bluuebunny/crossref_metadata_2025_split huggingface dataset:
Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells
Authors: Shih Chu-Chih , Hu Mickey C.-T. , Hu Jun , Medeiros Jeffrey , Forman Stephen J. 
Published: October 2019

AbstractWe have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.
Discordant Expression of Tissue Factor and Its Activity in Polarized Epithelial Cells. Asymmetry in Anionic Phospholipid Availability as a Possible Explanation
Authors: Hansen Carsten B. , van Deurs Bo , Petersen Lars C. , Rao L. Vijaya Mohan 
Published: October 2019

AbstractRecent studies have shown a discrepancy between the level of tissue factor (TF) expression and the level of TF procoagulant activity on the apical and basolateral surface domains of polarized epithelial cells. The present investigation was performed to elucidate possible reasons for the discordant expression of TF and its activity on the surface of polarized epithelial cells using a human intestinal epithelial cell line, Caco-2 and Madin-Darby canine kidney epithelial cells, type II (MDCK-II). Functional activity of coagulation factor VIIa (VIIa) in complex with TF was 6- to 7-fold higher on the apical than the basolateral surface in polarized Caco-2 cells. In contrast, no significant difference was found in the formation of TF/VIIa complexes between the apical and basolateral surface. Confocal microscopy of Caco-2 cells showed TF expression on both the apical and the basolateral surface domains. Studies with MDCK-II cells showed that the specific functional activity of TF expressed on the apical cell surface was 5-fold higher than on the basolateral surface. To test whether differential expression of TF pathway inhibitor (TFPI) on the apical and basolateral surface could account for differences in TF/VIIa functional activity, we measured cell-surface–bound TFPI activity in Caco-2 cells. Small but similar amounts of TFPI were found on both surfaces. Further, addition of inhibitory anti-TFPI antibodies induced a similar enhancement of TF/VIIa activity on both surface domains. Because the availability of anionic phospholipids on the outer leaflet of the cell membrane could regulate TF/VIIa functional activity, we measured the distribution of anionic phospholipids on the apical and basolateral surface by annexin V binding and thrombin generation. The results showed that the anionic phospholipid content on the basolateral surface, compared with the apical surface, was 3- to 4-fold lower. Mild acid treatment of polarized Caco-2 cells, which markedly increased the anionic phospholipid content on the basolateral surface membrane, increased the TF/VIIa activity on the basolateral surface without affecting the number of TF/VIIa complexes formed on the surface. Overall, our data suggest that an uneven expression of TF/VIIa activity between the apical and basolateral surface of polarized epithelial cells is caused by differences in anionic phospholipid content between the two surface domains and not from a polar distribution of TFPI.
Dichotomous Regulation of Myosin Phosphorylation and Shape Change by Rho-Kinase and Calcium in Intact Human Platelets
Authors: Bauer Markus , Retzer Michaela , Wilde Jonathan I. , Maschberger Petra , Essler Markus , Aepfelbacher Martin , Watson Steve P. , Siess Wolfgang 
Published: October 2019

AbstractBoth Rho-kinase and the Ca2+/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca2+. Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca2+ and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein–coupled platelet receptors, independent of an increase in cytosolic Ca2+. Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca2+/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).
Induction of Decay-Accelerating Factor by Cytokines or the Membrane-Attack Complex Protects Vascular Endothelial Cells Against Complement Deposition
Authors: Mason Justin C. , Yarwood Helen , Sugars Katharine , Morgan B. Paul , Davies Kevin A. , Haskard Dorian O. 
Published: October 2019

AbstractVascular endothelium is continuously exposed to complement-mediated challenge, and this is enhanced during inflammation. Although the complement-regulatory proteins decay-accelerating factor (DAF), CD59, and membrane cofactor protein (MCP) protect endothelial cells (ECs) against complement-mediated injury, the control of their expression and relative contributions to vascular protection is unclear. We explored the hypothesis that mechanisms exist which induce upregulation of complement-regulatory proteins on ECs to maintain vascular function in inflammation. Tumor necrosis factor alpha (TNF) and interferon gamma (IFNγ) each increased DAF expression but not CD59 or MCP expression, and a combination of these cytokines was more potent than either alone. Cytokine-induced expression depended on increased DAF mRNA and de novo protein synthesis and was maximal by 72 hours. In addition, assembly of the membrane-attack complex (MAC) on ECs induced a 3-fold increase in DAF expression, and this was enhanced by cytokines. DAF upregulation was not inhibited by protein kinase C (PKC) antagonists. The increase in DAF was functionally relevant since it reduced complement 3 (C3) deposition by 40%, and this was inhibited by an anti-DAF monoclonal antibody. These observations indicate that upregulation of DAF expression by cytokines or MAC may represent an important feedback mechanism to maintain the integrity of the microvasculature during subacute and chronic inflammatory processes involving complement activation.
Interleukin-9–Induced Expression of M-Ras/R-Ras3 Oncogene in T-Helper Clones
Authors: Louahed Jamila , Grasso Luigi , De Smet Charles , Van Roost Emiel , Wildmann Claude , Nicolaides Nicholas C. , Levitt Roy C. , Renauld Jean-Christophe 
Published: October 2019

AbstractIn an attempt to gain insight into the molecular mechanisms involved in interleukin-9 (IL-9) activities, representational difference analysis (RDA) was used to identify messages that are induced by IL-9 in a murine T-helper–cell clone. One of the isolated genes encodes for the newly described M-Ras or R-Ras3, which is part of the Ras gene superfamily. M-Ras expression was found to be induced by IL-9 but not IL-2 or IL-4 in various murine T-helper–cell clones, and this induction seems to be dependent on the JAK/STAT pathway. Contrasting with the potent upregulation of M-Ras expression, M-Ras was not activated by IL-9 at the level of guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding. However, IL-3 increased GTP binding to M-Ras, suggesting that M-Ras induction might represent a new mechanism of cooperativity between cytokines such as IL-3 and IL-9. Constitutively activated M-Ras mutants induced activation of Elk transcription factor by triggering the MAP kinase pathway and allowed for IL-3–independent proliferation of BaF3 cells. Taken together, these results show that cytokines such as IL-9 can regulate the expression of a member of the RAS family possibly involved in growth-factor signal transduction.
IκB Kinase Complex Is an Intracellular Target for Endotoxic Lipopolysaccharide in Human Monocytic Cells
Authors: Hawiger Jacek , Veach Ruth Ann , Liu Xue-Yan , Timmons Sheila , Ballard Dean W. 
Published: October 2019

AbstractEndotoxic lipopolysaccharide (LPS) is a proinflammatory agonist produced by gram-negative bacteria and a contributor to the majority of the 400,000 septic shock cases recorded annually in US hospitals. The primary target cells for LPS are monocytes and macrophages. Their response consists of massive production of proinflammatory cytokines, reactive oxygen- and nitrogen-intermediates, procoagulants, and cell adhesion molecules. In turn, expression of these LPS-responsive factors contributes to collapse of the circulatory system, to disseminated intravascular coagulation, and to a 30% mortality rate. A common intracellular mechanism responsible for the expression of septic shock genes in monocytes and macrophages involves the activation of NF-κB. This transcription factor is regulated by a family of structurally related inhibitors including IκB, IκBβ, and IκBɛ, which trap NF-κB in the cytoplasm. In this report, the investigators show that LPS derived from different gram-negative bacteria activates cytokine-responsive IκB kinases containing catalytic subunits termed IKK (IKK1) and IKKβ (IKK2). The kinetics of IKK and IKKβ activation in LPS-stimulated human monocytic cells differ from that recorded on their stimulation with tumor necrosis factor-, thereby implying a distinct activation mechanism. LPS-activated IKK complexes phosphorylate all 3 inhibitors of NF-κB: IκB, IκBβ, and IκBɛ. Moreover, LPS activates IKKβ preferentially, relative to IKK. Thus, IKK complex constitutes the main intracellular target for LPS-induced NF-κB signaling to the nucleus in human monocytic cells to activate genes responsible for septic shock.
Modulation of Caspase-8 and FLICE-Inhibitory Protein Expression as a Potential Mechanism of Epstein-Barr Virus Tumorigenesis in Burkitt’s Lymphoma
Authors: Tepper Clifford G. , Seldin Michael F. 
Published: October 2019

AbstractLigation of the Fas receptor induces death-inducing signaling complex (DISC) formation, caspase activation, and subsequent apoptotic death of several cell types. Epstein-Barr virus (EBV)-positive group III Burkitt’s lymphoma (BL) cell lines have a marked resistance to Fas-mediated apoptosis, although expressing each of the DISC components, Fas/ APO-1–associated death domain protein (FADD), and caspase-8 (FLICE/MACH/Mch5). The apoptotic pathway distal to the DISC is intact because ceramide analogs, staurosporine, and granzyme B activate caspase-3 and induce apoptosis. Fas resistance was not explained by the putative death-attenuating caspase-8 isoforms. However, while Fas-activated cytosolic extracts from sensitive cells were capable of processing both procaspase-8 and procaspase-3 into active subunit forms, resistant cell extracts did not possess either of these activities. Accordingly, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed higher transcript levels for the FLICE-inhibitory protein (FLIPL) in resistant cells and the ratio of caspase-8 to FLIPLmeasured by competition RT-PCR analysis directly correlated with susceptibility to Fas-mediated apoptosis of all cell lines. In addition, modification of the caspase-8/FLIPL ratio by caspase-8 or FLIPL overexpression was able to alter the susceptibility status of the cell lines tested. Our results imply that the relative levels of caspase-8 and FLIPL are an important determinant of susceptibility to Fas-mediated apoptosis.
VH Gene Sequences From Primary Central Nervous System Lymphomas Indicate Derivation From Highly Mutated Germinal Center B Cells With Ongoing Mutational Activity
Authors: Thompsett Andrew R. , Ellison David W. , Stevenson Freda K. , Zhu Delin 
Published: October 2019

AbstractPrimary central nervous system lymphoma (PCNSL) represents 1% to 3% intracranial tumors. Most PCNSL are located in the brain, and 75% are large B-cell lymphomas. The largest subgroup of these tumors contains cells that resemble centroblasts and has been labelled diffuse centroblastic (polymorphous) lymphoma. To investigate the cell of origin and the clonal history of these tumors, we have analyzed VH gene of 5 cases of PCNSL, all confirmed by histological studies to be Epstein-Barr virus (EBV)-negative, high-grade diffuse B-cell lymphomas. The V4-34 gene of the VH4 family was used in 4 of 5 cases. All VHgenes were found to have accumulated very high levels of somatic mutation (14% to 25%). In 3 of 5 cases, intraclonal nucleotide heterogeneity, including codon deletion in some clones in 1 case, was observed, indicating that the VH genes were still under the influence of the somatic hypermutation mechanism. Analysis of the distribution of silent and replacement mutations showed evidence for preservation of immunoglobulin structure in all cases. These results suggest that, although there is no evidence for germinal center formation in the brain tissue, PCNSL is derived from a B cell with features associated with location in a germinal center environment.
Neutrophil Antibody Specificity in Different Types of Childhood Autoimmune Neutropenia
Authors: Bruin Marrie C.A. , von dem Borne Albert E.G.Kr , Tamminga Rienk Y.J. , Kleijer Marion , Buddelmeijer Lica , de Haas Masja 
Published: October 2019

Abstract
Autoimmune neutropenia (AIN) in children can be divided into 2 forms. In primary AIN, neutropenia is the sole abnormality, and although neutrophil counts are generally below 500 μL−1, mild bacterial infections occur. Primary AIN is mostly seen in young children and shows a self-limited course. AIN occurring in association with autoimmune diseases (secondary AIN) often shows more severe infectious complications. We analyzed clinical and serological data from 28 pediatric patients with AIN to evaluate whether there is a possible relationship between specificity of the neutrophil autoantibodies and the clinical course of the disease. Specificity of the circulating antibodies was determined with the indirect granulocyte immunofluorescence test (GIFT) and a panel of phenotyped donor neutrophils. The samples were further analyzed in the monoclonal antibody immobilization of granulocyte antigens assay (MAIGA) for neutrophil antigen (NA)1, NA2, CD11a, and CD11b specificity. With the indirect GIFT, an antibody specificity was deduced in 26 of the 28 analyzed samples. In all but 3 sera from patients with primary AIN, NA1-(76%) or NA2-(10%) specific antibodies were detected with the indirect GIFT. In 2 samples, the reactivity in the indirect GIFT was too weak to draw conclusions, but the MAIGA showed NA1 and/or NA2 specificity of the antibodies. One serum, from a patient with primary AIN with a persistent neutropenia for more than 6 years, contained NA1, possibly pan-FcγRIIIb, and CD11a antibodies. In 4 sera from patients with primary AIN, weak antibodies with CD11a or CD11b specificity were detected with the MAIGA. Sera from 7 patients with secondary AIN contained in all cases antibodies with pan-FcγRIIIb specificity, as deduced from the indirect GIFT results and absorbance/elution experiments performed with 2 sera. The MAIGA confirmed this for only 1 of the 5 tested sera. Furthermore, CD11a antibodies were detected in 1 of the 5 tested sera. In conclusion, our results indicate that primary AIN is usually associated with NA-specific antibodies, whereas secondary AIN seems to be associated with pan-FcγRIIIb antibodies. Thus, characterization of the antibodies in sera from children with AIN discriminates patients with primary AIN from those with secondary AIN.
CCR5 Binds Multiple CC-Chemokines: MCP-3 Acts as a Natural Antagonist
Authors: Blanpain Cédric , Migeotte Isabelle , Lee Benhur , Vakili Jalal , Doranz Benjamin J. , Govaerts Cédric , Vassart Gilbert , Doms Robert W. , Parmentier Marc 
Published: October 2019

AbstractCCR5 was first characterized as a receptor for MIP-1, MIP-1β, and RANTES, and was rapidly shown to be the main coreceptor for M-tropic human immunodeficiency virus (HIV)-1 strains and simian immunodeficiency virus (SIV). Chemokines constitute a rapidly growing family of proteins and receptor-chemokine interactions are known to be promiscuous and redundant. We have therefore tested whether other CC-chemokines could bind to and activate CCR5. All CC-chemokines currently available were tested for their ability to compete with [125I]-MIP-1β binding on a stable cell line expressing recombinant CCR5, and/or to induce a functional response in these cells. We found that in addition to MIP-1β, MIP-1, and RANTES, five other CC-chemokines could compete for [125I]-MIP-1β binding: MCP-2, MCP-3, MCP-4, MCP-1, and eotaxin binding was characterized by IC50 values of 0.22, 2.14, 5.89, 29.9, and 21.7 nmol/L, respectively. Among these ligands, MCP-3 had the remarkable property of binding CCR5 with high affinity without eliciting a functional response, MCP-3 could also inhibit the activation of CCR5 by MIP-1β and may therefore be considered as a natural antagonist for CCR5. It was unable to induce significant endocytosis of the receptor. Chemokines that could compete with high affinity for MIP-1β binding could also compete for monomeric gp120 binding, although with variable potencies; maximal gp120 binding inhibition was 80% for MCP-2, but only 30% for MIP-1β. MCP-3 could compete efficiently for gp120 binding but was, however, found to be a weak inhibitor of HIV infection, probably as a consequence of its inability to downregulate the receptor.
Identification of a 14-3-3 Binding Sequence in the Common β Chain of the Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-3 (IL-3), and IL-5 Receptors That Is Serine-Phosphorylated by GM-CSF
Authors: Stomski F.C. , Dottore M. , Winnall W. , Guthridge M.A. , Woodcock J. , Bagley C.J. , Thomas D.T. , Andrews R.K. , Berndt M.C. , Lopez A.F. 
Published: October 2019

AbstractThe common β chain (βc) of the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 receptors is the major signaling subunit of these receptors coupling ligand binding to multiple biological activities. It is thought that these multiple functions arise as a consequence of the recruitment of specific signaling molecules to tyrosine-phosphorylated residues in the cytoplasmic domain of βc. However, the contribution of serine phosphorylation in βc to the recruitment of signaling molecules is not known. We show here the identification of a phosphoserine motif in the cytoplasmic domain of βc that interacts with the adaptor protein 14-3-3ζ. Coimmunoprecipitation and pull-down experiments with a glutathione S-transferase (GST):14-3-3ζ fusion protein showed that 14-3-3 directly associates with βc but not the GM-CSF receptor  chain. C-terminal truncation mutants of βcfurther showed that a region between amino acids 544 and 626 in βc was required for its association with 14-3-3ζ. This region contains the sequence 582HSRSLP587, which closely resembles the RSXSXP (where S is phosphorylated) consensus 14-3-3 binding site identified in a number of signaling molecules, including Raf-1. Significantly, substitution of582HSRSLP587 for EFAAAA completely abolished interaction of βc with GST–14-3-3ζ. Furthermore, the interaction of βc with GST–14-3-3 was greatly reduced in the presence of a peptide containing the 14-3-3 binding site, but only when 585Ser was phosphorylated. Direct binding experiments showed that the peptide containing phosphorylated 585Ser bound 14-3-3ζ with an affinity of 150 nmol/L. To study the regulation of 585S phosphorylation in vivo, we raised antibodies that specifically recognized 585Ser-phosphorylated βc. Using these antibodies, we showed that GM-CSF stimulation strongly upregulated 585Ser phosphorylation in M1 myeloid leukemic cells. The proximity of the SHC-binding site (577Tyr) to the 14-3-3–binding site (582HSRSLP587) and their conservation between mouse, rat, and human βc but not in other cytokine receptors suggest that they form a distinct motif that may subserve specialized functions associated with the GM-CSF, IL-3, and IL-5 receptors.
Simultaneous Antagonism of Interleukin-5, Granulocyte-Macrophage Colony-Stimulating Factor, and Interleukin-3 Stimulation of Human Eosinophils by Targetting the Common Cytokine Binding Site of Their Receptors
Authors: Sun Q. , Jones K. , McClure B. , Cambareri B. , Zacharakis B. , Iversen P.O. , Stomski F. , Woodcock J.M. , Bagley C.J. , D’Andrea R. , Lopez A.F. 
Published: October 2019

AbstractHuman interleukin-5 (IL-5), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-3 are eosinophilopoietic cytokines implicated in allergy in general and in the inflammation of the airways specifically as seen in asthma. All 3 cytokines function through cell surface receptors that comprise a ligand-specific  chain and a shared subunit (βc). Although binding of IL-5, GM-CSF, and IL-3 to their respective receptor  chains is the first step in receptor activation, it is the recruitment of βc that allows high-affinity binding and signal transduction to proceed. Thus, βc is a valid yet untested target for antiasthma drugs with the added advantage of potentially allowing antagonism of all 3 eosinophil-acting cytokines with a single compound. We show here the first development of such an agent in the form of a monoclonal antibody (MoAb), BION-1, raised against the isolated membrane proximal domain of βc. BION-1 blocked eosinophil production, survival, and activation stimulated by IL-5 as well as by GM-CSF and IL-3. Studies of the mechanism of this antagonism showed that BION-1 prevented the high-affinity binding of125I–IL-5, 125I–GM-CSF, and125I–IL-3 to purified human eosinophils and that it bound to the major cytokine binding site of βc. Interestingly, epitope analysis using several βc mutants showed that BION-1 interacted with residues different from those used by IL-5, GM-CSF, and IL-3. Furthermore, coimmunoprecipitation experiments showed that BION-1 prevented ligand-induced receptor dimerization and phosphorylation of βc, suggesting that ligand contact with βc is a prerequisite for recruitment of βc, receptor dimerization, and consequent activation. These results demonstrate the feasibility of simultaneously inhibiting IL-5, GM-CSF, and IL-3 function with a single agent and that BION-1 represents a new tool and lead compound with which to identify and generate further agents for the treatment of eosinophil-dependent diseases such as asthma.