bluuebunny/crossref_metadata_2025 huggingface dataset:
A novel beta-thalassemia frameshift mutation (codon 14/15), detectable by direct visualization of abnormal restriction fragment in amplified genomic DNA
Authors: Chan V , Chan TK , Kan YW , Todd D
Published: October 2019

A new frameshift mutation due to an insertion of G between codon 14/15 of the beta-globin gene was found in two unrelated Chinese patients with Cooley's anemia. The first patient (W.S.) was homozygous for haplotype 5 (Chinese) and carried a codon 41/42 (four base pair deletion) mutant, while the second patient (C.K.) was homozygous for haplotype 2 (Chinese), and also had a codon 17 (A----T) nonsense mutation. Molecular cloning and M13 sequencing of the beta gene in patient W.S. revealed that the new mutant was found in a beta-globin gene framework type 3 (Asian). Direct sequencing was performed on polymerase chain reaction-amplified genomic DNA from patient C.K. With the new mutation, an additional BstNI or EcoRII recognition site is generated and the abnormal restriction fragment (134 basepair) can be directly visualized on polyacrylamide gel electrophoresis of the amplified genomic DNA.
Antithrombin-III-Hamilton: a gene with a point mutation (guanine to adenine) in codon 382 causing impaired serine protease reactivity
Authors: Devraj-Kizuk R , Chui DH , Prochownik EV , Carter CJ , Ofosu FA , Blajchman MA
Published: October 2019

Antithrombin-III-Hamilton is a structural mutant of antithrombin III with defective serine protease reactivity, demonstrable in three members of a French Canadian family. The propositus, a 54-year-old man with a history of recurrent thromboembolic events, and his two asymptomatic grown children are heterozygous for the mutant antithrombin III gene. In all three individuals, the immunoreactive antithrombin III level is normal, while the antithrombin and antifactor Xa activity is approximately 50% of the control value. Two dimensional immunoelectrophoresis of antithrombin-III-Hamilton in the presence of heparin is normal. Purified antithrombin-III-Hamilton did not form thrombin-antithrombin III complex when incubated with thrombin for up to 30 minutes. The normal and mutant antithrombin III alleles of the propositus could be distinguished by linkage to Pstl restriction fragment length polymorphisms (RFLP). Genomic DNA from the propositus was cloned into EMBL 3 phage vectors and two clones containing nearly complete copies of the antithrombin-III-Hamilton allele were identified. Exon 6 of both clones was subcloned into M13 phage vector and sequenced, revealing a G----A point mutation in the first base of codon 382. Codon 382 codes for alanine in the normal allele and for threonine in the antithrombin-III-Hamilton allele. Alanine-382, 12 residues from the reactive center, is a highly conserved amino acid in the family of serine protease inhibitors known as the serpins. We postulate that, as a result of the substitution of threonine for alanine in antithrombin-III-Hamilton, either the tertiary structure or the hydrophobicity of the thrombin-binding region is altered, causing aberrant conformation of the Arg-393-Ser-394 bond at the reactive center impairing the interaction between antithrombin-III-Hamilton and the activated serine proteases.
Plasminogen interactions with platelets in plasma
Authors: Adelman B , Rizk A , Hanners E
Published: October 2019

In this report we used a fluorescent flow cytometry-based assay to examine plasminogen binding to platelets in plasma. Our data indicate that platelets activated in platelet-rich plasma (PRP) by adenosine-5′- diphosphate (ADP) or thrombin bind plasminogen to their surface. Fab fragments of the monoclonal antibody LJ-CP8 that are directed against the fibrinogen binding site on the glycoprotein (GP) IIb-IIIa complex inhibit both plasminogen and fibrinogen binding to ADP-stimulated platelets as does 5 mmol/L EDTA. Platelet aggregation and plasminogen and fibrinogen binding are also concurrently inhibited by the Gly-Arg- Asp (RGD) analogue Gly-Arg-Gly-Asp-Ser (GRGDS) when it is added to PRP before ADP stimulation. The scrambled peptide analogue SDGRG has no effect. The monoclonal antibody 6D1, directed against the von Willebrand factor binding site on GPIb, has no effect on plasminogen- platelet binding, nor does antithrombospondin antibody. epsilon- Aminocaproic acid (EACA), however, inhibits plasminogen binding to ADP- activated platelets. These data indicate that plasminogen binds to platelets activated in plasma, that binding occurs on platelet GPIIb/IIIa, and that binding may be mediated via plasminogen association with fibrinogen via lysine binding domains. Finally, we found both plasminogen and fibrinogen on resting platelets in PRP and demonstrated that they are equally displaced by EDTA, LJ-CP8, and 10E5 (an additional anti-GPIIb/IIIa monoclonal antibody). Plasminogen is also equally displaced by EACA. These data suggest that plasminogen is also bound to GPIIb/IIIa on resting platelets, possibly also via interaction with fibrinogen.
A novel monoclonal antibody based diagnostic test for alpha-thalassemia- 1 carriers due to the (-SEA/) deletion
Authors: Luo HY , Clarke BJ , Gauldie J , Patterson M , Liao SK , Chui DH
Published: October 2019

The presence of minute amounts of embryonic zeta-globin chains in adult hemolysates is a marker for carriers of alpha-thalassemia-1 resulting from (--SEA/) deletion. Recently, we developed a murine monoclonal antihuman embryonic zeta-globin chain antibody, 8E8. By using this antibody, we have now established a slot-blot immunobinding assay for the rapid detection of zeta-globin chains in adult hemolysates. zeta- globin chains were found to be present in 30 blood samples obtained from individuals who were carriers of alpha-thalassemia-1. In another 30 blood samples from individuals who were not carriers of the (--SEA/) deletion, zeta-globin chains were not detected. This simple diagnostic test can be used in appropriate populations to identify those couples at risk of conceiving fetuses afflicted with the Hb Bart's hydrops fetalis syndrome due to homozygous alpha-thalassemia.
Calcium transport and ultrastructure of red cells in beta-thalassemia intermedia
Authors: Bookchin RM , Ortiz OE , Shalev O , Tsurel S , Rachmilewitz EA , Hockaday A , Lew VL
Published: October 2019

Reported findings of elevated total calcium (Ca) contents in erythrocytes (RBCs) from patients with beta-thalassemia intermedia (beta-TI) prompted the question of whether the state and transport of Ca in these RBCs are similar to those in sickle cell anemia (SS) RBCs where the increased Ca is compartmentalized in endocytic inside-out vesicles and extracted by exposure of the cells to the Ca ionophore A23187 and a Ca chelator (ethylene glycol tetraacetic acid) and the levels of cytoplasmic free ionized Ca [( Ca2+]i) are normal. We confirmed a high total Ca content of 51 +/- 13 mumol/L RBCs in splenectomized (SPX) beta-TI and 24 +/- 1 mumol/L RBCs in non-SPX beta- TI. Unlike SS RBCs, however, most of the increased Ca was in the lighter, presumably younger beta-TI RBCs, and about half the Ca was not ionophore mobilizable but apparently firmly bound, possibly to remnants of organelles in nucleated and other young RBCs. In the denser RBCs from non-SPX beta-TI, total and extractable Ca amounts were normal. beta-TI RBCs loaded with the Ca chelator Benz 2 showed an initial influx of 45Ca in the normal range, which indicated normal Ca permeability, and near-steady-state levels of [Ca2+]i that were normal (22 +/- 7 nmol/L RBCs in non-SPX beta-TI) or minimally increased (40 +/- 19 nmol/L RBCs in SPX beta-TI). Serial-section electron microscopy of beta-TI ghosts from the denser cell fractions showed more fully enclosed vesicles in non-SPX ghosts than were seen in normal ghosts and many large vesicles and structured, electron-dense material in SPX ghosts. A delayed extrusion of ionophore-preloaded 45Ca only by the SPX beta-TI RBCs together with normal [Ca2+]i suggested compartmentalization of the loaded Ca in these RBCs, perhaps in endocytic inside-out vesicles, and normal Ca pumps. Since beta-TI RBCs show essentially normal levels of [Ca2+]i and normal Ca influx, their high total Ca content should not be associated with any of the deleterious effects observed in vitro with increased levels of [Ca2+]i.
In vitro inhibition of normal human hematopoiesis by marrow CD3+, CD8+, HLA-DR+, HNK1+ lymphocytes
Authors: Vinci G , Vernant JP , Nakazawa M , Zohair M , Katz A , Henri A , Rochant H , Breton- Gorius J , Vainchenker W
Published: October 2019

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.
Comparison of transferrin receptor-mediated endocytosis and drug- induced endocytosis in human neonatal and adult RBCs
Authors: Thatte HS , Schrier SL
Published: October 2019

Neonatal RBCs can undergo receptor-mediated endocytosis; normal adult RBCs cannot. Previously, we showed that drug-induced endocytosis, which can occur in adult RBCs exposed to amphipathic cations like primaquine, is greatly enhanced in all density-defined fractions of neonatal RBCs. To investigate the similarities and differences between receptor- mediated endocytosis and drug-induced endocytosis, we characterized transferrin receptor-mediated endocytosis in neonatal RBCs and compared it with drug-induced endocytosis. Primaquine drug-induced endocytosis is dependent on RBC ATP levels, is invariably preceded by stomatocytosis, and is inhibited by vanadate. In contrast, receptor- mediated endocytosis of transferrin is not preceded by stomatocytosis, is not nearly so dependent on ATP levels as is drug-induced endocytosis, and is not inhibited by vanadate. Furthermore, receptor- mediated endocytosis is quantitatively blocked by preincubation of neonatal RBCs with sodium cyanide, whereas cyanide does not inhibit drug-induced endocytosis in either adult or neonatal RBCs. Morphologic observation of the neonatal RBCs established the fact that only puckered RBCs that exhibited brilliant cresyl blue staining reticulum were capable of undergoing receptor-mediated endocytosis of transferrin. These characteristics identify them as motile R-1 reticulocytes. Reticulocytes in normal adult RBCs were incapable of exhibiting this phenomenon. Thus, receptor-mediated endocytosis, a property of motile reticulocytes in neonatal RBCs, differs from drug- induced endocytosis in its energy requirements, response to inhibitors, and morphologic concomitants.
Leukemia of non-T lineage natural killer cells
Authors: Sheridan W , Winton EF , Chan WC , Gordon DS , Vogler WR , Phillips C , Bongiovanni KF , Waldmann TA
Published: October 2019

An unusual case of an aggressive leukemia of natural killer (NK) cells occurred in a 65-year-old male. Clinical characteristics of this case included hepatosplenomegaly, ascites, marrow infiltrate with leukemic cells, and a WBC up to 82.8 X 10(9) before therapy. One year before his presentation he had been noted to have a WBC of 12.1 X 10(9) with 78% lymphocytes, and 6 months before had noted intermittent fever and weight loss. He and his brother had well documented hereditary cold urticaria. The patient was treated with a modification of ProMACE CYTABOM regimen and had prompt regression of the leukemia with associated acute tumor lysis. Renal, hepatic, and marrow failure predominated during a terminal course that ended 22 days after therapy was commenced, and at autopsy there was no evidence for leukemic cell infiltrate in the liver, spleen or marrow. The leukemic cells were large granular lymphocytes by light and electron microscopic criteria, and had the following immunophenotype: CD2+, DR+, Leu7+, NKH1+, CD11+, CD3-, CD5-, CD4-, CD8-, CD16-. The cells displayed high antibody- dependent cell-mediated cytotoxicity (ADCC) and NK activity, and had a high rate of spontaneous proliferation in vitro that was not augmented by phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). Southern analysis of DNA from leukemic cells revealed normal germline arrangements for the beta and gamma chains of the T cell antigen receptor and immunoglobulin heavy chain genes. The majority of metaphases were clonally abnormal revealing consistent rearrangements involving extra material attached to the long arms of chromosomes 5 and 11.
Recombinant human interleukin-3 stimulation of hematopoiesis in humans: loss of responsiveness with differentiation in the neutrophilic myeloid series
Authors: Lopez AF , Dyson PG , To LB , Elliott MJ , Milton SE , Russell JA , Juttner CA , Yang YC , Clark SC , Vadas MA
Published: October 2019

Recombinant human (rh) interleukin-3 (IL-3) stimulated the proliferation and differentiation of erythroid, granulocyte, macrophage, eosinophil (Eo), and mixed colonies as well as megakaryocytes from human bone marrow cells. rh IL-3 was a weaker stimulus than rh granulocyte-macrophage colony-stimulating factor (GM- CSF) for day 14 myeloid cell colonies. At day 7 of incubation, rh IL-3 stimulated a few G, M, and Eo clusters but no colonies. This loss of responsiveness of myeloid cells to rh IL-3 was accentuated with further differentiation of the cells. rh IL-3 stimulated very few or no clones after five-day incubation with enriched promyelocytes and myelocytes, whereas rh GM-CSF was an efficient stimulus. Responsiveness to rh IL-3 was completely lost in postmitotic mature neutrophils. Incubation of these cells with rh IL-3 did not result in enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells or superoxide anion production after stimulation with formyl-methyl-leucyl-phenylalanine (FMLP), although they could be stimulated by rh GM-CSF. In addition, preincubation of neutrophils with different concentrations of rh IL-3 failed to increase or decrease their response to rh GM-CSF. In contrast to neutrophils, mature Eos could be stimulated by rh IL-3 to kill antibody-coated tumor cells. These results show that cells of the neutrophilic myeloid series lose their responsiveness to h IL-3 as they differentiate and suggest that although h IL-3 may be an important therapeutic agent to use for hematopoietic regeneration in vivo, the lack of stimulation of mature neutrophil function makes it an unlikely sole candidate as adjunct therapy for treatment of infectious diseases.
Interleukin-3 and interleukin-1 alpha allow earlier bone marrow progenitors to respond to human colony-stimulating factor 1
Authors: Zhou YQ , Stanley ER , Clark SC , Hatzfeld JA , Levesque JP , Federici C , Watt SM , Hatzfeld A
Published: October 2019

By using human bone marrow cells enriched for early progenitors by selective immunoadsorption and plated at low cell density (10(3) to 10(4) cells/mL/9.6 cm2) in semisolid methylcellulose culture, we have analyzed the cooperative effects of human colony-stimulating factor 1 (CSF-1), granulocyte-macrophage-CSF (GM-CSF), interleukin-1 alpha (IL-1 alpha), and gibbon as well as human recombinant IL-3 on the formation of monocytic colonies. CSF-1 alone stimulated mature monocytic colony formation by human CFU-M. However, in the presence of IL-3 and erythropoietin, CSF-1 stimulated maximal immature monocytic colony formation at low concentrations and inhibited the formation of granulomonocytic, erythrocytic, and mixed colonies. Cultures with CSF-1 and IL-3 contained more immature monocytic colonies than did cultures with CSF-1 alone. IL-1 alpha alone had little effect. However, IL-1 alpha in combination with optimal concentrations of either CSF-1, GM- CSF, or IL-3 increased the number of colonies containing immature or mature monocytic colonies.
Clinical response to deoxycoformycin in chronic lymphoid neoplasms and biochemical changes in circulating malignant cells in vivo
Authors: Ho AD , Ganeshaguru K , Knauf WU , Dietz G , Trede I , Hunstein W , Hoffbrand AV
Published: October 2019

Deoxycoformycin (DCF), an adenosine deaminase (ADA) inhibitor, has been shown to be active in lymphoid neoplasms. The mechanism of cytotoxicity might involve accumulation of deoxyadenosine triphosphate (dATP), depletion of the nicotinamide adenine dinucleotide (NAD) and ATP pool, induction of double-stranded DNA strand breaks, or inhibition of S- adenosyl homocysteine hydrolase (SAH-hydrolase). We have investigated the biochemical changes in the circulating malignant cells of patients with chronic leukemia/lymphoma who were treated with DCF (4 mg/m2 weekly). Blood samples were taken from 17 patients with 60% or more circulating leukemic cells before, 4, 24, and 48 hours and five days after the first administration of DCF. Leukemic cells were separated and studied for changes in ADA, dATP, ATP, NAD, and SAH-hydrolase levels and DNA strand breaks and the data analyzed according to clinical response. Inhibition of ADA activity was found in all except one patient at 4 to 24 hours after the first administration of DCF. dATP started to accumulate at four hours, reached a maximum level between 24 and 48 hours, and returned to base values on the fifth day. Intracellular ATP and NAD levels were transiently reduced in some of the patients. However, no correlation between these changes and a clinical response could be found. DNA strand breaks could be studied in 13 patients. A significant increase in DNA breaks at 24 to 48 hours was found in six of the seven responders but only in one of the six nonresponders. At 24 hours, SAH-hydrolase levels were reduced in all seven responders studied, but only in two of the seven nonresponders. The difference in inhibition of SAH-hydrolase was statistically significant (P = .0023). These results suggest that DNA strand breaks and inhibition of SAH-hydrolase correlate with clinical response.
Comparison of genetic probe with immunophenotype analysis in lymphoproliferative disorders: a study of 87 cases
Authors: Henni T , Gaulard P , Divine M , Le Couedic JP , Rocha D , Haioun C , Henni Z , Marolleau JP , Pinaudeau Y , Goossens M
Published: October 2019

We examined 91 specimens (from 87 patients) for the expression of B- cell- and T-cell-associated differentiation antigens and rearrangements of the Ig and beta-chain of the T-cell (beta-TCR) genes. Of these, 74 were representative of various histologic subtypes of non-Hodgkin's lymphoma and related disorders, 11 of Hodgkin's disease, and 6 of reactive lymphoid hyperplasia. An Ig gene clonal rearrangement correlated to a monotypic (kappa/lambda) phenotype in 32 of 33 histologically defined lymphoma samples. The genotypic analysis also confirmed clonality in six of seven malignant diffuse lymphomas that were nonmonotypic but expressed pan-B antigens; in four, more than one clone was detected within individual tumors. A beta-TCR clonal rearrangement was found in 19 of 19 tumor samples considered as malignant T-cell lymphoma on the basis of histopathology and of the CD3- positive phenotype of tumoral cells, and in two cases of CD3-positive lymphomatoid disorders. A loss of pan-T antigens (CD7, CD5, CD2, CD4/CD8) was observed in all but three of these CD3-positive samples. Such an incomplete T-cell phenotype always correlated to the presence of a monoclonal process as revealed by genotypic analysis. DNA analysis was the only way to demonstrate clonality in other samples with either a polymorphous (partial involvement, pseudolymphoma, angioimmunoblastic lymphodenopathy [AILD]) or an undifferentiated (large cell anaplastic) phenotype. It is concluded that although in the majority of cases immunophenotyping alone provides criteria adequate for the diagnosis of lymphoid malignancy, in some, particularly polymorphous or large cell anaplastic processes, genetic probe analysis was additionally discriminative.
Platelets secrete a coagulation inhibitor functionally and antigenically similar to the lipoprotein associated coagulation inhibitor
Authors: Novotny WF , Girard TJ , Miletich JP , Broze GJ Jr
Published: October 2019

Stimulation with thrombin or the calcium ionophore, A23187 caused human platelets to release a coagulation inhibitor similar to the Lipoprotein Associated Coagulation Inhibitor (LACI). This was documented functionally, with clotting assays measuring tissue factor inhibition and factor Xa inhibition, as well as immunologically, in a competitive immunoassay. The total amount of LACI released by 3 x 10(8) platelets after two hours stimulation was 7% to 8% of the amount found in 1 mL of serum. Half of the LACI was released by five minutes. The LACI was present in the platelet supernatant and was not associated with the platelet membrane or shed vesicles. The tissue factor and factor Xa inhibitory activities that were released were neutralized by preincubating the platelet supernatants with specific rabbit polyclonal anti-LACI IgG. On Western blot, platelet LACI appeared to run as a doublet with a molecular weight (mol wt) 45,000 to 47,000. Blood samples obtained from the site of a wound (template bleeding time) demonstrated a progressive increase in LACI concentration. A cDNA probe, derived from endothelial cell LACI cDNA, hybridized selectively to 4.0 and 1.4 kb transcripts in a preparation of platelet mRNA.
Hypomethylation of ornithine decarboxylase gene and erb-A1 oncogene in human chronic lymphatic leukemia
Authors: Lipsanen V , Leinonen P , Alhonen L , Janne J
Published: October 2019

The methylation state of CCGG sites in and around the human ornithine decarboxylase gene, oncogenes c-myc and erb-A1, and actin genes were determined in human malignant leucocytes from patients with acute and chronic myeloid leukemia, chronic lymphatic leukemia, polycythemia vera, and multiple myeloma by means of isoschizomeric restriction endonuclease analysis. When compared with DNA from leucocytes of healthy controls, the ornithine decarboxylase and erb-A1 genes were substantially hypomethylated in all samples obtained from patients with chronic lymphatic leukemia. Hypomethylation of genes, particularly growth-related sequences, might be a crucial fact in the malignant transformation of human leucocytes. Its relatively simple detection from blood samples may prove clinically applicable in monitoring patients with chronic lymphatic leukemia.
Effects of recombinant erythropoietin on the concentration and cycling status of human marrow hematopoietic progenitor cells in vivo
Authors: Dessypris E , Graber SE , Krantz SB , Stone WJ
Published: October 2019

The concentration of human marrow progenitors CFU-E, BFU-E, CFU-GM, and CFU-Mk and the percentage of these progenitor cells in DNA synthesis were studied in nine patients with transfusion-dependent anemia of end- stage renal failure before and 2 weeks after treatment with human recombinant erythropoietin (Epo) at a dose of 150 to 300 U/kg intravenously three times per week. The concentration of CFU-E in the posttreatment marrow increased by a mean of 4.15-fold, BFU-E by 3.37- fold, CFU-GM by 1.86-fold, and CFU-Mk by 1.96-fold as compared with their respective concentrations in the pretreatment marrows. This increase in the concentrations of marrow progenitors was accompanied by almost a doubling of the percentage of these cells in DNA synthesis as assessed by the 3H-thymidine suicide technique. These observations demonstrate that at the progenitor cell level the human marrow responds to therapeutic doses of Epo as an organ rather than by a selective expansion of the erythroid cell line.
Proliferation and maturation of human erythroid progenitors in liquid culture
Authors: Fibach E , Manor D , Oppenheim A , Rachmilewitz EA
Published: October 2019

Hemopoiesis is studied in vitro mainly in semisolid culture, where hemopoietic progenitors develop into discrete colonies. We describe a liquid culture system that supports the proliferation and maturation of human erythroid progenitors. We seeded mononuclear cells from the peripheral blood (PB) of patients with beta-thalassemia in liquid medium in the presence of conditioned medium from human bladder carcinoma cells. Seven days later, RBCs, normoblasts, granulocytes, and monocytes disappeared, and the number of lymphocytes dropped considerably. In contrast, erythroid colony-forming cells increased fourfold to tenfold. The next step entailed the removal of colony- stimulating factor (CSF) and CSF-secreting cells, the exclusion of macrophages by harvesting nonadherent cells, and the lysis of T lymphocytes by treatment with monoclonal rat antihuman lymphocyte antibodies (CAMPATH-1) and complement. Reculture of the remaining cells in liquid medium supplemented with recombinant erythropoietin (EPO) resulted in the exclusive development of erythroid cells, with myeloid cells reduced to less than 2%. Stainable hemoglobin (Hb) appeared on day 3, with over 85% of the population containing hemoglobin by day 11 and the cell number increasing from 0.2 X 10(6) to 3 X 10(6) mL. By permitting the manipulation of culture conditions and components and increasing the cell yield, the liquid system may facilitate quantitative analysis of growth kinetics as well as biochemical and immunologic characterization of the developing erythroid cell.
In vivo hematologic effects of recombinant interleukin-6 on hematopoiesis and circulating numbers of RBCs and WBCs
Authors: Ulich TR , del Castillo J , Guo KZ
Published: October 2019

Interleukin-6 (IL-6) administered as a single intravenous (IV) injection caused the following changes in the peripheral circulation of rats: (a) a biphasic neutrophilia with an initial peak at 1.5 hours and a second sustained wave of neutrophilia between four and 12 hours, (b) a mild lymphocytosis at 0.5 hours and a mild lymphopenia between 1.5 and four hours, and (c) a reticulocytosis between 12 and 24 hours. The bone marrow showed no significant changes at 1.5 hours, suggesting that the peripheral neutrophilia at that time is caused by demargination of intravascular neutrophils and not by release of marrow neutrophils. The bone marrow at 12 hours showed a mild left-shifted myeloid hyperplasia of myeloblasts and promyelocytes and a tremendous erythroid hyperplasia of intermediate and late normoblasts. The bone marrow at 24 hours showed a continued mild myeloid hyperplasia and striking erythroid hyperplasia. In conclusion, IL-6 in vivo acts as a stimulus for myelopoiesis and erythropoiesis and causes accompanying peripheral changes in the number of neutrophils, lymphocytes, and RBCs.
Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure
Authors: Schuster SJ , Badiavas EV , Costa-Giomi P , Weinmann R , Erslev AJ , Caro J
Published: October 2019

Erythropoietin, a plasma glycoprotein produced primarily by the kidney, is a growth and differentiation factor for erythroid progenitor cells. Production of renal erythropoietin is regulated by modulation of mRNA levels in response to changes in tissue oxygenation. Exposure to cobalt, a nonphysiologic stimulus for erythropoietin production, also acts by inducing mRNA accumulation. To determine whether variations in erythropoietin mRNA levels result from enhanced transcription of the erythropoietin gene, in vitro transcription reactions were performed using isolated rat kidney cell nuclei. Quantitation of specific nuclear RNAs labeled during in vitro transcription revealed active erythropoietin gene transcription in kidney nuclei from anemic-hypoxic and cobalt-treated animals while erythropoietin transcriptional activity was undetectable in normal kidney nuclei. Time course studies showed that stimulation of transcription begins between two and four hours following cobalt treatment and parallels the kinetics of mRNA and plasma erythropoietin accumulation. These results indicate that tissue hypoxia and cobalt exposure specifically enhance erythropoietin gene expression. This increase in erythropoietin production is regulated at least in part at the level of gene transcription.
Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture
Authors: Moore KL , Esmon CT , Esmon NL
Published: October 2019

Tumor necrosis factor (TNF), a mediator of the inflammatory response, induces tissue factor and decreases the expression of thrombomodulin (TM) on endothelial cells, thus shifting the hemostatic properties of the endothelium. To determine the mechanism of TM downregulation, bovine aortic endothelial cells in culture were treated with TNF (2 nmol/L) and the fate of TM followed. Both surface expressed TM (antigen and activity), and the total TM pool (measured by radioimmunoassay and activity in detergent extracts) dropped to less than or equal to 20% of control values within 12 hours of TNF treatment. TM was not found in an immunologically recognizable form in the supernatants of treated cultures. Chloroquine (greater than or equal to 100 mumol/L) was able to abrogate the TNF effect on the total TM pool but not the effect on surface-expressed TM activity. We conclude that TNF induces the internalization and subsequent degradation of the TM molecule. None of the components of the protein C anticoagulant pathway, either alone or in combination, prevented the TNF-dependent downregulation of TM antigen.
Activation/inactivation of human factor V by plasmin
Authors: Lee CD , Mann KG
Published: October 2019

The effect of human plasmin on human coagulation factor V was studied using isolated proteins. Incubation of factor V with plasmin resulted in a rapid increase in procoagulant activity, followed by a subsequent decline in the ability of factor V to serve as a cofactor in the prothrombinase complex. Identical results were obtained when these reactions were conducted in the presence of dansylarginine-N-(3-ethyl- 1,5-pentanediyl) amide (DAPA), indicating that the changes observed could not have occurred as a consequence of cleavage by alpha-thrombin. Analysis of the products of the reaction by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) revealed a temporal correlation between the rise and fall in factor V activity and the presence of several transient intermediates. These fragments are distinct from the subunits of alpha-thrombin-activated factor V (factor Va). The activation phase of the reaction was not significantly affected by the presence of phospholipid. In contrast, the rate of degradation of active fragments of factor V and the accompanying loss of activity were markedly enhanced in the presence of phospholipid vesicles. These data suggest that the action of plasmin upon factor V results in the transient formation of proteolytic fragments which express significant procoagulant activity.
Histidine-rich glycoprotein does not interfere with interactions between antithrombin III and heparin-like compounds on vascular endothelial cells
Authors: Shimada K , Kawamoto A , Matsubayashi K , Ozawa T
Published: October 2019

The role of histidine-rich glycoprotein in controlling heparin-like compounds on the endothelial cell surface is still unclear. The effects of this heparin-neutralizing protein on the interaction between antithrombin III and cultured porcine aortic endothelial cells were examined. Displacement of 125I-labeled antithrombin III specifically bound to endothelial cells by unlabeled histidine-rich glycoprotein was much less potent than that by unlabeled antithrombin III. One hundred- fold molar excess of histidine-rich glycoprotein displaced specific 125I-antithrombin III binding only by 20%. Furthermore, the endothelial cell-mediated acceleration of thrombin inactivation by antithrombin III was diminished by protamine sulfate, but was not affected by histidine- rich glycoprotein even at a histidine-rich glycoprotein/antithrombin III molar ratio of approximately 7:1. These data indicate that histidine-rich glycoprotein does not interfere with the interaction of endothelial cell heparin-like compounds with antithrombin III. Thus, it may not play an important role in the modulation of anticoagulant activity of endothelial cells in vivo, suggesting that the commonly accepted view of the probable function of this protein is erroneous.
Increased serum CD8 antigen level in childhood Hodgkin's disease relates to advanced stage and poor treatment outcome
Authors: Pui CH , Ip SH , Thompson E , Dodge RK , Brown M , Wilimas J , Carrabis S , Kung P , Berard CW , Crist WM
Published: October 2019

Measurement of the soluble form of CD8 antigen, a surface membrane component of suppressor/cytotoxic T cells, has yielded useful information relevant to prognosis in the lymphoid malignancies of childhood. We therefore determined pretreatment levels of serum CD8 antigen in 90 children with newly diagnosed Hodgkin's disease. The findings ranged widely, from 220 to 2,585 U/mL (median, 556 U/mL). In patients with advanced disease (stage III or IV), the median serum CD8 level was significantly higher than in those with less disease extension (stage I or II): 675 v 477 U/mL, P = .003. It was also higher in children with B symptoms compared with all others: 622 v 494 U/mL, P = .005. Cases with a histologic classification of mixed cellularity had a significantly higher median level of the antigen than did those classified as nodular sclerosis: 847 v 509 U/mL, P = .005. Finally, higher serum CD8 levels (greater than 430 U/mL) were significantly associated with an increased probability of treatment failure (P = .02). In a multivariate analysis, serum CD8 level retained its impact on treatment outcome after adjustment for other potentially useful prognostic factors, including disease stage, presence of B symptoms, histology, erythrocyte sedimentation rate, sex, age, and race. The prognostic strength shown by soluble CD8 in this analysis suggests that the antigen has clinical value. We postulate that increased CD8 levels in serum indicate enhanced suppressor T-cell activity, which may compromise the host's antitumor immunity, leading to unusually aggressive disease.
Clonal analysis of myelodysplastic syndromes: evidence of multipotent stem cell origin
Authors: Janssen JW , Buschle M , Layton M , Drexler HG , Lyons J , van den Berghe H , Heimpel H , Kubanek B , Kleihauer E , Mufti GJ
Published: October 2019

Restriction fragment length polymorphisms (RFLPs) of the X-chromosome genes hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) were studied in 34 female patients with primary myelodysplastic syndromes (MDS). Twelve patients (35%) were heterozygous at the HPRT or PGK loci for BamHI or BglI RFLPs, respectively. In eight patients showing PGK polymorphisms, clonality was determined by X-chromosome inactivation analysis. These included patients from different morphologic subtypes: four with refractory anemia (RA), two with RA and ring sideroblasts (RARS), one patient with RA with excess of blasts (RAEB), and one with chronic myelomonocytic leukemia (CMML). A monoclonal pattern of X-chromosome inactivation was observed in seven cases. In a further case characterized by bone marrow hypoplasia, peripheral blood (PB) leukocytes were polyclonal in origin. Following low-dose cytarabine therapy, reversion to polyclonal hematopoiesis was observed in a case of RAEB indicating the presence of residual normal hematopoietic stem cells with the capacity for marrow reconstitution. The clonal relation of lymphoid and granulocyte/monocyte lineages was studied directly in two cases of CMML exhibiting somatic mutations of N-ras or Ki-ras oncogenes. By selective oligonucleotide hybridization to ras gene sequences amplified in vitro by the polymerase chain reaction, a mutated ras allele was demonstrated in PB granulocytes, monocytes, and B and T lymphocytes of both patients. We conclude that MDS arise from a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation.
Protooncogene expression and the clinical characteristics of acute nonlymphocytic leukemia: A Leukemia Intergroup pilot study
Authors: Preisler HD , Raza A , Larson R , LeBeau M , Browman G , Goldberg J , Grunwald H , Volger R , Verkh L , Singh P
Published: October 2019

Northern blot analysis was used to assess the level of expression of five protooncogenes and histone H3 in the bone marrow cells of patients with acute nonlymphocytic leukemia (ANLL). The relationship between the level of gene expression and the clinical characteristics of the disease and response to therapy was studied. The levels of expression of c-myc and c-myb are weakly correlated and are unrelated to French- American-British (FAB) type of ANLL. The levels of expression of c-fms, c-fes, and c-fos are highly correlated with each other and are highest in leukemia with a monocytic component (c-fms v FAB = .71, c-fes v FAB = .75). High levels of c-myc expression are associated with a high probability of not responding to remission induction therapy (P = .004). The converse is true for c-fms expression levels. High levels of expression of c-myc or c-myb are associated with short remissions (P = .059 and .065, respectively), perhaps because they are associated with a high capacity for leukemic cell self-renewal and/or an inability of leukemic cells to differentiate in response to chemotherapy.
Treatment of acute lymphoblastic leukemia in adults with intensive induction, consolidation, and maintenance chemotherapy
Authors: Hussein KK , Dahlberg S , Head D , Waddell CC , Dabich L , Weick JK , Morrison F , Saiki JH , Metz E , Rivkin SE
Published: October 2019

The Southwest Oncology Group conducted a study of acute lymphoblastic leukemia (ALL) in adults over a 5-year period, testing the utility of the L-10M regimen initially described by the group from Memorial Sloan- Kettering Cancer Center. One hundred sixty-eight eligible patients were treated with this intensive combination chemotherapy regimen. One hundred fifteen (68%) achieved complete remission. With the current median follow-up time of 34.5 months, the median durations of remission, relapse-free survival, and overall survival were 22.9, 20.9, and 17.7 months, respectively. Only 35% of the patients over 50 years of age achieved a complete remission. Age was a significant prognostic factor for complete response, survival, relapse-free survival, and remission duration. In addition, a low initial WBC count was found to have a statistically significant association with longer remission duration. Responders between the ages of 20 and 49 years with WBC counts of less than 15,000 appear to have an exceptionally good prognosis.
The in vivo metabolism of recombinant human erythropoietin in the rat
Authors: Spivak JL , Hogans BB
Published: October 2019

We compared the in vivo plasma clearance and organ accumulation in anesthetized rats of 125I-labeled, recombinant human erythropoietin and 125I-labeled, desialylated recombinant erythropoietin. The immediate volume of distribution of 125I-labeled, recombinant erythropoietin approximated that of the plasma volume. Its plasma clearance was multiexponential, with an initial rapid distribution phase (t1/2 = 53 minutes) and a slower elimination phase (t1/2 = 180 minutes). Organ accumulation of labeled recombinant erythropoietin, as compared with 125I-labeled human albumin, was negligible until 30 minutes after injection when small amounts appeared in the kidneys and bone marrow. Only 24% of the 125I-labeled, desialylated recombinant erythropoietin was recovered immediately after injection, and 96% of the hormone was cleared from the plasma with a t1/2 of 2.0 minutes. The bulk of the desialylated hormone accumulated in the liver where it was rapidly catabolized and its breakdown products released back into the plasma. Significantly, in contrast to unmodified erythropoietin, there was also early accumulation of desialylated hormone in the kidneys, marrow, and spleen. Desialylated orosomucoid but not orosomucoid, yeast mannan, or dextran sulfate 500 inhibited the rapid plasma clearance and hepatic accumulation of desialylated erythropoietin. Oxidation of the desialylated hormone restored its plasma recovery and clearance to normal but rendered it biologically inactive, and accumulation in organs other than the kidney was negligible.
Differential biologic effects resulting from bimodal binding of recombinant human tumor necrosis factor to myeloid leukemia cells
Authors: Ishikura H , Hori K , Bloch A
Published: October 2019

Human recombinant tumor necrosis factor (rTNF) bound to ML-1 and HL-60 human myeloid leukemia cells in a bimodal manner. Saturable high- affinity binding was maximal at approximately 3 nmol/L rTNF, whereas saturable low-affinity binding reached its maximum at approximately 30 nmol/L. As analyzed by computer program, the observed data fit a two- receptor site model, with p less than .05. High-affinity binding concentrations of rTNF caused the differentiation of both cell lines, whereas low-affinity binding concentrations abolished this effect in a concentration-dependent manner. Thus, the type of biologic response elicited with rTNF in these cells is a function of the concentration at which the factor is applied. If generally applicable, this bimodal effect may require consideration when rTNF is to be used therapeutically.
Interleukin-6 synergizes with M-CSF in the formation of macrophage colonies from purified human marrow progenitor cells
Authors: Bot FJ , van Eijk L , Broeders L , Aarden LA , Lowenberg B
Published: October 2019

We examined the in vitro stimulative effects of recombinant human interleukin-6 (IL-6, or interferon-beta 2) on purified human bone marrow progenitor cells. IL-6 alone or in combination with erythropoietin (Epo), IL-3, GM-CSF, or G-CSF did not induce colony formation. However, IL-6 strongly synergized with M-CSF in stimulating macrophage colony formation (colony numbers and size). The magnitude of IL-6 synergism with M-CSF was dose dependent; maximal potentiation of M- colony formation was evident at approximately 100 to 1,000 U/mL IL-6. When the addition of IL-6 to M-CSF-supplemented cultures was delayed for more than one day after the beginning of culture, enhancement of macrophage colony formation was lost. IL-6 stimulation of M-CSF- responsive colony formation was not apparent when nonpurified marrow cells were plated, most likely due to endogenous IL-6 release. These observations suggest that IL-6, in addition to playing a role in B- lymphocyte proliferation can potentiate the human immune defence mechanism by stimulating monocyte-macrophage development as well.
Inhibition and complexation of activated protein C by two major inhibitors in plasma
Authors: Heeb MJ , Espana F , Griffin JH
Published: October 2019

To determine the major physiologic inhibitors of activated protein C (APC), plasma was incubated with APC or with Protac C and subjected to immunoblotting. APC:inhibitor complexes gave two major bands reacting with antiprotein C antibodies when immunoblotted on nondenaturing gels, and additional minor bands that varied between serum and plasma. Formation of one of the two major bands of APC:inhibitor complex, but not the other, was stimulated by heparin and only this band reacted with antibodies to the previously described APC inhibitor that is here designated PCI-1. Plasma immunodepleted of PCI-1 formed complexes with APC as visualized with antiprotein C but not anti-PCI-1 antibodies, and exhibited heparin-independent inhibition of APC activity, providing evidence for the existence of a second major physiologic APC inhibitor, PCI-2. Formation of APC:PCI-2 complexes in PCI-1-depleted plasma paralleled inhibition of APC amidolytic activity. PCI-2 was separated from PCI-1 and partially purified using column chromatography. PCI-2 formed inactive complexes of approximately 110,000 molecular weight (mol wt) with APC suggesting PCI-2 has an approximate mol wt of 50,000. Thus, inhibition of APC in plasma involves two major distinct 50,000 mol wt inhibitors, the heparin-dependent PCI-1 and the heparin- independent PCI-2.
Identification of a F.VIII epitope recognized by a human hemophilic inhibitor
Authors: Lubahn BC , Ware J , Stafford DW , Reisner HM
Published: October 2019

Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.
Monocytoid B-cell lymphoma: its evolution and relationship to other low- grade B-cell neoplasms
Authors: Traweek ST , Sheibani K , Winberg CD , Mena RR , Wu AM , Rappaport H
Published: October 2019

Monocytoid B-cell lymphoma (MBCL) is a newly recognized B-cell neoplasm of uncertain histogenesis. The cytologic features of the neoplastic monocytoid B lymphocytes are virtually identical to those of hairy cell leukemia (HCL). As with HCL, progression of MBCL to a higher histologic grade is very unusual. However, whereas circulating leukemic cells are a characteristic feature of HCL, peripheral blood involvement has not been reported in MBCL. We recently studied a patient with MBCL of the spleen and axillary lymph nodes who developed peripheral blood involvement by MBCL cells. Unlike the cells of HCL, the circulating MBCL cells exhibited strong acid phosphatase activity that was tartrate sensitive. The leukemic cells had the antigenic phenotype IgM lambda, CD20+, CD11c+, CD5-, CD25(TAC)-, and PCA-1-. Immunogenetic studies of both lymph node and peripheral blood cells revealed identical immunoglobulin heavy-chain gene rearrangements. When compared with a series of HCL, the immunophenotype was similar except for the absence of PCA-1 and TAC. Progression of the MBCL to a large cell lymphoma, also expressing IgM lambda, was documented in an abdominal lymph node of this patient. Therefore, although rare, peripheral blood involvement by lymphoma cells may occur during the course of MBCL and should be distinguished from HCL with cytochemical and immunophenotypic studies. In addition, comparison of the clinical, pathologic, and immunologic features of MBCL with those of other low-grade B-cell neoplasms suggests that a close lineage relationship exists between MBCL and HCL.