bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor necrosis factor-alpha modulation of glycoprotein Ib alpha expression in human endothelial and erythroleukemia cells
Authors: Rajagopalan V , Essex DW , Shapiro SS , Konkle BA
Published: October 2019

Glycoprotein Ib alpha (GpIb alpha) is a platelet membrane Gp that binds von Willebrand factor and mediates platelet adhesion to subendothelium. We have found both GpIb alpha mRNA and protein in human umbilical vein endothelial cells (HUVEC). In previously published work we reported that combined treatment with interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) markedly increased the GpIb alpha mRNA level in HUVEC. We have now documented that TNF-alpha alone induces GpIb alpha mRNA and protein expression, studied the kinetics of this response, and investigated potential mechanisms of the TNF-alpha effect. GpIb alpha mRNA induction by TNF-alpha is detectable as early as 2 hours after exposure to this cytokine, and reaches a maximal level after 20 to 24 hours. Using a nuclear run-on assay we found that GpIb alpha gene transcription is increased approximately 10-fold after 2 hours of TNF-alpha treatment. Furthermore, using two monoclonal antibodies that recognize different epitopes of GpIb alpha, we found that the protein expression in endothelial cells is markedly increased by TNF-alpha. Interleukin-1 (IL-1) and the phorbol ester phorbol myristate acetate, which mimic many effects of TNF-alpha on endothelial cells, have no effect on endothelial or human erytholeukemia (HEL)-cell GpIb alpha mRNA. TNF-alpha treatment for 24 hours increases the HEL cell GpIb alpha mRNA level approximately fourfold, showing a time- and dose-dependent effect similar to that seen in HUVEC. TNF-alpha-induced GpIb alpha mRNA and protein synthesis may play a role in mediating platelet or other cell interaction with activated endothelium. Unlike other endothelial pro-thrombotic and pro-adhesive proteins induced by TNF-alpha, GpIb alpha is not induced by IL-1 treatment, which suggests a novel pathway for induction of this protein.
Measurement of basal levels of factor VIIa in hemophilia A and B patients [see comments]
Authors: Wildgoose P , Nemerson Y , Hansen LL , Nielsen FE , Glazer S , Hedner U
Published: October 2019

Previous results, presented in abstract form, indicate that replacement of thromboplastin with a mixture of phospholipid and truncated soluble tissue factor apoprotein results in a coagulation assay that can directly measure plasma factor VIIa levels without interference from zymogen factor VII (Atherosclerosis Thromb 11:1544a, 1991 [abstr]). We have exploited the specificity and sensitivity of such a factor VIIa specific coagulation assay to directly assess the in vivo relationship of factor VIII and factor IX on the production of factor VIIa levels under nonthrombotic and nonstimulatory conditions. Normal individuals (n = 20) were found to possess an average circulating factor VIIa level corresponding to 4.34 +/- 1.57 ng/mL, or approximately 1% of their total factor VII antigen. Severe factor VIII deficient patients (n = 13) possessed a slightly lower but statistically significant (P less than .01) decrease in their basal factor VIIa levels (2.69 +/- 1.52 ng/mL), corresponding to approximately 60% of that observed in normal individuals. On the other hand, severe factor IX deficient patients (n = 7) were found to possess even lower levels of factor VIIa corresponding to 0.33 +/- 0.15 ng/mL, or less than 10% of that observed in normal individuals. Measurement of total factor VII antigen levels shows that the variation in basal factor VIIa levels stems from differences in the degree of factor VII activation as opposed to differences in factor VII antigen levels. Our present data are consistent with the hypothesis that factor IXa is the principal in vivo activator of factor VII under basal conditions.
Compound heterozygosity in a complete erythrocyte bisphosphoglycerate mutase deficiency
Authors: Lemarchandel V , Joulin V , Valentin C , Rosa R , Galacteros F , Rosa J , Cohen- Solal M
Published: October 2019

Erythrocyte bisphosphoglycerate mutase (BPGM) deficiency is a rare disease associated with a decrease in 2,3-diphosphoglycerate concentration. A complete BPGM deficiency was described in 1978 by Rosa et al (J Clin Invest 62:907, 1978) and was shown to be associated with 30% to 50% of an inactive enzyme detectable by specific antibodies and resulting from an 89 Arg-->Cys substitution. The propositus' three sisters exhibited the same phenotype, while his two children had an intermediate phenotype. Samples from the family were examined using polymerase chain reaction and allele-specific oligonucleotide hybridization and sequencing techniques. Amplification of erythrocyte total RNA from the propositus' sister around the 89 mutation indicated the presence of two forms of messenger RNAs, a major form with the 89 Arg-->Cys mutation and a minor form with a normal sequence. Sequence studies of the propositus' DNA samples indicated heterozygosity at locus 89 and another heterozygosity with the deletion of nucleotide C 205 or C 206. Therefore, the total BPGM deficiency results from a genetic compound with one allele coding for an inactive enzyme (mutation BPGM Creteil I) and the other bearing a frameshift mutation (mutation BPGM Creteil II). Examination of the propositus' two children indicated that they both inherited the BPGM Creteil I mutation.
The ligand for c-kit, stem cell factor, stimulates the circulation of cells that engraft lethally irradiated baboons
Authors: Andrews RG , Bensinger WI , Knitter GH , Bartelmez SH , Longin K , Bernstein ID , Appelbaum FR , Zsebo KM
Published: October 2019

Recombinant human stem cell factor (SCF), the ligand for c-kit, has been shown to stimulate increased numbers of hematopoietic progenitor cells of multiple types to circulate in the blood of baboons, but it was not known if the cells stimulated to circulate by SCF contained cells capable of engrafting and rescuing lethally irradiated baboons. Peripheral blood mononuclear cells (PBMNC) were collected by leukapheresis from four untreated control baboons and from three baboons on the 10th or 11th day of treatment with SCF (200 micrograms/kg/d). All animals were transplanted with 1.00 to 1.04 x 10(8)/kg of cryopreserved autologous PBMNC after treatment with a single dose of 1,020 cGy total body irradiation (TBI). Three animals were transplanted with PBMNC that had been collected during SCF treatment, 24 to 38 days after the last dose of SCF. Rapid trilineage engraftment was documented by bone marrow biopsy in all three. The mean time to a total white blood cell count (WBC) > or = 500/microL, WBC > or = 1,000/microL, and an absolute neutrophil count (ANC) > or = 500/microL was 15 +/- 3 (mean +/- SD), 19 +/- 1, and 19 +/- 2 days, respectively. Two animals remain alive with stable engraftment more than 180 and 245 days posttransplant. The third died of sepsis 32 days posttransplant with a hypercellular marrow showing trilineage engraftment. The surviving animals were transfusion independent by 10 and 59 days posttransplant. Four control animals were transplanted with PBMNC collected in the absence of SCF stimulation. One was treated for 11 days with SCF (200 micrograms/kg/d) after PBMNC were collected. This animal was transplanted 25 days after the last dose of SCF. None of the four control animals engrafted and they died 13, 16, 28, and 38 days posttransplant with marrow aplasia. Treatment with SCF stimulates the circulation of cells that engraft and rescue lethally irradiated baboons. The characteristics of the transplantable cells present in the circulation are now amenable to direct study.
Potentiation of lipopolysaccharide-induced tumor necrosis factor-alpha expression by 1,25-dihydroxyvitamin D3
Authors: Prehn JL , Fagan DL , Jordan SC , Adams JS
Published: October 2019

The immunomodulatory hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to suppress T-cell proliferation and interleukin-2 synthesis as well as B-cell immunoglobulin synthesis, while stimulating many macrophage functions. We have previously shown increased synthesis of interleukin-1 beta in lipopolysaccharide (LPS)-stimulated U937 cells after pretreatment with 10 nmol/L 1,25-(OH)2D3. We now show that 1,25- (OH)2D3 also primes the increase in U937 cell tumor necrosis factor (TNF)-alpha-accumulated mRNA after activation with LPS; 50% effective concentration (EC50) for the LPS-induced expression of TNF-alpha mRNA was decreased by two orders of magnitude after incubation with 10 nmol/L 1,25-(OH)2D3. Pretreatment of U937 cells with 10 nmol/L 1,25- (OH)2D3 also increased subsequent LPS-induced TNF-alpha mRNA expression by twofold and cell-associated TNF protein levels by more than ninefold. This potentiation was steroid-specific for 1,25-(OH)2D3 because dexamethasone inhibited TNF-alpha mRNA. The potentiation required prior exposure to 1,25-(OH)2D3 for more than 6 hours and was clearly seen after 12 hours. The finding that the sensitivity of the U937 cell monokine response to LPS was dramatically increased by 1,25- (OH)2D3 and the delayed effect on the LPS-stimulated TNF-alpha gene transcript levels indicated that 1,25-(OH)2D3 may be altering the expression of a protein(s) in the U937 cell LPS-signal transduction pathway. In fact, 1,25-(OH)2D3 induced expression of the mRNA for CD14, the high affinity, cell-surface glycoprotein receptor for LPS, which could account for the enhancement of LPS-stimulated monokine gene expression by 1,25-(OH)2D3. Thus, local monokine gene expression may be regulated by both the amount and the temporal entry of the vitamin D hormone and activator(s) into the inflammatory microenvironment.
Expression of human recombination activating genes (RAG-1 and RAG-2) in Hodgkin's disease
Authors: Knecht H , Joske DJ , Emery-Goodman A , Bachmann E , Bachmann F , Odermatt BF
Published: October 2019

The differentiation status of Sternberg-Reed (SR) cells is still not well defined, primarily because of their scarcity in tumor biopsies of Hodgkin's disease (HD). In this study we have determined the genomic differentiation status of SR cells by quantitation of recombinase activating gene (RAG) expression. RAG genes are selectively transcribed in immature lymphoid cells. In B cells they are silent after genomic rearrangement has occurred, whereas in T cells they are downregulated during positive selection of double-positive thymocytes into single- positive cells. RNA from tumor biopsies either with numerous (11 cases) or a with few SR cells (16 cases) was assessed by a sensitive reverse transcriptase polymerase chain reaction (RT-PCR) and the results compared with established positive and negative controls. In all except two cases levels of RAG expression were within the range of those determined in negative controls. In both positive cases and in the positive control RAG mRNA was further quantitated by competitive PCR. In cases with abundant SR cells RAG expression was still below that observed in 10(-2) dilutions of positive controls. These results suggest that SR cells are derived from lymphoid cells, more differentiated than the pre-B or common thymocyte stage, which have already undergone genomic rearrangement. They show the value of assessing RAG expression by RT-PCR in the characterization of lymphoid malignancies.
Activation of the erythropoietin receptor by the Friend spleen focus- forming virus gp55 glycoprotein induces constitutive protein tyrosine phosphorylation
Authors: Showers MO , Moreau JF , Linnekin D , Druker B , D'Andrea AD
Published: October 2019

The erythropoietin receptor (EPO-R) can be activated to signal cell growth by binding either EPO or gp55, the Friend spleen focus-forming virus (SFFV) glycoprotein. EPO binding induces tyrosine kinase activity and rapid tyrosine phosphorylation of several cellular substrates. To test for gp55-induced tyrosine kinase activity, we performed immunoblots on two murine cell lines that stably express EPO-R and gp55. Stimulation of the parental cell line, Ba/F3, with murine interleukin-3 (IL-3) resulted in rapid, dose-dependent tyrosine phosphorylation of a 97-Kd substrate. Stimulation with IL-3 or EPO of the Ba/F3 cells expressing the recombinant EPO-R (Ba/F3-EPO-R) resulted in tyrosine phosphorylation of the same p97 substrate. These latter cells, when transformed to growth factor-independence by the Friend gp55 glycoprotein, exhibited constitutive tyrosine phosphorylation of the 97-Kd substrate. Other growth factor-independent Ba/F3 subclones, transformed with either the oncoprotein, v-abl, or with a constitutively activated EPO-R, also had constitutive phosphorylation of a 97-Kd substrate. In CTLL-2-EPO-R cells, a T-lymphocyte line stably transfected with the EPO-R, the 97-Kd substrate was tyrosine- phosphorylated in response to IL-2 or EPO. The 97-Kd protein was constitutively phosphorylated in CTLL-2-EPO-R-gp55 cells. In conclusion, a 97-Kd protein found in two murine cell lines is tyrosine-phosphorylated in response to multiple growth factors and viral oncoproteins, and appears to be a central phosphoprotein in signal transduction.
Regulatory effects of gallium on transferrin-independent iron uptake by human leukemic HL60 cells
Authors: Chitambar CR , Sax D
Published: October 2019

Gallium, a pharmacologically important metal, resembles iron with respect to transferrin (Tf) binding and Tf receptor-mediated cellular uptake. In the present study, we examined the effect of gallium on Tf- independent iron uptake by HL60 cells. In contrast to the inhibitory effect of Tf-gallium on Tf-iron uptake, gallium nitrate, in a time-, temperature-, and concentration-dependent manner, stimulated Tf- independent uptake of iron-nitrilotriacetic acid (Fe-NTA). Preexposure of cells to gallium followed by removal of gallium also resulted in sustained stimulation of iron uptake. The anti-Tf receptor monoclonal antibody 42/6 blocked Tf-iron uptake, but had no effect on gallium- induced stimulation of Tf-independent iron uptake. Gallium increased the number of cell membrane iron-binding sites, without a change in their affinity for iron. Ferric chloride stimulated Tf-independent gallium uptake. Although gallium nitrate inhibited cell growth in Tf- free medium, cellular proliferation was restored by Fe-NTA. Gallium and iron appear to share the same Tf-independent cellular uptake system in HL60 cells. Exposure of cells to gallium results in the activation of cell membrane non-Tf iron carriers that may play a role in overcoming the Tf-independent growth-inhibitory effects of gallium.
Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency by natural and amplification created restriction sites: five mutations account for most G6PD deficiency cases in Taiwan
Authors: Chang JG , Chiou SS , Perng LI , Chen TC , Liu TC , Lee LS , Chen PH , Tang TK
Published: October 2019

We have developed a rapid and simple method to diagnose the molecular defects of glucose-6-phosphate dehydrogenase (G6PD) deficiency in Chinese in Taiwan. This method involves the selective amplification of a DNA fragment from human G6PD gene with specific oligonucleotide primers followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. Ninety- four Chinese males with G6PD deficiency were studied. The results show that 50% (47 of 94) were G to T mutation at nucleotide (nt) 1376, 21.3% (20 of 94) were G to A mutation at nt 1388, 7.4% (7 of 94) were A to G mutation at nt 493, 7.4% (7 of 94) were A to G mutation at nt 95, 4.2% (4 of 94) were C to T mutation at nt 1024, 1.1% (1 of 94) was G to T mutation at nt 392, and 1.1% (1 of 94) was G to A mutation at nt 487. These results show that the former five mutations account for more than 90% of G6PD deficiency cases in Taiwan. Aside from showing that G to T change at nt 1376 is the most common mutation, our research indicates that nt 493 mutation is a frequent mutation among Chinese in Taiwan. We compared G6PD activity among different mutations, without discovering significant differences between them.
Kit ligand improves in vitro erythropoiesis in myelodysplastic syndrome
Authors: Backx B , Broeders L , Lowenberg B
Published: October 2019

Erythropoiesis in response to erythropoietin (Epo) in myelodysplastic syndrome (MDS) in vitro and in vivo is severely impaired. We investigated the stimulative effect of c-kit ligand (KL) on the erythroid colony-forming abilities of bone marrow cells from 17 patients with MDS. The effects of normal donor-derived marrow were examined in comparison. Suppression of erythroid colony formation in MDS in response to Epo could not be restored by the addition of interleukin-3 (IL-3) to culture. In cultures dishes supplemented with KL, erythroid colony formation was dramatically enhanced, regarding both colony number and size. Colony-forming abilities by MDS progenitors were improved following costimulation with KL, particularly in refractory anemia (RA) and refractory anemia with ring sideroblasts (RARS); however, little enhancement was apparent following KL stimulation of marrow from patients with refractory anemia with excess of blasts (RAEB), refractory anemia with excess of blasts in transformation (RAEB-t), and chronic myelomonocytic leukemia (CMML). These results suggest that KL responsiveness of patients with low-risk MDS may still be intact, and that with progression to high-risk MDS, erythroid progenitors lose proliferative reactivity to both KL and Epo stimulation. KL may have a therapeutic role in restoring erythropoiesis in a subset of patients with MDS.
Relevance of pretransfusion incubation of platelets at 37 degrees C
Authors: de Vries RA , Gerritsen JG , de Bruin M , Marx JJ , Hart HC , van de Wiel A
Published: October 2019

The effect of pretransfusion incubation of platelets at 37 degrees C was assessed because of the controversial reports about its relevance. A dual-label technique (111indium and 114mindium) was applied in 10 healthy subjects receiving warmed and unwarmed autologous platelets simultaneously. Fresh platelet concentrates were infused into five subjects, whereas the other five subjects received stored platelet concentrates. The mean platelet volume decreased in all platelet concentrates during incubation, reflecting the restoration of the discoid shape of the platelets. The mean decrease was 0.35 fL (P = .003). However, the initial recovery and the mean platelet life-span were not improved by this procedure. It was concluded that there is no evidence that brief warming of platelets has any beneficial effect on platelet viability in healthy volunteers.
Interaction of human bone marrow fibroblasts with megakaryocytes: role of the c-kit ligand
Authors: Avraham H , Scadden DT , Chi S , Broudy VC , Zsebo KM , Groopman JE
Published: October 2019

Human kit ligand (KL), also known as stem cell factor (SCF), steel factor, or mast cell growth factor, is a recently identified hematopoietic growth factor whose receptor is the product of the c-kit proto-oncogene. Alternative splicing of the pre-mRNA of KL/SCF results in secreted and membrane-bound forms of the protein. We and others have recently shown that the c-kit gene product is expressed on human megakaryocytes and that soluble KL/SCF in combination with granulocyte- macrophage colony-stimulating factor, interleukin-3 (IL-3), or IL-6 increased megakaryocyte progenitor colony formation (CFU-MEG) and stimulated mature megakaryocytes. Here we show that adhesion of human megakaryocytes to bone marrow stromal fibroblasts, which express the membrane-bound form of KL/SCF (mKL/SCF), is mediated in part by the interaction between mKL/SCF and the c-kit protein. This interaction also results in marrow fibroblast-stimulated proliferation but not an increase in ploidy of megakaryocytes; when the two cell types were separated by a transoluble membrane, proliferation did not occur. Adhesion and proliferation of human megakaryocytes to an immortalized murine stromal cell line SI/SI lacking the KL/SCF gene was impaired, whereas transfection of SI/SI cells with human mKL/SCF significantly increased both adhesion and proliferation. Marrow stromal fibroblast mKL/SCF may serve both as an adhesion structure and as a growth- potentiating factor for megakaryocytes in the bone marrow.
Hematopoietic cytokines inhibit apoptosis induced by transforming growth factor beta 1 and cancer chemotherapy compounds in myeloid leukemic cells
Authors: Lotem J , Sachs L
Published: October 2019

Transforming growth factor-beta 1 (TGF-beta 1) induces cell death in myeloid leukemia by apoptosis. In the M1 myeloid leukemia, this induction of apoptosis was inhibited by granulocyte colony-stimulating factor (G-CSF) or interleukin-6 (IL-6) and to a lesser extent by IL-1 alpha. IL-3 and stem cell factor/mast cell growth factor (SCF) showed only a marginal effect, and granulocyte-macrophage and macrophage CSFs (GM-CSF and M-CSF, respectively) were inactive. The induction of apoptosis by TGF-beta 1 in a different myeloid leukemia (7-M12) was inhibited by GM-CSF and IL-3 but not by the other cytokines. In the absence of TGF-beta 1, both M1 and 7-M12 leukemic cells were independent of hematopoietic cytokines for cell viability and growth. The cytotoxic compounds vincristine, vinblastine, adriamycin, cytosine arabinoside, cycloheximide, and sodium azide, some of which are used in cancer chemotherapy, induced cell death by apoptosis in both leukemias. As with TGF-beta 1, apoptosis induced by these cytotoxic compounds was inhibited by GM-CSF (7-M12 leukemia) and by G-CSF or IL-6 (M1 leukemia). Cyclosporine A decreased cell multiplication in M1 cells without inducing apoptosis, and G-CSF and IL-6 inhibited the cytostatic effect of cyclosporine A. It is suggested that the clinical use of cytokines to correct therapy-associated myelosuppression should be carefully timed to avoid protection of malignant cells from the cytotoxic action of the therapeutic compounds.
Heterogeneous immunophenotype of granular lymphocyte expansions: differential expression of the CD8 alpha and CD8 beta chains [see comments]
Authors: de Totero D , Tazzari PL , DiSanto JP , di Celle PF , Raspadori D , Conte R , Gobbi M , Ferrara GB , Flomenberg N , Lauria F
Published: October 2019

In this study, we have evaluated 14 large granular lymphocyte (LGL) expansions, 11 of which were CD8+. Analysis of the membrane expression of the alpha and beta chains of the CD8 antigen, using specific monoclonal antibodies (MoAbs), has shown that LGL expansions with the CD3+, CD4+, CD8+, CD57+ T-cell receptor (TcR) alpha beta phenotype bear the CD8 alpha/alpha isoform, while the CD3+, CD4-, CD8+, CD57+ TcR alpha beta samples were positive for both the CD8 alpha and CD8 beta chains. These data were confirmed also by messenger RNA analysis. One additional case, with a peculiar phenotype (CD3-, CD2-, CD4-, CD8+, CD57-) and a germline configuration of the TcR beta and gamma chain genes, expressed only the CD8 alpha chain. After additional phenotypic analysis with a wider panel of MoAbs, it was found that the beta chain of the interleukin-2 receptor was constitutively expressed on the majority of the samples tested, and that most of the monoclonal samples coexpressed CD45RA/R0 antigens. Using MoAbs directed against the variable regions of the TcR beta chain, we could show a preferential V beta region restriction in the CD8+ monoclonal cases. This more extensive characterization of CD8+ LGL expansions has further documented the marked heterogeneity within this rare condition and allowed a better phenotypic dissection between the monoclonal and polyclonal cases.
Progressive disease after high-dose therapy and autologous transplantation for lymphoid malignancy: clinical course and patient follow-up
Authors: Vose JM , Bierman PJ , Anderson JR , Kessinger A , Pierson J , Nelson J , Frappier B , Schmit-Pokorny K , Weisenburger DD , Armitage JO
Published: October 2019

Of 364 patients with lymphoid malignancy who underwent high-dose therapy with autologous bone marrow transplantation (ABMT) or peripheral stem cell transplantation (PSCT), 169 patients have had progressive disease after the procedure. The median survival from the time of relapse for patients with Hodgkin's disease (HD) who progressed after the transplant was 10.5 months. This compares with a median survival of 3 months for relapsed non-Hodgkin's lymphoma (NHL) patients (P = .0036). After failing transplantation, 56 patients were treated with further chemotherapy, 35 with involved field irradiation therapy, and 18 patients were treated with combination chemotherapy and irradiation. Seven patients received biologic therapy and seven patients underwent a second bone marrow transplant. The remainder of the patients were believed to be too ill for further therapy or chose not to receive further treatment for their recurrent lymphoid malignancy. Sixty of the 169 patients with progressive disease after the transplant are still alive; however, only 18 patients are alive off therapy without evidence of active disease after their relapse. Ten of the 18 patients are still less than 12 months past their posttransplant salvage therapy and are at high-risk for relapse. Five patients are progression free at 15 to 36 months after their posttransplant relapse. Only three patients (two NHL and one HD) treated with other modalities after autologous transplant failure are alive without evidence of disease and have been observed at least 4 years postrelapse. Although a few patients will have a durable response to subsequent therapy, the majority of patients who have progressive disease after an autologous transplant for lymphoid malignancy will succumb to recurrent disease within a short period of time.
A new in situ hybridization technique for spliced RNA species documents the bone marrow origin of pulmonary macrophages in chronic myelogenous leukemia
Authors: Ely P , Haase A , Wietgrefe S , Zupancic M , Beneke J , Hertz M , Miller W
Published: October 2019

Tissue macrophages derive from monocytes of bone marrow origin. Because monocytes from patients with chronic myelogenous leukemia (CML) contain the Philadelphia chromosome (Ph), it seemed probable that tissue macrophages in CML would originate from the malignant clone. Using powerful molecular techniques, we studied pulmonary alveolar macrophages (PAM) from two patients with CML. PAM from Patient 1, a patient in chronic phase studied before bone marrow transplantation (BMT), contained the Ph by Southern blot analysis. Patient 2, an accelerated phase patient, was studied after post-BMT relapse. PAM from this patient not only contained the Ph, but also expressed the BCR/ABL message documented by a new splice junction in situ hybridization technique. This new technique allows detection of BCR/ABL mRNA and determination of splice useage in individual cells. These data confirm the continued replenishing of PAM from peripheral blood monocytes in non-BMT settings and represent the first direct evidence that tissue macrophages are derived from the malignant clone in patients with CML.
Release of elastase from monocytes adherent to a fibronectin-gelatin surface
Authors: Xie DL , Meyers R , Homandberg GA
Published: October 2019

Fibronectin (Fn) is a circulating and extracellular matrix glycoprotein that may serve to facilitate phagocytosis because of its ability to bind many inflammatory ligands and to a monocyte receptor. Fn fragments have been shown in many systems to have augmented properties over those of native Fn. We show in this report that although Fn fragments did not cause elastase release from monocytes in suspension, fragments did cause elastase release from monocytes that were first bound to Fn- gelatin surfaces. An amino-terminal 29-Kd and a 140-Kd integrin-binding fragment were half-maximally active at 100 nmol/L, whereas the Arg-Gly- Asp-Ser integrin-recognition peptide was half-maximally active at 100 mumol/L. Fluid-phase Fn was ineffective yet blocked the activity of the Fn fragments. Complexing of Fn with gelatin or with heparin partially removed the blocking effect of Fn. Similar results were obtained with U- 937 cells. Substitution of the Fn-gelatin surface with bovine articular cartilage also promoted elastase release. Therefore, in conditions in vivo in which monocytes bind to tissue surface, a high ratio of Fn fragments to native Fn may upregulate certain monocyte activities such as protease release.
Clearance of human native, proteinase-complexed, and proteolytically inactivated C1-inhibitor in rats
Authors: de Smet BJ , de Boer JP , Agterberg J , Rigter G , Bleeker WK , Hack CE
Published: October 2019

C1-inhibitor is the only known inhibitor of the classical pathway of complement and the major inhibitor of the contact pathway of coagulation. Like other serine proteinase inhibitors, C1-inhibitor can exist in three conformations, ie, the native, the proteinase-complexed, and the proteolytically inactivated form. Here we studied the plasma elimination kinetics of these three forms of human C1-inhibitor in rats. The clearance of the complexed form of C1-inhibitor appeared to be the most rapid and depended in part on the proteinase involved (observed plasma t1/2 was 20 minutes for C1s-C1-inhibitor, 32 minutes for kallikrein-C1-inhibitor, and 47 minutes for beta XIIa-C1- inhibitor), whereas that of native C1-inhibitor was the slowest (observed plasma t1/2 4.5 hours). Inactivated C1-inhibitor was cleared with an apparent plasma t1/2 of 1.6 hours. Thus, the short plasma t1/2 of complexed relative to native C1-inhibitor explains why in patients only low concentrations of C1-inhibitor complexes may be observed despite activation of the contact and/or complement systems.
A bone marrow stromal cell line is a source and target for platelet- derived growth factor
Authors: Abboud SL
Published: October 2019

Platelet-derived growth factor (PDGF) stimulates multipotent and erythroid progenitors as well as stromal fibroblasts. Any of the three dimeric forms of PDGF (AA, AB, or BB) could potentially interact with these cells; however, the precise cellular origin of PDGF production in the bone marrow microenvironment is not known. In the present study, we found that medium conditioned by MBA-2, murine bone marrow-derived endothelial cells, contains PDGF activity that competes for [125I]PDGF binding to human foreskin fibroblasts and is mitogenic for these fibroblasts. Northern analysis of poly(A)+ RNA from MBA-2 shows the expression of both PDGF A-chain and B-chain mRNAs. Because cytokines such as transforming growth factor-beta (TGF-beta) regulate hematopoiesis and stimulate PDGF in certain mesenchymal cells, we determined whether TGF-beta influences PDGF secretion and gene expression in MBA-2. TGF-beta induced PDGF A-chain and B-chain mRNAs and the release of PDGF activity. Each of the three PDGF isoforms also stimulated DNA synthesis in MBA-2, but with different potency (BB = AB = AA). Ligand binding studies showed specific binding of labeled PDGF BB and, to a lesser extent, PDGF AA isoform, consistent with predominant expression of the PDGF-beta receptor in MBA-2. These data show that murine endothelial stromal cells release PDGF activity and respond to PDGF. Local production of PDGF in the marrow microenvironment may play an important role in regulating hematopoietic and stromal cell proliferation.
Role of protein kinase C in tumor necrosis factor induction of endothelial cell urokinase-type plasminogen activator
Authors: Niedbala MJ , Stein-Picarella M
Published: October 2019

Tumor necrosis factor (TNF) can promote endothelial cell transcription, synthesis, and secretion of urokinase plasminogen activator (uPA) augmenting extracellular matrix remodeling and influencing cellular differentiation. In this report, the role of the protein kinase C (PKC) pathway in mediating TNF induction of uPA in human umbilical vein endothelial cells is described. The PKC inhibitors (H-7, staurosporine, and calphostin C), but not HA-1004, inhibited TNF-induced uPA expression, synthesis, and secretion in a dose-dependent manner. Analysis of cell-free conditioned medium obtained from PKC inhibitor- treated cultures by micro-enzyme-linked immunosorbent assay methodologies using uPA- and plasminogen activator inhibitor type 1 (PAI-1)-specific monoclonal antibodies indicate that the decrease in uPA activity observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography was a direct result of decreased extracellular uPA antigen and not a consequence of increased PAI-1 antigen. The effect of PKC inhibitors was specific for TNF-mediated increased uPA expression because cytokine induction of PAI-1 was not influenced by these agents. Northern blot analyses also showed that PKC inhibitor treatment of endothelial cells resulted in a decreased steady- state level of uPA mRNA with no measurable change in PAI-1 mRNA in cultures incubated with TNF. Downregulation of cellular PKC by 18 hours of phorbol myristate acetate (PMA) pretreatment of endothelial cell cultures abolished TNF-mediated extracellular uPA induction. This effect was specific for PMA because 4-alpha PMA pretreatment of cells, which does not stimulate PKC, was ineffective in altering TNF induction of endothelial cell uPA. Induction of PKC directly with PMA, mezerein, and (-)-octylindolactam V increased endothelial cell levels of extracellular uPA in a time- and dose-dependent manner. In addition, this increase in endothelial cell extracellular uPA activity mediated by PKC agonists could be inhibited with PKC inhibitors. Endothelial cells treated with TNF acquire the ability to invade extracellular matrix and reorganize into tube-like structures when grown on Matrigel- coated culture dishes, a behavior blocked by H-7, but not by HA 1004. In summary, these data implicate a role for the PKC pathway in the TNF- mediated induction of uPA expression, subsequent matrix remodeling, and the formation of tube-like structures, a process important in neovascularization, wound healing, and leukocyte extravasation.
Adenosine diphosphate stimulation of cultured hematopoietic cell lines
Authors: Kalambakas SA , Robertson FM , O'Connell SM , Sinha S , Vishnupad K , Karp GI
Published: October 2019

Adenosine diphosphate (ADP) plays a critical role in platelet activation both by exogenous stimulation and the release of endogenous intracellular stores. As the platelet ADP receptor is not well defined, we have chosen to identify and characterize several cell lines that possess functional receptors for this nucleotide. Rat promegakaryoblasts (RPM), human erythroleukemia cells (HEL), U937, and K562 leukemia cells responded to ADP, as measured by a rapid increase in intracellular calcium. In the case of RPM cells, ADP was the only naturally occurring platelet agonist capable of eliciting this response. Binding studies with [3H]ADP and fixed cells showed 3.99 +/- 1.77 x 10(5) binding sites/cell for RPM cells (apparent dissociation constant [kd] = 7.75 +/- 2.3 x 10(-8) mol/L), 8.19 +/- 3.25 x 10(5) sites/cell for HEL cells (kd = 2.15 +/- 0.84 x 10(-7) mol/L, 1.15 +/- 0.23 x 10(6) sites/cell for U937 cells (kd = 2.20 +/- 0.53 x 10(-7) mol/L) and 5.39 +/- 2.80 x 10(5) sites/cell for K562 cells (kd = 1.37 +/- 0.39 x 10(-7) mol/L), Inhibition studies with unlabeled nucleotides and analogues showed that binding was approximately 85% specific and the inhibitory pattern was similar to that seen with mature platelets. The purine base adenosine resulted in little or no inhibition. These studies indicate that both human and rat hematopoietic cell lines possess intact ADP receptors and may be useful tools in future studies of the structure and function of this important platelet-activation system.
Human megakaryocytes have a CD4 molecule capable of binding human immunodeficiency virus-1
Authors: Kouri YH , Borkowsky W , Nardi M , Karpatkin S , Basch RS
Published: October 2019

Most human megakaryocytes (MGKs) express the CD4 antigen on their surface. Approximately 25% have a CD4 receptor density comparable to that of CD4+ T cells (Basch et al, Proc Natl Acad Sci USA 87:8085, 1990). In these studies, we show: (1) the presence of mRNA for CD4 in human MGKs; (2) the binding of human immunodeficiency virus-1 (HIV-1) to human MGKs; (3) the inhibition of binding by anti-CD4 (Leu3a) antibody or rCD4; (4) the infection of a human MGK line, CHRF-288 with HIV-1; and (5) inhibition of infection with anti-CD4. Human MGKs have mRNA for CD4 as shown by in situ hybridization with an RNA probe synthesized from a 3-kb cDNA sequence of plasmid pSP65.T4.8 containing the full-length CD4 sequence. MGKs (23% +/- 17%) bound HIV-1, as determined by anti-gp120 and anti-CD41 staining. Binding to human MGKs could be inhibited 55% to 75% with anti-CD4 or rCD4, respectively. Infection of a CD4+ MGK line (CHRF-288) could be accomplished with HIV- 1, as determined by proviral DNA polymerase chain reaction and p24 production. Preincubation with anti-CD4 inhibited apparent proviral DNA infection by 100% and p24 production by 65% to 70%. Thus, human MGKs have a CD4 receptor capable of binding HIV-1. Using this receptor, HIV- 1 can infect cells representative of the MGK lineage.
Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: potential role in gene transcription
Authors: Zon LI , Yamaguchi Y , Yee K , Albee EA , Kimura A , Bennett JC , Orkin SH , Ackerman SJ
Published: October 2019

The expression of the hematopoietic transcription factors GATA-1, GATA- 2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins.
Bone marrow transplantation with interleukin-1 plus kit-ligand ex vivo expanded bone marrow accelerates hematopoietic reconstitution in mice without the loss of stem cell lineage and proliferative potential
Authors: Muench MO , Firpo MT , Moore MA
Published: October 2019

Cytokine combinations were tested for their ability to expand murine bone marrow (BM) progenitors in short-term suspension cultures (delta- cultures) with the aim of providing an enriched source of progenitors for BM transplantation (BMT). In a comparison of the efficacy of the combinations interleukin-1 (IL-1) + IL-3, IL-1 + kit-ligand (KL), and IL-1 + IL-6 + KL, BMT with IL-1 + KL expanded progenitors was found to be most effective in accelerating the recovery of peripheral blood leukocytes, platelets, and erythrocytes in lethally irradiated mice. The ex vivo expansion of BM in IL-1 + KL-stimulated delta-cultures also greatly reduced the number of transplanted cells needed to provide radioprotection. All mice survived at least 30 days when receiving 5 x 10(3) delta-cultured d1 5-fluorouracil (5-FU) BM cells (BM cells harvested 1 day after 5-FU administration), whereas complete survival of mice receiving fresh d1 5-FU BM required BMT with a 200-fold greater number of cells. BMT with expanded BM lead to predominantly donor- derived hematopoietic reconstitution for 280 days postprimary BMT and another 71 days after secondary BMT. The expansion of BM did not adversely effect the proliferative capacity and lineage potential of the stem cell compartment.
Expression and functional role of tumor necrosis factor receptors on leukemic cells from patients with type B chronic lymphoproliferative disorders
Authors: Trentin L , Zambello R , Agostini C , Siviero F , Adami F , Marcolongo R , Raimondi R , Chisesi T , Pizzolo G , Semenzato G
Published: October 2019

Two receptors for tumor necrosis factor (TNF) with different molecular weight (75-Kd and 55-Kd) and binding affinity have been recently discovered. To investigate the distribution and the functional role of these receptors on leukemic B cells from hairy cell leukemia (HCL) and B-cell chronic lymphocytic leukemia (B-CLL) patients, we evaluated: (1) the cytofluorimetric pattern of uncultured and cultured leukemic B cells incubated with utr-1 and htr-9 monoclonal antibodies (MoAbs), which specifically recognize the 75-Kd and 55-Kd TNF receptors (TNFR), respectively; (2) the effect of TNF-alpha and TNF-beta on leukemic B cells in an in vitro proliferation assay; (3) the role of anti-TNFR MoAbs on TNF-alpha and TNF-beta-driven B-cell growth; and (4) the proliferative effect of utr-1 and htr-9 MoAbs on in vitro cultured leukemic cells. Our study shows that the high affinity (75-Kd) but not the low affinity (55-Kd) TNFR molecules are expressed on freshly isolated leukemic B cells recovered from HCL and B-CLL patients. The expression of these receptors was neither upregulated nor downregulated by different stimuli, including TNF-alpha, TNF-beta, B-cell growth factor, and interleukin-2. TNF-alpha efficiently triggers the proliferation of HC and, to a lesser extent, the growth of B-CLL cells. TNF-beta was also able to transduce the proliferative signal in HCL, but not in B-CLL patients. TNF-alpha- and TNF-beta-driven B-cell proliferation was inhibited by the preincubation of leukemic B cells with utr-1 but not htr-9 MoAb. Moreover, anti-75-Kd, but not anti-55-Kd TNFR MoAb, was able to trigger the proliferation of leukemic B cells, and in particular of HC. These results show that leukemic B cells from patients with HCL and B-CLL are equipped with a fully functional high affinity TNFR.
Dye-mediated photolysis is capable of eliminating drug-resistant (MDR+) tumor cells
Authors: Lemoli RM , Igarashi T , Knizewski M , Acaba L , Richter A , Jain A , Mitchell D , Levy J , Gulati SC
Published: October 2019

We evaluated the potential role of photoradiation therapy with a benzoporphyrin derivative, monoacid ring A (BPD-MA), and dihematoporphyrin ether (DHE), for the ex vivo purging of residual tumor cells from autologous bone marrow (BM) grafts. BPD-MA and DHE photosensitizing activity was tested against two human large-cell lymphoma cell lines and colony-forming unit-leukemia (CFU-L) derived from patients with acute myelogenous leukemia (AML). In mixing experiments, 4-log elimination of tumor cell lines was observed after 1 hour of incubation with 75 ng/mL of BPD-MA or 30 minutes of treatment with 12.5 micrograms/mL of DHE followed by white light exposure. By comparison, using the same concentration of BPD-MA, the mean recovery of normal BM progenitors was 4% +/- 0.8% (mean +/- SD) for granulocyte- macrophage colony-forming unit (CFU-GM) and 5% +/- 0.8% for burst- forming unit-erythroid (BFU-E). Similarly, DHE treatment resulted in the recovery of 5.2% +/- 2% and 9.8% +/- 3% of CFU-GM and BFU-E, respectively. Furthermore, equivalently cytotoxic concentrations of both DHE and BPD-MA and light were found not to kill normal pluripotent stem cells in BM, as demonstrated by their survival in two-step long- term marrow culture at levels equal to untreated controls. The T- lymphoblastic leukemia cell line CEM and its vinblastine (VBL)- resistant subline CEM/VBL, along with the acute promyelocyte leukemia cell line HL-60 and its vincristine (VCR)-resistant subline HL-60/VCR, were also tested. BPD-MA at 75 ng/mL was able to provide a greater than 4-log elimination of the drug-sensitive cell lines, but only a 34% and 55% decrease of the drug-resistant HL-60/VCR and CEM/VBL cell lines, respectively. On the contrary, 12.5 micrograms/mL of DHE reduced the clonogenic growth of all the cell lines by more than 4 logs. Further experiments demonstrated decreased uptake of both BPD-MA and DHE by the resistant cell lines. However, all the cell lines took up more DHE than BPD-MA under similar experimental conditions. Our results demonstrate the preferential cytotoxicity of BPD-MA and DHE toward neoplastic cell lines and CFU-L from AML patients. In addition, DHE was slightly more effective in purging tumor cells expressing the p-170 glycoprotein. These results suggest that photoradiation with DHE would be useful for in vitro purging of residual drug-resistant leukemia and lymphoma cells.
CD34 expression is regulated reciprocally with adhesion molecules in vascular endothelial cells in vitro
Authors: Delia D , Lampugnani MG , Resnati M , Dejana E , Aiello A , Fontanella E , Soligo D , Pierotti MA , Greaves MF
Published: October 2019

Freshly cultured vascular endothelial cells express the CD34 antigen in a diffuse cell surface pattern with some concentration on microvilli. Expression is downregulated with proliferation in continuous culture and undetectable after nine population doublings but can be maintained by restraining cell proliferation and promoting cell contact. Expression of CD34 at the antigen and mRNA levels on early passage cells is rapidly downregulated by interleukin-1 beta (IL-1 beta), interferon-gamma (INF-gamma), and tumor necrosis factor-alpha (TNF- alpha) under conditions in which these ligands upregulate the adhesion molecules: endothelial leukocyte adhesion molecule 1 (ELAM-1) and intracellular adhesion molecule 1 (ICAM-1). This reciprocal pattern of expression and the topographic distribution of CD34 molecules on the lumenal interdigitated microprocesses of adjacent endothelial cells in vivo suggest that CD34 might have a negative modulating role on adhesion functions of endothelia.
Downregulation of GATA-1 expression during phorbol myristate acetate- induced megakaryocytic differentiation of human erythroleukemia cells
Authors: Dai W , Murphy MJ Jr
Published: October 2019

Phorbol myristate acetate (PMA) induces the expression of megakaryocyte and/or platelet proteins during terminal differentiation of human erythroleukemia (HEL) cells. However, it is not established whether megakaryocytic differentiation is accompanied by the downregulation of the major erythroid transcription factor GATA-1 and the concomitant loss of the erythrocytic phenotype. Studies of the molecular mechanism of PMA-induced differentiation in HEL cells showed that when HEL cells are treated with PMA, they dramatically decrease the expression of the erythroid-specific gene glycophorin A at the mRNA level but apparently not at the steady-state protein level. In addition, a gel mobility shift assay was used to demonstrate that GATA-1, a major erythroid transcription factor normally present at high levels in HEL cells is downregulated after treatment with PMA. In contrast, the DNA-binding activities of transcription factors AP-1 and SP-1 are upregulated by PMA treatment of HEL cells. Furthermore, Northern blot analysis shows that PMA also downregulates the steady-state level of GATA-1 mRNA in HEL cells. The coordinated negative regulation of glycophorin A mRNA and GATA-1 expression after PMA treatment suggests that downregulation of GATA-1 expression may be partially responsible for the loss of the erythroid phenotype during megakaryocytic differentiation. The reported data also suggest that GATA-1 activity may not be essential for obtaining megakaryocytic phenotype during terminal differentiation in HEL cells.