bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of colony-stimulating factor genes by normal human mesothelial cells and human malignant mesothelioma cells lines in vitro
Authors: Demetri GD , Zenzie BW , Rheinwald JG , Griffin JD
Published: October 2019

We investigated normal human mesothelial cells and human malignant mesothelioma cell lines for the ability to produce hematopoietic colony- stimulating factors (CSFs) in culture. Early passage cultures of normal diploid human mesothelial cells spontaneously expressed detectable levels of M-CSF mRNA transcripts, but lacked detectable transcripts for GM-CSF or G-CSF. Exposure of normal mesothelial cells to epidermal growth factor (EGF), lipopolysaccharide (LPS), or tumor necrosis factor (TNF) induced expression of G-CSF mRNA. The combination of EGF and TNF induced threefold more G-CSF transcripts than did either factor alone. GM-CSF transcripts were induced only by the combination of TNF and EGF. Interleukin-1 beta (IL-1 beta) transcripts were induced by EGF, TNF, or LPS and were inhibited by hydrocortisone (HC). All malignant mesothelioma cell lines tested also spontaneously expressed M-CSF transcripts. However, in contrast to normal mesothelial cells, two of four malignant mesothelioma cell lines also autonomously expressed G- CSF and GM-CSF transcripts without TNF, EGF, or LPS stimulation. Secretion of biologically active CSFs was confirmed by testing media conditioned by the various cell types examined. The detection of biologically active CSFs correlated well with the presence of detectable CSF transcripts by Northern analysis. These data indicate that (a) normal human mesothelial cells spontaneously express detectable levels of M-CSF mRNA in culture; (b) EGF is an essential cofactor for optimal induction of G-CSF and GM-CSF expression; (c) exposure of normal mesothelial cells to inflammatory mediators such as LPS and TNF increases the levels of transcripts for CSFs and IL-1 beta; and (d) as compared with normal human mesothelial cells, some cell lines of human malignant mesothelioma exhibit aberrant gene expression for multiple cytokines, including G-CSF, GM-CSF, IL-1 beta, and IL-6.
Expression of the c-fos protooncogene by human and murine erythroblasts
Authors: Caubet JF , Mitjavila MT , Dubart A , Roten D , Weil SC , Vainchenker W
Published: October 2019

The expression of the c-fos protooncogene was investigated by in situ hybridization in normal murine bone marrow cells. A strong signal was found in murine marrow cells having the morphologic features of erythroblasts. This result was confirmed in human marrow cells using a double labeling technique (in situ hybridization and immunocytochemistry). A majority (70%) of the cells expressing c-fos mRNA were glycophorin A-positive. In contrast, granulocytic precursors (CD 15-positive) or monocytes and their precursors (CD 14-positive cells) did not significantly hybridize with the c-fos probe. In addition, c-fos mRNA (2.2Kb) was detected by Northern blotting in RNA extracted from homogeneous populations of erythroblasts obtained by immune panning from fetal liver and from adult blood BFU-E-derived colonies. Fos protein was also detected in erythroblasts by immunofluorescence. The high level of c-fos mRNA previously found in hematopoietic tissue should therefore be related to the transcription of the c-fos gene during terminal erythroid differentiation.
Biotinylated recombinant human erythropoietins: bioactivity and utility as receptor ligand [see comments]
Authors: Wojchowski DM , Caslake L
Published: October 2019

Recombinant human erythropoietin labeled covalently with biotin at sialic acid moieties has been prepared, and has been shown to possess high biological activity plus utility as a receptor ligand. Initially, the effects on biological activity of covalently attaching biotin to erythropoietin alternatively at carboxylate, amino, or sialic acid groups were compared. Biotinylation of erythropoietin at carboxylate groups using biotin-amidocaproyl hydrazide plus 1-ethyl-3-(3- dimethylaminopropyl) carbodiimide led to substantial biological inactivation, although biotinylated molecules retained detectable activity when prepared at low stoichiometries. Biotinylation at amino groups using sulfosuccinimidyl 6-(biotinamido) hexanoate resulted in a high level of biological inactivation with little, if any, retention of biological activity, regardless of labeling stoichiometries. Biotinylation at sialic acid moieties using periodate and biotinamidocaproyl hydrazide proceeded efficiently (greater than 95% and 80% labeling efficiencies for human urinary and recombinant erythropoietin, respectively) and yielded stably biotinylated erythropoietin molecules possessing comparably high biological activity (ie, 45% of the activity of unmodified hormone). Utility of recombinant biotin-(sialyl)-erythropoietin (in combination with 125I-streptavidin) in the assay of cell surface receptors was demonstrated using two distinct murine erythroleukemia cell lines, Friend 745 and Rauscher Red 1. The densities and affinities of specific hormone binding sites were 116 +/- 4 sites, 3.3 +/- 0.4 nmol/L kd and 164 +/- 5 sites, 2.7 +/- 0.4 nmol/L kd, respectively. It is predicted that the present development of biotin-(sialyl)-erythropoietin as a chemically and biologically stable, bioactive ligand will assist in advancing an understanding of the regulated expression and physicochemistry of the human and murine erythropoietin receptors.
Macrophage colony-stimulating factor (CSF-1) is expressed by spontaneously outgrown EBV-B cell lines and activated normal B lymphocytes
Authors: Reisbach G , Sindermann J , Kremer JP , Hultner L , Wolf H , Dormer P
Published: October 2019

Human B lymphocytes activated by mitogens or infected by Epstein Barr virus (EBV) have previously been shown to release colony-stimulating activity (CSA) supporting the growth of normal human bone marrow progenitors. We established five different human EBV-B cell lines spontaneously outgrown from nonmalignant peripheral blood cells and long-term bone marrow cultures. CSA derived from all of these lines induces the growth of murine macrophage colonies, whereas virtually no human bone marrow cell progenitors were stimulated. As observed in the tumor cell line MIA PaCa-2, a 4.3-kilobase (kb) transcript was detected in all cases using a human colony-stimulating factor (CSF)-1 probe. Expression of this transcript can be further stimulated within three hours upon addition of phorbol myristate acetate (PMA). The highly purified native protein exerting macrophage colony-stimulating activity (M-CSA) exhibits a molecular size of approximately 75 to 97 Kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The identity of EBV-B cell derived M-CSA with human urinary CSF-1 was confirmed by a complete neutralization of macrophage CSA by an antihuman urinary CSF-1 antiserum. Normal human B lymphocytes purified from tonsils or from mononuclear blood cells also express CSF-1 upon stimulation with Staphylococcus aureus Cowan I. No CSF-1 expression, however, could be detected in normal resting B lymphocytes or in the Burkitt lymphoma cell line RAJI.
Enhanced proteolysis of plasma von Willebrand factor in thrombotic thrombocytopenic purpura and the hemolytic uremic syndrome
Authors: Mannucci PM , Lombardi R , Lattuada A , Ruggenenti P , Vigano GL , Barbui T , Remuzzi G
Published: October 2019

To examine whether enhanced in vivo proteolysis of von Willebrand factor (vWF) would account for the reported loss of larger multimers in acute thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS), we studied eight patients with acute TTP/HUS whose blood samples were collected into an anticoagulant containing a cocktail of protease inhibitors to impede in vitro proteolysis. In all, enhanced proteolytic degradation of vWF was expressed as a relative decrease in the intact 225-Kd subunit of vWF and a relative increase in the 176-Kd fragment. However, instead of the loss of larger forms of normal multimers reported by other investigators, the plasma of all but one of our patients (whether they had TTP or HUS) contained a set of larger than normal (supranormal) multimers. Hence, although proteolytic fragmentation of vWF was enhanced during acute TTP/HUS, this phenomenon was not associated with the loss of larger multimers. In the five patients who survived the acute disease and underwent plasma exchange (three with HUS and two with chronic relapsing TTP), subunits and fragments returned to normal values, and supranormal multimers were no longer detected in plasma. In conclusion, even though vWF proteolysis is enhanced in acute TTP/HUS, it does not lead to loss of larger multimers.
Human plasma extrinsic pathway inhibitor activity: II. Plasma levels in disseminated intravascular coagulation and hepatocellular disease
Authors: Warr TA , Rao LV , Rapaport SI
Published: October 2019

Plasma or serum extrinsic pathway inhibitor (EPI) activity was measured in 24 patients with disseminated intravascular coagulation (DIC) and in 23 patients with severe hepatocellular disease. EPI was measured as activity in a test sample that inhibited factor VIIa/tissue factor (TF)- catalyzed activation of 3H-factor IX (activation peptide release) in the presence of factor X. Of the 24 patients with DIC, 13 had sepsis and five had metastatic carcinoma, disorders in which tissue factor is believed to initiate DIC. EPI activity ranged from 68% to 300% (mean 134% +/- 50%). Serial measurements in nine patients failed to show depletion of EPI activity coincident with worsening DIC. DIC induced by tissue factor or other activating materials may progress despite normal EPI levels. In the patients with liver disease, of whom 15 had decompensated chronic hepatocellular disease (two fatal cases) and eight had acute fulminant liver failure (seven fatal cases), plasma or serum EPI activity varied from less than 20% to 194%. Values were distributed in a bimodal fashion. EPI activity could not be correlated with either the etiology of the liver disease or the degree of prolongation of the prothrombin time. Patients with chronic hepatocellular disease who survived had normal or elevated EPI activity. Patients with fatal hepatic dysfunction had low, normal, or high values for EPI activity. This must mean that secretion of EPI from cells other than hepatocytes can maintain normal plasma EPI levels.
Malaria and red cell genetic defects
Authors: Nagel RL , Roth EF Jr
Published: October 2019

The study of inherited RBC resistance to malaria has increased our knowledge of the biochemistry and physiology of the host-parasite interaction and suggested potential sites for therapeutic intervention. Discovery by Jensen and Trager of the in vitro culture system for P falciparum has facilitated research in this area. Known RBC defects may affect invasion, growth, or merozoite liberation (Fig 1). Significant advances made in understanding mechanisms underlying protection against malaria should not obscure the fact that the data are far from complete. More knowledge is needed about the influence of the erythrocyte cytoskeleton on invasion and growth of parasites as well as the potential role of phospholipids, erythrocyte enzymes other than G6PD, or other metabolic products. Application of DNA analysis and recombinant technology may have an increasing impact on study of the interaction of RBC defects with malarial parasites.
Inhibition of platelet-von Willebrand factor binding to platelets by adhesion site peptides
Authors: Parker RI , Gralnick HR
Published: October 2019

Synthetic peptides containing the adhesion site recognition sequences present on the A alpha and gamma chains of fibrinogen were studied for their effect on the binding of endogenous platelet-von Willebrand factor (vWF) and exogenous plasma-vWf to thrombin-stimulated platelets. In agreement with previously reported data, the tetrapeptide consisting of the RGDS sequence was a more potent inhibitor of plasma-vWf binding to platelets than was the pentadecapeptide of the carboxy terminus of the fibrinogen gamma-chain (IC50 10.6 mumol/L for the RGDS tetrapeptide v 44.9 mumol/L for the gamma-chain pentadecapeptide). No apparent synergy in the inhibition of plasma-vWf binding was noted when the RGDS and gamma-chain peptides were used together (IC50 15.2 mumol/L). In contrast, the gamma-chain peptide was significantly more inhibitory than was the RGDS tetrapeptide on the binding of platelet-vWf to platelets (IC50 1.4 mumol/L for the gamma-chain pentadecapeptide v 4.5 mumol/L for the RGDS tetrapeptide, P less than .05), and there was significant synergy in the inhibition of platelet-vWf binding noted when the gamma-chain and RGDS peptides were used together (IC50 0.04 mumol/L). These results indicate that the binding of platelet-vWf to its receptor on the platelet glycoprotein IIb/IIIa complex involves both the RGDS and gamma-chain recognition sites. In contrast to the results with plasma-vWf binding, the gamma-chain recognition site appears to be more important than the RGDS recognition site in platelet- vWf binding to platelets.
Increased circulating CSF-1 (M-CSF) in myeloproliferative disease: association with myeloid metaplasia and peripheral bone marrow extension
Authors: Gilbert HS , Praloran V , Stanley ER
Published: October 2019

Myeloproliferative disease (MPD) is heterogeneous in phenotypic expression and may display features consistent with expansion and activation of the monocyte/macrophage population during its course. The role of colony-stimulating factor-1 (CSF-1) in the pathophysiology of MPD was investigated by measuring circulating CSF-1 levels and examining their relationship to disease phenotype. Serum CSF-1 concentrations, measured by radioimmunoassay, were elevated in all MPD phenotypes. CSF-1 levels differed significantly between groups of patients with essential thrombocythemia, polycythemia vera, and postpolycythemic or agnogenic myeloid metaplasia (in ascending order). CSF-1 serum levels were positively correlated with spleen size and the degree of peripheral bone marrow extension, determined by scintigraphy using a macrophage-seeking isotope. There was no correlation between CSF-1 concentration and circulating levels of erythrocytes, neutrophils or platelets, or the presence of bone marrow fibrosis. Elevated serum CSF-1 levels appear to be associated with an expanded monocyte/macrophage population in MPD. In view of the known cooperativity between CSF-1 and other growth factors in regulating hematopoiesis, the finding of increased serum CSF-1 concentrations and its association with myeloid metaplasia and bone marrow extension may indicate a pathophysiologic role for CSF-1 in determining the phenotypic expression of MPD.
Interleukin-6 is a potent thrombopoietic factor in vivo in mice
Authors: Ishibashi T , Kimura H , Shikama Y , Uchida T , Kariyone S , Hirano T , Kishimoto T , Takatsuki F , Akiyama Y
Published: October 2019

To determine the biologic activity of interleukin-6 (IL-6) on megakaryocytopoiesis and thrombocytopoiesis in vivo, the cytokine was administered intraperitoneally to mice every 12 hours at varying doses for five days or for varying time intervals, based on the kinetic analysis of IL-6 serum levels indicating the peak of 40 minutes following injection, with no detection at 150 minutes. A dose-response experiment showed that IL-6 increased platelet counts in a dose- dependent fashion at a plateau stimulation level of 5 micrograms. Administration of 5 micrograms of IL-6 reproducibly elevated platelet counts at five days by approximately 50% to 60% of increase. Moreover, a striking increase in megakaryocytic size in response to IL-6 was elicited by the treatment, but no change in megakaryocyte numbers; whereas IL-6 administration did not expand CFU-MK numbers. The in vivo studies in this manner had negligible effects on other hematologic parameters, with the minor exception of monocyte levels. These data show that IL-6 acts on maturational stages in megakaryocytopoiesis and promotes platelet production in vivo in mice, suggesting that IL-6 functions as thrombopoietin.
Changes in the plasma levels of type 1 and type 2 plasminogen activator inhibitors in normal pregnancy and in patients with severe preeclampsia
Authors: Estelles A , Gilabert J , Aznar J , Loskutoff DJ , Schleef RR
Published: October 2019

This report defines the nature of the molecules responsible for the increased plasma plasminogen activator inhibitor (PAI) activity in preeclamptic patients and the relationship of these inhibitors to the severity of placental damage in preeclampsia. Clinical groups consisting of pregnant women with either severe preeclampsia or chronic hypertension with superimposed severe preeclampsia, as well as normal pregnant and nonpregnant women, were analyzed in a panel of functional and immunologic assays for PAI-1 and PAI-2. Pure severe preeclamptic patients in their third trimester showed a significant increase in both antigenic (136 ng/mL) and functional (5.76 U/mL) type 1 PAI (PAI-1) as compared with normal third-trimester pregnant women (34.8 ng/mL and 2.57 U/mL, respectively). In contrast, antigenic (186 ng/mL) and functional (5.76 U/mL) levels of type 2 PAI (PAI-2) were significantly lower in the pure severe preeclampsia group as compared with the values of the normal pregnant group (269 ng/mL and 9.58 U/mL, respectively). The patients with chronic hypertension and superimposed severe preeclampsia exhibited PAI-2 levels comparable to those of the pure preeclamptic group, whereas their antigenic and functional PAI-1 levels were intermediate (94 ng/mL and 3.25 U/mL, respectively) between the normal pregnant and the pure preeclamptic groups. During early puerperium of both normal pregnant women and patients, plasma PAI-1 antigen and activity decreased within one day to approximately the levels detected in normal nonpregnant women, while PAI-2 levels remained elevated for over 11 days. Similar results were obtained in plasma samples obtained from citrated blood and blood collected with an anticoagulant/antiplatelet mixture, suggesting that increased PAI-1 levels in preeclamptic patients were not due to platelet activation in vitro. In preeclamptic patients, a positive correlation between birth weight and PAI-2 values was observed (r = .64, P less than .05), whereas birth weight was inversely correlated with both PAI-1 levels and total PAI activity (r = -.6, P less than .005 and r = -.76, P less than .005 respectively). Preeclamptic patients with extensive placental infarction exhibited higher plasma PAI activity (24.1 U/mL v 11.6 U/mL) and PAI-1 values (305 ng/mL v 80.9 ng/mL) than preeclamptic patients without extensive placental infarction. In contrast, PAI-2 levels were reduced in preeclamptic patients with infarction in comparison with those of patients without infarction (141 ng/mL v 212.9 ng/mL). Our data indicate that increases in the level of PAI-1 accounts for the high plasma PAI activity in severe preeclampsia as measured using single-chain t-PA.
Covalent modification of platelet proteins by palmitate
Authors: Muszbek L , Laposata M
Published: October 2019

Covalent attachment of fatty acid to proteins plays an important role in association of certain proteins with hydrophobic membrane structures. In platelets, the structure of many membrane glycoproteins (GPs) has been examined in detail, but the question of fatty acid acylation of platelet proteins has not been addressed. In this study, we wished to determine (a) whether platelet proteins could be fatty acid acylated; and, if so, (b) whether these modified proteins were present in isolated platelet membranes and cytoskeletal fractions; and (c) if the pattern of fatty acid acylated proteins changed on stimulation of the platelets with the agonist thrombin. We observed that in platelets allowed to incorporate 3H-palmitate, a small percentage (1.37%) of radioactivity incorporated into the cells became covalently bound to protein. Selective cleavage of thioester, thioester plus O-ester, and amide-linked 3H-fatty acids from proteins, and their subsequent analysis by high-performance liquid chromatography (HPLC) indicated that the greatest part of 3H-fatty acid covalently bound to protein was thioester-linked 3H-palmitate. By sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography, at least ten major radiolabeled proteins were detected. Activation of platelets by thrombin greatly increased the quantity of 3H- palmitoylated proteins associated with the cytoskeleton. Nearly all radiolabeled proteins were recovered in the membrane fraction, indicating that these proteins are either integral or peripheral membrane proteins or proteins tightly associated to membrane constituents. Components of the GPIIb-IIIa complex were not palmitoylated. Thus, platelet proteins are significantly modified posttranslationally by 3H-palmitate, and incorporation of palmitoylated proteins into the cytoskeleton is a prominent component of the platelet response to thrombin stimulation.
Distribution of integrins on human peripheral blood mononuclear cells
Authors: Klingemann HG , Dedhar S
Published: October 2019

The receptors for fibronectin (FN-R) and vitronectin (VN-R) belong to a family of integral membrane glycoproteins known to be involved in cell- extracellular matrix and cell-cell interactions named integrins (FN-R = beta 1 integrin and VN-R = beta 3 integrin). Adhesion studies using FN- coated plastic dishes and highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) showed a strong binding of monocytes and T lymphocytes to FN but virtually no binding of B cells to FN. Binding of monocytes and T cells to FN could be partially inhibited by a hexapeptide (GRGDSP) containing the adhesive peptide sequence Arg-Gly- Asp (RGD) as well as by an anti-FN-R antibody. The distribution of beta 1 and beta 3 integrin complexes on PBMCs was characterized by immunoprecipitation of detergent extracts of 125I-labeled cells using polyclonal antibodies against these two receptors. Two surface polypeptides corresponding to the alpha and beta chains of FN-R and VN- R were found on all three cell types. To characterize these receptors further, monoclonal antibodies (MoAbs) against the very late antigens (VLAs) 1, 3, and 5 were used for immunoprecipitation studies. Monocytes and T cells reacted with VLA 5 that was previously identified as the human FN receptor, whereas no labeling with anti-VLA 5 could be shown for B cells. When cell populations were cultured in 10% human serum for 24 hours, an increase in beta 1-integrin+ monocytes and T cells was observed. The number of beta 3-integrin+ cells remained essentially unchanged. The presence of beta 1 and beta 3 integrins on monocytes as well as on T and B lymphocytes may be of significance in the ability of these cells to interact with each other and participate in hematopoiesis and certain immune reactions.
CML-T1: a cell line derived from T-lymphocyte acute phase of chronic myelogenous leukemia
Authors: Kuriyama K , Gale RP , Tomonaga M , Ikeda S , Yao E , Klisak I , Whelan K , Yakir H , Ichimaru M , Sparkes RS
Published: October 2019

Most data suggest that malignant transformation in chronic myelogenous leukemia (CML) occurs in hematopoietic stem cell that is the progenitor of myelopoiesis and of B but not T lymphopoiesis. We established a T- lymphoid cell line (CML-T1) from a person with Ph-chromosome-negative CML in acute phase. Evidence of its T-lymphocyte origin includes the pattern cytochemical reactivity, reactivity with anti-T-cell monoclonal antibodies (MoAbs), and rearrangement of the beta-T-cell receptor (TCRB) gene. CML-T1 cells have features of type IV thymocytes. Cytogenetic analyses indicate a 47,XX, del(11), t(6;7)(q23;q24), +mar karyotype. CML-T1 cells exhibit molecular changes typical of CML, including translocation of the ABL protooncogene from chromosome 9 to 22, rearrangement of the BCR gene, and transcription of a chimeric BCR- ABL messenger RNA (mRNA). The ABL insertion on chromosome 22 appears interstitial, similar to other cases of Ph-chromosome-negative CML. These data clearly indicate that T cells can be involved in acute-phase CML. CML-T1 should be useful in studying this process as well as that underlying Ph-chromosome-negative CML.
Leg ulcers in patients with sickle cell disease [see comments]
Authors: Koshy M , Entsuah R , Koranda A , Kraus AP , Johnson R , Bellvue R , Flournoy-Gill Z , Levy P
Published: October 2019

During the entry examination, leg ulcers were present in 2.5% of 2,075 patients 10 years of age and older with sickle cell disease who entered into the Cooperative Study of Sickle Cell Disease (CSSCD) between 1979 and 1986. Prevalence rates were highest among patients with sickle cell anemia and sickle cell anemia with thalassemia genotypes. Among sickle cell anemia patients free of ulcers at entry, the overall incidence was 5.73 per 100 person years in those having associated alpha-thalassemia and 9.97 for those without. Among sickle cell anemia patients with two alpha genes, the estimated incidence of leg ulcers is 2.38 per 100 person years and 6.12 per 100 person years among sickle cell anemia patients with three alpha genes (P less than .05). In both groups, the incidence was highest among those patients over 20 years of age and considerably higher among males than females (P less than .001). Leg ulcers were nonexistent in patients with sickle beta plus thalassemia and sickle hemoglobin C disease. Low steady-state hemoglobin is associated with a higher incidence of ulcer formation (P less than .0001) in sickle cell anemia patients. The protective effect of hemoglobin F is apparent at all levels of total hemoglobin among sickle cell anemia patients and those with associated alpha-thalassemia.
Antibody-mediated marrow failure after allogeneic bone marrow transplantation
Authors: Barge AJ , Johnson G , Witherspoon R , Torok-Storb B
Published: October 2019

Marrow graft failure observed in association with histocompatibility differences between donor and recipient is often attributed to rejection mediated by host-derived cytolytic T lymphocytes. The data presented in this report indicate that persistent host antibodies specific for donor antigen may also mediate graft failure, either by antibody-dependent cell-mediated cytotoxicity (ADCC), or complement- mediated cytotoxicity. In the case of HLA Class I disparity, where all donor cells express the target antigen, the presence of alpha-donor antibody was associated with complete graft failure and death. In the case of ABO blood group antigen disparity, the presence of alpha-donor antibody resulted in erythroid hypoplasia. The latter cases proved informative insofar as they established that host antibodies could persist for more than 18 months after chemoradiotherapy and impair marrow function.
Molecular characterization of human factor XSan Antonio
Authors: Reddy SV , Zhou ZQ , Rao KJ , Scott JP , Watzke H , High KA , Jagadeeswaran P
Published: October 2019

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.
Nuclear oncoprotein expression as a function of lineage, differentiation stage, and proliferative status of normal human hematopoietic cells
Authors: Kastan MB , Stone KD , Civin CI
Published: October 2019

Relative levels of the nuclear oncoproteins c-myb, c-myc, and c-fos were determined in selected subpopulations of normal human bone marrow (BM) cells using a flow cytometric assay which simultaneously detects a cell-surface antigen (as a marker of lineage and stage of maturation) and levels of an intracellular protein. At least two monoclonal antibodies directed against each oncoprotein and specific peptide inhibition controls were used for these determinations. Hematopoietic progenitor cells (CD34+) express the highest levels of c-myb and c-myc, whereas c-fos levels in CD34+ progenitor cells are similar to c-fos levels in mature monocytes and granulocytes. Granulocytes are the only hematopoietic cells examined which do not express detectable levels of c-myb and c-myc. The levels of these oncoproteins in these normal, unstimulated BM cell populations were more closely linked to lineage and maturation stage than to the proliferative status of the given population, as determined by either DNA staining or expression of the cell-cycle specific nuclear protein, Ki67. This flow cytometric assay helps in interpreting the significance of oncoprotein levels in leukemia cells by allowing direct comparisons of a leukemia with the phenotypically similar “normal counterpart control” cell population in normal BM.
Effects of interleukin-3 and erythropoietin on in vivo erythropoiesis and F-cell formation in primates
Authors: Umemura T , al-Khatti A , Donahue RE , Papayannopoulou T , Stamatoyannopoulos G
Published: October 2019

To test the in vivo cooperativity between interleukin-3 (IL-3) and erythropoietin (Epo) in stimulating erythropoiesis and hemoglobin F (HbF) production in primates, we administered recombinant human IL-3 and recombinant human Epo to baboons and macaques. The effect of these treatments was assessed by serial bone marrow cultures and by measuring HbF production in the progeny of bone marrow progenitors and in peripheral-blood reticulocytes. Administration of IL-3 alone to hematologically normal or anemic baboons produced an early increase in erythroid colony-forming units (CFUe) and erythroid clusters (e- clusters) with an increase in reticulocyte counts and a late increment in the relative frequency of erythroid burst-forming units (BFUe). In parallel to the increase in peripheral-blood reticulocytes, IL-3 increased the frequency of F reticulocytes in the normal and anemic animals. When administration of IL-3 was followed by administration of Epo, expansion in all classes of erythroid progenitors and increase in reticulocytes occurred, beyond the levels observed when the animals were treated with Epo alone. The combination of IL-3 and Epo, however, did not increase consistently the rate of F reticulocytes beyond the level induced by Epo alone. These results suggest that IL-3 enhances the effect of Epo on erythropoiesis, but the combination of the two growth factors does not lead to a preferential and significant enhancement of HbF production.
Autoantibodies against platelet membrane glycoproteins in children with acute and chronic immune thrombocytopenic purpura
Authors: Berchtold P , McMillan R , Tani P , Sommerville-Nielsen S , Blanchette VS
Published: October 2019

The autoimmune nature of chronic immune thrombocytopenic purpura (ITP) in adults is widely accepted. In contrast, the pathogenetic mechanism in acute and chronic ITP in children is not known. In this report, we studied 39 children with destructive thrombocytopenia, 15 patients with acute ITP and 24 patients with chronic ITP. Platelet autoantibodies to platelet glycoprotein IIb/IIIa were detected in 14 of 24 patients (58.3%) in the chronic ITP group and in four of 15 (26.7%) with acute ITP. Binding ratios (+/- SD) of positive patients were significantly greater (P = .01) in chronic ITP (8.0 +/- 9.1) when compared with those of acute ITP where the binding ratios were only slightly above the normal range (1.9 +/- 0.4). The results show that autoantibodies against platelet glycoproteins are present in the majority of children with chronic ITP confirming the autoimmune nature of this disorder. The minimal elevation seen in the positive children with acute ITP suggests a different pathogenetic mechanism. These data suggest that this approach may be useful in differentiating acute from chronic ITP patients.
Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface
Authors: Duperray A , Troesch A , Berthier R , Chagnon E , Frachet P , Uzan G , Marguerie G
Published: October 2019

The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium- dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro- GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.
Localization of epitopes for human factor VIII inhibitor antibodies by immunoblotting and antibody neutralization
Authors: Scandella D , Mattingly M , de Graaf S , Fulcher CA
Published: October 2019

Human factor VIII(FVIII) inhibitors are pathologic, circulating antibodies that inactivate FVIII. We have examined the location of epitopes on the FVIII protein for inhibitors from hemophilia A and nonhemophilic individuals. The inhibitors were of type I or type II in the kinetics of their inactivation of FVIII. A cDNA clone of human FVIII was used to express defined FVIII protein fragments in Escherichia coli for immunoblotting with inhibitor plasma. An epitope for 18 heavy-chain inhibitors was localized to the aminoterminal 18.3 Kd of the A2 domain. Two of these inhibitors also recognized an epitope located between A1 and A2 domains. Similarly, an epitope for 23 light- chain inhibitors was localized to the C2 domain. Weaker epitopes for 13 of the same inhibitors within the C1 and C2 domains were also observed. Four of the 23 inhibitors in addition bound strongly to the A3 domain. Most inhibitors (22 of 23) were neutralized in vitro only by the FVIII fragments to which they bound on immunoblots; however, one inhibitor that was neutralized by a fragment containing the A1 domain did not bind to it on immunoblots. Conversely, 3 of 3 inhibitors that bound to the A3 domain and 5 of 15 that bound to the A2 domain were not neutralized by the corresponding fragments. The epitope specificity of an inhibitor did not depend on its source or type. Our results show that FVIII inhibitors bind to limited areas within the heavy and light chains of FVIII. Some inhibitor plasmas contain additional antibodies that may not be inhibitory.
Human leukocyte cathepsin G and elastase specifically suppress thrombin- induced prostacyclin production in human endothelial cells
Authors: Weksler BB , Jaffe EA , Brower MS , Cole OF
Published: October 2019

Polymorphonuclear leukocytes (PMN) when activated release products that can potentially injure endothelial cells or alter endothelial function. Exposure of cultured human umbilical vein endothelial cells to cathepsin G and elastase isolated from human PMN at concentrations reached in vivo (100 ng/mL to 10 micrograms/mL) selectively inhibited thrombin-induced prostacyclin production and the thrombin-induced rise in cytosolic free calcium ([Ca++]i) concentration. These proteases also blocked thrombin-induced release of arachidonic acid from prelabeled endothelial cells (EC). In contrast, induction of prostacyclin (PGI2) production by arachidonate, histamine, or the calcium ionophore A23187 was not altered by treatment of EC with these proteases. The effects of the proteases were concentration-dependent, were blocked by serum or serum protease inhibitors, and were reversed when the endothelial cells were further cultured for 24 hours in the absence of the proteases. Elastase, but not cathepsin G, also produced detachment of endothelial cells. Thus, the major leukocyte proteases selectively suppress thrombin-induced prostacyclin production by human vascular endothelial cells and may alter the hemostatic balance at sites of PMN activation.
Inhibition of tissue plasminogen activator activity by aspirin in vivo and its relationship to levels of tissue plasminogen activator inhibitor antigen, plasminogen activator and their complexes
Authors: Levin RI , Harpel PC , Harpel JG , Recht PA
Published: October 2019

The observation that aspirin inhibits the increment in tissue plasminogen activator (t-PA) activity induced by venous occlusion of the forearm became controversial with the publication of several nonconfirmatory studies. The current study was performed to confirm the original observation and determine the mechanism by which aspirin suppresses the incremental t-PA activity induced by venous occlusion. Aspirin (650 mg/d X 2) caused no change in resting levels of t-PA antigen (t-PA:Ag) or activity, plasminogen activator inhibitor 1 antigen (PAI-1:Ag), or activity or t-PA-PAI-1 complexes. In contrast, aspirin reduced the increments induced by venous occlusion as follows: t-PA:Ag by 45% (P = .001); t-PA activity (euglobulin lysis time, ELT) by 43% (P = .006); and t-PA activity (alpha 2-plasmin inhibitor-plasmin complexes, PIPC) by 41% (P = .003). The inhibition of incremental t-PA activity measured as ELT or PIPC was linearly correlated with the inhibition of incremental t-PA:Ag (respectively, r = .75, P less than .02; r = .67, P less than .05). Aspirin had no effect on the increment in PAI-1:Ag induced by venous occlusion, but similar to the effect on t- PA:Ag, aspirin induced a 51% inhibition of the increment in t-PA-PAI-1 complex formation. Aspirin did not alter the ability of alpha 2-plasmin inhibitor to bind plasmin, nor the ability of plasma to support the fibrin-catalyzed generation of plasmin by t-PA, nor the subsequent formation of PIPC. Aspirin inhibits the t-PA activity induced by venous occlusion primarily by inhibiting the release of t-PA antigen.
Proliferative pathways in CD1- CD3+ CD4+ CD8+ T-prolymphocytic leukemic cells: analysis with monoclonal antibodies and cytokines
Authors: Turco MC , De Felice M , Alfinito F , Lamberti A , Costanzo F , Giordano M , Martinelli V , Rotoli B , Ferrone S , Venuta S
Published: October 2019

The antigenic profile and the proliferative pathways in leukemic cells from the patient TRT with T-prolymphocytic leukemia (T-PLL) were analyzed using monoclonal antibodies (MoAbs) and cytokines. T-PLL cells expressed the phenotype CD1- CD3+ CD4+ CD8+. Incubation with the differentiating agent phorbol-12-myristate-13-acetate markedly increased the percentage of cells with the CD4- CD8+ phenotype, suggesting that leukemic cells were already committed towards a differentiated element with the CD4- CD8+ phenotype. T-PLL cells were induced to proliferate by anti-CD2 MoAb 9–1 + 9.6 and by anti-CD3 MoAb OKT3. The two pathways exhibited normal functional interactions and were susceptible to modulation by anti-HLA class I MoAbs. These results indicate that regulation of cell proliferation was preserved to a significant extent in the T-PLL cells analyzed. At variance with normal resting T cells that require previous activation to proliferate when incubated with interleukin-1 (IL-1) or interleukin-2 (IL-2), T-PLL cells proliferated vigorously when incubated with either interleukin. Furthermore, T-PLL cells proliferated when incubated with immune interferon (IFN-gamma). The latter finding parallels the enhancement by IFN-gamma of the proliferative response of lectin-activated murine T lymphocytes. These results suggest that T-PLL cells, which express a high constitutive level of c-myc mRNA, may be in an activated state. The antigenic phenotype and the characteristics of the proliferative pathways of T-PLL cells from the patient TRT are compatible with the possibility that they may be derived from an intermediate thymocyte.
Interleukin-2 induction of lymphokine-activated killer (LAK) activity in the peripheral blood and bone marrow of acute leukemia patients: II. Feasibility of LAK generation in children with active disease and in remission
Authors: Adler A , Albo V , Blatt J , Whiteside TL , Herberman RB
Published: October 2019

Activation and expansion in culture with rIL-2 of peripheral blood (PB) and/or bone marrow (BM) specimens derived from children with ALL and ANLL, with active disease (AP) and in remission were studied (RP). Baseline NK cytolytic activity from AP was found to be depressed, whereas RP-derived cells had normal NK activity, as assayed against K562 targets. Culture in rIL-2 significantly enhanced the NK activity of both AP- and RP-derived cells and generated LAK activity, as assayed by 4-hour 51Cr release, against NK-resistant Raji cell line and against fresh, allogeneic, and autologous tumor cells. Lytic activity against fresh, cryopreserved leukemia blasts was of lower than that found against cell lines. In three patients higher lytic activity against autologous than against allogeneic blasts was demonstrated. Expansion in culture with rIL-2 varied from twofold to 120-fold. rIL-2 activation and expansion was better in RP than in AP. The predominant phenotype of activated cells, as determined by flow cytometry, was [mean % (SD)]: CD3- = 54 (12), CD8+ = 55 (17), and NKH1+ = 26 (7). The consistently high level of CD8+ cells was accompanied by very low levels of CD4+ cells: mean = 11% (14). Double-marker analysis showed mean of 33% (10) for CD3+/NKH1+ cells and mean = 32 (11) for CD8+/NKH1+ cells, implying that these populations were overlapping. Kinetics of expression of cell surface markers during 2 to 3 weeks in culture showed that CD8+ and NKH1+ enrichment occurred during the first week and lasted for up to 4 weeks, whereas CD4+ expression decreased after the second week. A significant decrease in the expression of IL-2 receptors (CD25) was observed from the second week of culture. This study shows the feasibility of in vitro generation of killer cells from PB and BM of pediatric leukemia patients.
Immunoblotting characterization of neutrophil antigenic targets in autoimmune neutropenia
Authors: Rothko K , Kickler TS , Clay ME , Johnson RJ , Stroncek DF
Published: October 2019

We characterized neutrophil autoantigens using an immunoblotting technique with antibodies obtained from patients with autoimmune neutropenia. These results were correlated with serologic characterization of the antibodies, using indirect immunofluorescence and leukoagglutination. Of the 17 sera immunoblotted, 16 showed discrete bands in the molecular weight range of 30 to 112. Three patients with Felty's syndrome reacted with an antigenic target of 80 to 84 Kd molecular mass, a finding not seen in any of the other patients studied. By serologic testing, none of the autoimmune sera showed serologic specificity for any known neutrophil-specific alloantigen. Using an anti-NA-1 serum, we identified antigenic targets at 40, 50, and 101 Kd in both NA-1-positive and NA-1-negative neutrophils. Ten of 17 autoimmune sera showed reactivity in this corresponding range. These studies demonstrate that immunoblotting may be used to identify antigenic targets in autoimmune neutropenia and may suggest a specificity of these antibodies not definable by serologic techniques. Correlation of immunoblot reactivity with disease states associated with immune neutropenia may be useful in the study of the pathogenesis of the different forms of autoimmune neutropenia.
Sensitive inhibitory effect of interferon-alpha on M-protein secretion of human myeloma cells
Authors: Tanaka H , Tanabe O , Iwato K , Asaoku H , Ishikawa H , Nobuyoshi M , Kawano M , Kuramoto A
Published: October 2019

The effects of interferon-alpha (IFN alpha) on in vitro proliferation and M-protein secretion in human myeloma cells were investigated. Human myeloma cells were purified from bone marrow aspirates in 12 multiple myeloma patients. Purified myeloma cells were cultured for 48 hours with IFN alpha at its lower concentrations (0.1 to 100 U/mL). The cells were then pulsed with 3H-TdR for the last 12 hours and 3H-TdR uptake was measured (in vitro proliferation). After 48-hour culture, supernatants were harvested and the amount of M-protein in these fluids were measured by enzyme-linked immunosorbent assay (ELISA) (in vitro M- protein secretion). In vitro M-protein secretions of myeloma cells were significantly suppressed even at 0.1 U/mL of IFN alpha, while 3H-TdR uptakes were not so suppressed until 10 or 100 U/mL of IFN alpha were added. The expressions of secretory immunoglobulin (Ig) mRNA of these myeloma cells were also selectively suppressed by IFN alpha. Furthermore, after IFN alpha had been administered intramuscularly, 3 to 6 x 10(6) U/d for at least 1 month, in vitro M-protein secretions of these myeloma cells were decreased compared with those before IFN alpha administration. Therefore, these results suggest that IFN alpha has more sensitive inhibitory effect on M-protein secretion of human myeloma cells rather than on myeloma cell proliferation.
Peripheral acute leukemia: high peripheral but low-marrow blast count
Authors: Cason JD , Trujillo JM , Estey EH , Huh YO , Freireich EJ , Stass SA
Published: October 2019

We report five patients who had greater than 30% peripheral blasts and less than 30% marrow blasts. By the current standards these cases would be classified as myelodysplastic syndrome. Four of five patients progressed to acute leukemia within approximately 1 1/2 months of developing greater than 30% peripheral blasts. Two of these four patients had evidence of acute leukemia by criteria other than marrow involvement at the time of presentation: one patient had evidence of multifocal dermal involvement; and the other patient had a cytogenetic abnormality, t(8;21), found predominantly in acute leukemia. The fifth patient developed acute leukemia 2 years after initial presentation with greater than 30% peripheral blasts. Although our series of patients is small, it does suggest that patients who have greater than 30% peripheral blasts should be considered an acute leukemia, even with less than 30% marrow blasts.