bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of residual leukemia by the polymerase chain reaction and sequence-specific oligonucleotide probe hybridization after allogeneic bone marrow transplantation for AKR leukemia: a murine model for minimal residual disease
Authors: Drobyski WR , Baxter-Lowe LA , Truitt RL
Published: October 2019

Disease relapse after allogeneic bone marrow transplantation (BMT) is a major cause of treatment failure and is thought to evolve from clinically occult residual disease in the recipient. However, the demonstration of minimal residual disease (MRD) in individual patients is of uncertain prognostic significance because the detection of residual disease has not consistently correlated with subsequent relapse. Moreover, the optimal therapeutic approach in patients with MRD after allogeneic BMT is unknown. The study of these issues has been hindered by the lack of clinically relevant animal models. In this report, we characterize a novel murine model for the study of MRD after allogeneic BMT. This model was designed to simulate high-risk BMT in humans in which patients receive transplants in relapse and disease recurrence in the major cause of treatment failure. The H-2-compatible, mixed lymphocyte culture nonreactive murine strains, AKR (H-2k) and CBA (H-2k), were chosen to parallel marrow transplants from HLA-matched siblings, which represent the majority of allo-transplants in humans. Male AKR leukemia cells were used in female donor/host chimeras permitting the Y chromosome to serve as a leukemia-specific marker for MRD. Detection of residual male leukemia cells in the peripheral blood of the primary host was facilitated by use of the polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe hybridization (SSOPH). Use of PCR/SSOPH was highly predictive of clinical outcome (relapse or cure) in animals receiving transplants (P < .00002) and detected disease recurrence earlier than comparative flow cytometric analysis studies. This murine model will be useful in evaluating the efficacy of therapeutic strategies aimed at reducing disease relapse posttransplant and can be adapted to other transplant murine tumor systems for the study of MRD.
In vivo and in vitro regulation of erythropoietin mRNA: measurement by competitive polymerase chain reaction
Authors: Fandrey J , Bunn HF
Published: October 2019

The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.
Human interleukin-4 inhibits proliferation of megakaryocyte progenitor cells in culture
Authors: Sonoda Y , Kuzuyama Y , Tanaka S , Yokota S , Maekawa T , Clark SC , Abe T
Published: October 2019

We studied the effects of recombinant human interleukin-4 (rhIL-4) on megakaryocyte colony formation from enriched hematopoietic progenitors. IL-4 strongly inhibited pure and mixed megakaryocyte colony formation in a dose-dependent manner. Formation of erythroid bursts, eosinophil colonies, and erythrocyte-containing mixed colonies was not affected by the addition of IL-4 as reported previously (Sonoda Y, et al; Blood 75:1615, 1990). Delayed addition experiments suggested that IL-4 acts on an early stage of proliferation of megakaryocyte progenitors. Neutralizing antibodies (antisera) prepared against transforming growth factor beta, tumor necrosis factor alpha, interferon alpha (IFN alpha), and IFN gamma did not affect the inhibitory effects of IL-4 on pure and mixed megakaryocyte colony formation. In addition, the inhibitory effects of IL-4 was also seen in serum-free cultures and in cultures containing highly enriched CD34+, HLA-DR+ cells as a target population. These results indicate that IL-4 may function as one of the negative regulators in human megakaryocytopoiesis in vitro.
Basic fibroblast growth factor expression in human bone marrow and peripheral blood cells
Authors: Brunner G , Nguyen H , Gabrilove J , Rifkin DB , Wilson EL
Published: October 2019

We have shown previously that basic fibroblast growth factor (bFGF) is a mitogen for human bone marrow (BM) stromal cells and that bFGF stimulates myelopoiesis in primary BM cultures. In this article, we demonstrate the presence of bFGF in two cell lineages in human BM and peripheral blood as well as the deposition of bFGF into the extracellular matrix of BM stromal cell cultures. In immunofluorescence experiments on BM and peripheral blood smears, megakaryocytes and platelets stained strongly for bFGF, whereas weaker staining was observed in immature and mature cells of the granulocyte series. The presence of bFGF in platelets was confirmed by enzyme-linked immunosorbent assay as well as by immunoprecipitation followed by immunoblotting. bFGF was synthesized by BM stromal cell cultures and was found either cell associated or localized in the nucleus and the nucleoli, and its location was dependent on the fixation procedure used. Addition of exogenous bFGF to stromal cells showed the presence of extracellular binding molecules for this cytokine. bFGF could be released from these sites by soluble heparin or phosphatidylinositol- specific phospholipase C. This study supports the role of bFGF as a stromal cell mitogen and stimulator of myelopoiesis. The data indicate that the stromal cells produce bFGF and that their extracellular matrix can serve as a reservoir for this growth factor. In addition, the results suggest a possible involvement of bFGF in platelet function as well as in megakaryocytopoiesis.
Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin- specific antibody fused to single-chain urokinase
Authors: Holvoet P , Laroche Y , Stassen JM , Lijnen HR , Van Hoef B , De Cock F , Van Houtven A , Gansemans Y , Matthyssens G , Collen D
Published: October 2019

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single- chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady- state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody- targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.
Interferon-alpha alters spectrin organization in normal and leukemic human B lymphocytes
Authors: Evans SS , Wang WC , Gregorio CC , Han T , Repasky EA
Published: October 2019

Interferon-alpha (IFN-alpha) regulates the growth, differentiation, and recirculation of normal and malignant B lymphocytes. In this report we examine the effects of IFN-alpha on the distribution of the cytoskeletal protein spectrin in peripheral blood B lymphocytes from normal donors and patients diagnosed with chronic lymphocytic leukemia (CLL) and hairy cell leukemia (HCL). Exposure of normal and leukemic B cells to IFN-alpha in vitro was shown by immunofluorescence microscopy to cause a dose-dependent increase in the percentage of cells containing discrete focal accumulations of spectrin, ie, a single large aggregate or cap-like structure near the plasma membrane. Although the magnitude of this effect was variable among individual patient samples, in some experiments IFN-alpha induced a fourfold increase in the percentage of leukemic B cells exhibiting focal accumulations of spectrin. Spectrin reorganization induced by IFN-alpha was abrogated by the protein synthesis inhibitor cycloheximide. In addition, IFN-alpha increased the total cellular content of spectrin in B-CLL cells by approximately twofold to fourfold. Finally, a role for protein kinase C in mediating the effects of IFN-alpha on spectrin's organization is implicated by studies in which calphostin C inhibited the IFN-induced focal accumulation of spectrin. Taken together, these studies suggest that the immunomodulatory activities of IFN-alpha in normal and malignant B cells involve a change in the organization of the spectrin- based cytoskeleton.
Expression of syndecan regulates human myeloma plasma cell adhesion to type I collagen
Authors: Ridley RC , Xiao H , Hata H , Woodliff J , Epstein J , Sanderson RD
Published: October 2019

The syndecans comprise a family of integral membrane proteoglycans that regulate cell behaviors by binding to extracellular matrix and binding growth factors. In mouse blood cells, syndecan expression is restricted to cells of the B-cell lineage where it is expressed by pre-B cells and plasma cells, but is absent from circulating B cells. In the present study, we examined the expression, structure, and function of syndecan on human myeloma cell lines and myeloma patient bone marrow cells. On myeloma cells, syndecan is a small (modal relative molecular mass [M(r)] = 120 Kd) heparan sulfate proteoglycan localized at the cell surface. Syndecan was detected by immunodot blotting on 7 of 10 human myeloma cell lines and by reverse transcriptase polymerase chain reaction on 10 of 14 patient samples. Cell binding assays show that myeloma cells expressing syndecan bind to type I collagen via heparan sulfate chains, while those cell lines not expressing syndecan do not bind to collagen. Furthermore, the cell lines expressing syndecan were negative for CD19 and CD45 staining, indicating that syndecan expression is restricted to tumors having a well-differentiated phenotype. We conclude that syndecan acts as a matrix receptor on human myeloma cells but is not expressed by all tumors, suggesting that syndecan may participate in regulating myeloma cell adhesion to the bone marrow stromal matrix.
Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow
Authors: Leemhuis T , Leibowitz D , Cox G , Silver R , Srour EF , Tricot G , Hoffman R
Published: October 2019

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.
Analysis of mutant NADH-cytochrome b5 reductase: apparent "type III" methemoglobinemia can be explained as type I with an unstable reductase
Authors: Nagai T , Shirabe K , Yubisui T , Takeshita M
Published: October 2019

A patient in Kurobe, Japan, was previously reported to have a new class of hereditary methemoglobinemia, type III. In this patient, NADH cytochrome b5 reductase deficiency was observed in lymphocytes and platelets as well as in erythrocytes, but this was not associated with mental retardation. A base change was identified in the gene for NADH cytochrome b5 reductase, causing an amino acid substitution from Leu- 148 to Pro. In the present study, the mutant enzyme (Leu-148-->Pro) was expressed in Escherichia coli, purified, and characterized. The mutant enzyme retained about 60% of the catalytic activity of the wild type, but was remarkably heat unstable. By incubating the mutant enzyme at 42 degrees C for 10 minutes, 80% of the enzyme activity was lost, whereas the wild-type enzyme lost < 20% activity after incubation at 50 degrees C for 30 minutes. Another mutant in which Leu-148 was replaced by Ala was prepared to establish the role of the residue. This mutant was apparently less heat stable than the wild type, implying a structural role for Leu-148. Reinvestigation of the enzyme activity in the blood cells and fibroblasts of the type III Kurobe patient, revealed that about 40% of the normal activity was detected in these cells, in contrast to the previous report. Thus, this patient reported previously as having hereditary meth-hemoglobinemia type III was shown to have type I.
Human erythrocyte acetylcholinesterase bears the Yta blood group antigen and is reduced or absent in the Yt(a-b-) phenotype
Authors: Rao N , Whitsett CF , Oxendine SM , Telen MJ
Published: October 2019

The Cartwright (Yt) blood group antigens have previously been shown likely to reside on a phosphatidylinositol-linked erythrocyte membrane protein. In this study, an unusual individual whose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along with normal Yt(a+) cells, to investigate serologically and biochemically the relationship of the Yta antigen to known phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-) RBCs expressed normal amounts of various phosphatidyl-inositol-linked proteins except acetylcholinesterase. Further, human anti-Yta reacted with acetylcholinesterase in immunoprecipitation and immunoblotting studies. Thus, acetylcholinesterase is now identified as the protein bearing the Yt blood group antigens.
Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis
Authors: Simsek S , Faber NM , Bleeker PM , Vlekke AB , Huiskes E , Goldschmeding R , von dem Borne AE
Published: October 2019

Platelets from 200 random Dutch blood donors were typed for the human platelet alloantigens HPA-1 to -5 recognized at present and for Naka. Naka is an epitope on glycoprotein IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64% for HPA-1a and -1b, 100% and 13.15% for HPA-2a and -2b, 80.95% and 69.84% for HPA-3a and -3b, 100% and 0% for HPA-4a and -4b, 100% and 19.7% for HPA-5a and HPA-5b, respectively. Platelets from all donors reacted with the anti-Naka antibodies. To determine the gene frequencies for the HPA-1, HPA-2 and HPA-3 systems directly, DNA from 98 of these donors was isolated from peripheral blood mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments were analyzed using allele-specific restriction enzymes (ASRA). In all amplified PCR products an “internal control” for each assay, ie, a restriction site for the applied enzyme independent from the phenotype of the donor was present. In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the ASRA, were identical. Thus, the PCR-ASRA described in this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.
Quantitative assessment of posttransplant host-specific interleukin-2- secreting T-helper cell precursors in patients with and without acute graft-versus-host disease after allogeneic HLA-identical sibling bone marrow transplantation
Authors: Nierle T , Bunjes D , Arnold R , Heimpel H , Theobald M
Published: October 2019

Recent studies in mice and humans have emphasized an important contribution of host-reactive minor histocompatibility antigen (mH)- specific lymphokine-secreting donor T-helper cells (Th) for the induction of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). By using limiting dilution (LD) and clonal specificity analyses, we investigated in 14 patients with and without acute GVHD after non-T-depleted HLA-identical sibling BMT whether posttransplant host-reactive mH-specific interleukin-2 (IL-2)- secreting Th are involved in the development of clinically significant acute GVHD and the establishment of tolerance. At different time intervals posttransplant (I, days 0 through 45; II, days 45 through 90; III, days 90 through 180), host-specific IL-2-secreting Th-precursors (Th-p) were quantitatively assessed in six patients during clinically apparent grade II-III acute GVHD. Frequencies of responding Th-p ranged from 1/13,000 to 1 4,000. The presence of host-specific Th-p was significantly correlated with the development of grade II-III acute GVHD (P = .0003 by Fisher's exact test). The detectability of host- specific Th-p preceded the clinical onset of grade II-III acute GVHD. Host-specific Th-p were no longer detectable after the clinical resolution of grade II-III acute GVHD. No subsequent chronic GVHD was observed in these patients. However, prolonged occurrence of host- specific Th-p was accompanied by clinically persisting acute GVHD and the onset of secondary chronic GVHD. In patients with no acute GVHD (grade 0) (n = 7) and grade I (n = 1) acute GVHD, host-specific Th-p were not detectable at all. We conclude that host-reactive Th are critically involved in the development and maintenance of acute GVHD and may contribute to the establishment of tolerance after genotypically HLA-identical sibling BMT.
Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I (HTLV- I) tax and HTLV-II tax proteins
Authors: Ejima E , Rosenblatt JD , Massari M , Quan E , Stephens D , Rosen CA , Prager D
Published: October 2019

The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone- related protein (PTHrP) promoter is shown to be responsive to both HTLV- I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts. A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat (LTR). Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter. Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia.
Granulocyte colony-stimulating factor crosses the placenta and stimulates fetal rat granulopoiesis
Authors: Medlock ES , Kaplan DL , Cecchini M , Ulich TR , del Castillo J , Andresen J
Published: October 2019

We studied the effect of recombinant human granulocyte colony- stimulating factor (rhG-CSF) administration to pregnant rats upon fetal and neonatal myelopoiesis. Pregnant rats were treated with rhG-CSF twice daily for 2, 4, and 6 days before parturition. rhG-CSF crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. Peak fetal serum levels were 1,000-fold lower than levels detected in the dam. Hematopoietic effects of rhG-CSF were assessed by cytologic analysis of the newborn blood, spleen, bone marrow, thymus, and liver. White blood cell counts were increased twofold to fourfold in newborns. This increase was due to circulating numbers of polymorphonuclear cells (PMN). rhG-CSF induced a myeloid hyperplasia in the newborn marrow consisting of immature and mature myeloid cells in the day-2 and day-4 treated pups. Bone marrow of pups treated for 6 days contained mostly hyper-segmented PMN with little or no increase in myeloid precursors. An increase in the number of postmitotic (PMN, bands, and metamyelocytes) and mitotic (promyeloblasts, myeloblasts, and metamyeloblasts) myeloid cells in the spleen of neonates was observed. No change was detected in splenic lymphocytes or monocytes. No effect of rhG-CSF was noted in the newborn liver or thymus. These results demonstrate that maternally administered rhG-CSF crosses the placenta and specifically induces bone marrow and spleen myelopoiesis in the fetus and neonate. The significant myelopoietic effects of rhG-CSF at low concentrations in the fetus suggest an exquisite degree of developmental sensitivity to this cytokine and may provide enhanced defense mechanisms to the neonate.
Increased expression of platelet IgG Fc receptors in immune heparin- induced thrombocytopenia
Authors: Chong BH , Pilgrim RL , Cooley MA , Chesterman CN
Published: October 2019

Our previous finding that heparin-dependent antibodies in heparin- induced thrombocytopenia (HIT) bind to platelets via platelet IgG Fc receptors (FcRs) prompted this study. Platelet FcRs in 16 patients with HIT, 23 control patients, and 42 normal subjects were studied. Patients with HIT had substantially increased platelet FcRs during the acute illness. Those who suffered serious thrombotic complications or died shortly after diagnosis had significantly more FcRs per platelet than those with milder disease. Consistent with their increased FcRs, platelets of patients with HIT showed increased aggregation reactivity to aggregated IgG and heparin-dependent antibodies. Platelet FcRs in patients with HIT remained elevated for 1 to 3 months after the acute illness then stabilized to a mean value not significantly different from either control group. The increased expression of FcRs on HIT platelets and their increased reactivity to heparin-dependent antibodies may contribute to the pathogenesis of thrombocytopenia and thrombosis in HIT.
Defective signal transduction induced by thromboxane A2 in a patient with a mild bleeding disorder: impaired phospholipase C activation despite normal phospholipase A2 activation
Authors: Fuse I , Mito M , Hattori A , Higuchi W , Shibata A , Ushikubi F , Okuma M , Yahata K
Published: October 2019

A patient with a mild bleeding disorder whose platelets responded defectively to thromboxane A2 (TXA2) was identified, and the mechanism of this dysfunction was analyzed. The platelets were defective in shape change, aggregation, and release reaction in response to synthetic TXA2 mimetic (STA2). When the platelet TXA2 receptor was examined with both a 125I-labeled derivative of a TXA2 receptor antagonist ([125I]-PTAOH) and [3H]-labeled TXA2 agonist ([3H]U-46619), the equilibrium dissociation rate constants (kd) and the maximal concentrations of binding sites (Bmax) of the platelets to both ligands were within normal ranges, suggesting that the binding capacity of their TXA2 receptor was normal. STA2 could not induce IP3 formation and intracellular Ca2+ mobilization, whereas these responses to thrombin were within normal ranges. GTPase activity was also decreased when the patient's platelet membrane was challenged with STA2. On the other hand, lysophosphatidylinositol formation, which is a direct indicator of phospholipase A2 (PLA2) activation, was found to be normal when the [3H]-inositol-labeled platelets were challenged with STA2. Thromboxane B2 (TXB2) was also produced in response to STA2. These results suggested that the abnormality in these platelets was impaired coupling between TXA2 receptor and phospholipase C (PLC) activation. Furthermore, it is also suggested that the activation of PLA2 and PLC are separable events in thromboxane-induced platelet activation.
A serine/proline-rich protein is fused to HRX in t(4;11) acute leukemias
Authors: Morrissey J , Tkachuk DC , Milatovich A , Francke U , Link M , Cleary ML
Published: October 2019

Translocations involving chromosome band 11q23 in acute leukemias have recently been shown to involve the HRX gene that codes for a protein with significant similarity to Drosophila trithorax. HRX gene alterations are consistently observed in t(4;11) (q21;q23)-carrying leukemias and cell lines by Southern blot analyses and are accompanied by HRX transcripts of anomalous size on Northern blots. HRX-homologous cDNAs were isolated from a library prepared from t(4;11)-carrying acute leukemia cells. cDNAs representative of transcription products from the derivative 11 chromosome were shown to contain HRX sequences fused to sequences derived from chromosome band 4q21. Fragments of the latter were used to clone and analyze cDNAs for wild-type 4q21 transcripts that predicted a 140-Kd basic protein (named FEL) that is rich in prolines, serines, and charged amino acids. FEL contains guanosine triphosphate-binding and nuclear localization consensus sequences and uses one of two possible 5' exons encoding the first 12 or 5 amino acids. After t(4;11) translocations, 913 C-terminal amino acids of FEL are fused in frame to the N-terminal portion of HRX containing its minor groove DNA binding motifs. These features are similar to predicted t(11;19) fusion proteins, suggesting that HRX consistently contributes a novel DNA-binding motif to at least two different chimeric proteins in acute leukemias.
Treatment of myelodysplastic syndromes with all-trans retinoic acid. Leukemia Study Group of the Ministry of Health and Welfare
Authors: Ohno R , Naoe T , Hirano M , Kobayashi M , Hirai H , Tubaki K , Oh H
Published: October 2019

We treated 23 patients with myelodysplastic syndromes (MDS); 2 refractory anemia (RA) with prior therapy, 11 RA with excess of blasts (RAEB), and 10 RAEB in transformation (RAEB-T), with daily oral 45 mg/m2 all-trans retinoic acid (ATRA) in a multiinstitutional prospective study. In two patients with RAEB and one with RAEB-T, a more than 1,000/microL increase of peripheral neutrophil counts was observed with some reduction of blast percentage in the bone marrow 2 to 9 weeks after the start of ATRA. However, the effect was transient and did not last for more than 5 weeks despite the continuation of ATRA therapy. In one other patient with RA, one patient with RAEB, and one patient with RAEB-T, slight increase of hemoglobin levels or reduction of blast percentage in bone marrow was noted. Toxicities attributable to ATRA were minimal and included cheilitis, xerosis, dermatitis, gastrointestinal disorders, abnormal liver function tests, and high serum triglyceridemia. Although ATRA works remarkably as a differentiation therapy in acute promyelocytic leukemia, its effect in MDS included in this study was modest. Further study of this agent alone or in combination may be warranted in less advanced stages of this disease.
Zinc protoporphyrin in anemia of chronic disorders
Authors: Hastka J , Lasserre JJ , Schwarzbeck A , Strauch M , Hehlmann R
Published: October 2019

Hematofluorometric determination of zinc protoporphyrin (ZPP) is a screening method for the assessment of iron deficiency (ID). Chronic disorders are frequently accompanied by anemias of unclear origin, most probably caused by an impairment of iron metabolism. We investigated the relevance of ZPP for the detection of derangements of iron metabolism in anemias of chronic disorders (ACD). In 19 patients with ACD caused by chronic inflammatory non-neoplastic diseases, ZPP was determined and correlated with ferritin, transferrin saturation, and hemoglobin (Hb). Marrow sideroblast counts and semiquantitative grading of the marrow hemosiderin were performed in all patients to exclude ID and to show the decreased iron bioavailability. In all ACD patients who exhibited the typical laboratory findings of disturbed iron metabolism, such as hypoferremia, decreased transferrin saturation, decreased bone marrow sideroblasts, and increased marrow hemosiderin, strongly elevated ZPP levels were found (131 +/- 23 mumol/mol heme). ZPP returned to normal after successful treatment of the underlying disease. This is shown in three patients with polymyalgia rheumatica. We conclude that the fluorometric determination of ZPP allows detection and quantification of derangements of iron metabolism associated with chronic inflammatory disorders. By recording the derangements quantitatively, ZPP allows monitoring of therapy of chronic inflammatory diseases.
Autoantibodies against platelet glycoproteins in autoimmune thrombocytopenic purpura: their clinical significance and response to treatment
Authors: Berchtold P , Wenger M
Published: October 2019

Autoantibodies against platelet glycoproteins (GP) have been demonstrated in patients with autoimmune thrombocytopenic purpura (ATP). However, their clinical and pathogenetic significance as well as their response to immunosuppressive treatment is unknown. Using an immunobead assay capable of measuring autoantibodies against GPIIb-IIIa and GPIb-IX, we studied 58 adult patients with active ATP (platelet count < 150 x 10(9)/L) and 26 patients with ATP in remission (platelet count > 150 x 10(9)/L and without any therapy at time of investigation). Platelet-associated autoantibodies were detected in 39 of 53 patients with active ATP (73.6%) and in 2 of 26 patients in remission (7.7%). Circulating plasma autoantibodies were noted in 17 of 58 patients in the group with active disease (29.3%) and in none of the patients in remission. Twelve patients with active ATP and autoantibodies against GPIIb-IIIa were studied prospectively during treatment with corticosteroids. Of eight patients whose platelet count normalized during treatment, platelet-associated and plasma antibodies decreased significantly in two or became undetectable in six. In contrast, of four patients whose platelet counts were unchanged or increased moderately, we noted no significant change in antibodies. Moreover, autoantibodies reappeared in two responding patients at the time of relapse. The effect of high-dose intravenous immunoglobulin was studied in six active ATP patients with antiglycoprotein autoantibodies and refractoriness to prednisone. In one patient who developed a sustained remission after IvIgG, platelet-associated and plasma antibodies to GPIIb-IIIa decreased and became undetectable. In contrast, two patients who had only a transient rise of the platelet count after IvIgG showed no significant change in autoantibody. In three unresponsive patients, autoantibodies were without change in two and decreased transiently in one patient. We conclude that in ATP the presence of autoantibodies to GPIIb-IIIa and GPIb-IX is related to the activity of the disease. Corticosteroids may inhibit autoantibody formation in some ATP patients, whereas during the early response to IvIgG, autoantibody production may not be affected.
Kringle glycosylation in a modified human tissue plasminogen activator improves functional properties
Authors: Berg DT , Burck PJ , Berg DH , Grinnell BW
Published: October 2019

Native tissue plasminogen activator (ntPA) has a variable glycosylation site on its kringle-2 domain. We have examined the effects of kringle glycosylation on functional properties by studying the simplified tPA molecule, tPA-6. tPA-6 is composed of kringle-2 and the serine protease domains and, like ntPA, cells expressing tPA-6 process it into two glycoforms: the monoglycosylated tPA-6-primary (tPA-6P, type II) with N- linked glycosylation at Asn-448 in the serine protease domain and diglycosylated tPA-6-variant (tPA-6V, type I) with glycosylation at Asn- 448 and at Asn-184 in kringle-2. When the two glycoforms were separated, we found that purified tPA-6V had reduced fibrin-stimulated plasminogenolytic activity toward Glu-plasminogen when compared to purified tPA-6P. However, in the presence of fibrin, tPA-6V unexpectedly exhibited a sixfold increase in selectivity toward Lys- plasminogen. In addition, tPA-6V was less susceptible than tPA-6P to plasmin-mediated conversion to the two-chain form. By site-directed mutagenesis of tPA-6, we eliminated variable glycosylation at Asn-184 and engineered a new glycosylation signal at a remnant site in the kringle. This derivative, designated tPA-6D, was secreted with complete kringle glycosylation. Like the naturally occurring tPA-6V, tPA-6D had lower rates of fibrin-stimulated Glu-plasminogen activation, increased specificity toward Lys-plasminogen, and greater resistance to plasmin digestion. Although the activity of tPA-6D could be stimulated by fibrin, its activity was not stimulated significantly by fibrinogen, and in human plasma the rate of fibrinogen depletion was reduced threefold. Although fibrin binding to kringle-2 of tPA-6D was slightly improved, there was a substantial increase in the dissociation constant (kd) for lysine binding, demonstrating a lack of correlation between these ligand-binding sites. Overall, our data demonstrate the marked effect of kringle glycosylation on functional properties. In addition, we have generated a derivative with properties that potentially improve clot specificity and single-chain half-life and reduce the potential for plasminogen activation in the plasma.
Type II interleukin-1 receptor is not expressed in cultured endothelial cells and is not involved in endothelial cell activation
Authors: Colotta F , Sironi M , Borre A , Pollicino T , Bernasconi S , Boraschi D , Mantovani A
Published: October 2019

Interleukin-1 (IL-1) profoundly affects a number of functions of endothelial cells (EC). It was previously shown that EC express the type I 80-Kd IL-1 receptor (IL-1 RI). In this study we define the expression and functional significance of the type II IL-1R (IL-1 RII) in EC. Human umbilical vein EC did not express appreciable levels of IL- 1 RII mRNA as assessed by Northern analysis or reverse transcription and polymerase chain reaction. Exposure to various cytokines (including IL-4, which augments IL-1 RII in neutrophils) failed to induce IL-1 RII mRNA. The binding of radiolabeled IL-1 beta to EC was blocked by antitype I (M4) but not by antitype II (M22) monoclonal antibodies (MoAbs). MoAbs directed against the IL-1 RI (M1 and M4) inhibited the induction of IL-6 and adhesion molecules in EC by IL-1, whereas an anti- IL-1 RII (M22) was inactive. The human IL-1 receptor antagonist (IL- 1ra) preferentially interacts with IL-1 RI versus IL-1 RII in the mouse. IL-1 ra inhibited the response of mouse endothelial cells to IL- 1. We conclude that EC selectively express the IL-1 RI and that this is involved in the response of this cell type to IL-1.
Natural interferon-alpha in combination with melphalan/prednisone versus melphalan/prednisone in the treatment of multiple myeloma stages II and III: a randomized study from the Myeloma Group of Central Sweden
Authors: Osterborg A , Bjorkholm M , Bjoreman M , Brenning G , Carlson K , Celsing F , Gahrton G , Grimfors G , Gyllenhammar H , Hast R
Published: October 2019

Three hundred thirty-five previously untreated patients with multiple myeloma in clinical stages II and III entered a randomized trial comparing intermittent oral melphalan and prednisone (MP) therapy (n = 171) with MP in combination with natural (leukocyte-derived) alpha- interferon (MP/IFN) (n = 164). The treatment groups were comparable with regard to major prognostic factors. The response frequency was 42% in the MP group and 68% in the MP/IFN group (P < .0001). Eighty-five percent of IgA myelomas and 71% of Bence-Jones myelomas responded to MP/IFN compared with 48% and 27%, respectively, to MP treatment (P = .001). There was no difference in the overall survival between the two treatment groups. However, the survival of 72 patients with IgA or Bence-Jones myeloma randomized to receive MP/IFN was significantly longer (median 32 months) than that of 71 patients treated with MP (median 17 months) (p < .05). No statistically significant difference in response frequency (60% v 46%) or survival was found for patients with IgG myeloma. Hematologic toxicity, WHO grades III and IV, was higher in the MP/IFN group (48%) than in the MP group (33%) (P <.05) during the induction treatment period. Flulike syndrome was observed in 68% of patients receiving MP/IFN. The results show that MP/IFN is a well-tolerated treatment regimen, superior to MP for remission induction, and it improves significantly the overall survival for patients with IgA and Bence-Jones myelomas.
Cascade transactivation of growth factor receptors in early human hematopoiesis
Authors: Testa U , Pelosi E , Gabbianelli M , Fossati C , Campisi S , Isacchi G , Peschle C
Published: October 2019

Highly purified progenitors (including erythroid [BFU-E], granulo- monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony- stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross- reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down- modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the “distal” GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR (“pure” progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase- polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the “proximal” HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL- 6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain- potentiation mechanism.
Granulocyte colony-stimulating factor induces hFc gamma RI (CD64 antigen)-positive neutrophils via an effect on myeloid precursor cells
Authors: Kerst JM , van de Winkel JG , Evans AH , de Haas M , Slaper-Cortenbach IC , de Wit TP , von dem Borne AE , van der Schoot CE , van Oers RH
Published: October 2019

In this study we have examined hFc gamma RI expression during myelopoiesis. Normal bone marrow (BM) cells were found to express hFc gamma RI up to the metamyelocyte stage. A different Fc gamma RI expression pattern was observed in an in vitro model of myelopoiesis. Purified CD34-positive BM cells, cultured for 12 to 14 days with granulocyte colony-stimulating factor (G-CSF), differentiate into a population of mature granulocytic cells. In these cultures, in which hFc gamma RI was virtually absent on the initial CD34-positive BM cells, hFc gamma RI was strongly induced by G-CSF after only 5 days. During final maturation the cells remained hFc gamma RI positive. This expression was confirmed functionally by antibody-sensitized erythrocytes (EA)-rosette assays. Moreover, the mature myeloid cells were found to express mRNA encoding for hFc gamma RI, whereas reverse- transcriptase polymerase chain reaction analysis showed that both hFc gamma RIA and hFc gamma RIB genes were expressed. In contrast, on peripheral blood (PB) polymorphonuclear neutrophil leukocytes (PMN) the in vitro effect of G-CSF as to hFc gamma RI induction was limited. Therefore, we conclude that, with respect to hFc gamma RI expression on PMN, G-CSF acts on myeloid precursor cells rather than on mature cells. This conclusion could be strengthened by in vivo administration of a single dose of G-CSF to a healthy volunteer. After a 12-hour lag time, hFc gamma RI expressing PMNs were detected in the peripheral blood. This study shows that hFc gamma RI is an early myeloid differentiation marker that is lost during normal final maturation. However, committed myeloid progenitor cells can be strongly induced by G-CSF to express hFc gamma RI, ultimately resulting in mature granulocytic cells expressing the high-affinity receptor for IgG. This expression may have important consequences for the functional capacity of these cells.
Preclinical analysis of cytokine therapy in the SCID-hu mouse
Authors: Kyoizumi S , Murray LJ , Namikawa R
Published: October 2019

A severe combined immunodeficient (SCID)-hu mouse model implanted with human fetal bone was used to assess the effects of various recombinant human (rh) hematopoietic growth factors, administered either alone or in combination, on human hematopoiesis in vivo. Treatment with rh granulocyte colony-stimulating factor (G-CSF) elicited the expansion of mature neutrophilic granulocyte populations in human marrow. Administration of rh interleukin-3 (IL-3) induced significant increases of eosinophilic granulocyte and burst-forming unit, erythrocyte (BFU-E) activity. The rhIL-6 did not cause significant changes in the subpopulations of human hematopoietic cells within the grafts, but did increase the number of colony-forming unit, granulocyte-macrophage and BFU-E. Pretreatment with rhIL-3 followed by rh erythropoietin (Epo) administration enhanced Epo-induced human erythropoiesis significantly. No synergistic effects on myelopoiesis were observed using sequential treatment with rhIL-3 followed by rhG-CSF. Instead, these factors seemed to work independently, with rhG-CSF increasing the percentage of neutrophils and rhIL-3 increasing the percentage of eosinophils. When administered simultaneously with rhEpo, rhIL-6 showed dose-dependent inhibitory effects on in vivo Epo-induced human erythropoiesis. The rhIL-6 also caused a reduction in the percentage of human neutrophils induced by rhG-CSF. These results suggest that the SCID-hu mouse provides a useful small animal model to assess the in vivo effects of hematopoietic growth factors on human hematopoiesis.
Tyrosine phosphorylation of guanosine triphosphatase activating protein by activation via surface IgG in human B cells
Authors: Yagura H , Oyaizu N , Pahwa S
Published: October 2019

In this study, we analyzed tyrosine phosphorylation of guanosine triphosphatase (GTPase) activating protein in human B cells stimulated through surface IgG, using Western blot and immunoprecipitation. Stimulation through surface IgG induced the tyrosine phosphorylation of GTPase-activating protein (GAP) and two associated proteins, a 190-Kd protein and a 62-Kd protein, within 1 minute and in a dose-dependent manner. This tyrosine phosphorylation was blocked by Genistein (Extrasynthese, Genay, France). These data suggest that GTPase- activating protein is involved in a signal transduction pathway initiated from surface IgG in human B cells.
Only the HLA class I gene minimal promoter elements are required for transactivation by human cytomegalovirus immediate early genes
Authors: Burns LJ , Waring JF , Reuter JJ , Stinski MF , Ginder GD
Published: October 2019

The immediate early (IE) genes of human cytomegalovirus (HCMV) are expressed in lymphocytes and are known to transactivate both viral and cellular promoters. The mechanism by which IE gene products of HCMV transactivate expression of the HLA A2 gene promoter in Jurkat cells, a T-lymphocyte cell line, was investigated. Transient expression assays were performed using plasmids containing the HLA A2 promoter-regulatory region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and a plasmid expressing the CMV IE genes. The upregulation of the HLA A2 promoter by HCVM IE gene products was shown not to be secondary to either interferon-gamma or -alpha. Previously described MHC class I regulatory or enhancer elements such as the interferon-stimulated response element (ISRE), NF-kappa B and H2TF1 binding sequences, and the interferon consensus sequence (ICS) were not required for transactivation of the A2 promoter. Rather, the only known regulatory elements in the HLA A2 promoter necessary for both basal expression and transactivation by HCVM IE gene products are the CCAAT box and TATA box motifs. These results support a model in which HCVM IE gene products act through the minimal HLA A2 promoter elements to increase gene expression.