bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity
Authors: Pistoia V , Zupo S , Corcione A , Roncella S , Matera L , Ghio R , Ferrarini M
Published: October 2019

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.
Amino acid sequence of a platelet-binding human anti-DNA monoclonal autoantibody
Authors: Lampman GW , Furie B , Schwartz RS , Stollar BD , Furie BC
Published: October 2019

The complete amino acid sequences of the variable regions of the heavy and light chains of a human IgM monoclonal platelet-binding autoantibody have been determined. This antibody, HF2–1/17, produced by a human x human hybridoma prepared from lymphocytes of a patient with systemic lupus erythematosus and thrombocytopenia, is polyreactive with single-stranded DNA, synthetic polynucleotides, sulfated carbohydrates, and acidic glycolipids isolated from platelet membranes. The heavy chain is of the VHIII subgroup, and the light chain is of the VKI subgroup. The heavy chain is the expression product of the VH26 germline gene. The light chain bears significant homology to other immunoglobulins of known primary structure, including WEA, GAL, HAU, HK101, and DEE. These results suggest that HF2–1/17 may be an autoantibody derived with little or no modification from germline genes. A model of the antibody combining site suggests that arginine 24 and arginine 30 in the light chain (CDR1) interact with a surface defined by phosphate or sulfate groups of the antigen.
Cross-lineage gene rearrangements in human leukemic B-precursor cells occur frequently with V-DJ rearrangements of IgH genes
Authors: Nosaka T , Kita K , Miwa H , Kawakami K , Ikeda T , Ohno T , Matsuoka N , Arita Y , Doi S , Nishikori M
Published: October 2019

Gene configurations of immunoglobulin (Ig), T-cell antigen receptor (TCR) beta chain, and T-cell rearranging gene gamma (TRG gamma) were studied in human B-precursor lymphoblastic leukemic cells. These cells were phenotypically classified into three developmental stages (stages II through IV) according to Nadler's criteria. All of them showed the Ig heavy chain (IgH) gene rearrangement, and 67% of the cells in stage IV had the rearranged TRG gamma, albeit seldom in other stages. We further analyzed IgH gene rearrangements in detail using upstream to DH (diversity region of IgH) region probe to distinguish DJ from V-DJ recombination. All dual genotyped cells in each stage except one case in stage II showed the V-DJ rearrangements. This suggests the cross- lineage involvement of the putative recombinase, particularly in the process of V-DJ rearrangements. We next examined the transcriptional status of Ig genes as an indirect reflection of the accessibility of these genes to the recombinase. Properly spliced IgH transcripts of normal size were observed in stage IV and surface Ig positive stage, but not in stage II nor III. However, IgH transcripts of aberrant sizes were seen in stage II, III, and also IV. Cross-lineage gene rearrangements were shown to occur frequently when normally spliced IgH gene begins to be transcribed or just before this. These findings may implicate that V-DJ or V-VDJ gene rearrangements forming functional IgH genes, induce frequently TCR or TRG gene rearrangements. We propose these dual genotypes are different in origin from those observed in stem cell leukemia.
Hemoglobin Koln occurring in association with a beta zero thalassemia: hematologic and functional consequences
Authors: Galacteros F , Loukopoulos D , Fessas P , Kister J , Arous N , Bohn B , Loutradi A , Tsitrakis G , Poyart C
Published: October 2019

Hemoglobin (Hb) Koln-beta zero thalassemia compound heterozygosity was discovered in a young Greek patient. This gave us the unique opportunity for studying the functional properties of this unstable high-oxygen affinity hemoglobin variant in red cells containing almost pure Hb Koln. The red cells of the proposita exhibit morphological alterations and hematologic indices corresponding to the presence of an unstable Hb and beta thalassemia. Globin chain synthesis confirmed the association with a beta zero thalassemia gene. Oxygen-binding curves for these cells were biphasic, indicating the presence of both heme- saturated and of approximately 20% of non-cooperative Hb Koln. The major component exhibits an increased oxygen affinity, reduced cooperativeness, and normal alkaline Bohr effect. The 35-year-old proposita is active, has not been splenectomized, and has not been transfused in several years.
Induction of dependence on hematopoietic proteins for viability and receptor upregulation in differentiating myeloid leukemic cells
Authors: Lotem J , Sachs L
Published: October 2019

There are different types of myeloid leukemic cells that can be induced to differentiate to mature granulocytes or macrophages by different hematopoietic regulatory proteins. One type of leukemic clone can be induced to differentiate by recombinant macrophage and granulocyte differentiation-inducing protein-type 2 (MGI-2), which we have shown is Interleukin-6 (IL-6), and another type of leukemic clone can be differentiated by recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3. There was no subpopulation of growth factor- responsive or differentiation-defective cells before induction of differentiation in either type of clone. In both clones, induction of differentiation-induced requirement for a hematopoietic protein for cell viability. Viability of the cells was maintained by IL-6, IL-3, or macrophage colony-stimulating factor (M-CSF) but not by GM-CSF in the cells differentiated by IL-6, and by GM-CSF or IL-3 but not by IL-6 or M-CSF in the cells differentiated by GM-CSF or IL-3. The viable cells with a differentiated phenotype continued to multiply. In undifferentiated leukemic cells with no or few surface receptors for some of these proteins, there was an upregulation of the number of receptors during differentiation for the proteins to which the cells responded. But there were also differentiating leukemic cells with an upregulation of GM-CSF receptors although GM-CSF could not maintain the viability of the differentiating cells. The results indicate that induction of hormone responsiveness and upregulation of the hormone receptors can both occur in differentiating leukemic cells, and that the regulation of these two events can be separated.
Theta, zeta, and epsilon globin messenger RNAs are expressed in adults
Authors: Albitar M , Peschle C , Liebhaber SA
Published: October 2019

The theta globin gene is the most recently discovered member of the alpha globin gene family. Its pattern of expression during development is not fully defined, and its encoded protein has not yet been detected in vivo. The detection of theta globin messenger RNA (mRNA) in embryonic and fetal erythroid tissue but not in adults has suggested that theta is an embryonic globin gene. The present study further defines the pattern of theta globin gene expression. We use a modification of the highly sensitive polymerase chain reaction (PCR) technique to assess the levels of theta globin gene expression during development. We confirm the presence of the theta globin mRNA in embryonic and fetal erythroid tissue, and, in addition, we find theta mRNA in the peripheral reticulocytes of normal adults. Furthermore, using the same analytic approach, we detect low but significant levels of the embryonic zeta and epsilon mRNAs in reticulocytes of normal adults. Both zeta and theta gene expression appears erythroid specific in that neither mRNA species is detected in RNA isolated from brain tissue, peripheral blood mononuclear cells, or three nonerythroid cell lines (B-lymphocyte, T-lymphocyte, and hepatoma cell lines). The relative levels of zeta and theta gene expression were assayed during development by a coamplification technique. The results demonstrate the expected developmental regulation of zeta globin mRNA. In contrast, the level of theta globin mRNA fails to demonstrate the significant changes of the magnitude seen in other globin genes and remains low in embryonic, fetal, and adult life. The lack of zeta and epsilon globin proteins in normal adults using highly sensitive immunologic techniques, as reported by others, stands in contrast to these mRNA results and suggests a gap between mRNA and protein expression.
Impaired cytoplasmic ionized calcium mobilization in inherited platelet secretion defects
Authors: Rao AK , Kowalska MA , Disa J
Published: October 2019

Defects in platelet cytoplasmic Ca++ mobilization have been postulated but not well demonstrated in patients with inherited platelet secretion defects. We describe studies in a 42-year-old white woman, referred for evaluation of easy bruising, and her 23-year-old son. In both subjects, aggregation and 14C-serotonin secretion responses in platelet-rich plasma (PRP) to adenosine diphosphate (ADP), epinephrine, platelet activating factor (PAF), arachidonic acid (AA), U46619, and ionophore A23187 were markedly impaired. Platelet ADP and adenosine triphosphate (ATP), contents and thromboxane synthesis induced by thrombin and AA were normal. In quin2-loaded platelets, the basal intracellular Ca++ concentration, [Ca++]i, was normal; however, peak [Ca++]i measured in the presence of 1 mmol/L external Ca++ was consistently diminished following activation with ADP (25 mumol/L), PAF (20 mumol/L), collagen (5 micrograms/mL), U46619 (1 mumol/L), and thrombin (0.05 to 0.5 U/mL). In aequorin-loaded platelets, the peak [Ca++]i studied following thrombin (0.05 and 0.5 U/mL) stimulation was diminished. Myosin light chain phosphorylation following thrombin (0.05 to 0.5 U/mL) stimulation was comparable with that in the normal controls, while with ADP (25 mumol/L) it was more strikingly impaired in the propositus. We provide direct evidence that at least in some patients with inherited platelet secretion defects, agonist-induced Ca++ mobilization is impaired. This may be related to defects in phospholipase C activation. These patients provide a unique opportunity to obtain new insights into Ca++ mobilization in platelets.
Transcellular sulfidopeptide leukotriene biosynthetic capacity of vascular cells
Authors: Maclouf J , Murphy RC , Henson PM
Published: October 2019

Cells in the vasculature, including polymorphonuclear leukocytes, platelets, and endothelial cells, have been shown to be jointly involved in the biosynthesis of active lipid mediators derived from arachidonic acid. Stimulation of neutrophils with the calcium ionophore A23187 as a model for cell activation results in production of leukotriene (LT)A4 with subsequent intracellular conversion into LTB4. When platelets or endothelial cells were present in the incubation system, LTC4 was produced from the neutrophil-derived LTA4. Whereas production and release of LTA4 under resting conditions in vivo might be expected to be quite low, under pathologic conditions, LTA4 production could be markedly increased. Therefore, the metabolism of exogenous LTA4 by platelets and endothelial cells was studied at a wide range of LTA4 concentrations. The production of LTC4 during coincubation of neutrophils with platelets was found to be dependent on neutrophil number ranging from 2 x 10(5) to 2 x 10(7) cells/mL. When a fixed number of neutrophils were stimulated with platelets alone or with mixtures of platelets and endothelial cells, LTC4 production was observed to be dependent on both acceptor cell types. These results suggest that mixed cell populations, which are likely to occur in vivo, may be critical determinants of the profile of eicosanoids produced in pathophysiologic circumstances. We suggest that both endothelial cells and platelets, in the presence of neutrophils, contribute large quantities of sulfidopeptide leukotrienes to inflammatory and thrombotic situations. Furthermore, platelets, because of their quantity and reactivity, may play a pivotal role in transcellular biosynthesis of eicosanoids.
Complex formation between urokinase and plasma protein C inhibitor in vitro and in vivo
Authors: Geiger M , Huber K , Wojta J , Stingl L , Espana F , Griffin JH , Binder BR
Published: October 2019

Protein C inhibitor (PCI) and plasminogen activator inhibitor 3 (PAI-3; urinary urokinase inhibitor) are immunologically identical. The role of PCI for urokinase (uPA) inhibition in vivo was investigated. We therefore developed an enzyme-linked immunosorbent assay (ELISA) specific for uPA-PCI complexes: Rabbit anti-PCI IgG was immobilized on a microtiter plate and following incubation with uPA-PCI complex- containing samples, bound uPA-PCI complexes were quantified with a horseradish-peroxidase-linked monoclonal antibody (MoAb) to uPA. Using this assay, time, dose, and heparin-dependent complexes were detected when uPA was incubated with normal plasma or purified urinary PCI, whereas no complexes were measurable using PCI-immunodepleted plasma. Plasma samples (containing 20 mmol/L benzamidine to prevent complex formation ex vivo) from patients undergoing systemic urokinase therapy (1 x 10(6) IU/60 min intravenously [IV]) after myocardial infarction were also studied. uPA present in these plasma samples (up to 1,200 ng/mL) had only 43% to 70% of the specific activity of purified 2-chain uPA, suggesting that a major portion of uPA is complexed to inhibitors. In these plasma samples uPA-PCI complexes were present in a concentration corresponding to 21% to 25% of inactive uPA antigen. These data suggest that at high uPA concentrations, such as during uPA therapy, plasma PCI might contribute significantly to uPA inhibition in vivo.
Locus assignment of two alpha-globin structural mutants from the Caribbean basin: alpha Fort de France (alpha 45 Arg) and alpha Spanish Town (alpha 27 Val)
Authors: Cash FE , Monplaisir N , Goossens M , Liebhaber SA
Published: October 2019

Two alpha-globin structural mutants were mapped to their encoding loci by in vitro translation of hybrid-selected alpha 1- and alpha 2-globin mRNA. The more highly expressed mutant, alpha Spanish Town (alpha 27Val), is encoded at the alpha 2 locus and the less expressed mutant, alpha Fort de France (alpha 45Arg), is encoded at the alpha 1 locus. These results further define the distribution of alpha-globin structural mutations within the alpha-globin gene cluster and substantiate the dominant role of the alpha 2-globin locus in alpha- globin expression.
Characterization of a spontaneous mutation in beta-thalassemia associated with advanced paternal age
Authors: Chehab FF , Winterhalter KH , Kan YW
Published: October 2019

We characterized the molecular defect in a Swiss patient with a spontaneous beta-thalassemia mutation. Cloning and DNA sequencing of her beta-globin gene revealed a new frameshift mutation due to a single nucleotide deletion at codon 64 of the beta-globin gene. Restriction site polymorphism showed that the mutation arose on her paternal chromosome. Direct sequencing of a polymerase chain reaction amplified DNA segment showed absence of the lesion in both alleles of her father's beta-globin gene and confirmed the spontaneous nature of this mutation.
Marrow transplantation for acute lymphoblastic leukemia: factors affecting relapse and survival
Authors: Barrett AJ , Horowitz MM , Gale RP , Biggs JC , Camitta BM , Dicke KA , Gluckman E , Good RA , Herzig RH , Lee MB
Published: October 2019

Transplant outcome was analyzed in 690 recipients of bone marrow transplants (BMTs) for acute lymphoblastic leukemia (ALL) in first (n = 299) or second remission (n = 391). Actuarial 5-year leukemia-free survival was 42% +/- 9% (95% confidence interval) and 26% +/- 6%, respectively; relapse rates were 29% +/- 9% and 52% +/- 8%, respectively. Five-year leukemia-free survival was 56% +/- 18% in children and 39% +/- 10% in adults (P less than .02) transplanted in first remission. In first-remission adults, non-T-cell phenotype, male to female donor-recipient sex-match and graft-v-host disease (GVHD) were associated with decreased leukemia-free survival; inclusion of corticosteroids in the regimen to prevent GVHD was associated with increased leukemia-free survival. Variables associated with decreased leukemia-free survival after second-remission transplants were age greater than or equal to 16 years and relapse occurring while on therapy. Variables associated with increased probability of relapse were similar for first- and second-remission transplants and included GVHD prophylaxis without methotrexate and absence of GVHD. In first- remission transplants, leukocyte count greater than or equal to 50 x 10(9)/L at diagnosis was also associated with increased relapse; in second remission, relapse while receiving chemotherapy was also associated with increased posttransplant relapse. These data emphasize the importance of both disease- and transplant-related variables in predicting outcome after BMT. They may be used to explain differences between studies, design future trials, and identify persons most likely to benefit from BMT.
Expression of human adenosine deaminase from various strong promoters after gene transfer into human hematopoietic cell lines
Authors: Hock RA , Miller AD , Osborne WR
Published: October 2019

Adenosine deaminase (ADA) deficiency is associated with a fatal severe combined immunodeficiency. Because most patients do not have a suitable marrow donor, the introduction of a normal ADA gene into the patient's marrow cells is a potentially useful alternative therapy. To identify vectors that provide optimal gene expression in human hematopoietic cells, we investigated retroviral vectors containing the ADA gene under the transcriptional control of the promoter/enhancers of Moloney murine leukemia virus, the simian virus 40 early region, the cytomegalovirus immediate-early gene, the lymphotropic papovavirus, and the human beta- globin gene. ADA expression from these vectors was monitored in the ADA- human histiocytic lymphoma cell line DHL-9, and in the multipotential chronic myeloid leukemia cell line K562. ADA expression in infected K562 cells was also measured after induction of megakaryoblastic differentiation by phorbol ester, and after induction of erythroid differentiation by sodium n-butyrate or hemin. In these hematopoietic cell lines, the vectors that contained ADA controlled by either the Moloney murine leukemia virus promoter (LASN) or the cytomegalovirus promoter (LNCA) expressed ADA at much higher levels than the other vectors tested. Furthermore, in K562 cells infected with LASN and LNCA vectors, induction of terminal differentiation resulted in the same or higher level expression of ADA. These cell lines have permitted the evaluation of transduced gene expression in proliferating and differentiating hematopoietic cells that provide a model for bone marrow-targeted gene therapy.
Molecular defects in hemophilia A: identification and characterization of mutations in the factor VIII gene and family analysis
Authors: Higuchi M , Kochhan L , Schwaab R , Egli H , Brackmann HH , Horst J , Olek K
Published: October 2019

Hemophilia A is an X-linked bleeding disorder caused by a deficiency or abnormality of factor VIII, affecting approximately 1 male in 10,000. A subgroup of the patients develops inhibitors against factor VIII during substitution therapy. Because a considerable percentage of all cases is thought to result from de novo mutations, it is likely that many different molecular lesions lead to hemophilia A. In order to understand the molecular basis of this disorder, we examined 160 patients with different clinical features using factor VIII gene probes. We could identify six different deletions and seven nonsense mutations within the factor VIII gene. Family analysis revealed that five of these mutations occurred de novo within two generations; two of them arose in the maternal grandfather and three in the mother. In one of these mothers we could identify a mitotic origin. Mapping of the deletions showed no deletion-prone region within the gene. Furthermore, we could not find any correlation between the particular gene defects and “inhibitor” phenotypes.
T-cell receptor delta/alpha rearrangements in lymphoid neoplasms
Authors: Dyer MJ
Published: October 2019

Rearrangements within the T-cell receptor (TCR)delta/alpha locus were analyzed in a wide variety of lymphoid neoplasms by eight DNA probes specific for TCR J delta, J alpha and C alpha segments. In all 11 T- cell malignancies, rearrangement and/or deletion of TCR delta was detected irrespective of the stage of maturation of the tumor. The organization of TCR delta correlated with the phenotype of the tumor: In “prethymic” T-cell acute lymphocytic leukemia (ALL), TCR delta was the only TCR gene to be rearranged. More mature T cell malignancies expressing CD4 together with CD3 showed deletion of both alleles of TCR delta, suggestive of TCR V alpha-J alpha rearrangement. All 43 B-cell tumors expressing surface immunoglobulin (sIg), including two cases of adult B-cell ALL, had germline configuration of TCR delta/alpha. In contrast, all 17 B-cell precursor ALLs (null, common, and pre-B-cell ALLs) had rearrangement and/or deletion of TCR delta/alpha. A single case of “histiocytic” lymphoma also showed biallelic deletion of TCR delta. Oligoclonal rearrangements of Ig and TCR genes were observed in two cases of B-cell precursor ALL and in one case of T-cell lymphoblastic lymphoma. Patterns of such “aberrant” TCR rearrangement were similar to those observed in T-lineage malignancies. In particular, seven of eight cases of B-cell precursor ALL and the histiocytic lymphoma which demonstrated biallelic TCR delta deletion, (suggestive of a V alpha-J alpha rearrangement) had clonal TCR beta rearrangement. These data support the hypothesis that supposedly aberrant rearrangements of the TCR genes may follow the same developmental controls as found in T-cell differentiation, despite the lack of evidence for further commitment to the T-cell lineage. TCR delta rearrangement is a useful marker of clonality of immature T-cell tumors which may have only this gene rearranged but is not specific to the T-cell lineage.
Human platelet-derived mitogens. I. Identification of insulinlike growth factors I and II by purification and N alpha amino acid sequence analysis
Authors: Karey KP , Marquardt H , Sirbasku DA
Published: October 2019

Human platelet lysates contained potent mitogenic activities for MCF-7 human breast-cancer cells in serum-free-defined media. Because these activities were not replaced by known platelet mitogens, such as platelet-derived growth factor or transforming growth factor beta, we sought to identify the breast cancer cell mitogens by purification and N alpha amino-acid sequencing. Acetic acid extracts of outdated human platelets were concentrated by ammonium sulfate precipitation and fractionated on Sephadex G-50 and Bio-Gel P-10 columns in 0.5 mol/L acetic acid. Two major activities were resolved by molecular sieve methods and fractionated further by reverse-phase high-performance liquid chromatography (HPLC). Purifications (70,000 to 870,000-fold) were accomplished yielding mol wt 7,400 products that were homogeneous as determined by iodination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography. The factors were identified as insulinlike growth factor I (IGF-I) and II (IGF-II) and truncated IGF-I by N alpha amino acid microsequencing. In dose-response experiments, platelet-derived IGF-I and IGF-II promoted multiple divisions of the MCF-7 cells with ED50 values of 12 and 100 pg/mL, respectively. The specific activities and other bioassay characteristics of platelet-derived IGF-I and IGF-II were similar to those of recombinant-produced human growth factors. This is the first report of the purification of insulinlike growth factors from human platelet lysates.
Membrane assembly and remodeling during reticulocyte maturation
Authors: Chasis JA , Prenant M , Leung A , Mohandas N
Published: October 2019

Membrane skeletal and cytoskeletal remodeling occurs throughout erythroid maturation. Microtubules and microfilaments have been identified morphologically in the nucleated erythroblast but the functional capability of these cytoskeletal structures during reticulocyte maturation has not been studied. Reticulocytes are formed from orthochromatic normoblasts by the process of nuclear extrusion. Two recognizable stages of reticulocyte maturation follow. The least mature reticulocytes are motile and multilobular, while the more mature reticulocytes are cup-shaped and nonmotile. To study the respective roles of microtubules and microfilaments in nuclear extrusion and cell motility, experiments were performed with agents that perturb these structures. Following the injection into rats of colchicine, a microtubule-disrupting substance, the number of normoblasts arrested at the stage of nuclear extrusion increased linearly over four hours. Similar results were obtained when bone marrow cells were incubated in culture in the presence of colchicine. In contrast, cell motility was dramatically decreased by cytochalasin B, a microfilament-disrupting agent, but not by colchicine. These results imply that microtubules are essential for the nuclear extrusion process, while microfilaments are essential for cell motility. Simultaneous changes in membrane skeletal assembly were assessed by measuring membrane deformability and stability, two properties regulated by the skeletal proteins. In ektacytometric assays, membrane deformability and mechanical stability of immature reticulocytes were markedly decreased to approximately 10% of normal, while that of more mature reticulocytes were nearly normal. Since the skeletal protein organization regulates these membrane properties, our findings imply that substantial membrane skeletal remodeling occurs during reticulocyte maturation. Thus we have identified major remodeling of both skeletal and cytoskeletal components during reticulocyte maturation.
Autologous bone marrow transplantation with 4- hydroperoxycyclophosphamide purged marrows for acute nonlymphocytic leukemia in late remission or early relapse
Authors: Rosenfeld C , Shadduck RK , Przepiorka D , Mangan KF , Colvin M
Published: October 2019

Twenty-four patients with acute nonlymphocytic leukemia (ANLL) were treated with high-dose chemotherapy or chemoradiotherapy followed by infusion of autologous marrow purged with 100 micrograms/mL of 4- hydroperoxycyclophosphamide (4HC). The marrow harvests were performed when there were less than 5% blasts in the marrow. Seven patients were transplanted in second complete remission (CR), eight in third CR, one in fourth CR, and eight in early relapse. The median time to achieve 500 neutrophils/microL or 1,000 leukocytes/microL was 30 days. A platelet count of 20,000/microL and 50,000/microL was achieved at a median of 67 and 91 days, respectively. One patient failed to engraft by day 58. There were five other transplant-related deaths: sepsis (one), intracerebral hemorrhage (one), veno-occlusive disease (one), and interstitial pneumonia (two). Four of seven evaluable patients transplanted in early relapse obtained a CR lasting 112, 143, 189, and greater than 615 days. Eight of 11 evaluable patients transplanted in CR have relapsed at a median of 153 days (range, 104 to 311). The actuarial survival for all patients was 19%. There was a trend toward improved relapse-free survival for patients transplanted in remission as opposed to those transplanted in relapse (P = .11).
Mapping of monoclonal antibodies to human factor IX
Authors: Frazier D , Smith KJ , Cheung WF , Ware J , Lin SW , Thompson AR , Reisner H , Bajaj SP , Stafford DW
Published: October 2019

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF- like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.
Effect of recombinant human granulocyte colony-stimulating factor on hematopoiesis of normal dogs and on hematopoietic recovery after otherwise lethal total body irradiation
Authors: Schuening FG , Storb R , Goehle S , Graham TC , Appelbaum FR , Hackman R , Souza LM
Published: October 2019

This study was designed to test whether recombinant human G-CSF (rh G- CSF) affects hematopoiesis in normal dogs and, if so, to test the effects of G-CSF in dogs given otherwise lethal total body irradiation (TBI). Rh G-CSF given subcutaneously at 10 or 100 micrograms/kg/d for 14 days to two normal dogs increased peripheral blood neutrophils eight to tenfold and monocytes four to sixfold above controls. Lymphocyte counts remained unchanged at the lower dose and increased threefold at the higher dose of rh G-CSF. No significant changes were observed in eosinophil, platelet, reticulocyte, or hematocrit levels. After 2 weeks of treatment with rh G-CSF, bone marrow displayed myeloid hyperplasia and left-shifted granulocytopoiesis. After discontinuation of rh G-CSF, peripheral leukocyte counts returned to control levels within three days. Five dogs administered 400 cGy TBI at 10 cGy/min from two opposing 60Co sources and no marrow infusion or growth factor, all developed profound pancytopenia and died between 17 and 23 days after TBI with infections secondary to marrow aplasia. Four of five dogs treated within two hours after 400 cGy TBI with 100 micrograms rh G- CSF/kg/d subcutaneously twice a day for 21 days showed complete and sustained endogenous hematopoietic recovery. In contrast, five dogs irradiated with 400 cGy TBI and treated with 100 micrograms rh G- CSF/kg/d starting on day 7 after TBI, all died between days 17 and 20 after TBI with infections secondary to marrow aplasia. Rh G-CSF, if administered shortly after irradiation, can reverse the otherwise lethal myelosuppressive effect of radiation exposure.
Low molecular weight B-cell growth factor and recombinant interleukin-2 are together able to generate cytotoxic T lymphocytes with LAK activity from the bone marrow cells of children with acute lymphoblastic leukemia
Authors: Zhou MX , Findley HW Jr , Ragab AH
Published: October 2019

We are reporting here that low-mol wt B-cell growth factor (LMW-BCGF) and recombinant interleukin-2 (rIL-2) are together able to induce CD3+ cytotoxic T lymphocytes (CTL) with lymphokine-activated killer cell (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). Ficoll-Hypaque (FH)-separated BM cells were obtained from patients with active disease (at diagnosis N = 13, in relapse N = 15) and in complete remission (CR; N = 12). CD3+ cells were removed by Leu-4 antibody and immunobeads. Cells were cultured (10(5) cells/mL) in semisolid media with rIL-2 (100 mu/mL), LMW-BCGF (0.1 mu/mL), and the combination of rIL-2 plus LMW-BCGF, respectively, for seven to ten days. Pooled colonies were harvested for phenotyping. LMW-BCGF plus rIL-2 induced large numbers of CD3+ colonies from CD3- precursors. rIL-2 alone did not induce colony formation. In addition, cells were cultured in liquid media with LMW-BCGF, rIL-2, and the combination of LMW-BCGF plus rIL-2, respectively, for seven to 21 days. They were harvested for phenotyping, and cytotoxicity assays were performed v K562, Raji, and autologous leukemic cells. LMW-BCGF plus rIL-2 induced significant expansion of CD3+ cells from CD3- precursors, and these cells were activated to kill autologous leukemic cells in addition to Raji and K562 cell lines. LMW-BCGF or rIL-2 alone did not induce significant expansion or activation of cytotoxic CD3- cells. Our hypothesis is that LMW-BCGF plus rIL-2 stimulates the proliferation and activation of CD3- precursors from the BM cells of children with acute leukemia to become CD3+ cells that have LAK activity. This finding may have therapeutic implications.
Expression of the mdr-1/P-170 gene in patients with acute lymphoblastic leukemia
Authors: Rothenberg ML , Mickley LA , Cole DE , Balis FM , Tsuruo T , Poplack DG , Fojo AT
Published: October 2019

Increased expression of the multidrug resistance gene (mdr-1/P-170) and the dihydrofolate reductase (DHFR) gene have been implicated in the development of in vitro drug resistance. Overexpression, with or without gene amplification, is seen in the development of drug resistance in culture and it has been postulated that genetic modulation of mdr-1/P-170 and DHFR may also be involved in the development of clinical drug resistance. We screened lymphoblasts from 28 patients with acute lymphoblastic leukemia (ALL) for evidence of overexpression of mdr-1/P-170 using RNAse protection, RNA in situ hybridization and immunohistochemistry. Overexpression of mdr-1/P-170 without gene amplification was detected in samples from four patients (three after multiple relapses, one at presentation). Overexpression of mdr-1/P-170 was heterogeneous within the population of malignant lymphoblasts as demonstrated by RNA in situ hybridization, immunohistochemistry, and drug uptake using daunomycin autofluorescence analysis. There was no evidence of overexpression of DHFR in any of the eight patient samples tested by RNAse protection nor was there any evidence of gene amplification in 11 patient samples on Southern blot analysis. From these observations it appears that overexpression without gene amplification of mdr-1/P-170 may be one mechanism of clinical drug resistance in ALL.
Functional characteristics of receptor-bound urokinase on human monocytes: catalytic efficiency and susceptibility to inactivation by plasminogen activator inhibitors
Authors: Kirchheimer JC , Remold HG
Published: October 2019

We compared urokinase-type plasminogen activator (u-PA) in fluid phase and u-PA bound with its receptor on human blood monocytes with respect to proteolytic activity and susceptibility to inactivation by the plasminogen activator inhibitors PAI-1 and PAI-2. Receptor-bound u-PA is catalytically twice as efficient as fluid-phase u-PA. Fluid-phase u- PA is susceptible to rapid inhibition by PAI-1 and PAI-2 at an estimated PAI:u-PA molar ratio of 2:1. In contrast, u-PA bound to endogenously occupied receptors is inhibited by PAI-2 only at PAI:u-PA molar ratios of 20:1, but is not inhibited by PAI-1, u-PA/PAI-1 and u- PA/PAI-2 complexes bind to the receptor with a tenfold lower affinity than u-PA itself. Thus, competition of u-PA/PAI complexes with fluid- phase u-PA for binding to the receptor is unlikely to affect the overall plasminogen activator activity of the monocyte. These findings demonstrate that the activity of receptor-bound u-PA can be modulated by PAI-2, but not by PAI-1, to adjust the cell's proteolytic activity to different local situations.
Characterization and use of monoclonal antibodies directed against human erythropoietin that recognize different antigenic determinants
Authors: Goto M , Murakami A , Akai K , Kawanishi G , Ueda M , Chiba H , Sasaki R
Published: October 2019

We have established four hybridoma cells that produce monoclonal antibodies (MoAbs) R2, R4, R6, and R12 directed toward recombinant human erythropoietin (rHuEPO). MoAbs R2, R4, and R6 bound to EPO with high affinities (kd = approximately 2, 4, and 1 nmol/L, respectively) but MoAb R12 had a low affinity (240 nmol/L). These antibodies inhibited the biological activity of rHuEPO and EPOs from humans, rats, mice, and rabbits. This inhibition was due to the blocking of EPO binding to the target cells. The fully deglycosylated rHuEPO bound to the MoAbs, indicating that they recognized peptide sequences of the antigen but not the carbohydrates attached to the antigen. An immunosorbent column with the immobilized MoAb R2 was effective for the rapid purification of EPO. MoAb R6 bound to EPO at a site(s) different from those to which other MoAbs bound. Based on this finding, a sensitive and rapid enzyme-linked immunosorbent assay of EPO, in which EPO was sandwiched between two MoAbs (R2 and R6), was developed. The assay measured plasma levels of EPO as low as 5 mU/mL within several hours.
Enrichment of two glycosyl-phosphatidylinositol-anchored proteins, acetylcholinesterase and decay accelerating factor, in vesicles released from human red blood cells
Authors: Butikofer P , Kuypers FA , Xu CM , Chiu DT , Lubin B
Published: October 2019

Several proteins are attached to the cell membrane by a glycosyl- phosphatidylinositol (GPI) anchor. In this report, we show that during vesiculation of human RBCs in vitro, two of these proteins, acetylcholinesterase and decay accelerating factor, redistribute on the cell surface and become enriched in the released vesicles. As a result, the remnant cells are depleted of these proteins. We suggest that alterations in the architecture of the RBC membrane that precede vesiculation lead to selective polarization of GPI-anchored proteins within the domain of the membrane destined to become a vesicle. Since vesiculation occurs in many cell types, and if the loss of GPI-anchored proteins accompanies this process, it may have important biologic significance.
Differential regulation of tissue factor and plasminogen activator inhibitor by human mononuclear cells
Authors: Schwartz BS , Bradshaw JD
Published: October 2019

Fibrin is a hallmark of immune-mediated tissue lesions. The presence of fibrin in such lesions implies both the formation of fibrin via coagulation and the accompanying restriction of fibrinolysis, allowing fibrin to persist. Previous work has shown that human monocytes exposed to an inflammatory stimulus such as lipopolysaccharide (LPS) produce both tissue factor (TF) and plasminogen activator inhibitor--type 2 (PAI-2). These two proteins favor fibrin deposition, and evidence implies that cellular production of these two molecules may be linked. Another proinflammatory process pertinent to immune-mediated tissue damage and fibrin deposition is the response to alloantigen. Peripheral- blood mononuclear cells (PBM), consisting of lymphocytes and monocytes together, responded to alloantigen stimulation with differential expression of TF and PAI-2. PBM exposed to alloantigen developed high levels of TF activity, with no concomitant increase in PAI-2 activity or antigen. Alloantigen-stimulated PBM did not accumulate intracellular PAI-2, nor did they degrade PAI-2 added to cultures. This lack of PAI-2 production was not due to inadequate stimulation, as tritiated thymidine uptake and TF production demonstrated recognition of, and a vigorous reaction to, alloantigen. The divergent TF and PAI-2 responses of PBM exposed to alloantigen was maintained over 5 days and was reflected by mRNA profiles. These results imply that under specific physiologically relevant conditions, the procoagulant and antifibrinolytic effectors of inflammatory mononuclear cells can be independently regulated. This would imply more flexibility to monocyte mechanisms that favor fibrin deposition than previously thought.
Localization of the SRC oncogene to chromosome band 20q11.2 and loss of this gene with deletion (20q) in two leukemic patients
Authors: Morris CM , Honeybone LM , Hollings PE , Fitzgerald PH
Published: October 2019

In situ hybridization of the pHul-c-src probe to metaphase cells from three normal donors and two leukemic patients showed significant labeling in the proximal region of the long arm of chromosome 20q, with modal peaks of grains consistently at band 20q11.2. A secondary peak of grains was detected in the region 20q13.2-qter, the localization of SRC suggested by previous in situ studies. The exact localization of SRC is important for understanding the del(20q) chromosomal abnormality in myeloid neoplasias. Chromosome in situ hybridization and genomic studies showed loss of one allele of SRC in two patients with the deletion (20q). These results differ from previously published findings and suggest heterogeneity of the breakpoint at 20q11.2 in interstitial deletions of 20q, which characterize myeloid disorders.
High-efficiency gene transfer to human hematopoietic cells maintained in long-term marrow culture [see comments]
Authors: Hughes PF , Eaves CJ , Hogge DE , Humphries RK
Published: October 2019

We used a helper-free recombinant retrovirus carrying the neomycin resistance (neor) gene to investigate methods for improving gene transfer efficiencies to clonogenic hematopoietic progenitor cells of human origin and to assess the possibility of gene transfer to the more primitive cells from which clonogenic cells are derived after several weeks in long-term human marrow cultures. The proportion of neor CFU-GM in methylcellulose assays of infected fresh marrow was increased by six- to eightfold (mean 37.4%) by the addition of extra GM colony- stimulating factor and interleukin-1 beta or medium conditioned by a human marrow “stromal” cell line to medium conditioned by agar- stimulated human leukocytes both during the infection and the colony growth period. Similar increases were also noted in the proportion of neor BFU-E, although the efficiencies overall were somewhat lower (up to 25.7%, mean 16.3%). Initiation of long-term cultures with marrow exposed to virus under the same growth factor-supplemented conditions but without any immediate selection step resulted in sustained production of a high proportion of neor CFU-GM and BFU-E for 6 weeks in both the nonadherent and adherent fractions. Molecular analysis was used to confirm the presence of the neo gene after culture. These results demonstrate that stable, high-efficiency gene transfer can be accomplished to the most primitive class of human hematopoietic cells currently detectable that may also have in vivo reconstituting potential. Further use of this approach should provide new insights into human hematopoietic stem cell regulation and allow continued development and assessment of gene therapy procedures.
Human marrow stromal cells: response to interleukin-6 (IL-6) and control of IL-6 expression
Authors: Nemunaitis J , Andrews DF , Mochizuki DY , Lilly MB , Singer JW
Published: October 2019

Production of interleukin-6 (IL-6) by marrow stromal cells from human long-term marrow cultures and from stromal cells transformed with simian virus 40 was examined. As with other cultured mesenchymal cells, unstimulated stromal cells produced undetectable amounts of IL-6 mRNA when assayed by Northern blots. However, within 30 minutes after exposure of transformed marrow stromal cells to the inflammatory mediators, recombinant human interleukin-1 alpha (IL-1 alpha) or recombinant human tumor necrosis factor alpha (TNF alpha), significant increases in IL-6 expression were observed. The time course of IL-6 mRNA upregulation in transformed marrow stromal cells with IL-1 alpha and TNF alpha differed: The maximal response to TNF alpha was observed at 30 minutes whereas that to IL-1 alpha occurred at 8 hours. Although IL-6 at a concentration of 500 U/mL was inhibitory to adherent transformed marrow stromal cell proliferation, a concentration- dependent stimulation of anchorage-independent colony growth was observed when the cells were plated in semisolid medium with IL-6. The stromal cell colony-stimulating effect of IL-6 was abrogated by a neutralizing antibody to IL-6. Moreover, the heteroserum with anti-IL-6 activity and two anti-IL-6 monoclonal antibodies partially blocked autonomous and IL-1 alpha-induced colony formation, suggesting that colony formation by transformed marrow stromal cells may require IL-6. Clonal-transformed stromal cell lines were derived from the anchorage- independent stromal cell colonies. Both IL-6 mRNA and protein were constitutively produced at high levels. The addition of IL-6 to either long-term marrow culture adherent cells or transformed marrow stromal cells downregulated the expression of collagen I, a major stromal cell matrix protein. Thus, IL-6 affects proliferation of stromal cells and influences their production of extracellular matrix, suggesting that IL- 6 may have indirect as well as direct influences on hematopoietic cell proliferation.
DDAVP shortens the prolonged bleeding times of patients with severe von Willebrand disease treated with cryoprecipitate. Evidence for a mechanism of action independent of released von Willebrand factor
Authors: Cattaneo M , Moia M , Delle Valle P , Castellana P , Mannucci PM
Published: October 2019

After infusion of cryoprecipitate, the very prolonged bleeding time of patients with severe von Willebrand disease (vWD) is shortened but not always normalized in spite of normalization of plasma von Willebrand factor (vWF) levels. Therefore treatments that further improve primary hemostasis in severe vWD patients are needed. Since DDAVP shortens the bleeding time in a variety of bleeding disorders, we investigated in a double-blind, placebo-controlled crossover study the effects of the intravenous (IV) infusion of DDAVP (0.3 microgram/kg) on the bleeding times of 10 patients with severe vWD treated with cryoprecipitate. Their very prolonged bleeding times (greater than 30 minutes), partially corrected by the infusion of cryoprecipitate (14 +/- 2 minutes, mean +/- SEM), were further shortened by the administration of DDAVP (9 +/- 2 minutes, P less than .01) but not of saline (15 +/- 3 minutes, ns). Plasma vWF levels, raised from unmeasurable to normal values by cryoprecipitate, were not changed after DDAVP or saline. The defective deposition of platelets from eight patients onto human umbilical artery subendothelium was increased but not normalized by cryoprecipitate and was not significantly affected by DDAVP or saline. Therefore the infusion of DDAVP after cryoprecipitate may be of clinical benefit for management of bleeding episodes in severe vWD patients. Since severe vWD patients do not have releasable tissue stores of vWF, DDAVP must shorten their prolonged bleeding times independently of released vWF.