bluuebunny/crossref_metadata_2025 huggingface dataset:
Antitumor effects of human recombinant interleukin-6 on acute myeloid leukemia in mice and in cell cultures
Authors: Givon T , Slavin S , Haran-Ghera N , Michalevicz R , Revel M
Published: October 2019

Interleukin-6 (IL-6) has been shown to inhibit growth and induce differentiation of several myeloid leukemia cell lines. In this work, two in vivo models of acute myeloid leukemia (AML) in mice have been used to test the therapeutic potential of recombinant human IL-6. In mice inoculated by a transplantable AML tumor, IL-6 injections inhibited the development of leukemia and increased survival. The effect was related to dose and length of treatment. In a model of radiation-induced leukemogenesis in SJL/J mice, administration of low- dose IL-6 for 10 days, 4 months after irradiation, reduced the incidence of leukemia observed during 1 year, whereas granulocyte- macrophage colony-stimulating factor (GM-CSF) increased the incidence of leukemia. In vitro liquid cultures of leukemic blood cells obtained from AML patients showed that IL-6 slowed growth and decreased the proportion of blasts with an increase in more mature myeloid elements in 72% of M1, M2, M4 AML cases. In contrast, GM-CSF less often produced differentiation but stimulated leukemic cell growth in liquid cultures, without synergism by IL-6.
Expression of the interleukin-3 and granulocyte-macrophage colony- stimulating factor genes in Friend spleen focus-forming virus-induced erythroleukemia
Authors: Shimada Y , Migliaccio G , Ruscetti S , Adamson JW , Migliaccio AR
Published: October 2019

Friend spleen focus-forming virus (F-SFFV) is a replication-defective retrovirus that induces a multistage erythroleukemia in mice. In the first stage, expression of the SFFV envelope glycoprotein results in erythroid hyperplasia. Subsequently, the F-SFFV integrates near the Spi- 1 gene and activates its expression, resulting in immortalized cells that represent a second stage in the disease process. We report here that media conditioned by erythroleukemia cell lines or leukemic spleen cells induced by the polycythemia-inducing strain of F-SFFV (F-SFFVp), but not medium conditioned by SFFVp-induced hyperplastic spleens, promote the proliferation of normal granulocyte-macrophage progenitor cells and of granulocyte-macrophage colony-stimulating factor (GM-CSF)- and/or interleukin-3 (IL-3)-dependent cell lines. The colony- stimulating activity of the conditioned media from four of five of the lines studied was neutralized by antibodies specific for IL-3 and/or GM- CSF, and IL-3 and GM-CSF-specific mRNA could be detected in the cells after amplification by the polymerase chain reaction. No rearrangements of the IL-3 or GM-CSF genes were observed by Southern blot analysis. However, as previously shown for SFFV-induced cell lines, the Spi-1 gene was expressed in all of these cells. Because the Spi-1 gene encodes a transcription factor whose cognate sequences are present in the promoter region of many hematopoietic growth factor genes, including IL-3 and GM-CSF, Spi-1 activation may be inducing the expression of these genes.
N-glycolylneuraminic acid and N-acetylneuraminic acid define feline blood group A and B antigens
Authors: Andrews GA , Chavey PS , Smith JE , Rich L
Published: October 2019

Blood group incompatibility causes transfusion reactions and neonatal isoerythrolysis in cats. We investigated the molecular nature of the blood group antigens from cats that had blood type A, B, and AB erythrocytes. Naturally occurring anti-type B antibodies, Triticum vulgaris lectin, monoclonal antibody (MoAb) 32–27, and MoAb R-24 were used in agglutination tests, Western blots, and thin-layer chromatography (TLC) enzyme immunostaining. Type A erythrocytes had NeuGc-NeuGc-Galactose-Glucose-Ceramide ([NeuGc]2GD3) where NeuGc represents N-glycolylneuraminic acid, and NeuAc-NeuGc-GD3, where NeuAc represents N-acetylneuraminic acid, and may have [NeuGc]2 disialylparagloboside and NeuAc-NeuGc-disialylparagloboside. Type B erythrocytes only had [NeuAc]2GD3. Type AB erythrocytes had [NeuGc]2GD3, NeuAc-NeuGc-GD3, and [NeuAc]2GD3. Blood group antigens were also found on a 50-Kd membrane protein. We conclude that type B erythrocytes are characterized by [NeuAc]2GD3 as the only form of this ganglioside and the presence of NeuAc on a 50-Kd membrane protein. NeuGc is the major determinant of the A antigen; specifically, [NeuGc]2GD3 is the major glycolipid form. The A antigen is also present on a 50-Kd membrane protein.
Infection of hematopoietic progenitor cells by human cytomegalovirus
Authors: Maciejewski JP , Bruening EE , Donahue RE , Mocarski ES , Young NS , St Jeor SC
Published: October 2019

The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.
Merocyanine 540-sensitized photoinactivation of human erythrocytes parasitized by Plasmodium falciparum
Authors: Smith OM , Dolan SA , Dvorak JA , Wellems TE , Sieber F
Published: October 2019

The purpose of this study was to evaluate the photosensitizing dye merocyanine 540 (MC540) as a means for extracorporeal purging of Plasmodium falciparum-infected erythrocytes from human blood. Parasitized red blood cells bound more dye than nonparasitized cells, and exposure to MC540 and light under conditions that are relatively well tolerated by normal erythrocytes and normal pluripotent hematopoietic stem cells reduced the concentration of parasitized cells by as much as 1,000-fold. Cells parasitized by the chloroquine- sensitive HB3 clone and the chloroquine-resistant Dd2 clone of P falciparum were equally susceptible to MC540-sensitized photolysis. These data suggest the potential usefulness of MC540 in the purging of P falciparum-infected blood.
Clonal analysis of myelodysplastic syndrome: monosomy 7 is expressed in the myeloid lineage, but not in the lymphoid lineage as detected by fluorescent in situ hybridization
Authors: Gerritsen WR , Donohue J , Bauman J , Jhanwar SC , Kernan NA , Castro-Malaspina H , O'Reilly RJ , Bourhis JH
Published: October 2019

Conflicting results have been published on whether or not myelodysplastic syndromes (MDS) affect all cell lineages. Involvement of myeloid and erythroid cell lineages has been regularly observed, but it remains controversial whether the different lymphoid cell lineages are involved. In this study of eight patients with MDS associated with monosomy 7, fluorescent in situ hybridization (FISH) was used to enumerate the chromosomes 7 in interphase cells. With the probe D7Z1, the rate of false-positive detection of monosomy 7 was 3% +/- 2% in normal cells. T- and B-cell lines were established from eight patients with MDS and monosomy 7. As determined by FISH in interphase cells, 1.9% (0% to 3%) of the cells in the B-cell lines showed one fluorescent spot and 1.1% (0% to 2.9%) of the cells in the T-cell lines. These values do not differ from normal values. However, the possibility that normal cells were selected when the T- and B-cell lines were established could not be excluded. Therefore, peripheral blood cells were obtained, separated according to surface markers specific for lymphoid and myeloid cell lineage with a cell sorter, and analyzed for the expression of monosomy 7 by FISH. Antibodies recognizing T cells (CD3), B cells (CD20), natural killer (NK) cells (CD57), monocytes and granulocytes (low and high expression of CD11b antigen), and myeloid progenitors (CD33) were used to separate cells. The expression of monosomy 7 in the T cells, NK cells, and B cells did not differ from control values. These results in the lymphoid subpopulations are in stark contrast with the observations in the myeloid populations; the percentage of cells with monosomy 7 ranged from 9% to 78% (controls: 6% +/- 2%) in cells with low CD11b expression, 20% to 89% in cells with a high expression of the CD11b antigen (controls: 7% +/- 3%), and 23% to 91% in the CD33 positive cells (controls: 5% +/- 3%). The results of this study suggest that monosomy 7 does not usually affect lymphoid subpopulations but is restricted to committed progenitor cells with the capacity to differentiate into mature myeloid cells.
Interactions between recombinant human erythropoietin and serum factor(s) on murine megakaryocyte colony formation
Authors: Tsukada J , Misago M , Kikuchi M , Sato T , Ogawa R , Ota T , Oda S , Morimoto I , Chiba S , Eto S
Published: October 2019

We investigated the interactions between human erythropoietin (hEpo) and serum factor(s) on murine megakaryocyte (MK) colony formation. Serum-free cultures supported the growth of a large number of murine MK colonies in the presence of murine interleukin-3 (mIL-3). The addition of fetal calf serum (FCS) to mIL-3-containing cultures resulted in only a minimal increase in the number of murine MK colonies. In contrast, hEpo alone had no murine MK colony-stimulating activities in serum-free cultures. hEpo required the presence of FCS, murine serum, or human serum in cultures to promote murine MK colony growth and synergized with these sera to stimulate murine MK colony formation. Furthermore, sera from patients with aplastic anemia showed higher synergistic activities with hEpo than sera from hematologically normal persons (normal human serum). When normal human serum was fractionated by gel- filtration chromatography, two peaks with the synergistic activity were observed in the eluent. However, serum did not show any synergistic effects with hEpo on the growth of murine GM colonies or murine colony- forming unit-erythroid-derived colonies. Although human serum synergized with hEpo to stimulate murine MK colony formation, human cytokines such as IL-3, IL-4, IL-6, granulocyte-macrophage colony- stimulating factor (GM-CSF) and granulocyte-CSF (G-CSF) failed to induce murine MK colony formation in Epo-containing cultures. In cultures containing human IL-1 alpha + human IL-6 + hEpo as well as in cultures containing hEpo, human IL-3 and human GM-CSF failed to show stimulatory effects on murine MK colony formation. Moreover, the synergistic activity of human serum with hEpo could not be neutralized by antibodies such as antihuman IL-1 alpha, antihuman IL-3, antihuman IL-4, antihuman IL-6, antihuman G-CSF, and antihuman GM-CSF. Our data show that serum contains a growth factor(s) that synergizes with Epo to stimulate the proliferation and differentiation of MK precursors, and strongly suggest that this factor(s) is an unique growth factor(s) that is distinct from IL-1 alpha, IL-3, IL-4, IL-6, G-CSF, and GM-CSF.
Functional reconstitution of the human interleukin-3 receptor
Authors: Kitamura T , Miyajima A
Published: October 2019

The high-affinity receptors for human interleukin-3 (IL-3), GM-CSF, and IL-5 are composed of alpha and beta subunits. The alpha subunits are primary ligand binding proteins specific for each ligand, whereas the three human receptors share a common beta subunit (beta c). In contrast to humans mice have two closely related genes, AIC2A and AIC2B, which are homologous to human beta c. The AIC2A gene encodes a low-affinity murine IL-3 binding protein, and the AIC2B protein is the beta subunit shared between murine GM-CSF receptors (mGMR) and IL-5 receptors (mIL- 5R). To examine the function of these receptor components, we established various stable transfectants of murine IL-2-dependent CTLL- 2 cells. CTLL-2 transfectants expressing both the alpha and beta subunits of the human IL-3 receptor (hIL-3R) proliferated in response to physiologic concentrations of hIL-3. Coexpression of hIL-3R alpha with AIC2B but not with AIC2A in CTLL-2 cells conferred a growth response to hIL-3. Although CTLL-2 transfectants expressing hIL-3R alpha alone did not proliferate in the presence of hIL-3, hIL-3- responsive sublines were repeatedly isolated. These sublines expressed endogenous AIC2B but not AIC2A. These results indicate that human beta c is essential for hIL-3 signaling and that AIC2B is a murine equivalent of human beta c. We also showed that hIL-3 and hGM-CSF induced tyrosine phosphorylation of several proteins in CTLL transfectants, similar to those observed in human factor-dependent TF-1 cells stimulated with hIL-3 and hGM-CSF.
Bryostatin 1 modulates the proliferation and lineage commitment of human myeloid progenitor cells exposed to recombinant interleukin-3 and recombinant granulocyte-macrophage colony-stimulating factor
Authors: Li F , Grant S , Pettit GR , McCrady CW
Published: October 2019

The activity of protein kinase C (PK-C) has been implicated in the regulation of the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK- C-activating agents phorbol 12,13-dibutyrate (PDBu), mezerein, and bryostatin 1 on the proliferation and lineage commitment of CD34+ human myeloid progenitor cells stimulated by recombinant interleukin-3 (rIL- 3) and/or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Although each of the PK-C activators administered alone induced no colony formation, coadministration of these agents with plateau concentrations of each cytokine (eg, 50 ng/mL) increased the number of day 14 granulocyte-macrophage colony-forming units by 100% to 150%. The number of pure and mixed neutrophil and macrophage colonies was substantially enhanced in the presence of PK-C activators, whereas the percentage and, in most cases, the absolute number of eosinophilic colonies was significantly reduced. The inhibition of eosinophilic colony formation was not overcome by the addition of rIL-5. Although addition of bryostatin 1 24 hours before rIL-3 abrogated the increase in total colony formation observed with simultaneous administration of factors, the inhibition of eosinophilic colonies and the increase in neutrophil/macrophage colonies persisted under these conditions. The addition of bryostatin 1 for up to 144 hours after rIL-3 continued to potentiate total colony formation, whereas the inhibition of eosinophilic commitment was lost after 120 hours. Together, these results suggest that pharmacologic interventions at the level of PK-C may regulate both the proliferation as well as the lineage commitment of human hematopoietic progenitors exposed to rGM-CSF and rIL-3.
Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor
Authors: Kouns WC , Kirchhofer D , Hadvary P , Edenhofer A , Weller T , Pfenninger G , Baumgartner HR , Jennings LK , Steiner B
Published: October 2019

Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 4–5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 4–5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 4–5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 4– 5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 4–5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 4–5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 4–5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.
p55 and p75 tumor necrosis factor receptors in patients with chronic lymphocytic leukemia
Authors: Waage A , Liabakk N , Lien E , Lamvik J , Espevik T
Published: October 2019

We studied the expression of the two tumor necrosis factor (TNF) receptors, p55 and p75, on B cells from patients with chronic lymphocytic leukemia (CLL), and the presence of soluble TNF receptors in serum. Expression of membrane-associated receptors was quantified by double labeling of peripheral blood mononuclear cells (PBMC) with monoclonal antibodies against CD19 and p55/p75 TNF receptors and flow cytometry. A high fraction of the CD19+ cells expressed the p55 receptor (44% +/- 34% [SD]) and p75 receptor (61% +/- 31%). In healthy controls, 0% to 1% of the CD19+ cells expressed the p55 receptor and 0% to 10% expressed the p75 receptor. Incubation of CD19+ cells with 10 ng/mL of TNF increased the incorporation of thymidine in 11 patients tested, and this was decreased to 65% (P < .05) by antibodies to the p55 receptor or the p75 receptor, and to 35% +/- 7% (P < .001) when both antibodies were combined. With an enzyme-linked immunoassay, we measured soluble TNF receptors in serum from CLL patients. The mean level of p55 receptors was increased to 12.9 +/- 8.9 ng/mL (P < .000001 v normal). The mean level of p75 receptors was increased to 13 +/- 24 ng/mL (P = .01 v normal). The membrane expression of the two receptors was positively correlated (r +/- 97, P < .01); however, there was no correlation between membrane expression and serum concentration of either receptor. Autologous serum containing high levels of soluble TNF receptors inhibited TNF-induced proliferation of CD19+ cells. In conclusion, we have demonstrated that neoplastic cells from patients with CLL have increased expression of p55 and p75 TNF receptors, and that both receptors mediate signal to proliferation. Furthermore, serum from CLL patients has elevated levels of soluble TNF receptors, which may counteract the proliferative effect of TNF.
Interleukin-4 (IL-4) receptor expression on human T cells is affected by different intracellular signaling pathways and by IL-4 at transcriptional and posttranscriptional level
Authors: Dokter WH , Borger P , Hendriks D , van der Horst I , Halie MR , Vellenga E
Published: October 2019

Interleukin-4 (IL-4) modulates the survival, proliferation, and differentiation of a variety of hematopoietic cells. The effects are mediated through a single class of high-affinity receptors for IL-4. To understand the biologic effects of IL-4 on human T cells, we studied the regulation of IL-4 receptor (IL-4R) gene expression. We showed that IL-4R mRNA accumulation in human T cells is enhanced fourfold after activation of different secondary signaling pathways by concanavalin A (Con A), phorbol myristate acetate (PMA), the calcium ionophore A23187, and combinations of these factors. This could be ascribed to an increase in the IL-4R transcription rate and to stabilization of IL-4R mRNA resulting in a half-life of 80 to 90 minutes (v 35 to 40 minutes in resting T cells). IL-4 did enhance the IL-4R mRNA accumulation by a factor 10, which was caused by an increase in the IL-4R transcription rate and prolonging the half-life of IL-4R transcripts to 140 to 160 minutes. Finally, it was shown that A23187 induced IL-4R mRNA expression is a protein synthesis-dependent process. In contrast, Con A- , PMA-, Con A + PMA-, and Con A + A23187-induced expression of IL-4R mRNA is protein-synthesis independent. Cyclosporine A inhibited the A23187- and Con A + A23187-induced IL-4R mRNA accumulation, whereas Con A-, PMA-, and Con A + PMA-induced IL-4R mRNA expression was not affected by this drug. These data indicate that expression of IL-4 receptors on human T cells can be modulated by different intracellular signaling pathways at both transcriptional and posttranscriptional levels.
Expression and activity of the multidrug resistance P-glycoprotein in human peripheral blood lymphocytes [see comments]
Authors: Chaudhary PM , Mechetner EB , Roninson IB
Published: October 2019

P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance) gene, is a transmembrane efflux pump for different lipophilic compounds, including many anticancer drugs and fluorescent dyes. We have previously reported that the efflux of fluorescent dyes from lymphoid cells of human bone marrow was directly correlated with the cellular P-gp content. In the present study, we show that human peripheral blood lymphocytes (PBL) also express P-gp, and that P-gp expression correlates with the efflux of fluorescent dyes from PBL. This efflux was suppressed not only by chemical inhibitors of P-gp but also by a P-gp-specific monoclonal antibody UIC2, thus providing direct evidence that it was mediated by P-gp. We have also characterized dye efflux and UIC2 reactivity in specific PBL subsets. P-gp was expressed in the majority of CD56+, CD8+, and CD20+ lymphocytes, but in less than one half of CD4+ cells. P-gp-mediated dye efflux was highly heterogeneous relative to the expression of CD56RA, CD56RO, Leu-8, and HLA-DR antigens. No significant P-gp activity was detectable in CD14+ monocytes. MDR1 expression in normal lymphocytes may be a determinant of multidrug resistance in the corresponding malignancies.
In vivo effects of interleukin-6 on thrombopoiesis in healthy and irradiated primates [see comments]
Authors: Zeidler C , Kanz L , Hurkuck F , Rittmann KL , Wildfang I , Kadoya T , Mikayama T , Souza L , Welte K
Published: October 2019

We have studied the in vivo effects of recombinant human interleukin-6 (rhIL-6) on hematopoiesis in eight healthy and nine irradiated cynomolgus monkeys. Of the healthy animals, three received rhIL-6 alone (10 micrograms/kg/d, subcutaneously [SC]), one received rhIL-6 in combination with rhIL-3 (10 micrograms/kg/d, SC), one received rhIL-6 in combination with recombinant cynomolgus granulocyte-macrophage colony-stimulating factor (rcGM-CSF; 10 micrograms/kg/d, SC), two received rhIL-6 in combination with recombinant human granulocyte-CSF (rhG-CSF; 10 micrograms/kg/d, SC), and one received rhIL-6 in combination with recombinant human leukemia inhibitory factor (rhLIF; 10 micrograms/kg/d, SC). All animals were treated for at least 2 weeks with rhIL-6 or the above mentioned combinations. rhIL-6 alone significantly increased the peripheral blood platelet counts (2- to 3.5- fold). The platelets reached a plateau between days 10 and 15 of treatment. No synergistic effects on platelet numbers were observed when rhIL-6 was combined with rhIL-3, rcGM-CSF, rhG-CSF, or rhLIF. In addition to rhIL-6, only rhLIF increased the platelet numbers when administered alone. To test whether rhIL-6 might also protect the animal from thrombocytopenia or shorten the time of thrombocytopenia after irradiation, we treated nine animals with total body irradiation (3.8 Gy). Six of the animals were additional treated with rhIL-6 (4 with 10 micrograms/kg/d; and 2 with 100 micrograms/kg/d) from day -1 or +1 to day 28 post irradiation. In these animals, rhIL-6 at the same dose effective in healthy animals (10 micrograms/kg/d) was not capable of protecting the animals from platelet nadir. However, when pegylated rhIL-6 was used at a dosage of 100 micrograms/kg/d post irradiation, the mean of the nadirs was 71,000/microL as compared with 39,000/microL in control animals and the time of thrombocytopenia was shorter (3 v 5 days). In all animals (healthy and irradiated), rhIL-6 did not increase the number of bone marrow megakaryocytes but induced a right shift of DNA ploidy in megakaryocytes. These data suggest that IL-6 acts as “thrombopoietin”-like activity, but not as “megakaryocyte-CSF”-like activity.
15-Hydroxyeicosatetraenoic acid-mediated potentiation of thrombin- induced platelet functions occurs via enhanced production of phosphoinositide-derived second messengers--sn-1,2-diacylglycerol and inositol-1,4,5-trisphosphate
Authors: Setty BN , Werner MH , Hannun YA , Stuart MJ
Published: October 2019

We investigated whether biologically relevant concentrations of the mono-hydroxyeicosatetraenoic acids (mono-HETEs) modulate platelet functions. We report that 15-HETE, an eicosanoid produced by endothelial cells, granulocytes, and lymphocytes, potentiated platelet aggregation, nucleotide release, and elevation in intracellular calcium levels induced by a threshold concentration of thrombin (0.025 U/mL). Significant potentiation effects on these responses were observed at concentrations between 1 and 100 nmol/L. 15-HETE at these concentrations enhanced thrombin-induced platelet aggregation by 32% to 57%, nucleotide release by 40% to 65%, and elevation of intracellular calcium by 31% to 52% (P < .05 to .01). Both 12-HETE and 5-HETE, the structural isomers of 15-HETE, also potentiated thrombin-induced platelet aggregation and nucleotide release. While 12-HETE showed a small but significant effect at 100 pmol/L, 5-HETE had effects similar to those of 15-HETE at micromolar concentrations. To understand the mechanism of the HETE modulation of platelet functions, we studied the effect of 10 and 100 nmol/L 15-HETE on the production of sn-1,2- diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (1,4,5-IP3). 15- HETE enhanced thrombin-induced production of DAG and 1,4,5-IP3 in a time- and concentration-dependent manner. 15-HETE also potentiated agonist-induced phosphorylation of the 47-Kd platelet protein. These studies demonstrate an important modulatory role for 15-HETE on platelet functions. Since this eicosanoid is elevated in pathologic states associated with platelet hyperfunction, including diabetes mellitus and atherosclerosis, an elucidation of its mechanism(s) of action appears relevant to our understanding of the genesis of atherothrombotic vascular disease.
Human peritoneal mesothelial cells produce many cytokines (granulocyte colony-stimulating factor [CSF], granulocyte-monocyte-CSF, macrophage- CSF, interleukin-1 [IL-1], and IL-6) and are activated and stimulated to grow by IL-1
Authors: Lanfrancone L , Boraschi D , Ghiara P , Falini B , Grignani F , Peri G , Mantovani A , Pelicci PG
Published: October 2019

To investigate the role of peritoneal mesothelial cells in regulating hematopoiesis, as well as inflammation, healing, and tissue regeneration processes, long-term cultures of peritoneal mesothelial cells from human endocavitarian fluids were established. The purity of the cell population was assessed by morphologic and immunocytochemical criteria. Five peritoneal mesothelial cell cultures were analyzed for cytokine expression. Macrophage colony-stimulating factor (M-CSF), granulocyte-CSF (G-CSF), interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6 transcripts were constantly but variably detected throughout the culture period, while granulocyte-monocyte-CSF (GM-CSF) expression started as the cell culture aged. No IL-2, IL-3, IL-4, IL-5, or IL-7 transcripts were detected in the same samples. Corresponding cytokine activities were detected in the supernatants of the cultures. Peritoneal mesothelial cells proliferated after the addition of exogenous IL-1 beta or IL-1 alpha, whereas the addition of recombinant GM-CSF, G-CSF, M-CSF, or IL-6 failed to trigger proliferation. IL-1 receptor type I transcripts were detected in peritoneal mesothelial cells. Moreover, IL-1 was able to upregulate the expression of the genes that code for G-CSF, GM-CSF, IL-1 alpha, and IL-1 beta in these cells. These data indicate that peritoneal mesothelial cells produce many cytokines and suggest that IL-1 is a regulatory molecule for peritoneal mesothelial cells.
Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: a Cancer and Leukemia Group B Study (8762)
Authors: Westbrook CA , Hooberman AL , Spino C , Dodge RK , Larson RA , Davey F , Wurster- Hill DH , Sobol RE , Schiffer C , Bloomfield CD
Published: October 2019

The Philadelphia (Ph1) chromosome, or its molecular counterpart, the BCR-ABL fusion gene, is a rare but important prognostic indicator in childhood acute lymphoblastic leukemia (ALL), but its impact on adult ALL has not been well ascertained. A prospective study of the BCR-ABL fusion gene was begun on patients entered on clinical trials conducted by the Cancer and Leukemia Group B (CALGB). All patients received intensive, multiagent chemotherapy that included daunorubicin. Over 2 years, 56 patients were studied for molecular evidence of a BCR-ABL gene using Southern blot and pulsed-field gel hybridization analysis. Results were compared with cytogenetic detection of a Ph1 chromosome, and clinical features were compared for the BCR-ABL-positive and - negative groups. Molecular methods detected the BCR-ABL gene in 30% of cases compared with cytogenetic detection of the Ph1 chromosome in only 23%. The majority of cases (76%) showed the p190 gene subtype similar to pediatric ALL; the BCR-ABL-positive cases displayed a more homogeneous immunophenotype than the BCR-ABL-negative cases and were predominantly CALLA positive (86%) and B-cell surface antigen positive (82%). The rate of achieving complete remission was similar in the BCR- ABL-positive and -negative groups (71% and 77%, respectively, P = .72). There were more early relapses in the BCR-ABL-positive group, resulting in a shorter remission duration that was especially marked in the CALLA- positive and B-cell antigen-positive populations. These preliminary data suggest that the impact of the BCR-ABL gene on clinical outcome in ALL may be on maintenance of complete remission (CR) rather than achievement of CR when aggressive, multiagent chemotherapy is used. This study identifies the BCR-ABL gene as an important factor in adult ALL and demonstrates the utility of molecular methods for its accurate diagnosis.
Role of human immunodeficiency virus replication in defective in vitro growth of hematopoietic progenitors
Authors: Louache F , Henri A , Bettaieb A , Oksenhendler E , Raguin G , Tulliez M , Vainchenker W
Published: October 2019

A number of hematologic abnormalities, including cytopenias, have been observed in patients with human immunodeficiency virus (HIV) infection. To elucidate their mechanisms, a group of 27 patients with HIV-1 infection was studied. In all patients, a marked reduction of in vitro colony formation by erythroid, granulomacrophagic, and megakaryocytic bone marrow progenitors was observed in comparison to normal donors. HIV-1 infection of marrow progenitors was investigated in studying individual colonies with the polymerase chain reaction (PCR) technique. No HIV-1 DNA could be detected in these colonies, suggesting either that marrow progenitors were not infected or that infected progenitors were not able to generate colonies in vitro. The addition of antisense oligonucleotides directed against HIV tat or nef sequences in the culture medium led to a significant increase in colony formation, suggesting that HIV replication in hematopoietic progenitors could be responsible for their defective growth. However, no HIV-1-infected colonies could be detected by PCR after the antisense treatment, indicating that the increase in colony number was not due to the proliferation and differentiation of infected progenitors but to an inhibition of HIV replication in an accessory cell. This last hypothesis was further confirmed by the absence of effects of antisense oligomers on the plating efficiency of hematopoietic progenitors grown from CD34+ cells. These data indicate that hematologic abnormalities of HIV-infected patients cannot be explained by a direct infection of hematopoietic progenitor cells and suggest that a defective modulation of progenitor cell growth by HIV replication outside these cells might play a role in these abnormalities.
Over-expression of c-src or v-src in bone marrow stromal cells stimulates hematopoiesis in long-term bone marrow culture
Authors: Mladenovic J , Anderson SM
Published: October 2019

The S17 murine stromal cell line was infected with retroviral vectors encoding the v-src and c-src oncogenes and cells expressing high levels of either pp60v-src or pp60c-src were isolated. Long-term bone marrow cultures (LTBMCs) established with these different stromal cell lines showed that progenitor cells proliferated to a greater extent in cultures with stromal cells that over-expressed either c-src or v-src. An increase in the number of granulocytes, monocytes, and colony- forming units granulocyte-macrophage (CFU-GM) in the nonadherent cell population of LTBMCs prepared with S17/v-src or S17/c-src stromal cells was observed. Conditioned media from the S17/v-src and S17/src stromal cell lines stimulated the formation of CFU-GM in the absence of additional hematopoietic cell growth factors. Conditioned media from S17/v-src and S17/c-src stimulated proliferation of the granulocyte- macrophage colony-stimulating factor (GM-CSF)-responsive cell line FDCP-1 and this stimulation was inhibited by neutralizing antisera to murine GM-CSF. An increase in the concentration of GM-CSF was confirmed by enzyme-linked immunosorbent assay. No secretion of interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha was detected by any of the stromal cell lines. There was no increase in the secretion of either CSF-1 or IL-6 by either S17/v-src or S17/c-src. The addition of 1 micrograms/mL monoclonal anti-GM-CSF antibody to LTBMCs caused a decrease in the number of nonadherent cells in cultures established with each of the different stromal cell lines. Northern blot analysis showed no difference in the level of GM-CSF RNA among the different stromal cell lines. These studies suggest that the increased proliferation of hematopoietic progenitor cells in LTBMCs with S17/v-src or S17/c-src cells may result from a posttranscriptional event that elevates production of GM-CSF by the S17/c-src and S17/v-src stromal cells.
Dyskeratosis congenita fibroblasts are abnormal and have unbalanced chromosomal rearrangements
Authors: Dokal I , Bungey J , Williamson P , Oscier D , Hows J , Luzzatto L
Published: October 2019

Dyskeratosis congenita (DC) is a rare inherited disorder characterized by bone marrow failure, dystrophic changes in the skin and mucous membranes, and a predisposition to malignancy. The DC locus has been mapped to Xq28. The primary defect responsible for this disease remains unknown. We have studied four patients with this disease, three from one family and one from another. In all four patients, primary skin fibroblast cultures were abnormal both in morphology (polygonal cell shape, ballooning, and dendritic-like projections) and in growth rate (doubling time about twice normal). Fibroblast survival studies using four clastogens (bleomycin, diepoxybutane, mitomycin-c, and 4-nitroquinoline-1-oxide) and gamma radiation showed no significant difference between DC and normal fibroblasts. Cytogenetic studies performed on peripheral blood lymphocytes showed no difference between DC and normal lymphocytes with or without prior incubation with clastogens. However, bone marrow metaphases from one of three patients and fibroblasts from two of four patients (who were the eldest of the 4) showed numerous unbalanced chromosomal rearrangements (dicentrics, tricentrics, and translocations) in the absence of any clastogenic agents. Cell-specific differences and a higher rate of chromosomal rearrangements in the older patients appear to correlate with the clinical evolution of the disease. These findings suggest that the DC defect predisposes DC cells to developing chromosomal rearrangements.
Expression and downregulation of cytotoxic cell protease 1 or Granzyme 'B' transcripts during myeloid differentiation of interleukin-3- dependent murine stem cell lines
Authors: Hampson IN , Cross MA , Heyworth CM , Fairbairn L , Spooncer E , Cowling GJ , Dexter TM
Published: October 2019

Using the technique of differential cDNA library screening, we have molecularly cloned a gene that is highly expressed in an undifferentiated myeloid multipotent and growth factor-dependent stem cell line (FDCP-Mix) and that downregulates as these cells are induced to differentiate along monocytic, granulocytic, and erythroid cell lineages. Sequence analysis of this gene has shown homology with a previously cloned gene, cytotoxic cell protease 1 (CCP1 or Granzyme ‘B’), that has been shown to be expressed only in thymocytes, activated T cells, a mast cell line, and peritoneal exudate leukocytes. In situ hybridization, Northern blot analysis, and nuclear run-off assay has confirmed that expression of CCP1 is restricted to the phenotypically primitive multipotent undifferentiated. FDCP-Mix cells that are undergoing self-renewal in the presence of growth factors such as interleukin-3.
Expansion of a unique subpopulation of cytotoxic T cells that express a C alpha V delta 1 T-cell receptor gene in a patient with severe persistent neutropenia
Authors: Bank I , Book M , Cohen L , Kneller A , Rosental E , Pras M , Bassat IB , Ben-Nun A
Published: October 2019

CD8+ T-lymphocyte populations may be expanded in the peripheral blood of patients with chronic idiopathic neutropenia and may be involved in suppression of granulopoiesis. In this report, we have analyzed the T-cell receptor (TCR) used by the T lymphocytes of a patient with chronic severe neutropenia. Using specific oligonucleotides in the polymerase chain reaction (PCR) to amplify cDNA specific for the different families of the V alpha, V beta, and V delta TCR genes, and monoclonal antibodies (MoAbs) to examine T-lymphocyte subsets and their TCR, a persistent expansion of CD3+CD8+ T lymphocytes and a reduced repertoire of TCR V alpha and V beta genes were found in the patient's peripheral blood mononuclear cell (PBMC) preparations. A predominant portion of the T lymphocytes expressed a unique TCR structure. Thus, we found that, despite the fact that 98% of the T cells expressed alpha beta TCR on the surface membrane and less than 2% expressed tau delta TCR, nonetheless, 40% to 60% of the T cells stained positively with anti V delta 1 MoAb. Using the PCR analysis, the V delta 1 gene segment was found to be rearranged to C alpha, rather than to C delta genes. The expanded C alpha V delta 1+ cells, which are found only rarely in normal PB, expressed CD8 and were cytotoxic, and the C alpha V delta 1 receptor was functional in cytotoxicity. This constitutes the first description of an expansion of cytotoxic CD8+ lymphocytes expressing a functional “hybrid” C alpha V delta 1 gene in vivo, and suggests a pathogenic role for CD8+ C alpha V delta 1+ cells in some patients with idiopathic neutropenia.
Modulation of myeloid proliferation and differentiation by monoclonal antibodies directed against a protein that interacts with the interleukin-3 receptor
Authors: Tweardy DJ , Morel PA , Mott PL , Glazer EW , Zeh HJ , Sakurai M
Published: October 2019

Hematopoietic cells can be transformed through the acquisition of autocrine growth factor production. Because of their ability to inhibit autocrine growth, antibodies directed against the growth factor or its receptor may have therapeutic potential. However, these agents may also inhibit normal cell development. We have developed two monoclonal antibodies, 4G8 and 2F2, directed against a protein of 110 to 150 Kd that interacts with the interleukin-3 (IL-3) receptor (R) complex. These antibodies inhibit IL-3-induced proliferation of nonleukemic and leukemic IL-3-dependent cell lines, as well as the autonomous growth of WEHI-3B in vitro and in vivo. These results suggest the possibility that anti-IL-3R antibodies may be useful in the treatment of some leukemias. However, the effect of anti-IL-3R antibodies on normal myeloid development in vitro has not been examined. We examined the effect of 4G8 and 2F2 on the growth in vitro of colony-forming unit granulocyte-macrophage (CFU-GM) colonies induced by IL-3, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), and macrophage-CSF (M-CSF). Our results show that while 4G8 and 2F2 inhibited CFU-GM colony formation induced by IL-3, they augmented colony formation induced by the other hematopoietins. 4G8 and 2F2 also enhanced G-CSF-induced proliferation of 32Dc13 and GM-CSF-induced proliferation of PT18, confirming that the effect on CFU-GM was a direct effect. Finally, 4G8 and 2F2 inhibited G-CSF-induced differentiation of 32Dc13, similar to low levels of IL-3; yet, neither 4G8 nor 2F2 blocked binding of G-CSF to its receptor. These results indicate that, in the absence of IL-3 and in the presence of other hematopoietins, 4G8 and 2F2 can function as weak IL-3 agonists. These studies suggest that antibodies such as 4G8 and 2F2, directed against components of the IL-3R, could potentially augment myeloid growth in vivo, rather than inhibit myeloid growth.
Nonhematopoietic tumor cell lines express stem cell factor and display c-kit receptors
Authors: Turner AM , Zsebo KM , Martin F , Jacobsen FW , Bennett LG , Broudy VC
Published: October 2019

Human stem cell factor (SCF) acts in the presence of other growth factors to stimulate the growth of primitive hematopoietic progenitor cells. These effects are performed by activation of the SCF receptor, c- kit. Because of the potential use of SCF in patients undergoing chemotherapy and bone marrow transplantation, the effect of SCF on nonhematopoietic tumors requires investigation. To determine whether human tumor cell lines display c-kit receptors, we performed binding experiments with 125I-SCF on a breast carcinoma cell line (Du4475), a gastric carcinoma cell line (KATO III), a melanoma cell line (HTT144), as well as two small cell lung carcinoma cell lines (H69 and H128). The biologic effect of SCF on tumor cell lines was assessed by its ability to stimulate tritiated thymidine uptake and to enhance colony growth in methylcellulose. The breast carcinoma cell line, Du4475, as well as two small cell lung carcinoma cell lines, H69 and H128, exhibit high- affinity c-kit receptors with approximate binding affinities of 40, 100, and 90 pmol/L, respectively. The number of high-affinity receptors per cell ranged from 700 to 9,500. The gastric carcinoma cell line, as well as the melanoma cell line, showed trace binding of 125I-SCF. In the presence of SCF alone, or in combination with granulocyte- macrophage colony-stimulating factor or interleukin-3, there was less than a 17% increase in the colony growth of Du4475, H69, or H128 cell lines. Postulating that the lack of growth response could be secondary to endogenous SCF production by the tumor cell lines, we used an RNAse protection assay to determine whether the tumor cell lines contain SCF messenger RNA (mRNA). In addition, we tested tumor cell line supernatants for the presence of secreted SCF protein by enzyme immunoassay, and analyzed the tumor cell lines for membrane-bound SCF by indirect immunofluorescence. Our results show that the Du4475, H69, and H128 cell lines, as well as a melanoma cell line (HTT144), have multiple copies of SCF mRNA. Soluble SCF protein was detected in the cell supernatants in the Du4475 and H69 cell lines and SCF was found on the surface of all four cell lines. These data show that some human solid tumor cell lines display high-affinity c-kit receptors and produce SCF, which can be detected on the cell surface. These results suggest the possibility that autocrine production of SCF by c-kit receptor-bearing tumor cells may enhance cell growth in tumor cell lines.
Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical induction
Authors: Busfield SJ , Klinken SP
Published: October 2019

The J2E cell line is a novel erythroid cell line that differentiates in response to erythropoietin (Epo), the physiologic stimulus for erythropoiesis. After exposure to Epo, the cells synthesize hemoglobin, and we show here that this process is tightly linked to increases in cellular proliferation and DNA synthesis. The hormone-induced terminal differentiation also results in morphologic alterations and the accumulation of transcripts for alpha, beta maj, and beta min globins. c-myc messenger RNA levels increase rapidly after exposure to Epo and precede the increase in cell division, while c-myb undergoes a transient decrease. Differentiation of J2E cells can also be achieved with sodium butyrate, but, in contrast with Epo, this is associated with a retardation of replication and a sudden decrease in c-myc levels. These results show that, in this system, chemically induced differentiation differs from terminal maturation promoted by Epo and that the processes of proliferation and differentiation in J2E cells can be uncoupled.
Apoptotic cell death of primed CD45RO+ T lymphocytes in Epstein-Barr virus-induced infectious mononucleosis
Authors: Uehara T , Miyawaki T , Ohta K , Tamaru Y , Yokoi T , Nakamura S , Taniguchi N
Published: October 2019

The expansion of activated T cells, characterized by the expression of CD45RO as well as HLA-DR antigens, is a central feature in acute infectious mononucleosis (IM) induced by primary infection of Epstein- Barr virus (EBV). However, the fate of these activated T cells in this disease is not clearly understood. We found that, on simple culture, a large proportion of T cells isolated from acute IM patients died rapidly, but only a few T cells from normal individuals did. Morphologic observations and DNA fragmentation analysis showed that the loss of viability of IM T cells after incubation was mediated by apoptosis. IM T cells undergoing apoptosis resided exclusively in the CD45RO+ populations of both CD4+ and CD8+ T cells, most of which were shown to coexpress apoptosis-related Fas antigen. Some cytokines such as interleukin-2 (IL-2), IL-5, and IL-6 could rescue IM T cells from apoptotic cell death. The results seemed to imply that most of primed (CD45RO+) T cells in acute IM might be subject to apoptotic cell death, possibly when leaving from the local sites actively producing certain soluble factors required for their survival. Our studies suggest the programmed cell death of peripheral mature T cells as a mechanism of antigen-driven selection.