bluuebunny/crossref_metadata_2025 huggingface dataset:
Tissue Factor Pathway Inhibitor Gene Disruption Produces Intrauterine Lethality in Mice
Authors: Huang Zhong-Fu , Higuchi Darryl , Lasky Nina , Broze George J.
Published: October 2019

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa–dependent fashion, produces feedback inhibition of the factor VIIa/TF catalytic complex responsible for the initiation of coagulation. To further define the physiologic role of TFPI, gene-targeting techniques were used to disrupt exon 4 of the TFPI gene in mice. This exon encodes Kunitz domain-1 of TFPI, which is required for factor VIIa/TF inhibition. In mice heterozygous for TFPI gene-disruption, TFPIK1(+/−), an altered form of TFPI lacking Kunitz domain-1, circulates in plasma at a concentration ∼40% that of wild-type TFPI. TFPIK1(+/−) animals have plasma TFPI activity ∼50% that of wild-type mice, based on a functional assay that measures factor VIIa/TF inhibition, and have a normal phenotype. Sixty percent of TFPIK1(−/−) mice die between embryonic days E9.5 and E11.5 with signs of yolk sac hemorrhage. The extent of structural abnormalities within the yolk sac vascular system appears to mirror the condition of the embryo, suggesting that the embryonic and extra-embryonic tissues are both responding to same insult, presumably circulatory insufficiency. Organogenesis is normal in TFPIK1 null animals that progress beyond E11.5, but hemorrhage, particularly in the central nervous system and tail, is evident during later gestation and none of the TFPIK1(−/−) mice survive to the neonatal period. The presence of immunoreactive fibrin(ogen) in the liver and intravascular thrombi is consistent with the notion that unregulated factor VIIa/TF action and a consequent consumptive coagulopathy underlies the bleeding diathesis in these older embryos. Human TFPI-deficient embryos may suffer a similar fate because an individual with TFPI deficiency has not been identified.
In Vitro Behavior of Hematopoietic Progenitor Cells Under the Influence of Chemoattractants: Stromal Cell–Derived Factor-1, Steel Factor, and the Bone Marrow Environment
Authors: Kim Chang H. , Broxmeyer Hal E.
Published: October 2019

How multiple chemoattractants cooperate in directing the migration of hematopoietic progenitor cells (HPC) for homing and peripheral blood mobilization has not yet been established. We report here the behavior of HPC under the influence of two different chemoattractants, stromal cell-derived factor (SDF)-1 and steel factor (SLF), and the chemotactic nature of the bone marrow (BM) environment using a two-chamber in vitro migration system. Various formulae were adopted to quantitate these effects. Based on these quantitations, SDF-1 showed only chemotactic activity, while SLF showed both chemotactic and chemokinetic activities on factor-dependent MO7e cells. SLF, like SDF-1, attracted human HPC from a population of CD34+ cells and induced actin polymerization in MO7e cells. SLF and SDF-1 cooperated in attracting MO7e cells, as well as cord blood (CB) and BM CD34+cells. A negative concentration gradient of SLF and SDF-1, formed by the presence of chemoattractants in the upper chamber, showed potent inhibitory effects on MO7e cell migration induced by either of these chemoattractants in the lower chamber, and SDF-1 and SLF were synergistic in mobilizing cells to the lower chamber from this negative chemoattractant gradient. Plasma obtained from BM aspirates, but not CB or peripheral blood, showed strong chemotactic effects on BM and CB CD34+ cells, and an inhibitory effect in a negative gradient on SDF-1–dependent CD34+ cell migration. These in vitro migration experiments suggest that chemoattractants such as SDF-1 and SLF with other unidentified BM chemoattractants may be involved cooperatively in the migration of HPC to the BM and in preventing spontaneous mobilization of HPC out of the BM.
Stroma-Contact Prevents Loss of Hematopoietic Stem Cell Quality During Ex Vivo Expansion of CD34+ Mobilized Peripheral Blood Stem Cells
Authors: Breems Dimitri A. , Blokland Ellen A.W. , Siebel Karen E. , Mayen Angelique E.M. , Engels Lilian J.A. , Ploemacher Rob E.
Published: October 2019

Stroma-supported long-term cultures (LTC) allow estimation of stem cell quality by simultaneous enumeration of hematopoietic stem cell (HSC) frequencies in a graft using the cobblestone area forming cell (CAFC) assay, and the ability of the graft to generate progenitors in flask LTC (LTC-CFC). We have recently observed that the number and quality of mobilized peripheral blood stem cells (PBSC) was low in patients having received multiple rounds of chemotherapy. Moreover, grafts with low numbers of HSC and poor HSC quality had a high probability to cause graft failure upon their autologous infusion. Because ex vivo culture of stem cells has been suggested to present an attractive tool to improve hematological recovery or reduce graft size, we have studied the possibility that such propagation may affect stem cell quality. In order to do so, we have assessed the recovery of different stem cell subsets in CD34+ PBSC after a 7-day serum-free liquid culture using CAFC and LTC-CFC assays. A numerical expansion of stem cell subsets was observed in the presence of interleukin-3 (IL-3), stem cell factor, and IL-6, while stroma-contact, stromal soluble factors, or combined addition of FLT3-ligand and thrombopoietin improved this parameter. In contrast, ex vivo culture severely reduced the ability of the graft to produce progenitors in LTC while stromal soluble factors partly abrogated this quality loss. The best conservation of graft quality was observed when the PBSC were cultured in stroma-contact. These data suggest that ex vivo propagation of PBSC may allow numerical expansion of various stem cell subsets, however, at the expense of their quality. In addition, cytokine-driven PBSC cultures require stroma for optimal maintenance of graft quality.
Purine Nucleotides Induce Regulated Secretion of von Willebrand Factor: Involvement of Cytosolic Ca2+ and Cyclic Adenosine Monophosphate–Dependent Signaling in Endothelial Exocytosis
Authors: Vischer Ulrich M. , Wollheim Claes B.
Published: October 2019

von Willebrand factor (vWF) is stored and released from endothelial secretory granules called Weibel-Palade (WP) bodies. Acute release can be induced by thrombin, histamine, and other mediators of thrombosis or inflammation. Their effect is thought to be mediated by an increase in intracellular free calcium ([Ca2+]i). Purine nucleotides such as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) are released from platelet dense granules and from ischemic tissues and are important regulators of platelet function and vascular tone. In the present study, we investigated whether they could also induce exocytosis from cultured endothelial cells. ATP (1 to 100 μmol/L) induced a dose-related increase in vWF release, with a 2.3-fold maximal increase after 30 minutes. Similar responses were observed with ADP. ATP induced calcium mobilization from intracellular stores, an effect mimicked by 2-methylthio-ATP, a selective agonist for P2y receptors. However, 2-methylthio-ATP–induced vWF release was only 43% of the ATP response. ATP-induced vWF release was also associated with a twofold increase in cellular cyclic adenosine monophosphate (cAMP) content, and was potentiated by 3-isobutyl-1-methylxanthine ([IBMX] added to increase cAMP levels by blocking cellular phosphodiesterases) and 8-bromo-cAMP and inhibited by more than 50% by Rp-8-CPT-cAMPS, a competitive protein kinase A inhibitor. Adenosine but not 2-methylthio-ATP mimicked the ATP-induced increase in cAMP. ATP-induced vWF release was partly inhibited by adenosine deaminase, which degrades adenosine generated from ATP in the incubation medium. Adenosine (1 to 100 μmol/L) failed to induce vWF release, but potentiated the secretory response to 2-methylthio-ATP and thrombin without modifying the calcium response to these agents. Our results suggest that ATP/ADP can induce vWF release from endothelial cells via dual activation of P2y and adenosine A2 receptors. ATP/ADP-induced exocytosis could be involved in the regulation of thrombus formation and ischemia-reperfusion injuries. Further, we provide evidence that a receptor-mediated increase in cellular cAMP can potentiate the secretory response to calcium-mobilizing agents.
Identification of a Large Deletion, Spanning Exons 4 to 11 of the Human Factor XIIIA Gene, in a Factor XIII-Deficient Family
Authors: Anwar Rashida , Miloszewski Krzysztof J.A. , Markham Alexander F.
Published: October 2019

Inherited deficiency of factor XIIIA subunit (FXIIIA) is an autosomal recessive disorder that is characterized by a life-long bleeding tendency and complications in wound healing. Molecular genetic studies have shown the deficiency can be due to small sequence changes within the FXIIIA gene, such as point mutations or microdeletions. On molecular analysis of the FXIIIA gene in an FXIII-deficient patient, of United Kingdom origin, we identified a putative homozygous missense mutation, Arg408Gln. However, the father of this patient is homozygous normal for arginine at codon 408. Having proved paternity in this pedigree by microsatellite analysis, we examined the FXIIIA RNA of the patient by reverse transcriptase-polymerase chain reaction and found the paternal allele to lack exons 4 through 11 inclusive. Hence, a huge deletion extending from intron 3 to intron 11 and the Arg408Gln mutation are jointly responsible for FXIIIA deficiency in this family. This is the first finding of such a large deletion in the FXIIIA gene.
Diagnosis and Clinical Course of Autoimmune Neutropenia in Infancy: Analysis of 240 Cases
Authors: Bux Juergen , Behrens Georg , Jaeger Gudrun , Welte Karl
Published: October 2019

Primary autoimmune neutropenia (AIN) is caused by granulocyte-specific autoantibodies and occurs predominantly in infancy. Clinical presentation and diagnosis have not been well established, resulting in burdening diagnostic investigations and unnecessary treatment with granulocyte colony-stimulating factor (G-CSF). In the present study, clinical, laboratory, and immunologic data of 240 infants with primary AIN were evaluated. Suspected association with parvovirus B19 infection was investigated using serologic and DNA-based methods. Primary AIN was mainly diagnosed at the age of 5 to 15 months but was observed as early as day 33 of life. In 90% of the cases, AIN was associated with benign infections despite severe neutropenia. Spontaneous remission, shown by 95% of the patients, usually occurred within 7 to 24 months. Autoantibodies in the patient's sera were not always present, and screening had to be repeated several times until antibody detection succeeded. About 35% of the autoantibodies showed preferential binding to granulocytes from NA1 and NA2 homozygous donors. Bone marrow was typically normocellular or hypercellular, with a variably diminished number of segmented granulocytes. A significant association with parvovirus B19 infection was not found. Symptomatic treatment with antibiotics was sufficient in most patients. Eighty-nine percent of the patients received antibiotics (cotrimoxazole) for prophylaxis of infections. For severe infections or for surgical preparation, G-CSF, corticosteroids, and intravenous IgG were administered, resulting in increased neutrophil counts in 100%, 75%, and 50% of the patients treated, respectively. In combination with the detection of granulocyte-specific antibodies, the typical clinical picture allowed diagnosis of AIN without burdening investigations. Treatment with G-CSF was found to be a reliable alternative to temporarily increase the neutrophil count.
Lack of Interferon Consensus Sequence Binding Protein (ICSBP) Transcripts in Human Myeloid Leukemias
Authors: Schmidt Manuel , Nagel Stefan , Proba Jutta , Thiede Christian , Ritter Markus , Waring Jeffrey F. , Rosenbauer Frank , Huhn Dieter , Wittig Burghardt , Horak Ivan , Neubauer Andreas
Published: October 2019

Interferon consensus sequence binding protein (ICSBP) was first identified as a transcription factor of the interferon (IFN) regulatory factor family (IRF) which regulates expression of IFN-dependent genes by binding to DNA at specific sites, IFN-stimulated responsive elements. Analysis of ICSBP-deficient mice showed hematologic alterations similar to chronic myelogenous leukemia (CML) in humans and suggested a novel role for ICSBP in regulating proliferation and differentiation of hematopoietic progenitor cells. Here we show that ICSBP-mRNA expression is impaired in human myeloid leukemias: 27 of 34 CML patients (79%) and 21 of 32 patients with acute myeloid leukemia (AML) (66%) showed very low or absent transcript numbers of ICSBP. In contrast, only 2 of 33 normal volunteers (6%) showed low transcription of ICSBP(P < .0001 both for CML and AML values). The lack of expression was not associated with lack of lymphatic cells, which normally have been shown to express ICSBP at the highest level. More detailed analysis showed an absence of ICSBP-mRNA also in sorted B cells derived from CML patients. To analyze whetherICSBP may be induced in leukemic cells, ex vivoexperiments using a known inducer of ICSBP, IFN-γ, were performed. Ex vivo treatment of primary CML cells using IFN-γ resulted in induction of ICSBP transcripts. Furthermore, samples of CML patients during IFN-α treatment were analyzed. In 11 of 12 CML patients ICSBP-mRNA was inducible upon in vivo treatment with IFN-α, but decreased with progression of CML. Stable transfection of K-562 cell line with ICSBP led to no difference in bcr-abl expression in vitro, although two patients showed an inverse correlation between bcr-abl andICSBP in vivo. These data suggest that lack of ICSBPmay have an important role also in human myeloid leukemogenesis.
Somatic Triple Mosaicism in a Carrier of X-Linked Chronic Granulomatous Disease
Authors: de Boer Martin , Bakker Egbert , Van Lierde Stefaan , Roos Dirk
Published: October 2019

The X-linked form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene, which encodes the 91-kD subunit of the flavocytochrome b558, a component of the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytic leukocytes. Mutations in this gene are very heterogeneous and often unique for one family. Here we report on a family with two patients (brothers), one with a 3-kb deletion comprising exon 5 and the other with a 3.5-kb deletion comprising exons 6 and 7 of the CYBB gene. Sequence analysis of polymerase chain reaction (PCR)-amplified genomic DNA proved these deletions to be overlapping for 35 bp. Analysis by restriction fragment length polymorphism of genomic DNA from the mother's leukocytes showed her to be a carrier of both deletions in addition to the normal CYBB sequence. This triple somatic mosaicism was confirmed with PCR-amplified genomic and complementary DNA. The presence of the normal CYBB gene in the mother was also proven by the finding of normal superoxide-generating neutrophils in addition to cells lacking this ability. Triple X syndrome was excluded. These findings suggest that the mutations are the result of an event in early embryogenesis of the mother, possibly involving a mechanism like sister chromatid exchange.
Multilineage Hematopoietic Recovery by a Single Injection of Pegylated Recombinant Human Megakaryocyte Growth and Development Factor in Myelosuppressed Mice
Authors: Shibuya Kazunori , Akahori Hiromichi , Takahashi Kazumi , Tahara Emiko , Kato Takashi , Miyazaki Hiroshi
Published: October 2019

Previous studies have shown that daily multiple administration of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) markedly stimulates thrombopoiesis and effectively ameliorates thrombocytopenia, and in most cases anemia and neutropenia, in myelosuppressed animals. In this study, we evaluated the effects of a single intravenous injection of PEG-rHuMGDF on hematopoietic recovery after sublethal total-body irradiation in mice. A single injection of PEG-rHuMGDF (1 to 640 μg/kg) 1 hour after irradiation accelerated platelet, red blood cell (RBC), and white blood cell (WBC) recovery in a dose-dependent fashion. In the bone marrow of vehicle-treated mice, megakaryocytic, erythroid, and myeloid progenitors, as well as day 12 colony-forming unit–spleen (CFU-S), were dramatically decreased much earlier than the nadirs of peripheral blood cells, whereas megakaryocytes were modestly decreased. Treatment with PEG-rHuMGDF (80 μg/kg, an optimal dose) 1 hour after irradiation resulted in more rapid recovery of these four hematopoietic progenitors and also significantly facilitated megakaryocyte recovery. In addition, the same PEG-rHuMGDF administration schedule expanded bone marrow cells capable of rescuing lethally irradiated recipient mice. As the interval between irradiation and PEG-rHuMGDF treatment was longer, its effects on hematopoietic recovery were attenuated. In contrast to the effects of PEG-rHuMGDF, a single injection of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 1 hour after irradiation exclusively accelerated WBC recovery, but only to a similar extent as PEG-rHuMGDF (80 μg/kg) treatment even when rhG-CSF doses were escalated to 1,000 μg/kg. This appeared related to different pharmacokinetics of these two factors after a single injection in irradiated mice. The concentrations of PEG-rHuMGDF after injection persisted in the plasma for a longer time compared with rhG-CSF. These results indicate that a single injection of PEG-rHuMGDF at an early time after irradiation is able to effectively improve thrombocytopenia, anemia, and leukopenia with concomitant accelerated recovery of both primitive and committed hematopoietic progenitors in irradiated mice. Our data also show that compared with the rhG-CSF shown to exert multilineage effects on hematopoiesis, PEG-rHuMGDF has more wide-ranging effects on peripheral blood cell recovery.
Progressive Telomere Shortening in Aplastic Anemia
Authors: Ball Sarah E. , Gibson Frances M. , Rizzo Siân , Tooze Jennifer A. , Marsh Judith C.W. , Gordon-Smith Edward C.
Published: October 2019

Improved survival in aplastic anemia (AA) has shown a high incidence of late clonal marrow disorders. To investigate whether accelerated senescence of hematopoietic stem cells might underlie the pathophysiology of myelodysplasia (MDS) or paroxysmal nocturnal hemoglobinuria (PNH) occurring as a late complication of AA, we studied mean telomere length (TRF) in peripheral blood leukocytes from 79 patients with AA, Fanconi anemia, or PNH in comparison with normal controls. TRF lengths in the patient group were significantly shorter for age than normals (P < .0001). Telomere shortening was apparent in both granulocyte and mononuclear cell fractions, suggesting loss at the level of the hematopoietic stem cell. In patients with acquired AA with persistent cytopenias (n = 40), there was significant correlation between telomere loss and disease duration (r = −.685; P < .0001), equivalent to progressive telomere erosion at 216 bp/yr, in addition to the normal age-related loss. In patients who had achieved normal full blood counts (n = 20), the rate of telomere loss had apparently stabilised. There was no apparent association between telomere loss and secondary PNH (n = 13). However, of the 5 patients in the study with TRF less than 5.0 kb, 3 had acquired cytogenetic abnormalities, suggesting that telomere erosion may be relevant to the pathogenesis of MDS in aplastic anemia.
Dose-Intensive Melphalan With Blood Stem-Cell Support for the Treatment of AL (Amyloid Light-Chain) Amyloidosis: Survival and Responses in 25 Patients
Authors: Comenzo Raymond L. , Vosburgh Evan , Falk Rodney H. , Sanchorawala Vaishali , Reisinger Johann , Dubrey Simon , Dember Laura M. , Berk John L. , Akpek Gorgun , LaValley Michael , O'Hara Carl , Arkin Charles F. , Wright Daniel G. , Skinner Martha
Published: October 2019

AL (amyloid light-chain) amyloidosis is an uncommon plasma cell disorder in which depositions of amyloid light-chain protein cause progressive organ failure and death in a median of 13 months. Autologous stem-cell transplantation is effective therapy for multiple myeloma and therefore, we evaluated its efficacy for AL amyloidosis. Patients with adequate cardiac, pulmonary, and renal function had stem cells mobilized with granulocyte-colony stimulating factor and were treated with dose-intensive intravenous melphalan (200 mg/m2). Response to therapy was determined by survival and improvement of performance status, complete response or persistence of the clonal plasma cell disorder, and change in the function of organs involved with amyloid at baseline. We enrolled 25 patients with a median age of 48 years (range, 29-60), all of whom had biopsy-proven amyloidosis with clonal plasma cell disorders. Twenty-two (88%) were Southwest Oncology Group performance status 1 or 2 within a year of diagnosis, and 16 (64%) had received no prior therapy. Predominant amyloid-related organ involvement was cardiac (n = 8), renal (n = 7), hepatic (n = 6), neuropathic (n = 3), and lymphatic (n = 1). Fifteen patients had one or two organ systems involved, whereas 10 had three or more involved. With a median follow-up of 24 months (12-38), 17 of 25 patients (68%) are alive, and the median survival has not been reached. Thirteen of 21 patients (62%) evaluated 3 months posttransplant had complete responses of their clonal plasma cell disorders. Currently, two thirds of the surviving patients (11 of 17) have experienced improvements of amyloid-related organ involvement in all systems, whereas 4 of 17 have stable disease. The improvement in the median performance status of the 17 survivors at follow-up (0 [range, 0-3]) is statistically significant versus baseline (2 [range, 1-3]; P < .01). Significant negative prognostic factors with respect to overall survival include amyloid involvement of more than two major organ systems and predominant cardiac involvement. Three patients have experienced relapses of the clonal plasma cell disorder at 12 and 24 months. Dose-intensive therapy should currently be considered as the preferred therapy for patients with AL amyloidosis who meet functional criteria for autologous transplantation.
Myeloid Development Is Selectively Disrupted in PU.1 Null Mice
Authors: Anderson Karen L. , Smith Kent A. , Conners Kris , McKercher Scott R. , Maki Richard A. , Torbett Bruce E.
Published: October 2019

The ets family transcription factor PU.1 is expressed in monocytes/macrophages, neutrophils, mast cells, B cells, and early erythroblasts, but not in T cells. We have recently shown that PU.1 gene disruption results in mice with no detectable monocytes/macrophages and B cells but T-cell development is retained. Although neutrophil development occurred in these mice, it was delayed and markedly reduced. We now proceed to demonstrate that PU.1 null hematopoietic cells fail to proliferate or form colonies in response to macrophage colony-stimulating factor (M-CSF), granulocyte CSF (G-CSF), and granulocyte/macrophage CSF (GM-CSF). In contrast, PU.1 null cells did proliferate and form colonies in response to interleukin-3 (IL-3), although the response was reduced as compared with control littermates. Compared with control cells, PU.1 null cells had minimal expression of G- and GM-CSF receptors and no detectable M-CSF receptors. The size of individual myeloid colonies produced from PU.1 null primitive and committed myeloid progenitors in the presence of IL-3, IL-6, and stem cell factor (SCF) were reduced compared with controls. Under these conditions, PU.1 null progenitors produced neutrophils but not monocytes/macrophages. These observations suggest that PU.1 gene disruption induces additional cell-autonomous effects that are independent of the alterations in myeloid growth factor receptor expression. Our results demonstrate that PU.1 gene disruption affects a number of developmentally regulated hematopoietic processes that can, at least in part, explain the changes in myeloid development and reduction in myeloid and neutrophil expansion observed in PU.1 null mice.
Endomitosis of Human Megakaryocytes Are Due to Abortive Mitosis
Authors: Vitrat Natacha , Cohen-Solal Karine , Pique Claudine , LeCouedic Jean Pierre , Norol Françoise , Larsen Annette K. , Katz André , Vainchenker William , Debili Najet
Published: October 2019

During megakaryocyte differentiation, the promegakaryoblast (immature megakaryocyte) increases its ploidy to a 2x DNA content by a poorly understood process called endomitosis. This leads to the formation of a giant cell, the megakaryocyte (MK), which subsequently gives rise to platelets. In this report, we show that endomitotis of human MKs is due to abortive mitosis. Human MKs were obtained by a two-step purification of CD34+ blood or marrow precursors followed by in vitro culture in the presence of MK growth factors. Microcoscopic examination shows that a large number of centrosomes (up to 32) and centrioles are present in polyploid MKs. After nocodazole treatment, more than 20% of the MK are blocked in a typical pseudo-metaphase. Both spontaneous and nocodazole-induced endomitosis are associated with a breakdown of the nuclear envelope and possess a complex mitotic spindle composed of several asters. Spindle microtubules radiate from each aster, creating a spherical structure. At metaphase, expression of the kinetochore phosphoepitope recognized by the 3F3/2 antibody is lost, and the sister chromatides segregate moving toward the spindle poles. After limited segregation, the chromosomes decondense and the nuclear envelope reforms in the absence of cytokinesis, isolating all chromosomes in a single nucleus. It has been proposed that endomitosis could be due to an abnormal CDK1 activity or an absence of cyclin B1. Our results show that cyclin B1 can be detected in all MKs, including those with a ploidy of 8N or more. The cyclin B1 staining colocalizes with the mitotic spindle. Using flow cytometry, the level of cyclin B1 increased until 8N, but remained identical in 16N and 32N MKs. Cell sorting was used to separate the MKs into a 2N/4N and >4N population. Both cyclin B1 and CDK1 could be detected in the endomitotic polyploid MKs using Western blot analysis, and a histone H1 kinase activity was associated with immunoprecipitated cyclin B1. We conclude that endomitosis of human MKs is due to abortive mitosis, possibly due to alterations in the regulation of mitotic exit.
Human CD34+ Bone Marrow Cells Regulate Stromal Production of Interleukin-6 and Granulocyte Colony-Stimulating Factor and Increase the Colony-Stimulating Activity of Stroma
Authors: Gupta Pankaj , Blazar Bruce R. , Gupta Kalpna , Verfaillie Catherine M.
Published: October 2019

Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34+ cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34+cells for 3 to 5 weeks. Coculture of more mature CD15+/CD14− myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34+ nor CD15+/CD14−cells produced IL-6, G-CSF, IL-1β, or tumor necrosis factor α. When CD34+ cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34+ cells or when CD34+ cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit–granulocyte-macrophage–stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34+ progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant.
Human Hematopoietic Progenitors Express Erythropoietin
Authors: Stopka T. , Zivny J.H. , Stopkova P. , Prchal J.F. , Prchal J.T.
Published: October 2019

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34+lin− hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34+ cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34+ cells represent a heterogeneous cell population, we analyzed individual burst-forming units–erythroid (BFU-E) and nonerythroid colony-forming unit–granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.
Platelet Aggregation Induced by a Monoclonal Antibody to the A1 Domain of von Willebrand Factor
Authors: Depraetere Hilde , Ajzenberg Nadine , Girma Jean-Pierre , Lacombe Catherine , Meyer Dominique , Deckmyn Hans , Baruch Dominique
Published: October 2019

Shear-induced platelet aggregation (SIPA) involves von Willebrand Factor (vWF) binding to platelet glycoprotein (GP)Ib at high shear stress, followed by the activation of αIIbβ3. The purpose of this study was to determine the vWF sequences involved in SIPA by using monoclonal antibodies (MoAbs) to vWF known to interfere with its binding to GPIb and to αIIbβ3. Washed platelets were exposed to shear rates between 100 and 4,000 seconds−1 in a rotational viscometer. SIPA was quantitated by flow cytometry as the disappearance of single platelets (DSP) in the sheared sample in the presence of vWF, relative to a control in the absence of shear and vWF. At a shear rate of 4,000 seconds−1, DSP was increased from 5.9% ± 3.5% in the absence of vWF to 32.7% ± 6.3% in the presence of vWF. This increase in SIPA was not associated with an elevation of P-selectin expression. vWF-dependent SIPA was completely abolished by MoAb 6D1 to GPIb and partially inhibited by MoAb 10E5 to αIIbβ3. Three MoAbs to vWF were compared for their effect on SIPA at 4,000 seconds−1 in the presence of vWF: MoAb 328, known to block vWF binding to GPIb in the presence of ristocetin, MoAb 724 blocking vWF binding to GPIb in the presence of botrocetin, and MoAb 9, an inhibitor of vWF binding to αIIbβ3. Similar to the effect of MoAb 6D1, MoAb 328 completely inhibited the effect of vWF, whereas MoAb 9 had a partial inhibitory effect, as MoAb 10E5 did. In contrast, MoAb 724, as well as its F(ab′)2 fragments, promoted shear-dependent platelet aggregation (165% of the DSP value obtained in the absence of MoAb 724), indicating that MoAb 724 was responsible for an enhanced aggregation, which was independent of binding to the platelet Fcγ receptor. In addition, the enhancement of aggregation induced by MoAb 724 was abrogated by MoAb 6D1 or 10E5 to the level of SIPA obtained in the presence of vWF incubated with a control MoAb to vWF. Finally, the activating effect of MoAb 724 was also found under static conditions at ristocetin concentrations too low to induce platelet aggregation. Our results suggested that on binding to a botrocetin-binding site on vWF, MoAb 724 mimics the effect of botrocetin by inducing an active conformation of vWF that is more sensitive to shear stress or to low ristocetin concentration.
Expression of CD86 on Human Marrow CD34+ Cells Identifies Immunocompetent Committed Precursors of Macrophages and Dendritic Cells
Authors: Ryncarz Rita E. , Anasetti Claudio
Published: October 2019

Macrophages and dendritic cells derive from a hematopoietic stem cell and the existence of a common committed progenitor has been hypothesized. We have recently found in normal human marrow a subset of CD34+ cells that constitutively expresses HLA-DR and low levels of CD86, a natural ligand for the T cell costimulation receptor CD28. This CD34+ subset can elicit responses from allogeneic T cells. In this study, we show that CD34+/CD86+ cells can also present tetanus toxoid antigen to memory CD4+ T cells. CD86 is expressed at low levels in macrophages and high levels in dendritic cells. Therefore, we have tested the hypothesis that CD34+/CD86+ cells are the common precursors of both macrophages and dendritic cells. CD34+/CD86+ marrow cells cultured in granulocyte-macrophage colony-stimulating factor (GM-CSF)–generated macrophages. In contrast, CD34+/CD86− cells cultured in GM-CSF generated a predominant population of granulocytes. CD34+/CD86+ cells cultured in GM-CSF plus tumor necrosis factor-α (TNF-α) generated almost exclusively CD1a+/CD83+ dendritic cells. In contrast, CD34+/CD86− cells cultured in GM-CSF plus TNF-α generated a variety of cell types, including a small population of dendritic cells. In addition, CD34+/CD86+ cells cultured in granulocyte colony-stimulating factor failed to generate CD15+granulocytes. Therefore, CD34+/CD86+ cells are committed precursors of both macrophages and dendritic cells. The ontogeny of dendritic cells was recapitulated by stimulation of CD34+/CD86− cells with TNF-α that induced expression of CD86. Subsequent costimulation of CD86+cells with GM-CSF plus TNF-α lead to expression of CD83 and produced terminal dendritic cell differentiation. Thus, expression of CD86 on hematopoietic progenitor cells is regulated by TNF-α and denotes differentiation towards the macrophage or dendritic cell lineages.
Clinical Value of Soluble IgG Fc Receptor Type III in Plasma From Patients With Chronic Idiopathic Neutropenia
Authors: Koene Harry R. , de Haas Masja , Kleijer Marion , Huizinga Tom W.J. , Roos Dirk , von dem Borne Albert E.G.Kr.
Published: October 2019

Previous studies have shown that the plasma level of soluble IgG Fc receptor type III (sFcγRIII) is a measure of the total body neutrophil mass. The aim of this study was to determine whether the plasma level sFcγRIII is associated with the risk of contracting bacterial infections in patients with neutropenia. We collected blood from 66 patients suffering from acquired idiopathic neutropenia, whose blood was sent to our laboratory for diagnostic evaluation of neutropenia (neutrophil count <1,500 cells/μL). Soluble FcγRIII levels were measured in plasma. Genotype distibutions of FcγR polymorphisms were determined. Clinical data were obtained from the patient files. Patients were assessed as to whether or not they had suffered from a bacterial infection 3 months before to 3 months after a single sFcγRIII measurement. In addition, longitudinal data were obtained from 21 patients. Of the 66 neutropenic patients who were included, 15 had suffered from a bacterial infection in the period 3 months before to 3 months after sFcγRIII measurement. The age and sex distribution was equal among the groups with and without infections, as were the genotype frequencies of neutrophil FcγR polymorphisms. Both neutrophil count and plasma level sFcγRIII were significantly lower in the patient group with infections, compared with the noninfected group (P = .03 and P < .0001, respectively). No infections were reported for patients who had plasma sFcγRIII levels above 100 arbitrary units (AU; normal value, 30 to 200). After matching each infected patient with two noninfected patients having the same neutrophil count, sFcγRIII plasma levels remained significantly lower in the group with infections (P = .0001). For the patients who were followed in time, no infections were reported when sFcγRIII levels were above 100 AU. In conclusion, our population of patients with chronic idiopathic neutropenia with plasma sFcγRIII levels above 100 AU did not show an increased risk of contracting bacterial infections.
Thrombopoietin Enhances the Production of Myeloid Cells, but not Megakaryocytes, in Juvenile Chronic Myelogenous Leukemia
Authors: Sawai Nobukuni , Koike Kenichi , Higuchi Tsukasa , Ogami Kinya , Oda Megumi
Published: October 2019

We previously reported the aberrant growth of granulocyte-macrophage (GM) progenitors induced by a combination of stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in juvenile chronic myelogenous leukemia (JCML). We examined here the effects of thrombopoietin (TPO) on the proliferation and differentiation of hematopoietic progenitors in JCML. In serum-deprived single-cell cultures of normal bone marrow (BM) CD34+CD38high cells, the addition of TPO to the culture containing SCF + GM-CSF resulted in an increase in the number and size of GM colonies. In the JCML cultures, in contrast, the number of SCF + GM-CSF–dependent GM colonies was not increased by the addition of TPO. However, the TPO addition caused an enlargement of GM colonies in cultures from the JCML patients to a significantly greater extent compared with the normal controls. There was no difference in the type of the constituent cells of GM colonies with or without TPO grown by JCML BM cells. A flow cytometric analysis showed that the c-Mpl expression was found on CD13+ myeloid cells generated by CD34+CD38high BM cells from JCML patients, but was at an undetectable level in normal controls. The addition of TPO to the culture containing SCF or SCF + GM-CSF caused a significant increase in the production of GM colony-forming cells by JCML CD34+CD38neg/lowpopulation, indicating the stimulatory effects of TPO on JCML primitive hematopoietic progenitors. Normal BM cells yielded a significant number of megakaryocytes as well as myeloid cells in response to a combination of SCF, GM-CSF, and/or TPO. In contrast, megakaryocytic cells were barely produced by the JCML progenitors. Our results may provide a fundamental insight that the administration of TPO enhances the aberrant growth of GM progenitors rather than the recovery of megakaryocytopoiesis.
Methylenetetrahydrofolate Reductase C677T Mutation, Plasma Homocysteine, and Folate in Subjects From Northern Italy With or Without Angiographically Documented Severe Coronary Atherosclerotic Disease: Evidence for an Important Genetic-Environmental Interaction
Authors: Girelli Domenico , Friso Simonetta , Trabetti Elisabetta , Olivieri Oliviero , Russo Carla , Pessotto Renzo , Faccini Giovanni , Pignatti Pier Franco , Mazzucco Alessandro , Corrocher Roberto
Published: October 2019

Moderate elevation of plasma total homocysteine (tHcy) is a strong and independent risk factor for coronary artery disease (CAD). It can result from genetic or nutrient-related disturbances in the transsulfuration or remethylation pathways for Hcy metabolism. A point mutation (C677T; Ala-to-Val) in the gene encoding the 5,10-methylenetetrahydrofolate reductase (MTHFR) has been recently reported to render the enzyme thermolabile and less active. Studies on the role of this mutation as a risk factor for CAD have given conflicting results. We studied a total of 415 subjects, 278 with angiographically documented multivessel CAD and 137 with angiographically documented normal coronary arteries. The overall frequency of the MTHFR V/V homozygous genotype was 15.7% (with 52.5% heterozygous and 31.8% normal). Subgroup analysis showed no significant differences between CAD and CAD-free subjects. A genotype/phenotype correlation study showed a marked effect of folate on the association between MTHFR genotypes and tHcy. Among individuals with folate levels below the median (11.5 nmol/L), fasting tHcy was significantly increased not only in V/V homozygotes (by 59%) but also, at intermediate values, in A/V heterozygotes (by 21% on average). Conversely, the mutation resulted neutral with respect to tHcy levels in subjects with adequate folate levels. We conclude that, in our population, the MTHFR C677T mutation is rather common, but it does not appear to be associated per se to CAD. A genetic-environmental interaction may contribute to the vascular risk by elevating tHcy when folate status is low.
RhoA and the Function of Platelet Integrin αIIbβ3
Authors: Leng Lijun , Kashiwagi Hirokazu , Ren Xiang-Dong , Shattil Sanford J.
Published: October 2019

Integrins respond to “inside-out” signals, which enable them to bind adhesive ligands, and ligand binding initiates “outside-in” signals that mediate anchorage-dependent cellular responses. RhoA is a GTPase that regulates certain actin rearrangements and transcriptional events. It has also been implicated in integrin signaling, but the exact relationship is not understood. To examine this further, platelets were incubated with C3 exoenzyme to adenine diphosphate (ADP)-ribosylate and inactivate RhoA, and the function of integrin αIIbβ3 was studied. Despite inactivation of ≥ 90% of RhoA, platelets exhibited normal inside-out signaling, as monitored by agonist-induced binding of a fibrinogen-mimetic anti-αIIbβ3 antibody and normal fibrinogen-dependent aggregation. On the other hand, RhoA inactivation decreased the adhesion of agonist-stimulated platelets to fibrinogen (P < .04) and the formation of vinculin-rich focal adhesions in platelets that did adhere (P < .001). These effects were selective because fibrin clot retraction, a response also dependent on αIIbβ3 and actin contractility, was unaffected by C3, as was the content of F-actin in resting or agonist-stimulated platelets. Similar results were obtained in a Chinese hamster ovary (CHO) cell model system of αIIbβ3: C3 exoenzyme (or overexpression of dominant-negative N19RhoA) failed to influence integrin activation state, but it blocked the formation of focal adhesions in cells spread on fibrinogen. These studies establish that RhoA plays a highly selective role in αIIbβ3 signaling, and they identify a subset of responses to integrin ligation that may be uniquely dependent on the actin rearrangements regulated by this GTPase.
Correction of Abnormal T-Cell Receptor Repertoire During Interferon-α Therapy in Patients With Hairy Cell Leukemia
Authors: Kluin-Nelemans Hanneke C. , Kester Michel G.D. , van deCorput Lisette , Boor Patrick P.C. , Landegent Jim E. , van Dongen Jacques J.M. , Willemze Roel , Falkenburg J.H. Frederik
Published: October 2019

Patients with the B-cell malignancy hairy cell leukemia (HCL) exhibit a skewed T-cell repertoire with oligoclonal expression or absence of many members of the T-cell receptor (TCR) BV gene families. To evaluate whether interferon-α (IFN-α) therapy would not only restore normal hematopoiesis, but also the abnormal T-cell repertoire, we studied T lymphocytes from a cohort of HCL patients treated by IFN-α in the past, at initiation, and at several intervals up to 6 years of IFN-α treatment. The junctional regions from 22 TCRBV gene families were analyzed after polymerase chain reaction amplification of cDNA (RT-PCR) using family specific primers. In all seven patients improvement of the skewed T-cell repertoire was not seen until 2 years of treatment. It consisted of disappearance of oligoclonal subpopulations and (polyclonal) reappearance of absent TCRBV gene families. The RT-PCR results were correlated with the TCRBV protein expression using TCRBV-specific monoclonal antibodies. T lymphocytes from four patients with active HCL contained large expansions of particular TCRBV-expressing cells (up to 25% of the CD3+cells; 600 to 700/μL whole blood), which decreased during IFN-α therapy in both patients tested. Finally, restoration of the TCR repertoire matched normalization of the functional immune repertoire as measured by proliferative, helper, and cytotoxic T-lymphocyte precursor frequencies against major histocompatibility complex–unrelated individuals. In conclusion, oligoclonal bands of TCRBV gene families found by RT-PCR correspond with a dramatic increase in circulating T lymphocytes expressing the same TCRBV family. Moreover, IFN-α can restore the skewed T-cell repertoire and suppress persistent T-cell clones upon treatment of the accompanying malignancy.
Differential Sensitivity of CD4+ and CD8+T Lymphocytes to the Killing Efficacy of Fas (Apo-1/CD95) Ligand+ Tumor Cells in B Chronic Lymphocytic Leukemia
Authors: Tinhofer Inge , Marschitz Ingrid , Kos Marion , Henn Traudl , Egle Alexander , Villunger Andreas , Greil Richard
Published: October 2019

B-chronic lymphocytic leukemia (B-CLL) is characterized by cellular and humoral immune defects resulting in increased rates of infection and disturbed immune surveillance against cancer cells as well as by the expansion of slowly proliferating tumor cells. We found increased Fas receptor (FasR) expression in peripheral blood CD4+and CD8+ cells of B-CLL patients compared with the equivalent cells of healthy donors. Although increased Fas receptor expression was significant in both T-lymphocytic subsets, only CD4+ cells from B-CLL patients underwent apoptosis after treatment with the agonistic Fas antibody CH11. In CD4+cells of B-CLL patients, the Fas-sensitivity also correlated with a CD4+/CD8+ ratio below the lower threshold of healthy individuals (<1.0). By contrast, FasR expression in the CD19+ fraction of B-CLL patients was downregulated compared with normal controls, and this was associated with an insensitivity to CH11-induced apoptosis. The B-CLL cell line EHEB as well as CD19+ cells from B-CLL patients constitutively expressed Fas ligand (FasL). The FasL was functionally active, as the B-CLL cell line as well as T-cell–depleted CD19+ B-CLL fractions were able to kill target T-acute lymphatic leukemia (T-ALL) cells in vitro. This effect was inhibited by the antagonistic FasR-antibody ZB4, the neutralizing anti-FasL monoclonal antibody (MoAb) NOK-2 or by transfection of the caspase inhibitor crmA. These data point to the fact that expression of FasL on CD19+B-CLL cells, together with enhanced susceptibility of CD4+ T cells toward FasL-bearing effector cells, are causally linked to the relative reduction of CD4+ cells occurring during B-CLL progression. These findings could explain the inversion of the ratio of CD4+/CD8+ cell numbers, which may be causally linked to the immune deficiency observed in these patients and to the expansion of the neoplastic clone in B-CLL.