bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation
Authors: Zu YL , Ai Y , Gilchrist A , Labadia ME , Sha'afi RI , Huang CK
Published: October 2019

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.
Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHC-mismatched donor marrow grafts
Authors: Drobyski WR , Majewski D
Published: October 2019

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57– 597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H- 2d]) marrow grafts. Transplantation of H57–597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57–597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57–597. Donor T cells exposed to H57–597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.
Rapid and efficient selection of human hematopoietic cells expressing murine heat-stable antigen as an indicator of retroviral-mediated gene transfer
Authors: Conneally E , Bardy P , Eaves CJ , Thomas T , Chappel S , Shpall EJ , Humphries RK
Published: October 2019

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL- 3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony- forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony- forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.
Thrombin receptors activate potassium and chloride channels
Authors: Sullivan R , Kunze DL , Kroll MH
Published: October 2019

We used DAMI human megakaryocytic leukemia cells to study transmembrane ion currents activated through the G-protein-coupled thrombin receptor pathway. When the cells were stimulated by thrombin receptor-activating peptide, an increase in cytosolic Ca2+ ([Ca2+]i) developed as predicted by the known effect that thrombin exerts in the platelet. We then monitored the membrane potentials of individual DAMI cells during this response and observed complex, triphasic changes that could not be accounted for by Ca2+ fluxes alone. These consisted of rapid hyperpolarization, followed by depolarization to values more positive than the resting potential and then by slow repolarization. For the purpose of this study, we focused on the hyperpolarizing current that developed immediately after thrombin receptor activation. This proved to be composed of (1) a Ca(2+)-independent, outwardly rectifying Cl- current and (2) a strongly hyperpolarizing, inwardly rectifying, Ba(2+)- sensitive K+ current that required an increase of [Ca2+]i for activation. By analogy with their functions in other cell systems, it is logical to conclude that these prominent K+ and Cl- conductances may serve to regulate the complex volume changes that accompany thrombin receptor activation and/or to increase the electromotive drive that supports Ca2+ influx under these conditions through hyperpolarization of the cell membrane.
Anticoagulant effects of retinoic acids on leukemia cells
Authors: Saito T , Koyama T , Nagata K , Kamiyama R , Hirosawa S
Published: October 2019

We have recently found that all-trans retinoic acid (ATRA) upregulates thrombomodulin (TM) and downregulates tissue factor (TF) expression in acute myelogenous leukemia (AML) M3 cells (NB4) and acute monoblastic leukemia cells (U937) (Koyama et al, Blood 84:3001, 1994). We have further investigated the effects of ATRA on leukemic cells freshly isolated from patients at diagnosis. Increase of TM antigen was documented in all AML cells: M0 (n = 1), M2 (n = 5), M3 (n = 3), M4 (n = 3), M5 (n = 3), and M6 (n = 1). Decrease of TF antigen was observed in 4 M2, 1 M4, and all M3 and M5 patients. However, no TM and TF antigens were detected in all chronic lymphocytic leukemia cells (n = 3) with or without ATRA treatment. Changes of TM and TF antigen levels were associated with those of TM and TF cofactor levels on the cell surface. A stereoisomer of RA, 9-cis RA, is a high-affinity ligand for the RA receptors (RARs) and the retinoid X receptors, although ATRA and another isomer, 13-cis RA, solely bind to RARs. We have also studied the effects of 9-cis RA and 13-cis RA on the expressions of TM and TF in NB4 and U937 cells. A relatively wide range of 9-cis RA concentrations (0.01 to 1 mumol/L) compared with ATRA was optimal for prolongation of normal plasma-based recalcification time (reduction of cell surface TF activity), decrease of TF antigen, and increase of TM antigen on the surface and in the lysates of NB4 and U937 cells. Western blot analysis under nonreducing conditions showed that both ATRA and 9-cis RA markedly induced the prominent band at 75 kD of TM and reduced the band at 45 kD of TF. Northern blot analysis has shown similar changes of mRNA levels, which indicates that RAs regulate TM and TF expression in leukemic cells at transcriptional levels. Anticoagulant effects of ATRA, ie, upregulation of TM expression and downregulation of TF expression, are applied not only to established cell lines of specific subtypes (M3 and M5) but also to more universal AML (most cases of M3 and M5 and a part of the other types of AML) cells freshly isolated from patients. 9-cis RA may be more effective than ATRA as an inducer of differentiation of AML M3 cells and as an anticoagulant agent for patients with certain types of AML as well.
Interleukin-4 gene expression in activated human T lymphocytes is regulated by the cyclic adenosine monophosphate-dependent signaling pathway
Authors: Borger P , Kauffman HF , Postma DS , Vellenga E
Published: October 2019

In the present study, we have investigated the involvement of the cyclic adenosine monophosphate (cAMP)-dependent signaling pathway on interleukin-4 (IL-4) gene expression in freshly isolated human T lymphocytes. 2′–0-dibutyryl cAMP (db-cAMP) and prostaglandin E2 (PGE2) were used to directly and indirectly activate the protein kinase A pathway. Northern analysis showed that concanavalin A (Con A)-, anti- CD3 (alpha CD3)-, or anti-CD3 plus anti-CD28 (alpha CD3/alpha CD28)- induced accumulation of IL-4 mRNA was inhibited by db-cAMP (10(-3) mol/L). Db-cAMP showed a steep dose-dependent inhibition; concentrations or = 10(-4) mol/L did not affect IL-4 mRNA accumulation. In contrast, GM-CSF mRNA expression showed a wider dose- dependent range; 10(-5) mol/L db-cAMP still affected GM-CSF accumulation. PGE2 inhibited the Con A- and alpha CD3/alpha CD28- induced accumulation of IL-4 mRNA in a dose-dependent fashion. Con A- induced IL-4 mRNA was inhibited by 10(-4) to 10(-7) mol/L PGE2; alpha CD3/alpha CD28-induced IL-4 mRNA was inhibited by 10(-5) to 10(-8) mol/L PGE2. Nuclear run-on experiments showed that the inhibitory effects of db-cAMP and PGE2 were accomplished at transcriptional level in Con A-activated T cells, whereas changes at transcriptional and posttranscriptional level were involved in alpha CD3/alpha CD28- activated T lymphocytes. In contrast to Con A and alpha CD3/alpha CD28 activation, phorbol myristate acetate plus A23187-induced IL-4 mRNA expression was insensitive to the inhibitory effect of db-cAMP and PGE2. Moreover, it appeared that the sensitivity for cAMP-mediated downregulation could not be blocked by stimulation T lymphocytes with alpha CD3/alpha CD28 in the presence of IL-2, IL-7, IL-10, IL-12, or a combination of these cytokines. Finally, it was shown that, in accordance with the mRNA studies, db-cAMP and PGE2 suppressed the IL-4 secretion in Con A- and alpha CD3/alpha CD28-activated T cells. In conclusion, these data show that IL-4 expression is negatively regulated by the protein kinase A-dependent signaling pathway by transcriptional and posttranscriptional mechanisms that depend on costimulatory signals.
Ig VH gene mutational patterns indicate different tumor cell status in human myeloma and monoclonal gammopathy of undetermined significance
Authors: Sahota SS , Leo R , Hamblin TJ , Stevenson FK
Published: October 2019

Plasma cell tumors display a wide spectrum of clinical progression, ranging from aggressive multiple myeloma to a benign form known as monoclonal gammopathy of undetermined significance (MGUS), which requires no treatment. Because both diseases involve mature Ig- secreting plasma cells, the reason for this variation in malignant behavior is unclear. However, assessment of malignant potential is desirable for choice of treatment protocols. Ig variable (VH) gene sequences analysis has previously shown the tumor cell of multiple myeloma to be postfollicular, with mutated homogeneous clonal sequences indicating no continuing exposure to the somatic hypermutation mechanism, and this was confirmed in 7 of 7 patients. Comparison of the VH gene sequences in the monoclonal cells in MGUS yielded a different result, with 3 of 7 patients demonstrating mutated heterogeneous sequences consistent with the tumor cells remaining under the influence of the mutator. In 1 of 3 of these patients, an IgM-positive precursor cell was identified that expressed heterogeneous VH sequences similar to those of the isotype-switched plasma cell. These results indicate that the clonal cells in MGUS differ from those in myeloma and suggest that the difference may reflect malignant potential.
The defective interaction between von Willebrand factor and factor VIII in a patient with type 1 von Willebrand disease is caused by substitution of Arg19 and His54 in mature von Willebrand factor
Authors: Kroner PA , Foster PA , Fahs SA , Montgomery RR
Published: October 2019

In this report we describe the further investigation of the von Willebrand factor (vWF)/FVIII interaction in a type 1 von Willebrand disease patient characterized by discrepant VIII:C levels as determined by one-stage and two-stage VIII:C assays. A solid-phase binding assay shows that this patient's plasma vWF is moderately defective in capturing recombinant FVIII. Sequence analysis of the FVIII-binding domain encoded by the vWF mRNA of the affected individual identified mutations in both vWF alleles. In allele A, the mutations C2344T and T2451A result in the substitution of Trp for Arg19 (R19W) and of G1n for His54 (H54Q) in mature vWF, respectively. This allele also contains a reported polymorphism (A2365G, Thr26Ala). Allele B, which is underexpressed at the RNA level, contains a one-nucleotide deletion in the FVIII-binding domain (delta G2515) that results in the premature termination of translation. Analysis of the binding of FVIII by full- length vWF transiently expressed in COS-7 cells confirms that the combined R19W and H54Q substitutions are the cause of the defective vWF/FVIII interaction in this patient. The FVIII-binding defect of vWF containing either mutation alone is approximately half that of the double mutant, which suggests that the effect of these mutations is additive. The mutant proteins are recognized equally well by vWF monoclonal antibodies MBC105.4, 32B12, and 31H3, which block the binding of FVIII by vWF, indicating that amino acids Arg19, Thr26, and His54 are not critical residues in the epitopes of these antibodies.
Elevated intracellular level of basic fibroblast growth factor correlates with stage of chronic lymphocytic leukemia and is associated with resistance to fludarabine
Authors: Menzel T , Rahman Z , Calleja E , White K , Wilson EL , Wieder R , Gabrilove J
Published: October 2019

Chronic lymphocytic leukemia (CLL) is characterized by delayed senescence and slow accumulation of monoclonal, small lymphocytes. Basic fibroblast growth factor (bFGF) is a pleiotropic cytokine that plays a role in hematopoiesis and apoptosis. Elevated bFGF levels have been detected in urine from patients with a variety of neoplastic diseases including various leukemias; however, the cellular source of the bFGF has not been determined. In this study, the intracellular bFGF level in lymphocytes of 36 patients with B-CLL and 15 normal donors was determined using an enzyme-linked immunoassay. In cells derived from patients with high-risk disease, the median level of intracellular bFGF was 381.5 pg/2 x 10(5) cells, compared with a median of 90.5 pg/2 x 10(5) cells in patients with intermediate disease. In patients with low- risk disease, the median bFGF level was 4.9 pg/2 x 10(5) cells, and in normal controls, it was 6.0 pg/2 x 10(5) cells. The difference in the bFGF levels was significant for the comparison between low- and intermediate-risk (P = .00119), low- and high-risk (P < .0001), and intermediate- and high-risk disease (P = .0001). Immunofluorescent stains of peripheral blood mononuclear cells confirmed CLL lymphocytes as a cellular source of bFGF. To evaluate the potential contribution of elevated intracellular bFGF levels to the phenotype of CLL cells, leukemic cells were cultured in vitro with an apoptotic stimulus (fludarabine). CLL cells with high intracellular levels of bFGF appeared to be more resistant to fludarabine treatment. The addition of bFGF to fludarabine-treated CLL cells resulted in a delay of apoptosis and prolonged survival. These data suggest that bFGF may contribute to the resistance of CLL cells to an apoptotic stimulus.
Transforming growth factor-beta 1 cooperates with anti-immunoglobulin for the induction of apoptosis in group I (biopsy-like) Burkitt lymphoma cell lines
Authors: MacDonald I , Wang H , Grand R , Armitage RJ , Fanslow WC , Gregory CD , Gordon J
Published: October 2019

Group I Burkitt lymphoma (BL) cell lines, which retain the original biopsy phenotype, have been shown to enter apoptosis in response to a number of external stimuli including serum deprivation, thermal shock, addition of calcium ionophore, and ligation of surface immunoglobulin (Ig) by antibody. Transforming growth factor-beta 1 (TGF beta 1) is known to cause growth arrest in BL lines. Here we show that while it is by itself capable of promoting some degree of apoptosis in group IBL cells, TGF beta 1 cooperates with anti-immunoglobulin to this end. Trimeric soluble recombinant human CD40 ligand (sCD40L) was able to inhibit apoptosis induced by the combination of agonists to some degree, but such rescue proved to be short-lived. Both TGF beta 1 and anti-Ig individually caused BL cells to undergo growth arrest at the G1 phase of cell cycle before their entry into apoptosis: the consequence of sCD40L addition was to maintain the cells in cycle for longer. No induction of the apoptosis-protecting gene, bcl-2, occurred in the presence of sCD40L. These findings are discussed, particularly highlighting the relationship existing between survival and the cell cycle. The strong cooperative effects observed between anti-Ig and TGF beta 1 in promoting apoptosis and the inability of CD40 to signal for long-term rescue raise the potential for a novel therapeutic attack on B-cell lymphoma.
Suppression of apoptosis during cytokine deprivation of 32D cells is not sufficient to induce complete granulocytic differentiation
Authors: Rodel JE , Link DC
Published: October 2019

The role of cytokines in the control of hematopoietic cell differentiation remains controversial. Two general models for the cytokine control of hematopoietic differentiation have been proposed. In the stochastic model, cytokines provide proliferative and survival signals to the differentiating hematopoietic cell, but they do not provide specific lineage commitment signals. In the instructive model, cytokines transmit specific signals to multipotent hematopoietic cells, thereby directing lineage commitment. To distinguish between these two models with respect to granulocyte colony-stimulating factor (G-CSF) and granulocytic differentiation, we used the 32Dcl3 cell line, which is capable of differentiating into granulocytes in response to G-CSF, 32D cells transfected with either bcl-2 or bcl-XL showed prolonged survival in medium containing no cytokine supplement. Cells surviving in these cultures developed the segmented nuclei characteristic of mature neutrophils. However, no induction of myeloperoxidase activity or increase in cathepsin G transcripts were detected. These data support a hybrid model for the role of G-CSF in granulocytic differentiation; although some features of granulocytic differentiation, namely nuclear segmentation, do not require G-CSF and appear therefore to be preprogrammed in 32D cells, the complete maturation of these cells to granulocytes appears to be dependent on G- CSF.
Treatment of cutaneous T-cell lymphoma with chimeric anti-CD4 monoclonal antibody
Authors: Knox S , Hoppe RT , Maloney D , Gibbs I , Fowler S , Marquez C , Cornbleet PJ , Levy R
Published: October 2019

Chimeric anti-CD4 monoclonal antibody was administered intravenously as a single dose to eight patients with mycosis fungoides. The dose was escalated throughout the study between patients groups, and individual patients received 50, 100, or 200 mg per dose. Seven of eight patients responded to treatment with an average freedom from progression of 25 weeks (range, 6 to 52 weeks). The treatment was well tolerated, and there was no clinical evidence of immunosuppression. Following treatment, there was significant suppression of peripheral blood CD4 counts in all patients for 1 to 22+ weeks. Only one patient made a very low titer human antichimeric antibody response. All but two patients made primary antibody and T-cell proliferative responses to a foreign antigen administered 24 hours after antibody infusion. However, there was generally marked, but temporary suppression of T-cell proliferative responses in vitro to phytohemagglutinin (PHA), tetanus toxoid, and normal donor lymphocytes. We conclude that at the dose levels studied, this antibody (1) had clinical efficacy against mycosis fungoides; (2) was well tolerated; (3) had a low level of immunogenicity; (4) decreased T-cell proliferative responses in vitro, and (5) did not induce tolerance to a foreign antigen.
Phenotypic, molecular, and functional characterization of human peripheral blood CD34+/THY1+ cells
Authors: Humeau L , Bardin F , Maroc C , Alario T , Galindo R , Mannoni P , Chabannon C
Published: October 2019

A subset of mobilized CD34+ cells present in patient aphereses expresses Thy1 (CDw90). This population contains most long-term culture initiating cells, as assayed with a murine stromal cell line. It also contains a significant proportion of colony-forming unit granulocyte macrophage, but very few burst-forming unit erythroid. The limited differentiation towards the erythroid lineage is further confirmed by the absence of GATA-1 mRNA in the CD34+/Thy1+ subset, and by the low level of c-kit expression. The CD34+/Thy1+ subset appears phenotypically and functionally heterogeneous, a finding consistent with its high representation, compared to phenotypes such as CD34+/CD38- . Therefore, while at least some of CD34+/Thy1+ cells may be infectable by retroviral vectors, as shown by the presence of a transcript for the receptor for murine amphotropic retroviruses, the use of this selection strategy to specifically target human stem cells appears questionable.
Primary systemic CD30 (Ki-1)-positive anaplastic large cell lymphoma of the adult: sequential intensive treatment with the F-MACHOP regimen (+/- radiotherapy) and autologous bone marrow transplantation
Authors: Fanin R , Silvestri F , Geromin A , Cerno M , Infanti L , Zaja F , Barillari G , Savignano C , Rinaldi C , Damiani D , Buffoli A , Biffoni F , Baccarini M
Published: October 2019

Few series of adult patients with primary systemic CD30 (Ki-1)-positive anaplastic large cell lymphoma (ALCL) are reported in the literature; most of them have been treated with combination chemotherapy (CHT), with only an occasional patient being autotransplanted, mainly after relapsing. The remission rate ranges from 60% to 90%, but relapses are frequent (up to 60%) and precocious (mainly in the first 24 months). The aim of our study was to analyze the outcome of a series of adult patients affected by primary systemic ALCL that were treated at our institution with a sequential intensive therapeutic program including CHT, radiotherapy (RT), and autologous bone marrow transplantation (ABMT). Sixteen consecutive, unselected patients with ALCL were identified. All of them were treated with the 5-fluorouracil, methotrexate, cytosine arabinoside, cyclophosphamide, doxorubicin, vincristine, and prednisone (F-MACHOP) regimen; 9 of 16 (56.2%) reached a complete remission (CR). In six cases with residual mediastinal disease, involved-field RT was performed, allowing three additional patients to become free of disease. All 16 were then autotransplanted with bone marrow stem cells after conditioning with the cytosine arabinoside, etoposide, cyclophasphamide, and carmustine (BAVC) regimen. A present, 16 of 16 patients are alive and in CR. The actuarial overall survival is 100% at a median of 45.5 months, and the actuarial disease-free survival is 100% at a median of 33.5 months. These data suggest that ALCL can be successfully managed with a sequential intensive treatment (CHT +/- RT + ABMT) that prevents early relapses and projects these patients as long-term survivors.
Effects of fibroblast growth factor-4 (k-FGF) on long-term cultures of human bone marrow cells
Authors: Quito FL , Beh J , Bashayan O , Basilico C , Basch RS
Published: October 2019

Fibroblast growth factor-4 (FGF-4), a highly mitogenic protein encoded by the k-fgf/hst oncogene, stimulates the growth of a variety of cells of mesenchymal and neuroectodermal origin. Addition of FGF-4 to human long-term bone marrow cultures increased both the cell density of the stromal layer and the number of hematopoietic colony forming cells in the cultures in a dose-dependent manner. Hematopoiesis in the stromal layer persisted for up to 8 months. Erythropoiesis was maintained for up to 4 weeks, but granulocytes were the predominant nonadherent cell type. Cultures treated with FGF had increased numbers of monocytes compared with control cultures and some CD14+, CD45+ monocytes could still be detected after 8 months of continuous culture. The addition of the growth factor increased the rate of growth of the stromal layer and appeared to delay its senescence. Subcultures made in the presence of FGF-4 had up to 10-fold increases in plating efficiency and grew as relatively uniform monolayers. These subcultures retained the capacity to support hematopoiesis for several months, while untreated subcultures, made without FGF-4, grew erratically and generally lost the capacity to support hematopoiesis within 4 to 6 weeks. The improved growth after subculture greatly enhanced the reliability of limit- dilution assays of multipotential hematopoietic stem cells that use stromal cell monolayers. The primary effect of FGF-4 appeared to be on the stromal cells of the long-term bone marrow cultures, but a direct effect on hematopoietic progenitors could not be ruled out.
A case of hypereosinophilic syndrome is associated with the expansion of a CD3-CD4+ T-cell population able to secrete large amounts of interleukin-5
Authors: Brugnoni D , Airo P , Rossi G , Bettinardi A , Simon HU , Garza L , Tosoni C , Cattaneo R , Blaser K , Tucci A
Published: October 2019

Interleukin-5 (IL-5) is the major soluble factor able to mediate hypereosinophilia. We report a case of hypereosinophilic syndrome in which the presence of a population of CD3-CD4+ cells able to overproduce IL-5 was shown. The lack of CD3 and TCRAB membrane expression on otherwise phenotypically normal mature T lymphocytes together with the absence of detectable TCRBV mRNA and clonal rearrangement of TCRB gene suggested that the abnormal lymphocyte population was the expression of a peripheral T-cell lymphoma with an indolent clinical course. Peripheral blood lymphocytes enriched in this population were able to secrete high levels of IL-5 but not IL-4, and no IL-2 or interferon-gamma, when stimulated in vitro with phytohemagglutinin and phorbol myristate acetate. The serum contained eosinophil survival factors whose activity was partially neutralized by a specific antihuman IL-5 antibody. This observation further emphasized the relationship between hypereosinophilic syndrome. IL-5, and T-cell lymphoproliferative disorders.
Clinical features and outcome of children with first marrow relapse of acute lymphoblastic leukemia expressing BCR-ABL fusion transcripts. BFM Relapse Study Group
Authors: Beyermann B , Agthe AG , Adams HP , Seeger K , Linderkamp C , Goetze G , Ludwig WD , Henze G
Published: October 2019

Although the Philadelphia chromosome (Ph1) has been identified as an adverse prognostic factor in acute lymphoblastic leukemia (ALL), little is known about the incidence and clinical course of relapsed Ph1- positive ALL in children. The incidence was determined by screening of 170 consecutive children with first bone marrow relapse of ALL using the reverse transcriptase-polymerase chain reaction (RT-PCR) and comparison, with cytogenetic analysis. Among these 170 children, 20 (12%) were found to be BCR-ABL-positive, representing a rate that is about three times higher than that reported for newly diagnosed ALL. Ten of the cases were identified by RT-PCR only. In none of the 21 patients with T-cell immunophenotypes could an expression of the BCR- ABL mRNA be detected. BCR-ABL positivity was associated with a significantly shorter duration of first remission (P = .0086) and higher white blood cell (P = .0157) and blast cell counts (P = .0304) at relapse diagnosis. All patients were treated according to the ALL- REZ BFM 87 and 90 relapse trials of the BFM Relapse Study Group. The intensive multiagent chemotherapy induced a second complete remission in only 60% of children with BCR-ABL-positive ALL compared with in 91% of those without BCR-ABL expression (P = .0023). The prognosis of BCR- ABL-positive ALL in children is poor, with a probability of event-free survival at 2 years of 8% versus 50% in those without BCR-ABL mRNA or cytogenetic analysis should become part of the routine diagnostic panel for children with newly diagnosed ALL and is fundamental for children presenting with an early bone marrow relapse.
Improved red blood cell preservation correlates with decreased loss of bands 3, 4.1, acetylcholinestrase, and lipids in microvesicles
Authors: Dumaswala UJ , Dumaswala RU , Levin DS , Greenwalt TJ
Published: October 2019

In earlier studies we have shown that a final concentration of 0.69% glycerol in blood mixed with an experimental additive solution, EAS 25, improves the in vitro quality and in vivo survival of red blood cells (RBCs). The objective of this study was to determine if the better preservation of RBCs in EAS 25 is correlated with the improved maintenance of membrane lipids and proteins and decreased vesiculation. Split units of RBCs were stored in Adsol or EAS 25 (mmol/L: adenine 2/2, dextrose 122/110, mannitol 42/55, glycerol 0/150, NaCl 154/50). After 12 weeks storage, RBC and microvesicle membranes were analyzed for cholesterol, phospholipid, diphenyl hexatriene fluorescence anisotropy, and acetylcholinesterase (AchE) activity. Bands 3 and 4.1 were identified in the microvesicle membranes by immunoblotting. The RBC membrane cholesterol, phospholipids, and AchE remained higher in EAS 25 than in Adsol (P < .001). Vesicle membrane lipids and AchE in EAS 25 were significantly less than in Adsol (P < .001). The fluidity of stored cells in both the solutions was greater than the prestorage samples. Immunoblotting analyses showed that bands 3 and 4.1 were greatly reduced in the microvesicle membranes shed by the RBCs stored in EAS 25 compared with those formed in Adsol.
Deficiencies in progenitor cells of multiple hematopoietic lineages and defective megakaryocytopoiesis in mice lacking the thrombopoietic receptor c-Mpl
Authors: Alexander WS , Roberts AW , Nicola NA , Li R , Metcalf D
Published: October 2019

Mice with a null mutation in the thrombopoietin (TPO) receptor c-Mpl were generated by gene targeting. c-mpl-deficient mice developed normally but were deficient in megakaryocytes and severely thrombocytopenic. The hematocrit and numbers of mature circulating leukocytes were normal in mpl-/- mice, as was the distribution of morphologically identifiable precursors in hematopoietic tissues. Bone marrow and spleen cells of adult mpl-/- mice lacked specific binding sites for TPO, were unresponsive to TPO in culture, and displayed a marked deficiency in progenitor cells with megakaryocytic potential. Significantly, total hematopoietic progenitor cell numbers were also reduced in mpl-/- mice including multipotential, blast cell, and committed progenitors of multiple lineages. The megakaryocyte deficiency was evident as early as 14 days of gestation in mpl- deficient mice, although reductions in progenitor cell numbers arose only later in development. The data suggest that the critical function of c-Mpl signalling in megakaryocytopoiesis is in maintenance of mature megakaryocyte numbers through control of progenitor cell proliferation and maturation. Moreover, our results also imply an important role for TPO and c-Mpl in the production of primitive pluripotent progenitor cells as well as progenitor cells committed to nonmegakaryocytic lineages.
Long-term leukemia-initiating capacity of a CD34-subpopulation of acute myeloid leukemia
Authors: Terpstra W , Prins A , Ploemacher RE , Wognum BW , Wagemaker G , Lowenberg B , Wielenga JJ
Published: October 2019

Acute myeloid leukemia (AML) proliferation in vivo is maintained by a small fraction of progenitor cells. These cells have been assumed to express an immature phenotype and to produce most colony-forming units (CFU-AML). For one case of AML (French-American-British [FAB] M1, normal cytogenetics), we examined the capacity of the CD34+ (25% of unseparated AML cells) and CD34- fractions to initiate leukemia in severe combined immunodeficient (SCID) mice. In addition, the production of CFU-AML and nucleated cells (NC) of these subsets was investigated in long-term bone marrow culture (LTBMC). The frequencies of cobblestone area-forming cells (CAFC) were also estimated; early appearing cobblestone areas (CAs) are indicative of relatively mature progenitors and late CAs represent the progeny of primitive progenitors. In mice transplanted with CD34- (98% pure) or CD34+ (98% pure) grafts, similar AML cell growth was seen throughout an observation period of 106 days. The capacity to establish long-term growth from the CD34- cells was confirmed by renewed outgrowth after retransplantation. In vitro, the CD34- fraction contained both immature and mature CAFCs and produced high numbers of CFU-AML and NC in LTBMC. The CD34+ fraction produced only small numbers of CFU-AML, NC, and mature CAFCs. Therefore, the expression of CD34 and the content of CFU- AML were not associated with long-term growth of AML. However, similar frequencies of primitive CAFCs were observed in both fractions. Thus, both CD34- and CD34+ subsets of this AML sample contained immature progenitors with the capacity to initiate long-term AML growth as characterized in vivo (in SCID mice) as well as in vitro (in CAFC assay), indicating asynchrony between functional and immunophenotypical maturation of AML progenitor cell compartments.
Immobilized anti-KIT monoclonal antibody induces ligand-independent dimerization and activation of Steel factor receptor: biologic similarity with membrane-bound form of Steel factor rather than its soluble form
Authors: Kurosawa K , Miyazawa K , Gotoh A , Katagiri T , Nishimaki J , Ashman LK , Toyama K
Published: October 2019

Interaction of a tyrosine kinase type receptor and its ligand induces receptor-dimerization or -oligomerization followed by transphosphorylation and activation of its intrinsic kinase, which leads to a series of intracellular signals. We have previously reported that the membrane-bound form of Steel factor (SLF) induces more persistent tyrosine kinase activation and longer life span of c-kit encoded protein (KIT) than its soluble form (Miyazawa et al, Blood 85:641, 1995). In this study, we used YB5.B8 monoclonal antibody (MoAb) that recognizes the extracellular domain of KIT to investigate whether immobilized anti-KIT MoAb can substitute for SLF as a potent activator of KIT by cross-linking receptors and further compared its effect with each SLF isoform in a factor-dependent cell line M07e. YB5.B8 MoAb in a soluble state suppressed SLF-induced M07e cell proliferation in a dose- dependent manner. By contrast, once this antibody was immobilized on the goat-antimouse MoAb (GAM)-coated culture plates, it supported the growth of M07e cells in the absence of any growth factors, whereas culture the cells in GAM alone or YB5.B8 without GAM-coated plates resulted in rapid cell-death within 24 hours. As with the natural ligand SLF, immobilized YB5.B8 MoAb synergized with granulocyte- macrophage colony-stimulating factor (GM-CSF) in inducing cell proliferation compared with either YB5.B8 MoAb or GM-CSF alone. Immunoblotting with antiphosphotyrosine MoAb showed that interaction of M07e cells with immobilized YB5.B8 induced tyrosine phosphorylation of a series of intracellular proteins including KIT (145 kD). In addition, cross-linking studies using a water-soluble cross linking reagent bis- sulfosuccinimidyl-suberate showed that immobilized YB5.B8 MoAb induced dimerization and activation of KIT. However, as with stimulation by the membrane-bound form of SLF, the kinetics of KIT activation with YB5.B8 MoAb was more prolonged compared with the cells treated with recombinant soluble SLF. Flow cytometry showed that, unlike the cells treated with soluble SLF, no downmodulation of cell-surface KIT expression was observed in M07e cells cultured with immobilzed YB5.B8 MoAb. These data suggest that immobilized antibodies against hematopoietic receptors may replace their ligand-stimulators; however, their activities may resemble the membrane-bound form rather than the soluble form of natural ligands.
Antibody ligation of CD9 modifies production of myeloid cells in long- term cultures
Authors: Oritani K , Wu X , Medina K , Hudson J , Miyake K , Gimble JM , Burstein SA , Kincade PW
Published: October 2019

The KMC8.8 monoclonal antibody was made by immunizing rats with the BMS2 stromal cell clone, and was selected for further study because its ability to inhibit production of myeloid cells in Dexter cultures but not that of lymphoid cells in Whitlock-Witte cultures. The influence on myeloid progenitors might have been indirect, since the antibody did not prevent responsiveness to colony-stimulating factors in semisolid agar cultures. Furthermore, there was no inhibition, and some augmentation, of cell production when the antibody was added to established Dexter cultures. A cDNA clone that encoded the KMC8.8- recognized molecule was isolated by expression cloning and found to be identical in sequence to a previously published murine CD9 homologue. The antibody and cDNA clone were used to establish that CD9 is expressed by stromal cells, megakaryocytes, platelets, myeloid cells, and subpopulations of mature lymphocytes in mice. Treatment with the KMC8.8/CD9 antibody slightly augmented adhesion between myeloid cells and stromal cells, consistent with previous reports that this member of the tetraspan family of proteins can transmit proadhesive signals to human platelets and lymphoid cells. CD9 might participate in cell-cell interactions critical for correct orientation and movement of maturing myeloid cells in bone marrow.
Expression of cyclic ADP-ribose-synthetizing CD38 molecule on human platelet membrane
Authors: Ramaschi G , Torti M , Festetics ET , Sinigaglia F , Malavasi F , Balduini C
Published: October 2019

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP- ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP- ribose hydrolase activities. This finding indicates that the calcium- mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.
Inhibition of CD4 cross-linking-induced lymphocytes apoptosis by vesnarinone as a novel immunomodulating agent: vesnarinone inhibits Fas expression and apoptosis by blocking cytokine secretion
Authors: Oyaizu N , McCloskey TW , Than S , Pahwa S
Published: October 2019

Evidence is accumulating that T cells from human immunodeficiency virus type 1 (HIV-1)-infected individuals show accelerated cell death through apoptosis. We have recently demonstrated that the cross-linking of CD4 molecules (CD4XL) results in death of normal peripheral T cells through apoptosis and imbalanced cytokine secretion (ie, induction of tumor necrosis factor-alpha [TNF-alpha] and interferon-gamma [IFN-gamma] in the absence of interleukin-2 [IL-2] or IL-4 secretion). These upregulated cytokines (TNF-alpha/IFN-gamma) largely contributed to upregulation of the apoptosis-inducing cell surface molecule, Fas (APO- 1/CD95) and apoptosis induction. The present study investigated the effect of vesnarinone as a novel immunomodulating agent on CD4XL- induced T-cell apoptosis. The addition of vesnarinone to peripheral blood mononuclear cells (PBMC) significantly inhibited CD4XL-induced lymphocyte apoptosis. This apoptosis-inhibitory effect of vesnarinone was associated with the blocking of CD4XL-induced TNF-alpha IFN-gamma secretion and of Fas antigen upregulation. However, vesnarinone did not block effects of exogenously supplemented TNF-alpha/IFN-gamma on Fas induction. These data suggest that vesnarinone inhibits CD4XL-induced TNF-alpha/IFN-gamma secretion, thereby blocking subsequent Fas upregulation and apoptosis induction. Given the potent pathogenic role of imbalanced cytokine secretion observed in HIV-infection, an agent such as vesnarinone may be of therapeutic value in slowing disease progression.
The natural killer-related receptor for HLA-C expressed on T cells from CD3+ lymphoproliferative disease of granular lymphocytes displays either inhibitory or stimulatory function
Authors: Cambiaggi A , Orengo AM , Meazza R , Sforzini S , Tazzari PL , Lauria F , Raspadori D , Zambello R , Semenzato G , Moretta L , Ferrini S
Published: October 2019

Four patients with lymphoproliferative disease of granular lymphocytes (LDGL) coexpressing CD3 and the natural killer (NK)-related “p58” receptor for HLA-C alleles were studied. These CD3+p58+ LDGLs have been detected among a series of 44 CD3+ LDGLs analyzed. Two patients with LDGL (GI and BA) expressed only the p58 molecule defined by the GL-183 and CH-L monoclonal antibodies (MoAbs), while the cases of patients PU and MA also coexpressed the molecular form identified by EB6 anti-p58 MoAb. Three LDGL cases (GI, MA, and PU) displayed the CD8+4-CD16+ T- cell receptor (TCR)alpha/beta+ phenotype, while one patient (BA) was CD8+4+CD16+ TCRalpha/beta+. Freshly isolated granular lymphocytes (GL) from these cases displayed cytolytic activity in an anti-CD3 MoAb- triggered redirected killing assay against the Fcgamma-receptor+ (Fcgamma-R+) P815 target cell line. Lysis of P815 target cells, triggered by an anti-CD3 or by anti-CD16 MoAb, could be inhibited by the addition of anti-p58 MoAb in three fresh or interleukin (IL)-2- cultured GL tested (GI, MA, and PU). Triggering of cytotoxicity against the HLA-DR+ Fcgamma-R+ Daudi cell line induced by appropriate superantigens could also be inhibited by anti-p58 MoAb in patients PU and GI with LDGL. These data indicate that activation through the CD16, CD3, and TCR molecules can be modulated by p58 receptors in these LDGLs. On the contrary, IL-2-expanded cells of patient BA were induced to lyse P815 target cells by anti-p58 MoAb. In addition, anti-p58 MoAB enhanced anti-CD16 MoAb triggered lysis and did not inhibit activation via CD3. These data indicate that, in this particular patient with LDGL, p58 displays a stimulatory effect on cell triggering, rather than the typical inhibitory effect previously observed in p58+ T-cell clones derived from healthy donors. The anti-p58 MoAb did not induce CA++ mobilization in p58+ LDGLs and in a p58+CD3+ normal T-cell clone equipped with inhibitory p58 molecules, while Ca++ mobilization could be observed in cultured GL from patient BA, which could be activated by anti-58 MoAb. These findings suggest that stimulatory and inhibitory p58 molecules are equipped with different signal transducing properties, thus contributing to a better knowledge of the normal counterpart.