bluuebunny/crossref_metadata_2025 huggingface dataset:
Replication-Defective Canarypox (ALVAC) Vectors Effectively Activate Anti–Human Immunodeficiency Virus-1 Cytotoxic T Lymphocytes Present in Infected Patients: Implications for Antigen-Specific Immunotherapy
Authors: Ferrari G. , Berend C. , Ottinger J. , Dodge R. , Bartlett J. , Toso J. , Moody D. , Tartaglia J. , Cox William I. , Paoletti E. , Weinhold K.J.
Published: October 2019

In the attempt to develop immunotherapeutic strategies for acquired immunodeficiency syndrome capable of activating effector cells in an antigen-specific manner while maintaining the broadest possible T-cell repertoire, we evaluated two canarypox (ALVAC)-based vectors for their capacity to induce ex vivo activation/expansion of human immunodeficiency virus (HIV)-specific CD8+ cytotoxic lymphocyte precursors (CTLp) obtained from HIV-1–infected donors. These two vectors, vCP205 encoding HIV-1 gp120 + TM (28 amino acid transmembrane anchor sequence) in addition to Gag/protease and vCP300 encoding gp120 + Gag/protease as well as Nef and Pol CTL determinants, are pancytotropic but replication incompetent in mammalian cells. Bulk peripheral blood mononuclear cells (PBMCs) or enriched CD8+ T cells were stimulated for 10 days with autologous ALVAC-infected PBMCs in the presence of different cytokine combinations (interleukin-2 [IL-2], IL-4, IL-7, and IL-12). Activation by ALVAC constructs was highly antigen-specific, because vCP205 elicited only Env and Gag CTL, whereas vCP300 elicited broader reactivities against Env, Gag, Pol, and Nef determinants. The ALVAC activation of CTLp was IL-2 dependent and enhanced by the addition of IL-7, whereas IL-4 and IL-12 failed to augment cytotoxic reactivities elicited by these constructs. The expansion of enriched CD8+ T cells after activation with vCP300 was higher in patients with CD4 counts greater than 400 cells/μL. Two rounds of in vitro stimulation (IVS) with vCP300 resulted in nearly an eightfold expansion of CD8+ lymphocytes over a 25-day period. After the second IVS, an average 3.2-fold increase among the different antigen-specific CTL frequencies was achieved. These studies clearly show that HIV-recombinant ALVAC vectors represent powerful polyvalent antigenic stimuli for activation and expansion of the CD8 lymphocyte response that occurs as a result of HIV infection.
Use of α-2a-Interferon to Treat Cytogenetic Relapse of Chronic Myeloid Leukemia After Marrow Transplantation
Authors: Higano Celestia S. , Chielens Deborah , Raskind Wendy , Bryant Eileen , Flowers Mary E.D. , Radich Jerald , Clift Reginald , Appelbaum Frederick
Published: October 2019

Fourteen patients with cytogenetic relapse of chronic myeloid leukemia (CML) after transplantation with unmanipulated bone marrow were treated with α-2a–interferon. There were eight men and six women, median age, 33 years. Twelve patients received marrow from a related allogeneic donor and two received marrow from a syngeneic donor. The median percentage of Ph-positive metaphases at the time of starting interferon was 55% (10% to 87%). Daily interferon was started at a dose of 1 to 3 × 106 U/M2/d, depending on initial blood counts and was adjusted as tolerated to maintain the white blood count in the range of 2,000 to 3,000/μL and the platelet count greater than 60,000/μL. After a stable cytogenetic remission was achieved, the interferon dose was decreased to a maintenance level. Twelve patients achieved a complete cytogenetic remission on at least one occasion. Median time to achieve a complete cytogenetic remission was 7.5 months (range, 1.5 to 12). Eight patients remain in cytogenetic remission for 10+ to 54+ months from the time of first documented remission. After complete cytogenetic remission was established, nine patients were tested for the presence of the mRNA transcript of the bcr/abl fusion gene by polymerase chain reaction (PCR) testing. Four patients were PCR-negative on at least one occasion: two patients were PCR-negative on a single occasion; one patient had serial tests, which were PCR-negative; and one patient had serial PCR-negative peripheral blood tests with a single PCR-positive bone marrow obtained concurrently with a negative peripheral blood test. Median follow-up time for all patients is 44 months (range, 20 to 64). Interferon was generally well tolerated; only one responding patient was unable to continue interferon because of toxicity. Interferon induces durable cytogenetic remissions in a significant proportion (57%) of patients with cytogenetic relapse following bone marrow transplantation (BMT) without causing life-threatening toxicities.
The Human Antibacterial Cathelicidin, hCAP-18, Is Synthesized in Myelocytes and Metamyelocytes and Localized to Specific Granules in Neutrophils
Authors: Sørensen Ole , Arnljots Kristina , Cowland Jack B. , Bainton Dorothy F. , Borregaard Niels
Published: October 2019

hCAP-18 is the only human member of the antibacterial and endotoxin-binding family of proteins known as cathelicidins. The antibacterial and endotoxin binding domains reside in the C-terminal 37 amino acids of the protein (LL-37) and this is believed to be unleashed from the neutralizing N-terminus by proteases from peroxidase positive granules. In human neutrophils, peroxidase positive and peroxidase negative granules can be subdivided into granule subsets that differ in protein content and ability to be exocytosed. To determine the localization of hCAP-18, we performed high-resolution immuno-electron microscopy and subcellular fractionation on Percoll density gradients. Biosynthesis of hCAP-18 was investigated in isolated human bone marrow cells. hCAP-18 was found to colocalize and comobilize with lactoferrin, but not with gelatinase in subcellular fractions. This was confirmed by electron microscopy. hCAP-18 is synthesized at the same stage of myeloid cell maturation as lactoferrin, and is efficiently targeted to granules. Like the peroxidase negative granule's matrix metalloproteinases, collagenase and gelatinase, hCAP-18 is also stored in unprocessed form. hCAP-18 is a major protein of specific granules where it is present in equimolar ratio with lactoferrin.
Impaired Ability of Bone Marrow Stromal Cells to Support B-Lymphopoiesis With Age
Authors: Stephan Robert P. , Reilly Colette R. , Witte Pamela L.
Published: October 2019

B-lymphopoiesis decreases with age. We studied how aging affects bone marrow stromal cells, because they provide the growth factors and cell contacts required for B-lymphopoiesis. No differences were noted in the cell-surface phenotype of young and old primary-cultured stromal cells. Fluorescence-activated cell sorter-purified stromal cells from old mice were deficient in the ability to support the proliferation of interleukin-7 (IL-7)–specific B-lymphoid cell lines. The kinetics of this response indicated that IL-7 was not immediately available from stromal cells of either age and was further delayed on aged stromal cells. The levels of IL-7 protein within stromal cells were equivalent between young and old animals, suggesting that the production of IL-7 was not altered by aging. Negligible amounts of IL-7 were found either freely secreted or in the extracellular matrix of cultures of young and old marrow. Contact between the lymphoid cells and the primary stromal cells was required for detectable proliferation, suggesting that cell contact was required for the release of IL-7. We propose that stromal cells regulate B-lymphopoiesis by limiting the amount of IL-7 available to the developing precursors. Therefore, we conclude that the age-related decrease in the function of bone marrow stromal cells is related to the impaired release of IL-7.
Biologically Active Fas Antigen and Its Cognate Ligand Are Expressed on Plasma Membrane-Derived Extracellular Vesicles
Authors: Albanese Joseph , Meterissian Sarkis , Kontogiannea Maria , Dubreuil Catherine , Hand Arthur , Sorba Sandra , Dainiak Nicholas
Published: October 2019

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas–mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.
Transferrin Receptor-Dependent and -Independent Iron Transport in Gallium-Resistant Human Lymphoid Leukemic Cells
Authors: Chitambar Christopher R. , Wereley Janine P.
Published: October 2019

Recent studies showed that gallium and iron uptake are decreased in gallium-resistant (R) CCRF-CEM cells; however, the mechanisms involved were not fully elucidated. In the present study, we compared the cellular uptake of 59Fe-transferrin (Tf) and59Fe-pyridoxal isonicotinoyl hydrazone (PIH) to determine whether the decrease in iron uptake by R cells is caused by changes in Tf receptor (TfR)-dependent or TfR-independent iron uptake. We found that both 59Fe-Tf and 59Fe-PIH uptake were decreased in R cells. The uptake of 59Fe-Tf but not59Fe-PIH could be blocked by an anti-TfR monoclonal antibody. After 59Fe-Tf uptake, R cells released greater amounts of 59Fe than gallium-sensitive (S) cells. However, after 59Fe-PIH uptake 59Fe release from S and R cells was similar. 125I-Tf exocytosis was greater in R cells. At confluency, S and R cells expressed equivalent amounts of TfR; however, at 24 and 48 hours in culture, TfR expression was lower in R cells. Our study suggests that the decrease in Tf-Fe uptake by R cells is caused by a combination of enhanced iron efflux from cells and decreased TfR-mediated iron transport into cells. Furthermore, because TfR-dependent and -independent iron uptake is decreased in R cells, both uptake systems may be controlled at some level by similar regulatory signal(s).
The Molecular Basis for Cross-Reacting Material–Positive Hemophilia A Due to Missense Mutations Within the A2-Domain of Factor VIII
Authors: Amano Kagehiro , Sarkar Rita , Pemberton Susan , Kemball-Cook Geoffrey , Kazazian Haig H. , Kaufman Randal J.
Published: October 2019

Factor VIII (FVIII) is the protein defective in the bleeding disorder hemophilia A. Approximately 5% of hemophilia A patients have normal amounts of a dysfunctional FVIII protein and are termed cross-reacting material (CRM)-positive. The majority of genetic alterations that result in CRM-positive hemophilia A are missense mutations within the A2-domain. To determine the mechanistic basis of the genetic defects within the A2-domain for FVIII function we constructed six mutations within the FVIII cDNA that were previously found in five CRM-positive hemophilia A patients (R527W, S558F, I566T, V634A, and V634M) and one CRM-reduced hemophilia A patient (DeltaF652/3). The specific activity for each mutant secreted into the conditioned medium from transiently transfected COS-1 cells correlated with published data for the patients plasma-derived FVIII, confirming the basis of the genetic defect. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of immunoprecipitated FVIII protein radiolabeled in COS-1 cells showed that all CRM-positive mutant proteins were synthesized and secreted into the medium at rates similar to wild-type FVIII. The majority of the DeltaF652/3 mutant was defective in secretion and was degraded within the cell. All mutant FVIII proteins were susceptible to thrombin cleavage, and the A2-domain fragment from the I566T mutant had a reduced mobility because of use of an introduced potential N-linked glycosylation site that was confirmed by N-glycanase digestion. To evaluate interaction of FVIII with factor IXa, we performed an inhibition assay using a synthetic peptide corresponding to FVIII residues 558 to 565, previously shown to be a factor IXa interaction site. The concentration of peptide required for 50% inhibition of FVIII activity (IC50) was reduced for the I566T (800 μmol/L) and the S558F (960 μmol/L) mutants compared with wild-type FVIII (>2,000 μmol/L). N-glycanase digestion increased I566T mutant FVIII activity and increased its IC50 for the peptide (1,400 μmol/L). In comparison to S558F, a more conservative mutant (S558A) had a sixfold increased specific activity that also correlated with an increased IC50 for the peptide. These results provided support that the defects in the I566T and S558F FVIII molecules are caused by steric hindrance for interaction with factor IXa.
Distribution of Transferrin Saturations in the African-American Population
Authors: Gordeuk Victor R. , McLaren Christine E. , Looker Anne C. , Hasselblad Victor , Brittenham Gary M.
Published: October 2019

To determine if transferrin saturations in African Americans may reflect the presence of a gene that influences iron metabolism, we analyzed the distribution of these values in 808 African Americans from the second National Health and Nutrition Survey. We tested for a mixture of three normal distributions consistent with population genetics for a major locus effect in which the proportion of normal homozygotes is p2; of heterozygotes, 2pq; of affected homozygotes, q2; and in which p+q= 1. Three subpopulations based on transferrin saturation were present (P < .0001) and the fit to a mixture of three normal distributions was good (P = .2). A proportion of .009 was included in a subpopulation with a mean ± standard deviation transferrin saturation of 63.4% ± 5.7% (postulated homozygotes for a gene that influences iron metabolism), while a proportion of .136 had an intermediate saturation of 38.0% ± 5.7% (postulated heterozygotes) and .856 a saturation of 24.6% ± 5.7% (postulated normal homozygotes). These proportions were consistent with population genetics because the sum of the square roots of the proportions with the lowest mean transferrin saturation (P = .925) and the highest (q = 0.093) was approximately 1 (1.018). The results are consistent with the presence in African Americans of a common locus that influences iron metabolism.
Growth and Differentiation of Human Stem Cell Factor/Erythropoietin-Dependent Erythroid Progenitor Cells In Vitro
Authors: Panzenböck Birgit , Bartunek Petr , Mapara Markus Y. , Zenke Martin
Published: October 2019

Stem cell factor (SCF) and erythropoietin (Epo) effectively support erythroid cell development in vivo and in vitro. We have studied here an SCF/Epo-dependent erythroid progenitor cell from cord blood that can be efficiently amplified in liquid culture to large cell numbers in the presence of SCF, Epo, insulin-like growth factor-1 (IGF-1), dexamethasone, and estrogen. Additionally, by changing the culture conditions and by administration of Epo plus insulin, such progenitor cells effectively undergo terminal differentiation in culture and thereby faithfully recapitulate erythroid cell differentiation in vitro. This SCF/Epo-dependent erythroid progenitor is also present in CD34+ peripheral blood stem cells and human bone marrow and can be isolated, amplified, and differentiated in vitro under the same conditions. Thus, highly homogenous populations of SCF/Epo-dependent erythroid progenitors can be obtained in large cell numbers that are most suitable for further biochemical and molecular studies. We demonstrate that such cells express the recently identified adapter protein p62dok that is involved in signaling downstream of the c-kit/SCF receptor. Additionally, cells express the cyclin-dependent kinase (CDK) inhibitors p21cip1 and p27kip1 that are highly induced when cells differentiate. Thus, the in vitro system described allows the study of molecules and signaling pathways involved in proliferation or differentiation of human erythroid cells.
Flavopiridol Induces Apoptosis in Chronic Lymphocytic Leukemia Cells Via Activation of Caspase-3 Without Evidence of bcl-2 Modulation or Dependence on Functional p53
Authors: Byrd John C. , Shinn Charlotte , Waselenko Jamie K. , Fuchs Ephraim J. , Lehman Teresa A. , Nguyen Phuong L. , Flinn Ian W. , Diehl Louis F. , Sausville Edward , Grever Michael R.
Published: October 2019

Flavopiridol has been reported to induce apoptosis in lymphoid cell lines via downregulation of bcl-2. The in vitro activity of flavopiridol against human chronic lymphocytic leukemia (CLL) cells and potential mechanisms of action for inducing cytotoxicity were studied. The in vitro viability of mononuclear cells from CLL patients (n = 11) was reduced by 50% at 4 hours, 24 hours, and 4 days at a flavopiridol concentration of 1.15 μmol/L (95% confidence interval [CI] ±0.31), 0.18 μmol/L (95% CI ±0.04), and 0.16 μmol/L (95% CI ±0.04), respectively. Loss of viability in human CLL cells correlated with early induction of apoptosis. Exposure of CLL cells to 0.18 μmol/L of flavopiridol resulted in both decreased expression of p53 protein and cleavage of the caspase-3 zymogen 32-kD protein with the appearance of its 20-kD subunit. Contrasting observations of others in tumor cell lines, flavopiridol cytotoxicity in CLL cells did not correlate with changes in bcl-2 protein expression alterations. We evaluated flavopiridol’s dependence on intact p53 by exposing splenocytes from wild-type (p53+/+) and p53 null (p53−/−) mice that demonstrated no preferential cytotoxicity as compared with a marked differential with F-ara-a and radiation. Incubation of CLL cells with antiapoptotic cytokine interleukin-4 (IL-4) did not alter the LC50 of flavopiridol, as compared with a marked elevation noted with F-ara-a in the majority of patients tested. These data demonstrate that flavopiridol has significant in vitro activity against human CLL cells through activation of caspase-3, which appears to occur independently of bcl-2 modulation, the presence of IL-4, or p53 status. Such findings strongly support the early introduction of flavopiridol into clinical trials for patients with B-CLL.
Highly Efficient Transduction of the Green Fluorescent Protein Gene in Human Umbilical Cord Blood Stem Cells Capable of Cobblestone Formation in Long-Term Cultures and Multilineage Engraftment of Immunodeficient Mice
Authors: van Hennik Paula B. , Verstegen Monique M.A. , Bierhuizen Marti F.A. , Limón Ana , Wognum Albertus W. , Cancelas José A. , Barquinero Jordi , Ploemacher Rob E. , Wagemaker Gerard
Published: October 2019

Purified CD34+ and CD34+CD38− human umbilical cord blood (UCB) cells were transduced with the recombinant variant of Moloney murine leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope proteins. Using a 2-day growth factor prestimulation, followed by a 2-day, fibronectin fragment CH-296–supported transduction, CD34+ and CD34+CD38− UCB subsets were efficiently transduced using either vector. The use of the SF-EGFP/PG13 retroviral packaging cell combination consistently resulted in twofold higher levels of EGFP-expressing cells than the MFG-EGFP/Am12 combination. Transplantation of 105 input equivalent transduced CD34+ or 5 × 103input equivalent CD34+CD38− UCB cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages of 8% and 5%, respectively, which showed that the in vivo repopulating ability of the cells had been retained. In addition, mice engrafted after transplantation of transduced CD34+ cells using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP with median values of 2% and 23% of human CD45+ cells, respectively, which showed that the NOD/SCID repopulating cells were successfully transduced. EGFP+ cells were found in all human hematopoietic lineages produced in NOD/SCID mice including human progenitors with in vitro clonogenic ability. EGFP-expressing cells were also detected in the human cobblestone area–forming cell (CAFC) assay at 2 to 6 weeks of culture on the murine stromal cell line FBMD-1. During the transduction procedure the absolute numbers of CAFC week 6 increased 5- to 10-fold. The transduction efficiency of this progenitor cell subset was similar to the fraction of EGFP+ human cells in the bone marrow of the NOD/SCID mice transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced CD34+ cells, ie, 6% and 27%, respectively. The study thus shows that purified CD34+ and highly purified CD34+CD38− UCB cells can be transduced efficiently with preservation of repopulating ability. The SF-EGFP/PG13 vector/packaging cell combination was much more effective in transducing repopulating cells than the MFG-EGFP/Am12 combination.
The Second Exon-Encoded Factor XII Region Is Involved in the Interaction of Factor XII With Factor XI and Does Not Contribute to the Binding Site for Negatively Charged Surfaces
Authors: Citarella Franca , Fedele Giorgio , Roem Dorina , Fantoni Antonio , Hack C. Erik
Published: October 2019

Contact system activation, in vitro, is triggered by activation of factor XII (FXII) on binding to an activator, such as negatively charged surfaces. A putative surface-binding site of FXII has been located within the amino acid residues 1-28 by identifying the epitope recognized by a monoclonal antibody (MoAb), B7C9, which inhibits kaolin-induced clotting activity. To further elucidate the role of the amino terminal binding site in the regulation of FXII activation, we have characterized a FXII recombinant protein (rFXII-▵19) deleted of the amino acid residues 3-19, which are encoded by the second exon of FXII gene. A plasmid encoding for rFXII-▵19 was constructed and expressed in HepG2 cells by using vaccinia virus. Purified rFXII-▵19 migrated as a single band of Mr 77,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel, did not bind to MoAb B7C9 immobilized on Protein A-Sepharose, thus confirming that it lacked the epitope for this MoAb, and had no amidolytic activity towards the chromogenic substrate S-2302 in the absence of activator. rFXII-▵19 specific clotting activity was lower (44%) than that of native FXII. The activation rate of rFXII-▵19 by kallikrein in the absence of dextran sulfate was about four times higher than that of full-length FXII and was increased in the presence of dextran sulfate. However, rFXII-▵19 underwent autoactivation in the presence of dextran sulfate. Labeled rFXII-▵19 bound to kaolin, which binding was equally well inhibited by either, rFXII-▵19 or full-length FXII (IC50 = 7.2 ± 2.2 nmol/L for both proteins). Accordingly, a synthetic peptide corresponding to FXII amino acid residues 3-19 did not inhibit the binding of labeled full-length FXII to kaolin. rFXII-▵19 generated a similar amount of FXIIa- and kallikrein-C1–inhibitor complexes in FXII-deficient plasma in the presence of kaolin, as did full-length FXII; but generated less factor XIa-C1–inhibitor complexes (50%) than full-length FXII. This impaired factor XI activation by rFXII-▵19a was also observed in a purified system and was independent of the presence of high molecular weight kininogen. Furthermore, the synthetic peptide 3-19, preincubated with factor XI, inhibited up to 30% activation of factor XI both in the purified system as well as in plasma. These results together indicate that amino acid residues 3-19 of FXII are involved in the activation of factor XI and do not contribute to the binding of FXII to negatively charged surfaces.
In Vivo Treatment With Granulocyte Colony-Stimulating Factor Results in Divergent Effects on Neutrophil Functions Measured In Vitro
Authors: Leavey Patrick J. , Sellins Karen S. , Thurman Gail , Elzi David , Hiester Andrew , Silliman Christopher C. , Zerbe Gary , Cohen J. John , Ambruso Daniel R.
Published: October 2019

We have studied the effects of granulocyte colony-stimulating factor (G-CSF) administration to normal individuals on a variety of functional and biochemical neutrophil characteristics that relate to host defense. G-CSF adversely affected neutrophil (polymorphonuclear leukocyte [PMN]) chemotaxis. While this could be partially explained by reduced assembly of neutrophil F-actin, we also recognized an elevated cytosolic calcium mobilization and a normal upregulation of neutrophil CD11b. G-CSF resulted in reduced PMN killing of Staphylococcus aureus with a 10:1 (bacteria:neutrophil) ratio and normal killing with a 1:1 ratio. In association with this, we demonstrated divergent effects on the respiratory burst of intact cells and divergent effects on the content of marker proteins for neutrophil granules. While G-CSF may have resulted in increased content of cytochrome b558 in the cell membrane, it did not alter the amounts of cytosolic oxidase components. After therapy, there was normal content of the azurophilic granule marker, myeloperoxidase, decreased content of the specific granule marker, lactoferrin, and normal content of lysozyme (found in both granules classes). Finally, G-CSF therapy markedly reduced the apoptotic rate of the isolated neutrophil. Therefore, considering disparate functional and biochemical activities, the real benefit of G-CSF therapy may lie in enhanced number and survival of neutrophils.
A Critical Role for CD48 Antigen in Regulating Alloengraftment and Lymphohematopoietic Recovery After Bone Marrow Transplantation
Authors: Blazar Bruce R. , Taylor Patricia A. , Panoskaltsis-Mortari Angela , Yagita Hideo , Bromberg Jonathan S. , Vallera Daniel A.
Published: October 2019

The binding of CD2, present on T cells, to its counterreceptor CD48 facilitates adhesion, signaling, alloantigen-induced cytokine production, and cytotoxic T-lymphocyte responses. Because these T-cell functions have been implicated in graft-versus-host disease (GVHD) pathogenesis, we have analyzed the effects of the CD2:CD48 pathway on GVHD mediated by CD4+ and CD8+ T cells infused into sublethally irradiated recipients. CD4+ T-cell–mediated, and to a lesser extent, CD8+ T-cell–mediated GVHD was inhibited by CD2 + 48 monoclonal antibody (MoAb) infusion. To assess the effects of combined MoAb infusion on alloengraftment, two different alloengraftment bone marrow transplantation (BMT) models were used. In both, MoAb infusion markedly inhibited alloengraftment and hematopoietic recovery post-BMT. To determine if the adverse effects on lymphohematopoiesis in the allogeneic BMT recipients were caused by an immune or nonimmune mechanism, studies were performed in congenic BMT recipients to preclude an immune mechanism as the cause for delayed recovery post-BMT. MoAb infusion resulted in impaired lymphohematopoietic recovery in congenic BMT recipients and markedly reduced day 12 colony-forming unit–spleen formation in syngeneic BMT recipients, consistent with a nonimmune mediated mechanism. Because the spleen is a site of early hematopoietic recovery post-BMT, studies were performed using adult splenectomized syngeneic BMT recipients. MoAb infusion delayed recovery in both nonsplenectomized and splenectomized recipients post-BMT, indicating that the delayed hematopoietic recovery was not the consequence of an abnormal homing pattern of hematopoietic progenitors to the spleen early post-BMT. CD48 MoAb was necessary and sufficient for the inhibition of GVHD lethality and delayed lymphohematopoietic effects of the combined MoAb regimen. CD48 MoAb was found to induce a profound modulation of CD48 antigen expression on BM cells, suggesting that the CD48 antigen may have an important function in hematopoiesis in the BM compartment. Taken together, these data provide evidence that the CD48 antigen plays a critical role in regulating hematopoiesis in post-BMT.
Recombinant CD40L Treatment Protects Allogeneic Murine Bone Marrow Transplant Recipients From Death Caused by Herpes Simplex Virus-1 Infection
Authors: Beland Janice L. , Adler Heiko , Del-Pan Nadia C. , Kozlow Wende , Sung Janice , Fanslow William , Rimm Ilonna J.
Published: October 2019

Posttransplant infection associated with host immune deficiency is the major cause of nonrelapse mortality of human bone marrow transplant recipients. In a new murine model of posttransplant infection, allogeneic bone marrow transplant recipients were infected with herpes simplex virus-1 (HSV-1) via intraperitoneal inoculation 12 weeks after transplantation. Allogeneic transplant recipients with graft-versus-host disease (GVHD) had significantly increased mortality from HSV-1 encephalitis, with deficiencies of both specific anti–HSV-1 antibody and total serum IgG2a. GVHD mice displayed a Th2 cytokine profile (increased interleukin-4 [IL-4] and decreased interferon-γ) and decreased lipopolysaccharide (LPS) responses, suggesting that both T-cell and B-cell defects contributed to the impaired production of antibody. Because passive transfer of hyperimmune serum protected mice from HSV-1 infection, we hypothesized that CD40 ligand (CD40L), which induces B-cell maturation, would protect mice from HSV-1 infection. CD40L-treated GVHD mice showed elevated IgG2a levels and increased survival compared with vehicle-treated transplant recipients.
Improving the Outcome of Bone Marrow Transplantation by Using CD52 Monoclonal Antibodies to Prevent Graft-Versus-Host Disease and Graft Rejection
Authors: Hale Geoff , Zhang Mei-Jie , Bunjes Donald , Prentice H. Grant , Spence David , Horowitz Mary M. , Barrett A. John , Waldmann Herman
Published: October 2019

Graft-versus-host disease (GVHD) is a major cause of mortality and morbidity after allogeneic bone marrow transplantation, but can be avoided by removing T lymphocytes from the donor bone marrow. However, T-cell depletion increases the risk of graft rejection. This study examined the use of CD52 monoclonal antibodies to eliminate T cells from both donor marrow and recipient to prevent both GVHD and rejection. Seventy patients receiving HLA-identical sibling transplants for acute myelogenous leukemia (AML) in first remission were studied. An IgM (CAMPATH-1M) was used for in vitro depletion of the graft and an IgG (CAMPATH-1G) for in vivo depletion of the recipient before graft infusion. No posttransplant immunosuppression was given. Results were compared with two control groups: (1) 50 patients who received bone marrow depleted with CAMPATH-1M, but no CAMPATH-1G in vivo; and (2) 459 patients reported to the International Bone Marrow Transplant Registry (IBMTR) who received nondepleted grafts and conventional GVHD prophylaxis with cyclosporin A (CyA) and methotrexate (MTX). The incidence of acute GVHD was 4% in the treatment group compared with 35% in the CyA/MTX group (P < .001). Chronic GVHD was also exceptionally low in the treatment group (3% v 36%; P< .001). The problem of graft rejection, which had been frequent in the historic CAMPATH-1M group (31%), was largely overcome in the treatment group (6%). Thus, transplant-related mortality of the treatment group (15% at 5 years) was lower than for the CyA/MTX group (26%; P = .04). There was little difference in the risk of leukemia relapse between the treatment group (30% at 5 years) and the CyA/MTX group (29%). Survival of the treatment group at 6 months was better than the CyA/MTX group (92% v 78%), although at 5 years the difference was not significant (62% v 58%) and neither was the difference in leukemia-free survival (60% v52%). We conclude that T-cell depletion is a useful strategy to prevent GVHD, provided that measures are taken to ensure engraftment. Using CAMPATH-1G to deplete residual host lymphocytes is a simple and practical method to do this. At least in AML, the beneficial reduction in GVHD can be achieved without an increased risk of relapse.
Erythropoietin Induces Tyrosine Phosphorylation of Jak2, STAT5A, and STAT5B in Primary Cultured Human Erythroid Precursors
Authors: Oda Atsushi , Sawada Kenichi , Druker Brian J. , Ozaki Katsutoshi , Takano Hina , Koizumi Kazuki , Fukada Yoshikazu , Handa Makoto , Koike Takao , Ikeda Yasuo
Published: October 2019

We examined signaling by erythropoietin in highly purified human colony forming unit-erythroid cells, generated in vitro from CD34+ cells. We found that erythropoietin induces tyrosine phosphorylation of Jak2, STAT5A, and STAT5B. Tyrosine phosphorylation of Jak2 reaches a peak around 10 minutes after stimulation and is maximum at 5 U/mL of erythropoietin. Tyrosine phosphorylation of STAT5 is accompanied by the translocation of activated STAT5 to the nucleus as shown by electrophoretic mobility shift assay (EMSA) using 32Pi-labeled STAT5 binding site in the β-casein promoter. Tyrosine phosphorylation STAT1 or STAT3 was not detected in human erythroid precursors after stimulation with erythropoietin. Crkl, an SH2/SH3 adapter protein, becomes coimmunoprecipitated specifically with STAT5 from erythropoietin-stimulated erythroid cells; although it was shown to become associated with c-Cbl in the studies using cell lines. Thus, human erythroid precursors can be expanded in vitro in sufficient numbers and purity to allow its usage in signal transduction studies. This report sets a basis for further studies on signaling in primary cultured human erythroid precursors, which in turn contribute to our better understanding in the differentiation processes of erythrocytes and their precursors.
Apposition-Dependent Induction of Plasminogen Activator Inhibitor Type 1 Expression: A Mechanism for Balancing Pericellular Proteolysis During Angiogenesis
Authors: Bacharach Eran , Itin Ahuva , Keshet Eli
Published: October 2019

Plasminogen-activator inhibitor type I (PAI-1), the primary inhibitor of urinary-type plasminogen activator, is thought to play an important role in the control of stroma invasion by both endothelial and tumor cells. Using an in vitro angiogenesis model of capillary extension through a preformed monolayer, in conjunction with in situ hybridization analysis, we showed that PAI-1 mRNA is specifically induced in cells juxtaposed next to elongating sprouts. To further establish that PAI-1 expression is induced as a consequence of a direct contact with endothelial cells, coculture experiments were performed. PAI-1 mRNA was induced exclusively in fibroblasts (L-cells) contacting endothelial cell (LE-II) colonies. Reporter gene constructs driven by a PAI-1 promoter and stably transfected into L-cells were used to establish that both mouse and rat PAI-1 promoters mediate apposition-dependent regulation. This mode of PAI-1 regulation is not mediated by plasmin, as an identical spatial pattern of expression was detected in cocultures treated with plasmin inhibitors. Because endothelial cells may establish direct contacts with fibroblasts only during angiogenesis, we propose that focal induction of PAI-1 at the site of heterotypic cell contacts provides a mechanism to negate excessive pericellular proteolysis associated with endothelial cell invasion.

© 1998 by The American Society of Hematology.
Characterization of a Novel Human Natural Killer-Cell Line (NK-YS) Established From Natural Killer Cell Lymphoma/Leukemia Associated With Epstein-Barr Virus Infection
Authors: Tsuchiyama Junjiro , Yoshino Tadashi , Mori Masaharu , Kondoh Eisaku , Oka Takeshi , Akagi Tadaatsu , Hiraki Akio , Nakayama Hiroyuki , Shibuya Akira , Ma Yuxiang , Kawabata Teruyuki , Okada Shigeru , Harada Mine
Published: October 2019

A novel cell line was established from a patient with a leukemic-state nasal angiocentric natural killer (NK) cell lymphoma with systemic skin infiltration. The morphology of the leukemic cells was large-granular-lymphocyte (LGL), and their immunophenotype was CD2+, CD3−, CD5+, CD7+, CD16−, CD56+, and CD57−. The presence of Epstein-Barr viral (EBV) genome was shown in specimens from the patient’s nose, skin, and peripheral blood by in situ hybridization using an EBV-encoded small RNA-1 probe or by Southern blotting using a terminal-repeat probe of the EBV genome. Leukemic cells were cocultured with a mouse stromal cell line (SPY3-2) in the presence of 100 U/mL recombinant human interleukin-2 and a novel stromal cell-independent cell line, NK-YS, was established. The NK-YS cells showed LGL morphology and expressed surface CD2, CD5, CD7, CD25, CD56, and CD95. The NK-YS cells retained cytotoxicity against K562 and Jurkat cells. A Southern blotting using a terminal-repeat probe of EBV showed that NK-YS and fresh leukemic cells had a clonal EBV genome, whereas the T-cell receptor β and γ chain genes of NK-YS were not rearranged. In an immunocytochemical analysis, the NK-YS cells showed a type-II latent infection of EBV. The NK-YS cells preserved the original characteristics of NK cell lymphoma/leukemia and will be a useful tool for the study of biological characteristics of EBV-associated nasal angiocentric NK cell lymphoma/leukemia.

© 1998 by The American Society of Hematology.
Platelet Transfusion: A Dose-Response Study
Authors: Norol Françoise , Bierling Philippe , Roudot-Thoraval Françoise , Le Coeur Françoise Ferrer , Rieux Claire , Lavaux Anne , Kuentz Mathieu , Duedari Najib
Published: October 2019

Early recommendations on prophylactic transfusion of thrombocytopenic patients involved a standard platelet dose of about 0.5 × 1011/10 kg body weight. Given the lack of data supporting this dose, we prospectively studied the dose response to platelet transfusions in adults and children with hematologic malignancies. Each patient received, in similar clinical conditions, a medium, high, and very high dose of fresh (< 24 hours old) ABO-compatible platelets, in the form of apheresis platelet concentrates (APC). For the adults, the medium dose was defined as APC containing between 4 and 6 × 1011 platelets, the high dose between 6 and 8 × 1011, and the very high dose > 8 × 1011; for the children, the three doses corresponded to 2 to 4, 4 to 6, and > 6 × 1011 platelets. The end points were the platelet increment, platelet recovery, and the transfusion interval, and the results were compared with a paired t-test. Sixty-nine adults and 13 children could be assessed. Recoveries in the adults were similar with the three doses (from 28% to 30%), but the high and very high doses led to a significantly better platelet increment (52 and 61 × 109/L, respectively) than the medium dose (33 × 109/L, P < .01). The main difference was in the transfusion interval, which increased with the dose of platelets transfused, from 2.6 days with the medium dose to 3.3 and 4.1 days with the high and very high doses, respectively (P< .01). The positive effect of the high dose was observed regardless of pretransfusional clinical status, but was more marked in patients with no clinical factors known to impair platelet recovery. In these patients, a platelet dose of 0.07 × 1011 per kg of body weight led to a transfusion interval of more than 2 days in 95% of cases. In patients with clinical factors favoring platelet consumption, the proportion of transfusions yielding an optimal platelet increment and transfusion interval increased with the dose of platelets.The platelet dose-effect was also significant in the children, in whom the high and very high doses led to 1.5-fold to twofold higher posttransfusion platelet counts and transfusion intervals. We conclude that transfusion of high platelet doses can reduce the number of platelet concentrates required by thrombocytopenic patients and significantly reduce donor exposure.

© 1998 by The American Society of Hematology.
Hydrodynamic Shear Shows Distinct Roles for LFA-1 and Mac-1 in Neutrophil Adhesion to Intercellular Adhesion Molecule-1
Authors: Neelamegham Sriram , Taylor Andrew D. , Burns Alan R. , Smith C. Wayne , Simon Scott I.
Published: October 2019

The binding of neutrophil β2 integrin to intercellular adhesion molecule-1 (ICAM-1) expressed on the inflamed endothelium is critical for neutrophil arrest at sites of tissue inflammation. To quantify the strength and kinetics of this interaction, we measured the adhesion between chemotactically stimulated neutrophils and ICAM-1–transfected mouse cells (E3-ICAM) in suspension in a cone-plate viscometer at shear rates typical of venular blood flow (100 s−1 to 500 s−1). The kinetics of aggregation were fit with a mathematical model based on two-body collision theory. This enabled estimation of adhesion efficiency, defined as the probability with which collisions between cells resulted in firm adhesion. The efficiency of β2-integrin–dependent adhesion was highest (∼0.2) at 100 s−1 and it decreased to approximately zero at 400 s−1. Both LFA-1 and Mac-1 contributed equally to adhesion efficiency over the initial 30 seconds of stimulation, but adhesion was entirely Mac-1–dependent by 120 seconds. Two hydrodynamic parameters were observed to influence integrin-dependent adhesion efficiency: the level of shear stress and the intercellular contact duration. Below a critical shear stress (<2 dyn/cm2), contact duration predominantly limited adhesion efficiency. The estimated minimum contact duration for β2-integrin binding was approximately 6.5 ms. Above the critical shear stress (>2 dyn/cm2), the efficiency of neutrophil adhesion to E3-ICAM was limited by both the contact duration and the tensile stress. We conclude that at low shear, neutrophil adhesion is modulated independently through either LFA-1 or Mac-1, which initially contribute with equal efficiency, but differ over the duration of chemotactic stimulation.

© 1998 by The American Society of Hematology.
Secretable Human Platelet-Derived Factor V Originates From the Plasma Pool
Authors: Camire Rodney M. , Pollak Eleanor S. , Kaushansky Kenneth , Tracy Paula B.
Published: October 2019

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into -granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed.

© 1998 by The American Society of Hematology.
Endothelin-1 Induces Production of the Neutrophil Chemotactic Factor Interleukin-8 by Human Brain-Derived Endothelial Cells
Authors: Hofman F.M. , Chen P. , Jeyaseelan R. , Incardona F. , Fisher M. , Zidovetzki R.
Published: October 2019

Increased levels of endothelin-1 (Et-1), a potent vasoconstrictor, have been correlated with hypertension and neuronal damage in ischemic/reperfusion injury. The presence of polymorphonuclear cells (PMNs) in the brain has been shown to be directly responsible for this observed pathology. To address the question of whether Et-1 plays a role in this process, human brain-derived endothelial cells (CNS-ECs) were cultured with Et-1. The results demonstrate that Et-1 induces production of the neutrophil chemoattractant interleukin-8 (IL-8) twofold to threefold after 72 hours; mRNA was maximal after 1 hour of stimulation. Conditioned culture medium derived from Et-1–stimulated CNS-ECs induced a chemotactic response in the PMN migration assay. The inflammatory cytokines tumor necrosis factor- (TNF) and IL-1β functioned additively with Et-1 in increasing IL-8 production. In contrast, transforming growth factor-β (TGF-β), but not IL-10, completely abolished the effect of Et-1 on IL-8 production. However, Et-1 did not modulate intercellular adhesion molecule-1 (ICAM-1) expression. These data demonstrate that Et-1 may be a risk factor in ischemic/reperfusion injury by inducing increased levels of the neutrophil chemoattractant IL-8.

© 1998 by The American Society of Hematology.