bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemolysis of transfused group O red blood cells in minor ABO- incompatible unrelated-donor bone marrow transplants in patients receiving cyclosporine without posttransplant methotrexate
Authors: Gajewski JL , Petz LD , Calhoun L , O'Rourke S , Landaw EM , Lyddane NR , Hunt LA , Schiller GJ , Ho WG , Champlin RE
Published: October 2019

Hemolysis most commonly occurs following bone marrow transplant when there is “minor” ABO blood group incompatibility between donor and recipient. The hemolysis has been attributed to destruction of the patient's incompatible erythrocytes by donor-derived anti-A and/or anti- B antibody produced from “passenger” immunocompetent donor lymphocytes. Extraordinary transfusion requirements of group O erythrocytes in a series of patients receiving unrelated minor ABO-incompatible marrow grafts led us to investigate whether this mechanism could account for the extent of hemolysis observed. In seven consecutive minor ABO- incompatible unrelated-donor bone marrow transplant recipients receiving cyclosporine without posttransplant methotrexate, we observed excessive hemolysis. For cases in this index group, a strongly reactive donor-derived ABO blood group antibody was identified coincident with development of hemolysis. Transfusion requirements in the first three patients (26 U of group O erythrocytes each) greatly exceeded the recipient's volume of incompatible erythrocytes, indicating that lysis of transfused group O erythrocytes was also occurring. Pretransplant erythrocyte exchange transfusion with group O erythrocytes performed in the four subsequent patients decreased the severity of hemolysis, but did not prevent it. Among minor ABO-incompatible marrow graft recipients, an analysis of variance demonstrated effects on transfusion requirements due to donor-recipient relationship being unrelated (P less than .002) and the use of posttransplant methotrexate (P = .0001), and there was interaction between these two factors (P less than .001). Bone marrow transplants from unrelated donors resulted in an exaggerated immune response to ABO blood group antigens, which was associated with hemolysis of transfused group O erythrocytes, as well as the patient's ABO-incompatible erythrocytes. This serious complication may be prevented by posttransplant immunosuppression with methotrexate.
Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib alpha involved in platelet transfusion refractoriness
Authors: Murata M , Furihata K , Ishida F , Russell SR , Ware J , Ruggeri ZM
Published: October 2019

The platelet-specific alloantigen, Siba, located within the alpha- subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine/methionine dimorphism at position 145 of the GPIb alpha sequence, and determined that the Siba antigen corresponds to the molecule containing methionine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIb alpha gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A positive correlation exists between platelet reactivity with the anti-Siba antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIb alpha fragments differing only at residue 145, provided definitive evidence that the human anti-Siba antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Siba human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.
"Stem cell" origin of the hematopoietic defect in dyskeratosis congenita
Authors: Marsh JC , Will AJ , Hows JM , Sartori P , Darbyshire PJ , Williamson PJ , Oscier DG , Dexter TM , Testa NG
Published: October 2019

We have used the long-term bone marrow culture (LTBMC) system to analyze hematopoiesis in three patients with dyskeratosis congenita (DC), two of whom had aplastic anemia, and the third had a normal blood count (apart from mild macrocytosis) and normal BM cellularity. Hematopoiesis was severely defective in all three patients, as measured by a low incidence of colony-forming cells and a low level of hematopoiesis in LTBMC. The function of the marrow stroma was normal in its ability to support the growth of hematopoietic progenitors from normal marrows seeded onto them in all three cases, but the generation of hematopoietic progenitors from patients marrow cells inoculated onto normal stromas was reduced, thus suggesting the defect to be of stem cell origin. The parents and unaffected brother of one of the families have also been studied in LTBMC and all showed normal hematopoietic and stromal cell function. From this study we speculate that there are some similarities between DC and the defect in the W/Wv mouse.
Further phenotypic characterization and isolation of human hematopoietic progenitor cells using a monoclonal antibody to the c-kit receptor
Authors: Briddell RA , Broudy VC , Bruno E , Brandt JE , Srour EF , Hoffman R
Published: October 2019

A mouse antihuman monoclonal IgG2a antibody, termed stem cell receptor- 1 (SR-1), specific for a determinant of the c-kit ligand receptor (KR), was used as an immunologic probe to analyze KR expression by human bone marrow hematopoietic progenitor cells. Monoclonal antibodies to CD34 and HLA-DR were used in a multicolor staining protocol in conjunction with SR-1 to further define the phenotypes of various classes of hematopoietic progenitor cells. Expression of KR (SR-1+) on hematopoietic progenitor cells identified subpopulations of cells expressing CD34 (CD34+). While one-half of the CD34- and HLA-DR- expressing cells (CD34+ HLA-DR+) expressed the KR (SR-1+), one-third of the CD34+ cells that lacked HLA-DR expression (CD34+ HLA-DR-) were SR- 1+. The CD34+ HLA-DR+ SR-1+ cell population contained the vast majority of the more differentiated progenitor cells, including the colony- forming unit (CFU) granulocyte-macrophage; burst-forming unit- erythrocyte; CFU-granulocyte, erythrocyte, macrophage, megakaryocyte; and the CFU-megakaryocyte. The overall progenitor cell cloning efficiency of this subpopulation was greater than 31%. By contrast, the CD34+ HLA-DR- SR-1+ cell population contained fewer of these more differentiated progenitor cells but exclusively contained the more primitive progenitor cells, the BFU-megakaryocyte, high proliferative potential-colony-forming cell, and long-term bone marrow culture- initiating cell. The overall progenitor cell cloning efficiency of this subpopulation was greater than 7%. Both the CD34+ HLA-DR- and CD34+ HLA- DR+ cell subpopulations lacking KR expression contained few assayable hematopoietic progenitor cells. Long-term bone marrow cultures initiated with CD34+ HLA-DR- SR-1+ but not CD34+ HLA-DR- SR-1- cells, which were repeatedly supplemented with c-kit ligand (KL) and interleukin-3, generated assayable progenitor cells of at least 2 lineages for 10 weeks. These experiments demonstrate the expression of the KR throughout the hierarchy of human hematopoietic progenitor cell development. We conclude from our data that the KL and KR play a pivotal role in cytokine regulation of both the primitive and more differentiated human hematopoietic progenitor cells.
Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma
Authors: Kittler EL , McGrath H , Temeles D , Crittenden RB , Kister VK , Quesenberry PJ
Published: October 2019

The “stromal” or adherent cells of long-term murine Dexter explant bone marrow cultures provide the best in vitro model of the bone marrow microenvironment. Colony-stimulating factor-1 (CSF-1) is produced constitutively by these cells and is easily detected, but most investigators have not found constitutive production of the other hemolymphopoietic cytokines. We have previously reported the detection of granulocyte-macrophage-CSF (GM-CSF) in murine stromal cultures and its induction by the lectin Pokeweed mitogen. The present studies analyzing stromal cytokine messenger RNA (mRNA) production by standard Northern blot analysis show constitutive production of mRNAs for CSF-1, GM-CSF, granulocyte-CSF (G-CSF), c-kit ligand (KL), and interleukin-6 (IL-6), but not IL-3, IL-4, or IL-5 by 3-week irradiated or nonirradiated murine Dexter stromal cells. Exposure of stromal cells to Pokeweed mitogen or IL-1 16 hours before RNA harvest induces the messages for GM-CSF, G-CSF, KL, and IL-6, but not IL-3, IL-4, IL-5, or CSF-1. Polymerase chain reaction amplification of cDNA made with reverse transcriptase from stromal RNA using two separate sets of IL-3- specific primers shows the presence of IL-3 message in irradiated stromal cells, which is only detectable with this more sensitive technique. The factor-dependent cell lines FDC-P1 and 32D are supported by the stromal cells without the addition of exogenous growth factors, demonstrating a cytokine activity in these cultures that is inhibited by the addition of anti-IL-3 or anti-GM-CSF antibodies. These data indicate that murine Dexter stromal cells constitutively produce CSF-1, GM-CSF, G-CSF, IL-6, KL, and IL-3. This growth factor production could explain the support of granulocyte, macrophage, and megakaryocyte production and stem cell maintenance in Dexter-type long-term murine bone marrow cultures.
Interleukin-4 inhibits interleukin-1 alpha-induced granulocyte- macrophage colony-stimulating factor gene expression in a murine B- lymphocyte cell line via downregulation of RNA precursor
Authors: Akahane K , Pluznik DH
Published: October 2019

Interleukin-4 (IL-4) regulates the growth of B cells. When combined with colony-stimulating factors (CSFs) and selected cytokines, IL-4 has a synergistic effect on the clonal growth of bone marrow cells. Recently, we have shown that IL-1 alpha and lipopolysaccharide induce expression of the granulocyte-macrophage CSF (GM-CSF) gene in murine B- cell lines. In the present study, we show that IL-4 inhibits the production of GM-CSF in the IL-1 alpha-stimulated murine B-cell line M12.4.1. IL-4 did not change the transcription rate of the GM-CSF gene, and caused only a slight decrease in cytoplasmic GM-CSF messenger RNA (mRNA) half-life in cells treated with IL-1 alpha. PCR analysis of nuclear RNA with probes specific for GM-CSF intron sequences suggests that IL-1 alpha enhances accumulation of nuclear precursor RNA and that decreased GM-CSF expression after IL-4 treatment is mainly due to intranuclear destabilization of the primary transcript. Under the same experimental conditions, IL-4 did not affect expression of the IL-4 receptor mRNA and did increase the mRNA concentration of the low- affinity receptor for IgE (Fc epsilon RII). These data suggest that the suppressive effect of IL-4 is specific for GM-CSF mRNA expression, and thus provide evidence for an additional role of IL-4 in the regulation of GM-CSF expression in B cells.
Hyperdiploid (47-50) acute lymphoblastic leukemia in children
Authors: Raimondi SC , Roberson PK , Pui CH , Behm FG , Rivera GK
Published: October 2019

Among ploidy groups in childhood acute lymphoblastic leukemia (ALL), hyperdiploidy 47 to 50 is perhaps the least well known. From December 1979 to December 1990, we successfully studied banded karyotypes in 598 cases of newly diagnosed ALL, of which 86 (14.4%) had modal chromosome numbers of 47 to 50. In this group, the most frequently acquired numerical abnormalities were +21 (n = 34), +X (18), +8 (8), and +10 (7). The chromosomal regions most often affected by structural abnormalities were 1q (n = 13), 6q (12), 12p (18), and 19p (9). Analysis of event-free survival (EFS) for Studies X and XI among patients with hyperdiploid (47 to 50) ALL showed no significant differences in outcome according to the presence (n = 36) or absence (n = 35) of chromosomal translocations (P = .81) or the gain of specific chromosomes (P = .40). Patients with hyperdiploid (47 to 50) ALL treated in a contemporary program of multiagent chemotherapy had a significantly better outcome than did those in an earlier study using less intensive therapy (4-year EFS = 75% [95% confidence interval, 55% to 86%] v 41% [22% to 59%]; P = .006 by the logrank test). Our findings indicate that the adverse prognosis previously attributed to hyperdiploidy 47 to 50 improves significantly with more effective chemotherapy.
The translocation t(1;22)(p13;q13) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children
Authors: Lion T , Haas OA , Harbott J , Bannier E , Ritterbach J , Jankovic M , Fink FM , Stojimirovic A , Herrmann J , Riehm HJ
Published: October 2019

We present the nonrandom occurrence, frequency, and degree of immunophenotype association of the t(1;22)(p13;q13) in children with acute nonlymphocytic leukemia (ANLL). This karyotype anomaly occurred in leukemia cells from five of 445 (1.1%) children with newly diagnosed ANLL who were successfully studied by cytogenetic analysis at four European centers between January 1987 and January 1992. The occurrence of the t(1;22) was restricted to the French-American-British classification (FAB) subtype M7. The overall incidence in children with acute megakaryocytic leukemia (AMKL) was 27.8% (5/18 cases); in infants with AMKL, the frequency of the t(1;22) was 66.7% (4/6 cases). Three of the patients carrying this anomaly had a diploid karyotype, whereas in two cases a hyperdiploid karyotype was found. However, in all five patients, the t(1;22) was the only translocation event present at diagnosis. All patients received aggressive chemotherapy for acute myelogenous leukemia (AML). Two patients died within 15 months of diagnosis without entering remission. One of three patients who entered remission died 7 months after diagnosis, most likely from intramedullar hemorrhage. Only two of the five children with the t(1;22) who received autologous bone marrow transplantation (BMT) are alive and in complete remission (CR) 23 and 40 months after diagnosis, respectively. At the time of diagnosis, the age of the oldest child carrying the t(1;22) was 18 months. The cases with this chromosome anomaly were compared with an age-matched group of five children with AMKL lacking this translocation. The patients with the t(1;22) had a lower median value of the peripheral white blood cell (WBC) count and a higher median hemoglobin level than the patients from the matched group. In the latter cases, normocellular or hypercellular bone marrow (BM) was detected at diagnosis. In contrast, all children with the t(1;22) in our series had a hypocellular BM. Histological BM analyses were available in three of these patients and showed marked fibrosis. Other clinical and laboratory parameters showed no obvious differences between the matched groups. Despite intensive chemotherapy, AMKL in children appears to be associated with a poor prognosis. The clinical courses of the children with AMKL and the t(1;22) presented may be indicative of a beneficial effect of autologous BMT in this subset of patients.
Inhibition of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) messenger RNA (mRNA) expression in HL-60 leukemia cells by pentoxifylline and dexamethasone: dissociation of acivicin- induced TNF-alpha and IL-1 beta mRNA expression from acivicin-induced monocytoid differentiation
Authors: Weinberg JB , Mason SN , Wortham TS
Published: October 2019

We have previously noted that the glutamine antagonist acivicin (alpha S,5S-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid) induces monocytoid differentiation of freshly isolated human myeloid leukemia cells and cells of the myeloid leukemia cell line HL-60, and that the differentiation is accompanied by increases in expression of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). Because we also showed that TNF-alpha and IL-1 beta can act synergistically to cause monocytoid differentiation of HL-60 cells, we hypothesized that acivicin-induced TNF-alpha and IL-1 beta, in an autocrine manner, caused the differentiation. The purpose of the present study was to determine the causal roles of TNF-alpha and IL-1 beta in the acivicin-induced differentiation of HL-60 cells by the use of dexamethasone (DEX) and pentoxifylline (PTX), two drugs that effectively inhibit expression of TNF-alpha and IL-1 beta. Acivicin caused a monocytoid differentiation of the cells as manifest by diminished cell growth, morphologic maturation of the cells, increased ability to generate hydrogen peroxide in response to acute treatment with phorbol myristate acetate, and increased expression of nonspecific esterase and the surface antigens CD14 and CD11b. Acivicin treatment also caused the cells to have diminished steady-state expression of messenger RNA (mRNA) for c-myc and c-myb, and increased expression of mRNA for TNF-alpha and IL-1 beta. DEX and PTX did not alter cell growth, and did not block the acivicin-induced block in growth. PTX caused a slight increase in nonspecific esterase expression, but DEX had no effect on this, and neither drug diminished the acivicin-induced increase in nonspecific esterase. Although neither drug alone lessened the acivicin enhancement of hydrogen peroxide production, DEX and PTX together reduced this. DEX did not modify the acivicin-induced morphologic maturation of the cells, but PTX alone or PTX with DEX potentiated the acivicin-induced increase in mature cells. Basal CD14 and CD11b expression were slightly reduced by DEX and PTX, but neither drug modified the acivicin-induced increases. DEX and PTX reduced the acivicin-induced increases in TNF-alpha and IL-1 beta mRNA expression, but they had little or no effect on the acivicin-induced decreases in expression of mRNA for c-myc and c-myb. Thus, DEX and PTX effectively block the acivicin-induced expression of TNF-alpha and IL-1 beta, but they have little influence on the acivicin-induced differentiation process.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression of activin A/erythroid differentiation factor in murine bone marrow stromal cells
Authors: Yamashita T , Takahashi S , Ogata E
Published: October 2019

Accumulating evidence suggests that activin A/erythroid differentiation factor (EDF), a homodimer of beta A chain, is a physiologic hematopoietic factor, particularly of erythroid lineage. Media conditioned by phorbol myristate acetate (PMA)-stimulated murine bone marrow stromal cell lines, MC3T3-G2/PA6 and ST2, contained activin A/EDF, assayed as the activity inducing erythroid differentiation of an activin A/EDF-responsive murine erythroleukemia cell clone F5. Follistatin, a protein specifically binding to activin A/EDF, abolished this erythroid differentiation. Northern blot analysis showed that PMA rapidly increased beta A chain messenger RNA levels in MC3T3-G2/PA6 and ST2 cells. In a search for natural stimulators, we found that tumor necrosis factor-alpha, in itself and synergistically with interleukin-1 beta, induced activin A/EDF production in both cell lines. These results indicate that stromal cells produce activin A/EDF in bone marrow.
Cytarabine plus idarubicin or daunorubicin as induction and consolidation therapy for previously untreated adult patients with acute myeloid leukemia
Authors: Wiernik PH , Banks PL , Case DC Jr , Arlin ZA , Periman PO , Todd MB , Ritch PS , Enck RE , Weitberg AB
Published: October 2019

The purpose of this study was to determine the relative merits of idarubicin and daunorubicin in acute myeloid leukemia (AML) therapy. Thirty-two sites provided 214 previously untreated adults with AML aged 15 years or more who were randomized to receive for induction therapy cytarabine 100 mg/m2/d as a continuous 7-day infusion plus either daunorubicin 45 mg/m2/d (A + D) or idarubicin 13 mg/m2/d (A + I), daily on the first three days of treatment. Postremission therapy consisted of two courses of the induction regimen at the same daily doses, with the anthracycline administered for 2 days and cytarabine for 5. The complete response (CR) rates for evaluable patients were 70% (A + I) and 59% (A + D) (P = .08). The difference in CR rates was significant in patients aged 18 to 50 years (88% for A + I, 70% for A + D, P = .035). Resistant disease was a significantly more frequent cause of induction therapy failure with A + D than with A + I. Hyperleukocytosis (white blood cell count greater than 50,000/microL) unfavorably affected the attainment of CR with A + D but not with A + I. CR duration was significantly greater after A + I. CR duration was significantly greater after A + I treatment, and the survival of all randomized patients treated with A + I was significantly better than that observed after A + D treatment (median 12.9 months v 8.7 months, respectively, P = .038). Toxicity of the two treatments was similar, although A + I patients experienced more prolonged myelosuppression during consolidation therapy, and a greater incidence of mild chemical hepatitis was observed in the A + I group. It is concluded that, at the doses and schedule used in this study, A + I is superior to A + D for induction therapy of AML in adults.
Interleukin-11 enhances human megakaryocytopoiesis in vitro
Authors: Teramura M , Kobayashi S , Hoshino S , Oshimi K , Mizoguchi H
Published: October 2019

We investigated the effect of recombinant human interleukin-11 (rhIL- 11) on human megakaryocytopoiesis. Nonadherent and T-cell-depleted human bone marrow (BM) mononuclear cells were cultured in a serum-free agar culture system. rhIL-11 alone did not stimulate the growth of human megakaryocyte colonies. However, when rhIL-11 was combined with optimal or suboptimal doses of rhIL-3, the number and size of the megakaryocyte colonies increased. The same results were obtained when highly purified BM CD34-positive cells were used as target cells. Next, we investigated the effect of rhIL-11 on the ploidy of megakaryocytes. The ploidy distribution of individual cells in megakaryocyte colonies obtained by rhIL-11 in combination with rhIL-3 was significantly shifted towards higher values. Furthermore, when highly purified CD41- positive BM cells were cultured in the presence of rhIL-11, the ploidy distribution was shifted towards higher values. This effect was not suppressed by anti-IL-6 antibody. These results suggest that rhIL-11 acts directly as a megakaryocyte potentiator and may play a role in regulating human megakaryocytopoiesis.
Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro
Authors: Briddell RA , Brandt JE , Leemhuis TB , Hoffman R
Published: October 2019

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.
Isolation and characterization of a monoclonal antibody that recognizes the human c-kit receptor
Authors: Broudy VC , Lin N , Zsebo KM , Birkett NC , Smith KA , Bernstein ID , Papayannopoulou T
Published: October 2019

Stem cell factor (SCF) stimulates the growth of burst-forming unit- erythroid (BFU-E) and colony-forming unit granulocyte-macrophage (CFU- GM) by binding to a specific cell surface receptor. The receptor for SCF is encoded by the protooncogene c-kit. After immunizing mice with the human erythroleukemia cell line OCIM1, we obtained a monoclonal antibody (MoAb) that recognizes the human c-kit receptor. This MoAb, designated SR-1, blocks binding of 125I-human SCF to the c-kit receptor, and neutralizes the biologic effects of SCF in hematopoietic colony assays. With few exceptions, c-kit expression was identified on all hematopoietic and lymphoid cell lines tested by indirect immunofluorescent analysis using SR-1 and by binding studies with 125I- SCF. SR-1 recognizes a small fraction of normal bone marrow mononuclear cells, and these cells have the morphologic appearance of blasts. Colony assays show that BFU-E and CFU-GM display the c-kit receptor. SR- 1 does not cross-react with murine c-kit protein, indicating that the binding epitopes of the human and murine c-kit receptors are antigenically distinct. This MoAb may be useful to characterize the spectrum of cells that display the c-kit receptor and to further define the role of SCF in hematopoiesis.
Defective Ca(2+)-induced microvesiculation and deficient expression of procoagulant activity in erythrocytes from a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome
Authors: Bevers EM , Wiedmer T , Comfurius P , Shattil SJ , Weiss HJ , Zwaal RF , Sims PJ
Published: October 2019

The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.
Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation
Authors: Rawala-Sheikh R , Ahmad SS , Monroe DM , Roberts HR , Walsh PN
Published: October 2019

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.
Mutation of leucine-57 to phenylalanine in a platelet glycoprotein Ib alpha leucine tandem repeat occurring in patients with an autosomal dominant variant of Bernard-Soulier disease
Authors: Miller JL , Lyle VA , Cunningham D
Published: October 2019

The primary sequences of the three individual glycoprotein (GP) chains, GPIb alpha, GPIb beta, and GPIX, comprising the normal platelet GPIb/IX receptor for von Willebrand factor (vWF) have recently been determined, opening the possibility for characterization of disorders of this receptor at the molecular level. The presence of a leucine tandem repeat in each of these chains is of particular interest, because such repeats may be involved in associations between polypeptide segments. We now report an autosomal dominant variant of Bernard-Soulier disease associated with the heterozygous substitution of phenylalanine for a highly conserved leucine residue within the GPIb alpha leucine tandem repeat. Affected individuals experienced a moderate bleeding tendency, thrombocytopenia, and an increased mean platelet volume. Platelet aggregation was decreased only in response to ristocetin or to asialo- vWF. The kd for 125I-vWF binding to patients platelets was significantly increased over control values at 0.5 mg/mL ristocetin, but was normal at 1.0 or 1.5 mg/mL ristocetin. While sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an essentially normal complement of all components of the GPIb/IX complex, a minor amount of a putative proteolytic fragment was identified that migrated faster than GPIb and was immunoreactive with polyclonal anti-GPIb alpha antibody, but not with a monoclonal antibody directed against the 45-Kd amino-terminal region of GPIb alpha. However, because the great majority of patient GPIb alpha comigrates with normal GPIb alpha, the major functional abnormalities of the patient platelets are most likely a consequence of the altered structure of the nonproteolyzed protein. Full concordance within the studied family between phenotypic expression and a heterozygous single nucleotide substitution in genomic DNA coding for a phenylalanine in place of the wild-type leucine at residue 57 of the mature GPIb alpha, absence of this substitution in 266 alleles from the normal population, and the lack of any other abnormality of patient DNA throughout the entire coding sequence for GPIb alpha provide strong support that this substitution may constitute a pathologic point mutation responsible for the observed phenotypic abnormalities. While the roles that leucine tandem repeats may normally play within the GPIb/IX complex are not yet known, the perturbation of such a repeat in GPIb alpha may impair interaction with other components of the complex and/or with the binding of vWF.
Effect of recombinant human growth hormone on acute and chronic human immunodeficiency virus infection in vitro
Authors: Laurence J , Grimison B , Gonenne A
Published: October 2019

Growth hormone (somatotropin) is a potent anabolic protein currently being evaluated clinically in cachexia associated with malignancy and human immunodeficiency virus (HIV) disease. Growth hormone can also lead to enhancement of lectin-mediated cellular proliferation, macrophage activation, and cytokine induction, events linked to induction of latent HIV in vitro. We thus explored the ability of recombinant human growth hormone (rhGH) to affect viral replication in acute and chronic HIV infection, and to alter transcription at the HIV- 1 long terminal repeat (LTR). A clone of promonocytic cells, chronically infected with HIV-1 and susceptible to viral induction by a variety of cytokines and protein kinase C activators, was unperturbed by rhGH used over broad concentrations (10 to 500 ng/mL) and time intervals. This unresponsiveness paralleled the lack of effect of rhGH on HIV-associated trans-activation in both monocytic and CD4+ T-cell lines. In contrast, rhGH enhanced viral replication in acutely infected peripheral blood mononuclear cells (PBMC) by twofold to 20-fold, albeit having no adverse effect on the antiviral efficacy of zidovudine (AZT). Augmentation of HIV growth correlated with stimulation of cellular DNA synthetic responses and an increase in tumor necrosis factor-alpha (TNF- alpha) secretion. These data are discussed in the context of ongoing clinical trials of rhGH in HIV-seropositive individuals with wasting syndromes.
Mutations of the p53 gene in adult T-cell leukemia
Authors: Sakashita A , Hattori T , Miller CW , Suzushima H , Asou N , Takatsuki K , Koeffler HP
Published: October 2019

The p53 tumor suppressor gene was examined by direct sequencing of polymerase chain reaction-amplified DNA from fresh tumor cells of 10 patients with adult T-cell leukemia (ATL). Samples included nine patients with acute or lymphomatous ATL, and one patient in whom samples were examined in both his acute and chronic stages of ATL. Four missense mutations and one silent point mutation in the coding region of the p53 gene were found in cells from five patients with either acute or lymphomatous ATL. The missense mutations were homozygous and occurred in evolutionarily highly conserved regions of p53. One patient had no p53 mutation in his leukemic cells during chronic phase of ATL, but had a homozygous point mutation at codon 273 (Arg to His) when he progressed to acute ATL. In summary, we show that p53 is frequently mutated in the acute phase of ATL and one informative case suggests that p53 mutations may be associated with the transition from chronic to acute ATL.
The gene for B7, a costimulatory signal for T-cell activation, maps to chromosomal region 3q13.3-3q21
Authors: Freeman GJ , Disteche CM , Gribben JG , Adler DA , Freedman AS , Dougery J , Nadler LM
Published: October 2019

B7 is an activation antigen expressed on activated B cells and gamma- interferon-stimulated monocytes. The B7 antigen is the natural ligand for CD28 on T cells. After engagement of T-cell receptor with antigen in association with major histocompatibility complex class II, a second signal mediated through the binding of B7 to CD28 greatly upregulates the production of multiple lymphokines. We have now mapped the B7 gene to human chromosome 3 using the technique of polymerase chain reaction on a panel of hamster x human somatic cell hybrid DNAs. We have further localized the gene to 3q13.3–3q21 using in situ hybridization on human metaphase chromosomes. Trisomy of chromosome 3 is a recurrent chromosome change seen in various lymphomas and lymphoproliferative diseases, particularly diffuse, mixed, small, and large cell lymphomas, human T-cell lymphotropic virus type I-induced adult T-cell leukemia, and angioimmunoblastic lymphadenopathy. A number of chromosomal defects involving 3q21 have been described in acute myeloid leukemia and also in myelodysplastic and myeloproliferative syndromes. The mapping of B7 may permit further insight into disease states associated with aberrant lymphocyte activation and lymphokine synthesis.
A-T-rich scaffold attachment regions flank the hematopoietic serine protease genes clustered on chromosome 14q11.2
Authors: Hanson RD , Ley TJ
Published: October 2019

We have analyzed approximately 70 kb of the chromosome 14q11.2 hematopoietic serine protease gene cluster for the presence of nuclear scaffold attachment regions (SARs). At least 12 potential attachment sites were identified. SARs are present on both sides of the CGL-1/CSP- B and CGL-2/CCP-X genes and upstream from the cathepsin G (CG) gene. We have further characterized the SARs immediately flanking the cytotoxic lymphocyte-specific CGL-1/CSP-B gene. These 5′ and 3′ SARs are highly A- T-rich, contain multiple attachment sites, and are associated with the scaffolds of nuclei derived from both lymphoid and erythroid cell lines. These SARs contain multiple consensus elements frequently associated with A-T-rich sequences, including the vertebrate topoisomerase II (topo II) consensus sequence, the A-box and T-box elements, and the yeast autonomous replicating sequence (ARS). The potential role for the nuclear scaffold in the transcriptional regulation of CGL-1/CSP-B expression is discussed.
Increased erythropoietin-receptor expression on CD34-positive bone marrow cells from patients with chronic myeloid leukemia
Authors: Wognum AW , Krystal G , Eaves CJ , Eaves AC , Lansdorp PM
Published: October 2019

Erythropoietin-receptor (EpR) expression on bone marrow cells from normal individuals and from patients with chronic myeloid leukemia (CML) was examined by multiparameter flow cytometry after stepwise amplified immunostaining with biotin-labeled Ep, streptavidin- conjugated R-phycoerythrin, and biotinylated monoclonal anti-R- phycoerythrin. This approach allowed the detection of EpR-positive cells in all bone marrow samples studied. Most of the EpR-positive cells in normal bone marrow were found to be CD45-dull, CD34-negative, transferrin-receptor-positive and glycophorin-A-intermediate to - positive. This phenotype is characteristic of relatively mature erythroid precursors, ie, colony-forming units-erythroid and erythroblasts recognizable by classic staining procedures. Approximately 5% of normal EpR-positive cells displayed an intermediate expression of CD45, suggesting that these represented precursors of the CD45-dull EpR-positive cells. Some EpR-positive cells in chronic myeloid leukemia (CML) bone marrow had a phenotype similar to the major EpR-positive phenotype in normal bone marrow, ie, CD34-negative and CD45-dull. However, there was a disproportionate increase in the relative number of EpR-positive/CD45-intermediate cells in CML bone marrow. Even more striking differences between normal individuals and CML patients were observed when EpR-expression on CD34-positive marrow cells was analyzed. Very few EpR-positive cells were found in the CD34- positive fraction of normal bone marrow, whereas a significant fraction of the CD34-positive marrow cells from five of five CML patients expressed readily detectable EpR. These findings suggest that control of EpR expression is perturbed in the neoplastic clone of cells present in patients with CML. This may be related to the inadequate output of mature red blood cells typical of CML patients and may also be part of a more generalized perturbation in expression and/or functional integrity of other growth factor receptors on CML cells.
Necessity of extracellular domain of W (c-kit) receptors for attachment of murine cultured mast cells to fibroblasts
Authors: Adachi S , Ebi Y , Nishikawa S , Hayashi S , Yamazaki M , Kasugai T , Yamamura T , Nomura S , Kitamura Y
Published: October 2019

The receptor encoded by the W (c-kit) locus (W receptor) is expressed on the surface of cultured mast cells (CMC) derived from normal (+/+) mice, whereas its ligand encoded by the Sl locus (Sl ligand) is expressed on the surface of fibroblast cell lines derived from murine embryos. Involvement of W receptors and Sl ligands in attachment of CMC to fibroblasts was investigated. CMC were cocultured with fibroblasts; nonattaching CMC were removed and the remaining CMC were counted. CMC derived from mice of the W/W genotype did not express the extracellular domain of W receptors, and attachment of W/W CMC to +/+ fibroblasts was significantly impaired. Fibroblasts derived from embryos of the Sl/Sl genotype did not express Sl ligands, and the attachment of +/+ CMC to Sl/Sl fibroblasts was also impaired. The Wv and W42 alleles are point mutations at the intracellular tyrosine kinase domain. Attachment of either Wv/Wv, W/Wv, or W/W42 CMC to +/+ fibroblasts was comparable with that of +/+ CMC. Moreover, the addition of monoclonal antibody against the extracellular domain of W receptors inhibited the attachment of +/+ CMC to +/+ fibroblasts. Thus, the extracellular domain of W receptors appeared to be necessary for attachment of CMC to fibroblasts.
Myeloid and erythroid progenitor cells from normal bone marrow adhere to collagen type I
Authors: Koenigsmann M , Griffin JD , DiCarlo J , Cannistra SA
Published: October 2019

One of the mechanisms by which normal hematopoietic progenitor cells remain localized within the bone marrow microenvironment is likely to involve adhesion of these cells to extracellular matrix (ECM) proteins. For example, there is evidence that uncommitted, HLA-DR-negative progenitor cells and committed erythroid precursors (BFU-E) bind to fibronectin. However, fibronectin is not known to mediate binding of committed myeloid (granulocyte-macrophage) progenitors, raising the possibility that other ECM proteins may be involved in this process. We investigated the binding of the MO7 myeloid cell line to a variety of ECM proteins and observed significant specific binding to collagen type I (56% +/- 5%), minimal binding to fibronectin (18% +/- 4%) or to laminin (19% +/- 5%), and no binding to collagen type III, IV, or V. Similarly, normal bone marrow myeloid progenitor cells (CFU-GM) demonstrated significant specific binding to collagen type I (46% +/- 8% and 47% +/- 12% for day 7 CFU-GM and day 14 CFU-GM, respectively). The ability of collagen to mediate binding of progenitor cells was not restricted to the myeloid lineage, as BFU-E also showed significant binding to this ECM protein (40% +/- 10%). The binding of MO7 cells and CFU-GM was collagen-mediated, as demonstrated by complete inhibition of adherence after treatment with collagenase type VII, which was shown to specifically degrade collagen. Binding was not affected by anti-CD29 neutralizing antibody (anti-beta-1 integrin), the RGD-containing peptide sequence GRGDTP, or divalent cation chelation, suggesting that collagen binding is not mediated by the beta-1 integrin class of adhesion proteins. Finally, mature peripheral blood neutrophils and monocytes were also found to bind to collagen type I (25% +/- 8% and 29% +/- 6%, respectively). These data suggest that collagen type I may play a role in the localization of committed myeloid and erythroid progenitors within the bone marrow microenvironment.
Interactions of granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, and tumor necrosis factor alpha in the priming of the neutrophil respiratory burst
Authors: Khwaja A , Carver JE , Linch DC
Published: October 2019

Exposure of neutrophils to a range of cytokines augments their response to subsequent agonist-induced activation of the respiratory burst. We have examined the effects of several of these factors, both singly and in combination, on the priming of f-met-leu-phe (FMLP) and complement C5a-stimulated neutrophil H2O2 production, using a whole blood flow cytometric assay designed to minimize artefactual activation. Both granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha) produced a similar degree of priming of the FMLP-stimulated burst in vitro (558% +/- 86%, n = 41, and 581% +/- 95%, n = 21, of the response seen with FMLP alone, respectively), but with markedly different kinetics (half-maximal response 20 minutes and 7 minutes, respectively). Preincubation with granulocyte colony- stimulating factor (G-CSF) alone caused only modest priming (202% +/- 39%, n = 14). Priming with cytokine combinations of the FMLP-stimulated burst showed that the combinations of G-CSF and TNF alpha and GM-CSF and TNF alpha are highly synergistic, with recruitment of neutrophils unresponsive to priming by single agents. Priming with the combination of GM-CSF and G-CSF was not significantly different to priming with GM- CSF alone. Similar results were obtained using C5a as the respiratory burst stimulus. Significant priming of the FMLP-stimulated respiratory burst was seen in vivo in patients receiving an infusion of GM-CSF (332% +/- 50% of preinfusion response to FMLP, P less than .005, n = 8). Priming was also seen in patients receiving G-CSF (152% +/- 58%, n = 5), although this did not reach conventional significance levels (.05 less than P less than .1). Although GM-CSF infusion caused priming in vivo, this was 48% less than predicted by preinfusion in vitro responses. This result was not due to inadequate GM-CSF levels as addition of further GM-CSF ex vivo did not correct the response. However, these neutrophils were still able to respond appropriately to ex vivo priming with TNF alpha, with a doubling in H2O2 production.
Specific detection of monocytic lysozyme within normal and leukemic cells
Authors: Leculier C , Couprie N , Francina A , Archimbaud E , Adeleine P , Treille D , Denoyel G , Fiere D
Published: October 2019

A murine monoclonal antibody against human lysozyme (AHL MoAb) was produced and tested on normal and leukemic monocytes using flow cytometry. The antibody gave a positive reactivity on normal monocytes permeabilized by saponin (82% to 98% of positive cells) and a negative reactivity on normal permeabilized neutrophils. This monocyte-specific reactivity had not been observed using a polyclonal antibody. Nevertheless, immunoblotting detected lysozyme in both monocyte and polymorphonuclear leukocyte (PMNL) lysates. The AHL MoAb, in the presence of lysozyme substrate (Micrococcus lysodeikticus cell walls), strongly inhibited the enzymatic activity. Flow cytometric analysis of leukemic cells isolated from patients suffering from different subtypes of acute myeloid leukemia (French-American-British [FAB] classification FAB M1–5) showed a highly significant positivity of FAB M5 for lysozyme compared with the other subtypes. The present results were consistent with the detection of a lysozyme epitope by AHL MoAb located near the catalytic site in monocytes. The same epitope was probably masked in PMNL granules.