bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of interleukin-9 on clonogenic maturation and cell-cycle status of fetal and adult hematopoietic progenitors
Authors: Holbrook ST , Ohls RK , Schibler KR , Yang YC , Christensen RD
Published: October 2019

We assessed the effect of interleukin-9 (IL-9) on clonogenic maturation and cell-cycle status of hematopoietic progenitors of fetal (umbilical cord blood) and adult (bone marrow) origin. As a single agent IL-9 supported, in a concentration-dependent fashion, maturation of burst- forming units-erythroid (BFU-E) of adult and fetal origin. However, only 1/3 the number of adult BFU-E colonies developed, as did in response to granulocyte-macrophage colony-stimulating factor (GM-CSF), and only 1/6 the number developed as did in response to IL-3. In contrast, the effect of IL-9 on fetal BFU-E colonies was equal to that of GM-CSF and IL-3. Synergistic effects of IL-9 with low concentrations (0.1 ng/mL) of GM-CSF and IL-3 were seen on adult BFU-E colony formation, but no effect was apparent at higher concentrations (1.0 ng/mL). In contrast, using fetal cells, synergistic effects of IL-9 with low and high concentrations of GM-CSF and IL-3 were apparent. Addition of IL-9 to plates containing fetal cells plus GM-CSF and IL-3 not only resulted in more BFU-E colonies, but also in more multicentered (greater than or equal to 10 individual centers) colonies, and more cells per colony. IL-9 had a wider spectrum of action on progenitors of fetal origin than on progenitors of adult origin, supporting the generation of fetal multipotent colony-forming unit (CFU)-Mix and CFU-GM colonies. Incubation with IL-9 did not accelerate cycling of adult or fetal BFU-E, CFU-Mix, or CFU-GM to the extent observed after incubation with IL-6. Thus, IL-9 primarily supported maturation of erythroid progenitors of adult origin, and its addition to plates containing GM-CSF and IL-3 (1.0 ng/mL) did not result in maturation of additional clones. In contrast, IL-9 had a wider spectrum of action on fetal progenitors and, when combined with IL-3 and GM-CSF, resulted in clonogenic maturation of progenitors that did not undergo maturation after stimulation with IL-3 and GM-CSF.
Characterization of macrophage colony-stimulating factor in body fluids by immunoblot analysis
Authors: Suzu S , Yanai N , Sato-Somoto Y , Yamada M , Kawashima T , Hanamura T , Nagata N , Takaku F , Motoyoshi K
Published: October 2019

We characterized the molecular species of human macrophage colony- stimulating factor (hM-CSF) found in serum and urine, using immunoblot analysis after partial purification on an antibody-bound affinity column. Although antibodies were prepared using the recombinant product of the large form of hM-CSF with a molecular weight (MW) of 85 Kd as the antigen, this immunoblot system was also capable of detecting the small form of hM-CSF with a MW of 40 to 60 Kd. A single band with a MW of 43 Kd, which reacted with anti-recombinant hM-CSF IgG but not with control IgG, was found when serum and urine from normal adults underwent electrophoresis on reduced sodium dodecyl sulfate- polyacrylamide gel and subsequent immunoblotting. This band represented a subunit of the large form of hM-CSF, because the large form of hM-CSF is a homodimer of a subunit with a MW of 43 Kd and the small form of hM- CSF is a homodimer of a subunit with a MW of 20 to 30 Kd. Analysis of serum and urine from leukemic patients and pregnant women, who had higher serum levels of hM-CSF than normal adults, showed only a single band with a MW of 43 Kd as a hM-CSF-specific molecule. These results suggest that the large form of hM-CSF is the major species in human body fluids.
The sequence gamma-(312-324) is a fibrin-specific epitope
Authors: Schielen WJ , Adams HP , van Leuven K , Voskuilen M , Tesser GI , Nieuwenhuizen W
Published: October 2019

Fibrin accelerates the activation of plasminogen catalyzed by tissue- type plasminogen activator much stronger than fibrinogen. Detailed studies showed that (part of) this rate-enhancing effect of fibrin is brought about by two sites in the fibrin molecule: one in A alpha-(148- 160) and one in the gamma-chain stretch 311–379 (also known as FCB-5). During the fibrinogen-to-fibrin conversion, A alpha-(148–160) appears to become accessible, because a monoclonal antibody against synthetic A alpha-(148–160) reacts with fibrin, but not with fibrinogen. Because a similar situation may exist for (at least parts of) FCB-5, we have prepared a monoclonal antibody against a part (ie, gamma-[312–324]) of FCB-5, and found that this is fibrin-specific and does not bind fibrinogen. We conclude that gamma-(312–324) is hidden in fibrinogen and is exposed by the formation of fibrin.
A soluble factor released by CD8+CD57+ lymphocytes from bone marrow transplanted patients inhibits cell-mediated cytolysis
Authors: Autran B , Leblond V , Sadat-Sowti B , Lefranc E , Got P , Sutton L , Binet JL , Debre P
Published: October 2019

We report a new inhibitory activity of CD8+CD57+ peripheral blood lymphocytes from allo-bone marrow transplant patients. Positively selected CD8+CD57+ lymphocytes act as potent inhibitors of allospecific cytolytic T lymphocyte and lymphokine activated killer cell cytolytic activities. These suppressor cells are mature T cells expressing the CD2, 5, 7, CD3-TcR alpha beta complex, and lack natural killer cell markers as well as cytolytic function. Their inhibitory activity on both cytolytic processes and T-cell proliferation is mediated by a non- antigen-specific soluble factor released by CD8+CD57+ cells in culture supernatants. Preliminary characterization suggests that the CD8+CD57+ cells' inhibitory activity is mediated by a low molecular weight, glycosylated factor as indicated by its less than 1S coefficient of sedimentation and its binding to concanavalin A lectin.
Tumor necrosis factor-beta in the serum of adult T-cell leukemia with hypercalcemia
Authors: Ishibashi K , Ishitsuka K , Chuman Y , Otsuka M , Kuwazuru Y , Iwahashi M , Utsunomiya A , Hanada S , Sakurami T , Arima T
Published: October 2019

Serum tumor necrosis factor-beta (TNF-beta) from patients with adult T- cell leukemia (ATL) was studied by a sandwich enzyme-linked immunosorbent assay (ELISA) developed in our laboratory using biotinylated monoclonal anti-TNF-beta and recombinant TNF-beta. Seven of eight patients with hypercalcemia showed elevation of serum TNF- beta. On the other hand, TNF-beta could not be detected by the ELISA in 28 patients without hypercalcemia. The lower detection limit in this assay was 100 pg/mL, corresponding to 500 pg/mL by the conventional method. In two patients serum TNF-beta level decreased after treatment in association with the level of serum calcium. Furthermore, immuno- staining using anti-TNF-beta and avidin-biotin complex showed the presence of cytoplasmic TNF-beta in not only human T-cell leukemia virus type I infected cell lines, but also freshly isolated cells from ATL patients with hypercalcemia. The actual biologic activity of TNF- beta in serum was confirmed by a conventional bioassay in a patient with hypercalcemia, and its cytotoxic activity was inhibited by the addition of anti-TNF-beta antibody in the assay. These results suggested that serum TNF-beta might be one of the factors contributing to the hypercalcemia, at least in patients with ATL.
Direct correlation of cytogenetic findings with cell morphology using in situ hybridization: an analysis of suspicious cells in bone marrow specimens of two patients completing therapy for acute lymphoblastic leukemia
Authors: Anastasi J , Vardiman JW , Rudinsky R , Patel M , Nachman J , Rubin CM , Le Beau MM
Published: October 2019

Bone marrow cells from two pediatric patients completing therapy for acute lymphoblastic leukemia were studied using in situ hybridization with an alpha-satellite DNA probe specific for chromosome 17. Morphologic analysis of the end-therapy specimens from each patient had shown small numbers (7.5%, 8.5%) of cells that were suspicious for residual or recurrent disease. These cells could not be morphologically or immunophenotypically distinguished with certainty from immature lymphoid cells (hematogones), which may be present normally, sometimes in increased numbers, in the bone marrow specimens of children. In situ hybridization with a probe to chromosome 17 was used because the leukemic cells from each patient had originally been shown to have an extra copy of this chromosome. In one patient, in situ studies showed a population of cells (106 of 1,000 cells) with three hybridization signals indicating trisomy 17, and thus residual/recurrent leukemia. In the other patient trisomy 17 could not be detected. Additional hybridizations to previously stained bone marrow aspirate smears permitted a direct correlation of the cytogenetic findings with the suspicious cells on a cell-to-cell basis. The questionable cells were identified, photographed, and then re-examined after hybridization. In one patient, 13 of 18 (72%) of the suspicious cells were found to have trisomy 17, whereas in the other patient 0 of 24 (0%) demonstrated an extra copy of this chromosome. These cases illustrate a clinical application of interphase cytogenetic analysis and demonstrate how this technology can be used for direct correlation of cytogenetic findings with cell morphology. This technique should prove useful for the detection of minimal residual disease and for lineage studies in leukemia and myelodysplasia.
Epithelial membrane glycoprotein PAS-IV is related to platelet glycoprotein IIIb binding to thrombospondin but not to malaria-infected erythrocytes
Authors: Catimel B , McGregor JL , Hasler T , Greenwalt DE , Howard RJ , Leung LL
Published: October 2019

Glycoprotein (GP) IIIb (also termed GPIV or CD36) is an integral platelet membrane protein, and has been identified as a binding site for thrombospondin, collagen, and malaria-infected erythrocytes. PAS-IV is an integral membrane protein found in lactating mammary epithelial cells and capillary endothelial cells. The N-terminal sequence of PAS- IV is nearly identical to that of GPIIIb and monospecific anti-PAS-IV antibody reacts with GPIIIb, indicating that PAS-IV is structurally related to GPIIIb. In this study, human platelet GPIIIb and bovine epithelial PAS-IV were compared in terms of structural, immunologic, and functional characteristics. The two-dimensional tryptic peptide map of both intact and deglycosylated PAS-IV was highly similar but not identical to that of GPIIIb. PAS-IV and GPIIIb reacted to an equal extent with monoclonal antibodies OKM5 and OKM8 by enzyme-linked immunosorbent assay. GPIIIb bound to surface immobilized thrombospondin (TSP) in a concentration-dependent and saturable manner, with approximately 60% reduction in binding in the presence of EDTA. PAS-IV bound to TSP with similar characteristics except that maximum binding was consistently approximately 50% of that of GPIIIb and binding was not inhibited by EDTA. GPIIIb supported adhesion of Plasmodium falciparum-infected erythrocytes (PRBC) in a dose-dependent manner while no significant adhesion of PRBC to PAS-IV was observed. Our data demonstrate that while epithelial PAS-IV and platelet GPIIIb are structurally and immunologically related, there are significant differences in their functional properties. Whether this result is due to different posttranslational glycosylation modifications or that PAS- IV and GPIIIb represent a family of related cell adhesive protein receptors remains to be determined.
Transfected NA1 and NA2 forms of human neutrophil Fc receptor III exhibit antigenic and structural heterogeneity
Authors: Ory PA , Clark MR , Talhouk AS , Goldstein IM
Published: October 2019

Human neutrophils express two polymorphic forms (NA1 and NA2) of Fc receptor III (FcRIII), which differ structurally and antigenically. We recently isolated FcRIII cDNAs from NA1NA1 and NA2NA2 homozygotes and determined that they differ only at five nucleotides, predicting four amino acid substitutions. To determine whether the cDNAs that we isolated actually encode proteins that differ structurally and that react appropriately with anti-NA1 and anti-NA2 antibodies, we transfected Chinese hamster ovary (CHO) cells with constructs containing either the NA1 FcRIII cDNA or the NA2 FcRIII cDNA. The receptors on transfected CHO cells were then compared with the receptors on normal human neutrophils from an NA1NA2 heterozygote. After immunoprecipitation and treatment with N-glycanase, receptors isolated from surface-labeled CHO cells transfected with the NA1 FcRIII cDNA had an apparent molecular mass of 29 Kd after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), while the receptors isolated from CHO cells transfected with the NA2 FcRIII cDNA had an apparent molecular mass of 33 Kd. Identical 29-Kd and 33-Kd bands were observed when receptors isolated from surface-labeled neutrophils of an NA1NA2 heterozygote were treated similarly. Using a cell-free rabbit reticulocyte lysate system, we translated NA1 FcRIII and NA2 FcRIII RNAs in vitro and also found differences in the apparent molecular masses of the two forms of the receptor. Finally, reactivity of transfected CHO cells with anti-NA monoclonal and alloantibodies confirmed that the cDNAs we isolated actually encode the NA1 and NA2 forms of neutrophil FcRIII.
Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce rapid phosphorylation and activation of the proto-oncogene Raf-1 in a human factor-dependent myeloid cell line
Authors: Kanakura Y , Druker B , Wood KW , Mamon HJ , Okuda K , Roberts TM , Griffin JD
Published: October 2019

The product of the c-raf-1 proto-oncogene, Raf-1, is a 74,000-dalton cytoplasmic serine/threonine protein kinase that has been implicated as an intermediate in signal transduction mechanisms. In the human factor- dependent myeloid cell line MO7, both granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3) were found to induce rapid, dose-dependent phosphorylation of Raf-1, which resulted in altered Raf-1 mobility in sodium dodecyl sulfate-polyacrylamide gels. The increase in phosphorylation was due primarily to an increase in phosphoserine, with only a minor component (less than 2%) of phosphotyrosine. PMA (12-phorbol 13-myristic acid) also induced Raf-1 phosphorylation in MO7 cells, but the resulting alteration in electrophoretic mobility was different than that observed after GM-CSF or IL-3. GM-CSF and IL-3 rapidly and transiently increased Raf-1 kinase activity using Histone H1 as a substrate in an immune complex kinase assay in vitro. These results suggest that phosphorylation of Raf-1 could play a role in some aspect of GM-CSF and IL-3 signal transduction.
Retroviral transformation of cerebral microvascular endothelial cells: macrophage-like and microvascular endothelial cell properties
Authors: Robinson DH , Warren MK , Liang LT , Oprandy JJ , Nielsen TB , Kang YH
Published: October 2019

We report that L-cell-conditioned medium (LCM) transforms porcine cerebral microvascular (PCMV) endothelial cells into cells with macrophage-like properties. LCM is known to contain both cytokine(s) and the L-cell virus, a murine retrovirus found in the L929 cell and LCM. Our evidence suggests that both LCM cytokine(s) and the L-cell virus are involved in this PCMV endothelial cell transformation. Criteria for transformation include focus formation, decreased serum requirements for growth, changes in morphology including nonadherence, propagation in suspension culture, and a decreased growth response to stimulation with a known endothelial cell mitogen. Macrophage-like characteristics of this transformed cell, designated as RVTE, include pinocytosis of low-density lipoprotein, Fc receptor-mediated phagocytosis, phagocytosis of bacteria and zymosan, the expression of macrophage enzyme markers, and constitutive production of colony- stimulating factor 1. However, the transformed cell retains several properties of the nontransformed cell including the expression of FVIII:RAg and in vitro self-organization into capillary-like structures. Cloning of RVTE cells clearly shows that both macrophage- like and cerebral microvascular endothelial cell properties are present in the same cell. During self-organization, nontransformed cells express morphologic and functional characteristics classically associated with the macrophage. These findings suggest that some brain capillary pathophysiologies could involve macrophage-like cerebral microvascular endothelial cells. Furthermore, the “reticuloendothelial” phenotypic repertoire expressed by this transformed cerebral microvascular endothelial cell may show that the cerebral capillary endothelial cell in vivo is derived from a hematopoietic and/or phagocytic precursor.
Retinoic acid receptors in myeloid leukemia: characterization of receptors in retinoic acid-resistant K-562 cells
Authors: Robertson KA , Mueller L , Collins SJ
Published: October 2019

Although mRNA for the retinoic acid receptor alpha (RAR-alpha) is expressed in many different myeloid leukemias, most of these leukemia cells exhibit little if any phenotypic response when exposed to retinoic acid (RA). To determine whether such RA resistance is related to altered RA receptor structure or function, we performed a detailed analysis of nuclear RA receptors in RA-resistant K-562 cells. These cells exhibit RA receptors of the same approximate molecular weight and similar kd as those exhibited by the RA-sensitive HL-60 leukemia cell line, but the number of RA receptors in the RA-resistant K-562 cells (80 per cell) is significantly lower than that exhibited by RA- sensitive HL-60 cells (550 per cell). Retroviral-mediated transduction of RAR-alpha cDNA into K-562 significantly increased the number of RA receptors to 2,000 per cell. These RAR-alpha-transduced K-562 cells, when incubated with RA, exhibit diminished cell proliferation associated with decreased c-myc expression and an accumulation of cells in G0/G1. In addition, these RA-treated cells exhibit downregulation of the CD15 surface antigen and a slight increase in hemoglobin production but manifest no other evidence of significant erythroid, megakaryocytic, or myeloid differentiation. These results indicate that an elevated number of nuclear RA receptors can be involved in altering proliferation but not necessarily the differentiation of certain RA- treated myeloid leukemia cells.
Immunologic detection of endothelial cells in human whole blood
Authors: Sbarbati R , de Boer M , Marzilli M , Scarlattini M , Rossi G , van Mourik JA
Published: October 2019

In this report we show that human endothelial cells (EC) can be detected in circulating blood by means of the EC-specific monoclonal antibody (MoAb) designated as CLB-HEC 19 and expressed quantitatively as number of cells per milliliter of whole blood. We first developed a method that was able to recover cultured human EC added to whole blood by Percoll density gradient centrifugation. The final recovery of the EC was 91.6% (SE = 0.65%). The EC were identified in the gradient subfractions by indirect immunofluorescence with the MoAb CLB-HEC 19. This method was then applied to the separation and characterization of EC or EC remnants from the whole arterial and venous blood taken from two groups of patients subjected to heart catheterization. Firstly, a preliminary blood screening of random samples was performed in a group of eight patients (group I) using a scoring evaluation for the presence of EC and the results were expressed as positivity index. Secondly, the complete blood screening of a group of ten patients (group II) was performed for the detection of immunofluorescent cells and the results were expressed as number of EC per milliliter of whole blood. Our results show in both group I and II a significant presence of EC in the blood after catheterization compared with their basal values. The minimal detectable concentration of EC was 0.06 cells/mL (SE = 0.057) of whole blood. We consider this technique as a suitable clinical test for the detection of EC injury in cardiovascular pathology.
Requirement of heparin for arterial and venous thrombolysis with recombinant tissue-type plasminogen activator
Authors: Rapold HJ , Lu HR , Wu ZM , Nijs H , Collen D
Published: October 2019

The effect of concomitant intravenous (IV) heparin (200 U/kg bolus, followed by 100 U/kg/h) on the efficacy of arterial and venous thrombolysis with IV recombinant tissue-type plasminogen activator (rt- PA; 0.5 mg/kg over 1 hour) was investigated in a combined femoral arterial and venous thrombosis model in the dog. The arterial model consisted of a high-grade stenosis, endothelial damage, and a thrombotic occlusion, and the venous model consisted of a 125I-fibrin- labeled blood clot. After a dose-finding pilot study in four dogs, a randomized, prospective, and blind study was performed in 20 animals pretreated with 2.8 mg/kg IV acetyl salicylic acid (ASA). The combination of rt-PA and heparin (group I, n = 10) induced early (less than 30 minutes) arterial reperfusion in seven dogs, late (greater than 30 minutes) reflow in two dogs, and persistent occlusion in one dog. rt- PA alone (group II, n = 10) was associated with early reperfusion in one dog, late reflow in three dogs, and persistent occlusion in six dogs (P = .018). Reocclusion occurred in five of nine reperfused dogs of group I and in one of four reperfused dogs of group II (P = not significant). Venous clot lysis amounted to 81% +/- 4% (mean +/- SEM) for group I and to 49% +/- 7% for group II (P less than .001). Template bleeding times increased moderately, but significantly, in group I (from 2.2 +/- 0.2 minutes at baseline to 7.0 +/- 1.4 minutes at 30 minutes, P = .006), but only marginally in group II (from 2.2 +/- 0.2 minutes to 3.6 +/- 0.7 minutes, P = .09). No systemic fibrinogen depletion was observed. Thus, the concommitant use of heparin with rt- PA accelerates arterial reperfusion and enhances venous thrombolysis in dogs pretreated with ASA. These results, obtained in a randomized prospective study design, add to a growing body of experimental and clinical evidence, indicating that thrombolytic therapy with rt-PA requires concomitant adjunctive IV heparin for optimal efficacy, even in the face of treatment with ASA.
Identification of functionally distinct domains of human granulocyte- macrophage colony-stimulating factor using monoclonal antibodies
Authors: Kanakura Y , Cannistra SA , Brown CB , Nakamura M , Seelig GF , Prosise WW , Hawkins JC , Kaushansky K , Griffin JD
Published: October 2019

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein that is required for the survival, growth, and differentiation of hematopoietic progenitor cells. Although the primary structure of GM-CSF is known from cDNA cloning, the relationship between structure and function of GM-CSF is not fully understood. Fifteen different monoclonal antibodies (MoAbs) to human GM-CSF were generated to map immunologically distinct areas of the molecule. Each of the MoAbs was biotinylated and shown by enzyme-linked immunosorbent assay to bind to recombinant GM-CSF that had been affixed to a solid phase. Each of the 15 unconjugated MoAbs was then used to compete with each biotinylated MoAb for binding to GM-CSF. These cross-blocking studies identified eight distinct epitopes of native GM-CSF. Seven of these epitopes were also present in denatured GM-CSF by Western blotting, and four of the epitopes were at least partially conserved on GM-CSF that was reduced in beta-mercaptoethanol. MoAbs to four of eight epitopes neutralized both recombinant (glycosylated and nonglycosylated) and natural human GM-CSF in a GM colony-forming unit (CFU-GM) assay and blocked GM-CSF-induced activation of neutrophils. For most of the antibodies there was a good correlation between neutralizing activity and the capacity to block binding of 125I-GM-CSF to neutrophils or blasts. Non-neutralizing antibodies to one epitope partially blocked binding of 125I-GM-CSF to neutrophils. None of the MoAbs neutralized interleukin-3, G-CSF, or M-CSF. The locations of seven of the epitopes could be partially mapped with regard to the amino acid structure by determining reactivity to GM-CSF synthetic peptides or to human-mouse chimeric GM-CSFs. The neutralizing antibodies were found to map to amino acids 40–77, 78–94, or 110–127. Thus, these MoAbs are useful to identify functional domains of GM-CSF and in identifying regions that are likely to be involved in receptor interaction.
Continued blood cell formation in spherical bodies in a long-term mouse spleen culture
Authors: Matsuya Y , Yanai N , Ohtani H , Naganuma H , Obinata M
Published: October 2019

During the primary culture of spleen fragments of newborn mice, a spherical body (d = circa 200 to 300 microns) as a three-dimensional cellular organization was formed. Continued production of blood cells from the spherical body was observed without changing its size for about 2 months of culture. Without growth factor, the spherical bodies produced mainly lymphocytes and macrophages. Addition of interleukin-3 enhanced their granulocyte formation, and this enhancement was observed even after a prolonged maintenance without growth factors. The spherical bodies were composed of a uniform mixture of endothelial cells and fibroblasts within the body, and cell-cell contacts between lymphocytes and fibroblasts were notable in the periphery. With prolonged culture, the spherical bodies showed a definite change in their structure by sorting two cell types and the blood cell production gradually decreased. These results suggested that a three-dimensional structure was required for the maintenance, growth, and differentiation of blood cell progenitors in the long-term spleen culture.
Accessory receptors regulate coupling of the T-cell receptor complex to tyrosine kinase activation and mobilization of cytoplasmic calcium in T- lineage acute lymphoblastic leukemia
Authors: Ledbetter JA , Schieven GL , Kuebelbeck VM , Uckun FM
Published: October 2019

T-lineage acute lymphoblastic leukemia (T-ALL) cells have abundant cytoplasmic CD3/Ti but express low amounts on the cell surface and are deficient in CD3/Ti-mediated signal transduction. Nevertheless, plating T-ALL cells on dishes containing immobilized anti-CD3 monoclonal antibodies with a source of growth factors induced the expression of CD25 (interleukin-2 receptor alpha chain) and stimulated the formation of blast colonies in 12 of 14 cases studied. The proliferative response to CD3 ligation was modulated by the presence of antibodies to the CD2, CD4, or CD8 accessory T-cell receptors. The effect of these accessory receptors on signal transduction mediated by CD3/Ti was next investigated by monitoring cytoplasmic calcium concentration [( Ca2+]i) and by measuring tyrosine phosphorylation after stimulation. Crosslinking CD3, CD2, CD4, or CD8 alone did not induce cytoplasmic calcium mobilization in T-ALLs, but crosslinking the accessory receptors with CD3/Ti induced calcium responses in three of the T-ALLs and enhanced calcium responses in three of the T-ALL cell lines, including HPB-ALL, MOLT-4, and CEM. Crosslinking CD4 but not CD2 with CD3/Ti greatly enhanced tyrosine phosphorylation of multiple substrates in comparison with crosslinking either CD4 or CD3/Ti separately on both normal mature T cells and the CEM T-ALL cell line. Thus, CD4 regulates CD3/Ti signal transduction in T-ALL cells through the tyrosine phosphorylation of substrates whereas CD2 may regulate [Ca2+]i signal transduction through a separate mechanism.
Ultrastructural demonstration of peroxidase expression in acute unclassified leukemias: correlation to immunophenotype and treatment outcome
Authors: Heil G , Gunsilius E , Raghavachar A , Bartram CR , Ganser A , Kurrle E , Aydemir U , Loffler H , Hoelzer D , Thiel E
Published: October 2019

The lineage affinity of 57 cases of acute unclassified leukemias (AUL) was reevaluated by ultrastructural analysis of peroxidase expression (POEM) in combination with immunophenotyping and analysis of immunoglobulin gene configuration. Twenty-three cases of myeloid and three cases of megakaryocytic differentiation were identified by detection of ultrastructural myeloperoxidase (UMPO) and platelet peroxidase (UPPO). No significant correlation was noted between myeloid marker expression and POEM positivity, whereas presence of CD 19 or CD 24 antigen significantly correlated with POEM negativity (P = .001 and .023, respectively). Ig gene rearrangements including oligoclonal patterns were also recorded in 8 of 14 UMPO+ patients tested. Fourteen UMPO+ patients responded poorly to an ALL/AUL chemotherapy regimen with a low complete remission (CR) rate of 29% and a short median remission duration (MRD) of 5 months. The POEM- patients proved very heterogenous with respect to immunophenotype and Ig gene rearrangement. Seventeen of 21 patients tested had Ig gene rearrangements, including oligoclonal patterns. Combined data suggest that a proportion of these cases probably derive from a very immature lymphoid progenitor cell, particularly because 15 POEM- AUL patients showed a response to ALL/AUL chemotherapy comparable to that observed in patients with definitive acute lymphoblastic leukemia (ALL) (CR rate 80%, MRD 20 months). Thus, ultrastructural analysis of peroxidase expression can provide decisive prognostic information in AUL patients.
Locus control region-A gamma transgenic mice: a new model for studying the induction of fetal hemoglobin in the adult
Authors: Constantoulakis P , Josephson B , Mangahas L , Papayannopoulou T , Enver T , Costantini F , Stamatoyannopoulos G
Published: October 2019

All pharmacologic agents that induce fetal hemoglobin (Hb) have been discovered with in vivo studies of humans, macaques, and baboons. We tested whether transgenic mice carrying human fetal (gamma) globin genes provide a model for studying the pharmacologic induction of HbF in the adult. In initial studies, phenylhydrazine-induced hemolytic anemia, 5-azacytidine, butyrate, or combinations of these treatments failed to activate the human gamma-globin gene in a transgenic mouse line carrying a 4.4-kb G gamma globin gene construct that is expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human A gamma gene linked to the locus control region (LCR) regulatory sequences and expressing heterocellularly HbF (about 25%, gamma-positive cells) were used. Treatments with erythropoietin, 5- azacytidine, hydroxyurea, or butyrate resulted in induction of gamma gene expression as documented by measurement of F-reticulocytes, the gamma/gamma + beta biosynthetic ratio and the level of steady state gamma mRNA. Administration of erythropoietin or butyrate to transgenic mice carrying a muLCR-beta (human) globin construct, failed to increase human beta-globin expression. These results suggest that the muLCR-A gamma transgenic mice provide a new model for studying the induction of fetal Hb in the adult.
Calcium ionophore, A23187, induces commitment to differentiation but inhibits the subsequent expression of erythroid genes in murine erythroleukemia cells
Authors: Hensold JO , Dubyak G , Housman DE
Published: October 2019

Murine erythroleukemia (MEL) cells are a useful model for studying the processes that regulate erythroid differentiation because exposure of these cells to a variety of chemical inducing agents results in expression of erythroid-specific genes and the resultant loss of cellular immortality. Previously it has been suggested that the calcium ionophore, A23187, has effects on the early cellular events that lead to the commitment of these cells to differentiation, but was not in itself sufficient to induce differentiation. We demonstrate here that A23187, as well as another calcium ionophore, ionomycin, are capable of inducing commitment to differentiation. Unlike other inducing agents, continual exposure to A23187 inhibits transcription of the erythroid- specific genes, beta-globin and Band 3. This effect is not attributable to an increase in cytosolic calcium concentration, because cells induced by ionomycin produce normal amounts of hemoglobin. These effects of A23187 on MEL cells confirm that commitment to differentiation is a distinct event from the subsequent transcriptional activation of erythroid genes. The ability of both ionophores to induce commitment to differentiation suggests that an increase in cytosolic calcium can trigger commitment to differentiation. These agents should prove useful in investigating the cellular processes that are responsible for commitment to differentiation.
Growth factor requirements of childhood acute T-lymphoblastic leukemia: correlation between presence of chromosomal abnormalities and ability to grow permanently in vitro
Authors: O'Connor R , Cesano A , Lange B , Finan J , Nowell PC , Clark SC , Raimondi SC , Rovera G , Santoli D
Published: October 2019

Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL- 103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, - 105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.
Lack of effect of granulocyte-macrophage and granulocyte colony- stimulating factors on cultured human endothelial cells [see comments]
Authors: Yong K , Cohen H , Khwaja A , Jones HM , Linch DC
Published: October 2019

The hematopoietic growth factors, granulocyte-macrophage colony- stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF), enhance the effector functions of mature myeloid cells, including the interaction with vascular endothelium. We examined the direct effect of recombinant human GM-CSF (rhGM-CSF) and recombinant human G-CSF (rhG-CSF) on the growth and function of cultured human umbilical vein endothelial cells (HUVEC). Endothelial cell growth supplement (ECGS) increased the proliferation of passaged and primary cells by 305% +/- 45% (mean +/- SEM, n = 5, P less than .01) over control cells at 4 days; GM-CSF and G-CSF had no effect. Endothelial cell procoagulant activity was increased after 4-hour incubation with recombinant interleukin-1 beta (IL-1 beta) 10 U/mL and recombinant tumor necrosis factor (TNF) 10 U/mL to 1,721% +/- 376% (n = 7, P less than .005) and 247% +/- 71% (n = 4) of control levels, respectively. gamma-Interferon (gamma-IFN) 50 U/mL had no direct effect of its own but was able to prime the response to IL-1 beta. There was no direct or priming effect of GM-CSF (1 ng to 1 microgram/mL) on the expression of procoagulant activity in endothelial cells. GM-CSF and G-CSF (1 ng/mL to 1 microgram/mL) had no effect on the expression of either tissue plasminogen activator (tPA) or plasminogen activator inhibitor-1 (PAI- 1) by endothelial cells. The secretion of tPA by endothelial cells was increased, however, after 24-hour incubation with thrombin 4 U/mL (314% +/- 72% of control levels, n = 5, P less than .025). The production of PAI-1 was increased by TNF 200 U/mL (241% +/- 44% of control, n = 3, P less than .005), thrombin 4 U/mL (180% +/- 12% of control, n = 5, P less than .0005) and IL-1 beta 10 U/mL (275% +/- 44% of controls, n = 5, P less than .0005). In four experiments, endothelial cells showed no specific binding of 125I-GM-CSF, whereas peripheral blood (PB) neutrophils demonstrated the presence of 802 +/- 78 high-affinity receptors for GM-CSF. Thus, we found no effect of rhGM-CSF or rhG-CSF on the proliferation activities by these cells. These findings are in accordance with the lack of demonstrable receptors for GM-CSF on cultured HUVEC.
Differentiation of a human eosinophilic leukemia cell line (EoL-1) by a human T-cell leukemia cell line (HIL-3)-derived factor
Authors: Morita M , Saito H , Honjo T , Saito Y , Tsuruta S , Kim KM , Tanaka M , Mori KJ , Mayumi M , Mikawa H
Published: October 2019

Differentiation of a human eosinophilic leukemia cell line, EoL-1, induced by the culture supernatant of a human ATL cell line, HIL-3 (HIL- 3 sup) was compared with differentiation induced by defined cytokines. HIL-3 sup induced EoL-1 cells to express eosinophilic granules and segmented nuclei after 6 to 9 days of incubation. HIL-3 sup also induced the expression of Fc epsilon receptor II (Fc epsilon RII/CD23) and an eosinophil differentiation antigen EO-1 mainly on eosinophilic granule (+) cells. Furthermore, HIL-3 sup induced EoL-1 cells to respond to an eosinophil chemotactic factor, platelet activating factor. HIL-3 cells express messenger RNA (mRNA) of interleukin-5 (IL- 5), macrophage colony-stimulating factor (M-CSF), and IL-3 but not granulocyte CSF (G-CSF). Granulocyte-macrophage CSF (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) were detected in the HIL-3 sup. Recombinant IL-2 (rIL-2), rIL-3, rIL-4, rIL-5, rM-CSF, and rGM-CSF did not induce eosinophilic granules. rG-CSF induced a few eosinophilic granule (+) cells, and TNF-alpha, which did not induce eosinophilic granules by itself, enhanced the ability of G-CSF to induce them. However, G-CSF and TNF-alpha did not induce the expression of Fc epsilon RII and EO-1 antigen. Moreover, anti-G-CSF, anti-TNF-alpha, anti-GM-CSF, anti-IL-3, and anti-IL-5 antibodies did not suppress the effect of HIL-3 sup on the differentiation of EoL-1 cells. All the data suggest that HIL-3 sup contains an unidentified factor that induces differentiation of EoL-1 cells, and that EoL-1 cells and HIL-3 sup provide an important model for the examination of differentiation mechanisms and functions of eosinophils.
Autologous bone marrow transplantation in acute myelogenous leukemia: in vitro treatment with myeloid-specific monoclonal antibodies and drugs in combination
Authors: Lemoli RM , Gasparetto C , Scheinberg DA , Moore MA , Clarkson BD , Gulati SC
Published: October 2019

We report the results of a preclinical study comparing four different purging protocols using a promyelocytic human cell line HL-60 and myeloid leukemic progenitor cells (colony-forming unit-leukemic [CFU- L]) from acute myelogenous leukemia (AML) patients assayed in semisolid culture. We studied the antileukemic effect of (1) Single-cycle complement-mediated lysis by two different monoclonal antibodies (MoAbs) (M195 [CD33] and F23 [CD13] 40 micrograms/mL), reactive with distinct antigens found on early myeloid cells and monocytes, used alone and in combinations; (2) 4-Hydroperoxycyclophosphamide (4-HC) (80 mumol/L or 100 mumol/L) alone; or (3) combined with VP-16 (5 micrograms/mL) and (4) a cocktail of 1 through 3 as above (combined immunochemotherapy). More than 4 logs of HL-60 tumor cell elimination were observed after 1 hour of incubation with both MoAbs plus 4-HC + VP- 16 while the single treatment (immunotherapy or chemotherapy) provided 1.5 and 3.5 logs of colony-forming inhibition, respectively. When the same protocols were tested on cryopreserved leukemic cells from eight patients with AML, we observed a mean value of CFU-L inhibition of 92.3% +/- 2.5% SD, 95.5% +/- 1.4% SD, and 99% +/- 0.8% SD after MoAbs and complement lysis, 4-HC, and 4-HC + VP-16 treatment, respectively. The combined treatment of MoAbs and 4-HC + VP-16 produced more than 3- log reduction of CFU-L colony formation. By comparison, the mean recovery of committed normal bone marrow progenitors after incubation with MoAbs and complement was 12% for CFU-granulocyte-macrophage (CFU- GM), 22.9% for burst-forming unit erythroid (BFU-E), and the recovery following 4-HC + VP-16 treatment was 4.4% for CFU-GM and 5.6% BFU-E. In subsequent experiments, highly purified CD34+ blast cells, enriched by positive selection, and stimulated in liquid culture by cytokines (interleukin-1 [IL-1], IL-3, and combination of both) or MO-conditioned medium (MoCM), demonstrated that immunochemotherapy spares hematopoietic colony-forming cells earlier than day 14 CFU-GM, in vitro.
Hemolytic-uremic syndrome following bone marrow transplantation in adults for hematologic malignancies
Authors: Rabinowe SN , Soiffer RJ , Tarbell NJ , Neuberg D , Freedman AS , Seifter J , Blake KW , Gribben JG , Anderson KC , Takvorian T
Published: October 2019

One hundred and sixty eight adult patients with B-cell non-Hodgkin's lymphoma (NHL) and other hematologic malignancies who underwent autologous or allogeneic bone marrow transplantation (BMT) were investigated for the subsequent development of hemolytic-uremic syndrome (HUS). All patients were conditioned with cyclophosphamide and total body irradiation. When examined at 3-month intervals for the first year post-BMT, all patients had uniform measurements of hematocrit (Hct) and serum creatinine. Sixteen patients who initially exhibited Hct and creatinine values that were normal range for the BMT populations developed a sudden decrease in Hct and increase in creatinine between 3 and 11 months post-BMT and fulfilled the clinical and laboratory criteria for HUS. None of these patients had known active cytomegalovirus infection, graft-versus-host disease, or cyclosporine administration. The degree of decrease in Hct and creatinine elevation ranged from solely laboratory abnormalities to a clinically significant syndrome. Twelve of the 16 patients developed acute clinical complications of congestive heart failure, hypertension (HTN), or peripheral edema. Twelve patients required red blood cell support, whereas only four patients required platelet transfusions. Both hemolytic anemia and thrombocytopenia have resolved in virtually all cases. At a mean follow up of 18 months postdiagnosis, creatinine elevations have persisted along with HTN. All patients have survived without life-threatening long-term sequelae. With the increasing use of BMT as a curative modality for patients with hematologic malignancies, it becomes important to prospectively monitor patients for the development of HUS and its potential long-term impact on renal function.
In vivo administration of recombinant human interleukin-1 and macrophage colony-stimulating factor (M-CSF) induce a rapid loss of M- CSF receptors in mouse bone marrow cells and peritoneal macrophages: effect of administration route
Authors: Chen BD
Published: October 2019

Earlier studies suggested the existence of a blood-bone marrow barrier that significantly inhibits the transfer of plasma macrophage colony- stimulating factor (M-CSF) to responsive hematopoietic cells in vivo as indicated by its failure to induce a receptor downregulation in bone marrow cells. In this study, the effect of recombinant human interleukin-1 (rhuIL-1) was investigated. In vivo administration of rhuIL-1, either intraperitoneally (IP) or intravenously (IV), induced a rapid transient loss of M-CSF receptor binding activity in bone marrow cells, with a nadir occurring between 2 to 4 hours while loss of M-CSF receptors by cells in the peritoneal cavity occurred only in animals receiving rhuIL-1 via IP administration. The loss of M-CSF receptor activity after rhuIL-1 treatment was correlated with an elevated level of circulating M-CSF. However, the loss of M-CSF receptors in marrow cells was prevented by dexamethasone (Dex) treatment before rhuIL-1 administration. The fact that Dex treatment also reduced the level of circulating M-CSF after rhuIL-1 administration suggests that the inhibitory effects of IL-1 are mediated through locally produced M-CSF. Administration of rhuM-CSF at higher doses, either IV or IP, also induced a loss of M-CSF receptor of lesser degree in the marrow cells. However, the loss of M-CSF receptors by the peritoneal cells was induced only in mice receiving rhuM-CSF through IP administration. Taken together, these results indicate the existence of a unidirectional barrier that prevents the transfer of blood M-CSF and IL- 1 to peritoneal cavity but not vice versa.
A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope
Authors: Flug F , Espinola R , Liu LX , SinQuee C , DaRosso R , Nardi M , Karpatkin S
Published: October 2019

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.
Genomic organization of IgH gene compared with the expression of Bcl-2 gene in t(14;18)-positive lymphoma
Authors: Amakawa R , Fukuhara S , Ohno H , Matsuyama F , Kato I , Tanabe S , Sideras P , Mizuta TR , Honjo T , Okuma M
Published: October 2019

In three lymphoma cell lines carrying t(14;18), named FL-18, FL-218, and FL-318, the genomic organization of IgH gene was compared with the expression of bcl-2 gene; the t(14;18) of the FL-18 cells occurred downstream from the major breakpoint cluster region (mbr) of a bcl-2 gene, and that of the FL-218 and FL-318 cells within the mbr. The FL- 318 expressed the normal-sized bcl-2 transcript of 8.5-kb mRNA having the noncoding region 3 to the mbr, which was found in the FL-18, and the FL-218 lacking the intact bcl-2 gene did not. This finding suggests that in t(14;18)-positive lymphoma having the breakpoint within the mbr, transcription of the nontranslocated bcl-2 allele is not necessarily silent. In addition, the FL-218 and FL-318 expressed aberrant bcl-2 transcripts and heterogenous IgH transcripts lacking the VH region, and the bcl-2 transcripts each comigrated with parts of the sterile IgH mRNAs. The FL-318, which did not exhibit switch recombination on either IgH allele, contained abundant amounts of l gamma mRNAs, a prerequisite for the recombination into the C gamma locus. One of the I-mRNA species comigrated with the aberrant bcl-2 transcript. The FL-18 and FL-218 lacking the I gamma mRNAs had completed switch recombination of both IgH alleles. This result raises a possibility that deregulated bcl-2 transcription caused by t(14;18) is capable of playing a role in class switch recombination of IgH gene.