bluuebunny/crossref_metadata_2025 huggingface dataset:
Rosetting of Plasmodium falciparum-infected red blood cells with uninfected red blood cells enhances microvascular obstruction under flow conditions
Authors: Kaul DK , Roth EF Jr , Nagel RL , Howard RJ , Handunnetti SM
Published: October 2019

The occurrence of rosetting of Plasmodium falciparum-infected human red blood cells (IRBC) with uninfected red blood cells (RBC) and its potential pathophysiologic consequences were investigated under flow conditions using the perfused rat mesocecum vasculature. Perfusion experiments were performed using two knobby (K+) lines of P falciparum, ie, rosetting positive (K+R+) and rosetting negative (K+R-). The infusion of K+R+ IRBC resulted in higher peripheral resistance (PRU) than K+R- IRBC (P less than .0012). Video microscopy showed that under conditions of flow, in addition to cytoadherence of K+R+ IRBC to the venular endothelium, rosette formation was also restricted to venules, especially in the areas of slow flow. Rosettes were absent in arterioles and were presumably dissociated by higher wall shear rates. The presence of rosettes in the venules must therefore reflect their rapid reformation after disruption. Cytoadherence of K+R+ IRBC was characterized by formation of focal clusters along the venular wall. In addition, large aggregates of RBC were frequently observed at venular junctions, probably as a result of interaction between flowing rosettes, free IRBC, and uninfected RBC. In contrast, the infusion of K+R+ IRBC resulted in diffuse cytoadherence of these cells exclusively to the venular endothelium but not in rosetting or large aggregate formation. The cytoadherence of K+R+ IRBC showed strong inverse correlation with the venular diameter (r = -.856, P less than .00001). Incubation of K+R+ IRBC with heparin and with monoclonal antibodies to glycoprotein IV/CD36 abolished the rosette formation and resulted in decreased PRU and microvascular blockage. These findings demonstrate that rosetting of K+R+ IRBC with uninfected RBC enhances vasocclusion, suggesting an important in vivo role for rosetting in the microvascular sequestration of P falciparum-infected RBC.
Membrane surface antigen expression on neutrophils: a reappraisal of the use of surface markers for neutrophil activation
Authors: Kuijpers TW , Tool AT , van der Schoot CE , Ginsel LA , Onderwater JJ , Roos D , Verhoeven AJ
Published: October 2019

Neutrophil research relies largely on studies with highly purified cells. Yet the isolation procedures induce changes in surface expression of several proteins. We used a large panel of monoclonal antibodies (MoAbs) to characterize in detail the phenotypic changes during isolation and stimulation of human neutrophils. Centrifugation on density gradients appears to be the crucial step that causes an increase in expression of antigens not detectable on neutrophils in whole blood samples (cytochrome b558 recognized by MoAb 7D5; and CD10) or expressed at significantly lower levels (CD11a, CD11b, CD11c, CD13, CD16, CD45, and CD67). Other antigens were unaffected by the density gradient centrifugation step (CD32, CD54, CD58, Leu-8, HLA class I). Upregulation of antigens was also determined by stimulation of purified neutrophils. Upregulation of CD63 was an excellent marker for release from azurophil granules. We subsequently related the surface antigen expression to functional activities of purified neutrophils. From these experiments, we concluded that 7D5-as “early activation” marker--does not necessarily discriminate between primed or resting neutrophils with respect to NADPH oxidase activity.
Phase I trial of H65-RTA immunoconjugate in patients with cutaneous T- cell lymphoma
Authors: LeMaistre CF , Rosen S , Frankel A , Kornfeld S , Saria E , Meneghetti C , Drajesk J , Fishwild D , Scannon P , Byers V
Published: October 2019

H65-RTA is an immunoconjugate that consists of the A chain of ricin (RTA), a ribosomal-inhibiting protein, coupled to a murine monoclonal antibody (H65) directed against the pan-T-cell antigen CD5. The CD5 antigen is heterogeneously expressed on cutaneous T-cell lymphoma tumor cells, but is not expressed on normal cells except lymphocytes. A phase I trial was therefore conducted in which 14 patients with cutaneous T- cell lymphoma progressive on other therapies were treated with up to three cycles of H65-RTA. The maximal tolerated dose (MTD) of H65-RTA was 0.33 mg/kg/d administered intravenously for 10 days as defined by dyspnea at rest at higher doses. Other reversible side effects included myalgia, mild hypoalbuminemia with weight gain, pedal edema, fatigue, fevers, and chills. Six patients received more than one cycle of H65- RTA without increased side effects compared with the first cycle. Pharmacokinetic analysis showed that peak serum drug levels were dose- dependent, and ranged from 1.13 to 5.56 micrograms/mL, with a terminal half-life ranging from 1.0 to 2.9 hours. The development of antibodies against the immunoconjugate was associated with a lower peak drug level, but not with enhanced side effects. Partial responses lasting from 3 to 8 months were documented in four patients. Three of the responding patients received more than one cycle of H65-RTA in the presence of anti-immunoconjugate antibodies. The results from this phase I trial suggest that H65-RTA is an active drug in the treatment of cutaneous T-cell lymphoma. The immunoconjugate may be safely administered repeatedly, even in the presence of anti-immunoconjugate antibodies, with responses noted. Additional studies at the MTD are needed to define the response rate in this disease.
Immunodepletion of extrinsic pathway inhibitor sensitizes rabbits to endotoxin-induced intravascular coagulation and the generalized Shwartzman reaction
Authors: Sandset PM , Warn-Cramer BJ , Maki SL , Rapaport SI
Published: October 2019

We have reported earlier that immunodepletion of extrinsic pathway inhibitor (EPI) sensitizes rabbits to disseminated intravascular coagulation (DIC) induced by infusing a low concentration of tissue factor (TF). We now describe the effect of immunodepletion of EPI in rabbits administered endotoxin. Cortisone-treated rabbits were administered anti-rabbit EPI immunoglobulin (IgG) or Fab fragments or were administered control nonimmune material before an injection of endotoxin. In four of seven rabbits administered anti-EPI, plasma EPI activity levels were reduced by 70% to 80% of initial levels for 6 to 8 hours. In these rabbits the endotoxin induced extensive DIC, as evidenced by substantial decreases in fibrinogen, factor V, factor VIII, and platelets, and gross hemorrhagic necrosis of the kidneys due to massive deposition of fibrin in the glomerular microcirculation (the generalized Shwartzman reaction). In three rabbits administered anti- EPI, plasma EPI levels were only transiently reduced. In these rabbits and in four rabbits administered nonimmune IgG or Fab, endotoxin induced minimal to moderate intravascular clotting and deposits of fibrin were not found in the glomerular capillaries. Because it is believed that TF expressed on monocytes triggers endotoxin-induced coagulation, these data are taken as evidence that EPI functions as a natural anticoagulant that can regulate factor VIIa/TF activity expressed on cell surfaces in vivo. They support a hypothesis that EPI prevents thrombotic complications that might otherwise result from exposure of blood to cytokine-induced generation of small amounts of TF on cell surfaces in many inflammatory and infectious disease states.
Transferrin-independent iron uptake supports B lymphocyte growth
Authors: Seligman PA , Kovar J , Schleicher RB , Gelfand EW
Published: October 2019

Raji, a malignant B-lymphocyte cell line containing Epstein-Barr virus genomic elements, has been conditioned to proliferate optimally in transferrin (Tf)-free medium containing a very low concentration of an iron salt. We provide evidence that an Tf-independent iron uptake system is physiologically important for maintaining the growth of these cells. The data show that Raji cells take up iron from iron salts using a relatively high-capacity, low-affinity, temperature- and calcium- dependent uptake system. The apparent capacity of this system increases when: (1) cells are cultured in Tf-free medium containing high concentrations of iron salt as opposed to medium containing Tf; and (2) when the iron salt concentration of Tf-free medium is lowered to about 1.6 mumol/L. Cellular iron uptake also increases when a maximum number of cells are in S and G2 and M cell phases of the cell cycle. The cells are sensitive to growth inhibition by the addition of deferoxamine. This evidence supports the hypothesis that certain malignant lymphocytes, under iron deplete conditions, fulfill an iron requirement for proliferation by an adaptation such as Tf-independent iron uptake.
Administration of interleukin-2 (IL-2) results in increased plasma concentrations of IL-5 and eosinophilia in patients with cancer
Authors: van Haelst Pisani C , Kovach JS , Kita H , Leiferman KM , Gleich GJ , Silver JE , Dennin R , Abrams JS
Published: October 2019

Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.
VpreB gene expression in hematopoietic malignancies: a lineage- and stage-restricted marker for B-cell precursor leukemias
Authors: Bauer SR , Kubagawa H , Maclennan I , Melchers F
Published: October 2019

We show here that analysis of VpreB gene transcription can be a specific way to identify acute leukemias of cells at very early stages of B-cell development. Northern blot analysis of RNAs from 63 leukemia samples showed that VpreB RNA was present in malignancies of precursor B cells, the expression being a feature of both common acute lymphoblastic leukemia (ALL) (CD10+) and null ALL (CD10-). It was absent from malignancies of mature B cells (surface Ig positive), from acute leukemias of the T-cell lineage and granulocyte-macrophage lineages, and from normal tonsil B and T lymphocytes. Chronic myeloid leukemia blast crises of the B-precursor-cell type expressed the VpreB gene while myeloid blast crises did not. VpreB RNA was also expressed in the neoplastic cells of one of three patients with acute undifferentiated leukemias. These data show that VpreB RNA expression is a marker of the malignant forms of precursor B cells, and that it appears at least as early as cytoplasmic CD22 and CD19 in tumors of the B-cell lineage.
Molecular basis for elliptocytosis associated with glycophorin C and D deficiency in the Leach phenotype
Authors: Telen MJ , Le Van Kim C , Chung A , Cartron JP , Colin Y
Published: October 2019

Glycophorin C (GPC) and glycophorin D (GPD) are highly glycosylated integral membrane proteins of human erythrocytes encoded by the same gene and associated with expression of Gerbich blood group system antigens. GPC/D deficiency (the Leach phenotype) is a rare condition usually found after identification of Gerbich blood group system antibodies in persons undergoing prenatal or pretransfusion evaluation. In all cases, the Leach phenotype has been associated with elliptocytosis. Characterization of the molecular basis of this phenotype in three previously uninvestigated families has shown that the most common genetic basis of GPC/D deficiency is deletion of exons 3 and 4 of the GPC gene. However, in one family, the Leach phenotype appeared due to a deletion of one nucleotide in exon 3, causing a frameshift mutation in the messenger RNA and premature generation of a stop codon. The GPC and GPD protein sequences are therefore interrupted in the extracellular domain and probably intracellularly degraded.
Adducin in erythrocyte precursor cells of rats and humans: expression and compartmentalization
Authors: Nehls V , Drenckhahn D , Joshi R , Bennett V
Published: October 2019

Adducin is a calmodulin-binding protein involved in the assembly of the erythrocyte membrane skeleton. To investigate the expression of adducin during human erythropoiesis, we performed immunofluorescence studies on smears of cultured human erythroblasts. Adducin immunoreactivity was found in the early stages of erythropoiesis. Proerythroblasts were the first erythroid precursor cells positive for adducin. The adducin signal was very similar to the signal of erythroid beta-spectrin in that both proteins lined the membrane of erythroid precursor cells. Cell fractionation experiments were performed to further analyze the intracellular distribution of adducin in erythroid cells. In erythroblasts, about 60% of total cellular adducin appeared in the Triton-soluble fraction. In reticulocytes, the Triton-soluble fraction decreased to 30% of total reticulocyte adducin. Erythrocytes had no detectable amount of adducin in the Triton-soluble pool. Instead, adducin was quantitatively bound to the Triton-insoluble erythrocyte cytoskeleton. Our results suggest that adducin is expressed early in the development of the erythrocyte membrane skeleton, whereas stable assembly onto the membrane skeleton does not occur before the final stages of mammalian erythropoiesis.
Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura
Authors: Louache F , Bettaieb A , Henri A , Oksenhendler E , Farcet JP , Bierling P , Seligmann M , Vainchenker W
Published: October 2019

Twenty-one human immunodeficiency virus (HIV)-positive patients, including 11 acquired immunodeficiency syndrome (AIDS)-free patients with immune thrombocytopenic purpura (ITP), were studied to determine whether the megakaryocytic/platelet lineage was infected by HIV. Because purification of platelets did not reach a level sufficient for unequivocal results by the polymerase chain reaction, in situ hybridization was thus performed. Purified marrow megakaryocytes (MK) from 10 HIV-infected ITP patients were studied using a 35S HIV riboprobe, antisense of an HIV ENV sequence. HIV transcripts were clearly detected in MK from five of these 10 patients, although heterogeneity among MK was observed. In three of these five cases, small amounts of HIV glycoproteins were detected in MK by means of immunofluorescence. In addition anti-HIV antibodies could be eluted from platelets of all patients. In contrast, HIV transcripts were not detected in MK derived from colony-forming units-MK (CFU-MK) cultured in suspension, suggesting either that MK are infected by HIV during terminal differentiation or that HIV-infected CFU-MK are unable to differentiate in vitro. In conclusion, this study suggests that HIV infection of MK may be implicated in the pathogenesis of thrombocytopenia of HIV-positive patients.
Dynamics of leukocyte-platelet adhesion in whole blood
Authors: Rinder HM , Bonan JL , Rinder CS , Ault KA , Smith BR
Published: October 2019

The dynamics of leukocyte-platelet adhesion and platelet-platelet interaction in whole blood are not well understood. Using different platelet agonists, we have studied the whole blood kinetics of these heterotypic and homotypic interactions, the relative abilities of different leukocyte subsets to participate in platelet adhesion, and the ligands responsible for adhesion. When platelet aggregation was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide, thrombin stimulation of whole blood resulted in platelet expression of granule membrane protein 140 (GMP-140) and, simultaneously, a marked increase in the percentage of monocytes and neutrophils (PMN) binding platelets, as well as an increase in the number of platelets bound per monocyte and PMN. Lymphocytes were unaffected. Monocytes bound more platelets and at an initially faster rate than PMN. This increase in monocyte and PMN adhesion to platelets was completely inhibited by the blocking monoclonal antibody (MoAb), G1, to GMP-140. When the combination of epinephrine and adenosine diphosphate (epi/ADP) was used as a less potent agonist in the presence of RGDS, GMP-140 expression per platelet was less, and while monocyte-platelet conjugates formed, PMN-platelet conjugates did not. With epi/ADP in the absence of RGDS, there was an immediate, marked decrease in the percentage of all leukocytes with bound platelets, simultaneous with an increase in the percentage of unbound platelet aggregates. As these platelet aggregates dissociated, the percentage of monocytes and PMN with adherent platelets increased, with monocytes again binding at a faster initial rate than PMN. This recovery of monocyte and PMN adhesion to platelets was also inhibited by the G1 MoAb. We conclude that: (1) monocytes and PMN bind activated platelets in whole blood through GMP-140; (2) monocytes have a competitive advantage over PMN in binding activated platelets, particularly when less potent platelet agonists are used; and (3) platelet aggregate formation initially competes unactivated platelets off leukocytes; subsequent aggregate dissociation allows the now activated platelets to readhere to monocytes and PMN through GMP-140. These studies further elucidate the dynamic interaction of blood cells and possible links between coagulative and inflammatory processes.
Activated and unactivated platelet adhesion to monocytes and neutrophils
Authors: Rinder HM , Bonan JL , Rinder CS , Ault KA , Smith BR
Published: October 2019

To examine the possible receptor-ligand pairs mediating adhesion of activated and “unactivated” platelets to leukocytes and the kinetics of leukocyte-platelet binding, we developed a flow cytometric assay using isolated cell fractions to accurately measure heterotypic cell adhesion, including both total leukocyte-platelet conjugate formation as well as the number of platelets bound per leukocyte. We have shown that (1) activated platelet binding to both polymorphonuclear leukocytes (PMN) and monocytes is dependent on both a specific epitope (blocked by monoclonal antibody G1) of granule membrane protein-140 (GMP-140) and the presence of divalent cations; (2) unactivated platelets bind to 87% of viable, resting monocytes but to only 34% of PMN; (3) the receptor(s) on unactivated platelets that mediate adhesion to PMN and monocytes do not require divalent cations and become nonfunctional after thrombin activation; and (4) the kinetics of platelet adhesion to monocytes and PMN indicate that monocyte adhesion is favored over neutrophil adhesion. We conclude that platelet- heterotypic cell adhesion is a dynamic process reflecting the activation status of the platelet and differential binding abilities of leukocytes.
Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation
Authors: Toothaker LE , Gonzalez DA , Tung N , Lemons RS , Le Beau MM , Arnaout MA , Clayton LK , Tenen DG
Published: October 2019

We have isolated 5′ cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.
Inactivation of human immunodeficiency virus type 1 by ozone in vitro
Authors: Wells KH , Latino J , Gavalchin J , Poiesz BJ
Published: October 2019

A device was designed to deliver a constant source of given concentrations of ozone to fluids containing human immunodeficiency virus type 1 (HIV-1). Ozone was found to inactivate HIV-1 virions in a dose-dependent manner. Greater than 11 log inactivation was achieved within 2 hours at a concentration of 1,200 ppm ozone. Similar concentrations of ozone had minimal effect on factor VIII activity in both plasma and immunoaffinity-purified preparations of factor VIII treated for the same time period. The data indicate that the antiviral effects of ozone include viral particle disruption, reverse transcriptase inactivation, and/or a perturbation of the ability of the virus to bind to its receptor on target cells. Ozone treatment offers promise as a means to inactivate human retroviruses in human body fluids and blood product preparations.
Interleukin-6 supports human megakaryocytic proliferation and differentiation in vitro
Authors: Imai T , Koike K , Kubo T , Kikuchi T , Amano Y , Takagi M , Okumura N , Nakahata T
Published: October 2019

The effect of interleukin-6 (IL-6) on cells of human megakaryocyte (MK) lineage from cord blood was explored. In semisolid colony assays containing human plasma, a greater number of both MK colonies and cells per colony was seen in the presence of IL-6 and IL-3 than in the presence of IL-3 alone. This stimulatory effect of IL-6, observed on both small and large MK colonies, was completely eliminated by the addition of anti-IL-6 antibody to the culture. IL-6 alone had no effect on MK colony formation. In the primary culture, MKs showed larger cell size and DNA content in the presence of both IL-3 and IL-6 than IL-3 alone. The replating experiments using immature MKs grown in the presence of IL-3 showed that IL-6 significantly augmented both cell size and DNA content. This effect was also neutralized by an anti-IL-6 antibody. IL-3 had no tangible effect on MK differentiation. Synergism between IL-6 and IL-3 on MK differentiation was not confirmed. These results suggest that IL-6 is a synergistic factor in the proliferation of MK progenitors and a direct effector of differentiation of immature MKs on in vitro human megakaryocytopoiesis.
Selective depletion of bone marrow T lymphocytes with anti-CD5 monoclonal antibodies: effective prophylaxis for graft-versus-host disease in patients with hematologic malignancies
Authors: Antin JH , Bierer BE , Smith BR , Ferrara J , Guinan EC , Sieff C , Golan DE , Macklis RM , Tarbell NJ , Lynch E
Published: October 2019

Seventy-one patients with hematologic malignancies received bone marrow from a histocompatible sibling (n = 48) or a partially matched relative (n = 23) that had been depleted of CD5+ T cells with either an anti-CD5 mooclonal antibody (MoAb) plus complement (anti-Leu1 + C) or an anti- CD5 MoAb conjugated to ricin A chain (ST1 immunotoxin [ST1-IT]). These patients received intensive chemoradiotherapy consisting of cytosine arabinoside, cyclophosphamide, and fractionated total body irradiation. Both anti-Leu1 + C and ST1-IT ex vivo treatments effectively depleted bone marrow of T cells (97% and 95%, respectively). Overall, primary and late graft failure each occurred in 4% of evaluable patients. The diagnosis of myelodysplasia was a significant risk factor for graft failure (P less than .001), and if myelodysplastic patients were excluded, there were no graft failures in major histocompatibility complex (MHC)-matched patients and 2 of 23 (8.7%) in MHC-mismatched patients. The actuarial risk of grade 2 to 4 acute graft-versus-host disease (GVHD) was 23% in MHC-matched patients and 50% in MHC- mismatched patients. In MHC-matched patients, acute GVHD tended to be mild and treatable with corticosteroids. Chronic GVHD was observed in 6 of 36 (17%) MHC-matched patients and none of 11 MHC-mismatched patients. There were no deaths attributable to GVHD in the MHC-matched group. Epstein-Barr virus-associated lymphoproliferative disorders were observed in 3 of 23 MHC-mismatched patients. The actuarial event-free survival was 38% in the MHC-matched patients versus 21% in the MHC- mismatched patients. However, if outcome is analyzed by risk of relapse, low-risk patients had a 62% actuarial survival compared with 11% in high-risk patients. These data indicate that the use of anti-CD5 MoAbs can effectively control GVHD in histocompatible patients, and that additional strategies are required in MHC-mismatched and high-risk patients.
Rapid anticoagulation using ancrod for heparin-induced thrombocytopenia [see comments]
Authors: Demers C , Ginsberg JS , Brill-Edwards P , Panju A , Warkentin TE , Anderson DR , Turner C , Kelton JG
Published: October 2019

In order to determine the efficacy and safety of ancrod, a rapid acting defibrinogenating drug, for patients with heparin-induced thrombocytopenia, 11 consecutive patients who required anticoagulant therapy because of venous thromboembolism and who developed acute heparin-induced thrombocytopenia or had a history of heparin-induced thrombocytopenia were treated with ancrod. Heparin therapy was discontinued (in patients receiving heparin) and ancrod started at a dose of 1 to 2 U/kg every 24 hours with subsequent daily doses adjusted to maintain fibrinogen levels between 0.5 and 1.0 g/L. Ancrod was continued until warfarin had become effective. The platelet count increased to more than 150 x 10(9)/L within 2 to 10 days in all thrombocytopenic patients. Two patients with a history of heparin- induced thrombocytopenia maintained normal platelet counts while receiving ancrod. Two patients had recurrent venous thrombosis while receiving warfarin, 10 days after ancrod was discontinued: one of these patients had metastatic pancreatic carcinoma and developed phlegmasia cerulea dolens and the other patient developed a venographically proven extension of her deep venous thrombosis. One patient suffered a bleeding episode into the thigh with a 16-g/L decrease in her hemoglobin level while receiving ancrod therapy. No other side effects were noted. Our experience indicates that ancrod therapy is a reasonable approach for patients with heparin-induced thrombocytopenia who require anticoagulant therapy.
Diamond-Blackfan anemia: heterogenous response of hematopoietic progenitor cells in vitro to the protein product of the steel locus
Authors: Olivieri NF , Grunberger T , Ben-David Y , Ng J , Williams DE , Lyman S , Anderson DM , Axelrad AA , Correa P , Bernstein A
Published: October 2019

Diamond-Blackfan anemia is a congenital disorder of erythropoiesis in humans, characterized by a macrocytic anemia often associated with physical anomalies. Mutations at either the W or Steel loci in the mouse also leads to a severe macrocytic anemia, as well as other developmental abnormalities. The W locus encodes the proto-oncogene c- kit, a member of the receptor tyrosine kinase family, while the Steel locus encodes a potent hematopoietic growth factor that is the ligand for c-kit. Growth of clonogenic marrow erythroid progenitor cells in vitro in the presence of the recombinant hematopoietic growth factors interleukin-3 (IL-3) and Steel was used to characterize this disease at the cellular level. Three patterns of in vitro marrow response to both recombinant IL-3 or Steel were observed among 10 Diamond-Blackfan patients: those that responded quantitatively and qualitatively almost as well as cells from normal marrow, those that responded at an intermediate level, and those that did not respond at all. These results provide evidence for cellular heterogeneity underlying the pathogenesis of this disorder and therefore raise the possibility that there may be more than one underlying molecular basis for the disease. No gross abnormalities in the structure of either the c-kit or Steel loci were observed in these patients. The normal response in culture of the progenitor cells from at least some patients to Steel with or without IL-3 raises the possibility of using this novel growth factor as a therapeutic agent in Diamond-Blackfan anemia.
Molecular basis for antithrombin III type I deficiency: three novel mutations located in exon IV
Authors: Vidaud D , Emmerich J , Sirieix ME , Sie P , Alhenc-Gelas M , Aiach M
Published: October 2019

Antithrombin III (AT III) type I deficiencies are characterized by a 50% decrease of both immunoreactive and functional protein and carry a high risk of thrombotic complication. We have studied the molecular basis for such deficiencies by asymmetric polymerase chain reaction amplification and direct sequencing of the seven exons and of the intron-exon junction of the AT III gene. Three different mutations were observed in the exon IV: a 4-bp deletion, a 2-bp deletion, and a nucleotide insertion. Each of these mutations results in a frameshift introducing premature stop codons at positions 313, 309, and 232, respectively. These results were confirmed by dot-blot analysis with allele-specific oligonucleotide probes. Furthermore, no mutation was observed in the other six exons. The comparison of the type of mutations observed by our group in six cases of type I deficiencies and in 16 cases of type II heparin binding site variants deficiencies suggests that the former are caused by heterogeneous molecular abnormalities while the latter are caused by recurrent missense mutations.
Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1
Authors: Conboy JG , Shitamoto R , Parra M , Winardi R , Kabra A , Smith J , Mohandas N
Published: October 2019

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.
Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C
Authors: Durante W , Kroll MH , Vanhoutte PM , Schafer AI
Published: October 2019

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin- induced aggregation. These data indicate that EDRF blocks thrombin- induced platelet aggregation by inhibiting the activation of PIP2- specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.
IgM antibody response in acute hepatitis C viral infection
Authors: Clemens JM , Taskar S , Chau K , Vallari D , Shih JW , Alter HJ , Schleicher JB , Mimms LT
Published: October 2019

IgM antibody against hepatitis C virus (IgM anti-HCV) was measured in serial samples from 15 transfusion recipients in whom posttransfusion chronic non-A, non-B hepatitis (NANBH) developed and three plasmapheresis donors during acute HCV infection using recombinant proteins derived from three immunodominant regions: core, NS-3, and NS- 4 (c100). IgM anti-HCV core was detected in 13 of 15 posttransfusion patients. Nine of these patients had transient, acute-phase IgM anti- HCV core detected coincidentally or earlier than active IgG anti-HCV core response. The average duration of IgM anti-HCV core reactivity was 8.1 +/- 3.7 weeks. One patient lacking an IgM anti-HCV core response had detectable IgM anti-HCV NS-3 during the acute phase. Passive transfer of IgM anti-HCV was not observed in these posttransfusion cases, in contrast to the high frequency observed for IgG anti-HCV. Late IgM anti-HCV was detectable against core, c100, and NS-3 in three, two, and one posttransfusion patients, respectively. These data indicate that IgM anti-HCV core is a useful acute-phase marker in HCV infection.
Factor-dependent erythroid cell lines derived from mice transplanted with hematopoietic cells expressing the v-src oncogene
Authors: Kennedy M , Beug H , Wagner EF , Keller G
Published: October 2019

Transplantation of spleen cells from primary reconstituted mice expressing the v-src oncogene to secondary and tertiary irradiated recipients resulted in the emergence of erythroid precursors with a transformed phenotype. When cultured in methyl cellulose, these precursors generated colonies of undifferentiated cells that could be expanded into continuously growing factor-dependent cell lines in liquid culture. All lines tested had a similar phenotype and expressed the v-src oncogene. In addition they responded to factors that regulate normal erythroid development, namely erythropoietin (Epo), interleukin- 3 (IL-3), and mast cell growth factor (MGF), the ligand to the c-kit encoded receptor. When cells from one of the lines were maintained in the absence of factor, a “factor independent” subpopulation emerged that appeared to grow in an autocrine fashion. Conditioned medium from these cells stimulated their own growth as well as the growth of broad spectrum of normal precursors. Studies with neutralizing antibodies indicated that the predominant colony-stimulating factor produced by these cells is IL-3.
VH gene rearrangement events can modify the immunoglobulin heavy chain during progression of B-lineage acute lymphoblastic leukemia [see comments]
Authors: Wasserman R , Yamada M , Ito Y , Finger LR , Reichard BA , Shane S , Lange B , Rovera G
Published: October 2019

The presence of multiple VHDJH joinings in upwards of 30% of acute lymphoblastic leukemias (ALL) suggests a relative instability of the rearranged immunoglobulin heavy chain (IgH) gene, but the mechanisms involved are not completely understood. An investigation of the structure of the VHDJH joinings using complementarity determining region (CDR)3 polymerase chain reaction (PCR) in 12 leukemias at both diagnosis and relapse indicates that this instability may increase as a function of time. In only one of seven cases in which relapse occurred within 3 years from diagnosis was a new VHDJH joining identified and this coexisted with the original diagnostic joining. Most strikingly, new VHDJH joinings were identified in four of five cases in which relapse occurred more than 5 years from diagnosis. In this latter population, the instability of the joinings was generated from VH----VH gene replacement events in two cases, since the new joinings retained the original DJH sequences and partial N region homology at the VHD junction, and probably in a third case from a VH gene rearrangement to a common DJH precursor. Furthermore, in five of 23 (21.7%) additional cases studied at diagnosis, subclones were identified that had similar modifications of the VH-N region. These data indicate that VH gene replacement events and VH gene rearrangements to a common DJH joining contribute to the instability of the VHDJH joining in ALL. This phenomenon should be taken into consideration in those methodologies that exploit IgH rearrangements for detection of minimal residual disease.
Absence of abnormalities of c-kit or its ligand in two patients with Diamond-Blackfan anemia
Authors: Abkowitz JL , Broudy VC , Bennett LG , Zsebo KM , Martin FH
Published: October 2019

As Diamond-Blackfan anemia shares clinical features with W and Steel defects in mice, we investigated the possibility that this human disorder might result from an abnormality of the c-kit receptor or its ligand, stem cell factor (SCF). For these studies, full nucleotide sequences for coding regions of c-kit and SCF were generated for two Diamond-Blackfan anemia patients and were normal. Similarly, the kds of SCF receptors on their marrow cells (31 pmol/L, 43 pmol/L) were comparable with those found in three normal controls (50 pmol/L, 55 pmol/L, 27 pmol/L). Serum SCF concentrations were 6.9 ng/mL in patient A, 14.6 ng/mL in patient B, who has been in hematologic remission since adolescence, and 2.7 ng/mL in the 3-year-old daughter of patient B, who also has Diamond-Blackfan anemia but is transfusion-dependent. It is possible that the SCF level in patient B increased with puberty, leading to her remission. These data provide evidence that Diamond- Blackfan anemia does not result from structural abnormalities of c-kit or SCF.
Human lymphokine activated killer (LAK) cells suppress generation of allospecific cytotoxic T cells: implications for use of LAK cells to prevent graft-versus-host disease in allogeneic bone marrow transplantation
Authors: Uberti J , Martilotti F , Chou TH , Kaplan J
Published: October 2019

We have found that murine lymphokine activated killer (LAK) cells have veto and natural suppressor activities in vitro, and prevent graft- versus-host disease (GVHD) in vivo. To determine whether human LAK cells mediate veto and natural suppression we measured their ability to inhibit generation of allospecific cytotoxic T cells (CTL) in mixed lymphocyte culture (MLC). When added to MLCs at low concentrations LAK cells caused veto-type inhibition: stimulator-type LAK cells inhibited generation of CTL but responder or third-party LAK cells did not. At higher concentrations LAK cells caused nonspecific inhibition: all three LAK cell types inhibited generation of CTL. LAK cell veto and natural suppressor activities were largely eliminated by irradiation with 30 Gy and by depletion of CD56+ cells, but increased after depletion of CD3+ cells. LAK cell veto activity is not likely an artifact of cold-target inhibition by the LAK cells themselves or by proliferation of T cells contaminating LAK cell preparations: (1) veto only occurred when LAK cells were added to MLC on days 0 through 2, but not when added on day 5; (2) addition of saturating numbers of labeled targets to fixed numbers of allo-CTL effectors failed to overcome the inhibitory effects of adding stimulator-type LAK cells at the onset of MLC; and (3) CD3-depleted LAK cells showed greater veto activity than threefold greater numbers of control LAK cells. In light of our previous findings in mice, the current results imply that adoptive immunotherapy with LAK cells may be useful in preventing GVHD in human bone marrow transplant recipients.
Subcutaneous erythropoietin for treatment of refractory anemia in hematologic disorders. Results of a phase I/II clinical trial [see comments]
Authors: Cazzola M , Ponchio L , Beguin Y , Rosti V , Bergamaschi G , Liberato NL , Fregoni V , Nalli G , Barosi G , Ascari E
Published: October 2019

We have used recombinant human erythropoietin (rHuEPO) in a phase I/II clinical trial to evaluate its ability to reverse refractory anemia in hematologic disorders. rHuEPO was administered subcutaneously 5 days per week at escalating doses (50 to 150 U/kg per day). The aim of treatment was a hemoglobin (Hb) level greater than or equal to 10 g/dL without blood transfusion. Of 25 patients treated, 17 were evaluable, most of them with a regular need for transfusion. Eight of these had lymphoproliferative disorders (three cases of malignant lymphoma and five of monoclonal gammopathy) and were exposed to cytotoxic therapy. The other nine patients had hematopoietic stem cell disorders (four cases of myelodysplastic syndrome, three of idiopathic myelofibrosis, and two of chronic myelogenous leukemia). All patients with lymphoproliferative disorder had serum EPO levels inappropriately low for the degree of anemia, while patients with stem cell disorder showed variable values. Erythroid marrow activity was inadequate in all cases. Seven of eight patients with lymphoproliferative disorder responded to treatment maintaining Hb above 10 g/dL without transfusion. The median dose of rHuEPO required for correction of anemia was 75 U/kg. In four cases response was maintained with 50 U/kg, three times per week. There was no complete response among patients with hematopoietic stem cell disorder, although transfusion requirement was eliminated or reduced in four cases. Four patients developed functional iron deficiency during rHuEPO treatment and required iron supplementation to obtain response. Aggravation of splenomegaly was observed in two cases of myeloproliferative disorder. We conclude that: (1) subcutaneous administration of rHuEPO can be effective and safe in patients with lymphoproliferative disorder exposed to chemotherapy and showing inappropriate EPO response to anemia; (2) this is less likely in hematopoietic stem cell disorders, although favorable responses may be observed in occasional patients; and (3) functional iron deficiency as a cause of nonresponse to rHuEPO is frequent also in nonrenal anemia.
Phase II trial of recombinant human granulocyte-macrophage colony- stimulating factor in patients undergoing allogeneic bone marrow transplantation from unrelated donors
Authors: Nemunaitis J , Anasetti C , Storb R , Bianco JA , Buckner CD , Onetto N , Martin P , Sanders J , Sullivan K , Mori M
Published: October 2019

The safety and possible efficacy of recombinant human granulocyte- macrophage colony-stimulating factor (rhGM-CSF) were evaluated in 40 consecutive patients who received transplants from unrelated donors. rhGM-CSF was administered by 2-hour daily intravenous infusion from day 0 to day 20 or day 27 after the marrow infusion. These patients were compared with 78 historical patients who received transplants from unrelated donors who did not receive rhGM-CSF. The rhGM-CSF-treated patients were older (P = .037) and were treated less frequently in laminar air flow rooms (P = .005) than were control patients. However, the rhGM-CSF-treated group had a higher proportion of “good risk” patients with chronic myelogenous leukemia in chronic phase (P = .006) than did the comparison group (P = .017), rendering comparisons of transplant-related complications not meaningful. rhGM-CSF was well tolerated and did not adversely increase the incidence of graft rejection or increase the incidence and severity of acute graft-versus- host disease. The median day the absolute neutrophil count reached 500/mm3 in patients who received rhGM-CSF was day 21, which was not different from that of historical patients. Nevertheless, the numbers of febrile days and septicemic episodes within the first 28 days in patients who received rhGM-CSF were less than in historical patients. The probability of nonrelapse mortality at 1 year in patients who received rhGM-CSF was 22%. In view of the retrospective nature of the control group, we cannot conclusively determine whether rhGM-CSF administration was beneficial. A prospective, randomized controlled study of rhGM-CSF is required to confirm these suggestive data.
Cloning and analysis of the CD18 promoter
Authors: Rosmarin AG , Levy R , Tenen DG
Published: October 2019

CD18, the common beta chain of the leukocyte integrin adhesion proteins, is expressed exclusively by myeloid cells and lymphocytes. During myeloid differentiation, the increase in CD18 cell surface expression is paralleled by increased CD18 messenger RNA levels. Nuclear run-on studies show that CD18 expression is transcriptionally regulated during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL- 60 monocytic differentiation. The CD18 transcriptional start site was defined by primer extension and RNAse protection. The gene encoding CD18 was cloned and a fragment that overlaps the transcriptional start site was isolated. DNA sequence analysis of this promoter fragment identified potential AP-1 elements that may mediate the TPA transcriptional response. The CD18 promoter also contains a putative binding site for PU.1, a leukocyte-specific transcription factor. DNA elements resembling those found in other myeloid and integrin promoters were identified. The CD18 promoter fragment was linked to the luciferase reporter gene, electroporated into the U937 monocytic cell line, and its expression increased after exposure to TPA. Thus, CD18 may serve as a model for identifying the cis elements and trans-acting factors that regulate gene expression during myeloid differentiation.