thought to be due to the t(14;18) that results in the constitutive overexpression of the death-inhibiting protein bcl-2. However, the observation that FL cells are sensitive to treatment in vivo and prone to apoptosis on in vitro culture questions whether bcl-2 alone is responsible for the pathogenesis and clinical behavior of this disease. Therefore, multiple genes are likely to be involved in both the lymphomagenesis and the clinical course of FL. We examined whether expression of other bcl-2 family genes might also be operative. Here, we show that FL cells display a different pattern of expression of bcl-2 family proteins from normal germinal center (GC) B cells that are thought to be their normal counterpart. FL cells express the death-suppressor proteins bcl-2, bcl-xL, and mcl-1; whereas GC B cells express bcl-xL and mcl-1 but also the proapoptotic proteins bax-α and bad. Although maintaining constitutive levels of bcl-2 and mcl-1, FL cells are not protected from apoptosis when cultured in vitro. Their propensity to undergo apoptosis is temporally associated with downregulation of bcl-xL. More importantly, activation of FL cells via CD40 not only prevents downregulation but increases the level of bcl-xL expression and results in promotion of survival. These results support the hypothesis that the overexpression of bcl-2 is not the only antiapoptotic mechanism responsible for the pathogenesis of FL. Survival of FL cells is determined by a number of death-inhibiting proteins, among which bcl-xL appears to have the most critical role. Moreover, these findings are consistent with the hypothesis that, although FL cells are malignant, they respond to microenvironmental signals such as CD40L that appear to contribute to their survival through the upregulation of death-inhibiting proteins.
Long-Term Trial of Deferiprone in 51 Transfusion-Dependent Iron Overloaded Patients
Authors: Hoffbrand A. Victor , AL-Refaie Faris , Davis Bernard , Siritanakatkul Noppadol , Jackson Beverly F.A. , Cochrane John , Prescott Emma , Wonke Beatrix
Published: October 2019

Fifty-one transfusion-dependent iron-loaded adult patients (38 with thalassemia major) were treated with the orally active iron chelator deferiprone (1,2 dimethyl-3-hydroxypyrid-4-one, L1) at a dose of 75 mg/kg/d (range, 50 to 79). Twenty patients discontinued the drug and five died after a mean of 18.7 months (range, 4 to 35). Of the 20, 5 had arthropathy, 5 had gastrointestinal symptoms, 4 had a rising serum ferritin, 3 had agranulocytosis or neutropenia, 1 had tachycardia, 1 had renal failure, and 1 went abroad. Twenty-six patients continued deferiprone for a mean of 39.4 months (range, 12 to 49). Among these patients, there was no overall significant change in serum ferritin (initial mean, 2,937 μg/L; range, 980 to 5,970; final mean, 2,323 μg/L; range, 825 to 5,970) or in urine iron excretion (initial mean, 31.2 mg/24 h; range, 16.3 to 58.2; final mean, 32.1 mg/24 h; range, 9.4 to 75.8), implying no overall change in iron stores. When the patients who had received deferiprone for longer than 3 years were considered separately, there was also no significant change in serum ferritin or urinary iron excretion. The initial serum ferritin levels in the 26 patients who continued deferiprone treatment were significantly lower than in those who discontinued the drug (P < .01). The liver iron content in 17 patients who had received deferiprone for 24 to 48 months ranged from 5.9 to 41.2 mg/g dry weight, 50% having levels above 15.0 mg, a level associated with a high risk of cardiac disease due to iron overload. In this study the drug caused fewer side effects and was more effective at maintaining iron status among patients previously well chelated and with lower initial serum ferritin levels.
Gamma-Globin Gene Promoter Elements Required for Interaction With Globin Enhancers
Authors: Langdon Scott D. , Kaufman Russel E.
Published: October 2019

Normal expression of the human β-globin domain genes is dependent on at least three types of regulatory elements located within the β-globin domain: the locus control region (LCR), globin enhancer elements (3′β and 3′Aγ), and the individual globin gene promoter and upstream regions. It has been postulated that regulation occurs through physical interactions between factors bound to these elements, which are located at considerable distances from each other. To identify the elements required for promoter-enhancer interactions from a distance, we have investigated the expression of the wild-type, truncated, and mutated γ-globin promoters linked to the 5′HS2 enhancer. We show that in K562 cells, 5′HS2 increases activity approximately 20-fold from both a wild-type and truncated (-135 → +25) γ promoter and that truncation or site-directed mutagenesis of the tandem CCAAT boxes eliminated the enhancement by 5′HS2. Mutation of the γ-globin gene promoter GATA-1 binding sites did not decrease either promoter strength or enhancement of activity by 5′HS2. To determine if enhanced expression of γ-globin gene promoters carrying mutations associated with hereditary persistence of fetal hemoglobin (HPFH) was due to greater interactions with enhancers, we linked these HPFH γ-globin gene promoters to 5′HS2 and demonstrated a twofold to threefold higher expression than the corresponding wild-type promoter plus enhancer in MEL cells. Addition of the Aγ-globin gene 3′ enhancer to a plasmid containing the γ-globin gene promoter and 5′HS2 did not further enhance promoter strength. Furthermore, we have demonstrated that the previously identified core 5′HS2 enhancer (46-bp tandem AP-1/NF-E2 sites) increased expression only when located 5′, but not 3′, to the γ-globin-luciferase reporter gene, suggesting that its enhancer effect is not by DNA looping. Our results suggest that CCAAT boxes, but not GATA or CACCC binding sites, are required for interaction between the γ-globin promoter and the LCR/5′HS2 and that regulatory elements in addition to the core enhancer may be required for the enhancer to act from a distance.
Mechanisms of Long-Term Donor-Specific Allograft Survival Induced by Pretransplant Infusion of Lymphocytes
Authors: Yang Liming , Temple Barb Du , Khan Qasim , Zhang Li
Published: October 2019

Pretransplantation donor-specific transfusion (DST) can enhance allograft survival in man and animals. However, due to the lack of a specific marker to identify donor-reactive cells in vivo in man and normal (nontransgenic) animals, the underlying mechanism remains unknown. In this study, we use 2CF1 transgenic mice expressing a transgenic T-cell receptor (TCR) specifically recognizing Ld, a major histocompatibility complex (MHC) class I molecule, to delineate the role of DST in long-term skin allograft survival and its underlying mechanisms. Our main findings include: (1) in the absence of any other immunosuppressive treatment, a single dose pretransplantation infusion of viable splenocytes from an Ld+ donor is sufficient to induce permanent donor-specific skin allograft survival in 2CF1anti-Ld TCR transgenic mice; (2) DST leads to a deletion of the majority (>60%) of donor-reactive T cells in the periphery of the recipient. However, deletion does not necessarily result in tolerance; (3) remaining donor-reactive T cells from DST-treated mice are fully responsive to Ld in vitro, and can suppress the antidonor response of naive T cells in vitro only when exogenous interleukin (IL)-4 is provided; and (4) the sera level of IL-4 in DST-treated tolerant mice is significantly increased. These results suggest that the generation of a subset of T cells with the potential to specifically inhibit antidonor responses, together with promotion of IL-4 production in recipients, may be important mechanisms for the induction and maintenance of antigen-specific tolerance.
The Umbilical Cord Blood αβ T-Cell Repertoire: Characteristics of a Polyclonal and Naive but Completely Formed Repertoire
Authors: Garderet Laurent , Dulphy Nicolas , Douay Corinne , Chalumeau Nathalie , Schaeffer Véronique , Zilber Marie-Thérèse , Lim Annick , Even Jos , Mooney Nuala , Gelin Catherine , Gluckman Eliane , Charron Dominique , Toubert Antoine
Published: October 2019

Umbilical cord blood (CB) constitutes a promising alternative to bone marrow for allogeneic transplantation and is increasingly used because of the reduced severity of graft-versus-host disease after CB transplantation. We have compared the T-cell receptor β chain (TCRB) diversity of CB lymphocytes with that of adult lymphocytes by analyzing the complementarity determining region 3 (CDR3) size heterogeneity. In marked contrast to adult samples, we observed bell-shaped profiles in all of the 22 functional β-chain variable (BV) subfamilies that reflect the lack of prior antigenic stimulation in CB samples. However, the mean CDR3 size and BV usage were comparable between CB and adult samples. BJ2 (65%) segments were used preferentially to BJ1 (35%), especially BJ2S7, BJ2S5, BJ2S3, and BJ2S1, in both CB and in adult lymphocytes. We therefore conclude that although naive as reflected by the heterogeneity of the CDR3 size, the TCRBV repertoire appears fully constituted at birth. The ability to expand TCRB subfamilies was confirmed by stimulation with staphylococcal superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxin A. This study provides the basis for future analysis of the T-cell repertoire reconstitution following umbilical CB transplantation.
In Vitro and In Vivo Evidence That Ex Vivo Cytokine Priming of Donor Marrow Cells May Ameliorate Posttransplant Thrombocytopenia
Authors: Ratajczak Mariusz Z. , Ratajczak Janina , Machalinski Boguslaw , Mick Rosemarie , Gewirtz Alan M.
Published: October 2019

Thrombocytopenia is typically observed in patients undergoing hematopoietic stem cell transplantation. We hypothesized that delayed platelet count recovery might be ameliorated by increasing the number of megakaryocyte colony- forming units (CFU-Meg) in the hematopoietic cell graft. To test this hypothesis, we evaluated cytokine combinations and culture medium potentially useful for expanding CFU-Meg in vitro. We then examined the ability of expanded cells to accelerate platelet recovery in an animal transplant model. Depending on the cytokine combination used, we found that culturing marrow CD34+cells for 7 to 10 days in serum-free cultures was able to expand CFU-Meg ∼40 to 80 times over input number. Shorter incubation periods were also found to be effective and when CD34+ cells were exposed to thrombopoietin (TPO), kit ligand (KL), interleukin-1α (IL-1α), and IL-3 in serum-free cultures for as few as 48 hours, the number of assayable CFU-Meg was still increased ∼threefold over input number. Of interest, cytokine primed marrow cells were also found to form colonies in vitro more quickly than unprimed cells. The potential clinical utility of this short-term expansion strategy was subsequently tested in an in vivo animal model. Lethally irradiated Balb-C mice were transplanted with previously frozen syngeneic marrow mononuclear cells (106/mouse), one tenth of which (105) had been primed with [TPO, KL, IL-1a, and IL-3] under serum-free conditions for 36 hours before cryopreservation. Mice receiving the primed frozen marrow cells recovered their platelet and neutrophil counts 3 to 5 days earlier than mice transplanted with unprimed cells. Mice which received marrow cells that had been primed after thawing but before transplantation had similar recovery kinetics. We conclude that pretransplant priming of hematopoietic cells leads to faster recovery of all hematopoietic lineages. Equally important, donor cell priming before transplant may represent a highly cost-effective alternative to constant administration of cytokines during the posttransplant recovery period.
Membrane Translocation of 15-Lipoxygenase in Hematopoietic Cells Is Calcium-Dependent and Activates the Oxygenase Activity of the Enzyme
Authors: Brinckmann Roland , Schnurr Kerstin , Heydeck Dagmar , Rosenbach Thomas , Kolde Gerhard , Kühn Hartmut
Published: October 2019

Mammalian 15-lipoxygenases, which have been implicated in the differentiation of hematopoietic cells are commonly regarded as cytosolic enzymes. Studying the interaction of the purified rabbit reticulocyte 15-lipoxygenase with various types of biomembranes, we found that the enzyme binds to biomembranes when calcium is present in the incubation mixture. Under these conditions, an oxidation of the membrane lipids was observed. The membrane binding was reversible and led to an increase in the fatty acid oxygenase activity of the enzyme. To find out whether such a membrane binding also occurs in vivo, we investigated the intracellular localization of the enzyme in stimulated and resting hematopoietic cells by immunoelectron microscopy, cell fractionation studies and activity assays. In rabbit reticulocytes, the 15-lipoxygenase was localized in the cytosol, but also bound to intracellular membranes. This membrane binding was also reversible and the detection of specific lipoxygenase products in the membrane lipids indicated the in vivo activity of the enzyme on endogenous substrates. Immunoelectron microscopy showed that in interleukin-4 –treated monocytes, the 15-lipoxygenase was localized in the cytosol, but also at the inner side of the plasma membrane and at the cytosolic side of intracellular vesicles. Here again, cell fractionation studies confirmed the in vivo membrane binding of the enzyme. In human eosinophils, which constitutively express the 15-lipoxygenase, the membrane bound share of the enzyme was augmented when the cells were stimulated with calcium ionophore. Only under these conditions, specific lipoxygenase products were detected in the membrane lipids. These data suggest that in hematopoietic cells the cytosolic 15-lipoxygenase translocates reversibly to the cellular membranes. This translocation, which increases the fatty acid oxygenase activity of the enzyme, is calcium-dependent, but may not require a special docking protein.
Transient Thrombocytopenia Produced by Administration of Macrophage Colony-Stimulating Factor: Investigations of the Mechanism
Authors: Baker Georgiann R. , Levin Jack
Published: October 2019

Administration of macrophage colony-stimulating factor (M-CSF) to mice (2 to 8 mg/kg/d × 5d) produced dose-dependent thrombocytopenia, which reached its nadir on days 4 to 5, followed by rapid recovery. Surprisingly, when administration of M-CSF was prolonged, the thrombocytopenia completely resolved, despite continued treatment. Splenectomy did not prevent the thrombocytopenia. Readministration of M-CSF after various intervals continued to produce the thrombocytopenic effect, even after 35 days. Measurements of Meg-CFC and megakaryocyte ploidy during the periods of M-CSF treatment and recovery of normal platelet levels showed no evidence of bone marrow suppression. Platelet survival was markedly decreased after 5 days of M-CSF (at the platelet count nadir) and after 9 days of continued M-CSF treatment, when the platelet count had returned to normal. Platelets from M-CSF–treated donors demonstrated normal survival when transfused into normal recipients. We concluded that thrombocytopenia produced by M-CSF was not due to suppression of thrombopoiesis, but to increased activity of the monocyte/macrophage system, which caused shortened platelet survival, and that subsequently, increased platelet production compensated for ongoing platelet destruction and resulted in normal platelet levels.
Genetic Polymorphisms in the Tumor Necrosis Factor Locus Influence Non-Hodgkin's Lymphoma Outcome
Authors: Warzocha Krzysztof , Ribeiro Patricia , Bienvenu Jacques , Roy Pascal , Charlot Carole , Rigal Dominique , Coiffier Bertrand , Salles Gilles
Published: October 2019

Systemic release of tumor necrosis factor (TNF) and lymphotoxin-α (LTα) has been found to contribute to the severity of non-Hodgkin's lymphoma (NHL). We investigated whether genetic polymorphisms in the TNF locus, previously shown to influence TNF and LTα genes expression, might contribute to these cytokines production and to the clinical course of NHL. Genomic DNA from 273 lymphoma patients was typed for TNF (−308) polymorphism using an allele-specific polymerase chain reaction (PCR) and for LTα (+252) polymorphism with a PCR-based restriction fragment length polymorphism. The presence of the TNF allele involved in increased TNF gene transcription was associated with higher plasma levels of this cytokine at the time of lymphoma diagnosis (χ2 test, P = .013). An extended haplotype analysis showed that the presence of at least two TNF or LTα high-producer alleles constituted a risk factor for first-line treatment failure (χ2 test, P = .021), shorter progression-free survival (log-rank test, P = .0007), and overall survival (log-rank test, P = .012). In the subgroup of 126 patients with diffuse large-cell lymphoma, the presence of two or more TNF/LTα high producing alleles contributed significantly to a higher rate of relapse and progression (log-rank test, P = .045 and P = .027). In multivariate Cox regression models including the variables of the International Prognostic Index, the TNF/LTα haplotype status was found to be an independent risk factor for progression-free survival (relative risk 2.33, 95% confidence interval [1.17 to 4.64], P = .0053) and overall survival (relative risk 1.92, 95% confidence interval [0.63 to 5.80],P = .081) of large-cell lymphoma patients. These results indicate that genetic polymorphism leading to increased TNF production influences the outcome of NHL and suggest a pathophysiological role for the genetic control of the immune response in lymphoid malignancies.
Safety and Cost Effectiveness of a 10 × 109/L Trigger for Prophylactic Platelet Transfusions Compared With the Traditional 20 × 109/L Trigger: A Prospective Comparative Trial in 105 Patients With Acute Myeloid Leukemia
Authors: Wandt Hannes , Frank Markus , Ehninger Gerhard , Schneider Christiane , Brack Norbert , Daoud Ali , Fackler-Schwalbe Irene , Fischer Jürgen , Gäckle Ringfried , Geer Thomas , Harms Peter , Löffler Birgit , Öhl Siegfried , Otremba Burkhard , Raab Monika , Schönrock-Nabulsi Petra , Strobel Gerhard , Winter Rolf , Link Hartmut
Published: October 2019

In 105 consecutive patients with de novo acute myeloid leukemia (French-American-British M3 excluded), we compared prospectively the risk of bleeding complications, the number of platelet and red blood cell transfusions administered, and the costs of transfusions using two different prophylactic platelet transfusion protocols. Two hundred sixteen cycles of induction or consolidation chemotherapy and 3,843 days of thrombocytopenia less than 25 × 109/L were evaluated. At the start of the study, each of the 17 participating centers decided whether they would use a 10 × 109/L prophylactic platelet transfusion trigger (group A/8 centers) or a 20 × 109/L trigger (group B/9 centers). Bleeding complications (World Health Organization grade 2-4) during treatment cycles were comparable in the two groups: 20 of 110 (18%) in group A and 18 of 106 (17%) in group B (P = .8). Serious bleeding events (grade 3-4) were generally not related to the patient's platelet count but were the consequence of local lesions and plasma coagulation factor deficiencies due to sepsis. Eighty-six percent of the serious bleeding episodes occurred during induction chemotherapy. No patient died of a bleeding complication. There were no significant differences in the number of red blood cell transfusions administered between the two groups, but there were significant differences in the number of platelet transfusions administered per treatment cycle: pooled random donor platelet concentrates averaged 15.4 versus 25.4 (P < .01) and apheresis platelets averaged 3.0 versus 4.8 (P < .05) for group A versus group B, respectively. This resulted in the cost of platelet therapy being one third lower in group A compared with group B without any associated increase in bleeding risk.
SHP-1 Phosphatase C-Terminus Interacts With Novel Substrates p32/p30 During Erythropoietin and Interleukin-3 Mitogenic Responses
Authors: Yang Wentian , Tabrizi Mina , Berrada Karim , Yi Taolin
Published: October 2019

SHP-1 protein tyrosine phosphatase is a critical negative regulator of mitogenic signaling, as demonstrated by the heightened growth responses to hematopoietic growth factors in hematopoietic cells of motheaten mice, which lack functional SHP-1 expression due to mutations in the SHP-1 gene. The mitogenic signaling molecules dephosphorylated by SHP-1 have not been fully identified. We detected two proteins (p32/p30) that are hyperphosphorylated in a DA3/erythropoietin receptor (EpoR) cell line that expresses a mutant containing the SHP-1 C-terminus that suppresses the function of the endogenous phosphatase and induces hyperproliferative responses to interleukin-3 (IL-3) and Epo. Hyperphosphorylated p32/p30 are also detected in motheaten hematopoietic cells, demonstrating an association of p32/p30 hyperphosphorylation with SHP-1-deficiency and growth factor-hyperresponsiveness. The hyperphosphorylated p32/30 associate with SHP-1 via its C-terminus, because they coimmunoprecipitate with the phosphatase and the C-terminal mutant and they bind in vitro to a synthetic peptide of the mutant but not the GST fusion proteins of SHP-1 SH2 domains. Induction of p32/p30 phosphorylation by IL-3 or Epo occurs mainly at 2 to 18 hours poststimulation in the DA3/EpoR cell line, indicating p32/p30 as novel signaling molecules during cell cycle progression. These data demonstrate a function for the SHP-1 C-terminus in recruiting potential substrates p32/p30 and suggest that SHP-1 may regulates mitogenic signaling by dephosphorylating p32/p30.
Enhanced Megakaryocyte and Erythroid Development From Normal Human CD34+ Cells: Consequence of Enforced Expression of SCL
Authors: Elwood Ngaire J. , Zogos Helen , Pereira Daniel S. , Dick John E. , Begley C. Glenn
Published: October 2019

The product of the SCL gene is a basic helix-loop-helix (bHLH) transcription factor that is essential for the development of hematopoietic stem cells in both the embryo and the adult. However, once the stem cell compartment is established, the function of SCL in subsequent differentiation and commitment events within normal hematopoietic cells remains undefined. The aim of the current study was to investigate this role using purified normal human hematopoietic CD34+ cells. An SCL retrovirus was used to transduce CD34+ cells isolated from human bone marrow, peripheral blood, and umbilical cord blood. Enforced expression of SCL increased by a median of twofold the number of erythroid colonies, with an increase in both colony size and the rate of hemoglobinization. Unexpectedly, enforced expression in CD34+ cells also significantly increased the number of megakaryocyte colonies, but with no impact on the size of colonies. There was no consistent effect on the number nor size of granulocyte-macrophage (GM) colonies. The proliferative effect of enforced SCL expression on erythroid cells was attributed to a shortened cell cycle time; the self-renewal capacity of erythroid or GM progenitors was unchanged, as was survival of cells within colonies. These results demonstrate a role for SCL in determining erythroid and megakaryocyte differentiation from normal human hematopoietic CD34+ cells.
Novel Evidence of Expression and Activity of Ecto-Phospholipase C γ1 in Human T Lymphocytes
Authors: Miscia Sebastiano , Di Baldassarre Angela , Cataldi Amelia , Rana Rosa Alba , Di Valerio Valerio , Sabatino Giuseppe
Published: October 2019

Although much is known about the intracellular phospholipase C (PLC) specific for inositol phospholipids, few data are available about the presence of a less common PLC at the external side of the membrane bilayer of some cell types. This ectoenzyme seems to play particular roles in cellular function by hydrolyzing inositol lipids located on the outer leaflet of the plasma membrane. Here, we provide the first evidence that peripheral T lymphocytes express a discrete level of a PLCγ1 at the outer leaflet of the plasma membrane. Flow cytometry showed that the PLCγ1-positive (PLCγ1+) cells (∼37%) were CD8+ and CD45RA+. Biochemical evidence indicated that (1) this ectoenzyme displays a mass similar to the cytoplasmic form, (2) it is phosphorylated on tyrosine residues, and (3) its activity is Ca2+-dependent. In addition, this enzyme appeared to be correlated with the proliferative state of the cell, since stimulation with phytohemagglutinin (PHA) downregulated both its expression and activity, which were restored by treatment with an antiproliferative agent like natural interferon beta. Moreover, the different kinetics of formation of its hydrolytic products, inositol 1 phosphate and inositol 1:2 cyclic phosphate (Ins(1)P and Ins(1:2 cycl)P), formed upon incubation of the lymphocytes with [3H]-lyso-phosphatidylinositol (PI), allow the hypothesis of a selective involvement of the two inositol phosphates in the mechanisms regulating the metabolism of particular T-lymphocyte subsets.
Interleukin-13 Receptor α′ But Not α Chain: A Functional Component of Interleukin-4 Receptors
Authors: Murata Takashi , Taguchi Jun , Puri Raj K.
Published: October 2019

In hematopoietic cells, interleukin-2 receptor (IL-2R) γ chain (termed γc) is shown to be a component of the IL-4R system, whereas in nonhematopoietic cells, γc is absent and it is not a component of the IL-4R system. Here, we show that the IL-13R α′ chain (termed IL-13Rα′) but not the IL-13R α chain (termed IL-13Rα) can substitute for γc and, thus, IL-13Rα′ forms a novel component of the IL-4R system. This conclusion was drawn on the basis of chemical cross-linking, immunoprecipitation, the ability of IL-13Rα′ but not IL-13Rα to augment IL-4 binding affinity, and the requirement of IL-13Rα′ for IL-4–induced STAT6 activation in Chinese hamster ovary (CHO) cells transfected with various receptor subunits. Cotransfection of IL-4 receptor p140 (termed IL-4Rβ) with γc or IL-13Rα′ increased IL-4 binding affinity and allowed for STAT6 activation in response to IL-4. However, cotransfection of all three chains did not further increase IL-4 binding or alter the extent of STAT6 activation suggesting that all three chains together do not seem to participate in IL-4 function. Instead, IL-4Rβ heterodimerizes with γc or IL-13Rα′ and mediates STAT6 activation. Cotransfection of IL-4Rβ with IL-13Rα neither increased IL-4 binding affinity nor allowed for STAT6 activation in response to IL-4 indicating that IL-13Rα does not convert binding affinity nor transmit signals for IL-4. Because IL-4 phosphorylates JAK1 and JAK2 tyrosine kinases in nonhematopoietic cells, we investigated whether JAK1 and JAK2 are required for IL-4–induced STAT6 activation in various transfectants. Cotransfection experiments with different chains of IL-4R and kinase-deficient JAK1 and JAK2 mutants in CHO cells showed that JAK1 and JAK2 are required for optimal activation of STAT6 in the α′β transfectant but only partially in the βγc transfectant. Taken together, our results show that IL-13Rα′ is a novel functional component of the IL-4R system and that JAK1 and JAK2 mediate IL-4–induced optimal activation of STAT6 in nonhematopoietic cells.
Constitutive Chemokine Production Results in Activation of Leukocyte Function-Associated Antigen-1 on Adult T-Cell Leukemia Cells
Authors: Tanaka Yoshiya , Mine Shinichiro , Figdor Carl G. , Wake Atsushi , Hirano Hideyasu , Tsukada Junichi , Aso Megumi , Fujii Koichi , Saito Kazuyoshi , van Kooyk Yvette , Eto Sumiya
Published: October 2019

Adult T-cell leukemia (ATL) is characterized by massive infiltration of circulating ATL cells into a variety of tissues, a finding often associated with poor prognosis. Leukocyte migration from circulation into tissue depends on integrin-mediated adhesion to endothelium, and integrins are tightly regulated by several stimuli, such as inflammatory chemokines. However, the exact mechanisms that enhance adherence of leukemic cells to the endothelium and infiltration into tissues remain to be fully understood. We investigated the mechanisms of extravasation of leukemic cells using ATL cells and report the following novel features of endogenous chemokine-induced adhesion of ATL cells to the endothelium. ATL cells spontaneously adhered to endothelial cells without exogenous stimulation. Integrin leukocyte function-associated antigen-1 (LFA-1) on ATL cells was spontaneously activated. ATL cells produced high amounts of chemokines, macrophage inflammatory protein-1α (MIP-1α), and MIP-1β. Adhesion of ATL cells to endothelial cells and the expression of activated form of LFA-1 were reduced by pretreatment with pertussis toxin, wortmannin, or anti–MIP-1α and MIP-1β antibodies or transfection with antisense of MIP-1α or MIP-1β. Spontaneous polymerization of cytoskeletal F-actin was observed in ATL cells, which was also inhibited by pertussis toxin and wortmannin. We propose that ATL cells adhere to endothelial cells through an adhesion cascade similar to normal leukocytes and that the chemokines produced by ATL cells are involved in triggering integrin LFA-1 through cytoskeletal rearrangement induced by G-protein–dependent activation of phosphoinositide 3-kinases in an autocrine manner. These events result in a strong adhesion of ATL cells to the endothelium and spontaneous transendothelial migration.
CD95 (APO-1/Fas) Mutations in Childhood T-Lineage Acute Lymphoblastic Leukemia
Authors: Beltinger Christian , Kurz Elke , Böhler Thomas , Schrappe Martin , Ludwig Wolf-Dieter , Debatin Klaus-Michael
Published: October 2019

CD95 (APO-1/Fas)-mediated apoptosis is pivotal in normal lymphocyte homeostasis and mutations of CD95 cause a benign autoimmune lymphoproliferation syndrome (ALPS) in humans and mice. However, tumors only rarely develop in these patients, and no CD95 mutations have yet been directly implicated in tumorigenesis. We therefore examined 81 de novo childhood T-lineage acute lymphoblastic leukemias (T-ALL) including 54 steroid-poor responders, 10 relapsed T-ALL, and 10 leukemic T-cell lines, for the presence of CD95 mutations using single-strand confirmation polymorphism and sequence analysis. In leukemic blasts and normal T cells of one patient, a heterozygous mutation in exon 3 of CD95 causing a 68Pro → 68Leu change associated with decreased CD95-mediated apoptosis was found. In leukemic blasts and normal T cells of a second patient, a homozygous mutation in the promoter of CD95 causing disruption of a consensus sequence for AP-2 binding without decreasing constitutive CD95 expression was detected. No large intragenic alterations of CD95 were found, no homozygous loss was detected in the cell lines, and no CD95 mutations were detected in the relapses. The data presented here show that CD95 mutations occur in some T-ALL and may be of biological importance.
Inhibitory Anti–Factor V Antibodies Bind to the Factor V C2 Domain and Are Associated With Hemorrhagic Manifestations
Authors: Ortel Thomas L. , Moore Karen D. , Quinn-Allen Mary Ann , Okamura Takashi , Sinclair Allen J. , Lazarchick John , Govindan Ramaswamy , Carmagnol Françoise , Kane William H.
Published: October 2019

Factor V inhibitors may develop as spontaneous autoantibodies, as alloantibodies after exposure to bovine thrombin preparations, or in factor V–deficient patients after plasma therapy. Clinical manifestations range from asymptomatic laboratory abnormalities to life-threatening hemorrhage. We have characterized the anti–factor V antibodies from 12 patients diagnosed with factor V inhibitors. In 8 patients, hemorrhagic complications (5 autoantibodies and 3 bovine thrombin-induced alloantibodies) developed, and 4 were asymptomatic (2 autoantibodies and 2 alloantibodies). The IgG fractions from all 12 patients immunoprecipitated the factor Va light chain, but only the 8 IgG fractions associated with hemorrhage inhibited factor V activity in a prothrombinase assay. Nine IgG fractions, including the 8 patients with hemorrhage, immunoprecipitated the isolated second C-type domain (C2). The 8 IgG fractions from the symptomatic patients also immunoprecipitated recombinant chimeras containing only the N-terminal third of the factor V C2 domain, and isolated recombinant C2 domain abrogated the inhibitory effect of the antibodies. Five of the inhibitory IgG fractions blocked binding of factor V to phosphatidylserine. These results suggest that inhibitory anti–factor V antibodies are associated with hemorrhagic manifestations and frequently bind to a common region within the C2 domain, whether originating spontaneously or after exposure to bovine thrombin.
Whole Blood Tissue Factor Procoagulant Activity Is Elevated in Patients With Sickle Cell Disease
Authors: Key Nigel S. , Slungaard Arne , Dandelet Luke , Nelson Stephen C. , Moertel Christopher , Styles Lori A. , Kuypers Frans A. , Bach Ronald R.
Published: October 2019

We developed a simple assay for the measurement of tissue factor procoagulant activity (TF PCA) in whole blood samples that avoids the need for mononuclear cell isolation. This method combines convenience of sample collection and processing with a high degree of sensitivity and specificity for TF. Using this method, we have determined that TF PCA is detectable in whole blood samples from normal individuals, which is itself a novel observation. Essentially all PCA could be shown to be localized in the mononuclear cell fraction of blood. Compared with controls, whole blood TF levels were significantly (P < .000001) elevated in patients with sickle cell disease (SCD), regardless of the subtype of hemoglobinopathy (SS or SC disease). No significant difference in TF PCA was observed between patients in pain crisis compared with those in steady-state disease. Because TF functions as cofactor in the proteolytic conversion of FVII to FVIIa in vitro, it was expected that an increase in circulating TF PCA would lead to an increased in vivo generation of FVIIa. On the contrary, FVIIa levels were actually decreased in the plasma of patients with SCD. Plasma TF pathway inhibitor (TFPI) antigen levels were normal in SCD patients, suggesting that accelerated clearance of FVIIa by the TFPI pathway was not responsible for the reduced FVIIa levels. We propose that elevated levels of circulating TF PCA may play an important role in triggering the activation of coagulation known to occur in patients with SCD. Because TF is the principal cellular ligand for FVIIa, it is possible that increased binding to TF accounts for the diminished plasma FVIIa levels.
Characterization of a Novel CC Chemokine, HCC-4, Whose Expression Is Increased by Interleukin-10
Authors: Hedrick Joseph A. , Helms Allison , Vicari Alain , Zlotnik Albert
Published: October 2019

We have identified and characterized a human β (CC) chemokine, designated HCC-4, that is most closely related to HCC-1 and which demonstrates chemotactic activity for monocytes. Northern analysis of multiple tissue blots and of activated monocytes mRNA shows expression of a 500-bp mRNA. A 1,500-bp mRNA was highly expressed in monocytes activated 12 hours in the presence of interleukin-10 (IL-10) but was absent in monocytes activated for only 1 hour regardless of the presence or absence of IL-10. The upregulation of expression in the presence of IL-10 is in contrast to the downregulatory effects of IL-10 on expression of most other chemokines. Recombinant HCC-4 demonstrated chemotactic activity for human monocytes and THP-1 monocyte cells but not for resting lymphocytes or neutrophils. HCC-4 also induced a Ca2+ flux in THP-1 cells that was desensitized by prior exposure to RANTES. Taken together, these data indicate that HCC-4 is a novel chemokine whose expression is uniquely upregulated by IL-10.
Combined Arsenic and Retinoic Acid Treatment Enhances Differentiation and Apoptosis in Arsenic-Resistant NB4 Cells
Authors: Giannı̀ Maurizio , Koken Marcel H.M. , Chelbi-Alix Mounira K. , Benoit Gérard , Lanotte Michel , Chen Zhu , de Thé Hugues
Published: October 2019

In the acute promyelocytic leukemia (APL) cell line NB4, as well as in APL patients' cells, arsenic trioxide (As2O3) leads to incomplete cell maturation, induction of apoptosis, as well as to the degradation of the oncogenic PML/RARα fusion protein. We have isolated an arsenic-resistant NB4 subline (NB4-AsR), which fails to undergo apoptosis, but maintains the partial differentiation response to this drug. When grown in the presence of As2O3, NB4-AsRcells degrade PML/RARα, slightly differentiate, and become more sensitive to serum deprivation-induced apoptosis. Similarly, in RA-resistant NB4-R1 cells, RA induced a significant PML/RARα degradation and yet failed to induce cell maturation. Thus, As2O3- or retinoic acid (RA)-induced PML/RARα degradation may be a prerequisite, but is not sufficient for the full differentiative/apoptotic response to these drugs. Strikingly, RA-triggered differentiation and apoptosis were greatly accelerated in As2O3-treated NB4-AsR cells. The synergism between these two agents in this setting could provide an experimental basis for combined or sequential RA/As2O3 therapies.