bluuebunny/crossref_metadata_2025 huggingface dataset:
Immobilized Arg-Gly-Asp (RGD) peptides of varying lengths as structural probes of the platelet glycoprotein IIb/IIIa receptor
Authors: Beer JH , Springer KT , Coller BS
Published: October 2019

The interactions between ligands containing the recognition sequence arginine-glycine-aspartic acid (RGD) and integrin receptors are important in many cell-cell and cell-protein interactions. The platelet contains five integrin receptors and they contribute significantly to platelet adhesion and aggregation. To investigate the RGD binding domains on platelet integrins, we immobilized a series of RGD peptides containing variable numbers of glycine residues [(G)n-RGDF] on polyacrylonitrile beads and evaluated the ability of the beads to interact with platelets. With native platelets, virtually no interaction occurred with G1-RGDF beads, but the interactions increased as the number of glycine residues increased, plateauing with the G9- RGDF and G11-RGDF beads. ADP pretreatment enhanced the interactions with all of the beads, whereas prostaglandin E1 pretreatment eliminated the interactions with the shortest peptide beads, but only partially inhibited interactions with the longer peptide beads. Monoclonal antibodies to glycoprotein (GP) IIb/IIIa were most effective in inhibiting the interactions, but antibodies to GPIIb/IIIa with similar inhibitory effects on fibrinogen binding varied dramatically in their ability to inhibit the interaction between platelets and immobilized RGD peptides. Our data indicate that the majority of RGD binding sites on GPIIb/IIIa can be reached by peptides that extend out approximately 11 to 32 A from the surface of the bead, and these results are in accord with the dimensions of integrin receptors deduced from electron microscopy. Activation of GPIIb/IIIa facilitates the interactions, but platelet inhibition fails to eliminate the interactions with the longer peptide beads, suggesting that access to the RGD binding site on at least a fraction of the GPIIb/IIIa receptors is always possible for preferred ligands. Finally, we found that the G3-RGDF peptide beads were uniquely sensitive to the activation state of the GPIIb/IIIa receptor.
Cytochalasin D and E: effects on fibrinogen receptor movement and cytoskeletal reorganization in fully spread, surface-activated platelets: a correlative light and electron microscopic investigation
Authors: Olorundare OE , Simmons SR , Albrecht RM
Published: October 2019

This study investigates the involvement of actin microfilaments in fibrinogen receptor redistribution and cytoskeletal reorganization that takes place in fully spread, surface-activated platelets. Colloidal gold-labeled fibrinogen (Fgn-Au label) in conjunction with video- enhanced differential interference contrast light microscopy (VDIC) was used to identify fibrinogen binding sites, glycoprotein IIb/IIIb (GPIIb/IIIa), on fully spread platelets. Platelets were treated with cytochalasins D and E (5 x 10(-5) mol/L to 5 x 10(-8) mol/L) for 10 minutes, before or after incubation with Fgn-Au label. Results observed with VDIC were subsequently confirmed by high-voltage transmission and low voltage-high resolution scanning electron microscopic examination of the specimens. Preincubation of activated platelets with cytochalasin D or E (5 x 10(-5) and 5 x 10(-6) mol/L) inhibited fibrinogen receptor redistribution and abolished cytoskeletal reorganization in fully spread platelets. After surface-activated platelets were incubated with Fgn-Au label, treatment with the above concentrations of cytochalasin D or E disrupted cytoskeletal reorganization and caused random movement of previously redistributed receptor-ligand complexes. Incubation of platelets with cytochalasin E 5 x 10(-6) mol/L prevented platelet activation and spreading. Thus, actin filaments appear necessary for platelet spreading from the discoid to the fully spread stage. The ligand-triggered, cytoskeletally directed movement of fibrinogen receptors in fully spread platelets appears to be dependent on the presence of intact, polymerized actin. This movement is distinct from the cytochalasin-insensitive accumulation of GPIIb/IIIa-ligand in the channels of the open canalicular system.
Translocations involving band 3q27 and Ig gene regions in non-Hodgkin's lymphoma [see comments]
Authors: Bastard C , Tilly H , Lenormand B , Bigorgne C , Boulet D , Kunlin A , Monconduit M , Piguet H
Published: October 2019

We report a series of 20 non-Hodgkin's lymphomas (NHL) in which cytogenetic analysis showed a translocation involving band 3q27 and the site of one of the three Ig genes (14q32, 2p12, 22q11) in the neoplastic cells. These cases were found in a series of 319 patients with clonal chromosomal abnormalities studied over a 7-year period. Fourteen patients had diffuse lymphoma, mainly of large cell type and the remaining six were follicular lymphomas. All cases studied were of B-cell phenotype. A t(3;14)(q27;q32) was commonest, found in 15 patients (4.7%), with the two variant translocations, t(3;22)(q27;q11) and t(2;3)(p12;q27), being found in three and two patients, respectively. Additional chromosomal defects were present in most patients, but two patients had this type of translocation as the sole abnormality. These results indicate that translocations involving band 3q27 and Ig genes are not uncommon, and suggest that a novel oncogene, located at band 3q27, may be implicated in B-cell NHL.
Multicenter randomized study comparing cyclosporine-A alone and antithymocyte globulin with prednisone for treatment of severe aplastic anemia
Authors: Gluckman E , Esperou-Bourdeau H , Baruchel A , Boogaerts M , Briere J , Donadio D , Leverger G , Leporrier M , Reiffers J , Janvier M
Published: October 2019

We report the results of a randomized multicenter study comparing the efficacy of antithymocyte globulin (ATG) with that of cyclosporin A (CsA) as first-line therapy for severe aplastic anemia (SAA). Patients were randomized to receive ATG and prednisone (PDN) or CsA; hematologic response and toxicity were compared. At 3-month evaluation, patients who had no or minimal response received the alternative therapy to assess the value of a sequential immunosuppressive therapy for treatment of severe aplastic anemia. One hundred nineteen patients were randomized; 25 were excluded, of whom 3 were misdiagnosed and 22 did not follow the cross-over protocol. Ninety-four patients were analyzed; 46 received CsA, and 48 received ATG-PDN. The actuarial survival was 66.7%, with a median follow-up time of 19 months. There was no significant difference in survival between the groups with, at 3 months, an actuarial survival of 88% in the CsA group and 75% in the ATG group (NS); at 12 months, it was 70% in the CsA group and 64% in the ATG group (NS). The percentage of complete and partial response was 11.6% and 16%, respectively, at 3 months, and 31.6% and 30%, respectively, at 12 months (NS). The main prognostic factor was the absolute neutrophil count (ANC) at entry: Patients with ANC less than 0.2 x 10(9)/L had a significantly lower survival as compared with patients with more than 0.2 x 10(9)/L ANC (P = .0001). At 12 months, 62 evaluable patients were alive, with a complete or partial response in 36 patients. Patients who had responded to the first treatment had a better recovery of bone marrow failure than those who had sequential immunosuppression. The main complication was infection, which was more often observed and more often lethal during ATG and PDN therapy. In this study, initial treatment of SAA with either CsA or ATG-PDN followed by cross-over therapy for nonresponders produced comparable response and survival rates.
Interferon-gamma enhances factor-dependent myeloid proliferation of human CD34+ hematopoietic progenitor cells
Authors: Caux C , Moreau I , Saeland S , Banchereau J
Published: October 2019

Numerous studies have shown that interferon-gamma (IFN gamma) inhibits the proliferative effects of colony-stimulating factors (CSFs) on human bone marrow cells. In the present study we investigated the effects of IFN gamma and other described inhibitory factors on the proliferation of highly purified CD34+ human hematopoietic progenitor cells (HPC) in response to recombinant CSFs. While transforming growth factor-beta (TGF beta) and IFN alpha were highly inhibitory, IFN gamma strongly potentiated interleukin-3 (IL-3) and, to a lesser extent, granulocyte- macrophage-CSF (GM-CSF) induced growth of CD34+ HPC. IFN gamma had no significant proliferative effect per se, and did not affect granulocyte- CSF (G-CSF)-dependent cell proliferation. Within 10 days the number of viable cells generated in the presence of IL-3 + IFN gamma was two times higher than in the presence of IL-3 alone. Limiting dilution analysis showed that IFN gamma acts directly on its target cell to increase the frequency of IL-3-responding cells without affecting the average size of the IL-3-dependent clones. Enhanced frequency of IL-3- and GM-CSF-responding cells was also observed in colony assays where the addition of IFN gamma increased by twofold to threefold the number of granulocyte colony-forming units (CFU-G), macrophage CFUs (CFU-M), granulocyte-macrophage CFUs (CFU-GM), and mixed erythroid (E-MIX). In contrast, IFN gamma did not affect the generation of erythroid burst- forming units (BFU-e) in such cultures. In longer-term culture, the combination of IFN gamma and IL-3 did not alter the lineage distribution of the cells when compared with IL-3 alone. However, after 15 days, when mature cells were present in the cultures, IFN gamma displayed cell concentration-related growth-inhibitory effects. Thus, IFN gamma appears to stimulate the early stage of myelopoiesis by enhancing the frequency of growth factor-responding cells but, unlike tumor necrosis factor alpha (TNF alpha), does not alter cell differentiation.
Plasminogen activator inhibitor-1 messenger RNA expression is induced in rat hepatocytes in vivo by dexamethasone
Authors: Konkle BA , Schuster SJ , Kelly MD , Harjes K , Hassett DE , Bohrer M , Tavassoli M
Published: October 2019

Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue plasminogen activator (tPA), plays a crucial role in the regulation of fibrinolysis. Both hepatocytes and endothelial cells have been implicated as major sources of plasma PAI-1. To study the relative contribution of these cell types to hepatic PAI-1 production, we have separated hepatocytes and hepatic sinusoidal endothelial cells by fractionation of freshly isolated rat livers using metrizamide density gradients and centrifugal elutriation. In untreated animals, PAI-1 messenger RNA (mRNA) was detected only in the purified endothelial cell fraction, and not in the hepatocyte fraction or in unfractionated liver. However, when the animals were treated with dexamethasone, PAI-1 mRNA expression was transiently induced in the liver. This induction paralleled the appearance of PAI-1 mRNA in purified hepatocytes, while PAI-1 expression in sinusoidal endothelial cells was unchanged. Four hours after dexamethasone treatment, plasma PAI-1 levels were increased approximately twofold over levels measured in animals treated with the diluent alone. These data suggest that PAI- 1 production by hepatocytes may contribute to elevated plasma PAI-1 levels in the setting of acute injury and stress.
Mutations of p53 gene and their relation to disease progression in B- cell lymphoma
Authors: Ichikawa A , Hotta T , Takagi N , Tsushita K , Kinoshita T , Nagai H , Murakami Y , Hayashi K , Saito H
Published: October 2019

The alteration of p53 tumor suppressor gene was studied in 48 patients with B-cell lymphoma. A sequential combined technique of polymerase chain reaction-mediated single-strand conformational polymorphism (PCR- SSCP) or reverse transcription (RT)-PCR-SSCP and direct sequencing were used as a simple and sensitive approach to analyze nucleotide changes. By these methods, we identified 8 missense point mutations and 2 codon deletions in 9 of the 48 patients. These mutations were located in or close to the evolutionally highly conserved regions of the p53 gene. Eight of nine patients having p53 gene alterations were in advanced clinical stage (IV). It is the first report of p53 gene mutations in follicular and diffuse lymphoma. These observations suggest that the p53 gene alteration may play an important role in lymphomagenesis and/or disease progression in some types of B-cell lymphoma.
Marked increase in veno-occlusive disease of the liver associated with methotrexate use for graft-versus-host disease prophylaxis in patients receiving busulfan/cyclophosphamide
Authors: Essell JH , Thompson JM , Harman GS , Halvorson RD , Snyder MJ , Johnson RA , Rubinsak JR
Published: October 2019

The use of cyclosporine-A/methotrexate (CyA/MTX) for graft-versus-host disease (GVHD) prophylaxis is safe and effective for patients undergoing allogeneic bone marrow transplantation after preparation with cyclophosphamide and total body irradiation. We report 87 patients prepared for allogeneic transplant with busulfan 4 mg/kg/d orally for 4 days, followed by cyclophosphamide 60 mg/kg/d intravenously for 2 days (Bu4Cy2). A marked increase in hepatotoxicity was observed in 20 patients administered CyA/MTX, compared with 67 historical control patients who received CyA/methylprednisolone (CyA/MP) for GVHD prophylaxis with all other treatment and support variables remaining constant. The incidence of hyperbilirubinemia (bilirubin greater than or equal to 2 mg/dL) increased from 48% to 80% (P = .02), and the mean maximal bilirubin increased from 4.67 +/- 7.27 to 8.72 +/- 8.73 mg/dL (P = .04), when CyA/MTX was used in place of CyA/MP for GVHD prophylaxis. In addition, the incidence of veno-occlusive disease (VOD) increased from 18% to 70% (P = .0001), and death caused by VOD increased from 4.5% to 25% (P = .02). Survival was not significantly different for the two groups because of a higher non-VOD death rate in patients receiving CyA/MP for GVHD prophylaxis (P = .77). We suggest caution when using Bu4Cy2 in combination with CyA/MTX for GVHD prophylaxis.
Evidence for in vivo clonal proliferation of unique population of blood CD4-/CD8- T cells bearing T-cell receptor alpha and beta chains in two normal men
Authors: Kusunoki Y , Hirai Y , Kyoizumi S , Akiyama M
Published: October 2019

Rare T lymphocytes bearing CD3 surface antigen and T-cell receptor (TCR) alpha and beta chains, but lacking both CD4 and CD8 antigens, viz, TCR alpha beta+CD4–8- cells, appear at a frequency of 0.1% to 2% in peripheral blood TCR alpha beta+ cells of normal donors. Here we report two unusual cases, found among 100 healthy individuals studied, who showed an abnormally elevated frequency of these T cells, ie, 5% to 10% and 14% to 19%. Southern blot analyses of the TCR alpha beta+CD4–8- clones all showed the identical rearrangement patterns for each individual, demonstrating that these are derivatives of a single T cell. The same rearrangement patterns were also observed for the freshly isolated lymphocytes of TCR alpha beta+CD4-CD8- fraction, which excludes the possible bias in the processes of in vitro cloning. These TCR alpha beta+CD4–8- T cells were found to express other mature T-cell markers such as CD2, CD3, and CD5 antigens, as well as natural killer (NK) cell markers (CD11b, CD16, CD56, and CD57 antigens) for both individuals. Further, although lectin-dependent or redirected antibody- dependent cell-mediated cytotoxicities were observed for both freshly sorted lymphocytes of TCR alpha beta+CD4–8- fraction and in vitro established clones, NK-like activity was not detected.
Lactoferrin gene promoter: structural integrity and nonexpression in HL60 cells
Authors: Johnston JJ , Rintels P , Chung J , Sather J , Benz EJ Jr , Berliner N
Published: October 2019

Lactoferrin is a member of the transferrin family of iron-binding proteins. It is found in several glandular epithelial tissues and human neutrophils, where it is localized to secondary granules. To examine the mechanisms controlling lactoferrin gene expression in neutrophils and defects in its expression in acute leukemia, we have cloned a lactoferrin cDNA from a chronic myelogenous leukemia library, and used it to obtain genomic clones representing the chromosomal lactoferrin gene. Using polymerase chain reaction, primer extension, and S1 analysis, we have identified the 5′ end of the lactoferrin mRNA. We have defined a putative promoter region for the gene, and characterized its first two exons. In addition, we have examined the structure of these regions in DNA from HL60 cells. HL60 is a leukemic cell line that undergoes phenotypic neutrophil maturation on exposure to dimethyl sulfoxide (DMSO). However, the cells cannot be induced to express any secondary granule protein genes. We have shown that the 5′ end of the lactoferrin gene, including the putative promoter region, is entirely normal in HL60. By Northern analysis, nuclear run-on studies, and primer extension assays we have shown that the gene is not transcribed in DMSO-induced HL60 cells. This supports the hypothesis that the defect in HL60 is an abnormality in the production or activity of a transacting regulator of lactoferrin gene expression.
Identification of a 33-Kd protein associated with the alpha-granule membrane (GMP-33) that is expressed on the surface of activated platelets
Authors: Metzelaar MJ , Heijnen HF , Sixma JJ , Nieuwenhuis HK
Published: October 2019

To identify antigens on the platelet plasma membrane that are exposed after activation, we developed a monoclonal antibody (MoAb) designated RUU-SP 1.77. The RUU-SP 1.77 antigen is present on the membrane of resting platelets at a basal level and is strongly expressed on the plasma membrane after thrombin activation. Freshly fixed platelets bound 4,150 +/- 1,935 (mean +/- SD) RUU-SP 1.77 molecules per platelet; on fixed thrombin-stimulated platelets the number of binding sites was upregulated to 19,050 +/- 5,120 (kd 4.5 +/- 0.8 nmol/L). MoAb RUU-SP 1.77 recognized a major protein of 33 Kd and a minor 28-Kd protein, both under nonreduced and reduced conditions. Immunoelectron microscopic studies showed the presence of the protein associated with the membrane of alpha-granules. Due to the localization associated with the alpha-granule membrane, we have designated it GMP-33 (granule membrane protein with a molecular weight of 33 Kd). Based on structural properties, we conclude that GMP-33 is a protein associated with the alpha-granule membrane that has not been described before.
Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1
Authors: Li XK , Lijnen HR , Nelles L , Van Hoef B , Stassen JM , Collen D
Published: October 2019

A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296- G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296- G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt- PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS).
Changing antigen receptor gene rearrangements in a case of early pre-B cell leukemia: evidence for a tumor progenitor cell with stem cell features and implications for monitoring residual disease
Authors: Tycko B , Ritz J , Sallan S , Sklar J
Published: October 2019

A case of acute lymphoblastic leukemia (ALL) was encountered in which the two clonal gamma T-cell receptor gene (TCR gamma) rearrangements found in bone marrow (BM) samples at relapse both differed from the single clonal TCR gamma rearrangement present in BM obtained at diagnosis 5 years previously. In contrast, two clonal Ig heavy chain gene (IgH) rearrangements present at relapse were identical to those present at diagnosis. Comparison of the DNA sequences of the relapse TCR gamma rearrangements with that of the diagnostic TCR gamma rearrangement indicated that they must have been generated de novo from TCR gamma loci in germline configuration. By polymerase chain reaction using clonotypic N-region oligonucleotide primers (N-PCR), cells bearing the diagnosis or relapse TCR gamma rearrangements were undetectable in the sample from the opposite time point. Two BM samples obtained at different times in clinical remission were both devoid of detectable residual tumor when analyzed by N-PCR, indicating a depth of remission of less than 1 tumor cell per 4 x 10(5) BM mononuclear cells. The tumor cells showed a primitive phenotype: T-cell antigen-negative, CALLA/CD10-negative, CD20-negative, CD19-positive, and positive for the myeloid marker My9. This case, which appears to represent a tumor arising from a progenitor cell with both early B-lineage and certain stem cell features, has implications for monitoring residual ALL and possibly also for treatment of the disease.
Evidence for the involvement of distinct signal transduction pathways in the regulation of constitutive and interferon gamma-dependent gene expression of NADPH oxidase components (gp91-phox, p47-phox, and p22- phox) and high-affinity receptor for IgG (Fc gamma R-I) in human polymorphonuclear leukocytes
Authors: Amezaga MA , Bazzoni F , Sorio C , Rossi F , Cassatella MA
Published: October 2019

We recently showed that mRNA levels coding the high-affinity Fc gamma receptor for IgG (Fc gamma R-I, CD64) and two of the components of the phagocytic superoxide anion-generating system--the heavy-chain subunit of cytochrome b558 (gp91-phox) and the 47-Kd cytosolic factor (p47- phox)--are modulated by interferon gamma (IFN-gamma). In this study, we examined whether dexamethasone (DEX) affects gp91-phox and p47-phox mRNA expression of human polymorphonuclear leukocytes (PMN), treated or not with IFN-gamma. We also investigated whether staurosporine, a general inhibitor of protein kinases, influences gp91-phox, p47-phox, and Fc gamma R-I gene expression in PMN treated with or without IFN- gamma. We found that (1) gp91-phox mRNA steady-state levels, expressed in control or IFN-gamma-treated PMN, were significantly inhibited, in a dose-dependent fashion, by both DEX and staurosporine; (2) p47-phox mRNA steady-state levels, expressed in control or IFN-gamma-treated PMN, were not influenced by DEX, but were markedly depressed by staurosporine; (3) no changes of spectrophotometric cytochrome b558 were found in PMN treated for up to 20 hours with the inhibitors, regardless of the presence of IFN-gamma; (4) both DEX and staurosporine dose-dependently inhibited IFN-gamma-induced Fc gamma R-I mRNA and protein expression; and (5) stability of gp91-phox and Fc gamma R-I messages in IFN-gamma-treated PMN was not altered by the presence of DEX. Our results demonstrate that gp91-phox, p47-phox, and Fc gamma R-I gene expression of PMN is governed by specific and independent biochemical pathways. Moreover, IFN-gamma activates different signal transduction pathways to modulate mRNA expression of gp91-phox, p47- phox, and Fc gamma R-I.
Mechanisms of amphipath-induced stomatocytosis in human erythrocytes
Authors: Schrier SL , Zachowski A , Devaux PF
Published: October 2019

We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin- labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration- dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet.
Effect of M20 interleukin-1 inhibitor on normal and leukemic human myeloid progenitors
Authors: Peled T , Rigel M , Peritt D , Fibach E , Treves AJ , Barak V
Published: October 2019

This study aimed to assess the effect of the M20 interleukin-1 (IL-1) inhibitor on normal and leukemic hematopoietic cells. The M20-derived IL-1 inhibitor was found to inhibit the growth of various hematopoietic cells. The in vitro proliferation of myeloid cell lines in serum- containing medium or proliferation of these cells induced by IL-1 in serum-free medium (measured by 3H-TdR) were inhibited by the M20 IL-1 inhibitor. In addition, growth of normal progenitors and fresh leukemic cells stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF) (as measured by colony and liquid systems) was also inhibited by this factor. After the removal of the IL-1 inhibitor at the peak of growth inhibition, leukemic and normal progenitor cells retain their ability to grow and develop into GM-CSF colonies. These results show that the growth inhibition phenomena were reversible and did not result from a cytotoxic effect. Our data suggest that the M20-derived IL-1 inhibitor might function as a true negative growth regulator of normal and leukemic hematopoietic cells.
Cardiopulmonary bypass induces leukocyte-platelet adhesion
Authors: Rinder CS , Bonan JL , Rinder HM , Mathew J , Hines R , Smith BR
Published: October 2019

Cardiopulmonary bypass (CPB) has been demonstrated to activate platelets, producing an increased number of circulating platelets that have undergone alpha-granule release and express granule membrane protein-140 (GMP-140) on their surface. In vitro, GMP-140 mediates activated platelet adhesion to neutrophils (PMN) and monocytes, causing the formation of leukocyte-platelet conjugates. Using a newly developed assay that measures the percentage of circulating leukocyte-platelet conjugates in whole blood, we studied 17 patients undergoing CPB and have determined that (1) monocyte-platelet conjugates increased significantly during CPB, from 18% +/- 1.5% to 44% +/- 4.5% (mean +/- SEM) by the end of CPB, while PMN-platelet conjugates increased only slightly and lymphocyte-platelet conjugates decreased; (2) the time course of the increase in monocyte- and PMN-platelet conjugates paralleled that of the increase in circulating activated platelets, as determined by the presence of surface GMP-140; and (3) monocyte activation, as assessed by increased surface expression of CD11b, showed a gradual increase similar to the increase in monocyte-platelet conjugates, while PMN surface CD11b peaked immediately after the start of CPB. We conclude that CPB, through increased platelet GMP-140 expression, causes formation of monocyte-platelet, and to a lesser extent, PMN-platelet conjugates. The activation of monocytes and PMN on CPB, as evidenced by CD11b expression, occurs with differing time courses.
Hemophilia B caused by five different nondeletion mutations in the protease domain of factor IX
Authors: Ludwig M , Sabharwal AK , Brackmann HH , Olek K , Smith KJ , Birktoft JJ , Bajaj SP
Published: October 2019

Factor IX is a multidomain protein and is the proenzyme of a serine protease, factor IXa, essential for hemostasis. In this report, we describe the molecular basis of hemophilia B (deficiency of factor IX activity) in five patients who have neither deletions nor rearrangements of the factor IX gene. By enzymatic amplification and sequencing of all exons and promoter regions, the following causative mutation in the protease domain of factor IX was identified in each patient: IXSchmallenberg: nucleotide 31,215G----T, Ser365Ile; IXVarel: nucleotide 31,214A----G, Ser365Gly; IXMechtal: nucleotide 31,211G----C, Asp364His; IXDreihacken: nucleotide 30,864G----A, Arg248Gln; and IXMonschau: nucleotide 30,855A----T, Glu245Val. In IXVarel, nucleotide 31,213T was also replaced by C, which results in a silent mutation (GAT- ---GAC) at Asp-364. Thus, this patient has a double base-pair substitution of TA to CG at nucleotides 31,213 and 31,214 but only a single amino acid change of Ser-365 to Gly. This patient also developed an antibody to factor IX during replacement therapy, which suggests that deletion of the factor IX gene is not necessary for development of the antibody in hemophilia B patients. The levels of plasma factor IX antigen in the patients ranged from 40% to 100% except for IXDreihacken (Arg248Gln), in which case it was approximately 4% of normal. The Ser365Gly and Ser365Ile mutants are nonfunctional because of lack of the active site serine residue. Mutant Asp364His is inactive because it cannot form the hydrogen bond between the carboxylate group of Asp-364 and the alpha-amino group of Val-181 generated after activation. As observed in other homologous serine proteases, this hydrogen bond is essential for maintaining the correct active site conformation in normal factor IXa (IXaN). Purified Arg248Gln had approximately 41% and Glu245Val had approximately 17% of the activity of normal factor IX (IXN) in a partial thromboplastin time (aPTT) assay. In immunodot blot experiments, the isolated Glu245Val mutant did and the Arg248Gln mutant did not bind to an anti-IXN monoclonal antibody that has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. We have recently shown that a high-affinity calcium binding site exists in the protease domain of IXN; among the proposed Ca(2+)-binding ligands is the carboxyl group of Glu-245. Further, a part of the epitope for the above antibody was shown to be contained in the 231 to 265 residue segment of factor IX.(ABSTRACT TRUNCATED AT 400 WORDS).
Monosomy 7 myeloproliferative disease in children with neurofibromatosis, type 1: epidemiology and molecular analysis
Authors: Shannon KM , Watterson J , Johnson P , O'Connell P , Lange B , Shah N , Steinherz P , Kan YW , Priest JR
Published: October 2019

Loss of constitutional heterozygosity is a common molecular feature of cancers in which inactivation of one or more tumor suppressor genes is thought to contribute to tumorigenesis. Recent evidence suggests that the gene responsible for neurofibromatosis, type 1 (NF-1), belongs to this class of heritable cancer genes. Children with NF-1 show an increased incidence of myeloid leukemia, including juvenile chronic myelogenous leukemia (JCML) and, perhaps, the myeloproliferative syndrome (MPS) associated with bone marrow monosomy 7 (Mo 7). We have investigated five children with Mo 7: three with NF-1 and two others with suggestive evidence of NF-1. Southern blotting experiments performed in four patients showed no loss of heterozygosity in bone marrow specimens using probes linked to the NF-1 locus on the long arm of chromosome 17. Both of our patients with familial NF-1 inherited the disease from their mothers, as did 14 of 19 other cases of myeloid leukemia in children with familial NF-1. Seventeen of these 21 children were boys. Myeloid leukemia developed in 12 boys and four girls who inherited NF-1 from their mothers, and in five boys who inherited the disease from their fathers. Father-to-daughter transmission was not observed. Taken together, the presence of chromosome 7 deletions in the leukemias of children with NF-1, a pattern of inheritance favoring maternal transmission of NF-1, and the marked predilection for boys to develop JCML and Mo 7 suggest a multistep mechanism of oncogenesis in which epigenetic factors might play a role. Further investigation is required to determine if the NF-1 genes in the leukemic bone marrows of these patients have acquired point mutations or small deletions.
Two novel beta-thalassemia mutations in the 5' and 3' noncoding regions of the beta-globin gene [see comments]
Authors: Cai SP , Eng B , Francombe WH , Olivieri NF , Kendall AG , Waye JS , Chui DH
Published: October 2019

Two novel beta-thalassemia mutations are described. The first mutation, found in an Italian family, is a G----A substitution in nucleotide (nt) +22 relative to the beta-globin gene Cap site. This mutation creates a cryptic ATG initiation codon, the utilization of which for translation would result in premature termination 36 bp 3′ downstream. The second mutation, found in an Irish family, is a T----C substitution in nt +1570, or 12 bp 5′ upstream of the AATAAA polyadenylation signal in the 3′ noncoding region. It is postulated that this mutation leads to destabilization of the encoded beta-globin mRNA.
Recombinant human granulocyte-macrophage colony-stimulating factor accelerates neutrophil and monocyte recovery after allogeneic T-cell- depleted bone marrow transplantation
Authors: De Witte T , Gratwohl A , Van Der Lely N , Bacigalupo A , Stern AC , Speck B , Schattenberg A , Nissen C , Gluckman E , Fibbe WE
Published: October 2019

In a prospective randomized study, five European transplant centers compared recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; mammalian glycosylated) with placebo. rhGM-CSF was administered in a dose of 8 micrograms glycoprotein (5.5 micrograms protein)/kg/d, as a continuous intravenous (IV) infusion for 14 days, starting 3 hours after bone marrow infusion. Fifty-seven patients entered and completed the study. Median age of the recipients was 34 years (range, 17 to 51 y). All donors were HLA-identical, MLC- nonreactive siblings. Marrow grafts were depleted of T lymphocytes either by counterflow centrifugation (n = 42) or by immunological methods (n = 15). Twenty-nine patients received rhGM-CSF and 28 patients placebo. The leukocyte count and the absolute neutrophil count were significantly higher in the rhGM-CSF-treated group from day +9 to day +14 after bone marrow transplantation (BMT). This was also true for the monocyte count from day +12 to day +21. Early neutrophil (greater than 0.1 and greater than 0.3 x 10(9)/L) and early leukocyte (greater than 0.3 and greater than 0.5 x 10(9)/L) recovery was significantly faster for the patients given GM-CSF. The incidences of graft-versus- host disease (GVHD) and transplant-related mortality were not different in both groups. However, the number of bronchopneumonias was significantly lower in the rhGM-CSF-treated group (P = .03). Long-term follow-up showed a trend to better overall disease-free survival at 2 years and a trend to a lower relapse risk in patients treated with rhGM- CSF. This study shows that rhGM-CSF significantly increases neutrophil and monocyte counts during periods of 6 to 10 days in the second and third week after BMT. This shortened period until myeloid cell recovery after transplantation resulted in a decreased number of pneumonias, without an increase in incidence of GVHD or relapse.
An animal model of allogeneic donor platelet refractoriness: the effect of the time of leukodepletion
Authors: Blajchman MA , Bardossy L , Carmen RA , Goldman M , Heddle NM , Singal DP
Published: October 2019

Approximately 50% of multi-transfused individuals become refractory to random donor platelets. Recent clinical data suggest that those patients receiving leukocyte-depleted blood products are less likely to become refractory to random donor platelets than recipients of non- leukocyte-depleted products. Leukocyte depletion can be performed immediately after collection of a unit of whole blood before its storage (prestorage leukodepletion) or just before the transfusion of the blood product to a recipient, after its storage (poststorage leukodepletion). However, the most appropriate time for the leukodepletion of blood products has not been established. The present study was undertaken to establish an animal model of allogeneic platelet refractoriness, and to compare the effect of prestorage and poststorage leukodepletion on the frequency of refractoriness to allogeneic donor platelets. In this model, two strains of rabbits were used: California Black rabbits were used as blood donors, while New Zealand White rabbits were used as recipients. Eight weekly infusions of nonleukodepleted allogeneic fresh blood resulted in an allogeneic platelet refractory rate of 91.2% (31/34). The prestorage leukodepletion of the donor blood was associated with a significantly higher allogeneic platelet survival and lower refractory rate (33.3%) to allogeneic platelets than poststorage leukodepletion (66.7%). Furthermore, the data suggest that cell-free plasma products are capable of inducing refractoriness to allogeneic donor platelets; the stored plasma having a greater likelihood of inducing such refractoriness than fresh plasma. Thus, these data provide evidence that the prestorage leukodepletion of allogeneic donor blood is associated with a lower frequency of refractoriness and better allogeneic platelet survival than poststorage leukodepletion.
Antithrombin-III-Stockholm: a codon 392 (Gly----Asp) mutation with normal heparin binding and impaired serine protease reactivity
Authors: Blajchman MA , Fernandez-Rachubinski F , Sheffield WP , Austin RC , Schulman S
Published: October 2019

Antithrombin-III-Stockholm is a new structural variant of antithrombin- III (AT-III) with normal heparin affinity but defective serine protease inhibitory activity. The proposita, a white female born in 1966, was diagnosed to have developed a pulmonary embolus while on oral contraceptives at age 19. The proposita, as well as her father, were diagnosed to have a type 2 AT-III deficiency as they had normal levels of immunoreactive AT-III associated with decreased (approximately 60%) functional AT-III when measured with either alpha-thrombin or factor Xa as the substrate, either in the presence or absence of heparin. There was no evidence of abnormal electrophoretic mobility of AT-III from the proposita either in the presence or absence of heparin. Genomic DNA was prepared and all seven AT-III exons were polymerase chain reaction (PCR)-amplified and sequenced in both directions using nested primers. Only exon 7 provided evidence for the presence of a mutation, with the second base of codon 392 having a G----A substitution. Such a mutation would cause the substitution of aspartic acid at the site of the normally appearing glycine in the translated product. Furthermore, this mutation caused the destruction of an Hae III restriction site at this point in the AT-III gene. The absence of this Hae III site was confirmed using restriction fragment length polymorphism analysis of PCR-amplified material from the proposita. Experiments with AT-III from the proposita together with experiments with cell-free translated AT- III-Stockholm provided evidence that the mutant AT-III protein does not efficiently form a stable covalent inhibitory complex with alpha- thrombin, although it exhibits normal heparin affinity. The minimal thrombin-complexing ability of the mutant AT-III protein that was observed was accelerated by heparin, but to subnormal levels.
Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura
Authors: Fujisawa K , Tani P , O'Toole TE , Ginsberg MH , McMillan R
Published: October 2019

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to antiplatelet autoantibodies, many of which are directed against platelet membrane glycoprotein (GP) IIb-IIIa or GPIb-IX. In a recent study, we described plasma autoantibodies from 13 selected ITP patients, which required the presence of the putative GPIIIa cytoplasmic region for antibody binding. Since this region may not be available for antibody binding under physiologic conditions, we evaluated the frequency of binding to this or other regions of GPIIb- IIIa by platelet-associated and plasma autoantibody from a group of chronic ITP patients. We studied platelet-associated autoantibodies in 27 patients and plasma antibodies in 21 patients; in 15 patients, both were studied. To determine if autoantibodies were directed to the cytoplasmic portion of GPIIIa or to another portion of the GPIIb-IIIa molecule, antibody eluted from patient platelets or plasma antibody was tested in an antigen capture assay for binding to GPIIb-IIIa obtained from Chinese hamster ovary (CHO) cells transfected with GPIIb and either intact GPIIIa or GPIIIa lacking the carboxy terminal 35 residues. Of the 21 plasma autoantibodies tested, 13 bound primarily to the carboxy terminus of GPIIIa and eight to other epitopes. Conversely, all 26 platelet-associated autoantibodies, including eight of the 13 with anti-carboxy terminus antibodies, bound to epitopes in other regions of GPIIb-IIIa. Comparison of the degree of antibody adsorption by intact or lysed platelets indicated that epitopes on the c-terminal region of GPIIIa are relatively inaccessible on the surface of intact washed platelets when compared with other epitopes. We conclude that the importance of plasma autoantibodies in chronic ITP patients should be interpreted cautiously, since their specificity may differ from that of antibodies bound to the platelet. Whether antibodies against the c- terminus of GPIIIa are of pathogenetic importance remains to be determined, although patients with these antibodies have particularly severe disease.
Characterization of defensin precursors in mature human neutrophils
Authors: Harwig SS , Park AS , Lehrer RI
Published: October 2019

Human defensins HNP-1 and -3 are broad spectrum antimicrobial peptides that are synthesized by human neutrophils as 94 amino acid (aa) precursors that require proteolytic removal of 64 amino-terminal residues to produce the mature defensins. Recent studies have shown that the early proteolytic processing events include two sequential cleavages, each removing 19 amino-terminal aa residues, that yield 75 aa and 56 aa prodefensins, respectively. The subsequent processing steps that convert these 56 aa prodefensins to mature (30 aa) HNP-1 and HNP-3 are not yet known. We identified four new defensin precursors in mature normal neutrophils. The most abundant of these were two 39 aa forms that resulted from the monobasic endoproteolytic cleavage of proHNP-1 and proHNP-3. The presence of two proline residues in the vicinity of this newly defined scission site suggested that this cleavage might be “proline-directed.” Smaller amounts of the 34 aa and 32 aa prodefensin forms were also found. It remains to be established if these 39, 34, and 32 aa prodefensins are obligate intermediates in the prodefensin processing pathway, or arise from side reactions. In either event, because these prodefensin intermediates accounted for only 0.25% of the total defensin content, proteolytic conversion of 56 aa prodefensins to mature defensins appears to be a highly efficient process.
Rapid priming of human monocytes by human hematopoietic growth factors: granulocyte-macrophage colony-stimulating factor (CSF), macrophage-CSF, and interleukin-3 selectively enhance superoxide release triggered by receptor-mediated agonists
Authors: Yuo A , Kitagawa S , Motoyoshi K , Azuma E , Saito M , Takaku F
Published: October 2019

The effects of hematopoietic growth factors on human monocyte superoxide (O2-) release were investigated by using purified human monocytes in suspension. Among growth factors studied, granulocyte- macrophage colony-stimulating factor (GM-CSF), macrophage-CSF (M-CSF), and interleukin-3 (IL-3) primed human monocytes and enhanced O2- release stimulated by the receptor-mediated agonists, N-formyl- methionyl-leucyl-phenylalanine (FMLP) and concanavalin A (Con A), but not by phorbol myristate acetate, which bypasses the receptors to stimulate the cells. The optimal priming was obtained by pretreatment of cells with 1 to 5 ng/mL (0.07 to 0.34 nmol/L) GM-CSF, 50 to 100 ng/mL (0.5 to 1.1 nmol/L) M-CSF, or 10 to 20 ng/mL (0.6 to 1.3 nmol/L) IL-3 for 10 minutes at 37 degrees C. Potency of the maximal priming effects on FMLP- or Con A-induced O2- release was GM-CSF greater than M- CSF = IL-3. The combination of the optimal concentrations of any two CSFs resulted in the effect of more potent priming agent alone. Enhancement of O2- release by GM-CSF was observed over the complete range of effective concentrations of FMLP (10(-8) to 10(-6) mol/L). The pretreatment of monocytes with granulocyte-CSF (50 ng/mL), interferon- gamma (1,000 U/mL), or IL-4 (20 ng/mL) for 10 minutes at 37 degrees C had no effect on O2- release stimulated by FMLP or Con A. These findings show that GM-CSF, M-CSF, and IL-3 selectively enhance O2- release in human monocytes triggered by receptor-mediated agonists after short-term preincubation.
Isolation of the JMH antigen on a novel phosphatidylinositol-linked human membrane protein
Authors: Bobolis KA , Moulds JJ , Telen MJ
Published: October 2019

JMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospholipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI- linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.