bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia
Authors: Bedi A , Zehnbauer BA , Barber JP , Sharkis SJ , Jones RJ
Published: October 2019

BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR- ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.
Interleukin-2 treatment in lymphoma: a phase II multicenter study [see comments]
Authors: Gisselbrecht C , Maraninchi D , Pico JL , Milpied N , Coiffier B , Divine M , Tiberghien P , Bosly A , Tilly H , Boulat O
Published: October 2019

A phase II study was undertaken to assess whether continuous infusion of high-dose recombinant interleukin-2 (rIL-2) alone was active against different histologic subtypes of heavily pretreated lymphoma. Sixty one lymphomas were included in the study. rIL-2 (Roussel UCLAF, Romainville, France) was administered by continuous infusion at 20 x 10(6) IU/m2 for three cycles of 5 days, 4 days, and 3 days, during the first week, third week, and fifth week, respectively. Twenty-four low- grade non-Hodgkin's lymphomas (NHL) were resistant to an anthracycline- containing regimen. Twenty-three intermediate and high-grade NHL were refractory to initial treatment or to salvage therapy. Seven Hodgkin's diseases were refractory to at least three regimens or relapsed after autologous bone marrow transplantation. Seven mycosis fungoides were refractory to chemotherapy. In low-grade NHL, one complete response (CR) was observed. In aggressive NHL, there were three CRs and two partial responses (PRs). No response was noted in Hodgkin's disease. One CR and four PRs were observed in mycosis fungoides. Complete response durations were 23, 20, 17, 12, and 4 months. Grade 4 toxicity was observed in 29 patients leading to arrest of therapy in 12 patients. The response to rIL-2 therapy in lymphoma differs according to histologic subtypes. Five responses were observed in 23 aggressive lymphomas. Five of seven mycosis fungoides responded; these preliminary results warrant testing of a larger number of patients.
Long-term interleukin-6 administration stimulates sustained thrombopoiesis and acute-phase protein synthesis in a small primate-- the marmoset
Authors: Ryffel B , Car BD , Woerly G , Weber M , DiPadova F , Kammuller M , Klug S , Neubert R , Neubert D
Published: October 2019

Interleukin-6 (IL-6) has been ascribed significant roles in both hematopoiesis and the immune response, although its contribution to host defence as a whole is poorly understood. Because short-term IL-6 treatment was previously shown to stimulate megakaryocytopoiesis, we investigated the effect of long-term administration of IL-6 on megakaryocytopoiesis and other systemic parameters in nonhuman primates. We chose a small primate, the marmoset (Callithrix jacchus), which enabled long-term administration at high doses. Recombinant human IL-6 (rhIL-6) administered at doses of up to 1,000 micrograms/kg/d over 4 and 9 weeks caused a sustained twofold to threefold increase of thrombocyte counts, peaking at 4 weeks. Thrombocyte counts declined thereafter, despite continuing IL-6 administration. The number of bone marrow megakaryocytes at 4 and 9 weeks was not increased compared with controls, but the ploidy grade was augmented, suggesting that IL-6 effects are restricted to mature megakaryocytes in vivo. An acute-phase protein response was observed within 24 hours after the first IL-6 administration and reached a maximum after 1 week of IL-6 administration at 25 micrograms/kg. Serum C-reactive protein, haptoglobin, and ceruloplasmin were increased, whereas albumin and transferrin levels declined. The acute-phase protein response was not associated with any morphologic evidence of hepatocellular damage. The increased levels of Ig and soluble IL-2 receptor in the serum levels reflected systemic immunostimulation. There was no evidence of renal mesangioproliferative pathology. Antibodies against rhIL-6 developed within 2 weeks, continuously increasing during the course of the study. High titers of neutralizing antibodies appeared concomitantly with the decrease in platelet counts and decline in acute-phase proteins. Therefore, despite the pleiotropic effects of IL-6 observed in vitro, long-term administration of IL-6 caused a selective and sustained stimulation of thrombopoiesis in marmosets that was only ablated by the appearance of neutralizing antibodies, and high doses were well tolerated in marmosets. A long-term targeting of IL-6 to cells of the megakaryocytic lineage, without evoking general toxicity, confirms the potential therapeutic usefulness of rhIL-6 for the chronic treatment of thrombocytopenic patients.
Platelet coagulation factor Va: the major secretory platelet phosphoprotein
Authors: Rand MD , Kalafatis M , Mann KG
Published: October 2019

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.
Tumor necrosis factor (TNF)-induced protein phosphorylation in a human rhabdomyosarcoma cell line is mediated by 60-kD TNF receptors (TR60)
Authors: Mire-Sluis A , Meager A
Published: October 2019

In the present study, we used a cloned derivative, KYM-1D4, of the human rhabdomyosarcoma cell line, KYM-1, known to express high numbers of the two tumor necrosis factor (TNF) receptors, TR60 and TR80, and to be highly sensitive to TNF alpha-mediated cytotoxicity/antiproliferation, to investigate the role of TR60 and TR80 in protein phosphorylation. Using permeabilized KYM-1D4 cells, it was found that TNF alpha strongly induced phosphorylation of proteins of molecular weight 80, 65, 58, 42, and 30 kD. Addition of a monoclonal antibody (MoAb) against TR60 was shown to induce cytotoxicity/antiproliferation in KYM-1D4 cells and the same pattern of protein phosphorylation as TNF alpha, whereas addition of an MoAb against TR80 was both noncytotoxic and ineffective in inducing protein phosphorylation. In contrast, in a highly TNF alpha-resistant KYM-1- derived cell line, 37B8R, no protein phosphorylation was induced with either TNF alpha or the agonistic anti-TR60 MoAb. However, when 37B8R was allowed to revert to partial TNF sensitivity by culture in the absence of TNF alpha, the resultant cell line, 37B8S, was found to regain inducibility of protein phosphorylation by TNF alpha. These results indicate that expression of functional TR60 in KYM-1-related cell lines is principally involved in TNF-mediated cytotoxicity/antiproliferation and is necessary for the induction of protein phosphorylation. Nevertheless, the latter, although apparently strongly associated with cytotoxicity, was probably involved in protective mechanisms because protein kinase C inhibitors that inhibited TNF alpha and anti-TR60-induced phosphorylation increased the cytotoxic/antiproliferative response to these mediators.
Rescue from anti-MHC class II antibody-mediated marrow graft failure by c-kit ligand
Authors: Deeg HJ , Beckham C , Huss R , Myerson D , Greinix H , Appelbaum FR , Graham T , Schuening F , Storb R
Published: October 2019

Dogs given 920 cGy of total body irradiation (TBI) followed by autologous marrow infusion uniformly achieve sustained hematopoietic reconstitution. We have previously shown that administration of the anti-MHC class II monoclonal antibody (MoAb) H81.98.21 (IgG2a) at 0.6 mg/kg/d immediately after transplantation results in delayed graft failure. A second noncrossblocking anti-MHC class II MoAb, B1F6, of the same isotype, at the same dose, did not interfere with sustained engraftment, suggesting that the observed effect was epitope dependent. Although higher concentrations of B1F6 were required, in the present study both MoAbs interfered with the propagation of long-term marrow cultures. When MoAb B1F6 was given in vivo at 1.2 mg/kg/d, ie, twice the dose used previously, dogs so treated also developed delayed marrow graft failure. Marrow failure with either MoAb involved myeloid, erythroid, and megakaryocytic lineages. Administration of recombinant canine c-kit ligand/stem cell factor (SCF) for 7 or 21 days posttransplant resulted in reversal of graft failure. Although the short course did induce a broad transient early peak of granulocytes, the longer course of SCF was accompanied by earlier sustained recovery than the short course. In conclusion, therefore, marrow graft failure induced by anti-MHC class II MoAb does not appear to be epitope dependent, involves all hematopoietic lineages, and is overcome by the administration of c-kit ligand.
Discovery of novel hematopoietic cell adhesion molecules from human bone marrow stromal cell membrane protein extracts by a new cell- blotting technique
Authors: Seshi B
Published: October 2019

In an attempt to define the role of cell adhesion molecules (CAMs) within the bone marrow (BM) microenvironment in normal hematopoiesis and in leukemia development, a novel cell-blotting technique that involved cell adhesion to protein bands after separation by lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDS-PAGE) and blotting onto polyvinylidene difluoride (PVDF) membrane has been developed. Human BM stromal cell membrane fractions have been prepared from Dexter-type cultures after cell lysis by sonification and differential centrifugations of the sonification contents. The 20,000 g pellets representing membrane fractions have been solubilized by 2% Triton X-100, 0.575% LDS, and 8 mol/L urea in sequential order. The protein extracts are fractionated by LDS-PAGE and screened for CAMs by the new cell-blotting technique. This led to identification of nine protein bands in lanes containing LDS extracts showing adhesion of KG1a (CD34+ progenitor myeloid) cells. Evidence that the BM proteins exhibiting KG1a cell adhesion are novel CAMs is based on the observations that these proteins, in comparison with known CAMs, specifically VCAM-1, CD54, and CD44, show (1) contrasting detergent- solubility properties, (2) different temperature requirement for mediating cell adhesion function, and (3) markedly distinct electrophoretic mobilities. The various cell types tested, notably KG1a, NALM-6, WIL-2, Ramos, HS-Sultan, K562, JY B lymphoblastoid cells, and T lymphoblasts, showed distinctive patterns of binding to different subsets of BM CAMs. These results demonstrate a new approach to studies of molecular mechanisms that may determine specificity of hematopoietic cellular localization within BM microenvironment and may play an important role in controlling hematopoiesis.
Alteration of fibrin network by activated protein C
Authors: Gruber A , Mori E , del Zoppo GJ , Waxman L , Griffin JH
Published: October 2019

The antithrombotic plasma enzyme, activated protein C (APC), may play a role in thrombolysis. In vitro, acceleration of clot lysis by APC depends on its ability to inhibit the activation of prothrombin. The effect of APC on the assembly and dispersion of fibrin network was studied using turbidimetry, plasmin digestion of fibrin, and electron microscopy of plasma clots. The addition of APC before clotting but not after clotting accelerated clot lysis. The rate of increase in the turbidity of clotting plasma was reduced by APC. The turbidity of plasma clots containing APC was directly related to the clot lysis time. Fibrin from plasma clots that were formed in the presence of APC yielded less fibrin degradation products than fibrin from clots without added APC. Furthermore, APC reduced the diameter and relative number of fibrin fibers in plasma clots during gel assembly. We propose that APC may enhance the efficacy of thrombolysis by reducing the relative mass of fibrin within maturing thrombi.
Evidence for a role of glycoprotein IIb-IIIa, distinct from its ability to support aggregation, in platelet activation by ionophores in the presence of extracellular divalent cations
Authors: Lages B , Weiss HJ
Published: October 2019

Ionophore A23187-induced 14C-serotonin secretion and thromboxane B2(TxB2) formation were found to be absent in citrated platelet-rich plasma (PRP) from thrombasthenic subjects and in normal PRP treated with glycoprotein (GP) IIb-IIIa complex-specific monoclonal antibodies. Both responses were restored to normal levels when 5 mmol/L EDTA was present, indicating that their absence was not caused by the absence of aggregation per se. In gel-filtered platelets (GFP) incubated with various additions, the blockade or absence of GPIIb-IIIa resulted in reduced A23187-induced secretion and TxB2 formation in media containing 1 mmol/L Ca2+ with or without fibrinogen and 1 mmol/L Mg2+ plus fibrinogen, but not when Ca, Mg-free buffer alone, 1 mmol/L EDTA, or fibrinogen alone were present. In contrast, no such dependence or GPIIb- IIIa was seen in GFP stimulated with thrombin or phorbol myristate acetate in the presence of 1 mmol/L Ca2+, 1 mmol/L EDTA, or buffer alone. The inhibition of ionophore-induced responses seen in both normal GFP treated with antibodies and thrombasthenic GFP was not associated with any significant alteration of the ionophore-mediated [Ca2+]i increase, as measured in both aequorin-loaded GFP stimulated with A23187 and fura-2-loaded GFP stimulated with ionomycin. Incubation of normal GFP with either the monoclonal antibodies or the ligand binding site peptide RGDS in the presence of 1 mmol/L Ca2+ caused virtually complete inhibition of A23187-induced aggregation, measured as the loss of single platelets, but RGDS, in contrast to the antibodies, did not inhibit secretion or TxB2 formation. We conclude that platelet activation induced by ionophores in the presence, but not in the absence, of extracellular divalent cations involves a GPIIb-IIIa- dependent process that most likely involves a property of the ligand- occupied form of the complex distinct from its ability to support aggregation. This could represent another example of an aggregation- independent activity of the receptor-occupied state of the GPIIb-IIIa complex in signal transduction.
T lymphocytes in skin lesions of psoriasis and mycosis fungoides express B7-1: a ligand for CD28
Authors: Nickoloff BJ , Nestle FO , Zheng XG , Turka LA
Published: October 2019

The activation of T cells requires two distinct signals. One signal involves interaction of the antigen-specific T-cell receptor with major histocompatibility complex molecules plus antigenic peptide; a second signal, which is antigen nonspecific, is the interaction of CD28 with its natural ligands B7–1 and B7–2/B70. CD28 is expressed on 80% of T cells, is upregulated after activation, and binds to B7 gene-family members, found on antigen-presenting cells. Because of our interest in the immunologic basis of benign and malignant T-cell-mediated disorders of the skin, we investigated the cellular distribution of CD28 and B7 family members in lesions of psoriasis and mycosis fungoides. By immunostaining cryostat sections of skin, CD28 was found to be expressed on virtually all lymphocytes in the epidermis and dermis of both skin diseases. Surprisingly, B7–1 was also found to be expressed on virtually all lymphocytes in the epidermis and dermis of both skin diseases. B7–1 expression was confirmed on CD3+ T lymphocytes using flow cytometry of single cell suspensions of fresh, unfixed psoriatic lesional tissue. To exclude the possibility that this result was caused by a second reagent contaminating the monoclonal antibody (MoAb) preparation, two different lots were used, and the MoAb was absorbed onto Chinese hamster ovary (CHO) transfectants expressing B7–1, or vector-only transfected CHO cells. These procedures confirmed that a B7- 1-like epitope was being recognized on psoriatic lesional T cells. In contrast to B7–1 expression on lymphocytes, B7–3, as defined by anti-BB- 1 MoAb reactivity, was found primarily on epidermal keratinocytes in both skin diseases and was not found on T cells. These results indicate that within two common skin disorders, lesional T cells accumulate in the dermis and epidermis, which express B7–1. Such expression may permit self-costimulation involving the CD28-mediated activation pathway, and thereby contribute to the ongoing T-cell proliferation present in these chronic, benign, and malignant skin diseases.
Elevated levels of interleukin-1 beta (IL-1 beta) and IL-6 in serum and increased production of IL-1 beta mRNA in lymph nodes of patients with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome
Authors: Gherardi RK , Belec L , Fromont G , Divine M , Malapert D , Gaulard P , Degos JD
Published: October 2019

To evaluate a possible implication of cytokines in the pathogenesis of polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes (POEMS) syndrome, we studied five consecutive patients with this condition, of which four had sclerotic bone lesions and four had multicentric Castleman's disease. Interleukin-1 beta (IL-1 beta) and IL- 6 serum levels were determined in these patients (13 serum samples) and in patients with multiple myeloma (5) and Waldenstrom's macroglobulinemia (5). In situ hybridization of the relevant mRNAs was performed on lymph node specimens of two patients with POEMS syndrome who had Castleman's disease. Elevated serum levels of IL-1 beta (13/13 samples), and IL-6 (7/13 samples) were found in patients with POEMS syndrome. In the other patients, serum IL-1 beta was undetectable or slightly increased and IL-6 was elevated in a single patient with Waldenstrom's macroglobulinemia. Abundant IL-1 beta mRNA-producing cells were present in interfollicular spaces in the two tested patients, while IL-6 mRNA-producing cells were rare. We conclude that IL-1 beta and IL-6 serum levels may be chronically elevated in patients with POEMS syndrome, and that lymph node may be one site of IL-1 beta overproduction. These results are in keeping with the hypothesis that cytokines mediate systemic manifestations of POEMS syndrome.
Self-renewal and differentiation of stem cells in a biopotential murine leukemia: an in vitro model for differentiation therapy
Authors: Duhrsen U , Knieling G , Wu HX , Hossfeld DK
Published: October 2019

PGM-2 is a variant of the transplantable PGM-1 leukemia of strain C3H/HeJ. Freshly explanted cells had lymphoid morphology with a CD5+ CD45R (B220)- IgM- phenotype. They were not viable in unstimulated cultures, but formed IgM+ lymphoid colonies in response to interleukin- 2 (IL-2), IL-4, IL-5, IL-6, IL-7, and Steel factor, and macrophage colonies in response to IL-3. IL-3-stimulated colonies had no recloning potential, but colonies from IL-7 cultures gave rise to large numbers of secondary macrophage colonies in IL-3-stimulated cultures and secondary lymphoid colonies in IL-7-stimulated cultures. The latter ones could be serially transferred in vitro for several months, and formed typical PGM-2 tumors in vivo. IL-7-stimulated colonies could therefore be used to measure leukemic stem cells in vitro. Supramaximal IL-3 stimulation (2,500 U/mL) of suspension cultures was followed by an increase in overall cell numbers and a disappearance of leukemic stem cells, compatible with differentiation induction. This could not be counteracted by simultaneous stimulation with IL-7. However, lower IL-3 concentrations (500 U/mL) induced an expansion of the stem cell pool, possibly by facilitating density-dependent autostimulatory mechanisms involving endogenous production of IL-7. The system described is a simple in vitro model for differentiation therapy. It shows that leukemic stem cells can be induced by hematopoietic growth factors to undergo terminal differentiation, but the concentrations required for differentiation induction in stem cells are much higher than those required for other biologic effects. Submaximal stimulation may favor expansion rather than repression of the leukemic cell population.
Deformation of swollen erythrocytes provides a model of sickling- induced leak pathways, including a novel bromide-sensitive component
Authors: Sugihara T , Yawata Y , Hebbel RP
Published: October 2019

Deoxygenation-induced red blood cell (RBC) sickling probably activates multiple cation leak pathways. In an attempt to model this, we examined the net passive K efflux (“K leak”) from normal and sickle RBCs undergoing elliptical deformation in hypotonic media (200 mOsmol/L). This hypotonic deformation activates two deformation-dependent K leak pathways that are not detectable during the balanced leak (Kefflux = Nainflux) resulting from deformation of RBCs in isotonic medium. These are (1) a calcium-dependent leak component and (2) a novel leak pathway that is inhibited by substitution of bromide (but not sulfamate) for chloride, which converts the unbalanced K leak (Kefflux > Nainflux) of hypotonic deformation to a residual balanced leak. This dramatic effect of hypotonic deformation is reversible, is detected in both normal and sickle RBCs, and is inhibited significantly by 4,4′-diisothiocyano-2,2′- stilbene disulfonate. Remarkably, bromide also inhibits by 55% the K leak resulting from authentic deoxygenation-induced RBC sickling and, thereby, blunts the imbalance of accompanying monovalent cation leaks. The unique effect of bromide is not readily explainable on the basis of known behaviors of known ion leak/transport pathways. The mechanical threshold for triggering K leak during hypotonic deformation is at applied shear stress of 164 dyne/cm2, a value similar to the abnormal susceptibility we previously found for oxygenated sickle RBCs during isotonic deformation. These data suggest that membrane stretch accompanying hypotonic deformation activates the same multiple leak pathways that contribute to net K leak during authentic RBC sickling, including a previously unknown bromide-sensitive leak.
Effect of human immunodeficiency virus type-1 envelope glycoprotein gp160 on cytokine production from cord-blood T cells
Authors: Than S , Oyaizu N , Pahwa RN , Kalyanaraman VS , Pahwa S
Published: October 2019

We have recently demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp160 enhances the in vitro differentiation of hematopoietic myeloid progenitor cells derived from cord blood by inducing secretion of colony-stimulating factor(s) (CSF) in T cells, presumably through the interaction of gp160 with CD4 molecules. In this study, we investigated the gp 160-induced humoral CSFs in cord blood by enzyme-linked immunosorbent assay (ELISA) and by polymerase chain reaction on reverse-transcribed mRNA (RT-PCR). We demonstrate that gp160 can induce interleukin (IL)-3, IL-6, and granulocyte-macrophage CSF (GM-CSF) protein secretion only in purified cord-blood T cells (CB-T) and not in detectable amounts in whole cord blood cells (WCB); cytokine mRNA induction occurred in purified CB-T and WCB, but was significantly greater in the former. Treatment of gp160 with soluble CD4 (sCD4) abolished the secretion of all three cytokines in CB-T cells, which suggests that interaction of gp160 with CD4 molecules is required for the secretion of these cytokines from CB- T cells. However, in WCB cells, sCD4 treatment of gp160 resulted in inhibition of only IL-3 and GM-CSF mRNA, whereas IL-6 secretion was enhanced. Purified cord-blood monocytes secreted only IL-6 in response to gp160, and the gp160-induced IL-6 secretion by monocytes was also further increased by gp160 + sCD4 complex. Furthermore, monocyte culture supernatants suppressed gp160-induced IL-3 secretion from CB-T cells. These findings indicate that (1) CB-T cells are a potent source of gp160-induced hematopoietic cytokines, and (2) that different mechanisms are involved in the induction of IL-6 by gp160 in the T- and non-T-cell fractions of cord blood. The ability of HIV gp160 to induce hematopoietic CSFs in cord blood may be important in HIV pathogenesis.
Interleukin-2-dependent T-cell lines established from paroxysmal nocturnal hemoglobinuria patients
Authors: Nakakuma H , Nagakura S , Horikawa K , Hidaka M , Kawaguchi T , Iwamoto N , Sanada I , Kagimoto T , Takatsuki K
Published: October 2019

Peripheral blood T lymphocytes obtained from two patients with paroxysmal nocturnal hemoglobinuria (PNH) were immortalized with human T-lymphotropic virus type 1 (HTLV-1). These cells showed interleukin-2 (IL-2)-dependent cell growth in culture. Cell surface analysis showed that they had the phenotype of a helper/inducer T subset that was positive for CD2, CD3, and CD4, but negative for CD8, similar to adult T-cell leukemia cells induced by HTLV-1. These cell lines lacked glycosylphosphatidylinositol (GPI)-anchored proteins, CDw52, CD55 (decay-accelerating factor; DAF), and CD59 on the cell surface, whereas intracellular DAF protein was detected. These T-subset cell lines with a PNH phenotype did not synthesize GPI anchor, whereas a control cell line, similarly prepared from the T cells of a healthy volunteer, produced the anchor. The control cells expressed CDw52, DAF, and CD59 on the cell surface and showed the phenotype of a helper/inducer subset. Southern blot analysis confirmed the clonality of each cell line. These CD4+ T-cell lines with a PNH phenotype and a subset-matched control counterpart could be a useful model for PNH investigation.
Expression of interleukin-1 beta gene in candidate human hematopoietic stem cells
Authors: Watari K , Lansdorp PM , Dragowska W , Mayani H , Schrader JW
Published: October 2019

Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect interleukin-1 beta (IL-1 beta) mRNA in candidate human hematopoietic stem cells. The cells, obtained from adult bone marrow (BM) or umbilical cord blood, had a CD34+ CD45RAlo CD71lo phenotype and were further fractionated into CD38+ and CD38- or Thy-1+ and Thy-1- subpopulations. The purity of these fractions was always more than 99%. IL-1 beta and CD34 mRNA were detected in pools of 30 BM-derived CD34+ CD45RAlo CD71lo cells. To further exclude any contribution by contaminating cells, individual cells were analyzed for CD34 and IL-1 beta mRNA. Positive results were obtained with 2 of 5 individual BM- derived CD34+ CD45RAlo CD71lo CD38+ cells isolated by micromanipulation after overnight culture in serum-free medium without any exogenous cytokines, and 1 of 10 individual CD34+ CD45RAlo CD71lo CD38- cells isolated immediately after sorting. Moreover, of 10 pools of three BM- derived CD34+ CD45RAlo CD71lo cells cultured overnight in the presence of a mixture of various cytokines (Steel factor, IL-3, IL-6, macrophage colony-stimulating factor [M-CSF], erythropoietin, and IL-3/granulocyte- macrophage colony-stimulating factor [GM-CSF] fusion protein), 5 were positive for IL-1 beta mRNA. This result was compatible with more than 20% (95% confidence limit 0.06–0.61) of the BM cells with the CD34+ CD45RAlo CD71lo phenotype expressing IL-1 beta mRNA. IL-1 beta expression was also consistently observed from day 0 to day 9 in liquid cultures of cord-blood-derived CD34+ CD45RAlo CD71lo Thy-1+ or Thy-1- cells. The cultures contained the same combination of cytokines and resulted in an expansion of cell numbers of up to 400-fold. GM-CSF mRNA was not detected in the equivalent of 75 cells at any day, even though it could be detected with high sensitivity in control stromal cells. Because IL-1 beta is a powerful and pleiotropic biomodulator of cytokines and adhesion molecules, our observations suggest that at least some primitive hematopoietic cells do not merely respond passively to signals from their environment, but may themselves regulate the paracrine production of cytokines from neighboring stromal cells.(ABSTRACT TRUNCATED AT 400 WORDS).
Mast cell growth factor modulates CD36 antigen expression on erythroid progenitors from human bone marrow and peripheral blood associated with ongoing differentiation
Authors: de Wolf JT , Muller EW , Hendriks DH , Halie RM , Vellenga E
Published: October 2019

To study the differentiation process of erythroid progenitors from normal human bone marrow and peripheral blood, CD34/CD36 sorted cells were cultured in the presence of Erythropoietin (Epo) and Epo plus mast cell growth factor (MGF). The CD34+/CD36- cell fraction from bone marrow supported 74 +/- 33 erythroid burst forming units (BFU-E)/10(4) cells (mean +/- SD, n = 4) in the presence of Epo, which increased 2.1- fold by coculturing with MGF. However, erythroid colony-forming units (CFU-E) were not cultured from the CD34+/CD36- cell fraction. In contrast, the CD34-/CD36+ cell fraction supported CFU-Es in the presence of Epo (152 +/- 115/10(5)) or Epo plus MGF (180 +/- 112/10(5)), whereas BFU-Es were hardly noticed. However, the transition of the BFu-E to CFU-E was observed by incubating CD34+/CD36- cells (10(4)/100 microL) in suspension with Epo plus MGF for 7 days followed by Epo in the colony assay. This was reflected by the appearance of CD34-/CD36+/Glycophorin A+/CD14- cells. In addition high numbers of CFU- Es (1,000 +/- 150, n = 4) were cultured from this cell fraction. In contrast to bone marrow erythroid progenitors, no peripheral blood CFU- Es were cultured from either the CD36+ or CD36- fraction, whereas BFU- Es were predominantly present in the CD36+ fraction. However, the CD34+ progenitor cell from peripheral blood did have intrinsic capacity to differentiate to CFU-Es because CD34+/CD36- cells incubated with Epo plus MGF for 7 days and followed by Epo in the colony assay, supported high numbers of CFU-Es (1,200 +/- 400, n = 3). To study whether additional growth factors have similar effects on erythroid progenitors, experiments were performed with interleukin 1 (IL-1), IL- 3, and IL-6. IL-1 and IL-6 did not modulate the Epo supported proliferation and differentiation. In contrast, IL-3 in the presence of Epo did support CFU-Es, from CD34+/CD36- cells after 7 days in suspension culture. However, flow cytometry analysis showed that Epo plus IL-3 not only supported CD34-/CD36+/Glycophorin A+ cells but also CD36+/CD14+ cells, indicating the differentiation along different cell lineages. In summary, the data show a phenotypic distinction between bone marrow and peripheral blood erythroid progenitors with regard to CD36 expression. In addition, the results suggest that Epo plus MGF or IL-3 and preincubation in suspension culture are prerequisites for the transition of the BFU-E to the CFU-E.
Reduced thrombus formation in native blood of homozygous factor VII- deficient patients at high arterial wall shear rate
Authors: Barstad RM , Stormorken H , Orning L , Stephens RW , Petersen LB , Kierulf P , Sakariassen KS
Published: October 2019

Inhibition of thrombin formation in flowing native blood reduces thrombus formation on subendothelium, dacron, or collagen fibrils at arterial wall shear rates of 450 to 650 s-1. In the present study, we have investigated the role of low levels of factor VII (FVII) in thrombus formation on collagen fibrils at arterial wall shear rates of 650 s-1 (coronary arteries), 2,600 s-1 (mildly stenosed arteries), and 10,510 s-1 (severely stenosed arteries) in parallel-plate perfusion chambers. In the perfusion chamber with the highest wall shear rate, thrombus formation took place at the apex of an eccentric stenosis, which reduced the cross-sectional area of the blood flow channel by 80%, thus simulating thrombus formation at an atherosclerotic plaque rupture. Native blood from 21 healthy volunteers and 12 homozygous FVII- deficient patients was drawn by a pump directly from an antecubital vein over a surface of fibrillar collagen positioned in the respective perfusion chambers. The patients had FVII coagulant activities ranging from 1.3% to 4.5% and FVII antigen levels of 16% to 23% of normal. Immunoaffinity purification of the patients' FVII followed by electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]) and immunoblotting showed a protein with similar molecular mass as normal FVII. In the perfusion studies, a reduction in thrombus volume of 54% of normal (P < .007) at 10,510 s-1 was observed. The deposition of fibrin on the thrombogenic surface and the plasma level of fibrinopeptide A (FPA) in blood samples collected distal to the perfusion chamber were concomitantly reduced (P < .002 and P < .04, respectively). The plasma FPA level was also reduced at 2,600 s-1 (P < .04), but not at 650 s-1. However, at the lower shear conditions, the thrombus volume and the fibrin deposition were within the ranges observed in normal blood. The platelet-collagen adhesion was not affected at any of the three shear conditions. Thus, low plasma levels of FVII result in significantly less formation of thrombin and fibrin in and around growing platelet masses at high shear condition. This may weaken the thrombus stability and reduce platelet recruitment, thereby lowering thrombus volume. In support of this theory, one patient with afibrinogenemia had an 83% reduction in thrombus volume at this high shear condition.
Acquired von Willebrand disease caused by an autoantibody selectively inhibiting the binding of von Willebrand factor to collagen
Authors: van Genderen PJ , Vink T , Michiels JJ , van 't Veer MB , Sixma JJ , van Vliet HH
Published: October 2019

An 82-year-old man with a low-grade malignant non-Hodgkin lymphoma and an IgG3 lambda monoclonal gammopathy presented a recently acquired bleeding tendency, characterized by recurrent epistaxis, easy bruising, and episodes of melena, requiring packed red blood cell transfusions. Coagulation studies showed a von Willebrand factor (vWF) defect (Ivy bleeding time, > 15 minutes; vWF antigen [vWF:Ag], 0.08 U/mL; ristocetin cofactor activity [vWF:RCoF], < 0.05 U/mL; collagen binding activity [vWF:CBA], 0.01 U/mL; absence of the high molecular weight multimers of vWF on multimeric analysis). Mixing experiments suggested the presence of an inhibitor directed against the vWF:CBA activity of vWF without significantly inhibiting the FVIII:C, vWF:Ag, and vWF:RCoF activities. The inhibitor was identified as an antibody of the IgM class by immunoabsorption of vWF and inhibitor-vWF complexes from the plasma of the patient. Subsequent immunoprecipitation experiments using recombinant fragments of vWF showed that the inhibitor reacted with both the glycoprotein Ib binding domain (amino acids [aa] 422–826) and the A3 (aa 909–1112) domain of vWF, but not with the A2 (aa 716–908) or D4 (aa 1183–1535) domains. We conclude that the IgM autoantibody inhibits the vWF:CBA activity by reacting with an epitope present on both the glycoprotein Ib and A3 domains of vWF.
Genetic analysis is consistent with the hypothesis that NF1 limits myeloid cell growth through p21ras
Authors: Kalra R , Paderanga DC , Olson K , Shannon KM
Published: October 2019

Children with neurofibromatosis, type 1 (NF-1) are at increased risk of developing malignant myeloid disorders and their bone marrows frequently show loss of the normal allele of the NF1 tumor-suppressor gene. NF1 encodes a protein called neurofibromin, which accelerates guanosine triphosphate (GTP) hydrolysis on the p21ras (Ras) family of signaling proteins. We used a genetic approach to test the hypothesis that NF1 negatively regulates myeloid cell growth through its effect on Ras. This model predicts that, if RAS mutations and loss of NF1 function deregulate myeloid growth by the same biomechanical mechanism, then activating RAS mutations will be restricted to children with malignant myeloid disorders who do not have NF-1. We studied 71 children, including 28 with bone marrow monosomy 7 syndrome (Mo7), 35with juvenile chronic myelogenous leukemia (JCML), three with other forms of preleukemia, and five with acute myelogenous leukemia (AML), for activating mutations of KRAS and NRAS. The incidence of RAS mutations was 21% (12 of 55) in patients without NF-1 and 0% (zero of 16) in children with NF-1 (P = .04). Among the 55 patients who did not have NF-1, we found RAS mutations in four of 27 with Mo 7, in five of 24 with JCML, in two of 3 with AML, and in a patient with myeloproliferative syndrome (MPS). These data from primary human cancer cells provide strong genetic evidence that NF1 limits the growth of myeloid cells by regulating Ras.
Loxoribine induces chronic lymphocytic leukemia B cells to traverse the cell cycle
Authors: Goodman MG , Wormsley SB , Spinosa JC , Piro LD
Published: October 2019

Leukemic B cells from a majority of patients with chronic lymphocytic leukemia (CLL) enter the cell cycle upon stimulation in vitro with loxoribine, a potent 7,8-disubstituted guanine ribonucleoside immunostimulant. In the absence of added costimulants, a proportion of these cells become activated and undergo DNA synthesis and mitosis accompanied by a marked increase in expression of an array of cell surface activation antigens. The resultant activated B-CLL cells exhibit greatly enhanced sensitivity to cycle-active cytotoxic drugs. This approach may be of potential value in the therapy of CLL.
Expression of stem cell factor and c-kit in human neuroblastoma. The Children's Cancer Group
Authors: Cohen PS , Chan JP , Lipkunskaya M , Biedler JL , Seeger RC
Published: October 2019

During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Because melanocytes derive from neural crest cells, the role of SCF and c-kit was investigated in the neural crest-derived childhood tumor neuroblastoma. Using reverse transcription-polymerase chain reaction analysis, simultaneous expression of steady-state mRNA for the SCF ligand and its receptor c- kit was found in 14 of 14 (100%) human neuroblastoma cell lines and clones and in 8 of 18 (45%) human neuroblastoma tumor samples. Functional blockade of c-kit receptors in the cell lines SK-N-BE(2) and SH-SY5Y using the mouse monoclonal anti-c-kit antibody SR-1 resulted in a significant decrease in cellular growth rate when measured by either 3H-thymidine incorporation or clonogenicity. In addition, higher levels of c-kit mRNA expression were associated with parental neuroblastoma cell lines and subclones with a neuronal (N) differentiation phenotype, whereas lower levels of c-kit mRNA were associated with neuroblastoma cell line subclones having a schwannian/glial/melanocytic pattern of differentiation. However, the differentiation phenotype of neuroblastoma cell lines was not directly altered when c-kit expression was blocked using the SR-1 antibody. In summary, these data indicate that c-kit receptor expression may play a significant role in the growth regulation of the two neuroblastoma cell lines examined and suggest that c-kit may also play a similar role in neuroblastoma growth regulation in vivo. Simultaneous expression of SCF and c-kit mRNA in both neuroblastoma cell lines and tumors implies that c-kit may act as part of an autocrine growth loop in conjunction with endogenous production of SCF in this disease.
UVB irradiation of human platelet concentrates does not prevent HLA alloimmunization in recipients
Authors: Grijzenhout MA , Aarts-Riemens MI , de Gruijl FR , van Weelden H , van Prooijen HC
Published: October 2019

Exposure of platelet concentrates (PCs) to ultraviolet B radiation (UVB) has been advocated as an alternative method for prevention of the onset of HLA sensitization in recipients. In this study, pooled PCs were irradiated in a Haemonetics UV irradiator (Haemonetics Corp, Braintree, MA) at a dose that did not induce platelet activation. The effect of UVB irradiation on prevention of primary HLA sensitization was evaluated in a prospective controlled clinical study performed in cardiac patients undergoing cardiopulmonary bypass. Patients were treated with filtered red blood cells and a single transfusion of either standard (control group) or UVB-irradiated (UVB group) pooled platelets prepared from 12 donors. Five of 39 patients in the control group and 6 of 62 patients in the UVB group developed allo-antibodies against HLA antigens, which is not significantly different (P = .62). This unexpected finding prompted us to check the efficacy of UVB irradiation. We determined UVB-specific DNA damage in cells by measuring the fluorescence from a labeled specific monoclonal antibody against thymine dimers. With this novel flow cytometer technique, we estimated in UVB-irradiated leukocytes in saline that a mean fluorescence intensity (MFI) of 47 +/- 2 arbitrary units (n = 6) correlated with abolition of alloreactivity in mixed lymphocyte cultures and delayed cell death (within 72 hours). MFI in leukocytes suspended in plasma and exposed to the clinical dose of UVB was sixfold higher (310 +/- 41 arbitrary units) and resulted in early cell death (within 24 hours). We hypothesize that this high level of UVB radiation induces fragmentation of the leukocytes. As a consequence, the poor results of UVB irradiation may be explained by the onset of HLA- alloimmunization induced by soluble donor HLA class I antigens processed and presented by host antigen-presenting cells.
Highly sensitive polymerase chain reaction methods show the frequent survival of residual recipient multipotent progenitors after non-T-cell- depleted bone marrow transplantation
Authors: Petit T , Raynal B , Socie G , Landman-Parker J , Bourhis JH , Gluckman E , Pico J , Brison O
Published: October 2019

Twenty-four male patients grafted for various pathologies with the marrow of a female donor and presenting a complete donor-type hematopoiesis when analyzed by polymerase chain reaction (PCR) amplification of minisatellite sequences 33.6.3 and MS51 (0.1% to 1% sensitivity) were studied by the highly sensitive technique of PCR amplification of the Y-chromosome-specific DYZ1 sequence (0.01% sensitivity). Residual recipient male cells were detected in all peripheral blood samples collected within 1 year posttransplantation. These residual cells were present in both the lymphocyte and polymorphonuclear cell fractions when such a separation was performed by Ficoll gradient centrifugation and, for samples of 13 of 15 patients, at comparable levels in both fractions. In 3 samples collected from 3 patients 4 months or more posttransplantation, residual recipient cells were detected in the polymorphonuclear cell fraction but were present at a lower level or were undetectable in the lymphocyte fraction. These cells are of hematopoietic origin because they were detected at equivalent levels in whole blood and in B and T lymphocytes sorted with antibody-coated magnetic beads. They were not detected in samples collected more than 15 months posttransplantation for 6 of 7 patients. The persistence of residual recipient cells within 1 year posttransplantation is not restricted to male patients receiving a transplant from a female donor because they were also detected in 2 female patients using an allele-specific amplification method for the thyroid peroxydase gene that also has a high sensitivity (0.01%). Our results indicate that at least residual recipient myeloid progenitors and possibly totipotent hematopoietic stem cells may survive intensive pretransplant conditioning regimen and support a transient residual hematopoiesis of the host posttransplantation.
Prevention and treatment of meningeal leukemia in children
Authors: Pinkel D , Woo S
Published: October 2019

The prevention of meningeal leukemia has long been a keystone in its cure. The need was recognized when it became apparent in the 1950s and 1960s that meningeal relapse heralded hematologic relapse and a fatal course and that its incidence increased as systemic chemotherapy became more effective in controlling hematologic and visceral leukemia. Evasion of a biologic safety net, the blood-CSF barrier, is required to prevent meningeal leukemia. Three methods are used: meningeal radiotherapy, intrathecal administration of antileukemia drugs, and high-dosage intravenous antileukemia drugs. Recent and current clinical studies reflect a continuing dialogue about which methods are preferable and under what circumstances. For prevention of meningeal leukemia, extended intrathecal therapy and intensive systemic chemotherapy appear to be as effective as radiotherapy for most patients. For treatment of overt meningeal leukemia, meningeal radiotherapy may be necessary. However, its administration compromises subsequent systemic chemotherapy so that delay may be advisable to allow intensive systemic chemotherapy for control of concurrent hematologic and visceral leukemia, whether clinically evident or not. For patients with meningeal leukemia at diagnosis, cranial irradiation may be delayed or possibly omitted if evidence of disease is minimal and intrathecal and systemic chemotherapy are intensive. For those who develop meningeal leukemia while on therapy or after its completion, cranial or craniospinal irradiation is probably required as well as intensive intrathecal and systemic chemotherapy. Hopefully, current and future studies will dispel the uncertainties and better quantitate risks and benefits of alternative methods. Whatever method is used, careful attention to technical details is required to assure optimal efficacy at the least possible expense in immediate toxicity and late sequelae.