bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibody T-cell-depleted HLA-haploidentical bone marrow transplantation for Wiskott-Aldrich syndrome
Authors: Rumelhart SL , Trigg ME , Horowitz SD , Hong R
Published: October 2019

Four patients with Wiskott-Aldrich syndrome received bone marrow transplants (BMT) using monoclonal antibody T cell-depleted HLA- haploidentical marrow from a family member donor. The patients did not receive a significantly larger inoculum of mature T cells than other recipients of T cell-depleted marrow transplants. All four patients achieved quick engraftment, and three of the four patients are alive and well today. The three living patients have all had a complete return of normal T-cell and B-cell function. Infectious complications in the surviving patients were minimal; however, all three experienced some degree of graft-versus-host disease (GVHD). Two of these three patients received GVHD prophylaxis. The patient not receiving GVHD prophylaxis experienced severe GVHD and had a difficult posttransplant course. The patient who did not survive was chronically ill before BMT, whereas the other patients were in relatively good health at the time of BMT. Since the majority of individuals with this disease lack a matched bone marrow donor, our results using partially matched donors suggest that a greater number of patients can be successfully treated for Wiskott-Aldrich syndrome and that outcome is related to control of GVHD and state of health before BMT. Marrow transplantation should be offered earlier in the disease course before the onset of major infectious problems.
Incidence of response and long-term follow-up in patients with hairy cell leukemia treated with recombinant interferon alfa-2a [see comments]
Authors: Berman E , Heller G , Kempin S , Gee T , Tran LL , Clarkson B
Published: October 2019

Thirty-five evaluable patients with hairy cell leukemia (HCL) were treated with recombinant interferon alfa-2a (rIFN-alpha 2a), given at a dose of 3 X 10(6) units (U) intramuscularly (IM) daily for 6 months followed by 3 X 10(6) U IM three times a week for an additional 18 months in a single institution study. All treatment was stopped after 24 months. Sixty-nine percent of patients achieved a partial response, 11% a minor response, and 3% (one patient) had stable disease. Six patients (17%) did not respond to rIFN-alpha 2a. Two patients (6%) achieved a response but later progressed on treatment. A total of 23 patients completed 2 years of treatment and are evaluable for long-term follow-up at a median of 20 months postcompletion of therapy (range 9 to 32 months). Eleven patients (48%) have had progression of their disease at a median of 10 months (range .5 to 25 months) after treatment was discontinued. Statistical analysis of pretreatment patient characteristics did not reveal any factor(s) associated with a high probability of responding to rIFN-alpha 2a; however, analysis of post-treatment variables measured after 2 years of treatment suggested that a low platelet count was associated with a high rate of disease progression. These findings are compared with other published trials using rIFN-alpha 2b, a similar but not identical rIFN preparation. We conclude that while rIFN-alpha 2a has a high overall response incidence, the rate of disease progression after therapy is discontinued approaches 50%, and that a subset of patients can be identified who are at high risk for recurrence after completing 2 years of treatment.
Loss of attachment to fibronectin with terminal human erythroid differentiation
Authors: Vuillet-Gaugler MH , Breton-Gorius J , Vainchenker W , Guichard J , Leroy C , Tchernia G , Coulombel L
Published: October 2019

Human erythroblastic progenitors (colony-forming unit-erythroid [CFU-E] and burst-forming unit-erythroid [BFU-E]) have been shown to attach to fibronectin (Fn), a property that might be involved in the local regulation of erythropoiesis. In this study, we have investigated changes in cell attachment to Fn upon terminal erythroid differentiation. We first purified CFU-E from human marrow by avidin- biotin immune rosetting. This negative selection procedure yielded a cell population containing approximately 80% blasts that, after characterization by colony-assays and electron microscopy, appeared to consist of CFU-E (10% to 15%) and their immediate progeny (85% to 90%), here defined as “preproerythroblasts.” In the presence of erythropoietin, purified cells differentiated into reticulocytes in 7 to 10 days. Cell attachment to Fn was inversely correlated to the stage of differentiation of the erythroid cell: more than 50% of the CFU-E population reproducibly adhered to Fn, whereas at most 30% of the preproerythroblasts had the same capacity. Adhesion was further lost at late maturation stages, and a constant finding was the inability of reticulocytes to adhere to Fn. Finally, CFU-E adhesion to Fn was blocked by polyclonal lgG raised against the Fn receptor and by a monoclonal antibody against VLA-5. These results demonstrate that adhesion to Fn is developmentally regulated during normal human erythropoiesis. Restriction of its expression to CFU-E and its first divisions strikingly correlates with the migratory capacity of these cells.
Release of iron from phagocytosed Escherichia coli and uptake by neutrophil lactoferrin
Authors: Molloy AL , Winterbourn CC
Published: October 2019

Escherichia coli were labeled with 59Fe and then either treated with myeloperoxidase, H2O2, and chloride or opsonized and mixed with human neutrophils. The myeloperoxidase system at pH 7.4 caused release of most of the bacterial 59Fe. A similar result has been obtained by Rosen and Klebanoff (J Biol Chem 257:13731, 1982) but at pH 5. Iron release at pH 7.4 did not require the presence of a chelator, and the majority passed through a 10,000 relative molecular mass cut-off ultrafiltration membrane. When iron-poor lactoferrin was present during incubation with myeloperoxidase, 88% of the released 59Fe was precipitated with anti- lactoferrin antiserum, indicating that it was lactoferrin-bound. When the bacteria were mixed with neutrophils in a 10:1 ratio, approximately 50% were phagocytosed. About 40% of the 59Fe was released from the ingested bacteria over a 40-minute period. Initially, most remained associated with the neutrophil phagosomes, but with time, there was gradual transfer of some of the iron to the medium. Using anti- lactoferrin antiserum, 50% to 60% of phagosomal iron and 64% to 71% of iron in the medium was shown to be bound to lactoferrin. Thus, iron is released from phagocytosed E coli. Most becomes bound to lactoferrin, and some of this is released into the surroundings of the neutrophils. This suggests that neutrophil lactoferrin may function to trap iron from ingested microorganisms, enabling its removal from sites of inflammation. This may prevent iron from catalyzing undesirable oxidative reactions, as well as making it unavailable for growth of microorganisms that survive the killing process.
Retroviral-mediated transfer and amplification of a functional human factor VIII gene
Authors: Israel DI , Kaufman RJ
Published: October 2019

Hemophilia A results from a deficiency in factor VII (FVIII), a cofactor in the intrinsic pathway of blood coagulation. As an approach toward genetic therapy of this disease, we constructed a retroviral vector encoding human FVIII and a selectable and amplifiable genetic marker, human adenosine deaminase (Ada). A retrovirus packaging line was transfected with this vector and stable transformants were selected for Ada expression. Isolated transformants produced both FVIII activity in the conditioned medium and retrovirus capable of transferring the Ada selectable marker and FVIII expression to the mouse 3T3 fibroblasts. Selection of virus-producer cell lines for increasing levels of Ada expression yielded a 20-fold increase in both FVIII expression and viral titer. Similarly, selection of infected 3T3 fibroblasts for Ada gene amplification yielded a 20-fold increase in FVIII expression. The results demonstrate the feasibility of retrovirus- mediated transfer of human FVIII, and also the utility of selection for gene amplification to increase retrovirus titers in producer cell lines as well as expression levels in infected cells.
Effects of interleukin-4 on the in vitro growth of human lymphoid and plasma cell neoplasms
Authors: Taylor CW , Grogan TM , Salmon SE
Published: October 2019

Recombinant human interleukin-4 (rhIL-4) has pleiotropic biologic effects including stimulation of normal B-cell growth. We investigated the effects of rhIL-4 on the in vitro growth of 35 fresh human lymphoid and plasma cell malignancies. Inhibition of growth was seen in 52% and 60% of multiple myeloma and lymphoma specimens, respectively. Growth was stimulated relative to control in 8.6% (3 of 35) of the tumors tested. Immunophenotyping showed that tumors displaying growth stimulation expressed surface markers associated with a poor clinical prognosis. Our findings support the clinical evaluation of rhIL-4 as a therapeutic agent in selected patients with lymphoid and plasma cell neoplasms.
Synergistic effects of oxidation and deformation on erythrocyte monovalent cation leak
Authors: Ney PA , Christopher MM , Hebbel RP
Published: October 2019

The normal red blood cell (RBC) membrane is remarkable for its durability (eg, preservation of permeability barrier function) despite its need to remain deformable for the benefit of microvascular blood flow. Yet, it may be hypothesized that the membrane's tolerance of deformation might be compromised under certain pathologic conditions. We studied this by subjecting normal RBC in viscous suspending medium (20% dextran) to elliptical deformation induced by application of shear stress under physiologic conditions (290 mOsm/L, 37 degrees C, pH 7.40) in the presence of ouabain and furosemide. Measurement of resulting net passive K efflux (“K leak”) demonstrated that shear-induced RBC deformation causes K leak in a dose-dependent fashion at shear stresses far below the hemolytic threshold, an effect shown to be due to deformation per se. To model the specific hypothesis that oxidatively perturbed RBC membranes would be abnormally susceptible to this potentially adverse effect of deformation, we treated normal RBC with the lipid peroxidant t-butylhydroperoxide. Under conditions inducing only minimal K leak due to either oxidation alone or deformation alone, deformation of peroxidant-pretreated RBC showed a markedly enhanced K leak (P less than .001). This highly synergistic oxidation-plus- deformation leak pathway is less active at low pH, is neither chloride- dependent nor calcium-dependent, and allows K efflux to be balanced by Na influx so there is no change in total monovalent cation content or cell density. Moreover, it is fully reversible since deformation- induced K leak terminates on cessation of shear stress (even for oxidant-treated RBC). Control experiments showed that our results are not explained simply by hemolysis, RBC vesiculation, or development of prelytic pores. We conclude that oxidation and deformation individually promote passive leak of monovalent cation through RBC membranes and that a markedly synergistic effect is exerted when the two stresses are combined. We hypothesize that these findings may help explain the abnormal monovalent cation leak stimulated by deoxygenation of sickle RBC.
Effect of zidovudine and granulocyte-macrophage colony-stimulating factor on human immunodeficiency virus replication in alveolar macrophages
Authors: Hammer SM , Gillis JM , Pinkston P , Rose RM
Published: October 2019

The alveolar macrophage (AM), as a representative human tissue macrophage, was used in an in vitro system to examine the anti-human immunodeficiency virus type-1 (HIV-1) activity of zidovudine (AZT) and granulocyte-macrophage colony-stimulating factor (GM-CSF). AMs were infected with the IIIB strain of HIV-1 and exposed to AZT (1 mumol/L), GM-CSF (30 U/mL), a combination of AZT (1 mumol/L)/GM-CSF (30 U/mL), or medium control. At 10 or 20 days post-infection, phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear leukocytes (PBMLs) were added to the AM cultures as stimulated target cells. AZT effectively suppressed HIV replication and prevented transfer/amplification in target PBMLs as long as the drug was maintained in the medium. GM-CSF neither suppressed nor augmented HIV replication. The combination of AZT/GM-CSF was comparable with AZT alone in suppressing both the initial infection of AMs and the transfer to target PBMLs as long as the agents were maintained in the cultures. However, when the drugs were removed at the same time that PHA-stimulated PBMLs were added to the culture, the combination of AZT/GM-CSF was found to be more effective than AZT alone in preventing the transfer/amplification of HIV in the target lymphocytes. These results suggest that (1) AZT is effective in inhibiting HIV-1 infection in mononuclear phagocytes; (2) GM-CSF neither inhibits nor augments the replication of the IIIB strain of HIV in human AMs; and (3) the combination of AZT and GM-CSF may have an enhanced anti-HIV-1 activity compared with AZT alone. Clinical trials with the two agents in combination appear warranted.
Platelet factor 4 inhibits human megakaryocytopoiesis in vitro
Authors: Han ZC , Sensebe L , Abgrall JF , Briere J
Published: October 2019

Platelet factor 4 (PF4) is a multifunctional protein specific to platelets, synthesized in megakaryocytes and stored in alpha granules. This report of our work shows that PF4 potently inhibits human megakaryocyte colony formation in vitro. Colony formation by megakaryocyte progenitor cells from normal bone marrows was studied using the plasma clot culture system and indirect immunoperoxidase staining. Nonadherent mononuclear cells were co-cultured with various concentrations (0 to 20 micrograms/mL) of highly purified human PF4. Statistically significant inhibition of three classes of megakaryocyte progenitor cells, the mixed colony forming unit-megakaryocytes (mCFU- MK), the burst forming unit-megakaryocytes (BFU-MK), and the colony forming unit-megakaryocytes (CFU-MK), was seen at a PF4 concentration of 2.5 micrograms/mL or greater. PF4 had no effect on erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) colony formation except at high concentration (5 micrograms/mL for BFU-E and 10 micrograms/mL for CFU- GM). When a concentration of 5 micrograms/mL PF4 was added at various time points during marrow culture, a reduction of megakaryocyte colony formation also occurred. In the presence of PF4 2.5 micrograms or 5 micrograms/mL, the percentage of mature type of colonies was found to be decreased compared with cultures with no added PF4. These data demonstrate that PF4 inhibits both proliferation and maturation of megakaryocyte progenitor cells in vitro and suggest that PF4 may play a role in autoregulating human megakaryocytopoiesis.
Transplantation of hematopoietic stem cells obtained by a combined dye method fractionation of murine bone marrow
Authors: McAlister I , Wolf NS , Pietrzyk ME , Rabinovitch PS , Priestley G , Jaeger B
Published: October 2019

Hematopoietic stem cells were purified from murine bone marrow cells (BMC). Their characteristic density, size, internal complexity, Hoechst 33342 dye uptake, and wheat germ agglutinin (WGA) affinity were used to distinguish them from other cells in the bone marrow. BMC suspensions were centrifuged over Ficoll Lymphocyte Separation Media (Organon Teknika, Durham, NC; density 1.077 to 1.08). The lower-density cells were drawn off, stained with Hoechst and labeled with biotinylated WGA bound to streptavidin conjugated to phycoerythrin (WGA-B*A-PE) or with WGA conjugated to Texas Red. These cells were then analyzed and sorted by an Ortho Cytofluorograph 50-H cell sorter. The cells exhibiting medium to high forward light scatter, low to medium right angle light scatter, low Hoechst intensity, and high WGA affinity were selected. Sorted BMC (SBMC) were stained with Romanowsky-type stains for morphologic assay, and were assayed in lethally irradiated (LI) mice for their ability to produce colony-forming units in the spleen (CFU-S) and for their ability to produce survival. The spleen seeding factor for day 8 CFU-S upon retransplantation of the isolated cells was 0.1. The isolated cells were found to have consistent morphology, were enriched up to 135-fold as indicated by day 8 CFU-S assay, 195-fold as indicated by day 14 CFU-S assay, and 150 sorter-selected BMC were able to produce long-term survival in LI mice with retention of donor karyotype. When recipients of this first transplantation were themselves used as BMC donors, their number of day 8 and day 12 CFU-S were found to be reduced. However, 3 X 10(5) of their BMC provided 100% survival among secondary recipients. When the previously SBMC were competed after one transplantation against fresh nonsorted BMC in a mixed donor transplant, they showed the decline in hematopoietic potency normally seen in previously transplanted BMC. We conclude that the use of combinations of vital dyes for fluorescence-activated cell sorting (FACS) selection of survival-promoting murine hematopoietic stem cells provides results comparable with those produced by antibody- selected FACS and has the advantage of a method directly transferable to human BMC.
Hemorrhagic sequelae of immune thrombocytopenic purpura in human immunodeficiency virus-infected hemophiliacs
Authors: Ragni MV , Bontempo FA , Myers DJ , Kiss JE , Oral A
Published: October 2019

Clinical bleeding tendency and tests of immune function were studied prospectively in 11 human immunodeficiency virus (HIV)-infected hemophiliacs with immune thrombocytopenic purpura (ITP) and a platelet count less than 50,000/microL. These 11 patients represented 13% of a well-characterized cohort of 87 HIV + hemophiliacs. ITP developed a mean 3.5 years after seroconversion, mean platelet count at presentation was 36,000/microL (range 15,000 to 49,000/microL), and the mean age at seroconversion was 37.1 years. Nine patients (82%) suffered bleeding complications, including four with intracranial hemorrhage, which was fatal in three. At the onset of ITP, five had AIDS and six were asymptomatic. Mean T4 lymphocyte count at onset of ITP was 126 +/- 32/microL (range 5 to 267/microL). Sustained treatment responses occurred with intravenous gammaglobulin (2 of 2), one of whom spontaneously remitted, and with zidovudine (1 of 2), but not with steroids (0 of 6) or danazol (0 of 3). In conclusion, 13% of a cohort of HIV + hemophiliacs has developed ITP with platelets less than 50,000/microL, a significant proportion of whom (82%) have experienced bleeding complications. It is recommended that treatment for ITP in HIV + hemophiliacs be instituted once the platelet count falls below 50,000/microL in order to avoid serious hemorrhagic sequelae.
The role of membrane skeletal-associated alpha-globin in the pathophysiology of beta-thalassemia
Authors: Sorensen S , Rubin E , Polster H , Mohandas N , Schrier S
Published: October 2019

The beta-thalassemic mouse provides a useful model for testing hypotheses about the pathophysiology in human beta-thalassemia. The clinical picture of these mice and their red blood cell deformability characteristics are quite similar to those observed in human beta- thalassemia intermedia. The creation of transgenic mice that express human beta-globin (beta s) has provided an opportunity to study the effect of increasing the non-alpha-globin chain production on the thalassemic phenotype. A small increase in beta-globin production produces transgenic mice that are healthier, have almost normal hemoglobin values, and whose red blood cell deformability is increased. We quantified and characterized the membrane skeletal-associated globin in normal, transgenic thal/sickle, and thalassemic mice and showed that only alpha-globin was associated with the membrane skeleton in the pathologic red blood cells, and that the degree of rigidity as measured in the rheoscope correlated directly and closely with the amount of membrane skeletal-associated globin in these abnormal red blood cells.
Allogeneic bone marrow transplantation for leukemia with marrow grafts depleted of lymphocytes by counterflow centrifugation
Authors: Schattenberg A , De Witte T , Preijers F , Raemaekers J , Muus P , Van der Lely N , Boezeman J , Wessels J , Van Dijk B , Hoogenhout J
Published: October 2019

Eighty consecutive patients were transplanted with human leukocyte antigen (HLA)-identical sibling marrow for acute myelogenous leukemia (AML, N = 29), acute lymphoid leukemia (ALL, N = 23), or chronic myelogenous leukemia (CML, N = 28). Donor marrow was depleted of lymphocytes using counterflow centrifugation. Median age of the recipients was 31 years. Pretransplant conditioning consisted of cyclophosphamide and fractionated total body irradiation (TBI) with a low (4.1 +/- 0.3 cGy/min) or high (13.1 +/- 1.6 cGy/min) midline average dose rate. In 43 patients, cytosine-arabinoside or anthracyclines were added to the conditioning regimen. Immunoprophylaxis posttransplant consisted of methotrexate (MTX) alone, cyclosporine A (CsA) in combination with MTX, or CsA alone; two patients received no immunoprophylaxis at all. Graft failure occurred in 4 of 77 evaluable patients (5%). The probability of acute graft- versus-host disease (GVHD) greater than or equal to grade 2 at day 100 after transplantation was 15%. The projected 3-year estimate of extensive chronic GVHD was 12%. Only three patients died of cytomegalovirus-interstitial pneumonitis. The projected 3-year probability of relapse was 30% (95% confidence interval [CI], range 8% to 53%) in transplants for AML in first complete remission (CR1), 35% (95% CI, 1% to 69%) after transplantation for ALL in CR1, and 38% (95% CI, 2% to 74%) after transplantation for CML in first chronic phase (CP1). The projected 3-year probability of leukemia-free survival (LFS) was 56% (95% CI, 35% to 77%) after transplantation for AML-CR1, 42% (95% CI, 16% to 69%) in patients transplanted for ALL-CR1, and 49% (95% CI, 18% to 80%) after transplantation for CML-CP1. After transplantation for AML-CR1, ALL-CR1, or CML-CP1, the median follow-up time for leukemia-free survivors was 31+, 30+, and 21+ months, respectively. Probabilities of relapse, survival, and LFS in AML-CR1 and ALL-CR1 transplants were comparable with those reported in recipients of untreated grafts. In patients transplanted for CML-CP1, probability of relapse was higher and probability of LFS was lower than in recipients of untreated grafts. In transplants for leukemia in CR1 and CP1, preparative regimen and immunoprophylaxis posttransplant were not associated significantly with the probability of acute GVHD greater than or equal to grade 2, extensive chronic GVHD, relapse, survival, or LFS. In bone marrow transplantation for leukemia, counterflow centrifugation is a useful technique for the prevention of GVHD.(ABSTRACT TRUNCATED AT 400 WORDS)
A role for calmodulin in the growth of human hematopoietic progenitor cells
Authors: Katayama N , Nishikawa M , Komada F , Minami N , Shirakawa S
Published: October 2019

A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.
Inhibition of factor XIIIa in a canine model of coronary thrombosis: effect on reperfusion and acute reocclusion after recombinant tissue- type plasminogen activator
Authors: Shebuski RJ , Sitko GR , Claremon DA , Baldwin JJ , Remy DC , Stern AM
Published: October 2019

The effect of inhibition of factor XIIIa with 2-(l-acetonylthio)-5- methylthiazolo[2,3-b]1,3,4-thiadiazo lium perchlorate (L-722,151) on coronary thrombolysis and reocclusion was studied in an acute dog model of electrically induced coronary thrombosis. L-722,151 (0.1 mg/kg/min intravenously [IV] or placebo was administered 15 minutes before current initiation (150 microA) and for the duration of the experiment (270 minutes). Fifteen minutes after thrombus formation, heparin (300 U/kg, IV) was administered, followed 45 minutes later by recombinant tissue-type plasminogen activator (tPA) (10 micrograms/kg/min, IV for 90 minutes). Placebo-treated animals thrombosed at 48.9 +/- 8.1 minutes (mean +/- SEM) and reperfused in response to tPA at 49.1 +/- 9.3 minutes. L-722,151 pretreated animals thrombosed at 44.4 +/- 9.7 minutes and reperfused in response to tPA at 16.4 +/- 2.8 minutes (P less than .05 v vehicle). Furthermore, residual thrombus mass was reduced by L-722,151 from 6.9 +/- 1.9 mg in placebo-treated animals to 1.7 +/- 0.6 mg (P less than .05 v vehicle). Acute reocclusion occurred in 86% of placebo and in 75% of L-722,151-treated animals. The incidence of tPA-induced reperfusion in L-722,151-treated dogs was 100% (8 of 8), whereas only 70% (7 of 10) of placebo-treated dogs reperfused. These results demonstrate that pretreatment with L-722,151 hastens reperfusion time threefold and reduces residual thrombus mass. These effects occurred with no change in systemic blood pressure in response to L-722,151. When L-722,151 was administered 15 minutes after thrombus formation in a separate group of dogs (n = 5), no beneficial effect on thrombolysis time or thrombus mass was observed. Thus, the specific factor XIIIa catalyzed crosslinking reaction(s), which may determine(s) resistance to plasmin-mediated fibrin degradation, occur(s) rapidly. Inhibition of this crosslinking by pretreatment with L-722,151 promotes tPA-induced thrombolysis.
Multimer size dependence of von Willebrand factor binding to crosslinked or noncrosslinked fibrin
Authors: Ribes JA , Francis CW
Published: October 2019

von Willebrand factor (vWF) is synthesized in endothelial cells (EC) and may be either secreted constitutively or stored in Weibel-Palade bodies (WPB) for regulated release. Because fibrin stimulates rapid vWF release from EC, we examined the binding of EC synthesized vWF to fibrin. Culture medium containing constitutively secreted vWF was removed from metabolically labeled primary cultures of human umbilical vein EC, and vWF released from WPB was obtained after stimulation by A23187. vWF-deficient fibrinogen with or without factor XIII was added to releasate or media and clotted with thrombin to form crosslinked or noncrosslinked fibrin. vWF was immunopurified from releasate or media before and after clotting, and the amount and multimeric pattern of vWF bound was determined after sodium dodecyl sulfate agarose gel electrophoresis. High molecular weight multimers of vWF, whether secreted constitutively or released from WPB, bound preferentially to fibrin. Multimers of greater than 20 subunits represented 60% +/- 4% (SEM) of A23187 released vWF and 11% +/- 5% of media vWF, but binding to fibrin was similar, 96% +/- 1% and 94% +/- 2%, respectively. A progressively smaller proportion of vWF bound as multimer size decreased, and dimeric vWF binding was least, with 34% +/- 5% binding from A23187 releasate and 51% +/- 4% from media. The amount of vWF binding to crosslinked or noncrosslinked fibrin was similar, and preferential binding of high molecular weight multimers occurred with both. As measured by enzyme-linked immunosorbent assay, 45% +/- 2% of constitutively secreted vWF bound to crosslinked fibrin and 50% +/- 2% to noncrosslinked fibrin. The propolypeptide of vWF did not bind to fibrin. These findings indicate that binding of EC secreted vWF binding to fibrin depends on multimeric size but not on factor XIII crosslinking. This suggests that vWF released from EC in the presence of fibrin will bind locally, thereby facilitating platelet adhesion to the hemostatic plug or thrombus.
Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease [published erratum appears in Blood 1990 Nov 1;76(9):1901]
Authors: Ewenstein BM , Inbal A , Pober JS , Handin RI
Published: October 2019

Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12- myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.
High von Willebrand factor concentration compensates a relative adhesion defect in uremic blood
Authors: Zwaginga JJ , Ijsseldijk MJ , Beeser-Visser N , de Groot PG , Vos J , Sixma JJ
Published: October 2019

Uremia is associated with a bleeding diathesis. We investigated platelet adhesion as a cause for the impaired primary hemostasis and the role of von Willebrand factor (vWF) in this process in uremic patients. Perfusions with blood with standardized hematocrit, platelet count, and free Ca2+ ions were performed over inverted and deendothelialized artery segments from human umbilical cords in a modified Baumgartner perfusion chamber. Platelet adhesion in patient perfusates was comparable with control adhesion. However, the high vWF levels present in uremic whole blood did not increase adhesion above the adhesion in control blood with lower vWF levels. These results suggested that a relative adhesion defect was present in patient blood. Control blood in which vWF levels were raised to uremic levels showed the high adhesion that uremic whole blood failed to show. Additionally, in perfusions with uremic plasma in which the initially high vWF level was normalized by dilution with vWF-depleted uremic plasma, adhesion was clearly lower than in normal plasma. Washed patient platelets did not differ from normal platelets in their association with purified vWF, via their adhesion receptors glycoprotein Ib and IIb-IIIa. Patient platelets present in patient plasma showed a similar adhesion defect as control platelets, which were resuspended in the uremic plasma. Therefore, primary defects of uremic platelets were of minor importance for the observed adhesion defect in uremic whole blood. The adhesion defect was not dependent on the presence of uremic vWF; plasma of uremic patients depleted of vWF also inhibited adhesion, and the defect remained present when purified control vWF was added to vWF-depleted uremic plasma. The binding of uremic vWF to the vessel wall and its support of subsequent adhesion were not impaired. These results indicate that the observed adhesion defect was not due to abnormal vWF. Our current results suggest an unknown component present in uremic plasma that directly inhibits platelet interaction with artery segments; however, it has no effect on vWF binding to the vessel wall. High vWF levels in uremic plasma are able to compensate for the defect.
Fibrinogen induces endothelial cell adhesion and spreading via the release of endogenous matrix proteins and the recruitment of more than one integrin receptor
Authors: Dejana E , Lampugnani MG , Giorgi M , Gaboli M , Marchisio PC
Published: October 2019

We have previously shown that fibrinogen (fg) acts as a subendothelial matrix protein in promoting human endothelial cell (EC) adhesion and cytoskeletal organization. In this study we report that EC spreading on fg, at variance with other matrix proteins, requires endogenous matrix protein synthesis and secretion. ECs, upon seeding on fg, promptly released and organized a fibronectin (fn) matrix. Fg was more effective than vitronectin (vn) in promoting the deposition of this protein. ECs treated with monensin to block matrix protein secretion still adhered to fg but did not properly organize their cytoskeleton and adhesion structures. In contrast, monensin did not affect EC spreading either on vn or on fn. Using antibodies to the alpha and beta chains of fn (alpha 5 beta 1) and vn (alpha v beta 3) receptors, it was found that ECs adherent to fg show clustering and organization in adhesion structures of both type of receptors. A faint staining of adhesion structures with alpha 2 but not alpha 3 and alpha 6 antibodies was also observed. Antibodies either to vn or fn receptors were able to disrupt the EC monolayer and to induce EC retraction and detachment, thus indicating that both receptors are important in maintaining a sustained EC adhesion to fg. However, when ECs were treated with monensin only the vn receptor was organized in adhesion structures while the fn receptor was diffusely distributed. This suggests that clustering of the fn receptor is mediated by the release of endogenous matrix proteins induced by the exposure to fg. In conclusion, fg has a peculiar and complex type of interaction with ECs since it requires endogenous matrix protein release and the recruitment of more than one adhesive receptor. This suggests a specific way of response of ECs to each extracellular matrix component.
The production of transforming growth factor-beta in acute megakaryoblastic leukemia and its possible implications in myelofibrosis
Authors: Terui T , Niitsu Y , Mahara K , Fujisaki Y , Urushizaki Y , Mogi Y , Kohgo Y , Watanabe N , Ogura M , Saito H
Published: October 2019

Acute myelofibrosis is often associated with acute megakaryoblastic leukemia (AMKBL). Although the exact mechanism for the progression of myelofibrosis in AMKBL is unclear, certain humoral factors from megakaryoblastic cells, the precursors of platelets, may be involved in the enhancement of collagen synthesis by bone marrow fibroblasts. The present study, therefore, is an investigation of the possible pathogenic role of transforming growth factor-beta (TGF-beta), known to be a very potent collagen-stimulating factor found in platelets in the myelofibrosis of AMKBL. The results obtained were as follows: (1) Conditioned media from peripheral megakaryoblasts taken from an AMKBL patient and from established megakaryoblast cell lines (MEG-01) had much greater stimulatory effects on collagen synthesis in bone marrow fibroblasts than conditioned media from other leukemic cell types. (2) Based on an assessment of soft agar colony formation, there was greater TGF-beta activity in media that had been conditioned from megakaryoblasts than in media from other leukemic cell types. (3) When compared with other leukemic-cell types, megakaryoblasts showed substantially greater expression of TGF-beta mRNA that was hybridized at 2.5 kb with a TGF-beta cDNA probe, and TGF-beta polypeptides were detected at 13 Kd with anti-TGF-beta antibodies. (4) The addition of the anti-TGF-beta antibody inhibited the stimulatory effects of the megakaryoblast conditioned medium on collagen synthesis in bone marrow fibroblasts. These results clearly suggest that megakaryoblasts produce and secrete an active form of TGF-beta and stimulate collagen synthesis in bone marrow fibroblasts in a paracrine manner.
Artificial surface effect on red blood cells and platelets in laminar shear flow
Authors: Alkhamis TM , Beissinger RL , Chediak JR
Published: October 2019

Red blood cell (RBC) effects on platelet adhesion to a nonbiologic test surface (tetrafluoroethylene propylene copolymer) and platelet aggregation during laminar shear flow for shear rates to 5,680 s-1 (corresponding to shear stress to 200 dyne/cm2) were investigated. Results on hemoglobin (Hb) and adenosine diphosphate (ADP) release from RBCs, percent decrease of single platelets in the bulk, and percent of test surface covered with platelets were obtained in a cone-and-plate (CP) viscometer for samples of whole blood, suspensions of RBC ghosts in platelet-rich plasma (PRP), and suspensions of RBCs in either PRP or platelet-poor plasma. Results obtained over the shear rate range studied for samples of normal hematocrit indicated that low-stress shearing led to ADP and Hb release from intact RBCs; shear-induced release of ADP from RBCs was about twice that of platelets, and of the total ADP released, the ADP released from RBCs contributed about six times that of the platelets to single platelet reduction in the bulk and about twice that of the platelets to platelet adhesion, ie, coverage of the test surface with platelets. Results obtained for various hematocrits showed that above a threshold hematocrit of about 25% to 35% the RBCs (suspended in PRP) had a greater contribution to ADP release, platelet adhesion, and platelet aggregation than the platelets themselves. Single platelet reduction for samples of RBC ghosts suspended in PRP correlated with shear rate level and not with shear stress.
Partial purification and properties of nicotinamide adenine dinucleotide synthetase from human erythrocytes: evidence that enzyme activity is a sensitive indicator of lead exposure
Authors: Zerez CR , Wong MD , Tanaka KR
Published: October 2019

We have examined properties of nicotinamide adenine dinucleotide (NAD) synthetase from human erythrocytes. The enzyme was found to be cold labile and extremely unstable in crude hemolysate, with complete loss of activity occurring after 24 hours at 4 degrees C. However, maintenance of crude hemolysate at 20 to 25 degrees C in the presence of EDTA and KCl increased NAD synthetase stability substantially (half- life = 10 days). Using these conditions, NAD synthetase was purified 3,100-fold with a 29% yield using DEAE-cellulose column chromatography, ammonium sulfate fractionation, and dialysis. The apparent Michaelis- Menten constants for nicotinic acid adenine dinucleotide (NAAD), adenosine triphosphate, Mg2+, glutamine, and K+ were 0.108, 0.154, 1.36, 2.17, and 8.32 mmol/L, respectively. The pH optimum ranged between 6.8 and 7.4, and the molecular weight was estimated to be 483 +/- 5 Kd. The enzyme was markedly inhibited by Pb2+ and Zn2+, with concentrations necessary for 50% inhibition of activity of 1.3 and 2.0 mumol/L, respectively. The incubation of intact red blood cells with lead followed by rigorous washing to remove lead abolished nearly all NAD synthetase activity. In contrast, glucose-6-phosphate dehydrogenase activity, which is not sensitive to lead, was unaffected, whereas pyrimidine 5′-nucleotidase activity, which is sensitive to lead, was decreased 30% to 50% under these conditions. More importantly, patients with lead overburden (34 to 72 micrograms Pb2+/dL blood) all had markedly decreased NAD synthetase activity. These data together with other results suggest that erythrocyte NAD synthetase activity is a sensitive indicator of lead exposure in humans.
In vivo effects of recombinant human interleukin-6 in primates: stimulated production of platelets
Authors: Asano S , Okano A , Ozawa K , Nakahata T , Ishibashi T , Koike K , Kimura H , Tanioka Y , Shibuya A , Hirano T
Published: October 2019

In cynomolgus monkeys, twice daily subcutaneous injections of recombinant human interleukin-6 (rhIL-6) at doses of 5 to 80 micrograms/kg/d for 14 consecutive days caused dose-dependent increases in platelet count, usually continuing for more than 1 week after cessation of the injections. The count reached a level approximately twofold or more above the preinjection level even at 5 micrograms/kg/d, and at doses of more than 20 micrograms/kg/d, the increase became biphasic with a higher second peak 3 days after cessation of the injections. Morphologic analysis of the bone marrow after the 7 day- injections with 80 micrograms/kg/d revealed a marked increment in size of megakaryocytes compared with control, indicating the promotion of megakaryocyte maturation. Other changes attributable to the rhIL-6 treatment include dose-dependent loss of body weight, anemia, neutrophilia and monocytosis, elevation of serum C-reactive protein and alpha-1 acid glycoprotein levels, and decrease of serum albumin; all of which returned to normal within 1 week after cessation of the injections and were tolerable at doses of less than 10 micrograms/kg/d. These findings suggest that rhIL-6 may be an effective strategy for the treatment of thrombocytopenia.
Epidemiology of aplastic anemia in France: a prospective multicentric study. The French Cooperative Group for Epidemiological Study of Aplastic Anemia
Authors: Mary JY , Baumelou E , Guiguet M
Published: October 2019

Incidence rates of aplastic anemia (AA) are rare among defined populations. Since June, 1984, a cooperative group, including 83 University medical centers throughout metropolitan France, prospectively recorded new cases of AA and followed them up. Inclusion criteria were: at least two depressed blood cell lineages (hemoglobin less than or equal to 10 g/100 mL and reticulocytes less than or equal to 50 x 10(9)/L, granulocytes less than or equal to 1.5 x 10(9)/L, platelets less than or equal to 100 x 10(9)/L) and a bone marrow biopsy compatible with the disease. Between May, 1984, and April, 1987, 292 cases were recorded. After exclusion of constitutional disease, 27 patients did not satisfy the inclusion criteria with relation to either bone marrow or blood evaluations and seven patients were initially misdiagnosed (shown in the follow-up), leaving 250 confirmed AA cases in the register. The annual incidence in France appeared to be about 1.5 per million inhabitants. The sex ratio of AA cases was similar to that of the population. In men, two peaks of incidence were observed: one between 15 and 30 years and one after 60 years. In women, the only peak was observed after 60 years. An excess of cases was observed in small towns but not in rural areas. About two of every three cases had severe AA, with a possible excess in younger cases. Based on a minimum follow-up of 1 year for 238 patients, the fatality rate was estimated at 17% at 3 months after diagnosis and at 34% at 1 year. Among 243 suspected etiologies reported by the physicians, 74% were declared idiopathic, 13% presumably associated to drug toxicity, and 5% related to hepatitis. AA appears to be a rare and severe disease in metropolitan France, often of unknown origin, a fact that emphasizes the necessity of controlled etiologic studies.
Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma
Authors: Wilhelm O , Hafter R , Henschen A , Schmitt M , Graeff H
Published: October 2019

The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2- mercaptoethanol, the peptide chain components were separated by reverse- phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.
High prevalence of T-cell receptor V delta 2-(D)-D delta 3 or D delta 1/2-D delta 3 rearrangements in B-precursor acute lymphoblastic leukemias
Authors: Biondi A , Francia di Celle P , Rossi V , Casorati G , Matullo G , Giudici G , Foa R , Migone N
Published: October 2019

Rearrangement of the immunoglobulin (Ig) and T-cell receptor (TcR) genes generally has been considered a useful marker of B- and T-cell lineage in lymphoproliferative disorders. However, concomitant rearrangements of Ig and TcR genes have been commonly reported in the most immature lymphoid malignancies, mainly in B-cell precursor acute lymphoblastic leukemia (ALL). To better characterize the nature of this lineage promiscuity, we have analyzed the configuration of the TcR delta locus in 75 B-precursor ALL. The large majority of cases (87%) displayed a rearrangement or deletion at the delta locus. Among the 57 nondeletional rearrangements, two patterns were predominant and both appeared to derive from partial joining: a V delta-(D)-D delta 3 (32/57) and a D delta 1/2-D delta 3 (11/57) type. A single V delta gene (V delta 2) appeared to be involved in the first type of rearrangement. These findings are at variance with T-ALL, where rearrangements 5′ to V delta 2 are usually found. It remains to be elucidated whether this incomplete attempt of V delta 2 gene assembly is related to the neoplastic event or represents a physiologic predisposition occurring during early stages of the normal lymphocyte differentiation.
Chromosomal abnormalities in untreated patients with non-Hodgkin's lymphoma: associations with histology, clinical characteristics, and treatment outcome. The Nebraska Lymphoma Study Group
Authors: Schouten HC , Sanger WG , Weisenburger DD , Anderson J , Armitage JO
Published: October 2019

We describe the chromosomal abnormalities found in 104 previously untreated patients with non-Hodgkin's lymphoma (NHL) and the correlations of these abnormalities with disease characteristics. The cytogenetic method used was a 24- to 48-hour culture, followed by G- banding. Several significant associations were discovered. A trisomy 3 was correlated with high-grade NHL. In the patients with an immunoblastic NHL, an abnormal chromosome no. 3 or 6 was found significantly more frequently. As previously described, a t(14;18) was significantly correlated with a follicular growth pattern. Abnormalities on chromosome no. 17 were correlated with a diffuse histology and a shorter survival. A shorter survival was also correlated with a +5, +6, +18, all abnormalities on chromosome no. 5, or involvement of breakpoint 14q11–12. In a multivariate analysis, these chromosomal abnormalities appeared to be independent prognostic factors and correlated with survival more strongly than any traditional prognostic variable. Patients with a t(11;14)(q13;q32) had an elevated lactate dehydrogenase (LDH). Skin infiltration was correlated with abnormalities on 2p. Abnormalities involving breakpoints 6q11–16 were correlated with B symptoms. Patients with abnormalities involving breakpoints 3q21–25 and 13q21–24 had more frequent bulky disease. The correlations of certain clinical findings with specific chromosomal abnormalities might help unveil the pathogenetic mechanisms of NHL and tailor treatment regimens.