bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increasing the Affinity for Tumor Antigen Enhances Bispecific Antibody Cytotoxicity
Authors: McCall Adrian M. , Shahied Lillian , Amoroso Anne R. , Horak Eva M. , Simmons Heidi H. , Nielson Ulrick , Adams Gregory P. , Schier Robert , Marks James D. , Weiner Louis M. 
Published: April 2014

Abstract
We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10−7 to 10−11 M, were fused to the phage display-derived NM3E2 human scFv. NM3E2 associates with the extracellular domain of human FcγRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of 51Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu KD = 1.7 × 10−7 M, 14.5% with KD = 5.7 × 10−9 M, and 21.3% with KD = 1.7 × 10−10 M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG Bsab. Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.
Lupus Antibody Bivalency Is Required to Enhance Prothrombin Binding to Phospholipid
Authors: Field Susan L. , Chesterman Colin N. , Dai Yan-Ping , Hogg Philip J. 
Published: April 2014

Abstract
Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 μM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.
Fundamental Ca2+ Signaling Mechanisms in Mouse Dendritic Cells: CRAC Is the Major Ca2+ Entry Pathway
Authors: Hsu Shyue-fang , O’Connell Peta J. , Klyachko Vitaly A. , Badminton Michael N. , Thomson Angus W. , Jackson Meyer B. , Clapham David E. , Ahern Gerard P. 
Published: April 2014

AbstractAlthough Ca2+-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca2+ entry pathways are unknown. Therefore, we investigated Ca2+-signaling in mouse myeloid DC using Ca2+ imaging and electrophysiological techniques. Neither Ca2+ currents nor changes in intracellular Ca2+ were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca2+ channels. ATP, a purinergic receptor ligand, and 1–4 dihydropyridines, previously suggested to activate a plasma membrane Ca2+ channel in human myeloid DC, both elicited Ca2+ rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca2+ entry. In contrast, Ca2+ influx was activated by depletion of intracellular Ca2+ stores with thapsigargin, or inositol trisphosphate. This Ca2+ influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca2+ release-activated Ca2+ channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca2+ entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca2+ concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca2+ entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.
The Exon A (C77G) Mutation Is a Common Cause of Abnormal CD45 Splicing in Humans
Authors: Tchilian Elma Z. , Wallace Diana L. , Imami Nesrina , Liao Hua-Xin , Burton Catherine , Gotch Frances , Martinson Jeremy , Haynes Barton F. , Beverley Peter C. L. 
Published: April 2014

AbstractThe leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted.
A Prominent Role for Activator Protein-1 in the Transcription of the Human 2B4 (CD244) Gene in NK Cells
Authors: Chuang Samuel S. , Pham Hoang-Tuan K. , Kumaresan Pappanaicken R. , Mathew Porunelloor A. 
Published: April 2014

AbstractThe cell surface glycoprotein 2B4 (CD244) of the Ig superfamily is involved in the regulation of NK and T lymphocyte functions. We have recently identified CD48 as the high affinity counterreceptor for 2B4 in both mice and humans. The cytoplasmic domain of 2B4 associates with src homology 2 domain-containing protein or signaling lymphocyte activation molecule-associated protein, whose mutation is the underlying genetic defect in the X-linked lymphoproliferative syndrome. In this study, we report the molecular cloning and characterization of the human 2B4 (h2B4) promoter. Through primer extension analysis, we found that the transcription of the h2B4 gene initiates at multiple start sites. We isolated h2B4 genomic clones and PCR amplified the 5′ untranslated region containing the promoter elements. We have identified a functional AP-1 site that lies between (−106 to −100) through transient transfection analysis in YT cells, a human NK cell line. EMSAs with Abs specific for various protein factors of the AP-1 family revealed that multiple members of the Jun family are involved in the regulation of the h2B4 gene. Mutation of the AP-1 site not only abolishes protein/DNA interactions but also promoter activity. These results demonstrate a significant role for AP-1 in the transcriptional regulation of the h2B4 gene.
Culture at High Density Improves the Ability of Human Macrophages to Control Mycobacterial Growth
Authors: Boechat Neio , Bouchonnet Francine , Bonay Marcel , Grodet Alain , Pelicic Vladimir , Gicquel Brigitte , Hance Allan J. 
Published: April 2014

AbstractThe mechanisms through which granuloma formation helps control mycobacterial infection are poorly understood, but it is possible that the accumulation of macrophages at high density at sites of infection promotes the differentiation of macrophages into cells with improved mycobactericidal activity. To test this possibility, varying numbers of monocytes were cultured in 96-well plates for 3 days, infected with Mycobacterium bovis bacillus Calmette-Guérin, and mycobacterial number was assessed 7 days after infection based on the measurement of luciferase activity expressed by a mycobacterial reporter strain or by counting CFU. Mycobacterial growth was optimal in cultures containing 5 × 104 cells/well, but increasing the number of cells to 2 × 105 cells/well resulted in complete inhibition of mycobacterial growth. This effect could not be explained by differences in mycobacterial uptake, multiplicity of infection, acidification of the extracellular medium in high density cultures, enhanced NO production, or paracrine stimulation resulting from secretion of cytokines or other proteins. The morphology of cells cultured at high density was strikingly different from that of monocytes cultured at 5 × 104 cells/well, including the appearance of numerous giant cells. The bacteriostatic activity of monocyte-derived macrophages was also dependent on cell number, but fewer of these more mature cells were required to control mycobacterial growth. Thus, the ability of human macrophages to control mycobacterial infection in vitro is influenced by the density of cells present, findings that may help explain why the formation of granulomas in vivo appears to be a key event in the control of mycobacterial infections.
Vaccination with the T Cell Antigen Mtb 8.4 Protects Against Challenge withMycobacterium tuberculosis
Authors: Coler Rhea N. , Campos-Neto Antonio , Ovendale Pamela , Day Fiona H. , Fling Steven P. , Zhu Liqing , Serbina Natalya , Flynn JoAnne L. , Reed Steven G. , Alderson Mark R. 
Published: April 2014

AbstractThe development of an effective vaccine against Mycobacterium tuberculosis is a research area of intense interest. Mounting evidence suggests that protective immunity to M. tuberculosis relies on both MHC class II-restricted CD4+ T cells and MHC class I-restricted CD8+ T cells. By purifying polypeptides present in the culture filtrate of M. tuberculosis and evaluating these molecules for their ability to stimulate PBMC from purified protein derivative-positive healthy individuals, we previously identified a low-m.w. immunoreactive T cell Ag, Mtb 8.4, which elicited strong Th1 T cell responses in healthy purified protein derivative-positive human PBMC and in mice immunized with recombinant Mtb 8.4. Herein we report that Mtb 8.4-specific T cells can be detected in mice immunized with the current live attenuated vaccine, Mycobacterium bovis-bacillus Calmette-Guérin as well as in mice infected i.v. with M. tuberculosis. More importantly, immunization of mice with either plasmid DNA encoding Mtb 8.4 or Mtb 8.4 recombinant protein formulated with IFA elicited strong CD4+ T cell and CD8+ CTL responses and induced protection on challenge with virulent M. tuberculosis. Thus, these results suggest that Mtb 8.4 is a potential candidate for inclusion in a subunit vaccine against TB.
ATP-Mediated Killing ofMycobacterium bovisBacille Calmette-Guérin Within Human Macrophages Is Calcium Dependent and Associated with the Acidification of Mycobacteria-Containing Phagosomes
Authors: Stober Carmel B. , Lammas David A. , Li Cheuk M. , Kumararatne Dinikantha S. , Lightman Stafford L. , McArdle Craig A. 
Published: April 2014

AbstractWe previously demonstrated that extracellular ATP stimulated macrophage death and mycobacterial killing within Mycobacterium bovis Bacille Calmette-Guérin (BCG)-infected human macrophages. ATP increases the cytosolic Ca2+ concentration in macrophages by mobilizing intracellular Ca2+ via G protein-coupled P2Y receptors, or promoting the influx of extracellular Ca2+ via P2X purinoceptors. The relative contribution of these receptors and Ca2+ sources to ATP-stimulated macrophage death and mycobacterial killing was investigated. We demonstrate that 1) ATP mobilizes Ca2+ in UTP-desensitized macrophages (in Ca2+-free medium) and 2) UTP but not ATP fails to deplete the intracellular Ca2+ store, suggesting that the pharmacological properties of ATP and UTP differ, and that a Ca2+-mobilizing P2Y purinoceptor in addition to the P2Y2 subtype is expressed on human macrophages. ATP and the Ca2+ ionophore, ionomycin, promoted macrophage death and BCG killing, but ionomycin-mediated macrophage death was inhibited whereas BCG killing was largely retained in Ca2+-free medium. Pretreatment of cells with thapsigargin (which depletes inositol (1,4,5)-trisphosphate-mobilizable intracellular stores) or 1,2-bis-(2-aminophenoxy)ethane-N, N, N′,N′-tetraacetic acid acetoxymethyl ester (an intracellular Ca2+ chelator) failed to inhibit ATP-stimulated macrophage death but blocked mycobacterial killing. Using the acidotropic molecular probe, 3-(2,4-dinitroanilino)-3′-amino-N-methyl dipropylamine, it was revealed that ATP stimulation promoted the acidification of BCG-containing phagosomes within human macrophages, and this effect was similarly dependent upon Ca2+ mobilization from intracellular stores. We conclude that the cytotoxic and bactericidal effects of ATP can be uncoupled and that BCG killing is not the inevitable consequence of death of the host macrophage.
Cell Density Regulates Neutrophil IL-8 Synthesis: Role of IL-1 Receptor Antagonist and Soluble TNF Receptors
Authors: Hattar Katja , Fink Ludger , Fietzner Karin , Himmel Barbara , Grimminger Friedrich , Seeger Werner , Sibelius Ulf 
Published: April 2014

Abstract
Although cytokine synthesis in polymorphonuclear leukocytes (PMN) was shown to be modulated by soluble mediators, the impact of microenvironmental conditions has not been elucidated. In this study, we investigated the effect of cell density on cytokine release from human neutrophils. PMN were cultured at various cell densities (10 × 106 PMN/ml; 60 × 106 PMN/ml), and LPS-induced release of cytokines was quantified by ELISA technique. Upon an increase in PMN density, secretion of the CXC chemokine IL-8 was progressively reduced. This effect was paralleled by a decrease in IL-8 mRNA. In contrast, TNF-α and IL-1β rose proportionally with increasing cell density. The inhibition of IL-8 secretion was reproduced by conditioned media of PMN at high cell density, but was not affected by blocking β2 integrin-dependent adhesion. When analyzing the supernatant of LPS-challenged neutrophils, large amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R antagonist (IL-1RA), rising constantly with the cell density, were detected. Interestingly, combined blocking of the bioactivities of these mediators completely restored neutrophil IL-8 secretion at high cell densities, with the anti-IL-1RA Ab being the more potent agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to 10 × 106 PMN/ml reproduced the suppression of IL-8 generation. We conclude that neutrophil IL-8 synthesis is autoregulated, being suppressed under conditions of high cell density. IL-1RA and sTNFRs, accumulating under these circumstances, seem to be centrally involved in this regulatory mechanism by interfering with the IL-1β- and TNF-α-dependent IL-8 generation. This feedback mechanism may control further neutrophil recruitment and activation in a neutrophil-rich environment, thereby preventing tissue destruction.
Reactive Nitrogen Species Inhibit Alveolar Epithelial Fluid Transport After Hemorrhagic Shock in Rats
Authors: Pittet Jean-François , Lu Le N. , Morris David G. , Modelska Kathrin , Welch William J. , Carey Hannah V. , Roux Jeremie , Matthay Michael A. 
Published: April 2014

AbstractOur recent experimental work demonstrated that a neutrophil-dependent inflammatory response in the lung prevented the normal up-regulation of alveolar fluid clearance by catecholamines following hemorrhagic shock. In this study, we tested the hypothesis that the release of NO within the airspaces of the lung was responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Hemorrhagic shock was associated with an inducible NO synthase (iNOS)-dependent increase in the lung production of NO and a failure of the alveolar epithelium to up-regulate vectorial fluid transport in response to β-adrenergic agonists. Inhibition of iNOS restored the normal catecholamine-mediated up-regulation of alveolar liquid clearance. Airspace instillation of dibutyryl cAMP, a stable analog of cAMP, restored the normal fluid transport capacity of the alveolar epithelium after prolonged hemorrhagic shock, whereas direct stimulation of adenyl cyclase by forskolin had no effect. Pretreatment with pyrrolidine dithiocarbamate or sulfasalazine attenuated the iNOS-dependent production of NO in the lung and restored the normal up-regulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Based on in vitro studies with an alveolar epithelial cell line, A549 cells, the effect of sulfasalazine appeared to be mediated in part by inhibition of NF-κB activation, and the protective effect was mediated by the inhibition of IκBα protein degradation. In summary, these results provide the first in vivo evidence that NO, released within the airspaces of the lung probably secondary to the NF-κB-dependent activation of iNOS, is a major proximal inflammatory mediator that limits the rate of alveolar epithelial transport after prolonged hemorrhagic shock by directly impairing the function of membrane proteins involved in the β-adrenergic receptor-cAMP signaling pathway in alveolar epithelium.
Role of p190RhoGAP in β2 Integrin Regulation of RhoA in Human Neutrophils
Authors: Dib Karim , Melander Fredrik , Andersson Tommy 
Published: April 2014

Abstract
We found that engagement of β2 integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of β2 integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with β2 integrin-induced activation of p190RhoGAP. The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of β2 integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RhoGAP. Instead, the β2 integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the β2 integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from β2 integrin-stimulated cells. Thus, in neutrophils, β2 integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RhoA.
Antioxidants Inhibit Indoleamine 2,3-Dioxygenase in IFN-γ-Activated Human Macrophages: Posttranslational Regulation by Pyrrolidine Dithiocarbamate
Authors: Thomas Shane R. , Salahifar Houta , Mashima Ryuichi , Hunt Nicholas H. , Richardson Des R. , Stocker Roland 
Published: April 2014

Abstract
Induction of the heme-containing indoleamine 2,3-dioxygenase (IDO) by IFN-γ is implicated in anti-microbial and pro-inflammatory activities of human macrophages. Antioxidants can modulate the expression of immune and inflammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-κB. Here we show that IFN-γ treatment of human monocyte-derived macrophages (hMDMs) increased the proportion of oxidized glutathione. PDTC attenuated this increase and inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other antioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein expression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cellular IDO activity without affecting protein expression, whereas addition of hemin or the heme precursor δ-aminolevulinic acid increased IDO activity. Also, incubation of IFN-γ-activated hMDM with δ-[14C]-aminolevulinic acid resulted in the incorporation of label into immunoprecipitated IDO, a process inhibited by PDTC and SA. Furthermore, supplementation of lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activity to control levels, and hemin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requirement for de novo heme synthesis for IDO activity in IFN-γ-activated hMDM. They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes place posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.
ATP Mediates Calcium Signaling Between Astrocytes and Microglial Cells: Modulation by IFN-γ
Authors: Verderio Claudia , Matteoli Michela 
Published: April 2014

Abstract
Calcium-mediated intercellular communication is a mechanism by which astrocytes communicate with each other and modulate the activity of adjacent cells, including neurons and oligodendrocytes. We have investigated whether microglia, the immune effector cells involved in several diseases of the CNS, are actively involved in this communication network. To address this issue, we analyzed calcium dynamics in fura-2-loaded cocultures of astrocytes and microglia under physiological conditions and in the presence of the inflammatory cytokine IFN-γ. The intracellular calcium increases in astrocytes, occurring spontaneously or as a result of mechanical or bradykinin stimulation, induced the release of ATP, which, in turn, was responsible for triggering a delayed calcium response in microglial cells. Repeated stimulations of microglial cells by astrocyte-released ATP activated P2X7 purinergic receptor on microglial cells and greatly increased membrane permeability, eventually leading to microglial apoptosis. IFN-γ increased ATP release and potentiated the P2X7-mediated cytolytic effect. This is the first study showing that ATP mediates a form of calcium signaling between astrocytes and microglia. This mechanism of intercellular communication may be involved in controlling the number and function of microglial cells under pathophysiologic CNS conditions.