bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine 3′:5′-Cyclic Monophosphate (cAMP)-Inducible Pyrimidine 5′-Nucleotidase and Pyrimidine Nucleotide Metabolism of Chick Embryonic Erythrocytes
Authors: Dragon Stefanie , Hille Rainer , Götz Robert , Baumann Rosemarie
Published: October 2019

Terminally differentiating erythrocytes degrade most of their RNA with subsequent release of mononucleotides. Pyrimidine mononucleotides are preferentially cleaved by an erythrocyte-specific pyrimidine 5′-nucleotidase; deficiency of this enzyme causes hemolytic anemia in humans. Details of the regulation of its activity during erythroid differentiation are unknown. The present study arose from the observation that the immature red blood cells (RBCs) of mid-term chick embryos contain high concentrations of uridine 5′-triphosphate (UTP) (5 to 6 mmol/L), which decline rapidly from days 13 to 14 onward. We analyzed two key enzymes of RBC pyrimidine nucleotide metabolism: pyrimidine nucleoside phosphorylase (PNP) and pyrimidine 5′-nucleotidase (P-5′-N), to evaluate if changes of enzyme activity during embryonic development are correlated with changes of RBC UTP. Secondly, we tested if these enzymes are under hormonal control. The results show that embryonic RBCs contain only minimal activity of PNP. In contrast, P-5′-N increases from day 13 on, suggesting that the enzyme is a limiting factor in UTP degradation. Activation of β-adrenergic and A2A-adenosine receptors causes transcription-dependent de novo synthesis of P-5′-N. Because β-adrenergic and adenosine receptors are also found on adult erythroid cells, P-5′-N might be an enzyme of differentiating RBCs whose expression is in part controlled by adenosine 3′:5′-cyclic monophosphate (cAMP).
Genetic and Immunological Properties of Phage-Displayed Human Anti-Rh(D) Antibodies: Implications for Rh(D) Epitope Topology
Authors: Chang Tylis Y. , Siegel Don L.
Published: October 2019

Understanding anti-Rh(D) antibodies on a molecular level would facilitate the genetic analysis of the human immune response to Rh(D), lead to the design of therapeutically useful reagents that modulate antibody binding, and provide relevant information regarding the structural organization of Rh(D) epitopes. Previously, we described a Fab/phage display-based method for producing a large array of anti-Rh(D) antibodies from the peripheral blood lymphocytes of a single alloimmunized donor. In the current study, we present a detailed analysis of 83 randomly selected clones. Sequence analysis showed the presence of 28 unique γ1 heavy chain and 41 unique light chain gene segments. These paired to produce 53 unique Fabs that had specificity for at least half of the major Rh(D) epitopes. Surprisingly, despite this diversity, only 4 closely related heavy chain germline genes were used (VH3-30, VH3-30.3, VH3-33, and VH3-21). Similarly, nearly all Vκ light chains (15/18) were derived from one germline gene (DPK9). λ light chains showed a more diverse VL gene usage, but all (23/23) used the identical Jλ2 gene. Several Fabs that differed in epitope specificity used identical heavy chains but different light chains. In particular, 2 such clones differed by only 3 residues, which resulted in a change from epD2 to epD3 specificity. These results suggest a model in which footprints of anti-Rh(D) antibodies are essentially identical to one another, and Rh(D) epitopes, as classically defined by panels of Rh(D) variant cells, are not discrete entities. Furthermore, these data imply that the epitope specificity of an anti-Rh(D) antibody can change during the course of somatic mutation. From a clinical perspective, this process, which we term epitope migration, has significance for the design of agents that modulate antibody production and for the creation of mimetics that block antibody binding in the settings of transfusion reactions and hemolytic disease of the newborn.
Intracellular Cytokine Profile of Cord and Adult Blood Lymphocytes
Authors: Chalmers I.M.H. , Janossy G. , Contreras M. , Navarrete C.
Published: October 2019

Umbilical cord blood (CB) transplantation is thought to be associated with a reduced risk of severe graft-versus-host-disease (GVHD) compared with bone marrow transplantation (BMT). The cytokine cascade is known to be important in the pathogenesis of GVHD; however, previous studies investigating the cytokine secretion pattern of CB cells have been contradictory because of variations in experimental techniques. In this study, the cytokine profile of cord and adult blood lymphocytes and lymphocyte subsets has been assessed at the single-cell level by flow cytometry, using CD4/CD8 and CD45RA/CD45RO markers. Cord and adult blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin. After 4 to 24 hours of incubation, interleukin-2 (IL-2), IL-4, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) production was measured by three-color flow cytometry. The results show that cord blood lymphocytes (CBL) produce less IL-2, IL-4, IFN-γ, and TNF-α than adult peripheral blood lymphocytes (ABL). Further subset analysis showed that in CBL the majority of cytokine producing cells were CD4+CD45RA+, whereas in ABL the cytokine-producing cells were both CD4+CD45RO+ and CD8+CD45RO+. These results suggest that the reduced incidence of GVHD in CB transplantation may partly due to the altered cytokine profile seen in CBL.
Interleukin-6–Induced Inhibition of Multiple Myeloma Cell Apoptosis: Support for the Hypothesis That Protection Is Mediated Via Inhibition of the JNK/SAPK Pathway
Authors: Xu Feng-hao , Sharma Sanjesh , Gardner Agnes , Tu Yiping , Raitano Arthur , Sawyers Charles , Lichtenstein Alan
Published: October 2019

The mechanism by which interleukin-6 (IL-6) protects multiple myeloma (MM) plasma cells from apoptosis induced by anti-fas antibodies and dexamethasone was studied. Anti-apoptotic concentrations of IL-6 had no effect on cell-cycle distribution or activation of RAF-1 or ERK in dexamethasone- or anti–fas-treated 8226 and UCLA #1 MM cell lines. However, IL-6–dependent protection of viability correlated with an inhibition of dexamethasone- and anti–fas-induced activation ofjun kinase (JNK) and AP-1 transactivation. To test the hypothesis that cytokine-induced protection was mediated through inhibition of JNK/c-jun, we also inhibited c-junfunction in 8226 cells via introduction of a mutant dominant negative c-jun construct. Mutant c-jun–containing MM cells were also resistant to anti–fas-induced apoptosis but were significantly more sensitive to dexamethasone-induced apoptosis. These results support the notion that IL-6 protects MM cells against anti-fas through its inhibitory effects on JNK/c-junbut indicate protection against dexamethasone occurs through separate, yet unknown pathways.
Protease-Activated Receptor Genes Are Clustered on 5q13
Authors: Dupérat Véronique Guyonnet , Jacquelin Béatrice , Boisseau Pierre , Arveiler Benoı̂t , Nurden Alan T.
Published: October 2019

The serine protease, thrombin, is both a potent agonist for platelet aggregation and a mitogen inducing the proliferation of other cell types. Many cellular responses to thrombin are mediated by a G-protein–coupled thrombin receptor (protease-activated receptor-1, PAR-1). This represents the prototype of a new family of proteolytically cleaved receptors that includes PAR-2 and the recently identified PAR-3. Like PAR-1, PAR-3 is a potential thrombin receptor. Their similar gene structure, mechanism of activation, and colocalization to 5q13 raises the question of a common evolutionary origin and of their belonging to a clustered gene family. Construction of a physical map of the 5q13 region by pulsed-field gel electrophoresis (PFGE) has allowed us to identify six potential CpG islands and to establish a linkage of the PAR genes. Southern blot analysis showed that they were in a cluster on a 560-kb Asc I fragment, in the order PAR-2, PAR-1, and PAR-3. PAR-1 and PAR-2 genes were contained within the identical 240-kb Not I fragment, thus confirming a tight linkage between them. The localization of other CpG islands suggested that more PAR-family genes may be present.
Interleukin-15 Is an Autocrine/Paracrine Viability Factor for Cutaneous T-Cell Lymphoma Cells
Authors: Döbbeling Udo , Dummer Reinhard , Laine Elisabeth , Potoczna Natascha , Qin Jian-Zhong , Burg Günter
Published: October 2019

In this study we investigated the role of interleukin-15 (IL-15) in the immunobiology of cutaneous T-cell lymphoma (CTCL) cells. Using cell culture techniques, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunhistochemistry we found that IL-15, like IL-7, is a growth factor for the Sézary cell line SeAx and that both cytokines prolonged the survival of malignant T cells directly isolated from Sézary syndrome (SS) patients. Both IL-15 and IL-7 were more potent than IL-2. IL-4 and IL-9, whose receptors share the same gamma chain with the receptors of IL-2, IL-7, and IL-15, did not sustain the growth of CTCL cells, indicating that signaling through the common gamma chain (γc) is not sufficient for continuous growth. IL-13 and tumor necrosis factor-α (TNF-α) had no effect. IL-7 and IL-15 also supported the growth of SeAx cells in the presence of the apoptosis inducing agents dexamethasone and retinoic acid. The analysis of patient Sézary cells and three CTCL cell lines by RT-PCR showed that all these cells expressed IL-15 mRNA, but only a few (25%) produced IL-7 mRNA. Immunohistological analyses of skin biopsy samples of SS and Mycosis fungoides patients showed immunoreactivity for IL-15 in basal cell layer keratinocytes and in the infiltrating lymphocytes. We conclude that IL-15 is a growth or viability factor for CTCL-derived cell lines or shortly cultivated Sézary cells. The findings that IL-15 mRNA can be detected in Sézary syndrome peripheral blood mononuclear cells and that the IL-15 protein is detected in skin sections from CTCL patients suggest that IL-15 plays an important role in the biology of CTCL.
Prednisolone Resistance in Childhood Acute Lymphoblastic Leukemia: Vitro-Vivo Correlations and Cross-Resistance to Other Drugs
Authors: Kaspers G.J.L. , Pieters R. , Van Zantwijk C.H. , VanWering E.R. , Van Der Does-Van Den Berg A. , Veerman A.J.P.
Published: October 2019

As an important determinant of response to chemotherapy, accurate measurement of cellular drug resistance may provide clinically relevant information. Our objectives in this study were to determine the relationship between in vitro resistance to prednisolone (PRD) measured with the colorimetric methyl-thiazol-tetrazolium (MTT) assay, and (1) short-term clinical response to systemic PRD monotherapy, (2) long-term clinical outcome after combination chemotherapy within all patients and within the subgroups of clinical good and poor responders to PRD, and (3) in vitro resistance to 12 other drugs in 166 children with newly diagnosed acute lymphoblastic leukemia (ALL). The 12 clinical poor PRD responders had ALL cells that were median 88-fold more in vitro resistant to PRD than 131 good responders (P = .013). Within all patients, increased in vitro resistance to PRD predicted a significantly worse long-term clinical outcome, at analyses with and without stratification for clinical PRD response, and at multivariate analysis (P ≤ .001). Within both the clinical good and poor responder subgroups, increased in vitro resistance to PRD was associated with a worse outcome, which was significant within the group of clinical good responders (P < .001). LC50 values, ie, lethal concentrations to 50% of ALL cells, for PRD and each other drug correlated significantly with those of all other 12 drugs, with an average correlation coefficient of 0.44 (standard deviation 0.05). The highest correlations were found between structurally related drugs. In conclusion, in vitro resistance to PRD was significantly related to the short-term and long-term clinical response to chemotherapy, the latter also within the subgroup of clinical good responders to PRD. There was a more general in vitro cross-resistance between anticancer drugs in childhood ALL, although drug-specific activities were recognized.
An Erythroid-Specific Transcript Generates the Soluble Form of NADH-Cytochrome b5 Reductase in Humans
Authors: Bulbarelli Alessandra , Valentini Alessandra , DeSilvestris Marcella , Cappellini M. Domenica , Borgese Nica
Published: October 2019

Two forms of NADH-cytochrome b5 reductase (b5R), an erythrocyte-restricted soluble form, active in methemoglobin reduction, and a ubiquitous membrane-associated form involved in lipid metabolism, are produced from one gene. In the rat, the two forms are generated from alternative transcripts differing in the first exon, however, biogenesis of human b5R was less understood. Recently, two different transcripts (M and S), differing in the first exon were also described in humans. Here, we have investigated the tissue-specificity and the role of the S-transcript in the generation of soluble b5R. By RNase protection assays designed to simultaneously detect alternative b5R transcripts in the same sample, the S transcript was undetectable in nonerythroid and in erythroleukemic K562 cells induced to differentiate, but was present in terminal erythroblast cultures, and represented a major b5R transcript in reticulocytes. Analysis of the translation products of the M- and S-transcripts in HeLa cells transfected with the corresponding cDNAs demonstrated that the S-transcript generates soluble b5R, presumably from an internal initiation codon. Our results indicate that the S-transcript is expressed at late stages of erythroid maturation to generate soluble b5R.
Role of c-mpl in Early Hematopoiesis
Authors: Solar Gregg P. , Kerr William G. , Zeigler Francis C. , Hess Darren , Donahue Christopher , de Sauvage Frederic J. , Eaton Dan L.
Published: October 2019

Recently, several lines of evidence have indicated an expanded role for thrombopoietin (TPO) and its receptor, c-mpl, in hematopoiesis. In addition to being the primary physiological regulator of platelet production, it is now apparent that TPO also acts during early hematopoiesis. To futher define the role of TPO in early hematopoiesis we have identified discrete murine and human stem cell populations with respect to c-mpl expression and evaluated their potential for hematopoietic engraftment. Fluorescence-activated cell sorter analysis of enriched stem cell populations showed the presence of c-mpl expressing subpopulations. Approximately 50% of the murine fetal liver stem cell–enriched population, AA4+Sca+c-kit+, expressed c-mpl. Analysis of the murine marrow stem cell population LinloSca+c-kit+ showed that 70% of this population expressed c-mpl. Expression of c-mpl was also detected within the human bone marrow CD34+CD38− stem cell progenitor pool and approximately 70% of that population expressed c-mpl. To rigorously evaluate the role of TPO/c-mpl in early hematopoiesis we compared the repopulation capacity of murine stem cell populations with respect to c-mpl expression in a competitive repopulation assay. When comparing the fetal liver progenitor populations, AA4+Sca+c-kit+c-mpl+and AA4+Sca+c-kit+c-mpl−, we found that stem cell activity segregates with c-mpl expression. This result is complemented by the observation that the LinloSca+ population of c-mplgene-deficient mice was sevenfold less potent than LinloSca+ cells from wild-type mice in repopulating activity. The engraftment potential of the human CD34+CD38−c-mpl+ population was evaluated in a severe combined immunodeficient-human bone model. In comparison to the CD34+CD38−c-mpl− population, the CD34+CD38−c-mpl+ cells showed significantly better engraftment. These results demonstrate a physiological role for TPO and its receptor, c-mpl, in regulating early hematopoiesis.
The Redox State as a Correlate of Senescence and Wasting and as a Target for Therapeutic Intervention
Authors: Hack Volker , Breitkreutz Raoul , Kinscherf Ralf , Röhrer Helmut , Bärtsch Peter , Taut Friedemann , Benner Axel , Dröge Wulf
Published: October 2019

The loss of body cell mass (bcm) in senescence and wasting is poorly understood. We now show that the plasma cystine/acid soluble thiol ratio, ie, an indicator of the redox state, is increased in old age and cancer patients and correlated with a decrease in bcm and plasma albumin. A cause/effect relationship was suggested by two independent studies with N-acetyl-cysteine (NAC). NAC caused an increase in the bcm of healthy persons with high plasma cystine/thiol ratios, and treatment of cancer patients with NAC plus interleukin-2 caused an increase in bcm, plasma albumin, and functional capacity. Albumin levels below 680 μmol/L were associated with an increase in body water. Our studies suggest that the shift in the redox state may contribute to the loss of bcm and may provide a quantitative guideline for therapeutic intervention. Treatment of cancer patients with thiol-containing antioxidants may improve the quality of life.
Minimal Residual Disease Status Before Allogeneic Bone Marrow Transplantation Is an Important Determinant of Successful Outcome for Children and Adolescents With Acute Lymphoblastic Leukemia
Authors: Knechtli Christopher J.C. , Goulden Nicholas J. , Hancock Jeremy P. , Grandage Victoria L.G. , Harris Emma L. , Garland Russell J. , Jones Claire G. , Rowbottom Anthony W. , Hunt Linda P. , Green Ann F. , Clarke Emer , Lankester Alan W. , Cornish Jacqueline M. , Pamphilon Derwood H. , Steward Colin G. , Oakhill Anthony
Published: October 2019

The efficacy of allografting in acute lymphoblastic leukemia (ALL) is heavily influenced by remission status at the time of transplant. Using polymerase chain reaction (PCR)-based minimal residual disease (MRD) analysis, we have investigated retrospectively the impact of submicroscopic leukemia on outcome in 64 patients receiving allogeneic bone marrow transplantation (BMT) for childhood ALL. Remission BM specimens were taken 6 to 81 days (median, 23) before transplant. All patients received similar conditioning therapy; 50 received grafts from unrelated donors and 14 from related donors. Nineteen patients were transplanted in first complete remission (CR1) and 45 in second or subsequent CR. MRD was analyzed by PCR of Ig or T-cell receptor δ or γ rearrangements, electrophoresis, and allele-specific oligoprobing. Samples were rated high-level positive (clonal band evident after electrophoresis; sensitivity 10−2 to 10−3), low-level positive (MRD detected only after oligoprobing; sensitivity 10−3 to 10−5), or negative. Excluding 8 patients transplanted in CR2 for isolated extramedullary relapse (all MRD−), MRD was detected at high level in 12 patients, low level in 11, and was undetectable in 33. Two-year event-free survival for these groups was 0%, 36%, and 73%, respectively (P < .001). Follow-up in patients remaining in continuing remission is 20 to 96 months (median, 35). These results suggest that MRD analysis could be used routinely in this setting. This would allow identification of patients with resistant leukemia (who may benefit from innovative BMT protocols) and of those with more responsive disease (who may be candidates for randomized trials of BMT versus modern intensive relapse chemotherapy).
Reconstitution of the T-Cell Compartment After Bone Marrow Transplantation: Restoration of the Repertoire by Thymic Emigrants
Authors: Dumont-Girard Florence , Roux Etienne , van Lier René A. , Hale Geoff , Helg Claudine , Chapuis Bernard , Starobinski Michel , Roosnek Eddy
Published: October 2019

We have studied the reconstitution of the T-cell compartment after bone marrow transplantation (BMT) in five patients who received a graft-versus-host disease (GVHD) prophylaxis consisting of methotrexate, cyclosporin, and 10 daily injections (day −4 to day +5) of Campath-1G. This treatment eliminated virtually all T cells (7 ± 8 T cells/μL at day 14) which facilitated the analysis of the thymus-dependent and independent pathways of T-cell regeneration. During the first 6 months, the peripheral T-cell pool was repopulated exclusively through expansion of residual T cells with all CD4+ T cells expressing the CD45RO-memory marker. In two patients, the expansion was extensive and within 2 months, the total number of T cells (CD8>>CD4) exceeded 1,000/μL. In the other three patients, T cells remained low (87 ± 64 T cells/μL at 6 months) and remained below normal values during the 2 years of the study. In all patients, the first CD4+CD45RA+RO− T cells appeared after 6 months and accumulated thereafter. In the youngest patient (age 13), the increase was relatively fast and naive CD4+ T cells reached normal levels (600 T cells/μL) 1 year later. In the four adult patients (age 25 ± 5), the levels reached at that time-point were significantly lower (71 ± 50 T cells/μL). In all patients, the T-cell repertoire that had been very limited, diversified with the advent of the CD4+CD45RA+RO− T cells. Cell sorting experiments showed that this could be attributed to the complexity of the T-cell repertoire of the CD4+CD45RA+RO− T cells that was comparable to that of a normal individual and that, therefore, it is likely that these cells are thymic emigrants. We conclude that after BMT, the thymus is essential for the restoration of the T-cell repertoire. Because the thymic activity is restored with a lag time of approximately 6 months, this might explain why, in particular in recipients of a T-cell–depleted graft, immune recovery is delayed.
An Essential Role for NF-κB in Human CD34+ Bone Marrow Cell Survival
Authors: Pyatt David W. , Stillman Wayne S. , Yang Yanzhu , Gross Sherilyn , Zheng Jia hua , Irons Richard D.
Published: October 2019

The transcription factor, NF-κB, is important for T-cell activation, B-cell maturation, and human immunodeficiency virus transcription and plays a role in alternatively mediating and protecting against apoptosis in a variety of cell types. However, a role for NF-κB in human CD34+ bone marrow cells has not been described. We provide evidence here that virtually all human CD34+ bone marrow cells express NF-κB that can be activated by exposure to phorbol 12-myristate 13-acetate and a variety of cytokines, eg, tumor necrosis factor , interleukin-3, and granulocyte-macrophage colony-stimulating factor. In addition, we demonstrate that NF-κB may be required for human CD34+bone marrow cell clonogenic function and survival. These results offer insight into a new role for NF-κB in maintaining survival and function in hematopoietic stem and progenitor cells and suggest that proposed strategies involving inhibition of NF-κB activation as an adjunct to cancer chemotherapy should be approached with caution.
Two Common Functional Polymorphisms in the Promoter Region of the Coagulation Factor VII Gene Determining Plasma Factor VII Activity and Mass Concentration
Authors: van ’t Hooft Ferdinand M. , Silveira Angela , Tornvall Per , Iliadou Anastasia , Ehrenborg Ewa , Eriksson Per , Hamsten Anders
Published: October 2019

Recent studies have provided evidence for associations between common polymorphic markers in the coagulation factor VII (FVII) gene and plasma FVII levels. Here we describe two common, nonrelated, functional polymorphisms in the promoter region of the FVII gene, a G to T substitution at position −401 and a novel G to A substitution at position −402. Both polymorphisms strongly influence the binding properties of nuclear protein(s). The rare −401T allele is associated with a reduced basal rate of transcription of the FVII gene in human hepatoblastoma cells and with reduced plasma concentrations of total FVII (VIIag) and fully activated FVII molecules (VIIa). In contrast, the rare −402A allele confers increased transcriptional activity and is associated with increased plasma FVII levels. Together, the two polymorphisms explained 18% and 28% of the variation in VIIag and VIIa, respectively, in a group of 183 healthy, middle-aged men. It is concluded that these polymorphisms are important for the regulation of the plasma levels of FVII and that they are likely to be useful genetic markers to resolve the issue of whether a causal relationship exists between FVII levels and risk of coronary heart disease.
Elevated Levels of Circulating Procoagulant Microparticles in Patients With Paroxysmal Nocturnal Hemoglobinuria and Aplastic Anemia
Authors: Hugel Bénédicte , Socié Gérard , Vu Thi , Toti Florence , Gluckman Eliane , Freyssinet Jean-Marie , Scrobohaci Marie-Lorraine
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH), frequently occurring during suppressed hematopoiesis including aplastic anemia (AA), is a clonal disorder associated with an increased incidence of thrombotic events. Complement-mediated hemolysis, impairment of the fibrinolytic system, or platelet activation are thought to be responsible for the associated thrombotic risk. We investigated here the elevation of membrane-derived procoagulant microparticles in the blood flow of such patients. Elevated levels of circulating microparticles were in fact detected in both de novo PNH patients and AA subjects with a PNH clone, but not in those with AA without a PNH clone. The cellular origin of the microparticles was determined in PNH samples; most stemmed from platelets. Glycophorin A+ particles were rarely detected. Therefore, platelet activation, resulting in the dissemination of procoagulant phospholipids in the blood flow, could be one of the main causes for the elevated thrombotic risk associated with PNH. These observations suggest that shed membrane particles can be considered a valuable biological parameter for the assessment of possible thrombotic complications in patients with PNH.
Glass needle–mediated microinjection of macromolecules and transgenes into primary human blood stem/progenitor cells
Authors: Davis Brian R. , Yannariello-Brown Judith , Prokopishyn Nicole L. , Luo Zhongjun , Smith Mark R. , Wang Jue , Carsrud N. D. Victor , Brown David B.
Published: October 2019

A novel glass needle–mediated microinjection method for delivery of macromolecules, including proteins and larger transgene DNAs, into the nuclei of blood stem/progenitor cells was developed. Temporary immobilization of cells to extracellular matrix–coated dishes has enabled rapid and consistent injection of macromolecules into nuclei of CD34+, CD34+/CD38−, and CD34+/CD38−/Thy-1lo human cord blood cells. Immobilization and detachment protocols were identified, which had no adverse effect on cell survival, progenitor cell function (colony forming ability), or stem cell function (NOD/SCID reconstituting ability). Delivery of fluorescent dextrans to stem/progenitor cells was achieved with 52% ± 8.4% of CD34+ cells and 42% ± 14% of CD34+/CD38−cells still fluorescent 48 hours after injection. Single-cell transfer and culture of injected cells has demonstrated long-term survival and proliferation of CD34+ and CD34+/CD38−cells, and retention of the ability of CD34+/CD38− cells to generate progenitor cells. Delivery of DNA constructs (currently ≤ 19.6 kb) and fluorescently labeled proteins into CD34+ and CD34+/CD38− cells was achieved with transient expression of green fluorescent protein observed in up to 75% of injected cells. These data indicate that glass needle–mediated delivery of macromolecules into primitive hematopoietic cells is a valuable method for studies of stem cell biology and a promising method for human blood stem cell gene therapy.
Identification of the soluble granulocyte-macrophage colony stimulating factor receptor protein in vivo
Authors: Sayani Farzana , Montero-Julian Felix A. , Ranchin Valerie , Prevost Jay M. , Flavetta Sophie , Zhu Weibin , Woodman Richard C. , Brailly Herve , Brown Christopher B.
Published: October 2019

On the basis of the finding of alternatively spliced mRNAs, the -subunit of the receptor for GM-CSF is thought to exist in both a membrane spanning (tmGMR) and a soluble form (solGMR). However, only limited data has been available to support that the solGMR protein product exists in vivo. We hypothesized that hematopoietic cells bearing tmGMR would have the potential to also produce solGMR. To test this hypothesis we examined media conditioned by candidate cells using functional, biochemical, and immunologic means. Three human leukemic cell lines that express tmGMR (HL60, U937, THP1) were shown to secrete GM-CSF binding activity and a solGMR-specific band by Western blot, whereas a tmGMR-negative cell line (K562) did not. By the same analyses, leukapheresis products collected for autologous and allogeneic stem cell transplants and media conditioned by freshly isolated human neutrophils also contained solGMR. The solGMR protein in vivo displayed the same dissociation constant (Kd = 2-5 nmol) as that of recombinant solGMR. A human solGMR ELISA was developed that confirmed the presence of solGMR in supernatant conditioned by the tmGMR-positive leukemic cell lines, hematopoietic progenitor cells, and neutrophils. Furthermore, the ELISA demonstrated a steady state level of solGMR in normal human plasma (36 ± 17 pmol) and provided data suggesting that plasma solGMR levels can be elevated in acute myeloid leukemias.
Domain 5 of high molecular weight kininogen (kininostatin) down-regulates endothelial cell proliferation and migration and inhibits angiogenesis
Authors: Colman Robert W. , Jameson Bradford A. , Lin Yingzhang , Johnson Donald , Mousa Shaker A.
Published: October 2019

We have demonstrated that high molecular weight kininogen (HK) binds specifically on endothelial cells to domain 2/3 of the urokinase receptor (uPAR). Inhibition by vitronectin suggests that kallikrein-cleaved HK (HKa) is antiadhesive. Plasma kallikrein bound to HK cleaves prourokinase to urokinase, initiating cell-associated fibrinolysis. We postulated that HK cell binding domains would inhibit angiogenesis. We found that recombinant domain 5 (D5) inhibited endothelial cell migration toward vitronectin 85% at 0.27 μM with an IC50 (concentration to yield 50% inhibition) = 0.12 μM. A D5 peptide, G486-K502, showed an IC50 = 0.2 μM, but a 25-mer peptide from a D3 cell binding domain only inhibited migration 10% at 139 μM (IC50 > 50 μM). D6 exhibited weaker inhibitory activity (IC50 = 0.50 μM). D5 also potently inhibited endothelial cell proliferation with an IC50 = 30 nM, while D3 and D6 were inactive. Using deletion mutants of D5, we localized the smallest region for full activity to H441-D474. To further map the active region, we created a molecular homology model of D5 and designed a series of peptides displaying surface loops. Peptide 440-455 was the most potent (IC50 = 100 nM) in inhibiting proliferation but did not inhibit migration. D5 inhibited angiogenesis stimulated by fibroblast growth factor FGF2 (97%) in a chicken chorioallantoic membrane assay at 270 nM, and peptide 400-455 was also inhibitory (79%). HK D5 (for which we suggest the designation, “kininostatin”) is a potent inhibitor of endothelial cell migration and proliferation in vitro and of angiogenesis in vivo.
Inhibition of juvenile myelomonocytic leukemia cell growth in vitro by farnesyltransferase inhibitors
Authors: Emanuel Peter D. , Snyder Richard C. , Wiley Tonya , Gopurala Balaganesh , Castleberry Robert P.
Published: October 2019

Juvenile myelomonocytic leukemia (JMML) is an early childhood disease for which there is no effective therapy. Therapy with 13-cis retinoic acid or low-dose chemotherapy can induce some responses, but neither mode is curative. Stem cell transplantation can produce lasting remissions but is hampered by high rates of relapse. The pathogenesis of JMML involves deregulated cytokine signal transduction through the Ras signaling pathway, with resultant selective hypersensitivity of JMML cells to granulocyte-macrophage colony-stimulating factor (GM-CSF). A JMML mouse model, achieved through homozygous deletion of the neurofibromatosis gene, confirmed the involvement of deregulated Ras in JMML pathogenesis. With this pathogenetic knowledge, mechanism-based treatments are now being developed and tested. Ras is critically dependent on a prenylation reaction for its signal transduction abilities. Farnesyltransferase inhibitors are compounds that were developed specifically to block the prenylation of Ras. Two of these compounds, L-739,749 and L-744,832, were tested for their ability to inhibit spontaneous JMML granulocyte-macrophage colony growth. Within a dose range of 1 to 10 μmol/L, each compound demonstrated dose-dependent inhibition of JMML colony growth. An age-matched patient with a different disease and GM-CSF–stimulated normal adult marrow cells also demonstrated dose-dependent inhibitory effects on colony growth, but they were far less sensitive to these compounds than JMML hematopoietic progenitors. Even if the addition of L-739,749 were delayed for 5 days, significant inhibitory effects would still show in JMML cultures. These results demonstrate that a putative Ras-blocking compound can have significant growth inhibitory effects in vitro, perhaps indicating a potential treatment for JMML.
Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice
Authors: Gee Sherry L. , Aoyagi Kazuko , Lersch Robert , Hou Victor , Wu Michael , Conboy John G.
Published: October 2019

Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre–messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5′ splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step—excision of the downstream intron—and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain.
DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection
Authors: Vogt M. H. J. , de Paus R. A. , Voogt P. J. , Willemze R. , Falkenburg J. H. F.
Published: October 2019

Graft rejection after histocompatibility locus antigen (HLA)-identical stem cell transplantation results from the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes of recipient origin. T-lymphocyte clones that specifically recognize H-Y epitopes on male target cells have been generated during graft rejection after sex-mismatched transplantation. Previously, 2 human H-Y epitopes derived from the same SMCY gene have been identified that were involved in bone marrow graft rejection. We report the identification of a new male-specific transplantation antigen encoded by the Y-chromosome-specific gene DFFRY. The DFFRY-derived peptide was recognized by an HLA-A1 restricted CTL clone, generated during graft rejection from a female patient with acute myeloid leukemia who rejected HLA-phenotypically identical bone marrow from her father. The identification of this gene demonstrates that at least 2 genes present on the human Y-chromosome code for male-specific transplantation antigens.
Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells
Authors: Austin Timothy W. , Salimi Suzan , Veres Gabor , Morel Franck , Ilves Heini , Scollay Roland , Plavec Ivan
Published: October 2019

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.
High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo
Authors: Oostendorp Robert A. J. , Audet Julie , Eaves Connie J.
Published: October 2019

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.
Requirement of leucine-rich repeats of glycoprotein (GP) Ibα for shear-dependent and static binding of von Willebrand factor to the platelet membrane GP Ib–IX-V complex
Authors: Shen Yang , Romo Gabriel M. , Dong Jing-fei , Schade Alicia , McIntire Larry V. , Kenny Dermot , Whisstock James C. , Berndt Michael C. , López José A. , Andrews Robert K.
Published: October 2019

The platelet glycoprotein (GP) Ib–IX-V complex mediates adhesion to von Willebrand factor (vWf) in (patho)physiologic thrombus formation. The vWf-binding site on GP Ib–IX-V is within the N-terminal 282 residues of GP Ib, which consist of an N-terminal flanking sequence (His-1–Ile-35), 7 leucine-rich repeats (Leu-36–Ala-200), a C-terminal flank (Phe-201–Gly-268), and a sulfated tyrosine sequence (Asp-269–Glu-282). We have used mammalian cell expression of canine–human chimeras of GP Ib, corresponding to precise structural boundaries, to demonstrate the first specific requirement for individual leucine-rich repeats for binding of vWf either induced by a modulator, ristocetin, or under hydrodynamic flow. Implicit in this approach was that the GP Ib chimeras retained a functional conformation, a supposition confirmed by analyzing restoration of function to reversed human–canine chimeras and demonstrating that all chimeras bound vWf activated by botrocetin, a modulator that is indiscriminate between species. Leucine-rich repeats 2, 3, and 4 of GP Ib were identified as being critical for vWf adhesion to GP Ib–IX-V.
Cellular origin and procoagulant properties of microparticles in meningococcal sepsis
Authors: Nieuwland Rienk , Berckmans René J. , McGregor Sarah , Böing Anita N. , Th. M. Romijn Fred P. H. , Westendorp Rudi G. J. , Hack C. Erik , Sturk Augueste
Published: October 2019

Patients with meningococcal sepsis generally suffer from disseminated intravascular coagulation (DIC). The aim of this study was to address whether these patients have elevated numbers of circulating microparticles that contribute to the development of DIC. Plasma samples from 5 survivors, 2 nonsurvivors, and 5 healthy volunteers were analyzed for the presence of microparticles by flow cytometry. Ongoing coagulation activation in vivo was quantified by enzyme-linked immunosorbent assay of plasma prothrombin fragment F1 + 2, and procoagulant properties of microparticles in vitro were estimated by thrombin-generation assay. On admission, all patients had increased numbers of microparticles originating from platelets or granulocytes when compared with controls (P = .004 and P = .008, respectively). Patients had elevated levels of F1 + 2 (P = .004), and their microparticles supported thrombin generation more strongly in vitro (P = .003) than those of controls. Plasma from the patient with the most fulminant disease course and severe DIC contained microparticles that expressed both CD14 and tissue factor, and these microparticles demonstrated extreme thrombin generation in vitro. We conclude that patients with meningococcal sepsis have elevated numbers of circulating microparticles that are procoagulant. These findings may suggest a novel therapeutic approach to combat clinical conditions with excessive coagulation activation.
Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors
Authors: Peichev Mario , Naiyer Afzal J. , Pereira Daniel , Zhu Zhenping , Lane William J. , Williams Mathew , Oz Mehmet C. , Hicklin Daniel J. , Witte Larry , Moore Malcolm A. S. , Rafii Shahin
Published: October 2019

Emerging data suggest that a subset of circulating human CD34+ cells have phenotypic features of endothelial cells. Whether these cells are sloughed mature endothelial cells or functional circulating endothelial precursors (CEPs) is not known. Using monoclonal antibodies (MoAbs) to the extracellular domain of the human vascular endothelial receptor-2 (VEGFR-2), we have shown that 1.2 ± 0.3% of CD34+ cells isolated from fetal liver (FL), 2 ± 0.5% from mobilized peripheral blood, and 1.4 ± 0.5% from cord blood were VEGFR-2+. In addition, most CD34+VEGFR-2+ cells express hematopoietic stem cell marker AC133. Because mature endothelial cells do not express AC133, coexpression of VEGFR-2 and AC133 on CD34+ cells phenotypically identifies a unique population of CEPs. CD34+VEGFR-2+ cells express endothelial-specific markers, including VE-cadherin and E-selectin. Also, virtually all CD34+VEGFR-2+ cells express the chemokine receptor CXCR4 and migrate in response to stromal-derived factor (SDF)-1 or VEGF. To quantitate the plating efficiency of CD34+ cells that give rise to endothelial colonies, CD34+ cells derived from FL were incubated with VEGF and fibroblast growth factor (FGF)-2. Subsequent isolation and plating of nonadherent FL-derived VEGFR-2+ cells with VEGF and FGF-2 resulted in differentiation of AC133+VEGFR-2+ cells into adherent AC133−VEGFR-2+Ac-LDL+(acetylated low-density lipoprotein) colonies (plating efficiency of 3%). In an in vivo human model, we have found that the neo-intima formed on the surface of left ventricular assist devices is colonized with AC133+VEGFR-2+ cells. These data suggest that circulating CD34+ cells expressing VEGFR-2 and AC133 constitute a phenotypically and functionally distinct population of circulating endothelial cells that may play a role in neo-angiogenesis.
The CXC-chemokine platelet factor 4 promotes monocyte survival and induces monocyte differentiation into macrophages
Authors: Scheuerer Barbara , Ernst Martin , Dürrbaum-Landmann Iris , Fleischer Jens , Grage-Griebenow Evelin , Brandt Ernst , Flad Hans-Dieter , Petersen Frank
Published: October 2019

Unstimulated monocytes rapidly undergo physiological changes resulting in programmed cell death (apoptosis) while stimuli promoting differentiation of these cells into macrophages were shown to inhibit apoptotic processes. In the present study, we report that the platelet-derived -chemokine platelet factor 4 (PF4) induces the differentiation of monocytes into macrophages, as is evident from morphological changes as well as from the up-regulation of differentiation markers (carboxypeptidase M/MAX1 and CD71). Significant alterations of the phenotype were observed after 72 hours of culture in the presence of the chemokine and required a minimal concentration of 625 nmol/L PF4. PF4-induced macrophages were characterized by a lack of HLA-DR antigen on their surface but showed a strong increase in the expression of the CD28 ligand B7-2. Furthermore, PF4 stimulation prevented monocytes from undergoing spontaneous apoptosis during 72 hours of culture as determined in an annexin-V–binding assay. Although PF4 induced the secretion of relevant amounts of TNF-, neutralizing antibodies directed against TNF- or granulocyte-macrophage colony–stimulating factor (GM-CSF) did not revert PF4-induced rescue from programmed cell death, suggesting that PF4 exerts its antiapoptotic effects in a TNF-– or GM-CSF–independent fashion. On the basis of these results, we propose a novel role for PF4 in the control of monocyte differentiation during an inflammatory process in vivo.