bluuebunny/crossref_metadata_2025 huggingface dataset:
Erythropoietin synthesis by tumor cells in a case of meningioma associated with erythrocytosis
Authors: Bruneval P , Sassy C , Mayeux P , Belair MF , Casadevall N , Roux FX , Varet B , Lacombe C
Published: October 2019

While secondary erythrocytosis is often associated with tumors arising from the kidney, other tumors have been described to originate in the liver, uterus, ovary, adrenal gland, and central nervous system, among which cerebellar hemangioblastomas are involved in most instances. Two cases of meningioma associated with erythrocytosis have already been reported. We observed a 59-year-old female patient who had developed a frontal meningioma associated with erythrocytosis. Before surgery, she had a significantly elevated total red blood cell volume with a normal plasma volume. Serum erythropoietin (Epo) dosage assessed by radioimmunoassay was within the normal range. The tumor was removed and the pathologic study found a meningotheliomatous meningioma. Total RNA from the tumor was hybridized to a monkey cDNA Epo probe. A strong 1.6- kb messenger RNA (mRNA) signal was observed, which is the expected size of human Epo mRNA. In situ hybridization with the 35S-labeled Epo probe was performed on frozen tumor tissue sections. A significant hybridization was observed in all the tumor cells, whereas the stroma was negative. Therefore, in this meningioma associated with erythrocytosis, Epo was produced by the tumor cells themselves.
Hydroxyurea increases fetal hemoglobin in cultured erythroid cells derived from normal individuals and patients with sickle cell anemia or beta-thalassemia
Authors: Fibach E , Burke LP , Schechter AN , Noguchi CT , Rodgers GP
Published: October 2019

Hydroxyurea (HU), an inhibitor of DNA synthesis, has been shown to increase fetal hemoglobin (HbF) levels in patients with sickle cell anemia and in some patients with beta-thalassemia. However, until now there have not been good in vitro model systems that simulate this effect for study of the molecular and cellular mechanism(s) involved in perturbing the normal ontogeny of the globin genes. We analyzed the cellular effects of HU using a two-phase liquid culture procedure (Fibach et al: Blood 73:100, 1989) in which human peripheral blood- derived progenitor cells undergo proliferation and differentiation. HU was found to have multiple effects on these cultured cells: (1) an increase in the proportion of HbF produced; (2) a decrease in cell number due to inhibition of cell proliferation; (3) an increase in hemoglobin content per cell (mean corpuscular hemoglobin [MCH]); and (4) an increase in cell size (mean corpuscular volume). The extent of these effects was related to the HU dose and time of addition. When added to cell cultures from normal individuals, 4 days following their exposure to erythropoietin (EPO), 100 mumol/L HU caused a 1.3- to 3.5- fold increase in the proportion of HbF, from 0.4% to 5.2% (mean 1.6) in untreated to 1.5% to 8.2% (mean 3.1) in HU-treated cultures and a 45% +/- 10% increase in MCH but only a 25% +/- 7% decrease in cell number on day 13. Cultures of cells derived from five patients with sickle cell anemia have shown a twofold to fivefold increase in the percentage of Hb F following addition of HU while four patients with beta- thalassemia showed a 1.3- to 6.2-fold increase. We believe that this primary cell culture procedure should prove useful in studying the cellular and molecular mechanisms of pharmacologic induction of HbF and might provide a valuable predictive assay system for evaluation of the response of individual patients with hemoglobinopathies to HU and similar agents.
Human herpesvirus-6-associated suppression of growth factor-induced macrophage maturation in human bone marrow cultures
Authors: Burd EM , Knox KK , Carrigan DR
Published: October 2019

Cultures of marrow mononuclear cells were exposed to medium derived from cell cultures infected with several different strains of human herpesvirus-6 (HHV-6) immediately before the addition of either of two growth factors, granulocyte-macrophage colony-stimulating factor and interleukin-3. Exposure to any of the viral preparations suppressed the outgrowth of nonspecific esterase-positive adherent macrophages induced by the factors by more than 90%. The nonadherent cell populations in the infected cultures were numerically similar to those in uninfected control cultures, demonstrating the absence of a nonspecific cytotoxic effect of the viral materials. Infectious virus was not necessary for the macrophage outgrowth suppression. These findings suggest that HHV-6 either encodes or induces a soluble mediator or mediators that can interfere with the responses of bone marrow to growth factors and possibly block the normal differentiation of macrophages from marrow precursors.
Combination treatment of 2-chlorodeoxyadenosine and type I interferon on hairy cell leukemia-like cells: cytotoxic effect and MHC- unrestricted killer cell regulation
Authors: Reiter Z , Tomson S , Ozes ON , Taylor MW
Published: October 2019

Hairy cell leukemia (HCL) is a lymphoproliferative disorder of B lymphocytes. Interferons (IFNs), especially of the alpha (alpha) subtype, have shown a significant antitumor effect in HCL patients. However, the therapeutic effect of IFN-alpha is still rather limited. The purine analogue 2-chlorodeoxy-adenosine (2-CdA) was reported recently to be an effective agent in the treatment of HCL. In the present study, we find that the HCL cell lines HS-1 and HS-2 as well as Eskol and its IFN-resistant clone (IREs-4) are sensitive to the cytotoxic activity of 2-CdA. Combination treatment of IFN-Con1 and 2- CdA results in a synergistic effect at low doses but an additive inhibitory effect at higher concentrations. IREs-4 cells responded only to 2-CdA treatment. All the HCL cell lines are resistant to natural killer (NK) cell-mediated cytotoxicity (CMC) but are relatively sensitive to IFN-Con1-primed or interleukin-2 (IL-2)-primed NK-CMC activities. No inhibition in killing ability was measured when only the effector cells (NK) were treated with 2-CdA. Pretreatment of the HCL target cells with 2-CdA increases their susceptibility to NK-CMC. Pretreatment with IFN-Con1 can reduce the susceptibility of target cells to NK-CMC in HS-1, HS-2, and Eskol cells but not in the IFN- resistant clone IREs-4. 2-CdA abolished this IFN-induced protection against NK-CMC. Normal fibroblasts only responded to treatment with relatively high doses of 2-CdA, and only a moderate additive cell growth inhibitory effect was seen in combination of 2-CdA with IFN- Con1. Only high doses of 2-CdA increased the susceptibility of fibroblast culture to NK-CMC. Thus, combination of IFN-Con1 and 2-CdA results in an in vitro enhancement of the direct antiproliferative/cytotoxic activity of each treatment alone and increases the efficacy of the NK activity against the HCL cell lines.
Comparative effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) on priming peripheral blood progenitor cells for use with autologous bone marrow after high-dose chemotherapy
Authors: Peters WP , Rosner G , Ross M , Vredenburgh J , Meisenberg B , Gilbert C , Kurtzberg J
Published: October 2019

Two hematopoietic colony-stimulating factors, granulocyte colony- stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF), have been shown to accelerate leukocyte and neutrophil recovery after high-dose chemotherapy and autologous bone marrow (BM) support. Despite their use, a prolonged period of absolute leukopenia persists during which infections and other complications of transplantation occur. We collected large numbers of peripheral blood (PB) progenitors after CSF administration using either G-CSF or GM-CSF and tested their ability to affect hematopoietic reconstitution and resource utilization in patients undergoing high-dose chemotherapy and autologous BM support. Patients with breast cancer or melanoma undergoing high-dose chemotherapy and autologous BM support were studied in sequential nonrandomized trials. After identical high-dose chemotherapy, patients received either BM alone, with no CSF; BM with either G-CSF or GM-CSF; or BM with G-CSF or GM-CSF and G-CSF or GM-CSF primed peripheral blood progenitor cells (PBPC). Hematopoietic reconstitution, as well as resource utilization, was monitored in these patients. The use of CSF- primed PBPC led to a highly significant reduction in the duration of leukopenia with a white blood cell (WBC) count under 100 and 200 cells/mL, and neutrophil count under 100 and 200 cells/mL with both GM- and G-CSF primed PB progenitor cells, compared with the use of the CSF with BM or with historical controls using BM alone. In addition, the use of CSF-primed PBPC resulted in a significant reduction in median number of antibiotics used, days in the Bone Marrow Transplant Unit, and hospital resources used. Patients receiving G-CSF primed PBPC also experienced a reduction in the median number of days in the hospital, red blood cell (RBC) transfusions, platelet transfusions, days on antibiotics, and discounted hospital charges. Phenotypic analysis of the CSF-primed PBPC indicated the presence of cells bearing antigens associated with both early and late hematopoietic progenitor cells. The use of CSF-primed PBPC can significantly improve hematopoietic recovery after high-dose chemotherapy and autologous BM support. In addition, the use of G-CSF-primed PBPC was associated with a significant reduction in hospital resource utilization, and a reduction in hospital charges.
Bone marrow stroma in humans: anti-nerve growth factor receptor antibodies selectively stain reticular cells in vivo and in vitro
Authors: Cattoretti G , Schiro R , Orazi A , Soligo D , Colombo MP
Published: October 2019

Two anti-nerve growth factor receptor (LNGFR or p75NGFR) antibodies, Me20.4 and Me8211, label stromal cells with dendritic features in fresh smears and in formalin-fixed, paraffin-embedded human bone marrow (BM). The LNGFR+ cells have an oval nucleus, a scanty cytoplasm with long dendrites that intermingle with the hematopoietic cells, line the abluminal side of sinus endothelial cells, and provide the scaffold for the hematopoietic marrow. At the electron microscopy level, the immunogold tag labels the body and the long branching dendrites of fibroblast-like cells with scanty cytoplasm containing mitochondria, endoplasmic reticulum, and dense bodies. The LNGFR+ cells are positive for alkaline phosphatase, reticulin, collagen III, vimentin, TE-7, and CD13 but negative for endothelial (vWF, CD34, Pal-E), neural (CD56, neurofilament) and leukocyte markers (CD45, CD68). The LNGFR+ stromal cells appear in the fetal BM before the hematopoietic activity begins, originate from the vessel adventitia, and radiate in the Bm cavity. Long-term BM culture (LTBMC) in vitro contain LNGFR+ stromal cells. We document the presence of RNA message for the low- (LNGFR) and the high- affinity NGF receptor (NTRK1) by using RT-PCR on fresh BM aspirate and on LTBMC. BM biopsies from patients with hematologic fibrogenic diseases and in cytokine-treated cancer patients are evaluated for LNGFR+ cells: the amount of stained cells is correlated with the traditional reticulin stain in cases of myelofibrosis, therapy-related myelodysplasia, leukemia, and detected an increase of stromal cells in cytokine-treated patients. The anti-LNGFR antibodies represent a specific membrane marker for the adventitial reticular cells (ARC) of the human marrow and allow precise evaluation and quantitation of this important BM microenvironment component in vivo and in vitro.
Pre-CFU-S quiescence and stem cell exhaustion after cytostatic drug treatment: protective effects of the inhibitory peptide pGlu-Glu-Asp- Cys-Lys (pEEDCK)
Authors: Paukovits WR , Moser MH , Paukovits JB
Published: October 2019

Pre--CFU-S are characterized by their ability to generate spleen colony- forming cells (CFU-S) and by their ability to repopulate the hematopoietic system after damage. We have investigated their response to three consecutive injections of cytosine arabinoside (ara-C), given at t = 0, 12, and 20 hours. Nine hours after treatment, the number of CFU-S and pre--CFU-S was reduced to 10% or 30%, respectively. No pre-- CFU-S were in S-phase at this time, indicating that the pre--CFU-S losses were not caused by direct drug killing. Up to 1 year after treatment, pre--CFU-S were still depleted to 10% of normal, indicating that their proliferative quiescence was permanent. We have previously shown that inhibition of CFU-S recruitment with pGlu-Glu-Asp-Cys-Lys (pEEDCK) makes them ara-C resistant and prevents their decimation. We now found that this also prevented the excessive drainage of the pre-- CFU-S pool, suggesting that pre--CFU-S allocation into active hematopoiesis is triggered by the CFU-S deficit. pEEDCK may thus be applicable as a protector of the hematopoietic repopulation potential against cytostatic drug-induced aplasia. Postchemotherapeutic stimulator treatment with (pEEDCK)2-dimer did not ameliorate pre--CFU-S losses. Long-term culture-initiating cells (LTC-ICs) showed a similar pattern of irreversible reduction after cytostatic drug treatment, which could be prevented by pEEDCK. Our results suggest, that certain subclasses of hematopoietic stem cells (pre--CFU-S) are permanently quiescent and exhaustible and that the capacity for self-renewal is not a necessary property of all stem cell-like cells.
Pseudo-von Willebrand disease: a mutation in the platelet glycoprotein Ib alpha gene associated with a hyperactive surface receptor
Authors: Russell SD , Roth GJ
Published: October 2019

Pseudo (platelet-type)-von Willebrand disease is an autosomal dominant bleeding disorder caused by the hyperfunction of a receptor on the platelet surface. The abnormal receptor, glycoprotein Ib, displays increased affinity for its ligand, von Willebrand factor. Four members (normal mother/affected father/two affected daughters) of a family with pseudo-von Willebrand disease were studied to determine the molecular genetic basis for their congenital platelet defect. Segments of the platelet glycoprotein Ib alpha gene were amplified by means of the polymerase chain reaction, cloned, and sequenced. A point mutation (A to G, codon 239) was found in segments from the affected individuals but not from the normal. The mutation results in a single amino acid substitution (valine-mutant for methionine-normal) at residue 239 within the Ib alpha binding site for von Willebrand factor. Both the mutant and the normal sequence were found in affected individuals, suggesting a heterozygous state. Amplified DNA from family members and from 58 normal individuals was analyzed by allele-specific oligonucleotide hybridization. Only the normal sequence was found in the mother and the normal individuals, whereas both the normal and the mutant alleles were found in the affected family members. The described mutation is associated with the pseudo-von Willebrand disease phenotype seen in this kindred. The resultant single amino acid substitution in glycoprotein Ib alpha relates to increased receptor function and to excessive binding of von Willebrand factor to the platelet surface.
Induction of monocytic differentiation and tumorigenicity by v-Ha-ras in differentiation arrested hematopoietic cells
Authors: Hibi S , Lohler J , Friel J , Stocking C , Ostertag W
Published: October 2019

Activated ras genes are often associated with human myeloid leukemias with a monocytic differentiated phenotype. To investigate the role of the activated ras gene in leukemogenesis, a myeloid nontumorigenic cell line (FDC-P1) was infected with a selectable retroviral vector carrying the v-Ha-ras gene (H1neo). Infected FDC-P1 cells were not only tumorigenic, but also showed increased monocytic differentiation in vitro. Monocytic differentiation and tumorigenicity in vivo were correlated with several-fold increased levels of activated ras gene expression. Tumorigenic cells were arrested with respect to monocytic marker expression at a much later stage of macrophage differentiation than the parental noninfected FDC-P1 cells. These data thus suggest a model of how activated ras genes could be involved in the preferential induction of hematopoietic malignancies with a myelomonocytic phenotype.
Allogeneic blood transfusion-induced enhancement of tumor growth: two animal models showing amelioration by leukodepletion and passive transfer using spleen cells [see comments]
Authors: Blajchman MA , Bardossy L , Carmen R , Sastry A , Singal DP
Published: October 2019

Allogeneic blood transfusions have been reported to induce immunomodulation in recipients of blood products. While the mechanism(s) of this immunomodulatory effect is unknown, it has been suggested that this effect of allogeneic blood transfusions could adversely affect patients with a malignant disorder. These concerns have been supported by a number of nonrandomized, mainly retrospective, clinical studies which indicate that allogeneic blood transfusions can adversely affect prognosis following the surgical treatment of oncology patients. Recently, we have shown that allogeneic blood transfusions enhance primary tumor growth and increase metastatic pulmonary nodule formation in inbred mice. The tumor growth-promoting activity of allogeneic blood transfusions was studied also using outbred rabbits. In this present study, we demonstrate that the tumor growth-promoting effect of allogeneic blood transfusions is mediated by donor leukocytes and that this effect can be abolished by their removal before transfusion. We show also that the allogeneic blood transfusion tumor growth-promoting effect can be passively transferred to naive animals (both mice and rabbits) using spleen cells from allogeneically transfused animals. In these experiments, numbers of metastatic pulmonary nodules were significantly increased in both mice and rabbits that had received spleen cells from allogeneically transfused animals compared with those that had received spleen cells from syngeneically transfused animals, or from animals that had been transfused with leukodepleted allogeneic blood.
Serum interleukin-3 levels following autologous or allogeneic bone marrow transplantation: effects of T-cell depletion, blood stem cell infusion, and hematopoietic growth factor treatment
Authors: Mangan KF , Mullaney MT , Barrientos TD , Kernan NA
Published: October 2019

Engraftment of marrow following autologous or allogeneic bone marrow transplantation (BMT) may be influenced by quantity and function of stem cells. T lymphocytes, supporting microenvironmental cells, and hematopoietic growth factors (HGF). To elucidate the physiologic role of interleukin-3 (IL-3) in the engraftment process, serum IL-3 levels were measured in over 400 samples from 77 transplant recipients before and for up to 3 weeks following transplantation using a novel enzyme- linked immunoabsorbent assay (ELISA) with a sensitivity of > or = 78 pg/mL. Thirty-seven patients received two to three log T-cell-depleted allografts. In the remaining 40 patients (18 autologous marrow, 12 allogeneic marrow, and 10 autologous peripheral blood [PB] stem cell), T cells were not depleted (non-TCD) from the grafts. A burst of IL-3 (peak levels, 1,500 to 6,000 pg/mL) was detected in the immediate posttransplant period between day 0 and day 14 in all non-TCD recipients and in 21 of 37 (57%) of TCD recipients. A strong inverse relationship between IL-3 levels and absolute neutrophil count (ANC) was observed in both non-TCD recipients (r = -.796) and in TCD recipients (r = -.897). However, both peak IL-3 levels and mean IL-3 levels from day 0 through 14 were significantly lower in TCD recipients compared with either autologous or unmodified allogeneic marrow recipients (P < .01). The lowest peak or mean day 0 through 14 IL-3 levels were observed in matched related recipients undergoing the most aggressive (2.5 to 3.0 log) T-cell-depleted BMT. Autografted patients receiving blood stem cell transplants alone or posttransplant granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony stimulating factor (GM-CSF) also had significantly lower peak IL- 3 levels (P < .01). In patients receiving TCD grafts, administration of antithymocyte globulin (ATG) posttransplant significantly increased peak IL-3 levels compared with patients not treated with ATG (P < .04). This study shows that endogenous release of IL-3 is strongly associated with myeloid engraftment and inversely related to ANC. Removal of T lymphocytes from donor marrow or acceleration of engraftment by use of stem cells or growth factors appears to blunt the endogenous release of IL-3 whereas use of ATG posttransplant increases IL-3 release.
Autoimmune neutropenia [see comments]
Authors: Shastri KA , Logue GL
Published: October 2019

There have been several new developments in the field of autoimmune neutropenia over the past decade. Neutropenia caused by antibodies directed against granulocyte precursor cells, the oligoclonal nature of antineutrophil antibodies, and the expanding knowledge of neutrophil antigens, particularly in relationship to autoantibodies, are exciting new areas of investigation. Knowledge has also been advanced in the effector mechanisms of neutrophil autoantibodies and the effect of autoantibodies on the neutrophil function. In addition, some clinical syndromes of immune neutropenia have been better defined over the past decade, such as autoimmune neutropenia of infancy and chronic idiopathic neutropenia in adults. The past decade also saw interesting developments in the treatment of immune neutropenia, particularly in the use of gammaglobulin preparations and more recently in the advent of hematopoietic growth factors. This review focuses on these newer aspects of autoimmune neutropenia.
Differential activation of the endogenous leukotriene biosynthesis by two different preparations of granulocyte-macrophage colony-stimulating factor in healthy volunteers
Authors: Denzlinger C , Tetzloff W , Gerhartz HH , Pokorny R , Sagebiel S , Haberl C , Wilmanns W
Published: October 2019

Results from in vitro investigations and recent data obtained in patients with drug-induced cytopenia or myelodysplasia suggest that leukotrienes may be involved in mediating some of the actions of granulocyte-macrophage colony-stimulating factor (GM-CSF). In the present study, the possible role of leukotrienes was further characterized in 21 healthy individuals to avoid modification of response to GM-CSF by disease-specific variables. The effects of two different preparations of human recombinant GM-CSF, ie, glycosylated GM- CSF as expressed in a Chinese hamster ovary carcinoma (CHO) cell line and nonglycosylated GM-CSF obtained from Escherichia coli, were compared. GM-CSF was administered subcutaneously at a single dose of 0.7 nmol/kg body weight. Pharmacokinetic parameters and hematopoietic and adverse effects were monitored by blood analyses or physical examination, respectively. Leukotriene generation in vivo was evaluated by determination of leukotriene E4 and N-acetyl-leukotriene E4 in urine. After the injection of GM-CSF from E coli, serum concentrations increased and decreased more rapidly and reached a 2.3-fold higher maximum compared with GM-CSF from CHO. GM-CSF induced a biphasic change in leukocyte counts that proceeded considerably faster after the E coli preparation than after GM-CSF from CHO. The urinary leukotriene concentration increased 1.3- to 14-fold or 2.1- to 44-fold after the administration of GM-CSF from CHO or E coli, respectively. Urinary leukotriene concentrations correlated significantly with the maximum of basophil counts and correlated with the occurrence of some adverse reactions, ie, flu-like symptoms, bone pain, or dyspnoea. Our data confirm the conception that leukotrienes may play a significant role in GM-CSF action in vivo. They especially direct attention to the possible relevance of leukotrienes to untoward effects of GM-CSF treatment.
Relationship between tissue factor expression and deposition of fibrin, platelets, and leukocytes on cultured endothelial cells under venous blood flow conditions
Authors: Kirchhofer D , Sakariassen KS , Clozel M , Tschopp TB , Hadvary P , Nemerson Y , Baumgartner HR
Published: October 2019

Endothelial cell-mediated coagulation and leukocyte adhesion are processes that might be connected by the generation of thrombin. To examine the interaction of procoagulant and proadhesive activity, cultures of endothelial cells were stimulated with tumor necrosis factor-alpha, which resulted in the surface expression of tissue factor. Subsequent exposure to human nonanticoagulated blood at a shear rate of 100 s-1 in a parallel plate perfusion device led to the deposition of polymerized fibrin, which covered 63% of the endothelial surface. In addition, numerous platelet aggregates (71 per 10 mm cross- section) and leukocytes (53 +/- 6/mm2) were deposited on stimulated endothelial cells, whereas no fibrin and only a few platelet aggregates (4 +/- 1 per 10 mm cross-section) and leukocytes (6 +/- 1/mm2) were detected on control cells. A significant portion of the adherent leukocytes bound to fibrin and platelets. However, when the deposition of fibrin and platelet aggregates was inhibited with the anti-tissue factor antibody HTFI-7B8 by 100% and 86%, respectively, leukocyte adherence remained unchanged (68 +/- 6/mm2). This indicated that leukocytes could efficiently adhere to endothelial cells through direct cell-cell contact independent of both thrombin and deposited fibrin. Moreover, this direct adhesion of leukocytes to the endothelial surface was reduced twofold to threefold when fibrin deposition occurred. These data suggest a relationship between endothelial procoagulant and proadhesive properties in that tissue factor-initiated coagulation may contribute to leukocyte adhesion through the formation of an adhesive fibrin/platelet meshwork but concurrently prevents the adhesive endothelial surface to bind leukocytes at its full capacity.
Interleukin-6 inhibits the proliferation of B-chronic lymphocytic leukemia cells that is induced by tumor necrosis factor-alpha or -beta
Authors: Aderka D , Maor Y , Novick D , Engelmann H , Kahn Y , Levo Y , Wallach D , Revel M
Published: October 2019

Tumor necrosis factor (TNF-alpha) acts as a growth stimulatory factor on leukemic B lymphocytes from many patients with chronic lymphocytic leukemia (CLL). Because TNF induces production of interleukin-6 (IL-6), which has been shown to be a growth factor for myeloma and other transformed B cells, we examined the possibility that IL-6 mediates the growth-stimulatory effect of TNF on B-CLL cells. In fact, we found that IL-6 is an inhibitor of B-CLL growth. The addition of recombinant human IL-6 markedly decreased the TNF-induced B-CLL growth, and this decrease was even greater when soluble IL-6 receptor, known to act as IL-6 agonist, was added with recombinant IL-6. Conversely, neutralizing monoclonal antibodies to IL-6 and to the IL-6 receptor potentiated the growth stimulation of TNF on B-CLL cells, in line with the possibility that IL-6 functions as a negative feedback regulator of an autocrine TNF action on these B-leukemic cells. Evidence is presented that production of IL-6 by monocytes and B cells of CLL patients is low, suggesting that administration of IL-6 may be beneficial in CLL to reduce the eventual growth stimulation by TNF and, possibly, also the deficiency in platelets and Ig production in this disease.
Recombinant human stem cell factor enhances myeloid colony growth from human peripheral blood progenitors
Authors: Sekhsaria S , Malech HL
Published: October 2019

Peripheral blood hematopoietic progenitors (PBHP) are capable of colony growth in vitro. The effect of stem cell factor (SCF), interleukin-6 (IL-6), and basic fibroblast growth factor (bFGF) on myeloid colony proliferation of PBHP was determined. PBHP purified by positive selection with CD34-specific antibody were plated in semisolid agarose with reported plateau doses of interleukin-3 (IL-3), granulocyte- macrophage colony-stimulating factor (GM-CSF), and granulocyte colony- stimulating factor (G-CSF) to enhance myeloid colony growth. Experiments then were done to examine colony growth in response to SCF or with SCF and bFGF and/or IL6. SCF alone in the absence of any other growth factors did not support colony growth. SCF at a determined optimum concentration of 100 ng/mL added to the combination of IL-3, GM- CSF, and G-CSF enhanced colony growth and size relative to proliferation in response to the latter three factors alone (from 78 to 188 total colonies/10(4) PBHP plated and from 10 to 93 large [> 200 cells] colonies/10(4) PBHP plated). Furthermore, addition of bFGF and/or IL-6 to the combination of optimum concentrations of SCF, IL-3, GM-CSF, and G-CSF further enhanced colony number and size in a dose- dependent fashion. Using the optimum combination of all growth factors, we determined that the number of myeloid colony-forming PBHP in whole blood was similar between individuals at about three colonies per milliliter whole blood. We conclude that progenitors capable of responding to the early-acting growth factor, SCF, are represented in PBHP and that the number of circulating myeloid colony-forming PBHP is likely a regulated parameter that may have an important biologic function.
Improvement of superoxide production in monocytes from patients with chronic granulomatous disease by recombinant cytokines
Authors: Jendrossek V , Peters AM , Buth S , Liese J , Wintergerst U , Belohradsky BH , Gahr M
Published: October 2019

Cytokines have been shown to modulate the respiratory burst of polymorphonuclear leukocytes and monocytes from normal controls. We have examined whether monocytes from children with chronic granulomatous disease (CGD) can be primed by cytokines other than interferon-gamma (IFN gamma), which has been demonstrated to improve the production of reactive oxygen species in vivo and in vitro. Monocytes isolated from peripheral blood were cultured without and with IFN gamma (500 U/mL), tumor necrosis factor-alpha (500 U/mL), interleukin-1 beta (IL-1 beta) (100 U/mL), and IL-3 (100 U/mL). After 3 days of culture, the phorbolmyristate acetate (2 ng/mL) and the formyl- methionyl-leucyl-phenylalanine (0.1 mumol/L)-stimulated superoxide- production was determined in a microtiter system. In nearly all of the 14 patients examined (5 autosomal, 5 X-chromosomal, and 4 of unknown inheritance), an improvement of superoxide production could be demonstrated. The most impressive effect with the cytokines newly tested was seen with monocytes from autosomal CGD patients treated with IL-3 and stimulated by phorbolmyristate acetate. In single patients cultivation of monocytes with IL-6 and granulocyte-macrophage colony- stimulating factor resulted in only slight improvement of superoxide production. Our findings indicate that cytokines other than IFN gamma can positively modulate the defective respiratory burst in CGD and that each patient reacts with an individual pattern to different cytokines.
A sudden increase in factor VIII inhibitor development in multitransfused hemophilia A patients in The Netherlands. Dutch Hemophilia Study Group
Authors: Rosendaal FR , Nieuwenhuis HK , van den Berg HM , Heijboer H , Mauser- Bunschoten EP , van der Meer J , Smit C , Strengers PF , Briet E
Published: October 2019

The development of antibodies to factor VIII (inhibitors) in response to clotting-factor concentrates administration in hemophilia is common during the first few years of treatment but rare in multitransfused patients. We have investigated the possible association of a recently introduced factor VIII concentrate (Factor VIII CPS-P) in The Netherlands with the occurrence of inhibitors. To this effect, we conducted two studies. First, we performed a national multicenter study in which clinical information and inhibitor test results were obtained for 447 hemophilia A patients over the period 1988 through 1991. Secondly, for a baseline comparison we estimated the frequency of inhibitor development in a closely followed cohort of 144 patients, from 1984 through 1989. Before the introduction of Factor VIII CPS-P, the incidence of new inhibitors was 4.4/1,000 patient-years in the national study from March 1988 through May 1990, and 3.9/1,000 patient- years in the cohort followed from 1984 through 1989. These figures are similar to the incidence of new inhibitors that was found in a large cohort of patients in the United States followed in the 1970s. In the period that the new concentrate Factor VIII CPS-P was on the market, from June 1990 through November 1991, 11 clinically relevant inhibitors were detected, which yielded an incidence over this interval of 20.1/1,000 patient-years, a 4.5-fold increase compared with the previous interval (C195: 1.4 to 14.3). Nine of these 11 patients had in their lifetime received over 250 infusions with factor VIII preparations. whereas all of the inhibitors detected in the previous time interval, and all of the 24 inhibitor patients described in the US study, had received less than 250 infusions in their lifetime. All patients who developed inhibitors after June 1990 had been exposed to Factor VIII CPS-P, whereas only 75% of the patients who did not develop an inhibitor had been exposed to this product. In a prospective extension of the study, with a second inhibitor measurement after 3 months, we found that one additional inhibitor had developed during 52.5 patient-years of Factor VIII CPS-P use. In conclusion, there has been a sudden increase in the frequency of inhibitor patients, for a large part among multitransfused patients. It seems more than likely that this increase is associated with the introduction of a new factor VIII concentrate in The Netherlands.(ABSTRACT TRUNCATED AT 400 WORDS)
Correlation of minor histocompatibility antigen-specific cytotoxic T lymphocytes with graft-versus-host disease status and analyses of tissue distribution of their target antigens
Authors: Niederwieser D , Grassegger A , Aubock J , Herold M , Nachbaur D , Rosenmayr A , Gachter A , Nussbaumer W , Gaggl S , Ritter M
Published: October 2019

Peripheral blood mononuclear cells (PBMC) from 17 patients receiving HLA-identical sibling bone marrow grafts were stimulated with host pretransplant PBMC. Cytotoxic T-cell lines (TCL) with specificity for host pretransplant PBMC were obtained from 9 of these patients, all presenting with severe graft-versus-host disease (GVHD), but from none of the remaining cases lacking evidence of disease. Cytotoxic TCL were specific for host targets and failed to lyse donor cells. Monoclonal antibodies (MoAbs) blocking experiments and donor population screening analyses demonstrated that minor histocompatibility antigen (MiHA)- specific lysis of host targets was restricted by class I major histocompatibility complex (MHC) determinants. Whereas hematopoietic cells such as phytohemagglutinin (PHA) blasts or lymphoblastoid cell lines were susceptible to lysis by MiHA-specific TCL, keratinocytes (K) representing the natural targets of GVHD were quite resistant. Quantitative radioimmunometric measurements indicated very low constitutive expression of class I MHC antigens on K targets, which was readily increased by treatment with interferon-gamma (IFN-gamma). IFN- gamma treatment at the same time rendered these cells susceptible to lysis by MiHA-specific TCL. Host leukemic cells of 3 patients were recognized by MiHA-specific TCL in a chromium release assay and in one experiment host leukemic cells were effectively killed and their growth specifically inhibited in a leukemia colony assay by a clone. These data demonstrate that (1) host-specific cytotoxic TCL are detected exclusively in the PB of patients with acute GVHD grades II through IV after allogeneic matched bone marrow transplantation, and (2) their target antigens are simultaneously expressed on several host cell lines, including lymphoblastoid cell lines, PHA blasts, leukemic cells, and K. We also extend previous findings by showing that, besides the expression of the nominal MiHA, the density of the restricting class I MHC elements also crucially determines the extent of TCL lysis. Because of its capacity to enhance class I MHC antigen expression, IFN-gamma represents a key cytokine for determining the susceptibility of MiHA targets for lysis by TCL and clones, and in one patient an MiHA- specific clone recognized host leukemic cells and also inhibited host leukemic cell growth in a colony inhibition assay.
Regulation of human B-cell precursor adhesion to bone marrow stromal cells by cytokines that exert opposing effects on the expression of vascular cell adhesion molecule-1 (VCAM-1)
Authors: Dittel BN , McCarthy JB , Wayner EA , LeBien TW
Published: October 2019

Self-renewal and differentiation of B-cell precursors is dependent on interactions with bone marrow (BM) stromal cells and associated extracellular matrix. We have recently developed an interleukin (IL)-7- dependent, BM-derived stromal cell culture that supports the growth of normal human B-cell precursors. In the current study, we have characterized the constitutive expression, cytokine-regulated expression, and function of adhesion molecules on BM stromal cells that are critical for adhesion of B-cell precursors. Flow cytometric analysis showed that cultured adult BM stromal cells expressed higher constitutive levels of vascular cell adhesion molecule (VCAM)-1 than intercellular adhesion molecule (ICAM)-1 (CD54). IL-1 beta upregulated VCAM-1 and CD54 in a dose-dependent manner, whereas IL-4 upregulated VCAM-1, but had no effect on CD54. In contrast, transforming growth factor (TGF)-beta decreased the level of BM stromal cell VCAM-1. Using an assay to measure the adhesion of 51Cr-labeled B-cell precursors to BM stromal cells, we observed a direct correlation between cytokine- regulated levels of VCAM-1 and the capacity of stromal cells to support the adhesion of B-cell precursors. Blocking studies using a panel of monoclonal antibodies (MoAb) showed that adhesion of B-cell precursors to untreated and cytokine-treated (IL-1 beta, IL-4) BM stromal cells was mediated by very late antigen (VLA)-4 (CD49d/CD29) and VCAM-1. Adhesion of B-cell precursors could also be enhanced by direct stimulation with MoAb to the CD29 subunit. Our collective results indicate that B-cell precursor/BM stromal cell adhesion is mediated by a VLA-4-VCAM-1 interaction, which in turn can be regulated at the level of the BM stromal cell by cytokines that specifically increase or decrease cell surface VCAM-1.
Vanadate activates platelets by enhancing arachidonic acid release
Authors: McNicol A , Robertson C , Gerrard JM
Published: October 2019

Human platelet activation is associated with, and regulated by, the phosphorylation of a number of proteins. Recently, attention has been focused on tyrosine phosphorylation of proteins and their function in platelet activation. Here vanadate, an inhibitor of tyrosine phosphohydrolase, was used to examine the role that tyrosine phosphorylation plays in platelet activation. Vanadate (7.5 to 100 mumol/L) stimulated the dose-dependent aggregation of saponin- permeabilized, but not intact, platelets. Electron-microscopic studies indicated small degranulated aggregates. Vanadate-induced aggregation was inhibited by pretreatment with prostacyclin (1 to 10 nmol/L), genistein (1 to 10 micrograms/mL), aspirin (100 mumol/L), or BW755C (80 mumol/L). Aggregation was associated with the aspirin-sensitive formation of [32P]phosphatidic acid and the phosphorylation of platelet proteins, notably pleckstrin and myosin light chain. Immunoblotting studies indicated that vanadate caused the tyrosine phosphorylation of proteins of approximate molecular weights 26, 29, 32, 40, 42, 80, and 90 Kd. Preincubation with BW755C abolished the phosphorylation of the 26-, 29-, 32-, 40-, and 42-Kd proteins but not the 80- and 90-Kd proteins. Vanadate stimulated the release of [3H]-arachidonic acid that was not affected by pretreatment with BW755C. The subsequent conversion of [3H]-arachidonic acid to [3H]-thromboxane A2 was significantly inhibited. These findings show that vanadate stimulates platelets by promoting arachidonic acid release from phospholipids. Tyrosine phosphorylation, potentially of the 80- or 90-Kd proteins, may regulate a platelet phospholipase A2. The release arachidonic acid was converted to thromboxane A2 that produced secondary effects such as phospholipase C activation, protein phosphorylation, and aggregation, and was associated with the tyrosine phosphorylation of the 26-, 29-, 32-, 40-, and 42-Kd proteins.
Granulocyte-macrophage colony-stimulating factor induces sequential activation and deactivation of binding via a low-affinity IgG Fc receptor, hFc gamma RII, on human eosinophils
Authors: Koenderman L , Hermans SW , Capel PJ , van de Winkel JG
Published: October 2019

Eosinophils are important in antibody-mediated immune defense against parasites based on interaction with Ig receptors (FcR). Of the three classes of IgG FcR in humans, hFc gamma RI, II, and III, solely hFc gamma RII (CD32) is expressed on freshly isolated eosinophils. Despite an expression level similar to that found on monocytes and polymorphonuclear granulocytes, binding activity of hFc gamma RII on eosinophils is constitutively low. Freshly isolated eosinophils had a negligible ability to form rosettes with IgG-sensitized erythrocytes (EA-IgG). Addition of granulocyte-macrophage colony-stimulating factor (GM-CSF) caused an approximately threefold increase in EA-IgG rosettes. This increase was maximal after 35 minutes, and declined upon further incubation at 37 degrees C. Analysis of hFc gamma RII expression levels showed no significant changes and neither was the expression of other hFc gamma R classes induced. Blocking studies with anti-Fc gamma receptor monoclonal antibody (MoAb) proved hFc gamma RII specificity of enhanced IgG complex binding. These phenomena were not restricted to GM- CSF action, because the addition of interleukin-3 or interleukin-5 similarly enhanced EA-IgG binding. The kinetics of activation of hFc gamma RII binding activity were paralleled by the binding of EA-C3bi to CR3 on eosinophils. In contrast to the stable expression of hFc gamma RII during activation with GM-CSF, CR3 expression increased slowly. Ligand binding via both types of opsonin receptors proved receptor specific. However, the kinetics of enhanced binding via hFc gamma RII and CR3 suggested the possibility of a common mechanism underlying the enhancement of ligand binding via hFc gamma RII and CR3. This hypothesis was supported by the fact that binding via hFc gamma RII proved sensitive to both high concentrations of F(ab')2 fragments of anti-CD11b MoAb MO1 and chelation of bivalent cations with EDTA. In conclusion, our studies indicate that cytokines can induce a transient enhancement of hFc gamma RII binding activity. Qualitative, and not quantitative, changes in this receptor appear to underly the modulation of binding activity, which may be linked to changes in CR3 activity.
Low substrate affinity of pyruvate kinase variant (PK Sapporo) caused by a single amino acid substitution (426 Arg-->Gln) associated with hereditary hemolytic anemia
Authors: Kanno H , Fujii H , Miwa S
Published: October 2019

A point mutation (1277 CGG to CAG) was identified in the R-type pyruvate kinase (PK) cDNA of a PK variant, PK Sapporo, associated with hereditary non-spherocytic hemolytic anemia. The mutation causes a single amino acid substitution from Arg to Gln at the 426th amino acid residue of human R-type PK; consequently, the hydrophobicity around the mutated site is drastically decreased. The amino acid change occurred in the eighth alpha helix of A domain (A alpha 8) of PK, and it has been proposed that this region as well as A alpha 7, A beta 7, and A beta 8 is a potassium (K+) binding site. Because K+ binding to the PK subunit is considered to be essential for substrate binding, the mutation might account for the decreased affinity for phosphoenolpyruvate (PEP). This is compatible with the fact that all the reported PK variants carrying point mutations in those area have a high Michaelis constant (Km) for PEP.
Origin of a soluble truncated transferrin receptor
Authors: Ahn J , Johnstone RM
Published: October 2019

It has recently become evident that elevation of reticulocytes in the circulation of several species, including humans, leads to the formation of a noncellular transferrin receptor (TFR). In humans, the majority of the released receptor is in truncated form (Shih et al: J Biol Chem 265:19077, 1990). In other species (sheep, rat, chicken) the receptor is associated with a vesicle (exosome) and is full length (Johnstone et al: J Cell Physiol 147:27, 1991). In this report we show that in sheep reticulocytes incubated in vitro, the majority (approximately 75%) of the released receptor is of native size and is exosome associated. A fraction (approximately 25%) is a truncated form of approximately 80 Kd corresponding to the exofacial domain of the TFR. Herein we also address the question of whether the truncated receptor originates by proteolytic cleavage directly from the cell surface or by cleavage from exosomes. Using surface 125I-labeled sheep reticulocytes as the experimental model, we show that during in vitro maturation, 125I-TFR of native size appears in exosomes before the soluble, truncated, exofacial domain of the receptor is detected in the medium. Because cleavage and release of the exofacial domain would likely leave the truncated cytoplasmic and transmembrane domains in the originating membrane (plasma membranes or exosomes), both fractions were probed with antibodies specifically generated against the cytoplasmic domain of the receptor. Only exosomes, not plasma membranes, show the presence of a approximately 17-Kd peptide recognized by the antibody to the cytoplasmic domain of the transferrin receptor. Thus, it is concluded that the truncated, soluble receptor originates from exosomes in sheep. A 17-Kd cytoplasmic domain of the TFR was also detected in exosomes from the reticulocytes of an anemic man, suggesting that the truncated receptor in man may also originate from exosomes. Using in vitro cultures of surface 125I-labeled sheep reticulocytes, it is concluded that exosome formation is the principal route for maturation-associated loss of the TFR. A similar conclusion was made earlier (Johnstone et al: J Cell Physiol 147:27, 1991) for the nucleoside transporter of maturing sheep reticulocytes.
Deletions within the LMP1 oncogene of Epstein-Barr virus are clustered in Hodgkin's disease and identical to those observed in nasopharyngeal carcinoma
Authors: Knecht H , Bachmann E , Brousset P , Sandvej K , Nadal D , Bachmann F , Odermatt BF , Delsol G , Pallesen G
Published: October 2019

This study of 52 European patients with Hodgkin's disease (HD) expressing the latent membrane protein 1 (LMP1) oncogene within diagnostic Hodgkin and Reed-Sternberg (HRS) cells was performed to detect LMP1 isolates carrying deletions and to characterize them at a molecular and histologic level. Deletions were identified in 5 cases, clustered near the 3′ end of the LMP1 gene, and histologically associated with numerous HRS cells. DNA sequencing showed homology with the deletions seen in the Asian nasopharyngeal carcinoma (NPC) isolates CAO and 1510. Our findings suggest that partial deletions of the LMP1 oncogene, associated with aggressive behavior in NPC CAO and NPC 1510, occur at a particular localization and confer a proliferative phenotype to lymphoid cells in HD.
A gene for a myosin peptide is disrupted by the inv(16)(p13q22) in acute nonlymphocytic leukemia M4Eo
Authors: van der Reijden BA , Dauwerse JG , Wessels JW , Beverstock GC , Hagemeijer A , van Ommen GJ , Breuning MH
Published: October 2019

Chromosome 16 aberrations are well known in acute nonlymphocytic leukemia (ANLL). The most frequent chromosome 16 aberration in ANLL subtype M4Eo is the inv(16)(p13q22). Recently, we showed that in 5 inv(16) patients with ANLL M4Eo the short arm breakpoints are clustered within a 14-kb genomic EcoRI fragment. We report here the identification of a gene situated in the 14-kb fragment. The gene, which codes for a myosin peptide, is disrupted by the inversion of chromosome 16 in the 5 patients. To the best of our knowledge, this is the first report of a myosin gene disrupted in leukemia.