Spontaneous food allergy in Was-/- mice occurs independent of FcεRI-mediated mast cell activation.

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Lexmond, W S; Goettel, J A; Sallis, B F; McCann, K; Rings, E H H M; Jensen-Jarolim, E; Nurko, S; Snapper, S B; Fiebiger, E

2017-12-01

Food allergies are a growing health problem, and the development of therapies that prevent disease onset is limited by the lack of adjuvant-free experimental animal models. We compared allergic sensitization in patients with food allergy or Wiskott-Aldrich syndrome (WAS) and defined whether spontaneous disease in Was -/- mice recapitulates the pathology of a conventional disease model and/or human food allergy. Comparative ImmunoCAP ISAC microarray was performed in patients with food allergy or WAS. Spontaneous food allergy in Was -/- mice was compared to an adjuvant-based model in wild-type mice (WT-OVA/alum). Intestinal and systemic anaphylaxis was assessed, and the role of the high-affinity IgE Fc receptor (FcεRI) in allergic sensitization was evaluated using Was -/- Fcer1a -/- mice. Polysensitization to food was detected in both WAS and food-allergic patients which was recapitulated in the Was -/- model. Oral administration of ovalbumin (OVA) in Was -/- mice induced low titers of OVA-specific IgE compared to the WT-OVA/alum model. Irrespectively, 79% of Was -/- mice developed allergic diarrhea following oral OVA challenge. Systemic anaphylaxis occurred in Was -/- mice (95%) with a mortality rate >50%. Spontaneous sensitization and intestinal allergy occurred independent of FcεRI expression on mast cells (MCs) and basophils. Was -/- mice provide a model of food allergy with the advantage of mimicking polysensitization and low food-antigen IgE titers as observed in humans with clinical food allergy. This model will facilitate studies on aberrant immune responses during spontaneous disease development. Our results imply that therapeutic targeting of the IgE/FcεRI activation cascade will not affect sensitization to food. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Sirt1 negatively regulates FcεRI-mediated mast cell activation through AMPK- and PTP1B-dependent processes.

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Li, Xian; Lee, Youn Ju; Jin, Fansi; Park, Young Na; Deng, Yifeng; Kang, Youra; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Chang, Young-Chae; Ko, Hyun-Jeong; Kim, Cheorl-Ho; Murakami, Makoto; Chang, Hyeun Wook

2017-07-25

Sirt1, a key regulator of metabolism and longevity, has recently been implicated in the regulation of allergic reactions, although the underlying mechanism remains unclear. Here we show that Sirt1 negatively regulates FcεRI-stimulated mast cell activation and anaphylaxis through two mutually regulated pathways involving AMP-activated protein kinase (AMPK) and protein tyrosine phosphatase 1B (PTP1B). Mast cell-specific knockout of Sirt1 dampened AMPK-dependent suppression of FcεRI signaling, thereby augmenting mast cell activation both in vitro and in vivo. Sirt1 inhibition of FcεRI signaling also involved an alternative component, PTP1B, which attenuated the inhibitory AMPK pathway and conversely enhanced the stimulatory Syk pathway, uncovering a novel role of this phosphatase. Moreover, a Sirt1 activator resveratrol stimulated the inhibitory AMPK axis, with reciprocal suppression of the stimulatory PTP1B/Syk axis, thus potently inhibiting anaphylaxis. Overall, our results provide a molecular explanation for the beneficial role of Sirt1 in allergy and underscore a potential application of Sirt1 activators as a new class of anti-allergic agents.

Clinical efficacy of omalizumab in chronic spontaneous urticaria is associated with a reduction of FcεRI-positive cells in the skin.

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Metz, Martin; Staubach, Petra; Bauer, Andrea; Brehler, Randolf; Gericke, Janine; Kangas, Michael; Ashton-Chess, Joanna; Jarvis, Philip; Georgiou, Panayiotis; Canvin, Janice; Hillenbrand, Rainer; Erpenbeck, Veit J; Maurer, Marcus

2017-01-01

Background. Treatment with omalizumab, a humanized recombinant monoclonal anti-IgE antibody, results in clinical efficacy in patients with Chronic Spontaneous Urticaria (CSU). The mechanism of action of omalizumab in CSU has not been elucidated in detail. Objectives. To determine the effects of omalizumab on levels of high affinity IgE receptor-positive (FcεRI + ) and IgE-positive (IgE + ) dermal cells and blood basophils. Treatment efficacy and safety were also assessed. Study design. In a double-blind study, CSU patients aged 18‑75 years were randomized to receive 300 mg omalizumab (n=20) or placebo (n=10) subcutaneously every 4 weeks for 12 weeks. Changes in disease activity were assessed by use of the weekly Urticaria Activity Score (UAS7). Circulating IgE levels, basophil numbers and levels of expression of FcεRI + and IgE + cells in the skin and in blood basophils were determined. Results. Patients receiving omalizumab showed a significantly greater decrease in UAS7 compared with patients receiving placebo. At Week 12 the mean difference in UAS7 between treatment groups was -14.82 (p=0.0027), consistent with previous studies. Total IgE levels in serum were increased after omalizumab treatment and remained elevated up to Week 12. Free IgE levels decreased after omalizumab treatment. Mean levels of FcεRI + skin cells in patients treated with omalizumab 300 mg were decreased at Week 12 compared with baseline in the dermis of both non-lesional and lesional skin, reaching levels comparable with those seen in healthy volunteers (HVs). There were no statistically significant changes in mean FcɛRI + cell levels in the placebo group. Similar results were seen for changes in IgE + cells, although the changes were not statistically significant. The level of peripheral blood basophils increased immediately after treatment start and returned to Baseline values after the follow-up period. The levels of FcεRI and IgE expression on peripheral blood basophils were

Importance of neonatal FcR in regulating the serum half-life of therapeutic proteins containing the Fc domain of human IgG1: a comparative study of the affinity of monoclonal antibodies and Fc-fusion proteins to human neonatal FcR.

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Suzuki, Takuo; Ishii-Watabe, Akiko; Tada, Minoru; Kobayashi, Tetsu; Kanayasu-Toyoda, Toshie; Kawanishi, Toru; Yamaguchi, Teruhide

2010-02-15

The neonatal FcR (FcRn) binds to the Fc domain of IgG at acidic pH in the endosome and protects IgG from degradation, thereby contributing to the long serum half-life of IgG. To date, more than 20 mAb products and 5 Fc-fusion protein products have received marketing authorization approval in the United States, the European Union, or Japan. Many of these therapeutic proteins have the Fc domain of human IgG1; however, the serum half-lives differ in each protein. To elucidate the role of FcRn in the pharmacokinetics of Fc domain-containing therapeutic proteins, we evaluated the affinity of the clinically used human, humanized, chimeric, or mouse mAbs and Fc-fusion proteins to recombinant human FcRn by surface plasmon resonance analysis. The affinities of these therapeutic proteins to FcRn were found to be closely correlated with the serum half-lives reported from clinical studies, suggesting the important role of FcRn in regulating their serum half-lives. The relatively short serum half-life of Fc-fusion proteins was thought to arise from the low affinity to FcRn. The existence of some mAbs having high affinity to FcRn and a short serum half-life, however, suggested the involvement of other critical factor(s) in determining the serum half-life of such Abs. We further investigated the reason for the relatively low affinity of Fc-fusion proteins to FcRn and suggested the possibility that the receptor domain of Fc-fusion protein influences the structural environment of the FcRn binding region but not of the FcgammaRI binding region of the Fc domain.

Subthreshold Desensitization of Human Basophils Re-capitulates the Loss of syk and FcεRI expression Characterized by Other Methods of Desensitization

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MacGlashan, Donald

2012-01-01

Background Clinical desensitization of patients to drugs involves progressive exposure to escalating doses of drug over a period of 24 hours. In prior studies, this method was recapitulated in vitro to also demonstrate loss of mast cell or basophil responsiveness. However, most signaling studies of human basophils have identified changes in signaling by using other methods of inducing cellular desensitization. Objective This study examined two well-described endpoints of basophil desensitization, loss of syk or FcεRI expression, under conditions of subthreshold desensitization. Methods The loss of FceRI and syk was examined in human basophils. Results It was shown that both loss of syk and FcεRI/IgE occurred during an escalating series of stimulation (anti-IgE Ab) and that expression loss occurred despite the presence of little histamine release. If basophils were first cultured for 3 days in 10 ng/ml IL-3, the concentration-dependence of histamine release shifted to 100 fold lower concentrations of stimulus. However, loss of syk did not show any change in its EC50 while loss of FcεRI also shifted 100 fold. From the perspective of early signal element activation, the marked shift in the EC50 for histamine release was not accompanied by similar shifts in the EC50s for several signaling elements. The EC50s for phospho-Src, phospho-SHIP1, phospho-Syk, or phospho-Cbl did not change while the EC50s for phospho-Erk and the cytosolic calcium response did shift 100 fold. Conclusions These studies show that under normal conditions, subthreshold desensitization leads to loss of two critical signaling molecules (FcεRI and syk) but under at least one condition, treatment with IL-3, it is possible to markedly blunt the loss of syk, but not FcεRI, while executing a proper subthreshold titration. These data also suggest that IL-3 modifies only the sensitivity of signaling elements that are downstream of syk activation. PMID:22702505

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

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Abdiche, Yasmina Noubia; Yeung, Yik Andy; Chaparro-Riggers, Javier; Barman, Ishita; Strop, Pavel; Chin, Sherman Michael; Pham, Amber; Bolton, Gary; McDonough, Dan; Lindquist, Kevin; Pons, Jaume; Rajpal, Arvind

2015-01-01

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Davallia mariesii Moore Improves FcεRI-Mediated Allergic Responses in the Rat Basophilic Leukemia Mast Cell Line RBL-2H3 and Passive Cutaneous Anaphylaxis in Mice

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Hyun Ju Do

2017-01-01

Full Text Available Davallia mariesii Moore (Drynaria rhizome extract (DRE is widely known for its efficacy in treating inflammation, arteriosclerosis, and bone injuries. This study evaluated whether treatment with DRE inhibited FcɛRI-mediated allergic responses in the RBL-2H3 mast cells and investigated the early- and late-phase mechanisms by which DRE exerts its antiallergic effects. IgE anti-DNP/DNP-HSA-sensitized RBL-2H3 mast cells were tested for cytotoxicity to DRE, followed by the assessment of β-hexosaminidase release. We measured the amounts of inflammatory mediators (e.g., histamine, PGD2, TNF-α, IL-4, and IL-6 and examined the expression of genes involved in arachidonate and FcεRI signaling pathways. In addition, we confirmed the antiallergic effects of DRE on passive cutaneous anaphylaxis (PCA in mice. DRE inhibited RBL-2H3 mast cell degranulation and production of allergic mediators in them. In early allergic responses, DRE reduced expression of FcεRI signaling-related genes (e.g., Syk, Lyn, and Fyn and extracellular signal-regulated kinase phosphorylation in mast cells. In late allergic responses, DRE reduced PGD2 release and COX-2 expression and cPLA2 phosphorylation in FcɛRI-mediated mast cells. Lastly, 250–500 mg/kg DRE significantly attenuated the IgE-induced PCA reaction in mice. These findings provide novel information on the molecular mechanisms underlying the antiallergic effects of DRE in FcɛRI-mediated allergic responses.

Rapid polyclonal desensitization with antibodies to IgE and FcεRIα.

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Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Lewkowich, Ian; Morris, Suzanne C; Finkelman, Fred D

2013-06-01

Rapid desensitization, a procedure in which persons allergic to an antigen are treated at short intervals with increasing doses of that antigen until they tolerate a large dose, is an effective, but risky, way to induce temporary tolerance. We wanted to determine whether this approach can be adapted to suppress all IgE-mediated allergies in mice by injecting serially increasing doses of monoclonal antibodies (mAbs) to IgE or FcεRIα. Active and passive models of antigen- and anti-IgE mAb-induced IgE-mediated anaphylaxis were used. Mice were desensitized with serially increasing doses of anti-IgE mAb, anti-FcεRIα mAb, or antigen. Development of shock (hypothermia), histamine and mast cell protease release, cytokine secretion, calcium flux, and changes in cell number and FcεRI and IgE expression were evaluated. Rapid desensitization with anti-IgE mAb suppressed IgE-mediated immediate hypersensitivity; however, some mice developed mild anaphylaxis during desensitization. Rapid desensitization with anti-FcεRIα mAb that only binds FcεRI that is not occupied by IgE suppressed both active and passive IgE-mediated anaphylaxis without inducing disease. It quickly, but temporarily, suppressed IgE-mediated anaphylaxis by decreasing mast cell signaling through FcεRI, then slowly induced longer lasting mast cell unresponsiveness by removing membrane FcεRI. Rapid desensitization with anti-FcεRIα mAb was safer and longer lasting than rapid desensitization with antigen. A rapid desensitization approach with anti-FcεRIα mAb safely desensitizes mice to IgE-mediated anaphylaxis by inducing mast cell anergy and later removing all mast cell IgE. Rapid desensitization with an anti-human FcεRIα mAb may be able to prevent human IgE-mediated anaphylaxis. Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Rapid desensitization of mice with anti-FcγRIIb/FcγRIII mAb safely prevents IgG-mediated anaphylaxis.

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Khodoun, Marat V; Kucuk, Zeynep Yesim; Strait, Richard T; Krishnamurthy, Durga; Janek, Kevin; Clay, Corey D; Morris, Suzanne C; Finkelman, Fred D

2013-12-01

Stimulatory IgG receptors (FcγRs) on bone marrow-derived cells contribute to the pathogenesis of several autoimmune and inflammatory disorders. Monoclonal antibodies that block FcγRs might suppress these diseases, but they can induce anaphylaxis. We wanted to determine whether a rapid desensitization approach can safely suppress IgG/FcγR-mediated anaphylaxis. Mice were injected with serially increasing doses of 2.4G2, a rat mAb that blocks the inhibitory FcγR, FcγRIIb, and the stimulatory receptor, FcγRIII. Rectal temperature was used to detect the development of anaphylaxis. Passive and active IgG-mediated anaphylaxis were evaluated in mice that had been rapidly desensitized with 2.4G2 or mock-desensitized in mice in which monocyte/macrophages, basophils, or neutrophils had been depleted or desensitized and in mice in which FcγRI, FcγRIII, and/or FcγRIV had been deleted or blocked. Rapid desensitization with 2.4G2 prevented 2.4G2-induced shock and completely suppressed IgG-mediated anaphylaxis. Rapid desensitization of ovalbumin-sensitized mice with 2.4G2 was safer and more effective than rapid desensitization with ovalbumin. 2.4G2 treatment completely blocked FcγRIII and removed most FcγRI and FcγRIV from nucleated peripheral blood cells. Because IgG(2a)-mediated anaphylaxis was partially FcγRI and FcγRIV dependent, the effects of 2.4G2 on FcγRI and FcγRIV were probably crucial for its complete inhibition of IgG(2a)-mediated anaphylaxis. IgG(2a)-mediated anaphylaxis was partially inhibited by depletion or desensitization of monocyte/macrophages, basophils, or neutrophils. IgG-mediated anaphylaxis can be induced by ligation of FcγRI, FcγRIII, or FcγRIV on monocycte/macrophages, basophils, or neutrophils and can be safely suppressed by rapid desensitization with anti-FcγRII/RIII mAb. A similar approach may safely suppress other FcγR-dependent immunopathology. Published by Mosby, Inc.

Multiple Plasmodium falciparum erythrocyte membrane protein 1 variants per genome can bind IgM via its Fc fragment Fcμ

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Jeppesen, Anine; Ditlev, Sisse Bolm; Soroka, Vladyslav

2015-01-01

with severe clinical manifestations, such as cerebral malaria in children and placental malaria in pregnant women. PfEMP1 that can bind the Fc part of IgM (Fcμ) characterizes one such type, although the functional significance of this IgM binding to PfEMP1 remains unclear. In this study, we report...... resemble the rosette-mediating and IgM-binding PfEMP1 HB3VAR06, but none of them mediated formation of rosettes. We could map the capacity for Fc-specific IgM binding to DBLε domains near the C terminus for three of the four PfEMP1 proteins tested. Our study provides new evidence regarding Fc...

Lin- CD34hi CD117int/hi FcεRI+ cells in human blood constitute a rare population of mast cell progenitors.

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Dahlin, Joakim S; Malinovschi, Andrei; Öhrvik, Helena; Sandelin, Martin; Janson, Christer; Alving, Kjell; Hallgren, Jenny

2016-01-28

Mast cells are rare tissue-resident immune cells that are involved in allergic reactions, and their numbers are increased in the lungs of asthmatics. Murine lung mast cells arise from committed bone marrow-derived progenitors that enter the blood circulation, migrate through the pulmonary endothelium, and mature in the tissue. In humans, mast cells can be cultured from multipotent CD34(+) progenitor cells. However, a population of distinct precursor cells that give rise to mast cells has remained undiscovered. To our knowledge, this is the first report of human lineage-negative (Lin(-)) CD34(hi) CD117(int/hi) FcεRI(+) progenitor cells, which represented only 0.0053% of the isolated blood cells in healthy individuals. These cells expressed integrin β7 and developed a mast cell-like phenotype, although with a slow cell division capacity in vitro. Isolated Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells had an immature mast cell-like appearance and expressed high levels of many mast cell-related genes as compared with human blood basophils in whole-transcriptome microarray analyses. Furthermore, serglycin, tryptase, and carboxypeptidase A messenger RNA transcripts were detected by quantitative reverse transcription-polymerase chain reaction. Altogether, we propose that the Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood cells are closely related to human tissue mast cells and likely constitute an immediate precursor population, which can give rise to predominantly mast cells. Furthermore, asthmatics with reduced lung function had a higher frequency of Lin(-) CD34(hi) CD117(int/hi) FcεRI(+) blood mast cell progenitors than asthmatics with normal lung function. © 2016 by The American Society of Hematology.

Down-regulation of FcεRI-mediated CD63 basophil response during short-term VIT determined venom-nonspecific desensitization.

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Nina Čelesnik Smodiš

Full Text Available BACKGROUND: We recently showed a desensitization of FcεRI-mediated basophil response after short-term VIT. Our aim was to evaluate the allergen specificity of this desensitization. METHODS: In 11 Hymenoptera-venom double positive subjects, basophil threshold sensitivity (CD-sens to anti-FcεRI, honeybee, and Vespula venom was assessed at the beginning and just before the first maintenance dose (MD of single ultra-rush VIT. In some patients we also monitored CD-sens to rApi m 1 and/or rVes v 5 or other co-sensitizations (i.e., grass pollen. In additional 7 patients, basophils were stripped and sensitized with house dust mite (HDM IgEs at the same time points. RESULTS: We demonstrated a marked reduction of CD-sens to anti-FcεRI and VIT-specific venom before the first MD in all 18 subjects included. Furthermore, in 10 out of 11 double positive subjects, a significant and comparable decrease before the first MD was also evident for non-VIT venom; this nonspecific decrease was further supported by the opposite recombinant species-specific major allergen. In one subject with additional grass pollen allergy, a decrease of CD-sens to grass allergen was also demonstrated. Similarly, in 7 cases of patients with passively HDM-sensitized basophils, a significant reduction of CD-sens was also evident to de novo sensitized HDM allergen. CONCLUSIONS: Short-term VIT induced basophil desensitization to VIT-specific as well as to VIT-nonspecific venom. As opposed to long-term VIT, which induces venom-specific changes, the effect of short-term VIT seems to be venom-nonspecific.

Effects of altered FcγR binding on antibody pharmacokinetics in cynomolgus monkeys

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Leabman, Maya K; Meng, Y Gloria; Kelley, Robert F; DeForge, Laura E; Cowan, Kyra J; Iyer, Suhasini

2013-01-01

Antibody interactions with Fcγ receptors (FcγRs), like FcγRIIIA, play a critical role in mediating antibody effector functions and thereby contribute significantly to the biologic and therapeutic activity of antibodies. Over the past decade, considerable work has been directed towards production of antibodies with altered binding affinity to FcγRs and evaluation of how the alterations modulate their therapeutic activity. This has been achieved by altering glycosylation status at N297 or by engineering modifications in the crystallizable fragment (Fc) region. While the effects of these modifications on biologic activity and efficacy have been examined, few studies have been conducted to understand their effect on antibody pharmacokinetics (PK). We present here a retrospective analysis in which we characterize the PK of three antibody variants with decreased FcγR binding affinity caused by amino acid substitutions in the Fc region (N297A, N297G, and L234A/L235A) and three antibody variants with increased FcγRIIIA binding affinity caused by afucosylation at N297, and compare their PK to corresponding wild type antibody PK in cynomolgus monkeys. For all antibodies, PK was examined at a dose that was known to be in the linear range. Since production of the N297A and N297G variants in Chinese hamster ovary cells results in aglycosylated antibodies that do not bind to FcγRs, we also examined the effect of expression of an aglycosylated antibody, without sequence change(s), in E. coli. All the variants demonstrated similar PK compared with that of the wild type antibodies, suggesting that, for the six antibodies presented here, altered FcγR binding affinity does not affect PK. PMID:24492343

Fc-Binding Ligands of Immunoglobulin G: An Overview of High Affinity Proteins and Peptides

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Weonu Choe

2016-12-01

Full Text Available The rapidly increasing application of antibodies has inspired the development of several novel methods to isolate and target antibodies using smart biomaterials that mimic the binding of Fc-receptors to antibodies. The Fc-binding domain of antibodies is the primary binding site for e.g., effector proteins and secondary antibodies, whereas antigens bind to the Fab region. Protein A, G, and L, surface proteins expressed by pathogenic bacteria, are well known to bind immunoglobulin and have been widely exploited in antibody purification strategies. Several difficulties are encountered when bacterial proteins are used in antibody research and application. One of the major obstacles hampering the use of bacterial proteins is sample contamination with trace amounts of these proteins, which can invoke an immune response in the host. Many research groups actively develop synthetic ligands that are able to selectively and strongly bind to antibodies. Among the reported ligands, peptides that bind to the Fc-domain of antibodies are attractive tools in antibody research. Besides their use as high affinity ligands in antibody purification chromatography, Fc-binding peptides are applied e.g., to localize antibodies on nanomaterials and to increase the half-life of proteins in serum. In this review, recent developments of Fc-binding peptides are presented and their binding characteristics and diverse applications are discussed.

Human FcγRIIA induces anaphylactic and allergic reactions.

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Jönsson, Friederike; Mancardi, David A; Zhao, Wei; Kita, Yoshihiro; Iannascoli, Bruno; Khun, Huot; van Rooijen, Nico; Shimizu, Takao; Schwartz, Lawrence B; Daëron, Marc; Bruhns, Pierre

2012-03-15

IgE and IgE receptors (FcεRI) are well-known inducers of allergy. We recently found in mice that active systemic anaphylaxis depends on IgG and IgG receptors (FcγRIIIA and FcγRIV) expressed by neutrophils, rather than on IgE and FcεRI expressed by mast cells and basophils. In humans, neutrophils, mast cells, basophils, and eosinophils do not express FcγRIIIA or FcγRIV, but FcγRIIA. We therefore investigated the possible role of FcγRIIA in allergy by generating novel FcγRIIA-transgenic mice, in which various models of allergic reactions induced by IgG could be studied. In mice, FcγRIIA was sufficient to trigger active and passive anaphylaxis, and airway inflammation in vivo. Blocking FcγRIIA in vivo abolished these reactions. We identified mast cells to be responsible for FcγRIIA-dependent passive cutaneous anaphylaxis, and monocytes/macrophages and neutrophils to be responsible for FcγRIIA-dependent passive systemic anaphylaxis. Supporting these findings, human mast cells, monocytes and neutrophils produced anaphylactogenic mediators after FcγRIIA engagement. IgG and FcγRIIA may therefore contribute to allergic and anaphylactic reactions in humans.

Multivalent Fcγ-receptor engagement by a hexameric Fc-fusion protein triggers Fcγ-receptor internalisation and modulation of Fcγ-receptor functions.

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Qureshi, O S; Rowley, T F; Junker, F; Peters, S J; Crilly, S; Compson, J; Eddleston, A; Björkelund, H; Greenslade, K; Parkinson, M; Davies, N L; Griffin, R; Pither, T L; Cain, K; Christodoulou, L; Staelens, L; Ward, E; Tibbitts, J; Kiessling, A; Smith, B; Brennan, F R; Malmqvist, M; Fallah-Arani, F; Humphreys, D P

2017-12-06

Engagement of Fcγ-receptors triggers a range of downstream signalling events resulting in a diverse array of immune functions. As a result, blockade of Fc-mediated function is an important strategy for the control of several autoimmune and inflammatory conditions. We have generated a hexameric-Fc fusion protein (hexameric-Fc) and tested the consequences of multi-valent Fcγ-receptor engagement in in vitro and in vivo systems. In vitro engagement of hexameric-Fc with FcγRs showed complex binding interactions that altered with receptor density and triggered the internalisation and degradation of Fcγ-receptors. This caused a disruption of Fc-binding and phagocytosis. In vivo, in a mouse ITP model we observed a short half-life of hexameric-Fc but were nevertheless able to observe inhibition of platelet phagocytosis several days after hexameric-Fc dosing. In cynomolgus monkeys, we again observed a short half-life, but were able to demonstrate effective FcγR blockade. These findings demonstrate the ability of multi-valent Fc-based therapeutics to interfere with FcγR function and a potential mechanism through which they could have a sustained effect; the internalisation and degradation of FcγRs.

Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression in U937 cells.

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Chihara, Kazuyasu; Kato, Yuji; Yoshiki, Hatsumi; Takeuchi, Kenji; Fujieda, Shigeharu; Sada, Kiyonao

2017-09-13

The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.

FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody.

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Zhang, Di; Whitaker, Brian; Derebe, Mehabaw G; Chiu, Mark L

2018-04-01

Immunostimulatory antibodies against the tumor necrosis factor receptors (TNFR) are emerging as promising cancer immunotherapies. The agonism activity of such antibodies depends on crosslinking to Fc gamma RIIB receptor (FcγRIIB) to enable the antibody multimerization that drives TNFR activation. Previously, Fc engineering was used to enhance the binding of such antibodies to Fcγ receptors. Here, we report the identification of Centyrins as alternative scaffold proteins with binding affinities to homologous FcγRIIB and FcγRIIA, but not to other types of Fcγ receptors. One Centyrin, S29, was engineered at distinct positions of an anti-OX40 SF2 antibody to generate bispecific and tetravalent molecules named as mAbtyrins. Regardless of the position of S29 on the SF2 antibody, SF2-S29 mAbtyrins could bind FcγRIIB and FcγRIIA specifically while maintaining binding to OX40 receptors. In a NFκB reporter assay, attachment of S29 Centyrin molecules at the C-termini, but not the N-termini, resulted in SF2 antibodies with increased agonism owing to FcγRIIB crosslinking. The mAbtyrins also showed agonism in T-cell activation assays with immobilized FcγRIIB and FcγRIIA, but this activity was confined to mAbtyrins with S29 specifically at the C-termini of antibody heavy chains. Furthermore, regardless of the position of the molecule, S29 Centyrin could equip an otherwise Fc-silent antibody with antibody-dependent cellular phagocytosis activity without affecting the antibody's intrinsic antibody-dependent cell-meditated cytotoxicity and complement-dependent cytotoxicity. In summary, the appropriate adoption FcγRII-binding Centyrins as functional modules represents a novel strategy to engineer therapeutic antibodies with improved functionalities.

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

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Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia

2017-01-01

-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer......-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment...... of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4...

Site-selective conjugation of an anticoagulant aptamer to recombinant albumins and maintenance of neonatal Fc receptor binding

Science.gov (United States)

Schmøkel, Julie; Voldum, Anders; Tsakiridou, Georgia; Kuhlmann, Matthias; Cameron, Jason; Sørensen, Esben S.; Wengel, Jesper; Howard, Kenneth A.

2017-05-01

Aptamers are an attractive molecular medicine that offers high target specificity. Nucleic acid-based aptamers, however, are prone to nuclease degradation and rapid renal excretion that require blood circulatory half-life extension enabling technologies. The long circulatory half-life, predominately facilitated by engagement with the cellular recycling neonatal Fc receptor (FcRn), and ligand transport properties of albumin promote it as an attractive candidate to improve the pharmacokinetic profile of aptamers. This study investigates the effect of Cys34 site-selective covalent attachment of a factor IXa anticoagulant aptamer on aptamer functionality and human FcRn (hFcRn) engagement using recombinant human albumin (rHA) of either a wild type (WT) or an engineered human FcRn high binding variant (HB). Albumin-aptamer conjugates, connected covalently through a heterobifunctional succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate linker, were successfully prepared and purified by high performance liquid chromatography as confirmed by gel electrophoresis band-shift analysis and matrix-assisted laser desorption/ionization time of flight. Minimal reduction (∼25%) in activity of WT-linked aptamer to that of aptamer alone was found using an anticoagulant activity assay measuring temporal levels of activated partial thrombin. Covalent albumin-aptamer conjugation, however, substantially compromized binding to hFcRn, to 10% affinity of that of non-conjugated WT, determined by biolayer interferometry. Binding could be rescued by aptamer conjugation to recombinant albumin engineered for higher FcRn affinity (HB) that exhibited an 8-fold affinity compared to WT alone. This work describes a novel albumin-based aptamer delivery system whose hFcRn binding can be increased using a HB engineered albumin.

Coordinate expression of activating Fc gamma receptors I and III and inhibiting Fc gamma receptor type II in the determination of joint inflammation and cartilage destruction during immune complex-mediated arthritis.

NARCIS (Netherlands)

Nabbe, K.C.A.M.; Blom, A.B.; Holthuysen, A.E.M.; Boross, P.; Roth, J.; Verbeek, S.; Lent, P.L.E.M. van; Berg, W.B. van den

2003-01-01

OBJECTIVE: To study the role of the activating Fc gamma receptor types I and III (Fc gamma RI and Fc gamma RIII, respectively) and the inhibiting Fc gamma receptor II (Fc gamma RII) in inflammation and in various aspects of cartilage destruction during arthritis that is solely induced by immune

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

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Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

2017-10-01

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

The importance of human FcgammaRI in mediating protection to malaria.

Directory of Open Access Journals (Sweden)

Richard S McIntosh

2007-05-01

Full Text Available The success of passive immunization suggests that antibody-based therapies will be effective at controlling malaria. We describe the development of fully human antibodies specific for Plasmodium falciparum by antibody repertoire cloning from phage display libraries generated from immune Gambian adults. Although these novel reagents bind with strong affinity to malaria parasites, it remains unclear if in vitro assays are predictive of functional immunity in humans, due to the lack of suitable animal models permissive for P. falciparum. A potentially useful solution described herein allows the antimalarial efficacy of human antibodies to be determined using rodent malaria parasites transgenic for P. falciparum antigens in mice also transgenic for human Fc-receptors. These human IgG1s cured animals of an otherwise lethal malaria infection, and protection was crucially dependent on human FcgammaRI. This important finding documents the capacity of FcgammaRI to mediate potent antimalaria immunity and supports the development of FcgammaRI-directed therapy for human malaria.

Conformational plasticity at the IgE-binding site of the B-cell receptor CD23.

Science.gov (United States)

Dhaliwal, Balvinder; Pang, Marie O Y; Yuan, Daopeng; Yahya, Norhakim; Fabiane, Stella M; McDonnell, James M; Gould, Hannah J; Beavil, Andrew J; Sutton, Brian J

2013-12-01

IgE antibodies play a central role in allergic disease. They recognize allergens via their Fab regions, whilst their effector functions are controlled through interactions of the Fc region with two principal cell surface receptors, FcɛRI and CD23. Crosslinking of FcɛRI-bound IgE on mast cells and basophils by allergen initiates an immediate inflammatory response, while the interaction of IgE with CD23 on B-cells regulates IgE production. We have determined the structures of the C-type lectin "head" domain of CD23 from seven crystal forms. The thirty-five independent structures reveal extensive conformational plasticity in two loops that are critical for IgE binding. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

The binding affinity of a soluble TCR-Fc fusion protein is significantly improved by crosslinkage with an anti-C{beta} antibody

Energy Technology Data Exchange (ETDEWEB)

Ozawa, Tatsuhiko; Horii, Masae; Kobayashi, Eiji [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jin, Aishun [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Immunology, College of Basic Medical Sciences, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin 150081 (China); Kishi, Hiroyuki, E-mail: immkishi@med.u-toyama.ac.jp [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Muraguchi, Atsushi [Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

2012-06-01

Highlights: Black-Right-Pointing-Pointer A novel soluble TCR composed of TCR V and C regions with Ig Fc region is generated. Black-Right-Pointing-Pointer TCR-Fc protein immobilized by an anti-C{beta} antibody bound to a p/MHC tetramer. Black-Right-Pointing-Pointer Binding affinity of TCR-Fc was markedly increased by binding with anti-C{beta} antibody. -- Abstract: The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-C{beta} antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 Multiplication-Sign 10{sup -5} M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-C{beta} antibody, its binding affinity for p/MHC increased by 5-fold (2.2 Multiplication-Sign 10{sup -6} M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-C{beta} antibody, which is probably due to the stabilization of the V

Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

International Nuclear Information System (INIS)

Mueller, H.; Fehr, J.

1986-01-01

The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

Mycoplasma infection of cell lines can simulate the expression of Fc receptors by binding of the carbohydrate moiety of antibodies.

Science.gov (United States)

Lemke, H; Krausse, R; Lorenzen, J; Havsteen, B

1985-05-01

During the production of Fc receptor (FcR)-bearing hybridomas it was observed with a particular monoclonal anti-sheep red blood cell antibody (anti-SRBC 1/5, IgG1) that the contamination with Mycoplasma arginini of in vitro cultured cell lines leads to an apparent FcR activity. This property did not correspond with the serological typing since other antibodies of the same isotype could not support FcR rosette formation. Another mycoplasma strain M. orale lacked this property. Analysis of the binding reaction revealed that M. arginini contains a lectin which binds the carbohydrate moiety of the anti-SRBC 1/5 antibody, i.e. anti-SRBC 1/5 synthesized under the influence of tunicamycin or deglycosylated by NaIO4 oxidation did not support rosette formation. These data suggest that binding of antibodies to certain mycoplasma strains may be a pathogenic factor during mycoplasma infections by masking the microorganisms with the host's own defense molecules. The experiments with M. arginini-infected cell lines gain immunological importance since we obtained identical results with staphylococcal protein A, as another bacteriological FcR, and cell lines expressing intrinsic membrane FcR. Although it is an open question whether the glycoconjugates are directly bound by the FcR or else by influencing the three-dimensional structure of the antibodies, it seems possible that FcR in general may be lectins.

House dust mite-specific immunotherapy alters the basal expression of T regulatory and FcεRI pathway genes.

Science.gov (United States)

Pevec, Branko; Radulovic Pevec, Mira; Stipic Markovic, Asja; Batista, Irena; Rijavec, Matija; Silar, Mira; Kosnik, Mitja; Korosec, Peter

2012-01-01

Regulatory T (Treg) cells and IgE-mediated signaling pathways could play important roles in the induction of allergen tolerance during house dust mite-specific subcutaneous immunotherapy (HDM-SCIT). Our aim was to compare the basal expression levels of Treg, T helper 1 (Th1) and Th2 transcription factors and components involved in IgE-mediated signaling in healthy subjects with those in HDM-allergic patients both untreated and successfully treated with HDM-SCIT. Thirty-nine HDM-allergic patients who completed a 3- to 5-year course of mite extract SCIT, 20 mite-allergic controls and 25 healthy controls participated in this study. The efficacy of SCIT was monitored using skin-prick tests (SPTs), total immunoglobulin E (tIgE), specific IgE (sIgE), sIgG(4), nasal challenge and visual analog scale (VAS) scores at several time points. The mRNA levels of forkhead box protein 3 (FOXP3), T-BET, GATA-3, FcεRI, spleen tyrosine kinase (Syk), phosphatidylinositol 3 kinase (PI3K) and SH2 domain-containing inositol phosphatase (SHIP) were quantified by real-time RT-PCR using nonstimulated whole blood samples. Decreased wheal sizes and VAS scores, negative challenges and increased sIgG(4) levels indicated that SCIT was effective in the treated patients. Basal expression levels of FOXP3 and GATA-3 decreased and T-BET levels increased in both treated patients and in healthy controls compared to untreated patients. The IgE-mediated pathway kinases Syk and PI3K exhibited reduced expression, whereas SHIP phosphatase levels were elevated in both treated patients and healthy controls relative to untreated patients. The expression levels of FcεRI were not significantly altered. Immunotherapy using HDM extracts results in a modification of the basal expression levels of several IgE-related signaling factors and induces a highly significant upregulation of Th1-response and downregulation of Th2-response transcription factors. Interestingly, this therapy also appears to reduce the basal

Allosteric mechanism of action of the therapeutic anti-IgE antibody omalizumab.

Science.gov (United States)

Davies, Anna M; Allan, Elizabeth G; Keeble, Anthony H; Delgado, Jean; Cossins, Benjamin P; Mitropoulou, Alkistis N; Pang, Marie O Y; Ceska, Tom; Beavil, Andrew J; Craggs, Graham; Westwood, Marta; Henry, Alistair J; McDonnell, James M; Sutton, Brian J

2017-06-16

Immunoglobulin E and its interactions with receptors FcϵRI and CD23 play a central role in allergic disease. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcϵRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23 on B cells and antigen-presenting cells. We solved the crystal structure of the complex between an omalizumab-derived Fab and IgE-Fc, with one Fab bound to each Cϵ3 domain. Free IgE-Fc adopts an acutely bent structure, but in the complex it is only partially bent, with large-scale conformational changes in the Cϵ3 domains that inhibit the interaction with FcϵRI. CD23 binding is inhibited sterically due to overlapping binding sites on each Cϵ3 domain. Studies of omalizumab Fab binding in solution demonstrate the allosteric basis for FcϵRI inhibition and, together with the structure, reveal how omalizumab may accelerate dissociation of receptor-bound IgE from FcϵRI, exploiting the intrinsic flexibility and allosteric potential of IgE. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Embryo-fetal transfer of bevacizumab (Avastin) in the rat over the course of gestation and the impact of neonatal Fc receptor (FcRn) binding.

Science.gov (United States)

Thorn, Mitchell; Piche-Nicholas, Nicole; Stedman, Donald; Davenport, Scott W; Zhang, Ning; Collinge, Mark; Bowman, Christopher J

2012-10-01

There is concern about embryo-fetal exposure to antibody-based biopharmaceuticals based on the increase of such therapies being prescribed to women of childbearing potential. Therefore, there is a desire to better characterize embryo-fetal exposure of these molecules. The pregnant rat is a standard model for evaluating the potential consequences of exposure but placental transfer of antibody-based biopharmaceuticals is not well understood in this model. The relative embryo-fetal distribution of an antibody-based biopharmaceutical was evaluated in the rat. Bevacizumab (Avastin) was chosen as a tool antibody since it does not have significant target binding in the rat that might influence embryo-fetal biodistribution. Avastin was labeled with a fluorescent dye, characterized, and injected into pregnant rats at different gestation ages. Labeled Avastin in fetal tissues was visualized ex vivo using an IVIS 200 (Caliper, A PerkinElmer Company, Alameda, CA). Avastin localized to the fetus as early as 24-hr post intravenous injection of the dam, and was taken up by the fetus in a dose-dependent manner. Avastin was detectable in the developing embryo as early as gestation day 13 and continued to be transferred until the end of gestation. Fetal transfer of Avastins mutated in the portion of the antibody that binds the neonatal Fc receptor (FcRn) was tested in late gestation and was found to correlate with affinities of the mutant Avastin antibody to FcRn. The novel application of this imaging technology was used to characterize the onset and duration of Avastin maternal-fetal transfer in rats and the importance of FcRn binding. © 2012 Wiley Periodicals, Inc.

Low-affinity FcγR interactions can decide the fate of novel human IgG-sensitised red blood cells and platelets

Science.gov (United States)

Armour, Kathryn L; Smith, Cheryl S; Turner, Craig P; Kirton, Christopher M; Wilkes, Anthony M; Hadley, Andrew G; Ghevaert, Cedric; Williamson, Lorna M; Clark, Michael R

2014-01-01

G1Δnab is a mutant human IgG1 constant region with a lower ability to interact with FcγR than the natural IgG constant regions. Radiolabelled RBCs and platelets sensitised with specific G1Δnab Abs were cleared more slowly from human circulation than IgG1-sensitised counterparts. However, non-destructive splenic retention of G1Δnab-coated RBCs required investigation and plasma radioactivities now suggest this also occurred for platelets sensitised with an IgG1/G1Δnab mixture. In vitro assays with human cells showed that G1Δnab-sensitised RBCs did not cause FcγRI-mediated monocyte activation, FcγRIIIa-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) or macrophage phagocytosis although they did adhere to macrophages. Thus, FcγRII was implicated in the adhesion despite the Δnab mutation reducing the already low-affinity binding to this receptor class. Additional contacts via P-selectin enhance the interaction of sensitised platelets with monocytes and this system provided evidence of FcγRII-dependent activation by G1Δnab. These results emphasise the physiological relevance of low-affinity interactions: It appears that FcγRII interactions of G1Δnab allowed splenic retention of G1Δnab-coated RBCs with inhibitory FcγRIIb binding preventing RBC destruction and that FcγRIIb engagement by G1Δnab on IgG1/G1Δnab-sensitised platelets overcame activation by IgG1. Considering therapeutic blocking Abs, G1Δnab offers lower FcγR binding and a greater bias towards inhibition than IgG2 and IgG4 constant regions. PMID:24285214

Signal transduction by FcεRI: Analysis of the early molecular events

Directory of Open Access Journals (Sweden)

Henry Metzger

1999-01-01

Full Text Available We are analysing the initial molecular events stimulated by the high-affinity receptor for IgE, FcεRI. Earlier studies have shown that the first response when the receptor-bound IgE interacts with a multivalent antigen is a transphosphorylation of receptor tyrosines, induced by the approximation of two or more receptors by a constitutively associated src-family kinase (Lyn. The amount of weakly associated kinase regulates the intensity of the response. Several aspects are being analyzed: (i the sites on Lyn and the receptor that account for the constitutive interaction; (ii how the intrinsic affinity of a ligand for the receptor-bound IgE influences the responses; and (iii the mechanism(s by which low-affinity ligands can act as antagonists. In the latter studies, mast cell responses were followed by monitoring the phosphorylation of tyrosines on several proteins and secretion. At equivalent levels of receptor phosphorylation, a ligand with high affinity stimulated vigorous phosphorylation of downstream components, whereas a low-affinity ligand was unable to stimulate phosphorylation of the same components effectively. Cells stimulated with a mixture of high- and low-affinity ligands, under a protocol where simple displacement of one by the other was prevented, remarkably showed that excess low-affinity ligand inhibited the phosphorylation as well as degranulation by the high-affinity ligand. This antagonism results from a competition for the limiting amount of the constitutive initiating kinase. Related receptors that depend on recruitment of initiating kinases may be subject to similar regulatory mechanisms.

FcγRll: Characterisation of novel Fc receptor interactions and a new receptor form.

OpenAIRE

JESSICA CLAIRE ANANIA

2018-01-01

Leukocyte Fc receptors (FcR) bind to immunogloulins (Ig) to link the innate and humoral immune system to help balance the immune system and clear infections. We have characterised a novel FcR for IgG (FcγR) form, designated FcγRIIa3, which contains a 19 amino acid insert. This insert interacts with cytoskeletal structures allowing the receptor to be retained for longer periods of time at the cells surface upon activation, higher cell signalling which causes greater cellular activation. Theref...

Tanshinone IIA suppresses FcεRI-mediated mast cell signaling and anaphylaxis by activation of the Sirt1/LKB1/AMPK pathway.

Science.gov (United States)

Li, Xian; Park, Soon Jin; Jin, Fansi; Deng, Yifeng; Yang, Ju Hye; Chang, Jae-Hoon; Kim, Dong-Young; Kim, Jung-Ae; Lee, Youn Ju; Murakami, Makoto; Son, Kun Ho; Chang, Hyeun Wook

2018-06-01

AMP-activated protein kinase (AMPK) and its upstream mediators liver kinase B1 (LKB1) and sirtuin 1 (Sirt1) are generally known as key regulators of metabolism. We have recently reported that the AMPK pathway negatively regulates mast cell activation and anaphylaxis. Tanshinone IIA (Tan IIA), an active component of Salvia miltiorrhiza extract that is currently used for the treatment of cardiovascular and cerebrovascular diseases, shows anti-diabetic activity and improves insulin resistance in db/db mice through activation of AMPK. The aim of this study was to evaluate the anti-allergic activity of Tan IIA in vivo and to investigate the underlying mechanism in vitro in the context of AMPK signaling. The anti-allergic effect of Tan IIA was evaluated using mouse bone marrow-derived mast cells (BMMCs) from AMPKα2 -/- or Sirt1 -/- mice, or BMMCs transfected with siRNAs specific for AMPKα2, LKB1, or Sirt1. AMPKα2 -/- and Sirt1 -/- mice were used to confirm the anti-allergic effect of Tan IIA in anaphylaxis in vivo. Tan IIA dose-dependently inhibited FcεRI-mediated degranulation and production of eicosanoids and cytokines in BMMCs. These inhibitory effects were diminished by siRNA-mediated knockdown or genetic deletion of AMPKα2 or Sirt1. Moreover, Tan IIA inhibited a mast cell-mediated local passive anaphylactic reaction in wild-type mice, but not in AMPKα2 -/- or Sirt1 -/- mice. In conclusion, Tan IIA suppresses FcεRI-mediated mast cell activation and anaphylaxis through activation of the inhibitory Sirt1-LKB1-AMPK pathway. Thus, Tan IIA may be useful as a new therapeutic agent for mast cell-mediated allergic diseases. Copyright © 2018 Elsevier Inc. All rights reserved.

A Two-pronged Binding Mechanism of IgG to the Neonatal Fc Receptor Controls Complex Stability and IgG Serum Half-life

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Schoch, Angela; Larraillet, Vincent

2017-01-01

The success of recombinant monoclonal immunoglobulins (IgG) is rooted in their ability to target distinct antigens with high affinity combined with an extraordinarily long serum half-life, typically around 3 weeks. The pharmacokinetics of IgGs is intimately linked to the recycling mechanism...... half-life of ∼8 days. Here we dissect the molecular origins of excessive FcRn binding in therapeutic IgGs using a combination of hydrogen/deuterium exchange mass spectrometry and FcRn affinity chromatography. We provide experimental evidence for a two-pronged IgG-FcRn binding mechanism involving direct...

Correlation between CD16a binding and immuno effector functionality of an antigen specific immunoglobulin Fc fragment (Fcab).

Science.gov (United States)

Kainer, Manuela; Antes, Bernhard; Wiederkum, Susanne; Wozniak-Knopp, Gordana; Bauer, Anton; Rüker, Florian; Woisetschläger, Max

2012-10-15

Antigen binding immunoglobulin Fc fragments (Fcab) are generated by engineering loop regions in the CH3 domain of human IgG1 Fc. Variants of an Fcab specific for Her-2 were designed to display either enhanced (S239D:A330L:I332E) or diminished (L234A:L235A) binding affinities to the Fc receptor CD16a based on mutations described previously. The two mutant Fcab proteins demonstrated the expected modulation of CD16a binding. Interaction with recombinant or cell surface expressed Her-2 was unaffected in both mutants compared to the parental Fcab. Binding affinities for CD16a correlated with the ADCC-potencies of the Fcab variants. Additional studies indicated that the L234A:L235A variant Fcab had equivalent structural features as the unmodified Fcab since their DSC profiles were similar and antigen binding after re-folding upon partial heat denaturation had not changed. Introduction of the S239D:A330L:I332E mutations resulted in a significant reduction of the CH2 domain melting temperature, a moderate decrease of the thermal transition of the CH3 domain and lower antigen binding after thermal stress compared to the parental Fcab. We conclude that the known correlation between CD16a binding affinity and ADCC potency is also valid in Fcab proteins and that antigen specific Fcab molecules can be further engineered for fine tuning of immuno effector functions. Copyright © 2012 Elsevier Inc. All rights reserved.

At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody

OpenAIRE

Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R.; Rudd, Pauline M.; Wang, Junzhi

2016-01-01

Two non-human glycan epitopes, galactose-Į-1,3-galactose (Į-gal) and Neu5Gc-Į-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while Į-gal attached to Fc glycans were not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Ne...

Oxidation of M252 but not M428 in hu-IgG1 is responsible for decreased binding to and activation of hu-FcγRIIa (His131).

Science.gov (United States)

Cymer, Florian; Thomann, Marco; Wegele, Harald; Avenal, Cecile; Schlothauer, Tilman; Gygax, Daniel; Beck, Hermann

2017-11-01

Oxidation of monoclonal therapeutic antibodies (mAbs) can affect binding to Fc-receptors and potentially influence pharmacokinetics or effector functions like e.g. antibody dependent cellular phagocytosis (ADCP). Recently, it has been demonstrated that binding to FcγRIIa (H131) is affected by methionine oxidation of the Fc-portion but it is currently unknown which methionine is responsible for decreased binding. We separated an oxidized IgG1 monoclonal antibody based on the oxidation state of methionine 252 and analyzed fractionated material in receptor binding experiments as well as in functional (cell-based) assays. Although the unfractionated mixture demonstrated weaker interaction/activation of the receptor, differently oxidized isolated subspecies can lead both to stronger as well as weaker binding and activation of the histidine variant of FcγRIIa. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Increase in neutrophil Fc gamma receptor I expression following interferon gamma treatment in rheumatoid arthritis.

Science.gov (United States)

Goulding, N J; Knight, S M; Godolphin, J L; Guyre, P M

1992-04-01

The therapeutic potential of interferon gamma (IFN gamma) in a number of disease states is still being explored, but progress is hampered by the lack of a suitable measure of in vivo biological activity. To assess the in vivo biological effects of recombinant human IFN gamma (rhIFN gamma), 14 patients were studied in a randomised, prospective, double blind, placebo controlled trial of this cytokine for the treatment of rheumatoid arthritis. The levels of Fc gamma receptors on peripheral blood neutrophils were measured at baseline and after 21 days of once daily, subcutaneous injections of rhIFN gamma or placebo. An induction of neutrophil Fc gamma receptor type I (Fc gamma RI) was seen in the group of patients receiving recombinant human rhIFN gamma but not in those receiving placebo. No change in the expression of Fc gamma RII or Fc gamma RIII was detected. The amount of induction of Fc gamma RI detected on the neutrophils of patients receiving rhIFN gamma did not correlate with clinical measures of response at either 21 days or at the end of the study (24 weeks). No significant clinical responses were observed in the rhIFN gamma group at these times. These data confirm that the reported in vitro effect of IFN gamma on human neutrophil Fc receptor expression can be reproduced in vivo.

TTI-621 (SIRPαFc): A CD47-Blocking Innate Immune Checkpoint Inhibitor with Broad Antitumor Activity and Minimal Erythrocyte Binding.

Science.gov (United States)

Petrova, Penka S; Viller, Natasja Nielsen; Wong, Mark; Pang, Xinli; Lin, Gloria H Y; Dodge, Karen; Chai, Vien; Chen, Hui; Lee, Vivian; House, Violetta; Vigo, Noel T; Jin, Debbie; Mutukura, Tapfuma; Charbonneau, Marilyse; Truong, Tran; Viau, Stephane; Johnson, Lisa D; Linderoth, Emma; Sievers, Eric L; Maleki Vareki, Saman; Figueredo, Rene; Pampillo, Macarena; Koropatnick, James; Trudel, Suzanne; Mbong, Nathan; Jin, Liqing; Wang, Jean C Y; Uger, Robert A

2017-02-15

Purpose: The ubiquitously expressed transmembrane glycoprotein CD47 delivers an anti-phagocytic (do not eat) signal by binding signal-regulatory protein α (SIRPα) on macrophages. CD47 is overexpressed in cancer cells and its expression is associated with poor clinical outcomes. TTI-621 (SIRPαFc) is a fully human recombinant fusion protein that blocks the CD47-SIRPα axis by binding to human CD47 and enhancing phagocytosis of malignant cells. Blockade of this inhibitory axis using TTI-621 has emerged as a promising therapeutic strategy to promote tumor cell eradication. Experimental Design: The ability of TTI-621 to promote macrophage-mediated phagocytosis of human tumor cells was assessed using both confocal microscopy and flow cytometry. In vivo antitumor efficacy was evaluated in xenograft and syngeneic models and the role of the Fc region in antitumor activity was evaluated using SIRPαFc constructs with different Fc tails. Results: TTI-621 enhanced macrophage-mediated phagocytosis of both hematologic and solid tumor cells, while sparing normal cells. In vivo , TTI-621 effectively controlled the growth of aggressive AML and B lymphoma xenografts and was efficacious in a syngeneic B lymphoma model. The IgG1 Fc tail of TTI-621 plays a critical role in its antitumor activity, presumably by engaging activating Fcγ receptors on macrophages. Finally, TTI-621 exhibits minimal binding to human erythrocytes, thereby differentiating it from CD47 blocking antibodies. Conclusions: These data indicate that TTI-621 is active across a broad range of human tumors. These results further establish CD47 as a critical regulator of innate immune surveillance and form the basis for clinical development of TTI-621 in multiple oncology indications. Clin Cancer Res; 23(4); 1068-79. ©2016 AACR . ©2016 American Association for Cancer Research.

Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis

DEFF Research Database (Denmark)

Thiele, Stefanie; Mungalpara, J; Steen, A

2014-01-01

have previously been suggested based on molecular docking guided by structure-activity relationship (SAR) data; however, none of these have been verified by in vitro experiments. EXPERIMENTAL APPROACH: Heterologous (125) I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated...... activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. KEY RESULTS: The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic......-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity. CONCLUSIONS AND IMPLICATIONS: Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes...

[Ala12]MCD peptide: a lead peptide to inhibitors of immunoglobulin E binding to mast cell receptors.

Science.gov (United States)

Buku, A; Condie, B A; Price, J A; Mezei, M

2005-09-01

An effort was made to discover mast cell degranulating (MCD) peptide analogs that bind with high affinity to mast cell receptors without triggering secretion of histamine or other mediators of the allergic reaction initiated by immunoglobulin E (IgE) after mast cell activation. Such compounds could serve as inhibitors of IgE binding to mast cell receptors. An alanine scan of MCD peptide reported previously showed that the analog [Ala12]MCD was 120-fold less potent in histamine-releasing activity and fivefold more potent in binding affinity to mast cell receptors than the parent MCD peptide. Because this analog showed marginal intrinsic activity and good binding affinity it was subsequently tested in the present study as an IgE inhibitor. In contrast to MCD peptide, [Ala12]MCD showed a 50% inhibition of IgE binding to the Fc epsilon RI alpha mast cell receptor by using rat basophilic leukemia (RBL-2H3) mast cells and fluorescence polarization. Furthermore, in a beta-hexosaminidase secretory assay, the peptide also showed a 50% inhibition of the secretion of this enzyme caused by IgE. An attempt was made to relate structural changes and biologic differences between the [Ala12]MCD analog and the parent MCD peptide. The present results show that [Ala12]MCD may provide a base for designing agents to prevent IgE/Fc epsilon RI alpha interactions and, consequently, allergic conditions.

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

Science.gov (United States)

Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

2014-11-13

To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

Traces of pFc' in IVIG interact with human IgG Fc domains and counteract aggregation

NARCIS (Netherlands)

Rispens, Theo; Himly, Martin; Ooievaar-de Heer, Pleuni; den Bleker, Tamara H.; Aalberse, Rob C.

2010-01-01

To prevent multimer formation, intravenous immunoglobulin (IVIG) is often treated with traces of pepsin. So far, the mechanism behind this treatment has been unclear. Recently, we reported that human IgG4 binds other IgG molecules via Fc-Fc interactions. Here we show that IVIG treated with traces of

At least two Fc Neu5Gc residues of monoclonal antibodies are required for binding to anti-Neu5Gc antibody.

Science.gov (United States)

Yu, Chuanfei; Gao, Kai; Zhu, Lei; Wang, Wenbo; Wang, Lan; Zhang, Feng; Liu, Chunyu; Li, Meng; Wormald, Mark R; Rudd, Pauline M; Wang, Junzhi

2016-01-29

Two non-human glycan epitopes, galactose-α-1,3-galactose (α-gal) and Neu5Gc-α-2-6-galactose (Neu5Gc) have been shown to be antigenic when attached to Fab oligosaccharides of monoclonal antibodies (mAbs) , while α-gal attached to Fc glycans was not. However, the antigenicity of Neu5Gc on the Fc glycans remains unclear in the context that most mAbs carry only Fc glycans. After studying two clinical mAbs carrying significant amounts of Fc Neu5Gc, we show that their binding activity with anti-Neu5Gc antibody resided in a small subset of mAbs carrying two or more Fc Neu5Gc, while mAbs harboring only one Neu5Gc showed no reactivity. Since most Neu5Gc epitopes were distributed singly on the Fc of mAbs, our results suggest that the potential antigenicity of Fc Neu5Gc is low. Our study could be referenced in the process design and optimization of mAb production in murine myeloma cells and in the quality control of mAbs for industries and regulatory authorities.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Science.gov (United States)

Mittal, Rahul; Sukumaran, Sunil K; Selvaraj, Suresh K; Wooster, David G; Babu, M Madan; Schreiber, Alan D; Verbeek, J Sjef; Prasadarao, Nemani V

2010-11-18

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Fc-fusion Proteins in Therapy: An Updated View.

Science.gov (United States)

Jafari, Reza; Zolbanin, Naime M; Rafatpanah, Houshang; Majidi, Jafar; Kazemi, Tohid

2017-01-01

Fc-fusion proteins are composed of Fc region of IgG antibody (Hinge-CH2-CH3) and a desired linked protein. Fc region of Fc-fusion proteins can bind to neonatal Fc receptor (FcRn) thereby rescuing it from degradation. The first therapeutic Fc-fusion protein was introduced for the treatment of AIDS. The molecular designing is the first stage in production of Fc-fusion proteins. The amino acid residues in the Fc region and linked protein are very important in the bioactivity and affinity of the fusion proteins. Although, therapeutic monoclonal antibodies are the top selling biologics but the application of therapeutic Fc-fusion proteins in clinic is in progress and among these medications Etanercept is the most effective in therapy. At present, eleven Fc-fusion proteins have been approved by FDA. There are novel Fc-fusion proteins which are in pre-clinical and clinical development. In this article, we review the molecular and biological characteristics of Fc-fusion proteins and then further discuss the features of novel therapeutic Fc-fusion proteins. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Structural characterization of the Man5 glycoform of human IgG3 Fc

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Shah, Ishan S.; Lovell, Scott; Mehzabeen, Nurjahan; Battaile, Kevin P.; Tolbert, Thomas J. (Kansas); (HWMRI)

2017-12-01

Immunoglobulin G (IgG) consists of four subclasses in humans: IgG1, IgG2, IgG3 and IgG4, which are highly conserved but have unique differences that result in subclass-specific effector functions. Though IgG1 is the most extensively studied IgG subclass, study of other subclasses is important to understand overall immune function and for development of new therapeutics. When compared to IgG1, IgG3 exhibits a similar binding profile to Fcγ receptors and stronger activation of complement. All IgG subclasses are glycosylated at N297, which is required for Fcγ receptor and C1q complement binding as well as maintaining optimal Fc conformation. We have determined the crystal structure of homogenously glycosylated human IgG3 Fc with a GlcNAc2Man5 (Man5) high mannose glycoform at 1.8 Å resolution and compared its structural features with published structures from the other IgG subclasses. Although the overall structure of IgG3 Fc is similar to that of other subclasses, some structural perturbations based on sequence differences were revealed. For instance, the presence of R435 in IgG3 (and H435 in the other IgG subclasses) has been implicated to result in IgG3-specific properties related to binding to protein A, protein G and the neonatal Fc receptor (FcRn). The IgG3 Fc structure helps to explain some of these differences. Additionally, protein-glycan contacts observed in the crystal structure appear to correlate with IgG3 affinity for Fcγ receptors as shown by binding studies with IgG3 Fc glycoforms. Finally, this IgG3 Fc structure provides a template for further studies aimed at engineering the Fc for specific gain of function.

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs.

Science.gov (United States)

Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-06-15

Ab-mediated rejection (AMR) of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor-specific Ab binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the Ab. We investigated the mechanisms underlying monocyte recruitment by HLA class I (HLA I) Ab-activated endothelium. We used a panel of murine mAbs of different subclasses to crosslink HLA I on human aortic, venous, and microvascular endothelial cells and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine (m)IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. mIgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during AMR. We confirmed these observations using human HLA allele-specific mAbs and IgG purified from transplant patient sera. HLA I Abs universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during AMR. Importantly, the subclass of donor-specific Ab may influence its pathogenesis. These results imply that human IgG1 and human IgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions.

HLA class I antibodies trigger increased adherence of monocytes to endothelial cells by eliciting an increase in endothelial P-selectin and, depending on subclass, by engaging FcγRs1

Science.gov (United States)

Valenzuela, Nicole M; Mulder, Arend; Reed, Elaine F

2013-01-01

Antibody-mediated rejection of solid organ transplants is characterized by intragraft macrophages. It is incompletely understood how donor specific antibody binding to graft endothelium promotes monocyte adhesion, and what, if any, contribution is made by the Fc region of the antibody. We investigated the mechanisms underlying monocyte recruitment by HLA class I antibody-activated endothelium. We used a panel of murine monoclonal antibodies of different subclasses to crosslink HLA I on human aortic, venous and microvascular endothelial cells, and measured the binding of human monocytic cell lines and peripheral blood monocytes. Both anti-HLA I murine IgG1 and mIgG2a induced endothelial P-selectin, which was required for monocyte adhesion to endothelium irrespective of subclass. Mouse IgG2a but not mIgG1 could bind human FcγRs. Accordingly, HLA I mIgG2a but not mIgG1 treatment of endothelial cells significantly augmented recruitment, predominantly through FcγRI, and, to a lesser extent, FcγRIIa. Moreover, HLA I mIgG2a promoted firm adhesion of monocytes to ICAM-1 through Mac-1, which may explain the prominence of monocytes during antibody mediated rejection. We confirmed these observations using human HLA allele specific monoclonal antibodies and IgG purified from transplant patient sera. HLA I antibodies universally elicit endothelial exocytosis leading to monocyte adherence, implying that P-selectin is a putative therapeutic target to prevent macrophage infiltration during antibody-mediated rejection. Importantly, the subclass of donor specific antibody may influence its pathogenesis. These results imply that hIgG1 and hIgG3 should have a greater capacity to trigger monocyte infiltration into the graft than IgG2 or IgG4 due to enhancement by FcγR interactions. PMID:23690477

Comparative studies on Fc receptors for IgG on resting and activated T lymphocytes

International Nuclear Information System (INIS)

Hueckel, C.; Jensen, H.L.; Rychly, J.; Sandor, M.; Erdei, A.; Gergely, J.

1986-01-01

Fc-receptors for IgG (FcγR) on resting (i.e. freshly prepared) and mitogen (Con A) or alloantigen-activated mouse spleen T cells were compared using binding of different markers such as 125 I-labelled immune complexes, 125 I-labelled anti FcγR monoclonal antibody, FITC-labelled aggr. IgG and sheep erythrocytes covered with specific antibody (EA rosetting). C3b receptors were detected by rosetting with sheep erythrocytes covered with antibody and complement (EAC rosetting). The electrophoretic mobility of the cells without or after binding of aggr. IgG was also tested. Differences between resting and activated T cells were found: (1) After activation of T cells by mitogen or alloantigen, a proportion of FcγR-positive cells increased two to four times. (2) FcγR number per FcγR-positive cell seemed to be higher on activated then on resting cells. (3) FcγR-positive resting cells did not shed their FcγR upon incubation at 4 0 C followed by incubation at 37 0 C, but FcγR-positive activated cells shed a remarkable proportion of their FcγR on the same conditions. (4) Binding of aggr. IgG caused a decrease of electrophoretic mobility of activated but not resting cells. (5) FcγR-positive resting cells were also C3b receptor-positive, whereas FcγR-positive activated cells had no detectable C3b receptors. (author)

Acidic environment augments FcεRI-mediated production of IL-6 and IL-13 in mast cells

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Kamide, Yosuke, E-mail: m08702012@gunma-u.ac.jp [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi (Japan); Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Sagamihara (Japan); Ishizuka, Tamotsu [Third Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Tobo, Masayuki [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Tsurumaki, Hiroaki [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi (Japan); Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Aoki, Haruka; Mogi, Chihiro [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, Tokyo (Japan); Yatomi, Masakiyo; Ono, Akihiro; Koga, Yasuhiko [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi (Japan); Sato, Koichi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan); Hisada, Takeshi [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi (Japan); Dobashi, Kunio [Gunma University Graduate School of Health Sciences, Maebashi (Japan); Yamada, Masanobu [Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi (Japan)

2015-08-28

Although blood pH is maintained in a narrow range of around pH 7.4 in living organisms, inflammatory loci are characterized by acidic conditions. Mast cells tend to reside close to the surface of the body in areas such as the mucosa and skin where they may be exposed to exogenous acids, and they play an important role in immune responses. However, little is known about the effects of extracellular acidification on the functions of mast cell. Here, we found that extracellular acidification increased the dinitrophenyl-conjugated human serum albumin (DNP-HSA)-induced production of interleukin (IL)-6 and IL-13 in MC/9 cells or bone marrow-derived mouse mast cells sensitized with anti-DNP IgE. Extracellular acidification also inhibited migration of MC/9 cells toward DNP-HSA. In addition, acidic pH stimulated antigen-induced activation of p38 mitogen-activated protein kinase (MAPK) and protein kinase B (Akt). These findings suggest that extracellular acidification augmented antigen/IgE-induced and FcεRI-mediated production of IL-6 and IL-13 in mast cells, and that this was associated with the enhancement of p38 MAPK and Akt activation. - Highlights: • Antigen-induced IL-6 and IL-13 production was augmented by acidic pH in mast cells. • Acidic pH-induced actions were associated with activation of p38 MAPK and Akt. • Inhibition of p38 MAPK and Akt attenuated cytokine responses to acidic pH. • Acidic pH effects are not attributable to actions of known proton-sensing GPCRs.

Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties.

Science.gov (United States)

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas Ali; Rezaee, S A R

2017-05-01

Affinity tags are vital tools for the production of high-throughput recombinant proteins. Several affinity tags, such as the hexahistidine tag, maltose-binding protein, streptavidin-binding peptide tag, calmodulin-binding peptide, c-Myc tag, glutathione S-transferase and FLAG tag, have been introduced for recombinant protein production. The fragment crystallizable (Fc) domain of the IgG1 antibody is one of the useful affinity tags that can facilitate detection, purification and localization of proteins and can improve the immunogenicity, modulatory effects, physicochemical and pharmaceutical properties of proteins. Fcγ recombinant forms a group of recombinant proteins called Fc-fusion proteins (FFPs). FFPs are widely used in drug discovery, drug delivery, vaccine design and experimental research on receptor-ligand interactions. These fusion proteins have become successful alternatives to monoclonal antibodies for drug developments. In this review, the physicochemical, biochemical, immunological, pharmaceutical and therapeutic properties of recombinant FFPs were discussed as a new generation of bioengineering strategies.

Generation and Characterization of an IgG4 Monomeric Fc Platform.

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Lu Shan

Full Text Available The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.

Structural basis for pH-insensitive inhibition of immunoglobulin G recycling by an anti-neonatal Fc receptor antibody

Energy Technology Data Exchange (ETDEWEB)

Kenniston, Jon A.; Taylor, Brandy M.; Conley, Gregory P.; Cosic, Janja; Kopacz, Kris J.; Lindberg, Allison P.; Comeau, Stephen R.; Atkins, Kateri; Bullen, Jameson; TenHoor, Christopher; Adelman, Burt A.; Sexton, Daniel J.; Edwards, Thomas E.; Nixon, Andrew E. (Beryllium); (Dyax)

2017-09-06

The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn–DX-2507 Fab complex, revealing a nearly complete overlap of the IgG–Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.

Human IgG4 binds to IgG4 and conformationally altered IgG1 via Fc-Fc interactions

NARCIS (Netherlands)

Rispens, Theo; Ooievaar-de Heer, Pleuni; Vermeulen, Ellen; Schuurman, Janine; van der Neut Kolfschoten, Marijn; Aalberse, Rob C.

2009-01-01

The Fc fragment of IgG4 can interact with the Fc fragment of another IgG molecule. This interaction is a confounding factor when measuring IgG4 rheumatoid factor levels. Recently, we demonstrated that half-molecules of IgG4 can exchange to form a bispecific Ab. We expected these two phenomena to be

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA.

Science.gov (United States)

Suzuki, Mami; Yamanoi, Ayaka; Machino, Yusuke; Kobayashi, Eiji; Fukuchi, Kaori; Tsukimoto, Mitsutoshi; Kojima, Shuji; Kohroki, Junya; Akimoto, Kazunori; Masuho, Yasuhiko

2013-07-05

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs - A-RTX (alkylated) and S-RTX (S-sulfonated) - were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA. Copyright © 2013 Elsevier Inc. All rights reserved.

Imaging and measuring the biophysical properties of Fc gamma receptors on single macrophages using atomic force microscopy

Energy Technology Data Exchange (ETDEWEB)

Li, Mi [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Liu, Lianqing, E-mail: lqliu@sia.cn [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xi, Ning [Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong (China); Wang, Yuechao [State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016 (China); Xiao, Xiubin [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China); Zhang, Weijing, E-mail: zhangwj3072@163.com [Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071 (China)

2013-09-06

Highlights: •Nanoscale cellular ultra-structures of macrophages were observed. •The binding affinities of FcγRs were measured directly on macrophages. •The nanoscale distributions of FcγRs were mapped on macrophages. -- Abstract: Fc gamma receptors (FcγR), widely expressed on effector cells (e.g., NK cells, macrophages), play an important role in clinical cancer immunotherapy. The binding of FcγRs to the Fc portions of antibodies that are attached to the target cells can activate the antibody-dependent cell-mediated cytotoxicity (ADCC) killing mechanism which leads to the lysis of target cells. In this work, we used atomic force microscopy (AFM) to observe the cellular ultra-structures and measure the biophysical properties (affinity and distribution) of FcγRs on single macrophages in aqueous environments. AFM imaging was used to obtain the topographies of macrophages, revealing the nanoscale cellular fine structures. For molecular interaction recognition, antibody molecules were attached onto AFM tips via a heterobifunctional polyethylene glycol (PEG) crosslinker. With AFM single-molecule force spectroscopy, the binding affinities of FcγRs were quantitatively measured on single macrophages. Adhesion force mapping method was used to localize the FcγRs, revealing the nanoscale distribution of FcγRs on local areas of macrophages. The experimental results can improve our understanding of FcγRs on macrophages; the established approach will facilitate further research on physiological activities involved in antibody-based immunotherapy.

High Affinity IgE-Fc Receptor alpha and gamma Subunit Interactions

International Nuclear Information System (INIS)

Rashid, A.; Housden, J. E. M.; Sabban, S.; Helm, B.

2014-01-01

Objective: To explore the relationships between the subunits (alpha, beta and gamma) of the high affinity IgE receptor (Fc and RI) and its ability to mediate transmembrane signaling. Study Design: Experimental study. Place and Duration of Study: Department of Molecular Biology and Biotechnology, University of Sheffield, UK, from 2008 to 2009. Methodology: The approach employed was to create a chimera (human alpha-gamma-gamma) using the extracellular (EC) domain of the human high affinity IgE receptor. The alpha subunit (huFc and RIalpha) of IgE receptor was spliced onto the rodent gamma TM and cytoplasmic domain (CD). This was transfected into the Rat Basophilic Leukemia cell line in order to assess the possibility of selectively activating cells transfected with this single pass construct for antigen induced mediator release. Results: The RBLs cell lines transfected with the huFc and RIalpha/gamma/gamma cDNA constructs were assessed for the cell surface expression of the huFc and RIalpha subunit and the response to the antigenic stimulus by looking for degranulation and intracellular Ca2+ mobilisation. The results obtained showed the absence of huFc and RIalpha subunit expression on the surface of transfected cells as seen by flowcytometric studies, beta-hexosaminidase assays and intracellular calcium mobilisation studies. Conclusion: In the present study the grounds for non-expression of huFc and RIalpha/gamma/gamma cDNA remains elusive but may be due to the fact that the human-rodent chimeric receptors are assembled differently than the endogenous rodent receptors as seen in study in which COS 7 cells were transfected with human/rat chimeric complexes. (author)

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

Science.gov (United States)

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Chimeric immunoglobulin E reactive with tumor-associated antigen activates human Fc epsilon RI bearing cells

NARCIS (Netherlands)

Luiten, R. M.; Warnaar, S. O.; Schuurman, J.; Pasmans, S. G.; Latour, S.; Daëron, M.; Fleuren, G. J.; Litvinov, S. V.

1997-01-01

Crosslinking of immunoglobulin E molecules that are bound to the Fc epsilon receptors expressed on mast cells or basophils triggers activation of these cells, resulting in the development of a type I hypersensitivity. Targeting this potent immune reaction towards tumors by using IgE that reacts with

The production of KIR-Fc fusion proteins and their use in a multiplex HLA class I binding assay.

Science.gov (United States)

Hilton, Hugo G; Moesta, Achim K; Guethlein, Lisbeth A; Blokhuis, Jeroen; Parham, Peter; Norman, Paul J

2015-10-01

Soluble recombinant proteins that comprise the extracellular part of a surface expressed receptor attached to the Fc region of an IgG antibody have facilitated the determination of ligand specificity for an array of immune system receptors. Among such receptors is the family of killer cell immunoglobulin-like receptors (KIR) that recognize HLA class I ligands. These receptors, expressed on natural killer (NK) cells and T cells, play important roles in both immune defense and placental development in early pregnancy. Here we describe a method for the production of two domain KIR-Fc fusion proteins using baculovirus infected insect cells. This method is more scalable than traditional mammalian cell expression systems and produces efficiently folded proteins that carry posttranslational modifications found in native KIR. We also describe a multiplex binding assay using the Luminex platform that determines the avidity and specificity of two domain KIR-Fc for a panel of microbeads, each coated with one of 97 HLA class I allotypes. This assay is simple to perform, and represents a major improvement over the assays used previously, which were limited in the number of KIR and HLA class I combinations that could be assayed at any one time. The results obtained from this assay can be used to predict the response of NK cell and T cells when their KIR recognize HLA class I. Copyright © 2015 Elsevier B.V. All rights reserved.

Decreased Fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically HIV-1 infected individuals.

Science.gov (United States)

Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T; Ackerman, Margaret E; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

2011-07-05

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. Copyright © 2011 Elsevier Inc. All rights reserved.

Decreased Fc-Receptor expression on innate immune cells is associated with impaired antibody mediated cellular phagocytic activity in chronically HIV-1 infected individuals

Science.gov (United States)

Dugast, Anne-Sophie; Tonelli, Andrew; Berger, Christoph T.; Ackerman, Margaret E.; Sciaranghella, Gaia; Liu, Qingquan; Sips, Magdalena; Toth, Ildiko; Piechocka-Trocha, Alicja; Ghebremichael, Musie; Alter, Galit

2011-01-01

In addition to neutralization, antibodies mediate other antiviral activities including antibody-dependent cellular-phagocytosis (ADCP), antibody dependent cellular-cytotoxicity (ADCC), as well as complement deposition. While it is established that progressive HIV infection is associated with reduced ADCC and ADCP, the underlying mechanism for this loss of function is unknown. Here we report considerable changes in FcR expression over the course of HIV infection on both mDCs and monocytes, including elevated FcγRI expression in acute HIV infection and reduced expression of FcγRII and FcγRIIIa in chronic HIV infection. Furthermore, selective blockade of FcγRII alone was associated with a loss in ADCP activity, suggesting that FcγRII plays a central role in modulating ADCP. Overall, HIV infection is associated with a number of changes in FcR expression on phagocytic cells that are associated with changes in their ability to respond to antibody-opsonized targets, potentially contributing to a failure in viral clearance in progressive HIV-1 infection. PMID:21565376

A novel TNFα antagonizing peptide-Fc fusion protein designed based on CDRs of TNFα neutralizing monoclonal antibody

International Nuclear Information System (INIS)

Qin Weisong; Feng Jiannan; Zhang Wei; Li Yan; Shen, Beifen

2004-01-01

The variable regions of antibody molecules bind antigens with high affinity and specificity. The binding sites are imparted largely to the hypervariable portions (i.e., CDRs) of the variable region. Peptides derived from CDRs can bind antigen with similar specificity acting as mimic of antibody and become drug-designing core, although with markedly lower affinity. In order to increase the affinity and bioactivity, in this study, a novel peptide (PT) designed on CDRs of a TNFα neutralizing monoclonal antibody Z12 was linked with Fc fragment of human IgG1. The interaction mode of PT-linker-Fc (PLF) with TNFα was analyzed with computer-guided molecular modeling method. After expression in Escherichia coli and purification, recombinant PT-linker-Fc could bind directly with the TNFα coated on the ELISA plates. Furthermore, PLF could competitively inhibit the binding of Z12 to TNFα and also inhibit the TNFα-induced cytotoxicity on L929 cells. The TNFα antagonizing activity of PLF was significantly higher than that of the free peptide. This study highlights the potential of human Fc to enhance the potency of peptides designed on the CDRs of antibodies and could be useful in developing new TNFα antagonists

Activatory and Inhibitory Fcγ Receptors Augment Rituximab-mediated Internalization of CD20 Independent of Signaling via the Cytoplasmic Domain*

Science.gov (United States)

Vaughan, Andrew T.; Chan, Claude H. T.; Klein, Christian; Glennie, Martin J.; Beers, Stephen A.; Cragg, Mark S.

2015-01-01

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes. PMID:25568316

Activation/Inhibition of mast cells by supra-optimal antigen concentrations.

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Huber, Michael

2013-01-22

Mast cells (MCs) are tissue resident cells of hemopoietic origin and are critically involved in allergic diseases. MCs bind IgE by means of their high-affinity receptor for IgE (FcεRI). The FcεRI belongs to a family of multi-chain immune recognition receptors and is activated by cross-linking in response to multivalent antigens (Ags)/allergens. Activation of the FcεRI results in immediate release of preformed granular substances (e.g. histamine, heparin, and proteases), generation of arachidonic acid metabolites, and production of pro-inflammatory cytokines. The FcεRI shows a remarkable, bell-shaped dose-response behavior with weak induction of effector responses at both low and high (so-called supra-optimal) Ag concentrations. This is significantly different from many other receptors, which reach a plateau phase in response to high ligand concentrations. To explain this unusual dose-response behavior of the FcεRI, scientists in the past have drawn parallels to so-called precipitin curves resulting from titration of Ag against a fixed concentration of antibody (Ab) in solution (a.k.a. Heidelberger curves). Thus, for high, supra-optimal Ag concentrations one could assume that every IgE-bound FcεRI formed a monovalent complex with "its own Ag", thus resulting in marginal induction of effector functions due to absence of receptor cross-linking. However, this was never proven to be the case. More recently, careful studies of FcεRI activation and signaling events in MCs in response to supra-optimal Ag concentrations have suggested a molecular explanation for the descending part of this bell-shaped curve. It is obvious now that extensive FcεRI/IgE/Ag clusters are formed and inhibitory molecules and signalosomes are engaged in response to supra-optimal cross-linking (amongst them the Src family kinase Lyn and the inositol-5'-phosphatase SHIP1) and they actively down-regulate MC effector responses. Thus, the analysis of MC signaling triggered by supra

Conformational destabilization of Immunoglobulin G increases the low pH-binding affinity with the Neonatal Fc Receptor

DEFF Research Database (Denmark)

Walters, Benjamin T; Jensen, Pernille Foged; Larraillet, Vincent

2016-01-01

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the ......H-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.......Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain...... the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and Fc...

Induction of functional Fc receptors in P388 leukemia cells. Requirement for multiple differentiation signals.

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Cohen, D A; Stotelmyer, N L; Kaplan, A M

1985-04-01

The development of functional Fc receptors (FcR) during induced differentiation with the tumor promoter, phorbol myristate acetate (PMA), was studied in the murine tumor cell line, P388. PMA induced the appearance of FcR on the membranes of P388 cells as indicated by the binding of IgG-coated sheep red blood cells (IgG-SRBC). Concentrations of PMA as low as 1 ng/ml were sufficient to induce the expression of FcR as well as to inhibit cellular division and to induce adherence in the P388 tumor cell line; however, optimal FcR induction occurred at PMA concentrations of 10-100 ng/ml. Immunofluorescent analysis with heat-aggregated myeloma proteins indicated that PMA induced FcR which were capable of binding IgG2a and IgG2b immunoglobulins, but not IgG1. Adherence to a substratum was determined to be a second required signal for expression of FcR, since PMA induction of P388 tumor cells in teflon dishes failed to fully develop FcR and adherence of P388 cells to poly-L-lysine-coated culture dishes in the absence of PMA was insufficient for FcR expression. FcR which appeared after PMA induction were non-functional in the sense that membrane-bound IgG-SRBC were not ingested to any significant extent by the tumor cells. However, if FcR induction occurred in the presence conA-induced rat spleen cell culture supernatants, phagocytosis of membrane-bound erythrocytes occurred. These findings suggest that for the expression of FcR which are capable of particle internalization, at least three identifiable membrane-transmitted signals are required during differentiation.

Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC

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Markus Roucka

2016-12-01

Full Text Available Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC-optimized variant (MB314. MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL, Pisum sativum agglutinin (PSA, Lens culinaris agglutinin (LCA, and Aleuria aurantia lectin (AAL were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody—whose affinity is mediated through core fucosylation—and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

Activatory and inhibitory Fcγ receptors augment rituximab-mediated internalization of CD20 independent of signaling via the cytoplasmic domain.

Science.gov (United States)

Vaughan, Andrew T; Chan, Claude H T; Klein, Christian; Glennie, Martin J; Beers, Stephen A; Cragg, Mark S

2015-02-27

Type I anti-CD20 mAb such as rituximab and ofatumumab engage with the inhibitory FcγR, FcγRIIb on the surface of B cells, resulting in immunoreceptor tyrosine-based inhibitory motif (ITIM) phosphorylation. Internalization of the CD20·mAb·FcγRIIb complex follows, the rate of which correlates with FcγRIIb expression. In contrast, although type II anti-CD20 mAb such as tositumomab and obinutuzumab also interact with and activate FcγRIIb, this interaction fails to augment the rate of CD20·mAb internalization, raising the question of whether ITIM phosphorylation plays any role in this process. We have assessed the molecular requirements for the internalization process and demonstrate that in contrast to internalization of IgG immune complexes, FcγRIIb-augmented internalization of rituximab-ligated CD20 occurs independently of the FcγRIIb ITIM, indicating that signaling downstream of FcγRIIb is not required. In transfected cells, activatory FcγRI, FcγRIIa, and FcγRIIIa augmented internalization of rituximab-ligated CD20 in a similar manner. However, FcγRIIa mediated a slower rate of internalization than cells expressing equivalent levels of the highly homologous FcγRIIb. The difference was maintained in cells expressing FcγRIIa and FcγRIIb lacking cytoplasmic domains and in which the transmembrane domains had been exchanged. This difference may be due to increased degradation of FcγRIIa, which traffics to lysosomes independently of rituximab. We conclude that the cytoplasmic domain of FcγR is not required for promoting internalization of rituximab-ligated CD20. Instead, we propose that FcγR provides a structural role in augmenting endocytosis that differs from that employed during the endocytosis of immune complexes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues

DEFF Research Database (Denmark)

Andersen, Jan T; Justesen, Sune; Fleckenstein, Burkhard

2008-01-01

knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251......) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding approximately 70 mg.L(-1) of fermentation of refolded soluble human FcRn. The structural and functional integrity...... was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response...

Comparative analysis of biological activities of Der p I-derived peptides on Fc epsilon receptor-bearing cells from Dermatophagoides pteronyssinus-sensitive patients.

Science.gov (United States)

Jeannin, P; Pestel, J; Bossus, M; Lassalle, P; Tartar, A; Tonnel, A B

1993-01-01

The ability of four uncoupled synthetic peptides (p52-71, p117-133, p176-187, p188-199) derived from Der p I, a major allergen from the house dust mite Dermatophagoides pteronyssinus (Dpt) to stimulate Fc epsilon R+ cells from Dpt-sensitive patients was comparatively analysed. Each free peptide may specifically stimulate basophils (Fc epsilon RI+ cells) and platelets (Fc epsilon RII+ cells) from patients with significant levels of anti-Der p I IgE antibodies; p52-71 and p117-133 appear the best cell stimulation inducers. Both concentration-dependent biological activities of Der p I-peptide on Fc epsilon R+ cells are enhanced by coupling peptide to a carrier (as human serum albumin). Interestingly each Der p I-sensitive patient tested presents an individual pattern of response to peptide. Thus, from our results it appears that different Der p I sequences could be involved in the immune response to Der p I. PMID:7682161

Comparative analysis of biological activities of Der p I-derived peptides on Fc epsilon receptor-bearing cells from Dermatophagoides pteronyssinus-sensitive patients.

Science.gov (United States)

Jeannin, P; Pestel, J; Bossus, M; Lassalle, P; Tartar, A; Tonnel, A B

1993-04-01

The ability of four uncoupled synthetic peptides (p52-71, p117-133, p176-187, p188-199) derived from Der p I, a major allergen from the house dust mite Dermatophagoides pteronyssinus (Dpt) to stimulate Fc epsilon R+ cells from Dpt-sensitive patients was comparatively analysed. Each free peptide may specifically stimulate basophils (Fc epsilon RI+ cells) and platelets (Fc epsilon RII+ cells) from patients with significant levels of anti-Der p I IgE antibodies; p52-71 and p117-133 appear the best cell stimulation inducers. Both concentration-dependent biological activities of Der p I-peptide on Fc epsilon R+ cells are enhanced by coupling peptide to a carrier (as human serum albumin). Interestingly each Der p I-sensitive patient tested presents an individual pattern of response to peptide. Thus, from our results it appears that different Der p I sequences could be involved in the immune response to Der p I.

Emerging functions of natural IgM and its Fc receptor FCMR in immune homeostasis

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Hongsheng eWang

2016-03-01

Full Text Available Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions.

Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

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Yijun Shen

Full Text Available Blocking proprotein convertase subtilisin kexin type 9 (PCSK9 binding to low-density lipoprotein receptor (LDLR can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs, alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

Plant-expressed Fc-fusion protein tetravalent dengue vaccine with inherent adjuvant properties.

Science.gov (United States)

Kim, Mi Young; Copland, Alastair; Nayak, Kaustuv; Chandele, Anmol; Ahmed, Muhammad S; Zhang, Qibo; Diogo, Gil R; Paul, Matthew J; Hofmann, Sven; Yang, Moon-Sik; Jang, Yong-Suk; Ma, Julian K-C; Reljic, Rajko

2017-12-09

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly-immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor-targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell-mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen-specific CD4 + and CD8 + T-cell proliferation, IFN-γ and antibody production. The purified polymeric fraction of dengue PIGS (D-PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low-affinity Fcγ receptors on antigen-presenting cells. These results show that the plant-expressed D-PIGS have the potential for translation towards a safe and easily scalable single antigen-based tetravalent dengue vaccine. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Clearance of Human IgG1-Sensitised Red Blood Cells In Vivo in Humans Relates to the In Vitro Properties of Antibodies from Alternative Cell Lines

Science.gov (United States)

Armour, Kathryn L.; Smith, Cheryl S.; Ip, Natasha C. Y.; Ellison, Cara J.; Kirton, Christopher M.; Wilkes, Anthony M.; Williamson, Lorna M.; Clark, Michael R.

2014-01-01

We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC) were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance. PMID:25302805

Clearance of human IgG1-sensitised red blood cells in vivo in humans relates to the in vitro properties of antibodies from alternative cell lines.

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Kathryn L Armour

Full Text Available We previously produced a recombinant version of the human anti-RhD antibody Fog-1 in the rat myeloma cell line, YB2/0. When human, autologous RhD-positive red blood cells (RBC were sensitised with this IgG1 antibody and re-injected, they were cleared much more rapidly from the circulation than had been seen earlier with the original human-mouse heterohybridoma-produced Fog-1. Since the IgG have the same amino acid sequence, this disparity is likely to be due to alternative glycosylation that results from the rat and mouse cell lines. By comparing the in vitro properties of YB2/0-produced Fog-1 IgG1 and the same antibody produced in the mouse myeloma cell line NS0, we now have a unique opportunity to pinpoint the cause of the difference in ability to clear RBC in vivo. Using transfected cell lines that express single human FcγR, we showed that IgG1 made in YB2/0 and NS0 cell lines bound equally well to receptors of the FcγRI and FcγRII classes but that the YB2/0 antibody was superior in FcγRIII binding. When measuring complexed IgG binding, the difference was 45-fold for FcγRIIIa 158F, 20-fold for FcγRIIIa 158V and approximately 40-fold for FcγRIIIb. The dissimilarity was greater at 100-fold in monomeric IgG binding assays with FcγRIIIa. When used to sensitise RBC, the YB2/0 IgG1 generated 100-fold greater human NK cell antibody-dependent cell-mediated cytotoxicity and had a 103-fold advantage over the NS0 antibody in activating NK cells, as detected by CD54 levels. In assays of monocyte activation and macrophage adherence/phagocytosis, where FcγRI plays major roles, RBC sensitised with the two antibodies produced much more similar results. Thus, the alternative glycosylation profiles of the Fog-1 antibodies affect only FcγRIII binding and FcγRIII-mediated functions. Relating this to the in vivo studies confirms the importance of FcγRIII in RBC clearance.

Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening.

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Hye-Ji Choi

Full Text Available Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A with mutations in one CH3 domain (CH3A on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B with mutations in the other CH3 domain (CH3B. In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80-90% with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.

Antiallergic Activity of Ethanol Extracts of Arctium lappa L. Undried Roots and Its Active Compound, Oleamide, in Regulating FcεRI-Mediated and MAPK Signaling in RBL-2H3 Cells.

Science.gov (United States)

Yang, Woong-Suk; Lee, Sung Ryul; Jeong, Yong Joon; Park, Dae Won; Cho, Young Mi; Joo, Hae Mi; Kim, Inhye; Seu, Young-Bae; Sohn, Eun-Hwa; Kang, Se Chan

2016-05-11

The antiallergic potential of Arctium lappa L. was investigated in Sprague-Dawley rats, ICR mice, and RBL-2H3 cells. Ethanol extract (90%) of A. lappa (ALE, 100 μg/mL) inhibited the degranulation rate by 52.9%, determined by the level of β-hexosaminidase. ALE suppressed passive cutaneous anaphylaxis (PCA) in rats and attenuated anaphylaxis and histamine release in mice. To identify the active compound of ALE, we subsequently fractionated and determined the level of β-hexosaminidase in all subfractions. Oleamide was identified as an active compound of ALE, which attenuated the secretion of histamine and the production of tumor necrosis factor (TNF)-α and interleukin-4 (IL-4) in cells treated with compound 48/80 or A23187/phorbol myristate acetate (PMA). Oleamide suppressed FcεRI-tyrosine kinase Lyn-mediated pathway, c-Jun N-terminal kinases (JNK/SAPK), and p38 mitogen-activated protein kinases (p38-MAPKs). These results showed that ALE and oleamide attenuated allergic reactions and should serve as a platform to search for compounds with antiallergic activity.

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

International Nuclear Information System (INIS)

Tang, Jin-Bao; Sun, Xi-Feng; Yang, Hong-Ming; Zhang, Bao-Gang; Li, Zhi-Jian; Lin, Zhi-Juan; Gao, Zhi-Qin

2013-01-01

Graphical abstract: -- Highlights: •A versatile platform for immobilizing functionally intact IgG is proposed. •The mechanism relies on properly oriented ZZ–PS-tag onto a hydrophilic PS surface. •The oriented ZZ–PS-tag presents ∼fivefold higher IgG-binding activity. •The platform shows tenfold higher sensitivity and a wider linear range in ELISA. -- Abstract: The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′) 2 anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used

Well-oriented ZZ–PS-tag with high Fc-binding onto polystyrene surface for controlled immobilization of capture antibodies

Energy Technology Data Exchange (ETDEWEB)

Tang, Jin-Bao, E-mail: tangjinbao@yahoo.com.cn [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China); Sun, Xi-Feng [Clinical Laboratory, Weifang People' s Hospital, Weifang 261041 (China); Yang, Hong-Ming [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China); Zhang, Bao-Gang [School of Basic Medicine, Weifang Medical University, Weifang 261053 (China); Li, Zhi-Jian [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China); Lin, Zhi-Juan [School of Basic Medicine, Weifang Medical University, Weifang 261053 (China); Gao, Zhi-Qin, E-mail: zhiqingao@yahoo.cn [School of Pharmacy and Biology, Weifang Medical University, Weifang 261053 (China)

2013-05-07

Graphical abstract: -- Highlights: •A versatile platform for immobilizing functionally intact IgG is proposed. •The mechanism relies on properly oriented ZZ–PS-tag onto a hydrophilic PS surface. •The oriented ZZ–PS-tag presents ∼fivefold higher IgG-binding activity. •The platform shows tenfold higher sensitivity and a wider linear range in ELISA. -- Abstract: The site specificity and bioactivity retention of antibodies immobilized on a solid substrate are crucial requirements for solid phase immunoassays. A fusion protein between an immunoglobulin G (IgG)-binding protein (ZZ protein) and a polystyrene-binding peptide (PS-tag) was constructed, and then used to develop a simple method for the oriented immobilization of the ZZ protein onto a PS support by the specific attachment of the PS-tag onto a hydrophilic PS. The orientation of intact IgG was achieved via the interaction of the ZZ protein and the constant fragment (Fc), thereby displayed the Fab fragment for binding antigen. The interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP was analyzed. Results showed that the oriented ZZ–PS-tag yielded an IgG-binding activity that is fivefold higher than that produced by the passive immobilization of the ZZ protein. The advantage of the proposed immunoassay strategy was demonstrated through an enzyme-linked immunosorbent assay, in which monoclonal mouse anti-goat IgG and HRP-conjugated rabbit F(ab′){sub 2} anti-goat IgG were used to detect goat IgG. The ZZ–PS-tag presented a tenfold higher sensitivity and a wider linear range than did the passively immobilized ZZ protein. The proposed approach may be an attractive strategy for a broad range of applications involving the oriented immobilization of intact IgGs onto PS supports, in which only one type of phi-PS (ZZ–PS-tag) surface is used.

Investigating the Interaction between the Neonatal Fc Receptor and Monoclonal Antibody Variants by Hydrogen/Deuterium Exchange Mass Spectrometry

DEFF Research Database (Denmark)

Jensen, Pernille Foged; Larraillet, Vincent; Schlothauer, Tilman

2015-01-01

The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest...... in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we studied the interaction between a full-length human IgG1 and human FcRn via hydrogen/deuterium exchange mass spectrometry and targeted electron transfer dissociation...... to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation, in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we found...

Enhanced antibody-dependent cellular phagocytosis by chimeric monoclonal antibodies with tandemly repeated Fc domains.

Science.gov (United States)

Nagashima, Hiroaki; Ootsubo, Michiko; Fukazawa, Mizuki; Motoi, Sotaro; Konakahara, Shu; Masuho, Yasuhiko

2011-04-01

We previously reported that chimeric monoclonal antibodies (mAbs) with tandemly repeated Fc domains, which were developed by introducing tandem repeats of Fc domains downstream of 2 Fab domains, augmented binding avidities for all Fcγ receptors, resulting in enhanced antibody (Ab)-dependent cellular cytotoxicity. Here we investigated regarding Ab-dependent cellular phagocytosis (ADCP) mediated by these chimeric mAbs, which is considered one of the most important mechanisms that kills tumor cells, using two-color flow cytometric methods. ADCP mediated by T3-Ab, a chimeric mAb with 3 tandemly repeated Fc domains, was 5 times more potent than that by native anti-CD20 M-Ab (M-Ab hereafter). Furthermore, T3-Ab-mediated ADCP was resistant to competitive inhibition by intravenous Ig (IVIG), although M-Ab-mediated ADCP decreased in the presence of IVIG. An Fcγ receptor-blocking study demonstrated that T3-Ab mediated ADCP via both FcγRIA and FcγRIIA, whereas M-Ab mediated ADCP exclusively via FcγRIA. These results suggest that chimeric mAbs with tandemly repeated Fc domains enhance ADCP as well as ADCC, and that Fc multimerization may significantly enhance the efficacy of therapeutic Abs. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Vesicular stomatitis virus expressing a chimeric Sindbis glycoprotein containing an Fc antibody binding domain targets to Her2/neu overexpressing breast cancer cells

International Nuclear Information System (INIS)

Bergman, Ira; Whitaker-Dowling, Patricia; Gao Yanhua; Griffin, Judith A.; Watkins, Simon C.

2003-01-01

Vesicular stomatitis virus (VSV) is a candidate for development for cancer therapy. It is an oncolytic virus that is safe in humans. Recombinant virus can be made directly from plasmid components. We attempted to create a virus that targeted specifically to breast cancer cells. Nonreplicating and replicating pseudotype VSV were created whose only surface glycoprotein (gp) was a Sindbis gp, called Sindbis-ZZ, modified to severely reduce its native binding function and to contain the Fc-binding domain of Staphylococcus aureus protein A. When titered on Her2/neu overexpressing SKBR3 human breast cancer cells, pseudotype VSV coated with Sindbis-ZZ had 5 /ml. This work demonstrates the ability to easily create, directly from plasmid components, an oncolytic replicating VSV with a restricted host cell range

Altered polymorphonuclear leukocyte Fc gamma R expression contributes to decreased candicidal activity during intraabdominal sepsis

International Nuclear Information System (INIS)

Simms, H.H.; D'Amico, R.; Monfils, P.; Burchard, K.W.

1991-01-01

We investigated the effects of untreated intraabdominal sepsis on polymorphonuclear leukocyte (PMN) candicidal activity. Two groups of swine were studied. Group I (n=6) underwent sham laparotomy, group II (n=7) underwent cecal ligation and incision. Untreated intraabdominal sepsis resulted in a progressive decrease in PMN candicidal activity. Concomitant rosetting and phagocytosis assays demonstrated a decrease in both the attachment and phagocytosis of Candida albicans opsonized with both normal and septic swine serum by PMNs in group II. Iodine 125-labeled swine immunoglobulin G (IgG) and fluorescein isothioalanate (FITC)-labeled swine IgG were used to investigate Fc gamma receptor ligand interactions. Scatchard analyses demonstrated a progressive decline in both the binding affinity constant and number of IgG molecules bound per PMN. Stimulation of the oxidative burst markedly reduced 125I-labeled IgG binding in both group I and group II, with a greater decrement being seen in animals with intraabdominal sepsis. Further, in group II, PMN recycling of the Fc gamma receptor to the cell surface after generation of the oxidative burst was reduced by postoperative day 4. Binding of monoclonal antibodies to Fc gamma receptor II, but not Fc gamma receptor I/III markedly reduced intracellular candicidal activity. Immunofluorescence studies revealed a homogeneous pattern of FITC-IgG uptake by nearly all group I PMNs, whereas by postoperative day 8 a substantial number of PMNs from group II failed to internalize the FITC-IgG. These studies suggest that untreated intraabdominal sepsis reduces PMN candicidal activity and that this is due, in part, to altered PMN Fc gamma receptor ligand interactions

Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

International Nuclear Information System (INIS)

Chotiwan, Nunya; Roehrig, John T.; Schlesinger, Jacob J.; Blair, Carol D.; Huang, Claire Y.-H.

2014-01-01

Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection

Molecular determinants of dengue virus 2 envelope protein important for virus entry in FcγRIIA-mediated antibody-dependent enhancement of infection

Energy Technology Data Exchange (ETDEWEB)

Chotiwan, Nunya; Roehrig, John T. [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States); Schlesinger, Jacob J. [Department of Medicine, University of Rochester, Rochester, NY 14642 (United States); Blair, Carol D. [Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523 (United States); Huang, Claire Y.-H., E-mail: yxh0@cdc.gov [Arboviral Diseases Branch, Division of Vector-Borne Disease, Centers for Disease Control and Prevention, Fort Collins, CO 80521 (United States)

2014-05-15

Antibody-dependent enhancement (ADE) of infection may cause severe illness in patients suffering a secondary infection by a heterologous dengue virus (DENV) serotype. During ADE of infection, cross-reactive non- or poorly-neutralizing antibodies form infectious virus-Ab complexes with the newly infecting serotype and enhance virus infection by binding to the Fcγ receptors (FcγR) on FcγR-bearing cells. In this study, we determined that molecular determinants of DENV2 envelope protein critical for virus entry during non-ADE infection are also required for ADE infection mediated by FcγRIIA, and binding of virus-Ab complexes with FcγRIIA alone is not sufficient for ADE of infection. The FcγRIIA mainly plays an auxiliary role in concentrating the virus–Ab complex to the cell surface, and other primary cellular receptors are required for virus entry. Understanding the viral entry pathway in ADE of DENV infection will greatly facilitate rational designs of anti-viral therapeutics against severe dengue disease associated with ADE. - Highlights: • KKK305/307/310 in DENV2 E-DIII is critical for virus attachment in ADE and non-ADE infection. • Binding of DENV2–Ab complex with FcγRII alone is not sufficient for virus entry in ADE infection. • Other primary receptors were required for DENV2 internalization during FcγRII–mediated ADE. • G104 and L135 of DENV2 E are critical for virus-mediated membrane fusion. • DENV2 virus-mediated membrane fusion is required for both ADE and non-ADE infection.

Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNF-α in RBL-2H3 cells.

Science.gov (United States)

Chihara, Kazuyasu; Nakashima, Kenji; Takeuchi, Kenji; Sada, Kiyonao

2011-12-01

Adaptor protein 3BP2, a c-Abl Src homology 3 (SH3) domain-binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen-induced degranulation in rat basophilic leukemia RBL-2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain-binding protein. In this report, to further understand the functions of 3BP2 in FcεRI-mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain-containing phosphatase-1 (SHP-1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr(564) in the carboxy (C)-terminal tail region of SHP-1 is required for the direct interaction of SHP-1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP-1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP-1-mediated tumor necrosis factor-α (TNF-α) production without any effects on the degranulation in antigen-stimulated RBL-2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr(564) -phosphorylated form of SHP-1 and positively regulates the function of SHP-1 in FcεRI-mediated signaling in mast cells. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Relationship between Fc receptors and Ia alloantigens: analysis with a sensitive radioimmunoassay

International Nuclear Information System (INIS)

Rieber, E.P.; Wernet, P.

1977-01-01

This paper describes the successful use of 125 I-labeled lgG aggregate to detect la-type alloantibodies in pregnancy sera. The blockade of aggregate uptake of a variety of normal mononuclear and leukemic cell types by anti-Ia alloantibodies is analyzed. Fc receptors and la alloantigens are clearly two distinct molecular entities. The association between Fc receptors and Ia alloantigens on a quantitative level seems to depend on a ligand-binding mechanism to control their interaction rather than the presence of a topographical molecular tandem arrangement. (Auth.)

Anti-influenza Hyperimmune Immunoglobulin Enhances Fc-functional Antibody Immunity during Human Influenza Infection.

Science.gov (United States)

Vanderven, Hillary A; Wragg, Kathleen; Ana-Sosa-Batiz, Fernanda; Kristensen, Anne B; Jegaskanda, Sinthujan; Wheatley, Adam K; Wentworth, Deborah; Wines, Bruce D; Hogarth, P Mark; Rockman, Steve; Kent, Stephen J

2018-05-31

New treatments for severe influenza are needed. Passive transfer of influenza-specific hyperimmune pooled immunoglobulin (Flu-IVIG) boosts neutralising antibody responses to past strains in influenza-infected subjects. The effect of Flu-IVIG on antibodies with Fc-mediated functions, which may target diverse influenza strains, is unclear. We studied the capacity of Flu-IVIG, relative to standard IVIG, to bind to Fc receptors and mediate antibody-dependent cellular cytotoxicity in vitro. The effect of Flu-IVIG infusion, compared to placebo infusion, was examined in serial plasma samples from 24 subjects with confirmed influenza infection in the INSIGHT FLU005 pilot study. Flu-IVIG contains higher concentrations of Fc-functional antibodies than IVIG against a diverse range of influenza hemagglutinins. Following infusion of Flu-IVIG into influenza-infected subjects, a transient increase in Fc-functional antibodies was present for 1-3 days against infecting and non-infecting strains of influenza. Flu-IVIG contains antibodies with Fc-mediated functions against influenza virus and passive transfer of Flu-IVIG increases anti-influenza Fc-functional antibodies in the plasma of influenza-infected subjects. Enhancement of Fc-functional antibodies to a diverse range of influenza strains suggests that Flu-IVIG infusion could prove useful in the context of novel influenza virus infections, when there may be minimal or no neutralising antibodies in the Flu-IVIG preparation.

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

Science.gov (United States)

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

Directory of Open Access Journals (Sweden)

Se Jin Im

Full Text Available Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc consisting of IgD and IgG4, and tested its function using erythropoietin (EPO conjugate, EPO-hyFc. Despite low amino acid homology (20.5% between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H, a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last. Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

Science.gov (United States)

Im, Se Jin; Yang, Sang In; Yang, Se Hwan; Choi, Dong Hoon; Choi, So Young; Kim, Hea Sook; Jang, Do Soo; Jin, Kyeong Sik; Chung, Yo-Kyung; Kim, Seung-Hee; Paik, Sang Hoon; Park, Yoo Chang; Chung, Moon Koo; Kim, Yong Bum; Han, Kang-Hyun; Choi, Kwan Yong; Sung, Young Chul

2011-01-01

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

The neonatal Fc receptor, FcRn, as a target for drug delivery and therapy.

Science.gov (United States)

Sockolosky, Jonathan T; Szoka, Francis C

2015-08-30

Immunoglobulin G (IgG)-based drugs are arguably the most successful class of protein therapeutics due in part to their remarkably long blood circulation. This arises from IgG interaction with the neonatal Fc receptor, FcRn. FcRn is the central regulator of IgG and albumin homeostasis throughout life and is increasingly being recognized as an important player in autoimmune disease, mucosal immunity, and tumor immune surveillance. Various engineering approaches that hijack or disrupt the FcRn-mediated transport pathway have been devised to develop long-lasting and non-invasive protein therapeutics, protein subunit vaccines, and therapeutics for treatment of autoimmune and infectious disease. In this review, we highlight the diverse biological functions of FcRn, emerging therapeutic opportunities, as well as the associated challenges of targeting FcRn for drug delivery and disease therapy. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitation of Fc receptors and surface immunoglobulin is affected by cell isolation procedures using plasmagel and ficoll-hypaque.

Science.gov (United States)

Alexander, E L; Titus, J A; Segal, D M

1978-01-01

When mononuclear leukocytes are isolated directly from whole human blood using Ficoll-Hypaque or Plasmagel, cytophilic immunoglobulin is detected on cell surfaces. Upon incubation at 37 degrees C, this cell-associated immunoglobulin is shed slowly into the medium. However, when cells are prewashed in phosphate-buffered saline prior to isolation, they appear to be free of cytophilic immunoglobulin. Compared to prewashed cells, populations retaining cytophilic immunoglobulin on their surfaces demonstrate a decreased binding of soluble immune complexes and radiolabelled trimeric rabbit IgG. The data suggest that Ficoll-Hypaque and Plasmagel cause serum IgG to bind with abnormally high affinity to human mononuclear leukocytes, probably via Fc receptors. This artifact of preparation can lead to erroneous estimates of the numbers of cells bearing Fc receptors or intrinsic membrane immunoglobulin within a given population of cells and to an inaccurate assessment of the average number of Fc receptors per cell.

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

Science.gov (United States)

Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Purification and identification of the fusicoccin binding protein from oat root plasma membrane

Science.gov (United States)

de Boer, A. H.; Watson, B. A.; Cleland, R. E.

1989-01-01

Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

Stabilizing the CH2 Domain of an Antibody by Engineering in an Enhanced Aromatic Sequon.

Science.gov (United States)

Chen, Wentao; Kong, Leopold; Connelly, Stephen; Dendle, Julia M; Liu, Yu; Wilson, Ian A; Powers, Evan T; Kelly, Jeffery W

2016-07-15

Monoclonal antibodies (mAbs) exhibiting highly selective binding to a protein target constitute a large and growing proportion of the therapeutics market. Aggregation of mAbs results in the loss of their therapeutic efficacy and can result in deleterious immune responses. The CH2 domain comprising part of the Fc portion of Immunoglobulin G (IgG) is typically the least stable domain in IgG-type antibodies and therefore influences their aggregation propensity. We stabilized the CH2 domain by engineering an enhanced aromatic sequon (EAS) into the N-glycosylated C'E loop and observed a 4.8 °C increase in the melting temperature of the purified IgG1 Fc fragment. This EAS-stabilized CH2 domain also conferred enhanced stability against thermal and low pH induced aggregation in the context of a full-length monoclonal IgG1 antibody. The crystal structure of the EAS-stabilized (Q295F/Y296A) IgG1 Fc fragment confirms the design principle, i.e., the importance of the GlcNAc1•F295 interaction, and surprisingly reveals that the core fucose attached to GlcNAc1 also engages in an interaction with F295. Inhibition of core fucosylation confirms the contribution of the fucose-Phe interaction to the stabilization. The Q295F/Y296A mutations also modulate the binding affinity of the full-length antibody to Fc receptors by decreasing the binding to low affinity Fc gamma receptors (FcγRIIa, FcγRIIIa, and FcγRIIIb), while maintaining wild-type binding affinity to FcRn and FcγRI. Our results demonstrate that engineering an EAS into the N-glycosylated reverse turn on the C'E loop leads to stabilizing N-glycan-protein interactions in antibodies and that this modification modulates antibody-Fc receptor binding.

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

Science.gov (United States)

Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

Advances in therapeutic Fc engineering - modulation of IgG associated effector functions and serum half-life

Directory of Open Access Journals (Sweden)

Abhishek Saxena

2016-12-01

Full Text Available Today monoclonal immunoglobulin gamma (IgG antibodies have become a major option in cancer therapy especially for the patients with advanced or metastatic cancers. Efficacy of monoclonal antibodies (mAbs are achieved through both its antigen binding fragment (Fab and crystallizable fragment (Fc. Fab can specifically recognize tumor associated antigen (TAA and thus modulate TAA-linked downstream signaling pathways that may lead to inhibition of tumor growth, induction of tumor apoptosis and differentiation. The Fc region can further improve mAbs’ efficacy by mediating effector functions such as antibody-dependent cellular cytotoxicity (ADCC, complement-dependent cytotoxicity (CDC and antibody dependent cell-mediated phagocytosis (ADCP. Moreover, Fc is the region interacting with the neonatal Fc receptor (FcRn in a pH-dependent manner that can slow down IgG’s degradation and extend its serum half-life. Loss of the antibody Fc region dramatically shortens its serum half-life and weakens its anti-cancer effects. Given the essential roles that the Fc region plays in the modulation of the efficacy of mAb in cancer treatment, Fc engineering has been extensively studied in the past years. This review focuses on the recent advances in therapeutic Fc engineering that modulates its related effector functions and serum half-life. We also discuss the progress made in aglycosylated mAb development that may substantially reduce cost of manufacture but maintain similar efficacies as conventional glycosylated mAb. Finally, we highlight several Fc engineering based mAbs under clinical trials.

DIFFERENTIAL BINDING OF HUMAN INTERLEUKIN-1 (IL-1) RECEPTOR ANTAGONIST TO NATURAL AND RECOMBINANT SOLUBLE AND CELLULAR IL-1 TYPE-I RECEPTORS

DEFF Research Database (Denmark)

Svenson, Morten; Nedergaard, Susanne; Heegaard, Peter M. H.

1995-01-01

antagonist (IL-1ra). Recombinant soluble human IL-1RI expressed in COS cells (sIL-1RI) consists of the extracellular part of the receptor and binds all three known IL-1 species but preferentially to IL-1ra. We further characterized the sizes and binding of IL-1raBF and sIL-1RI to IL-1ra by polyacrylamide gel...... electrophoresis in the presence of sodium dodecylsulfate, ligand binding interference analyses, N-glycosidase treatment, concanavalin A affinity chromatography, and with the use of monoclonal antibodies (mAb) to human recombinant IL-1ra. We also evaluated the binding of IL-1ra to cellular IL-1RI on MRC5...... binding of both molecules to IL-1ra. Both factors blocked binding of IL-1ra to cellular IL-1RI, as did mAb to IL-1ra, but the sites on IL-1ra which bound to the mAb, and to IL-1raBF and sIL-1RI, differed. We conclude that there are important differences between the natural and recombinant forms of soluble...

Dicty_cDB: FC-BS11 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS11 (Link to dictyBase) - - - Contig-U16517-1 FC-BS11Z (Li...nk to Original site) - - FC-BS11Z 683 - - - - Show FC-BS11 Library FC (Link to library) Clone ID FC-BS11 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS11Q.Seq.d/ Representative seq. ID FC-BS...11Z (Link to Original site) Representative DNA sequence >FC-BS11 (FC-BS11Q) /CSM/FC/FC-BS/FC-BS11Q.Seq....s producing significant alignments: (bits) Value FC-BS11 (FC-BS11Q) /CSM/FC/FC-BS/FC-BS

Dicty_cDB: FC-BS10 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS10 (Link to dictyBase) - - - Contig-U16283-1 FC-BS10Z (Li...nk to Original site) - - FC-BS10Z 720 - - - - Show FC-BS10 Library FC (Link to library) Clone ID FC-BS10 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS10Q.Seq.d/ Representative seq. ID FC-BS...10Z (Link to Original site) Representative DNA sequence >FC-BS10 (FC-BS10Q) /CSM/FC/FC-BS/FC-BS10Q.Seq....E Sequences producing significant alignments: (bits) Value FC-BS10 (FC-BS10Q) /CSM/FC/FC-BS/FC-BS10Q.Seq.d/

Dicty_cDB: FC-BS21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS21 (Link to dictyBase) - - - Contig-U15764-1 FC-BS21Z (Li...nk to Original site) - - FC-BS21Z 723 - - - - Show FC-BS21 Library FC (Link to library) Clone ID FC-BS21 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS21Q.Seq.d/ Representative seq. ID FC-BS...21Z (Link to Original site) Representative DNA sequence >FC-BS21 (FC-BS21Q) /CSM/FC/FC-BS/FC-BS21Q.Seq.... Score E Sequences producing significant alignments: (bits) Value FC-BS21 (FC-BS21Q) /CSM/FC/FC-BS/FC-BS21Q.

Dicty_cDB: FC-BS19 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS19 (Link to dictyBase) - - - Contig-U16583-1 FC-BS19E (Li...nk to Original site) - - - - - - FC-BS19E 604 Show FC-BS19 Library FC (Link to library) Clone ID FC-BS19 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS19Q.Seq.d/ Representative seq. ID FC-BS...19E (Link to Original site) Representative DNA sequence >FC-BS19 (FC-BS19Q) /CSM/FC/FC-BS/FC-BS19Q.Seq....E Sequences producing significant alignments: (bits) Value FC-BS19 (FC-BS19Q) /CSM/FC/FC-BS/FC-BS19Q.Seq.d/

Dicty_cDB: FC-AI01 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI01 (Link to dictyBase) - - - Contig-U15592-1 FC-AI01E (Li...nk to Original site) - - - - - - FC-AI01E 307 Show FC-AI01 Library FC (Link to library) Clone ID FC-AI01 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI01Q.Seq.d/ Representative seq. ID FC-AI...01E (Link to Original site) Representative DNA sequence >FC-AI01 (FC-AI01Q) /CSM/FC/FC-AI/FC-AI01Q.Seq....uences producing significant alignments: (bits) Value FC-AI01 (FC-AI01Q) /CSM/FC/FC-AI/FC-AI01Q.Seq.d/ 68 8e

Dicty_cDB: FC-BS09 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS09 (Link to dictyBase) - - - Contig-U16215-1 FC-BS09Z (Li...nk to Original site) - - FC-BS09Z 626 - - - - Show FC-BS09 Library FC (Link to library) Clone ID FC-BS09 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS09Q.Seq.d/ Representative seq. ID FC-BS...09Z (Link to Original site) Representative DNA sequence >FC-BS09 (FC-BS09Q) /CSM/FC/FC-BS/FC-BS09Q.Seq....ignments: (bits) Value SSF360 (SSF360Q) /CSM/SS/SSF3-C/SSF360Q.Seq.d/ 854 0.0 FC-BS09 (FC-BS09Q) /CSM/FC/FC-BS/FC-BS

Dicty_cDB: FC-AI13 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI13 (Link to dictyBase) - - - Contig-U16263-1 FC-AI13F (Li...nk to Original site) FC-AI13F 507 - - - - - - Show FC-AI13 Library FC (Link to library) Clone ID FC-AI13 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI13Q.Seq.d/ Representative seq. ID FC-AI...13F (Link to Original site) Representative DNA sequence >FC-AI13 (FC-AI13Q) /CSM/FC/FC-AI/FC-AI13Q.Seq....nificant alignments: (bits) Value FC-AI13 (FC-AI13Q) /CSM/FC/FC-AI/FC-AI13Q.Seq.d/ 963 0.0 VSA730 (VSA730Q)

The herpes virus Fc receptor gE-gI mediates antibody bipolar bridging to clear viral antigens from the cell surface.

Directory of Open Access Journals (Sweden)

Blaise Ndjamen

2014-03-01

Full Text Available The Herpes Simplex Virus 1 (HSV-1 glycoprotein gE-gI is a transmembrane Fc receptor found on the surface of infected cells and virions that binds human immunoglobulin G (hIgG. gE-gI can also participate in antibody bipolar bridging (ABB, a process by which the antigen-binding fragments (Fabs of the IgG bind a viral antigen while the Fc binds to gE-gI. IgG Fc binds gE-gI at basic, but not acidic, pH, suggesting that IgG bound at extracellular pH by cell surface gE-gI would dissociate and be degraded in acidic endosomes/lysosomes if endocytosed. The fate of viral antigens associated with gE-gI-bound IgG had been unknown: they could remain at the cell surface or be endocytosed with IgG. Here, we developed an in vitro model system for ABB and investigated the trafficking of ABB complexes using 4-D confocal fluorescence imaging of ABB complexes with transferrin or epidermal growth factor, well-characterized intracellular trafficking markers. Our data showed that cells expressing gE-gI and the viral antigen HSV-1 gD endocytosed anti-gD IgG and gD in a gE-gI-dependent process, resulting in lysosomal localization. These results suggest that gE-gI can mediate clearance of infected cell surfaces of anti-viral host IgG and viral antigens to evade IgG-mediated responses, representing a general mechanism for viral Fc receptors in immune evasion and viral pathogenesis.

Dicty_cDB: FC-BR23 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BR23 (Link to dictyBase) - - - Contig-U15008-1 FC-BR23Z (Li...nk to Original site) - - FC-BR23Z 641 - - - - Show FC-BR23 Library FC (Link to library) Clone ID FC-BR23 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BR/FC-BR23Q.Seq.d/ Representative seq. ID FC-BR2...3Z (Link to Original site) Representative DNA sequence >FC-BR23 (FC-BR23Q) /CSM/FC/FC-BR/FC-BR23Q.Seq....9 0.0 SLA211 (SLA211Q) /CSM/SL/SLA2-A/SLA211Q.Seq.d/ 1029 0.0 FC-BR23 (FC-BR23Q) /CSM/FC/FC-BR/FC-BR2

Dicty_cDB: FC-BS14 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS14 (Link to dictyBase) - - - Contig-U16399-1 FC-BS14E (Li...nk to Original site) - - - - - - FC-BS14E 534 Show FC-BS14 Library FC (Link to library) Clone ID FC-BS14 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BS/FC-BS14Q.Seq.d/ Representative seq. ID FC-BS...14E (Link to Original site) Representative DNA sequence >FC-BS14 (FC-BS14Q) /CSM/FC/FC-BS/FC-BS14Q.Seq.... vs CSM-cDNA Score E Sequences producing significant alignments: (bits) Value FC-BS14 (FC-BS

Dicty_cDB: FC-AI17 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI17 (Link to dictyBase) - - - Contig-U15690-1 FC-AI17P (Li...nk to Original site) FC-AI17F 587 FC-AI17Z 626 FC-AI17P 1212 - - Show FC-AI17 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI17Q.Seq.d/ Representative seq. ID FC-AI...17P (Link to Original site) Representative DNA sequence >FC-AI17 (FC-AI17Q) /CSM/FC/FC-AI/FC-AI...slated Amino Acid sequence ANIATVGDFLKADTVVPKMIITYNKRKQGTDYLKAVIGPILSNVIKQELNLELKPNLVYA AIISEQEIRTGEKSTLDRNV

Dicty_cDB: FC-AI18 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI18 (Link to dictyBase) - - - Contig-U15590-1 FC-AI18P (Li...nk to Original site) FC-AI18F 621 FC-AI18Z 703 FC-AI18P 1324 - - Show FC-AI18 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI18Q.Seq.d/ Representative seq. ID FC-AI...18P (Link to Original site) Representative DNA sequence >FC-AI18 (FC-AI18Q) /CSM/FC/FC-AI/FC-AI...AVWPLIPGYERA DGEKQYPVAAMLCNFTKPTPTTPSLLTHDEVVTFFHEFGHVMHNMSTKVHYSMFSGTSVE RDFVECPSQLFEFWCWNKDVLVNKLSGHXKDHSKKLPTDLVERMIAAKNLNVAI

RI scintigraphy in myasthenia gravis

International Nuclear Information System (INIS)

Kuroda, Yoshikazu; Miyamoto, Mariko; Maki, Masako; Yamazaki, Toshiro.

1982-01-01

35 cases of myasthenia gravis were studied with RI scintigraphy. 67 Ga-citrate was used in 34 patients and 76 Se-selenomethionine in 5 patients for thymus scintigraphy. RI scintigram was negative in non-tumorous thymus, regardless of the severity of illness and it was positive in seven of nine patients with thymomas. RI accumulation in thymus was found both in benign and malignant thymomas. RI seems to accumulated in lymphocytic and mixed thymus more than epithelial type. (author)

Dicty_cDB: FC-AI04 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI04 (Link to dictyBase) - - - Contig-U15121-1 FC-AI04E (Li...nk to Original site) - - - - - - FC-AI04E 772 Show FC-AI04 Library FC (Link to library) Clone ID FC-AI04 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI04Q.Seq.d/ Representative seq. ID FC-AI...04E (Link to Original site) Representative DNA sequence >FC-AI04 (FC-AI04Q) /CSM/FC/FC-AI/FC-AI04Q.Seq....qvnkhqqvvtktvsd vlvphqvhnqvfphipqqmtlvnkhqpvvtktvsdvlvphqvhnqvfphtpqlkiqvylq vfqvvvvtiisai

Dicty_cDB: FC-AI10 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI10 (Link to dictyBase) - - - Contig-U16270-1 FC-AI10F (Li...nk to Original site) FC-AI10F 405 - - - - - - Show FC-AI10 Library FC (Link to library) Clone ID FC-AI10 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI10Q.Seq.d/ Representative seq. ID FC-AI...10F (Link to Original site) Representative DNA sequence >FC-AI10 (FC-AI10Q) /CSM/FC/FC-AI/FC-AI10Q.Seq.... sequence RKKRKSDYTSFSTYIHKLLKQITPPTNAKSNEKGDRKFTISSKAMSVMNSFVHDIFDRIA TEASGLAKKKKRQTLHSRDIQVAVRIILTGELAXHAI

Dicty_cDB: FC-AI23 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI23 (Link to dictyBase) - - - Contig-U15308-1 FC-AI23Z (Li...nk to Original site) - - FC-AI23Z 603 - - - - Show FC-AI23 Library FC (Link to library) Clone ID FC-AI23 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI23Q.Seq.d/ Representative seq. ID FC-AI...23Z (Link to Original site) Representative DNA sequence >FC-AI23 (FC-AI23Q) /CSM/FC/FC-AI/FC-AI23Q.Seq....LNTLAKKNEQVVEGEILAKQLTGVTAEELSEFKACFSHFDKDN DNKLNRLEFSSCLKSIGDELTEEQLNQVISKIDTDGNGTISFEEFIDYMVSSRKGTDSVE STKAAFKVMAEDKDFITEAQIRAAI

Dicty_cDB: FC-AI05 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI05 (Link to dictyBase) - - - Contig-U15516-1 FC-AI05E (Li...nk to Original site) - - - - - - FC-AI05E 1189 Show FC-AI05 Library FC (Link to library) Clone ID FC-AI05 (L...//dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI05Q.Seq.d/ Representative seq. ID FC-AI...05E (Link to Original site) Representative DNA sequence >FC-AI05 (FC-AI05Q) /CSM/FC/FC-AI/FC-AI05Q.Seq...KIVGEASLKNKGKMSRVLAAKAALSARFD ALCEVSDTSYGIAYKGAVDRRAAAIEGREVRKSLNAVKPEKSGNVAKYDHTKSATTNTTR DVATKSSKESSIKQEKQ

Dicty_cDB: FC-AI21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI21 (Link to dictyBase) - - - Contig-U16254-1 FC-AI21Z (Li...nk to Original site) - - FC-AI21Z 696 - - - - Show FC-AI21 Library FC (Link to library) Clone ID FC-AI21 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI21Q.Seq.d/ Representative seq. ID FC-AI...21Z (Link to Original site) Representative DNA sequence >FC-AI21 (FC-AI21Q) /CSM/FC/FC-AI/FC-AI21Q.Seq....YPGYMYTDLSTIYERAGRIQGRNGSITQI PILTMPNDDITHPIPDLTGYITEGQIFIDRQINNRQIYPPINVLPSLSRLMKSAI

Revisiting Field Capacity (FC: variation of definition of FC and its estimation from pedotransfer functions

Directory of Open Access Journals (Sweden)

Theophilo Benedicto Ottoni Filho

2014-12-01

Full Text Available Taking into account the nature of the hydrological processes involved in in situ measurement of Field Capacity (FC, this study proposes a variation of the definition of FC aiming not only at minimizing the inadequacies of its determination, but also at maintaining its original, practical meaning. Analysis of FC data for 22 Brazilian soils and additional FC data from the literature, all measured according to the proposed definition, which is based on a 48-h drainage time after infiltration by shallow ponding, indicates a weak dependency on the amount of infiltrated water, antecedent moisture level, soil morphology, and the level of the groundwater table, but a strong dependency on basic soil properties. The dependence on basic soil properties allowed determination of FC of the 22 soil profiles by pedotransfer functions (PTFs using the input variables usually adopted in prediction of soil water retention. Among the input variables, soil moisture content θ (6 kPa had the greatest impact. Indeed, a linear PTF based only on it resulted in an FC with a root mean squared residue less than 0.04 m³ m-3 for most soils individually. Such a PTF proved to be a better FC predictor than the traditional method of using moisture content at an arbitrary suction. Our FC data were compatible with an equivalent and broader USA database found in the literature, mainly for medium-texture soil samples. One reason for differences between FCs of the two data sets of fine-textured soils is due to their different drainage times. Thus, a standardized procedure for in situ determination of FC is recommended.

Dicty_cDB: FC-AI03 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI03 (Link to dictyBase) - - - Contig-U15833-1 FC-AI03P (Li...nk to Original site) FC-AI03F 690 FC-AI03Z 651 FC-AI03P 1341 - - Show FC-AI03 Library FC (Link to library) Clone ID FC-AI...nal site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI03Q.Seq.d/ Representative seq. ID FC-AI...03P (Link to Original site) Representative DNA sequence >FC-AI03 (FC-AI03Q) /CSM/FC/FC-AI/FC-AI...li*l*ivtlskv*qswyqvfcslmrftcwi*nv fht*ivhwsqhwhql*flqpivaiv*skapitifnlhmaslwif*ivl*sfvpfhiitmk sfkfspfvpqlki

Autoantibody-induced internalization of CNS AQP4 water channel and EAAT2 glutamate transporter requires astrocytic Fc receptor.

Science.gov (United States)

Hinson, Shannon R; Clift, Ian C; Luo, Ningling; Kryzer, Thomas J; Lennon, Vanda A

2017-05-23

Aquaporin-4 (AQP4) water channel-specific IgG distinguishes neuromyelitis optica (NMO) from multiple sclerosis and causes characteristic immunopathology in which central nervous system (CNS) demyelination is secondary. Early events initiating the pathophysiological outcomes of IgG binding to astrocytic AQP4 are poorly understood. CNS lesions reflect events documented in vitro following IgG interaction with AQP4: AQP4 internalization, attenuated glutamate uptake, intramyelinic edema, interleukin-6 release, complement activation, inflammatory cell recruitment, and demyelination. Here, we demonstrate that AQP4 internalization requires AQP4-bound IgG to engage an astrocytic Fcγ receptor (FcγR). IgG-lacking Fc redistributes AQP4 within the plasma membrane and induces interleukin-6 release. However, AQP4 endocytosis requires an activating FcγR's gamma subunit and involves astrocytic membrane loss of an inhibitory FcγR, CD32B. Interaction of the IgG-AQP4 complex with FcγRs triggers coendocytosis of the excitatory amino acid transporter 2 (EAAT2). Requirement of FcγR engagement for internalization of two astrocytic membrane proteins critical to CNS homeostasis identifies a complement-independent, upstream target for potential early therapeutic intervention in NMO.

Dicty_cDB: FC-AI12 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI12 (Link to dictyBase) - - - Contig-U15484-1 FC-AI12Z (Li...nk to Original site) - - FC-AI12Z 614 - - - - Show FC-AI12 Library FC (Link to library) Clone ID FC-AI12 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI12Q.Seq.d/ Representative seq. ID FC-AI...12Z (Link to Original site) Representative DNA sequence >FC-AI12 (FC-AI12Q) /CSM/FC/FC-AI/FC-AI12Q.Seq....EKIVRRI ELLDGITCYRNEKAKDEIVLTGNSLELLSQSCATIQLRSAIKYKDVRKFLDGIYVSERNV LESN*in*riys

Dicty_cDB: FC-AI19 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI19 (Link to dictyBase) - - - Contig-U15115-1 FC-AI19Z (Li...nk to Original site) - - FC-AI19Z 661 - - - - Show FC-AI19 Library FC (Link to library) Clone ID FC-AI19 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI19Q.Seq.d/ Representative seq. ID FC-AI...19Z (Link to Original site) Representative DNA sequence >FC-AI19 (FC-AI19Q) /CSM/FC/FC-AI/FC-AI19Q.Seq....lmrqswvkkiesi*lvl krrkkkknnkkkkkkkkkkklfn*lvnkkn*ik*kkllcnqkk Frame B: ---*ekaieilsklfsin*kfn**ysiiigkkstkyq

Dicty_cDB: FC-AI14 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI14 (Link to dictyBase) - - - Contig-U16280-1 FC-AI14Z (Li...nk to Original site) - - FC-AI14Z 671 - - - - Show FC-AI14 Library FC (Link to library) Clone ID FC-AI14 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI14Q.Seq.d/ Representative seq. ID FC-AI...14Z (Link to Original site) Representative DNA sequence >FC-AI14 (FC-AI14Q) /CSM/FC/FC-AI/FC-AI14Q.Seq....nqrllv*lvvlskklqllnsnqsfkfkkvq rmkknsvkntkn*rfvllt*nlkslkrmpksknsptkliifilkly Frame B: ---lkdl*krtphl*stcfhhptlcssrrfclwslnai

Dicty_cDB: FC-AI06 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI06 (Link to dictyBase) - - - Contig-U16465-1 FC-AI06E (Li...nk to Original site) - - - - - - FC-AI06E 1138 Show FC-AI06 Library FC (Link to library) Clone ID FC-AI06 (L...//dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI06Q.Seq.d/ Representative seq. ID FC-AI...06E (Link to Original site) Representative DNA sequence >FC-AI06 (FC-AI06Q) /CSM/FC/FC-AI/FC-AI06Q.Seq...FGRGIDIERVNVVINYDMAESADTYLHRVGRAGRFGTK GLAISFVPSKEDPVLEQVQSKFVVSIKELVATPDPSTYMSG*kkkkkkkknlfvlksikk k*kkk*in

Dicty_cDB: FC-AI09 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI09 (Link to dictyBase) - - - Contig-U16149-1 FC-AI09Z (Li...nk to Original site) - - FC-AI09Z 591 - - - - Show FC-AI09 Library FC (Link to library) Clone ID FC-AI09 (Li.../dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI09Q.Seq.d/ Representative seq. ID FC-AI...09Z (Link to Original site) Representative DNA sequence >FC-AI09 (FC-AI09Q) /CSM/FC/FC-AI/FC-AI09Q.Seq....*tkl ik*ilifykiknnkkkkkk Frame B: ---gt*kvpeflailfkrmasrsvlwy*rcltkakkglkapqtltik

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

Directory of Open Access Journals (Sweden)

Simone I Richardson

2018-04-01

Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

RI scintigraphy in myasthenia gravis

Energy Technology Data Exchange (ETDEWEB)

Kuroda, Yoshikazu; Miyamoto, Mariko (Tokyo Metropolitan Fuchu Hospital (Japan)); Maki, Masako; Yamazaki, Toshiro

1982-10-01

35 cases of myasthenia gravis were studied with RI scintigraphy. /sup 67/Ga-citrate was used in 34 patients and /sup 76/Se-selenomethionine in 5 patients for thymus scintigraphy. RI scintigram was negative in non-tumorous thymus, regardless of the severity of illness and it was positive in seven of nine patients with thymomas. RI accumulation in thymus was found both in benign and malignant thymomas. RI seems to accumulated in lymphocytic and mixed thymus more than epithelial type.

Dicty_cDB: FC-AI15 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI15 (Link to dictyBase) - G01730 DDB0214993 Contig-U15123-1 FC-AI...15E (Link to Original site) - - - - - - FC-AI15E 856 Show FC-AI15 Library FC (Link to library) Clone ID FC-AI...3-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI15Q.Se...q.d/ Representative seq. ID FC-AI15E (Link to Original site) Representative DNA sequence >FC-AI15 (FC-AI15Q) /CSM/FC/FC-AI/FC-AI...AAAAAAAATA sequence update 1996.12.24 Translated Amino Acid sequence kt*riyi*KMMIKYITIAILFIASLVKADLQFSLCPTCV

Dicty_cDB: FC-AI24 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI24 (Link to dictyBase) - - - - FC-AI24Z (Link to Original site) - - FC-AI...24Z 693 - - - - Show FC-AI24 Library FC (Link to library) Clone ID FC-AI24 (Link to dictyBas...e) Atlas ID - NBRP ID - dictyBase ID - Link to Contig - Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...24Q.Seq.d/ Representative seq. ID FC-AI24Z (Link to Original s...ite) Representative DNA sequence >FC-AI24 (FC-AI24Q) /CSM/FC/FC-AI/FC-AI24Q.Seq.d/ XXXXXXXXXXAAATTAGAAAACAAA

Potential of Murine IgG1 and Human IgG4 to Inhibit the Classical Complement and Fcγ Receptor Activation Pathways

Directory of Open Access Journals (Sweden)

Gina-Maria Lilienthal

2018-05-01

Full Text Available IgG antibodies (Abs mediate their effector functions through the interaction with Fcγ receptors (FcγRs and the complement factors. The main IgG-mediated complement activation pathway is induced through the binding of complement C1q to IgG Abs. This interaction is dependent on antigen-dependent hexamer formation of human IgG1 and IgG3 to increase the affinity for the six-headed C1q molecule. By contrast, human IgG4 fails to bind to C1q. Instead, it has been suggested that human IgG4 can block IgG1 and IgG3 hexamerization required for their binding to C1q and activating the complement. Here, we show that murine IgG1, which functionally resembles human IgG4 by not interacting with C1q, inhibits the binding of IgG2a, IgG2b, and IgG3 to C1q in vitro, and suppresses IgG2a-mediated complement activation in a hemolytic assay in an antigen-dependent and IgG subclass-specific manner. From this perspective, we discuss the potential of murine IgG1 and human IgG4 to block the complement activation as well as suppressive effects of sialylated IgG subclass Abs on FcγR-mediated immune cell activation. Accumulating evidence suggests that both mechanisms seem to be responsible for preventing uncontrolled IgG (autoAb-induced inflammation in mice and humans. Distinct IgG subclass distributions and functionally opposite IgG Fc glycosylation patterns might explain different outcomes of IgG-mediated immune responses and provide new therapeutic options through the induction, enrichment, or application of antigen-specific sialylated human IgG4 to prevent complement and FcγR activation as well.

Fc engineering of anti-Nectin-2 antibody improved thrombocytopenic adverse event in monkey.

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Tsutomu Oshima

Full Text Available Nectin-2 is a transmembrane glycoprotein which is involved in the process of Ca2+-independent cell-cell adhesion. In our previous study, we have demonstrated that Nectin-2 is over-expressed in breast and ovarian cancer tissues by using gene expression analysis and immunohistochemistry. Furthermore, we discovered multiple anti-Nectin-2 fully human monoclonal antibodies which inhibited tumor growth in in vivo subcutaneous xenograft models with antibody-dependent cellular cytotoxicity (ADCC as the principal mechanism of action. In this report, we assessed the toxicity of Y-443, a fully human IgG1/kappa anti-Nectin-2 monoclonal antibody exhibiting strong in vitro ADCC and in vivo anti-tumor activity in cynomolgus monkeys (Macaca fascicularis (Cynos. Unexpectedly, upon administration, Y-443 induced strong thrombocytopenia through Nectin-2 expressed on Cyno platelets, presumably followed by phagocytosis in the mononuclear phagocytic system. To mitigate the adverse safety profile, we mutated the Fc region of Y-443 to reduce the Fc binding activity to Fcγ receptor I, which is the primary receptor for phagocytosis on macrophages. Moreover, we further engineered the Fc through defucosylation to maintain ADCC activity. The resultant Fc engineered antibody, termed Y-634, demonstrated diminished thrombocytopenia in Cyno toxicological studies and maintained anti-tumor activity in a mouse xenograft model. These findings suggest that Y-634 may have a therapeutic potential for the treatment of Nectin-2 positive cancers, and moreover, Fc engineering is a potential mitigation strategy to ameliorate safety liabilities in antibody induced thrombocytopenia while maintaining antibody potency.

Dicty_cDB: FC-AI08 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI08 (Link to dictyBase) - G01729 DDB0233148 Contig-U14939-1 FC-AI...08P (Link to Original site) FC-AI08F 654 FC-AI08Z 563 FC-AI08P 1217 - - Show FC-AI08 Library FC (Link... to library) Clone ID FC-AI08 (Link to dictyBase) Atlas ID - NBRP ID G01729 dictyBase ID DDB0233148 Link to ...Contig Contig-U14939-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...08Q.Seq.d/ Representative seq. ID FC-AI08P (Link to Original site) Representative DNA sequence >FC-AI08 (FC-AI

Crystallization and preliminary X-ray diffraction studies of an RNA aptamer in complex with the human IgG Fc fragment

International Nuclear Information System (INIS)

Sugiyama, Shigeru; Nomura, Yusuke; Sakamoto, Taiichi; Kitatani, Tomoya; Kobayashi, Asako; Miyakawa, Shin; Takahashi, Yoshinori; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Nakamura, Yoshikazu; Matsumura, Hiroyoshi

2008-01-01

An RNA aptamer in complex with the human IgG Fc fragment have been crystallized. The stirring technique with a rotary shaker was used to improve the crystals and to ensure that they were of high quality and single, resulting in crystals that diffracted to 2.2 Å resolution. Aptamers, which are folded DNA or RNA molecules, bind to target molecules with high affinity and specificity. An RNA aptamer specific for the Fc fragment of human immunoglobulin G (IgG) has recently been identified and it has been demonstrated that an optimized 24-nucleotide RNA aptamer binds to the Fc fragment of human IgG and not to other species. In order to clarify the structural basis of the high specificity of the RNA aptamer, it was crystallized in complex with the Fc fragment of human IgG1. Preliminary X-ray diffraction studies revealed that the crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 83.7, b = 107.2, c = 79.0 Å. A data set has been collected to 2.2 Å resolution

Dicty_cDB: FC-BR21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BR21 (Link to dictyBase) - - - Contig-U15384-1 | Contig-U16443-1 FC-BR2...1P (Link to Original site) FC-BR21F 551 FC-BR21Z 122 FC-BR21P 673 - - Show FC-BR21 Library FC (L...ink to library) Clone ID FC-BR21 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Cont...ig-U15384-1 | Contig-U16443-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-BR/FC-BR2...1Q.Seq.d/ Representative seq. ID FC-BR21P (Link to Original site) Representative DNA sequence >FC-BR21 (FC-BR2

Dicty_cDB: FC-AI07 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI07 (Link to dictyBase) - - - Contig-U15296-1 | Contig-U15756-1 FC-AI...07P (Link to Original site) FC-AI07F 580 FC-AI07Z 723 FC-AI07P 1303 - - Show FC-AI07 Library FC (...Link to library) Clone ID FC-AI07 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Con...tig-U15296-1 | Contig-U15756-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...07Q.Seq.d/ Representative seq. ID FC-AI07P (Link to Original site) Representative DNA sequence >FC-AI07 (FC-AI

Dicty_cDB: FC-AI22 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AI22 (Link to dictyBase) - - - Contig-U15369-1 | Contig-U15732-1 FC-AI...22P (Link to Original site) FC-AI22F 583 FC-AI22Z 683 FC-AI22P 1266 - - Show FC-AI22 Library FC (...Link to library) Clone ID FC-AI22 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Con...tig-U15369-1 | Contig-U15732-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC/FC-AI/FC-AI...22Q.Seq.d/ Representative seq. ID FC-AI22P (Link to Original site) Representative DNA sequence >FC-AI22 (FC-AI

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

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Gerco den Hartog

Full Text Available Respiratory syncytial virus (RSV infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins.To investigate whether IgG purified from bovine milk (bIgG can modulate immune responses against human RSV.ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated.bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV.The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

Specificity and Effector Functions of Human RSV-Specific IgG from Bovine Milk.

Science.gov (United States)

den Hartog, Gerco; Jacobino, Shamir; Bont, Louis; Cox, Linda; Ulfman, Laurien H; Leusen, Jeanette H W; van Neerven, R J Joost

2014-01-01

Respiratory syncytial virus (RSV) infection is the second most important cause of death in the first year of life, and early RSV infections are associated with the development of asthma. Breastfeeding and serum IgG have been shown to protect against RSV infection. Yet, many infants depend on bovine milk-based nutrition, which at present lacks intact immunoglobulins. To investigate whether IgG purified from bovine milk (bIgG) can modulate immune responses against human RSV. ELISAs were performed to analyse binding of bIgG to human respiratory pathogens. bIgG or hRSV was coated to plates to assess dose-dependent binding of bIgG to human Fcγ receptors (FcγR) or bIgG-mediated binding of myeloid cells to hRSV respectively. S. Epidermidis and RSV were used to test bIgG-mediated binding and internalisation of pathogens by myeloid cells. Finally, the ability of bIgG to neutralise infection of HEp2 cells by hRSV was evaluated. bIgG recognised human RSV, influenza haemagglutinin and Haemophilus influenza. bIgG bound to FcγRII on neutrophils, monocytes and macrophages, but not to FcγRI and FcγRIII, and could bind simultaneously to hRSV and human FcγRII on neutrophils. In addition, human neutrophils and dendritic cells internalised pathogens that were opsonised with bIgG. Finally, bIgG could prevent infection of HEp2 cells by hRSV. The data presented here show that bIgG binds to hRSV and other human respiratory pathogens and induces effector functions through binding to human FcγRII on phagocytes. Thus bovine IgG may contribute to immune protection against RSV.

Protein tyrosine phosphatase-PEST (PTP-PEST) regulates mast cell-activating signals in PTP activity-dependent and -independent manners.

Science.gov (United States)

Motohashi, Satoru; Koizumi, Karen; Honda, Reika; Maruyama, Atsuko; Palmer, Helen E F; Mashima, Keisuke

2014-01-01

Aggregation of the high-affinity IgE receptor (FcεRI) in mast cells leads to degranulation and production of numerous cytokines and lipid mediators that promote allergic inflammation. Tyrosine phosphorylation of proteins in response to FcεRI aggregation has been implicated in mast cell activation. Here, we determined the role of PTP-PEST (encoded by PTPN12) in the regulation of mast cell activation using the RBL-2H3 rat basophilic leukemia cell line as a model. PTP-PEST expression was significantly induced upon FcεRI-crosslinking, and aggregation of FcεRI induced the phosphorylation of PTP-PEST at Ser39, thus resulting in the suppression of PTP activity. By overexpressing a phosphatase-dead mutant (PTP-PEST CS) and a constitutively active mutant (PTP-PEST SA) in RBL-2H3 cells, we showed that PTP-PEST decreased degranulation and enhanced IL-4 and IL-13 transcription in FcεRI-crosslinked RBL-2H3 cells, but PTP activity of PTP-PEST was not necessary for this regulation. However, FcεRI-induced TNF-α transcription was increased by the overexpression of PTP-PEST SA and suppressed by the overexpression of PTP-PEST CS. Taken together, these results suggest that PTP-PEST is involved in the regulation of FcεRI-mediated mast cell activation through at least two different processes represented by PTP activity-dependent and -independent pathways. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy.

Science.gov (United States)

Singh, Surinder M; Bandi, Swati; Jones, David N M; Mallela, Krishna M G

2017-12-01

We examined how polysorbate 20 (PS20; Tween 20) and polysorbate 80 (PS80; Tween 80) affect the higher-order structure of a monoclonal antibody (mAb) and its antigen-binding (Fab) and crystallizable (Fc) fragments, using near-UV circular dichroism and 2D nuclear magnetic resonance (NMR). Both polysorbates bind to the mAb with submillimolar affinity. Binding causes significant changes in the tertiary structure of mAb with no changes in its secondary structure. 2D 13 C- 1 H methyl NMR indicates that with increasing concentration of polysorbates, the Fab region showed a decrease in crosspeak volumes. In addition to volume changes, PS20 caused significant changes in the chemical shifts compared to no changes in the case of PS80. No such changes in crosspeak volumes or chemical shifts were observed in the case of Fc region, indicating that polysorbates predominantly affect the Fab region compared to the Fc region. This differential effect of polysorbates on the Fab and Fc regions was because of the lesser thermodynamic stability of the Fab compared to the Fc. These results further indicate that PS80 is the preferred polysorbate for this mAb formulation, because it offers higher protection against aggregation, causes lesser structural perturbation, and has weaker binding affinity with fewer binding sites compared to PS20. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Cross-Linking GPVI-Fc by Anti-Fc Antibodies Potentiates Its Inhibition of Atherosclerotic Plaque- and Collagen-Induced Platelet Activation

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Janina Jamasbi, RPh

2016-04-01

Full Text Available To enhance the antithrombotic properties of recombinant glycoprotein VI fragment crystallizable (GPVI-Fc, the authors incubated GPVI-Fc with anti-human Fc antibodies to cross-link the Fc tails of GPVI-Fc. Cross-linking potentiated the inhibition of human plaque- and collagen-induced platelet aggregation by GPVI-Fc under static and flow conditions without increasing bleeding time in vitro. Cross-linking with anti-human-Fc Fab2 was even superior to anti-human-Fc immunoglobulin G (IgG. Advanced optical imaging revealed a continuous sheath-like coverage of collagen fibers by cross-linked GPVI-Fc complexes. Cross-linking of GPVI into oligomeric complexes provides a new, highly effective, and probably safe antithrombotic treatment as it suppresses platelet GPVI-plaque interaction selectively at the site of acute atherothrombosis.

Associations between an IgG3 polymorphism in the binding domain for FcRn, transplacental transfer of malaria-specific IgG3, and protection against Plasmodium falciparum malaria during infancy: A birth cohort study in Benin.

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Celia Dechavanne

2017-10-01

Full Text Available Transplacental transfer of maternal immunoglobulin G (IgG to the fetus helps to protect against malaria and other infections in infancy. Recent studies have emphasized the important role of malaria-specific IgG3 in malaria immunity, and its transfer may reduce the risk of malaria in infancy. Human IgGs are actively transferred across the placenta by binding the neonatal Fc receptor (FcRn expressed within the endosomes of the syncytiotrophoblastic membrane. Histidine at position 435 (H435 provides for optimal Fc-IgG binding. In contrast to other IgG subclasses, IgG3 is highly polymorphic and usually contains an arginine at position 435, which reduces its binding affinity to FcRn in vitro. The reduced binding to FcRn is associated with reduced transplacental transfer and reduced half-life of IgG3 in vivo. Some haplotypes of IgG3 have histidine at position 435. This study examines the hypotheses that the IgG3-H435 variant promotes increased transplacental transfer of malaria-specific antibodies and a prolonged IgG3 half-life in infants and that its presence correlates with protection against clinical malaria during infancy.In Benin, 497 mother-infant pairs were included in a longitudinal birth cohort. Both maternal and cord serum samples were assayed for levels of IgG1 and IgG3 specific for MSP119, MSP2 (both allelic families, 3D7 and FC27, MSP3, GLURP (both regions, R0 and R2, and AMA1 antigens of Plasmodium falciparum. Cord:maternal ratios were calculated. The maternal IgG3 gene was sequenced to identify the IgG3-H435 polymorphism. A multivariate logistic regression was used to examine the association between maternal IgG3-H435 polymorphism and transplacental transfer of IgG3, adjusting for hypergammaglobulinemia, maternal malaria, and infant malaria exposure. Twenty-four percent of Beninese women living in an area highly endemic for malaria had the IgG3-H435 allele (377 women homozygous for the IgG3-R435 allele, 117 women heterozygous for the Ig

Antibody Responses with Fc-Mediated Functions after Vaccination of HIV-Infected Subjects with Trivalent Influenza Vaccine

DEFF Research Database (Denmark)

Kristensen, Anne B; Lay, William N; Ana-Sosa-Batiz, Fernanda

2016-01-01

to immunize this at-risk group. IMPORTANCE: Infection with HIV is associated with increasing disease severity following influenza infections, and annual influenza vaccinations are recommended for this target group. However, HIV-infected individuals respond relatively poorly to vaccination compared to healthy......This study seeks to assess the ability of seasonal trivalent inactivated influenza vaccine (TIV) to induce nonneutralizing antibodies (Abs) with Fc-mediated functions in HIV-uninfected and HIV-infected subjects. Functional influenza-specific Ab responses were studied in 30 HIV-negative and 27 HIV......-positive subjects immunized against seasonal influenza. All 57 subjects received the 2015 TIV. Fc-mediated antihemagglutinin (anti-HA) Ab activity was measured in plasma before and 4 weeks after vaccination using Fc-receptor-binding assays, NK cell activation assays, and phagocytosis assays. At baseline, the HIV...

RI/MOM and RI/SMOM renormalization of overlap quark bilinears on domain wall fermion configurations

Science.gov (United States)

Bi, Yujiang; Cai, Hao; Chen, Ying; Gong, Ming; Liu, Keh-Fei; Liu, Zhaofeng; Yang, Yi-Bo; χ QCD Collaboration

2018-05-01

Renormalization constants (RCs) of overlap quark bilinear operators on 2 +1 -flavor domain wall fermion configurations are calculated by using the RI/MOM and RI/SMOM schemes. The scale independent RC for the axial vector current is computed by using a Ward identity. Then the RCs for the quark field and the vector, tensor, scalar, and pseudoscalar operators are calculated in both the RI/MOM and RI/SMOM schemes. The RCs are converted to the MS ¯ scheme and we compare the numerical results from using the two intermediate schemes. The lattice size is 4 83×96 and the inverse spacing 1 /a =1.730 (4 ) GeV .

Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

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Bingchun Zhao

2018-05-01

Full Text Available Epstein–Barr virus (EBV was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications.

Hydrodynamic delivery of plasmid DNA encoding human FcγR-Ig dimers blocks immune-complex mediated inflammation in mice.

Science.gov (United States)

Shashidharamurthy, R; Machiah, D; Bozeman, E N; Srivatsan, S; Patel, J; Cho, A; Jacob, J; Selvaraj, P

2012-09-01

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcγ receptor-Ig fusion molecules (FcγR-Igs) in mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness with purified molecules in blocking immune-complex (IC)-mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16A(F)-Ig, CD32A(R)-Ig and CD32A(H)-Ig) reached a maximum of 130 μg ml(-1) of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with the FcγR-Ig gene can be used to study the consequences of blocking IC binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.

Surveillance for Intracellular Antibody by Cytosolic Fc Receptor TRIM21

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William A. McEwan

2016-11-01

Full Text Available TRIM21 has emerged as an atypical Fc receptor that is broadly conserved and widely expressed in the cytoplasm of mammalian cells. Viruses that traffic surface-bound antibodies into the cell during infection recruit TRIM21 via a high affinity interaction between Fc and TRIM21 PRYSPRY domain. Following binding of intracellular antibody, TRIM21 acts as both antiviral effector and sensor for innate immune signalling. These activities serve to reduce viral replication by orders of magnitude in vitro and contribute to host survival during in vivo infection. Neutralization occurs rapidly after detection and requires the activity of the ubiquitin-proteasome system. The microbial targets of this arm of intracellular immunity are still being identified: TRIM21 activity has been reported following infection by several non-enveloped viruses and intracellular bacteria. These findings extend the sphere of influence of antibodies to the intracellular domain and have broad implications for immunity. TRIM21 has been implicated in the chronic auto-immune condition systemic lupus erythematosus and is itself an auto-antigen in Sjögren’s syndrome. This review summarises our current understanding of TRIM21’s role as a cytosolic Fc receptor and briefly discusses pathological circumstances where intracellular antibodies have been described, or are hypothesized to occur, and may benefit from further investigations of the role of TRIM21.

Imaging of injured and atherosclerotic arteries in mice using fluorescence-labeled glycoprotein VI-Fc

Energy Technology Data Exchange (ETDEWEB)

Bigalke, Boris, E-mail: boris.bigalke@med.uni-tuebingen.de [Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universitaet Tuebingen (Germany); Division of Imaging Sciences, St Thomas' Hospital, King' s College London (United Kingdom); Pohlmeyer, Ilka; Schoenberger, Tanja [Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universitaet Tuebingen (Germany); Griessinger, Christoph M. [Labor fuer Praeklinische Bildgebung und Bildgebungstechnologie der Werner-Siemens-Stiftung, Radiologische Klinik, Eberhard-Karls-Universitaet Tuebingen (Germany); Ungerer, Martin [Corimmun GmbH, Martinsried (Germany); Botnar, Rene M. [Division of Imaging Sciences, St Thomas' Hospital, King' s College London (United Kingdom); Pichler, Bernd J. [Labor fuer Praeklinische Bildgebung und Bildgebungstechnologie der Werner-Siemens-Stiftung, Radiologische Klinik, Eberhard-Karls-Universitaet Tuebingen (Germany); Gawaz, Meinrad [Medizinische Klinik III, Kardiologie und Kreislauferkrankungen, Eberhard-Karls-Universitaet Tuebingen (Germany)

2011-08-15

Purpose: To assess endothelial injury and repair using fluorescence-labeled glycoprotein VI (GPVI)-Fc in a murine model. Materials and methods: Three 4-week-old male ApoE-deficient (ApoE{sup -/-})-mice were fed with a 1.25% cholesterol diet over 16 weeks and compared to three wild type (WT) C57BL/6J-mice in a wire-induced vascular injury model. Another group of WT mice (n = 10) were mechanically injured by carotid ligation. Fluorescence-labeled GPVI-Fc (150 {mu}g/mouse) was administered and assessed by optical imaging 24 h after injury and compared to another group (n = 3) which was injected two days after injury and sacrificed another day later. Results: After denudation, all injured carotids of WT mice showed a higher mean fluorescence signal than the corresponding intact carotids of the same animals (48.4 {+-} 18.9 vs. 10.4 {+-} 1.0; P = 0.028). Injection of unlabeled GPVI-Fc 20 h and 3 h before injecting GPVI-Fc-FITC significantly reduced the fluorescence signal in injured carotids to 14.6 {+-} 4.6, while intact carotids showed a signal of 9.2 {+-} 1.1; P = 0.046. Ligation injury resulted with an increased GPVI-Fc-binding to injured carotids compared to intact carotids (31.53 {+-} 6.18 vs. 16.48 {+-} 5.15; P = 0.039). Three days after injury and 24 h after GPVI-Fc-FITC injection, differences between intact and injured carotids have vanished (12.51 {+-} 2.76 vs. 14.76 {+-} 1.59; P = 0.519). Conclusions: A GPVI-based plaque imaging system could help to identify vascular lesions and to take a precautionary measure as necessary.

Endogenous IL-1 in cognitive function and anxiety: a study in IL-1RI-/- mice.

Directory of Open Access Journals (Sweden)

Carol L Murray

Full Text Available Interleukin-1 (IL-1 is a key pro-inflammatory cytokine, produced predominantly by peripheral immune cells but also by glia and some neuronal populations within the brain. Its signalling is mediated via the binding of IL-1α or IL-1β to the interleukin-1 type one receptor (IL-1RI. IL-1 plays a key role in inflammation-induced sickness behaviour, resulting in depressed locomotor activity, decreased exploration, reduced food and water intake and acute cognitive deficits. Conversely, IL-1 has also been suggested to facilitate hippocampal-dependent learning and memory: IL-1RI(-/- mice have been reported to show deficits on tasks of visuospatial learning and memory. We sought to investigate whether there is a generalised hippocampal deficit in IL-1RI(-/- animals. Therefore, in the current study we compared wildtype (WT mice to IL-1RI(-/- mice using a variety of hippocampal-dependent learning and memory tasks, as well as tests of anxiety and locomotor activity. We found no difference in performance of the IL-1RI(-/- mice compared to WT mice in a T-maze working memory task. In addition, the IL-1RI(-/- mice showed normal learning in various spatial reference memory tasks including the Y-maze and Morris mater maze, although there was a subtle deficit in choice behaviour in a spatial discrimination, beacon watermaze task. IL-1RI(-/- mice also showed normal memory for visuospatial context in the contextual fear conditioning paradigm. In the open field, IL-1RI(-/- mice showed a significant increase in distance travelled and rearing behaviour compared to the WT mice and in the elevated plus-maze spent more time in the open arms than did the WT animals. The data suggest that, contrary to prior studies, IL-1RI(-/- mice are not robustly impaired on hippocampal-dependent memory and learning but do display open field hyperactivity and decreased anxiety compared to WT mice. The results argue for a careful evaluation of the roles of endogenous IL-1 in hippocampal

Dicty_cDB: FC-AC21 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AC21 (Link to dictyBase) - - - Contig-U15104-1 FC-AC21E (Li...nk to Original site) - - - - - - FC-AC21E 527 Show FC-AC21 Library FC (Link to library) Clone ID FC-AC21 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15104-1 Original site URL http://dict...ce KDSLDVIIFPEMVKLVGLTPNTMEKVLTYFQDNDTIDLSTFPMEIQVEQLSGKYIFICTH KQKDQRCGYCGPILVDQLRDQIKERSLEKEIQVFGTSHVGGHKY... Frames) Frame A: KDSLDVIIFPEMVKLVGLTPNTMEKVLTYFQDNDTIDLSTFPMEIQVEQLSGKYIFICTH KQ

Polydatin (PD) inhibits IgE-mediated passive cutaneous anaphylaxis in mice by stabilizing mast cells through modulating Ca{sup 2+} mobilization

Energy Technology Data Exchange (ETDEWEB)

Yuan, Meichun [Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen 518060 (China); Department of Physiology, Hubei University of Medicine, Shiyan (China); Li, Jianjie [State Key Laboratory of Respiratory Disease for Allergy at Shengzhen University, Shenzhen 518060 (China); Lv, Jingzhang [Shenzhen Entry-Exit Inspection and Quarantine Bureau, Shenzhen 518045 (China); Mo, Xucheng; Yang, Chengbin [State Key Laboratory of Respiratory Disease for Allergy at Shengzhen University, Shenzhen 518060 (China); Chen, Xiangdong [Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen 518060 (China); Liu, Zhigang [State Key Laboratory of Respiratory Disease for Allergy at Shengzhen University, Shenzhen 518060 (China); Liu, Jie, E-mail: ljljz@yahoo.com [Department of Pathophysiology, School of Medicine, Shenzhen University, Shenzhen 518060 (China)

2012-11-01

Mast cells play a key role in the pathogenesis of asthma and are a promising target for therapeutic intervention in asthma. This study investigated the effects of polydatin (PD), a resveratrol glucoside, on mast cell degranulation upon cross-linking of the high-affinity IgE receptors (FcεRI), as well as the anti-allergic activity of PD in vivo. Herein, we demonstrated that PD treatment for 30 min suppressed FcεRI-mediated mast cell degranulation in a dose-dependent manner. Concomitantly, PD significantly decreased FcεRI-mediated Ca{sup 2+} increase in mast cells. The suppressive effects of PD on FcεRI-mediated Ca{sup 2+} increase were largely inhibited by using LaCl{sub 3} to block the Ca{sup 2+} release-activated Ca{sup 2+} channels (CRACs). Furthermore, PD significantly inhibited Ca{sup 2+} entry through CRACs evoked by thapsigargin (TG). Knocking down protein expression of Orai1, the pore-forming subunit of CRACs, significantly decreased PD suppression of FcεRI-induced intracellular Ca{sup 2+} influx and mast cell degranulation. In a mouse model of mast cell-dependent passive cutaneous anaphylaxis (PCA), in vivo PD administration suppressed mast cell degranulation and inhibited anaphylaxis. Taken together, our data indicate that PD stabilizes mast cells by suppressing FcεRI-induced Ca{sup 2+} mobilization mainly through inhibiting Ca{sup 2+} entry via CRACs, thus exerting a protective effect against PCA. -- Highlights: ► Polydatin can prevent the pathogenesis of passive cutaneous anaphylaxis in mice. ► Polydatin stabilizes mast cells by decreasing FcεRI-mediated degranulation. ► Polydatin suppresses Ca{sup 2+} entry through CRAC channels in mast cells.

Polydatin (PD) inhibits IgE-mediated passive cutaneous anaphylaxis in mice by stabilizing mast cells through modulating Ca2+ mobilization

International Nuclear Information System (INIS)

Yuan, Meichun; Li, Jianjie; Lv, Jingzhang; Mo, Xucheng; Yang, Chengbin; Chen, Xiangdong; Liu, Zhigang; Liu, Jie

2012-01-01

Mast cells play a key role in the pathogenesis of asthma and are a promising target for therapeutic intervention in asthma. This study investigated the effects of polydatin (PD), a resveratrol glucoside, on mast cell degranulation upon cross-linking of the high-affinity IgE receptors (FcεRI), as well as the anti-allergic activity of PD in vivo. Herein, we demonstrated that PD treatment for 30 min suppressed FcεRI-mediated mast cell degranulation in a dose-dependent manner. Concomitantly, PD significantly decreased FcεRI-mediated Ca 2+ increase in mast cells. The suppressive effects of PD on FcεRI-mediated Ca 2+ increase were largely inhibited by using LaCl 3 to block the Ca 2+ release-activated Ca 2+ channels (CRACs). Furthermore, PD significantly inhibited Ca 2+ entry through CRACs evoked by thapsigargin (TG). Knocking down protein expression of Orai1, the pore-forming subunit of CRACs, significantly decreased PD suppression of FcεRI-induced intracellular Ca 2+ influx and mast cell degranulation. In a mouse model of mast cell-dependent passive cutaneous anaphylaxis (PCA), in vivo PD administration suppressed mast cell degranulation and inhibited anaphylaxis. Taken together, our data indicate that PD stabilizes mast cells by suppressing FcεRI-induced Ca 2+ mobilization mainly through inhibiting Ca 2+ entry via CRACs, thus exerting a protective effect against PCA. -- Highlights: ► Polydatin can prevent the pathogenesis of passive cutaneous anaphylaxis in mice. ► Polydatin stabilizes mast cells by decreasing FcεRI-mediated degranulation. ► Polydatin suppresses Ca 2+ entry through CRAC channels in mast cells.

RI management by personal computer

International Nuclear Information System (INIS)

Ono, Isamu; Hiyoshi, Katsunori; Ono, Kazuhiko; Morimitsu, Wataru

1983-01-01

For RI-handling facilities up to medium scale, it has been studied whether a personal computer is applicable to the practical management of radioisotopes. In the present system, the number of writing in mini floppy disks is 1280 articles per diskette, which is the sufficient capacity for a storage medium for one year in variety of books. The correction of radioactivity decay as well as various totalizations can be made easily, so that the state of RI storage and use for the whole RI-handling facility can be grasped. Further, by the improvement of the output formats, the transfer to the books as obligated to write is possible. From the above reason, a personal computer is practically applicable with the management system and also leads to labor saving in RI management personnel. (Mori, K.)

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

Science.gov (United States)

Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

2015-09-01

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Comparison of the Fc fragment from a human IgG1 and its CH2, pFc', and tFc' subfragments. A study using reductive methylation and 13C NMR

International Nuclear Information System (INIS)

Jentoft, J.E.; Rayford, R.

1989-01-01

The Fc fragment of a human monoclonal IgG1 was compared with subfragments containing (a) the intact CH2 domain (CH2 fragment) or (b) the intact CH3 domain (pFc' and tFc' fragments). All fragments were reductively 13 C-methylated and their resulting dimethyllysyl resonances characterized in 0.1 M KCl as a function of pH by 13 C NMR spectroscopy. Seven resonances were characterized for the 18 lysine residues of the Fc fragment, eight for the 12 lysines of the CH2 fragment, and five each for the 9 lysines of the pFc' and the 6 lysines of the tFc' fragments, respectively. The multiplicity of resonances indicates that the lysine residues in each fragment exist in a variety of microenvironments and that the fragments are all highly structured. The correspondence between 6 of the 12 or 13 perturbed lysine residues in the Fc fragment and the smaller subfragments indicates that the conformation of the CH2 and CH3 domains is largely unchanged in the smaller fragments. However, in addition to three lysines at the CH2-CH3 domain interface, whose environments were known to be disrupted in the smaller fragments, three or four lysine residues have somewhat different properties in the Fc fragment and in the subfragments, indicating that some local perturbations are included in the domain structure in the subfragments

RiArsB and RiMT-11: Two novel genes induced by arsenate in arbuscular mycorrhiza.

Science.gov (United States)

Maldonado-Mendoza, Ignacio E; Harrison, Maria J

Plants associated with arbuscular mycorrhizal fungi (AMF) increase their tolerance to arsenic-polluted soils. This study aims to investigate the genes involved in the AMF molecular response to arsenic pollution. Genes encoding proteins involved in arsenic metabolism were identified and their expression assessed by PCR or RT-qPCR. The As-inducible gene GiArsA (R. irregularis ABC ATPase component of the ArsAB arsenite efflux pump) and two new genes, an arsenate/arsenite permease component of ArsAB (RiArsB) and a methyltransferase type 11 (RiMT-11) were induced when arsenate was added to two-compartment in vitro monoxenic cultures of R. irregularis-transformed carrot roots. RiArsB and RiMT-11 expression in extraradical hyphae in response to arsenate displayed maximum induction 4-6 h after addition of 350 μM arsenate. Their expression was also detected in colonized root tissues grown in pots, or in the root-fungus compartment of two-compartment in vitro systems. We used a Medicago truncatula double mutant (mtpt4/mtpt8) to demonstrate that RiMT-11 and RiArsB transcripts accumulate in response to the addition of arsenate but not in response to phosphate. These results suggest that these genes respond to arsenate addition regardless of non-functional Pi symbiotic transport, and that RiMT-11 may be involved in arsenate detoxification by methylation in AMF-colonized tissues. Copyright © 2017 British Mycological Society. All rights reserved.

Dicty_cDB: FC-AY04 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AY04 (Link to dictyBase) - - - Contig-U16521-1 FC-AY04Z (Li...nk to Original site) - - FC-AY04Z 583 - - - - Show FC-AY04 Library FC (Link to library) Clone ID FC-AY04 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16521-1 Original site URL http://dict...10 4 CA753827 |AF402814_1 ( AF402814 ) 40S ribosomal protein S6 [Ictalurus puncta...0.00 m_ : 1.00 92.0 %: cytoplasmic 4.0 %: mitochondrial 4.0 %: nuclear >> prediction for FC-AY04 is cyt 5' e

Dicty_cDB: FC-AK01 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AK01 (Link to dictyBase) - - - Contig-U15038-1 FC-AK01E (Li...nk to Original site) - - - - - - FC-AK01E 996 Show FC-AK01 Library FC (Link to library) Clone ID FC-AK01 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15038-1 Original site URL http://dict...s clone omyk-evn... 172 6e-79 AF401557_1( AF401557 |pid:none) Ictalurus punctatus...reticulum 4.0 %: vacuolar >> prediction for FC-AK01 is mit 5' end seq. ID - 5' end seq. - Length of 5' end s

Development of Biochips and Kits using RI Detection Technology for Life Science

International Nuclear Information System (INIS)

Park, Sang Hyun; Park, H. J.; Choi, S. M.

2010-04-01

Microarrays have recently become a precious research tool for proteomics and clinical investigation. In this study, we report on an efficient strategy for the detection of phosphorylation of a substrate catalyzed by kinase, using the radioisotope (RI) detection technique for a protein biochip. The biochip for the detection of phosphorylation using a RI facilitates the detection of phosphorylation with minimum amount of a sample by simple processes, compared with the conventional method using an antibody. It was more than 30 times detection sensitivity compared to conventional fluorescence detection technique. This technique does not employ the use of the blocking step which is commonly used in conventional methods to prevent non-specific binding. ELSA method was more than seven steps, but the usage of RI detection technique reduces the overall steps. Consequently, this technique is very simple, and considerably reduces the experiment time thus facilitating in an easy detection. Furthermore, it is highly sensitivity, thus facilitating the detection of even minor quantities of protein and rapid data analysis. It can be a useful tool for a high throughput screening in biological and medical research, and for studying protein-protein interactions

NEW ROLES FOR FC RECEPTORS IN NEURODEGENERATION-THE IMPACT ON IMMUNOTHERAPY FOR ALZHEIMER’S DISEASE

Directory of Open Access Journals (Sweden)

James P. Fuller

2014-08-01

Full Text Available There are an estimated 18 million Alzheimer’s disease (AD sufferers worldwide and with no disease modifying treatment currently available, development of new therapies represents an enormous unmet clinical need. AD is characterised by episodic memory loss followed by severe cognitive decline and is associated with many neuropathological changes. AD is characterised by deposits of amyloid beta (Aβ, neurofibrillary tangles, and neuroinflammation. Active immunisation or passive immunisation against Aβ leads to the clearance of deposits in transgenic mice expressing human Aβ. This clearance is associated with reversal of associated cognitive deficits, but these results have failed to translate to humans, with both active and passive immunotherapy failing to improve memory loss. One explanation for these observations is that certain anti-Aβ antibodies mediate damage to the cerebral vasculature limiting the top dose and potentially reducing efficacy. Fc gamma receptors (Fcγ are a family of immunoglobulin like receptors which bind to the Fc portion of IgG, and mediate the response of effector cells to immune complexes. Data from both mouse and human studies suggest that cross-linking Fc receptors by therapeutic antibodies and the subsequent pro-inflammatory response mediates the vascular side effects seen following immunotherapy. Increasing evidence is emerging that Fc receptor expression on CNS resident cells, including microglia and neurons, is increased during aging and functionally involved in the pathogenesis of age-related neurodegenerative diseases. We propose that increased expression and ligation of Fc receptors in the CNS, either by endogenous IgG or therapeutic antibodies, has the potential to induce vascular damage and exacerbate neurodegeneration. To produce safe and effective immunotherapies for AD and other neurodegenerative diseases it will be vital to understand the role of Fc receptors in the healthy and diseased brain.

Dicty_cDB: FC-BF01 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BF01 (Link to dictyBase) - - - Contig-U15105-1 FC-BF01Z (Li...nk to Original site) - - FC-BF01Z 674 - - - - Show FC-BF01 Library FC (Link to library) Clone ID FC-BF01 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15105-1 Original site URL http://dict...402813_1( AF402813 |pid:none) Ictalurus punctatus 40S ribosomal ... 260 3e-68 AJ783868_1( AJ783868 |pid:none...lasmic 8.0 %: mitochondrial 4.0 %: nuclear >> prediction for FC-BF01 is cyt 5' end seq. ID - 5' end seq. - L

Dicty_cDB: FC-BC16 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BC16 (Link to dictyBase) - - - Contig-U15105-1 FC-BC16Z (Li...nk to Original site) - - FC-BC16Z 620 - - - - Show FC-BC16 Library FC (Link to library) Clone ID FC-BC16 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15105-1 Original site URL http://dict...4 2e-66 AY961522_1( AY961522 |pid:none) Lysiphlebus testaceipes ribosomal ... 254 2e-66 AF402813_1( AF402813 |pid:none) Ict...hondrial 4.0 %: nuclear >> prediction for FC-BC16 is cyt 5' end seq. ID - 5' end seq. - Length of 5' end seq

Use of RI-cone penetrometer in clay foundations

International Nuclear Information System (INIS)

Mimura, Mamoru; Shibata, Toru; Shrivastava, A.K.

1993-01-01

RI cone penetrometer tests are carried out at four different sites. The foundation grounds discussed here mainly consist of clayey materials. The measured results by RI cone penetrometers are shown for Kyobashi, Hachirougata, Kurihama and Kinkai Bay site. According to comparison of water content and density profiles by RI cone measurement with the conventional testing results, RI cone penetrometers are proved to be versatile tools for site investigation. Settlement assessment by RI cone penetrometer is also discussed by exemplifying the embankment at Kinkai Bay site. Elasto-vis-coplastic finite element analysis correspondingly performed strongly supports the RI cone based assessment. Repeated use of RI cone penetrometer with the advance of construction enables us to assess the consolidation process of the clay foundation. (author)

Contributions of F-BAR and SH2 domains of Fes protein tyrosine kinase for coupling to the FcepsilonRI pathway in mast cells.

Science.gov (United States)

McPherson, Victor A; Everingham, Stephanie; Karisch, Robert; Smith, Julie A; Udell, Christian M; Zheng, Jimin; Jia, Zongchao; Craig, Andrew W B

2009-01-01

This study investigates the roles of Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) and SH2 domains of Fes protein tyrosine kinase in regulating its activation and signaling downstream of the high-affinity immunoglobulin G (IgE) receptor (FcepsilonRI) in mast cells. Homology modeling of the Fes F-BAR domain revealed conservation of some basic residues implicated in phosphoinositide binding (R113/K114). The Fes F-BAR can bind phosphoinositides and induce tubulation of liposomes in vitro. Mutation of R113/K114 to uncharged residues (RK/QQ) caused a significant reduction in phosphoinositide binding in vitro and a more diffuse cytoplasmic localization in transfected COS-7 cells. RBL-2H3 mast cells expressing full-length Fes carrying the RK/QQ mutation show defects in FcepsilonRI-induced Fes tyrosine phosphorylation and degranulation compared to cells expressing wild-type Fes. This correlated with reduced localization to Lyn kinase-containing membrane fractions for the RK/QQ mutant compared to wild-type Fes in mast cells. The Fes SH2 domain also contributes to Fes signaling in mast cells, via interactions with the phosphorylated FcepsilonRI beta chain and the actin regulatory protein HS1. We show that Fes phosphorylates C-terminal tyrosine residues in HS1 implicated in actin stabilization. Thus, coordinated actions of the F-BAR and SH2 domains of Fes allow for coupling to FcepsilonRI signaling and potential regulation the actin reorganization in mast cells.

HAL/S-FC compiler system specifications

Science.gov (United States)

1976-01-01

This document specifies the informational interfaces within the HAL/S-FC compiler, and between the compiler and the external environment. This Compiler System Specification is for the HAL/S-FC compiler and its associated run time facilities which implement the full HAL/S language. The HAL/S-FC compiler is designed to operate stand-alone on any compatible IBM 360/370 computer and within the Software Development Laboratory (SDL) at NASA/JSC, Houston, Texas.

Bispecific engineered antibody domains (nanoantibodies that interact noncompetitively with an HIV-1 neutralizing epitope and FcRn.

Directory of Open Access Journals (Sweden)

Rui Gong

Full Text Available Libraries based on an isolated human immunoglobulin G1 (IgG1 constant domain 2 (CH2 have been previously diversified by random mutagenesis. However, native isolated CH2 is not very stable and the generation of many mutations could lead to an increase in immunogenicity. Recently, we demonstrated that engineering an additional disulfide bond and removing seven N-terminal residues results in an engineered antibody domain (eAd (m01s with highly increased stability and enhanced binding to human neonatal Fc receptor (FcRn (Gong et al, JBC, 2009 and 2011. We and others have also previously shown that grafting of the heavy chain complementarity region 3 (CDR-H3 (H3 onto cognate positions of the variable domain leads to highly diversified libraries from which a number of binders to various antigens have been selected. However, grafting of H3s to non-cognate positions in constant domains results in additional residues at the junctions of H3s and the CH2 framework. Here we describe a new method based on multi-step PCR that allows the precise replacement of loop FG (no changes in its flanking sequences by human H3s from another library. Using this method and limited mutagenesis of loops BC and DE we generated an eAd phage-displayed library. Panning of this library against an HIV-1 gp41 MPER peptide resulted in selection of a binder, m2a1, which neutralized HIV-1 isolates from different clades with modest activity and retained the m01s capability of binding to FcRn. This result provides a proof of concept that CH2-based antigen binders that also mimic to certain extent other functions of full-size antibodies (binding to FcRn can be generated; we have previously hypothesized that such binders can be made and coined the term nanoantibodies (nAbs. Further studies in animal models and in humans will show how useful nAbs could be as therapeutics and diagnostics.

Imaging of injured and atherosclerotic arteries in mice using fluorescence-labeled glycoprotein VI-Fc

International Nuclear Information System (INIS)

Bigalke, Boris; Pohlmeyer, Ilka; Schoenberger, Tanja; Griessinger, Christoph M.; Ungerer, Martin; Botnar, Rene M.; Pichler, Bernd J.; Gawaz, Meinrad

2011-01-01

Purpose: To assess endothelial injury and repair using fluorescence-labeled glycoprotein VI (GPVI)-Fc in a murine model. Materials and methods: Three 4-week-old male ApoE-deficient (ApoE -/- )-mice were fed with a 1.25% cholesterol diet over 16 weeks and compared to three wild type (WT) C57BL/6J-mice in a wire-induced vascular injury model. Another group of WT mice (n = 10) were mechanically injured by carotid ligation. Fluorescence-labeled GPVI-Fc (150 μg/mouse) was administered and assessed by optical imaging 24 h after injury and compared to another group (n = 3) which was injected two days after injury and sacrificed another day later. Results: After denudation, all injured carotids of WT mice showed a higher mean fluorescence signal than the corresponding intact carotids of the same animals (48.4 ± 18.9 vs. 10.4 ± 1.0; P = 0.028). Injection of unlabeled GPVI-Fc 20 h and 3 h before injecting GPVI-Fc-FITC significantly reduced the fluorescence signal in injured carotids to 14.6 ± 4.6, while intact carotids showed a signal of 9.2 ± 1.1; P = 0.046. Ligation injury resulted with an increased GPVI-Fc-binding to injured carotids compared to intact carotids (31.53 ± 6.18 vs. 16.48 ± 5.15; P = 0.039). Three days after injury and 24 h after GPVI-Fc-FITC injection, differences between intact and injured carotids have vanished (12.51 ± 2.76 vs. 14.76 ± 1.59; P = 0.519). Conclusions: A GPVI-based plaque imaging system could help to identify vascular lesions and to take a precautionary measure as necessary.

Dicty_cDB: FC-IC0102 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC-IC (Link to library) FC-IC0102 (Link to dictyBase) - - - Contig-U16527-1 FC-IC01...02F (Link to Original site) FC-IC0102F 434 - - - - - - Show FC-IC0102 Library FC-IC (Link to library) Clone ...ID FC-IC0102 (Link to dictyBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16527-1 Original site URL http://dict... (bits) Value N AB088483 |AB088483.1 Dictyostelium discoideum gene for gamete and mating-type specific prote...oducing significant alignments: (bits) Value AB088483_1( AB088483 |pid:none) Dictyostelium discoideum gmsA g

Dicty_cDB: FC-AA02 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AA02 (Link to dictyBase) - - - Contig-U16527-1 FC-AA02Z (Li...nk to Original site) - - FC-AA02Z 458 - - - - Show FC-AA02 Library FC (Link to library) Clone ID FC-AA02 (Link to dic...linum small subunit ribosomal RNA gene, partial sequence. 149 2e-47 2 AY179984 |AY179984.1 Uncultured alveo... CP000930_2283( CP000930 |pid:none) Heliobacterium modesticaldum Ic... 33 2.3 AP009386_1894( AP009386 |pid:none) Burkholderia multi... EU955514 |pid:none) Zea mays clone 1535262 hypothetica... 50 2e-05 BA000023_1285

Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column.

Science.gov (United States)

Kiyoshi, Masato; Caaveiro, Jose M M; Tada, Minoru; Tamura, Hiroko; Tanaka, Toru; Terao, Yosuke; Morante, Koldo; Harazono, Akira; Hashii, Noritaka; Shibata, Hiroko; Kuroda, Daisuke; Nagatoishi, Satoru; Oe, Seigo; Ide, Teruhiko; Tsumoto, Kouhei; Ishii-Watabe, Akiko

2018-03-02

The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.

A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

International Nuclear Information System (INIS)

Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan; Li, Lin; Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen; Jiang, Shibo

2010-01-01

Research highlights: → One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. → N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. → These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

Biologically active, magnICON®-expressed EPO-Fc from stably transformed Nicotiana benthamiana plants presenting tetra-antennary N-glycan structures.

Science.gov (United States)

Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Zabeau, Lennart; Tavernier, Jan; Delanghe, Joris R; Boets, Annemie; Castilho, Alexandra; Weterings, Koen

2012-08-31

In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized. Here, we report the transient magnICON(®) expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (Nagels et al., 2011). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (Bitonti et al., 2004). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants. Copyright © 2012 Elsevier B.V. All rights reserved.

Restricted processing of CD16a/Fc γ receptor IIIa N-glycans from primary human NK cells impacts structure and function.

Science.gov (United States)

Patel, Kashyap R; Roberts, Jacob T; Subedi, Ganesh P; Barb, Adam W

2018-03-09

CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates ( N -glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N -glycans (23%). These proportions indicated restricted N -glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N -glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N -glycoforms similar to NK cell-derived CD16a but yielded N -glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N -glycan composition affected antibody binding: CD16a with oligomannose N -glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N -glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N -glycan composition and antibody-binding affinity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

Science.gov (United States)

Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

2018-01-01

Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

Dicty_cDB: FC-BS24 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-BS24 (Link to dictyBase) - - - Contig-U16209-1 FC-BS24Z (Li...nk to Original site) - - FC-BS24Z 721 - - - - Show FC-BS24 Library FC (Link to library) Clone ID FC-BS24 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16209-1 Original site URL http://dict...nce. 52 9e-12 4 BQ096846 |BQ096846.1 IfHdk00487 Ictalurus furcatus head kidney cDNA library Ict...N SA ;, mRNA sequence. 44 1e-10 4 BM438323 |BM438323.1 IpLvr01076 Liver cDNA library Ictalurus punctatus cDN

Experimental investigation of small diameter two-phase closed thermosyphons charged with water, FC-84, FC-77 and FC-3283

International Nuclear Information System (INIS)

Jouhara, Hussam; Robinson, Anthony J.

2010-01-01

An experimental investigation of the performance of thermosyphons charged with water as well as the dielectric heat transfer liquids FC-84, FC-77 and FC-3283 has been carried out. The copper thermosyphon was 200 mm long with an inner diameter of 6 mm, which can be considered quite small compared with the vast majority of thermosyphons reported in the open literature. The evaporator length was 40 mm and the condenser length was 60 mm which corresponds with what might be expected in compact heat exchangers. With water as the working fluid two fluid loadings were investigated, that being 0.6 ml and 1.8 ml, corresponding to approximately half filled and overfilled evaporator section in order to ensure combined pool boiling and thin film evaporation/boiling and pool boiling only conditions, respectively. For the Fluorinert TM liquids, only the higher fill volume was tested as the aim was to investigate pool boiling opposed to thin film evaporation. Generally, the water-charged thermosyphon evaporator and condenser heat transfer characteristics compared well with available predictive correlations and theories. The thermal performance of the water-charged thermosyphon also outperformed the other three working fluids in both the effective thermal resistance as well as maximum heat transport capabilities. Even so, FC-84, the lowest saturation temperature fluid tested, shows marginal improvement in the heat transfer at low operating temperatures. All of the tested Fluorinert TM liquids offer the advantage of being dielectric fluids, which may be better suited for sensitive electronics cooling applications and were all found to provide adequate thermal performance up to approximately 30-50 W after which liquid entrainment compromised their performance.

The effect of FcγRIIA and FcγRIIB on coronary artery lesion formation and intravenous immunoglobulin treatment responses in children with Kawasaki disease

Science.gov (United States)

Chang, Ling-Sai; Lo, Mao-Hung; Li, Sung-Chou; Yang, Ming-Yu; Hsieh, Kai-Sheng; Kuo, Ho-Chang

2017-01-01

Previous research has found patients with the FcγRIIIB NA1 variant having increased risk of intravenous immunoglobulin (IVIG) resistance in Kawasaki disease (KD). Our previous studies revealed that elevated FcγRIIA expression correlated with the susceptibility of KD patients. We conducted this research to determine whether and how Fcγ receptors affect the susceptibility, IVIG treatment response, and coronary artery lesions (CAL) of KD patients. The activating FcγRIIA and inhibitory FcγRIIB methylation levels of seven patients with KD and four control subjects were examined using HumanMethylation27 BeadChip. We enrolled a total of 44 KD patients and 10 control subjects with fevers. We performed real-time RT-PCR to determine the FcγRIIA and FcγRIIB expression levels, as well as a luciferase assay of FcγRIIA. We found a considerable increase in methylation of both FcγRIIA and FcγRIIB in KD patients undergoing IVIG treatment. Promoter methylation of FcγRIIA inhibited reporter activity in K562 cells using luciferase assay. The FcγRIIB mRNA expression levels were not found to increase susceptibility, CAL formation, or IVIG resistance. FcγRIIA mRNA expression levels were significantly higher in IVIG-resistant patients than in those that responded to IVIG during the pre-treatment period. Furthermore, the FcγRIIA/IIB mRNA expression ratio was considerably higher in KD patients with CAL than in those without CAL. FcγRIIA and FcγRIIB both demonstrated increased methylation levels in KD patients that underwent IVIG treatment. FcγRIIA expression influenced the IVIG treatment response of KD patients. The FcγRIIA/IIB mRNA expression ratio was greater in KD patients with CAL formation. PMID:27893416

Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.

Science.gov (United States)

Hasenhindl, Christoph; Traxlmayr, Michael W; Wozniak-Knopp, Gordana; Jones, Phil C; Stadlmayr, Gerhard; Rüker, Florian; Obinger, Christian

2013-10-01

Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

Presence of pRI1:

DEFF Research Database (Denmark)

Migura, Lourdes Garcia; Hasman, Henrik; Jensen, Lars Bogø

2009-01-01

This study focused on the molecular characterization of a small cryptic, mobilizable plasmid (6038 bp) sequenced from an E. faecium 9631160-1 of poultry origin. Sequence analysis of pRI1 revealed seven open reading frames. pRI1 contained an IS 100% identical to ISEfa4. This insertion element...... almost identical to the repA from the pEFNP1 and pKQ10 plasmids from E. faecium was also identified. Presence of the pRI1 replication initiation gene (rep) was analyzed in a panel of 159 E. faecium isolates of human and animal origin from different European countries, of which 60 tested positive...

Discussion on the tropospheric concentrations of FC21

Energy Technology Data Exchange (ETDEWEB)

Crescentini, G.; Mangani, F.; Mastrogiamcomo, A.R.; Cappiello, A.; Bruner, F.

1986-01-01

FC21 tropospheric mixing ratios measured in air samples collected at different locations in the world are presented. Results of a campaign carried out at two locations in the Sahara desert where FC21 and FC11 mixing ratios were simultaneously determined in 180 samples are also shown. Though scattered high values have been found, the average background concentration of FC21 ranged between 0 and 1 pptv. 9 references, 1 figure, 2 tables.

The number of FceRI receptors on basophils decreases during subcutaneous immunotherapy

DEFF Research Database (Denmark)

Schmid, J. M.; Dahl, R.; Hoffmann, H. J.

2015-01-01

Background: Allergen specific immunotherapy is the only disease modifying treatment of allergic diseases. It induces complex cellular and humoral changes leading to an inhibition of type-1 allergic reactions. Method: Twenty four young grass pollen allergic adults suffering from seasonal rhino......-conjunctivitis were randomized to receive standard subcutaneous immunotherapy (n = 18) or to an open control group (n = 6). The number of FceRI receptors on basophils was measured by quantitative analysis of indirect immunofluorescence by flow cytometry. Grass binding to basophils was measured by staining with grass......)/basophil in the treatment group (n = 0.016), while it remained constant in the control group (pre: 102424; post: 103753). We found only minor changes in maximal grass binding on the basophils. The allergen concentration leading to halfmaximum grass binding decreased slightly during SCIT, whereas the EC50...

Fc-fusion technology and recombinant FVIII and FIX in the management of the hemophilias.

Science.gov (United States)

Mancuso, Maria Elisa; Mannucci, Pier Mannuccio

2014-01-01

Prophylaxis with regular infusions of factor VIII (FVIII)- or factor IX (FIX)- containing products is the mainstay of modern hemophilia care. However, this therapeutic regimen is inconvenient, requiring repeated intravenous injections from childhood. Approaches meant to prolong the half-life of FVIII and FIX in plasma have been developed in order to improve the feasibility and acceptability of replacement therapy, extending protection from bleeding, reducing infusion frequency and hence the need for venous access devices in young children. Several strategies have been implemented to enhance the pharmacokinetics of clotting factors, including conjugation with polyethylene glycol and the production by genetic engineering of fusion proteins containing the coagulation factors linked to a long-lived plasma protein such as albumin or the Fc fragment of immunoglobulin (Ig)G. The latter technology is one of the most promising, since the prolongation of FVIII and FIX half-life is obtained by exploiting the physiological binding of the Fc domain to the neonatal Fc receptor. Fc fusion monomers have been obtained with both recombinant FVIII (rFVIIIFc) and FIX (rFIXFc), and data from preclinical and clinical studies showed improved pharmacokinetics for both factors, which are produced in human embryonic kidney (HEK) 293 cells, thus ensuring full human post-translational modifications. In Phase I/IIa studies, rFVIIIFc and rFIXFc showed 1.5-1.7 fold and 3.0-4.0 fold longer elimination half-life, respectively. Similar data have been obtained in the Phase III clinical studies with rFVIIIFc and rFIX-Fc published recently. Both drugs were satisfactorily safe, particularly with respect to immunogenicity, and no serious adverse event was observed.

Szathmári, a great documentary artist

Directory of Open Access Journals (Sweden)

Ionescu, Adrian-Silvan

2014-01-01

Full Text Available Carol Pop de Szathmári was born in Cluj, Transylvania, on 11 January 1812. His talent for painting shone out from an early age. Being a passionate traveller, Szathmári journeyed through Europe and often crossed the Carpathian Mountains to visit Wallachia and its capital, Bucharest, where he eventually settled in 1843. An accomplished landscape and portrait painter, at ease with both watercolours and oil paints, Szathmári obtained commissions from the wealthy Wallachian boyars. Szathmári kept up constant, good relations with the successive ruling princes of Wallachia for whom he painted portraits and various other compositions. By 1848, Szathmári began to experiment with photography. The outbreak of the Russian-Ottoman War in late June 1853 saw the Romanian principalities occupied by the Russian army. In April 1854, Szathmári filled a van with his cameras and glass plates and went to the Danube to document the fighting between the Russian and Turkish armies.The result of Szathmári's bravery and hard work was a photographic album. His album, containing some two hundred images, became famous due to its presentation at the 1855 Paris World's Fair and Szathmári was awarded the Second Class Medal for his work.From that time on, photography, painting and lithography were always closely connected in Szathmári's career. In 1864 he became a member of the Société Française de Photographie in Paris and in 1870 of the one in Vienna. In 1863, he was appointed Court Painter and Photographer, a position he held for the rest of his life. In that capacity, he followed his patron, Prince Carol I, on the battlefield during the Russian-Romanian-Ottoman War of 1877, which was waged south of the Danube. Alongside martial compositions and albums, Szathmári had long been attracted by folk types and produced a large series of pictures of peasants, gypsies, postillions, merchants and artisans. He toured the fairs and the crowded streets of the town in search

Equilibrium and kinetics of Sin Nombre hantavirus binding at DAF/CD55 functionalized bead surfaces.

Science.gov (United States)

Buranda, Tione; Swanson, Scarlett; Bondu, Virginie; Schaefer, Leah; Maclean, James; Mo, Zhenzhen; Wycoff, Keith; Belle, Archana; Hjelle, Brian

2014-03-10

Decay accelerating factor (DAF/CD55) is targeted by many pathogens for cell entry. It has been implicated as a co-receptor for hantaviruses. To examine the binding of hantaviruses to DAF, we describe the use of Protein G beads for binding human IgG Fc domain-functionalized DAF ((DAF)₂-Fc). When mixed with Protein G beads the resulting DAF beads can be used as a generalizable platform for measuring kinetic and equilibrium binding constants of DAF binding targets. The hantavirus interaction has high affinity (24-30 nM; k(on) ~ 10⁵ M⁻¹ s⁻¹, k(off) ~ 0.0045 s⁻¹). The bivalent (DAF)₂-Fc/SNV data agree with hantavirus binding to DAF expressed on Tanoue B cells (K(d) = 14.0 nM). Monovalent affinity interaction between SNV and recombinant DAF of 58.0 nM is determined from competition binding. This study serves a dual purpose of presenting a convenient and quantitative approach of measuring binding affinities between DAF and the many cognate viral and bacterial ligands and providing new data on the binding constant of DAF and Sin Nombre hantavirus. Knowledge of the equilibrium binding constant allows for the determination of the relative fractions of bound and free virus particles in cell entry assays. This is important for drug discovery assays for cell entry inhibitors.

Jüri Talveti luulevalimik hispaania keeles

Index Scriptorium Estoniae

2003-01-01

Talvet, Jüri. Elegia Estonia y otros poemas : [luulevalimik] / tõlkinud Jüri Talvet ja Albert Lázaro Tinaut ; järelsõna Janika Kronberg. Valencia : Palmart Capitelum, 2002; Vt. ka Eesti Päevaleht, 13. jaan., lk. 16

Development of RI Target Production Technology

International Nuclear Information System (INIS)

Jeong, Do Young; Ko, Kwang Hoon; Kim, Cheol Jung; Kim, Taek Soo; Rho, Si Pyo; Park, Hyun Min; Lim, Gwon; Cha, Yong Ho; Han, Jae Min

2010-04-01

This project was accomplished with an aim of productive technical development on the 'enriched target' which is used essentially in radioisotope production. The research was advanced systematically with target production pilot system configuration and core technical development. We composed Yb-176 productive pilot system which equip the chemical purification technique of medical treatment level and proved its capability. Possibilities to separate Zn-67 by the method of using the polarizing light in principle and to separate Zn-70 by the method of using the double optical pumping in theory were also proved. RI target production technologies are recognized excessively with monopolistic techniques of part atomic energy advanced nations such as Russia and US and they are come, but we prepared the opportunity will be able to complete a full cycle of like (RI material production -> RI target production -> RI application) with this project accomplishment. When considering only the direct demand of stable isotope which is used in various industrial, we forecast with the fact that RI target markets will become larger with the approximately 5 billion dollars in 2020 and this technology will contribute in the domestic rising industry creation with high value added

Riigi äriühingu juhtorgani liige: kas ametiisik või ärijuht? / Jüri Raidla, Toomas Vaher, Kristel Urke

Index Scriptorium Estoniae

Raidla, Jüri, 1957-

2017-01-01

Riigi äriühingu põhijoontest, riigi äriühingu juhtorgani liikme kohustustest ja nõuetest juhtorgani liikmetele. Riigi äriühingute juhtorganite liikmete pidamisest ametiisikuteks korruptsioonivastase seaduse § 2 lõike 1 ja karistusseadustiku § 288 lõike 1 mõttes. Sisaldab asjakohast kohtupraktikat

A potent human neutralizing antibody Fc-dependently reduces established HBV infections.

Science.gov (United States)

Li, Dan; He, Wenhui; Liu, Ximing; Zheng, Sanduo; Qi, Yonghe; Li, Huiyu; Mao, Fengfeng; Liu, Juan; Sun, Yinyan; Pan, Lijing; Du, Kaixin; Ye, Keqiong; Li, Wenhui; Sui, Jianhua

2017-09-26

Hepatitis B virus (HBV) infection is a major global health problem. Currently-available therapies are ineffective in curing chronic HBV infection. HBV and its satellite hepatitis D virus (HDV) infect hepatocytes via binding of the preS1 domain of its large envelope protein to sodium taurocholate cotransporting polypeptide (NTCP). Here, we developed novel human monoclonal antibodies that block the engagement of preS1 with NTCP and neutralize HBV and HDV with high potency. One antibody, 2H5-A14, functions at picomolar level and exhibited neutralization-activity-mediated prophylactic effects. It also acts therapeutically by eliciting antibody-Fc-dependent immunological effector functions that impose durable suppression of viral infection in HBV-infected mice, resulting in reductions in the levels of the small envelope antigen and viral DNA, with no emergence of escape mutants. Our results illustrate a novel antibody-Fc-dependent approach for HBV treatment and suggest 2H5-A14 as a novel clinical candidate for HBV prevention and treatment of chronic HBV infection.

Macrophage colony stimulating factor (M-CSF) induces Fc receptor expression on macrophages

International Nuclear Information System (INIS)

Magee, D.M.; Wing, E.J.; Waheed, A.; Shadduck, R.K.

1986-01-01

M-CSF is a glycoprotein that stimulates bone marrow progenitor cells to proliferate and differentiate into macrophages (M theta). In addition, M-CSF can modulate the function of mature M theta. In this study, the authors determined the effect of M-CSF on expression of receptors for IgG (Fc receptors). Murine resident peritoneal M theta monolayers were incubated with either M-CSF, recombinant gamma interferon (IFN), or left untreated for 48 hrs. Expression of Fc receptors was assessed by microscopy using an antibody coated sheet erythrocytes (EA) rosette assay. The results indicated that M-CSF treated M theta had significantly higher numbers of bound EA (7.1 erythrocytes/M theta), than IFN M theta (4.4), or untreated M theta (2.5) (p 51 Cr labelled EA assay, CSF M theta (16,411 cpm), IFN M theta (10,887), untreated M theta (6897) (p < 0.001). Additionally, the maximal response was noted between 10 and 500 units M-CSF. Purified anti-M-CSF IgG, when included in the cultures, ablated the enhancement of EA binding, whereas normal rabbit IgG did not. These findings indicate that M-CSF is a potent inducer of Fc receptor expression on M theta and supports other data concerning the role of M-CSF as a biological response modifier

Peptidomimetic ligands for the tandem SH2 domain of Syk kinase

NARCIS (Netherlands)

Kuil, J.

2009-01-01

The Spleen tyrosine kinase (Syk) protein functions as a switch in a number of receptor signaling cascades. One of these cascades is the high affinity IgE receptor (Fc?RI) signaling pathway. Fc?RI consists of an ?-, ?- and two ?-chains. The ?- and ?-chains have intracellular an Immunoreceptor

A Peptide-Fc Opsonin with Pan-Amyloid Reactivity

Directory of Open Access Journals (Sweden)

James S. Foster

2017-09-01

Full Text Available There is a continuing need for therapeutic interventions for patients with the protein misfolding disorders that result in systemic amyloidosis. Recently, specific antibodies have been employed to treat AL amyloidosis by opsonizing tissue amyloid deposits thereby inducing cell-mediated dissolution and organ improvement. To develop a pan-amyloid therapeutic agent, we have produced an Fc-fusion product incorporating a peptide, p5, which binds many if not all forms of amyloid. This protein, designated Fcp5, expressed in mammalian cells, forms the desired bivalent dimer structure and retains pan-amyloid reactivity similar to the p5 peptide as measured by immunosorbent assays, immunohistochemistry, surface plasmon resonance, and pulldown assays using radioiodinated Fcp5. Additionally, Fcp5 was capable of opsonizing amyloid fibrils in vitro using a pH-sensitive fluorescence assay of phagocytosis. In mice,125 I-labeled Fcp5 exhibited an extended serum circulation time, relative to the p5 peptide. It specifically bound AA amyloid deposits in diseased mice, as evidenced by biodistribution and microautoradiographic methods, which coincided with an increase in active, Iba-1-positive macrophages in the liver at 48 h postinjection of Fcp5. In healthy mice, no specific tissue accumulation was observed. The data indicate that polybasic, pan-amyloid-targeting peptides, in the context of an Fc fusion, can yield amyloid reactive, opsonizing reagents that may serve as next-generation immunotherapeutics.

Medical RI development plan of KOMAC

Science.gov (United States)

Kim, Kye-Ryung; Jung, Myung-Hwan; Yoon, Sang-Pil; Min, Yi-Sub; Cho, Yong-Sub

2017-12-01

Many kinds of radioisotopes (RIs) produced by the high energy (100 200 MeV) proton accelerators are developed by the foreign R&D institutes and the worldwide demands are being increased continuously. The RI production using high energy proton beam higher than 50 MeV was not considerable because of the limit of the proton beam energy from existing proton accelerator facilities in Korea before 2013. The available maximum proton energy was 50 MeV from MC-50 cyclotron of Korea Institute of Radiological and Medical Sciences (KIRAMS) at that time. After the construction of a 100 MeV high-current and high-energy proton accelerator and a new irradiation facility for the RI production in 2013 and 2016 by the Korea Multi-purpose Accelerator Complex (KOMAC) at Korea Atomic Energy Research Institute (KAERI), we can make a plan for the new RI production of Cu-67, Sr-82 and so on. In the medical application fields, the worldwide demand of Sr-82 is being increased rapidly during last several years and the domestic demand of Cu-67 is also expected to be increased in near future. And alpha-emitters, such as Ac-225 and Ra-223, are becoming attractive to the users in the medical science fields in the future. The RI development plan of KOMAC was specified recently reflecting the recent environment changes and requirements from the users. In this paper, the results and present status of RI production and R&D facilities, calculation results related to the RI production yields, and future plans is presented.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

Science.gov (United States)

Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F; Mo, Xiaokui; Byrd, John C; Carson, William E; Butchar, Jonathan P; Tridandapani, Susheela

2016-02-05

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. © 2016 by The American Society for Biochemistry

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function*

Science.gov (United States)

Ren, Li; Campbell, Amanda; Fang, Huiqing; Gautam, Shalini; Elavazhagan, Saranya; Fatehchand, Kavin; Mehta, Payal; Stiff, Andrew; Reader, Brenda F.; Mo, Xiaokui; Byrd, John C.; Carson, William E.; Butchar, Jonathan P.; Tridandapani, Susheela

2016-01-01

The irreversible Bruton's tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function. PMID:26627823

Mannan-binding lectin is involved in the protection against renal ischemia/ reperfusion injury by dietary restriction

NARCIS (Netherlands)

Shushimita; P. van der Pol (Pieter); R.W.F. de Bruin (Ron); J.N.M. IJzermans (Jan); C. van Kooten (Cees); F.J.M.F. Dor (Frank)

2015-01-01

textabstractPreoperative fasting and dietary restriction offer robust protection against renal ischemia/ reperfusion injury (I/RI) in mice.We recently showed that Mannan-binding lectin (MBL), the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on

Optimization on Fc for Improvement of Stability and Aggregation Resistance.

Science.gov (United States)

Chen, Xiaobo; Zeng, Fang; Huang, Tao; Cheng, Liang; Liu, Huan; Gong, Rui

2016-01-01

Fc-based therapeutics including therapeutic full-size monoclonal antibodies (mAbs) and Fcfusion proteins represent fastest-growing market in biopharmaceutical industrial. However, one major challenge during development of Fc-based therapeutics is how to maintain their efficacy in clinic use. Many factors may lead to failure in final marketing. For example, the stability and aggregation resistance might not be high enough for bearing the disadvantages during fermentation, purification, formulation, storage, shipment and other steps in manufacture and sale. Low stability and high aggregation tendency lead to decreased bioactivity and increased risk of immunogenicity resulting in serious side effect. Because Fc is one of the major parts in monoclonal antibodies and Fc-fusion proteins, engineering of Fc to increase its stability and reduce or eliminate aggregation due to incorrect association are of great importance and could further extend the potential of Fc-based therapeutics. Lots of studies focus on Fc optimization for better physical and chemical characteristics and function by structured-based computer-aid rational design, high-throughput screening expression system selection and other methods. The identification of optimized Fc mutants increases the clinic potential of currently existed therapeutics mAbs and Fc-fusion proteins, and accelerates the development of new Fc-based therapeutics. Here we provide an overview of the related field, and discuss recent advances and future directions in optimization of Fc-based therapeutics with modified stability and aggregation resistance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Dicty_cDB: FC-AJ15 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available FC (Link to library) FC-AJ15 (Link to dictyBase) - G01152 DDB0232964 Contig-U16520-...to library) Clone ID FC-AJ15 (Link to dictyBase) Atlas ID - NBRP ID G01152 dictyBase ID DDB0232964 Link to C...ontig Contig-U16520-1 Original site URL http://dictycdb.biol.tsukuba.ac.jp/CSM/FC...KIEFPLPDIKTKRKIFEI HTAKMNLSEDVNLEEFVMSKDDLSGADIKAICTESGLLALRERRMRVTHTDFKKAKEKVL YRKTAGAPEGLYM*kkknqnqk Trans...vih*qlviwrrlsmxitpschqp*tralcpyhvfv--- ---ELLNQLDGFDASTDVKVIMATNRIETLDPALIRPGRIDRKIEFPLPDIKTKRKIFEI HTAKMNLSEDVNLEEFVMSKDDLSGADIKAICT

Jüri Ratas : Tallinnasse rahvuspark! / Jüri Ratas ; interv. Ilmar Palli

Index Scriptorium Estoniae

Ratas, Jüri, 1978-

2005-01-01

Tallinna uus linnapea Jüri Ratas rohelisest vööndist, keskkonnakaitsest, ohtlikest veostest, teede olukorrast, linna elukeskkonnast, asotsiaalide probleemist ning kohaliku omavalitsuse ja riigi koostööst

SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G.

Science.gov (United States)

Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, Manfred; Fulde, Marcus

2017-01-01

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm -positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis , respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis . The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.

Immunization with avian metapneumovirus harboring chicken Fc induces higher immune responses.

Science.gov (United States)

Paudel, Sarita; Easwaran, Maheswaran; Jang, Hyun; Jung, Ho-Kyoung; Kim, Joo-Hun; Shin, Hyun-Jin

2016-07-15

In this study, we evaluated the immune responses of avian metapneumovirus harboring chicken Fc molecule. Stable Vero cells expressing chicken Fc chimera on its surface (Vero-cFc) were established, and we confirmed that aMPV grown in Vero-cFc incorporated host derived chimera Fc into the aMPV virions. Immunization of chicken with aMPV-cFc induced higher level of antibodies and inflammatory cytokines; (Interferon (IFN)-γ and Interleukin (IL)-1β) compared to those of aMPV. The increased levels of antibodies and inflammatory cytokines in chicken immunized with aMPV-cFc were statistically significantly (p<0.05) to that of aMPV and control. The aMPV-cFc group also generated the highest neutralizing antibody response. After challenges, chickens immunized with aMPV-cFc showed much less pathological signs in nasal turbinates and trachea so that we could confirm aMPV-cFc induced higher protection than that of aMPV. The greater ability of aMPV harboring chicken Fc to that of aMPV presented it as a possible vaccine candidate. Copyright © 2016 Elsevier B.V. All rights reserved.

RIKEN RI Beam Factory project

Energy Technology Data Exchange (ETDEWEB)

Yano, Yasushige; Goto, Akira; Katayama, Takeshi [Institute of Physical and Chemical Research, Wako, Saitama (Japan)

1997-03-01

The RARF proposes `RIKEN RI Beam Factory` as a next facility-expanding project. The factory makes it the primary aim to provide RI (Radioactive Isotope) beams covering over the whole atomic-mass range with the world-highest intensity in a wide energy range up to several hundreds MeV/nucleon. These RI beams are generated by the fragmentation of high-intensity heavy-ion beams. For the efficient production heavy-ion energies will be boosted up to over 100 MeV/nucleon even for very heavy ions by a K2500-MeV superconducting ring cyclotron serving as a post accelerator of the existing K540-MeV ring cyclotron. A new type of experimental installation called `MUSES` (Multi-USe Experimental Storage rings) will be constructed as well. With MUSES, various types of unique colliding experiments will become possible. (author)

Policy Activities in Europlanet 2020 RI

Science.gov (United States)

Giacomini, L.; Heward, A.; Mason, N.

2017-09-01

The Europlanet 2020 Research Infrastructure (RI) has received 9.945 million Euros from the European Commission to integrate planetary science across Europe, provide access to facilities, develop tools and build community cohesion. To help these processes and to increase engagement between our policy makers and the planetary science community, part of Europlanet 2020 RI's efforts are dedicated to building connections and organising activities for and within the European Parliament. Since September 2015, Europlanet 2020 RI has contacted all 134 Members of the European Parliament (MEPs) on the ITRE Committee. More than 20 individual briefings have been held to date with MEPS and/or their representatives. In November 2016, Europlanet 2020 RI organized a very successful exhibition in the European Parliament as part of the 8th European Innovation Summit and the STOA Annual Lecture, and a dinner debates was held in the on the 'Impact of the EU on planetary science' in April 2016. These events enable members of the Europlanet community, politicians and interested parties to come together and discuss views on topics of interest or concern to the space and planetary sectors. Efforts in recent years have led to important opportunities for our community to feed into reporting and consultative processes. In this talk we will discuss the results achieved in the last two years of activities and the next steps foreseen by Europlanet 2020 RI.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

Science.gov (United States)

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

2016-10-01

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

Science.gov (United States)

Schlothauer, Tilman; Rueger, Petra; Stracke, Jan Olaf; Hertenberger, Hubert; Fingas, Felix; Kling, Lothar; Emrich, Thomas; Drabner, Georg; Seeber, Stefan; Auer, Johannes; Koch, Stefan; Papadimitriou, Apollon

2013-01-01

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity. PMID:23765230

Feasibility study on RI biochip Application to detection of risk factors of atherosclerosis

International Nuclear Information System (INIS)

Ko, Kyong Cheol; Choi, Mi Hee; Park, Sang Hyun; Cho, Kyung Hyun; Lee, Ki Teak

2009-01-01

Microarrays can be used to screen thousands of binding events in a parallel and high throughput fashion and are of major importance in discase diagnosis and drug discovery. The use of radioisotope is conventionally regarded as one of the most sensitive detection methods. Atherosclerosis is a common disorder affecting arterial blood vessels. It happens when fat, cholesterol, and other substances made in the arterial blood vessels form a hard substances called plaque. Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), a phospholipase A 2 enzyme, is used as a marker for cardiac disease. The detection of Lp-PLA 2 was accomplished by using radioactive [ 3 H-acetyl] PAF as a substrate and a feasibility study on RI biochip application to detection of Lp-PLA 2 , a risk factors of atherosclerosis was performed. Inhibitive activity of a native plant extract was also determined by using the RI biochip. It was found to be applicable to a high-throughput screening of inhibitors for developing atherosclerosis therapeutic agents

Application of RI-counting method for posttraumatic CSF rhinorrhea

International Nuclear Information System (INIS)

Itoh, Takahiko; Terai, Yoshinori; Fujimoto, Shunichiro; Kawauchi, Masamitsu.

1987-01-01

Numerous techniques and procedures have been reported for the evaluation of CSF fistulas. Especially metrizamide CT cisternography and radioisotope (RI) cisternography have been reported to be reliable for localizing the site of CSF leakage, however, it has been difficult to diagnose the existence and the site of CSF leakages in some cases. RI-counting method, measuring RI-count of intranasal cotton pledgets after the intrathecal injection of RI ( 111 In-DTPA) is thought to be the most reliable method for detecting the presence of CSF leakage in these cases. We used six cotton pledgets which were inserted into upper, middle, and lower meatuses on both side. Because the site of pledgets with ghest RI-count has anatomical relationship to the opening of the paranasal sinus, we can surmise the leakage of dural defect and CSF leakage. RI counting method was applied to two patients in whom it was difficult to diagnose the presence of CSF leakage with other procedures. The patients were free in position for four hours after the intrathecal injection of RI, and in the prone position for 30 minutes before RI-counting of intranasal cotton pledgets. After measuring the RI-counts of six pledgets, the counts were compared with each other. The cotton pledget inserted into left middle meatus showed the highest count in both cases. From this result and finding of conventional skull tomography we speculated the site of CSF leakage to be frontal sinus or ethmoid sinus. Operation demonstrated the opening of dura into the frontal sinus in one case, and ethmoid sinus in another case. As mentioned above, RI-counting method has the advantages of detecting the existence and the site of CSF leakage. (author)

Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

Science.gov (United States)

Liu, Liming

2015-06-01

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Spallation RI beam facility and heavy element nuclear chemistry

Energy Technology Data Exchange (ETDEWEB)

Nagame, Yuichiro [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment

1997-11-01

An outline of the spallation RI (Radioactive Ion) beam facility is presented. Neutron-rich nuclides are produced in the reaction of high intensity (10-1000 {mu}A) protons with energy of 1.5 GeV and an uranium carbide target. Produced nuclides are ionized in an isotope separator on-line (ISOL) and accelerated by the JAERI tandem and the booster linac. Current progress and a future project on the development of the RI beam facility are given. Studies of transactinide elements, including the synthesis of superheavy elements, nuclear structure far from stability, and RI-probed material science are planned with RI beams. An outlook of the transactinide nuclear chemistry studies using neutron-rich RI beams is described. (author)

Glycosylation of immunoglobulin A influences its receptor binding.

Science.gov (United States)

Basset, C; Devauchelle, V; Durand, V; Jamin, C; Pennec, Y L; Youinou, P; Dueymes, M

1999-12-01

Immunoglobulin A (IgA), which is heavily glycosylated, interacts with a variety of receptors, e.g. the asialoglycoprotein receptor (ASGP-R), which binds terminal galactose residues, and the Fcalpha receptor (FcalphaRI). It has thus been proposed that elevated serum levels of IgA in primary Sjögren's syndrome (pSS) are caused by its defective clearance. To test this hypothesis, we developed a method (based on sialyl transferases eluted from a hepatoma cell line) to increase the amount of sialic acid (SA) on IgA, and used a battery of IgA1- and IgA2-specific glycosidases to reduce this amount. Binding of IgA1 and IgA2 to ASGP-R and FcalphaRI was found to be sugar dependent because oversialylated IgA bound less than native or desialylated IgA. However, individual sugars did not play a direct role in this binding. Given that IgA are oversialylated in pSS, defective clearance of IgA may indeed be ascribed to an excess of SA in IgA1 and IgA2.

The Fc-receptor III of cultured human monocytes. Structural similarity with FcRIII of natural killer cells and role in the extracellular lysis of sensitized erythrocytes

NARCIS (Netherlands)

Klaassen, R. J.; Ouwehand, W. H.; Huizinga, T. W.; Engelfriet, C. P.; von dem Borne, A. E.

1990-01-01

FcRIII is not present on peripheral blood monocytes, but becomes expressed upon culturing and can be demonstrated on tissue macrophages. We studied the expression of FcRIII of cultured monocytes in detail and compared its structure with FcRIII of neutrophils and NK cells. The cell density of FcRIII

Glucagon-like Peptide 1 Conjugated to Recombinant Human Serum Albumin Variants with Modified Neonatal Fc Receptor Binding Properties. Impact on Molecular Structure and Half-Life

DEFF Research Database (Denmark)

Bukrinski, Jens T.; Sønderby, Pernille; Antunes, Filipa

2017-01-01

Glucagon-like peptide 1 (GLP-1) is a small incretin hormone stimulated by food intake, resulting in an amplification of the insulin response. Though interesting as a drug candidate for the treatment of type 2 diabetes mellitus, its short plasma half-life of less than 3 minutes limits its clinical...... use. A strategy to extend the half-life of GLP-1 utilizes the long half-life of human serum albumin (HSA) by combining the two via chemical conjugation or genetic fusion. HSA has a plasma half-life of around 21 days owing to its interaction with the neonatal Fc receptor (FcRn) expressed in endothelial...... with the available structural information on the FcRn and GLP-1 receptor (GLP-1R) obtained from X-ray crystallography, we can explain the observed in-vitro and in-vivo behaviour. We conclude that the conjugation of GLP-1 to rHSA does not affect the interaction between rHSA and FcRn, while the observed decrease...

Nooruslik kaupmeheemand kavatseb äri laiendada / Hille Tänavsuu

Index Scriptorium Estoniae

Tänavsuu, Hille, 1941-2014

2006-01-01

Vt. ka Postimees : na russkom jazõke 23. märts lk. 8. Tallinnas Vana-Kalamaja tänaval paikneva Alma Äri omanik Alma Sooäär avas oma esimese poe enam kui 15 aastat tagasi Nõmmel, praegu plaanib poeomanik majapidamistarvete äri laiendamist. Kaart: Alma Äri

Optimal football strategies: AC Milan versus FC Barcelona

OpenAIRE

Papahristodoulou, Christos

2012-01-01

In a recent UEFA Champions League game between AC Milan and FC Barcelona, played in Italy (final score 2-3), the collected match statistics, classified into four offensive and two defensive strategies, were in favour of FC Barcelona (by 13 versus 8 points). The aim of this paper is to examine to what extent the optimal game strategies derived from some deterministic, possibilistic, stochastic and fuzzy LP models would improve the payoff of AC Milan at the cost of FC Barcelona.

Decontamination manual of RI handling laboratory

International Nuclear Information System (INIS)

Wadachi, Yoshiki

2004-01-01

Based on experiences in Japan Atomic Energy Research Institute (JAERI), the essential and practical knowledge of radioactive contamination and its decontamination, and the method and procedure of floor decontamination are described for researcher and managing person in charge of handling radioisotopes (RI) in RI handling laboratories. Essential knowledge concerns the uniqueness of solid surface contamination derived from RI half lives and quantities, surface contamination density limit, and mode/mechanism of contamination. The principle of decontamination is a single conduct with recognition of chemical form of the RI under use. As the practical knowledge, there are physical and chemical methods of solid surface decontamination. The latter involves use of inorganic acids, chelaters and surfactants. Removal and replacement of contaminated solid like floor material are often effective. Distribution mapping of surface contamination can be done by measuring the radioactivity in possibly contaminated areas, and is useful for planning of effective decontamination. Floor surface decontamination is for the partial and spread areas of the floor. It is essential to conduct the decontamination with reagent from the highly to less contaminated areas. Skin decontamination with either neutral detergent or titanium oxide is also described. (N.I.)

Thermal decomposition of FC(O)OCH3 and FC(O)OCH2CH3.

Science.gov (United States)

Berasategui, M; Argüello, G A; Burgos Paci, M A

2018-05-09

The thermal decomposition of methyl and ethyl formates has been extensively studied due to their importance in the oxidation of several fuels, pesticidal properties and their presence in interstellar space. We hitherto present the study of the thermal decomposition of methyl and ethyl fluoroformates, which could help in the elucidation of the reaction mechanisms. The reaction mechanisms were studied using FTIR spectroscopy in the temperature range of 453-733 K in the presence of different pressures of N2 as bath gas. For FC(O)OCH3 two different channels were observed; the unimolecular decomposition which is favored at higher temperatures and has a rate constant kFC(O)OCH3 = (5.3 ± 0.5) × 1015 exp[-(246 ± 10 kJ mol-1/RT)] (in units of s-1) and a bimolecular channel with a rate constant kFC(O)OCH3 = (1.6 ± 0.5) × 1011 exp[-(148 ± 10 kJ mol-1/RT)] (in units of s-1 (mol L)-1). However for ethyl formate, only direct elimination of CO2, HF and ethylene operates. The rate constants of the homogeneous first-order process fit the Arrhenius equation kFC(O)OCH2CH3 = (2.06 ± 0.09) × 1013 exp[-(169 ± 6 kJ mol-1/RT)] (in units of s-1). The difference between the mechanisms of the two fluoroformates relies on the stabilization of a six-centered transition state that only exists for ethyl formate. First principles calculations for the different channels were carried out to understand the dynamics of the decomposition.

Identification of Novel Molecular Targets for Pleckstrin Homology (PH) Domains Found in Oncogenes Implicated in Breast Cancer

Science.gov (United States)

2008-03-01

roles as a positive regulator of IL- 2 gene induction in T cells (Deckert et al, 1998), NK cell-mediated cytotoxicity (Jevremovic et al, 2001), and FcRI...Identification of a gene encoding a human oxysterol-binding protein-homologue: a potential general molecular marker for blood dissemination of solid...Biol. 6(5), 384-385. Scanlan, MJ, Gout , I, Gordon, CM, Williamson, B, Stockert, E, Gure, AO, Jager, D, Chen, YT, Mackay, A, O’Hare, MJ, and Old LJ

LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

Energy Technology Data Exchange (ETDEWEB)

Kawamoto, Eiji [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Takagi, Yoshimi [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Honda, Goichi [Medical Affairs Department, Asahi Kasei Pharma Corporation, 1-105 Kanda Jinbo-cho, Chiyoda-ku, Tokyo 101-8101 (Japan); Suzuki, Koji [Faculty of Pharmaceutical Science, Suzuka University of Medical Science, 3500-3, Minamitamagaki-cho, Suzuka, Mie 513-8679 (Japan); Imai, Hiroshi [Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Shimaoka, Motomu, E-mail: shimaoka@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan)

2016-05-13

LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

Tüüri vioolakontserdi esiettekanne

Index Scriptorium Estoniae

2008-01-01

24. okt. toovad Taanis Sonderborgis maailmaesiettekandele Erkki-Sven Tüüri Vioolakontserdi "Illuminatio" norra vioolamängija Lars Anders Tomter ja Lõuna- Jüütimaa Sümfooniaorkester, mida juhatab Vladimir Ziva. Evelyn Glennie ja New Jersey Sümfooniaorkester Neeme Järvi juhatusel esitavad 10. okt. USAs Newarkis ja 12. okt. New Brunswickis Tüüri 4. sümfoonia "Magma". Sellega seoses esineb Tüür loengutega oma loomingust järgmistes kõrgkoolides: New York Univerdity, Juilliard Scool of Music, Princeton University ja Montclair State University

Sequence specific DNA binding by P53 is enhanced by ionizing radiation and is mediated via DNA-PK activity

International Nuclear Information System (INIS)

Kachnic, L.A.; Wunsch, H.; Mekeel, K.L.; De Frank, J.S.; Powell, S.N.

1996-01-01

Purpose: P53 is known to be involved in the cellular response to DNA damage. It mediates many of its effects by acting as a transcription factor via sequence-specific DNA binding. The half-life of p53 is prolonged following DNA damage, and this results in elevated levels of p53 for a period of 2-8 hours. The increase in p53 is often relatively small, but this produces significant stimulation of a downstream gene such as p21(WAF1/cip1). We investigated post-translational modification of p53 following ionizing radiation damage. Materials and Methods: The response of normal Balb-C mouse fibroblasts (FC) to ionizing radiation (IR, 8 Gy) was measured at 0,3,6,9 and 24 hours, by the levels of p53, p21, flow cytometry and the electrophoretic mobility shift assay (EMSA). EMSA utilized a 26 bp consensus sequence end-labeled oligonucleotide to measure sequence-specific p53 binding. P53 specificity was confirmed by an enhanced mobility shift (retardation) when using p53 antibody. Comparison was made with scid fibroblasts (FS) and FC cells transfected with a plasmid (CX3) containing mutant p53 (alanine-143) or infected with a retrovirus containing the E6 protein of human papilloma virus type 16. Results: The response of p53 to DNA damage shows a 3-fold increase at 3-6 hours, and was not significantly different between FC and FS. FC-CX3 showed detectable basal levels of p53, and a 2-fold further induction of p53 after IR. FC-E6 showed no detectable levels of p53 before or after IR. No induction of p21 or G1/S arrest was seen in FC-CX3 or FC-E6, as has been observed previously. The induction of p21 in FS cells was attenuated and delayed: a 2-3-fold increase seen maximally at 9 hours, compared with a 5-fold increase seen maximally at 3-6 hours in FC cells. The accumulation of cells at the G1/S junction after IR showed the same kinetics as p21 induction: the peak of cells in G1 occurs at 3-6 hours in FC, but not until 9-24 hours in FS. The response is reminiscent of that seen in

Improving Forecast Skill by Assimilation of AIRS Cloud Cleared Radiances RiCC

Science.gov (United States)

Susskind, Joel; Rosenberg, Robert I.; Iredell, Lena

2015-01-01

ECMWF, NCEP, and GMAO routinely assimilate radiosonde and other in-situ observations along with satellite IR and MW Sounder radiance observations. NCEP and GMAO use the NCEP GSI Data Assimilation System (DAS).GSI DAS assimilates AIRS, CrIS, IASI channel radiances Ri on a channel-by-channel, case-by-case basis, only for those channels i thought to be unaffected by cloud cover. This test excludes Ri for most tropospheric sounding channels under partial cloud cover conditions. AIRS Version-6 RiCC is a derived quantity representative of what AIRS channel i would have seen if the AIRS FOR were cloud free. All values of RiCC have case-by-case error estimates RiCC associated with them. Our experiments present to the GSI QCd values of AIRS RiCC in place of AIRS Ri observations. GSI DAS assimilates only those values of RiCC it thinks are cloud free. This potentially allows for better coverage of assimilated QCd values of RiCC as compared to Ri.

Fc-mediated immune precipitation. III. Visualization by electron microscopy

DEFF Research Database (Denmark)

Møller, NPH; Christiansen, Gunna

1983-01-01

with either rabbit anti-KLH IgG or anti-KLH F(ab')2 fragments. The Fc-Fc interactions were investigated by reacting these surface-adsorbed antibody-rich KLH immune complexes with soluble, antigen-rich ferritin-anti-ferritin complexes using either rabbit anti-ferritin IgG or the corresponding isomolar F(ab')2...... fragments as antibody. Fc-Fc interactions were indicated by the formation of clusters or ring structures of ferritin molecules, which were only seen when using KLH anti-KLH IgG and ferritin-anti-ferritin IgG complexes. When F(ab')2 fragments were used as antibody, no reaction between KLH anti-KLH complexes...

75 FR 15343 - Regulated Navigation Area: Narragansett Bay, RI and Mount Hope Bay, RI and MA, Including the...

Science.gov (United States)

2010-03-29

...: Narragansett Bay, RI and Mount Hope Bay, RI and MA, Including the Providence River and Taunton River AGENCY... River and Mount Hope Bay in the vicinity of the two Brightman Street bridges have not been adopted and... Island and Mt. Hope Bay, MA.'' The notice was prompted primarily by two events: (1) The U.S. Army Corps...

Epigenetic regulation of RELN and GAD1 in the frontal cortex (FC) of autism spectrum disorder (ASD) subjects.

Science.gov (United States)

Zhubi, Adrian; Chen, Ying; Guidotti, Alessandro; Grayson, Dennis R

2017-11-01

Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains. We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP). We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments. Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged

Resolution of Disulfide Heterogeneity in Nogo Receptor 1 Fusion Proteins by Molecular Engineering

Energy Technology Data Exchange (ETDEWEB)

P Weinreb; D Wen; F Qian; C Wildes; E Garber; L Walus; M Jung; J Wang; J Relton; et al.

2011-12-31

NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys{sup 266} and Cys{sup 309} with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.

Modulating Cytotoxic Effector Functions by Fc Engineering to Improve Cancer Therapy.

Science.gov (United States)

Kellner, Christian; Otte, Anna; Cappuzzello, Elisa; Klausz, Katja; Peipp, Matthias

2017-09-01

In the last two decades, monoclonal antibodies have revolutionized the therapy of cancer patients. Although antibody therapy has continuously been improved, still a significant number of patients do not benefit from antibody therapy. Therefore, rational optimization of the antibody molecule by Fc engineering represents a major area of translational research to further improve this potent therapeutic option. Monoclonal antibodies are able to trigger a variety of effector mechanisms. Especially Fc-mediated effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement- dependent cytotoxicity (CDC) are considered important in antibody therapy of cancer. Novel mechanistic insights into the action of monoclonal antibodies allowed the development of various Fc engineering approaches to modulate antibodies' effector functions. Strategies in modifying the Fc glycosylation profile (Fc glyco-engineering) or approaches in engineering the protein backbone (Fc protein engineering) have been intensively evaluated. In the current review, Fc engineering strategies resulting in improved ADCC, ADCP and CDC activity are summarized and discussed.

Space and Salvation in Colette's Chéri and La Fin de Chéri

Directory of Open Access Journals (Sweden)

Ann Leone Philbrick

1984-01-01

Full Text Available Colette's critics often seem to dismiss all but her autobiographical creatures as whimsical and inarticulate. Her characters are frequently less eloquent than the spaces they create and inhabit; this observation offers an approach to Chéri and La Fin de Chéri that invites us to read them as two of Colette's most ambitious and authentic works. Here are stories of compromises with the containers of one's life and identity: streets, salons, boudoirs, and, ultimately, the body. Indeed, the self and its containers function symbiotically. Chéri makes no effort to direct this relationship, and kills himself when the world finally seems inscrutable and formless; his older mistress, Léa, responds joyfully—or with melancholic respect—to the surfaces and limits of her world. We are left with a harsher insight into Colette's vision than we are accustomed to. She suggests that survival lies not in the endless definition of one's place in the world, but in its recognition and a loving, even fearing, homage.

Jüri Talveti luulepiiriületamised / Jüri Talvet ; intervjueerinud Rein Veidemann

Index Scriptorium Estoniae

Talvet, Jüri, 1945-

2010-01-01

IX Moncayo luulefestivalist Aragoonias 10. aug. 2010 ja Jüri Talveti luule tõlgetest: Del sueno, de la nieve / tõlkinud Albert Lázaro Tinaut (Olifante, 2010) ; Of Snow, of Soul / tõlkinud H. L. Hix (Guernica, 2010)

Targeting allergen to FcgammaRI reveals a novel T(H)2 regulatory pathway linked to thymic stromal lymphopoietin receptor.

Science.gov (United States)

Hulse, Kathryn E; Reefer, Amanda J; Engelhard, Victor H; Patrie, James T; Ziegler, Steven F; Chapman, Martin D; Woodfolk, Judith A

2010-01-01

The molecule H22-Fel d 1, which targets cat allergen to FcgammaRI on dendritic cells (DCs), has the potential to treat cat allergy because of its T-cell modulatory properties. We sought to investigate whether the T-cell response induced by H22-Fel d 1 is altered in the presence of the T(H)2-promoting cytokine thymic stromal lymphopoietin (TSLP). Studies were performed in subjects with cat allergy with and without atopic dermatitis. Monocyte-derived DCs were primed with H22-Fel d 1 in the presence or absence of TSLP, and the resulting T-cell cytokine repertoire was analyzed by flow cytometry. The capacity for H22-Fel d 1 to modulate TSLP receptor expression on DCs was examined by flow cytometry in the presence or absence of inhibitors of Fc receptor signaling molecules. Surprisingly, TSLP alone was a weak inducer of T(H)2 responses irrespective of atopic status; however, DCs coprimed with TSLP and H22-Fel d 1 selectively and synergistically amplified T(H)2 responses in highly atopic subjects. This effect was OX40 ligand independent, pointing to an unconventional TSLP-mediated pathway. Expression of TSLP receptor was upregulated on atopic DCs primed with H22-Fel d 1 through a pathway regulated by FcgammaRI-associated signaling components, including src-related tyrosine kinases and Syk, as well as the downstream molecule phosphoinositide 3-kinase. Inhibition of TSLP receptor upregulation triggered by H22-Fel d 1 blocked TSLP-mediated T(H)2 responses. Discovery of a novel T(H)2 regulatory pathway linking FcgammaRI signaling to TSLP receptor upregulation and consequent TSLP-mediated effects questions the validity of receptor-targeted allergen vaccines. Copyright 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

76 FR 50770 - Submission for Review: Financial Resources Questionnaire (RI 34-1, RI 34-17, and RI 34-18) and...

Science.gov (United States)

2011-08-16

... OFFICE OF PERSONNEL MANAGEMENT Submission for Review: Financial Resources Questionnaire (RI 34- 1...) 3206-0167, Financial Resources Questionnaire and Notice of Amount Due Because of Annuity Overpayment... techniques or other forms of information technology, e.g., permitting electronic submissions of responses...

N-glycan engineering of a plant-produced anti-CD20-hIL-2 immunocytokine significantly enhances its effector functions.

Science.gov (United States)

Marusic, Carla; Pioli, Claudio; Stelter, Szymon; Novelli, Flavia; Lonoce, Chiara; Morrocchi, Elena; Benvenuto, Eugenio; Salzano, Anna Maria; Scaloni, Andrea; Donini, Marcello

2018-03-01

Anti-CD20 recombinant antibodies are among the most promising therapeutics for the treatment of B-cell malignancies such as non-Hodgkin lymphomas. We recently demonstrated that an immunocytokine (2B8-Fc-hIL2), obtained by fusing an anti-CD20 scFv-Fc antibody derived from C2B8 mAb (rituximab) to the human interleukin 2 (hIL-2), can be efficiently produced in Nicotiana benthamiana plants. The purified immunocytokine (IC) bearing a typical plant protein N-glycosylation profile showed a CD20 binding activity comparable to that of rituximab and was efficient in eliciting antibody-dependent cell-mediated cytotoxicity (ADCC) of human PBMC against Daudi cells, indicating its fuctional integrity. In this work, the immunocytokine devoid of the typical xylose/fucose N-glycosylation plant signature (IC-ΔXF) and the corresponding scFv-Fc-ΔXF antibody not fused to the cytokine, were obtained in a glyco-engineered ΔXylT/FucT N. benthamiana line. Purification yields from agroinfiltrated plants amounted to 20-35 mg/kg of leaf fresh weight. When assayed for interaction with FcγRI and FcγRIIIa, IC-ΔXF exhibited significantly enhanced binding affinities if compared to the counterpart bearing the typical plant protein N-glycosylation profile (IC) and to rituximab. The glyco-engineered recombinant molecules also exhibited a strongly improved ADCC and complement-dependent cytotoxicity (CDC). Notably, our results demonstrate a reduced C1q binding of xylose/fucose carrying IC and scFv-Fc compared to versions that lack these sugar moieties. These results demonstrate that specific N-glycosylation alterations in recombinant products can dramatically affect the effector functions of the immunocytokine, resulting in an overall improvement of the biological functions and consequently of the therapeutic potential. © 2017 Wiley Periodicals, Inc.

Construction of a bimolecular fluorescence complementation (BiFC ...

African Journals Online (AJOL)

Protein–protein interactions are essential for signal transduction in cells. Bimolecular fluorescence complementation (BiFC) is a novel technology that utilises green fluorescent proteins to visualize protein–protein interactions and subcellular protein localisation. BiFC based on pSATN vectors are a good system for ...

[The role of Cd-binding proteins and phytochelatins in the formation of cadmium resistance in Nicotiana plumbaginifolia cell lines].

Science.gov (United States)

Fenik, S I; Solodushko, V G; Kaliniak, T B; Blium, Ia B

2007-01-01

Nicotiana plumbaginifolia callus lines with the equal resistance to cadmium have been produced under different selective conditions--either without inhibition of the phytochelatin synthesis (line Cd-R) or in the presence of the inhibitor butionine sulfoximine (line Cd-Ri). The level of phytochelatin synthesis in the line Cd-R five-fold exceeded the control value and in the line Cd-Ri it was twice as much as in the control. It was shown that in the control line mainly three cadmium-binding proteins are expressed of the molecular weihgts 41, 34 and 19 kD. The common feature of the both resistant lines is the expression of the cadmium-binding proteins of 40, 37 and 19 kD. The resistant lines differ with respect to the synthesis of relatively low-molecular cadmium-binding proteins. The proteins of the molecular weights 12.5, 11.5 and 9 kD are expressed in the line Cd-R, while the proteins of 13 and 10 kD are expressed in the line Cd-Ri. It was supposed that both the phytochelatins and the Cd-binding proteins contribute to the resisitance of N. plumbaginifolia callus lines to cadmium and the lack of the phytochelatins can be equilibrated by the changes in the low-molecular Cd-binding protein synthesis.

Effective management of regulator RI/FS comments

International Nuclear Information System (INIS)

Wolinsky, S.M.; Lojek, D.; George, R.D.; Houser, S.M.; Strimbu, M.J.

1995-01-01

This paper describes a successful strategy that facilitates regulatory approval of CERCLA documents required by compliance agreement and CERCLA, based on the experience of Operable Unit 1, Waste Storage Area, of the Fernald Environmental Management Project (FEMP). This strategy, which has become the site standard at the FEMP, was instrumental in obtaining regulator approval of the OU1 RI and FS, and early approval of the Record of Decision during a very tight compliance agreement-driven schedule. This strategy can be applied at any DOE Superfund site, especially where there is need to recover lost schedule, an incentive to meet milestones early, a need to improve the relationship between the DOE and the regulators, or where the regulatory agencies have historically provided a large volume of comments on CERCLA documents. The strategy focuses on early identification and resolution of issues relating to draft RI/FS documents, as raised in regulatory agency review comments. This pro-active strategy has the potential for schedule and cost savings, as well as for improved communication between DOE and the regulators. The strategy includes preparation of a separate comment response document, integration of comment responses with RI/FS documents, development of a database of agency comments and their resolution, and sharing lessons learned with preparers of subsequent RI/FS documents. The paper provides background on the FEMP and describes the FEMP comment response strategy; DOE and regulator interface; the Sitewide Comment Database; networked electronic file management; the process for classifying, analyzing, and responding to comments; integration with base RI/FS documents; and a conclusion

Effective management of regulator RI/FS comments

Energy Technology Data Exchange (ETDEWEB)

Wolinsky, S.M.; Lojek, D.; George, R.D.; Houser, S.M.; Strimbu, M.J.

1995-12-31

This paper describes a successful strategy that facilitates regulatory approval of CERCLA documents required by compliance agreement and CERCLA, based on the experience of Operable Unit 1, Waste Storage Area, of the Fernald Environmental Management Project (FEMP). This strategy, which has become the site standard at the FEMP, was instrumental in obtaining regulator approval of the OU1 RI and FS, and early approval of the Record of Decision during a very tight compliance agreement-driven schedule. This strategy can be applied at any DOE Superfund site, especially where there is need to recover lost schedule, an incentive to meet milestones early, a need to improve the relationship between the DOE and the regulators, or where the regulatory agencies have historically provided a large volume of comments on CERCLA documents. The strategy focuses on early identification and resolution of issues relating to draft RI/FS documents, as raised in regulatory agency review comments. This pro-active strategy has the potential for schedule and cost savings, as well as for improved communication between DOE and the regulators. The strategy includes preparation of a separate comment response document, integration of comment responses with RI/FS documents, development of a database of agency comments and their resolution, and sharing lessons learned with preparers of subsequent RI/FS documents. The paper provides background on the FEMP and describes the FEMP comment response strategy; DOE and regulator interface; the Sitewide Comment Database; networked electronic file management; the process for classifying, analyzing, and responding to comments; integration with base RI/FS documents; and a conclusion.

Structure of the omalizumab Fab

Science.gov (United States)

Jensen, Rasmus K.; Plum, Melanie; Tjerrild, Luna; Jakob, Thilo; Spillner, Edzard; Andersen, Gregers Rom

2015-01-01

Omalizumab is a humanized anti-IgE antibody that inhibits the binding of IgE to its receptors on mast cells and basophils, thus blocking the IgE-mediated release of inflammatory mediators from these cells. Omalizumab binds to the Fc domains of IgE in proximity to the binding site of the high-affinity IgE receptor Fc∊RI, but the epitope and the mechanisms and conformations governing the recognition remain unknown. In order to elucidate the molecular mechanism of its anti-IgE activity, the aim was to analyse the interaction of omalizumab with human IgE. Therefore, IgE Fc C∊2–4 was recombinantly produced in mammalian HEK-293 cells. Functionality of the IgE Fc was proven by ELISA and mediator-release assays. Omalizumab IgG was cleaved with papain and the resulting Fab was purified by ion-exchange chromatography. The complex of IgE Fc with omalizumab was prepared by size-exclusion chromatography. However, crystals containing the complex were not obtained, suggesting that the process of crystallization favoured the dissociation of the two proteins. Instead, two structures of the omalizumab Fab with maximum resolutions of 1.9 and 3.0 Å were obtained. The structures reveal the arrangement of the CDRs and the position of omalizumab residues known from prior functional studies to be involved in IgE binding. Thus, the structure of omalizumab provides the structural basis for understanding the function of omalizumab, allows optimization of the procedure for complex crystallization and poses questions about the conformational requirements for anti-IgE activity. PMID:25849503

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Science.gov (United States)

Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

2013-01-01

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

Trastuzumab triggers phagocytic killing of high HER2 cancer cells in vitro and in vivo by interaction with Fcγ receptors on macrophages.

Science.gov (United States)

Shi, Yun; Fan, Xuejun; Deng, Hui; Brezski, Randall J; Rycyzyn, Michael; Jordan, Robert E; Strohl, William R; Zou, Quanming; Zhang, Ningyan; An, Zhiqiang

2015-05-01

Trastuzumab has been used for the treatment of HER2-overexpressing breast cancer for more than a decade, but the mechanisms of action for the therapy are still being actively investigated. Ab-dependent cell-mediated cytotoxicity mediated by NK cells is well recognized as one of the key mechanisms of action for trastuzumab, but trastuzumab-mediated Ab-dependent cellular phagocytosis (ADCP) has not been established. In this study, we demonstrate that macrophages, by way of phagocytic engulfment, can mediate ADCP and cancer cell killing in the presence of trastuzumab. Increased infiltration of macrophages in the tumor tissue was associated with enhanced efficacy of trastuzumab whereas depletion of macrophages resulted in reduced antitumor efficacy in mouse xenograft tumor models. Among the four mouse FcγRs, FcγRIV exhibits the strongest binding affinity to trastuzumab. Knockdown of FcγRIV in mouse macrophages reduced cancer cell killing and ADCP activity triggered by trastuzumab. Consistently, an upregulation of FcγRIV expression by IFN-γ triggered an increased ADCP activity by trastuzumab. In an analogous fashion, IFN-γ priming of human macrophages increased the expression of FcγRIII, the ortholog of murine FcγRIV, and increased trastuzumab-mediated cancer cell killing. Thus, in two independent systems, the results indicated that activation of macrophages in combination with trastuzumab can serve as a therapeutic strategy for treating high HER2 breast cancer by boosting ADCP killing of cancer cells. Copyright © 2015 by The American Association of Immunologists, Inc.

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D.

Science.gov (United States)

Kapur, Rick; Della Valle, Luciana; Verhagen, Onno J H M; Hipgrave Ederveen, Agnes; Ligthart, Peter; de Haas, Masja; Kumpel, Belinda; Wuhrer, Manfred; van der Schoot, C Ellen; Vidarsson, Gestur

2015-03-01

RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction. Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female Dutch HIDs and RhIG were analyzed with mass spectrometry. The Fc-glycosylation profiles of HIDs were evaluated with regard to their immunization history. HID sera demonstrated clearly lowered anti-D Fc-fucosylation compared to normal IgG fucosylation (93%); this was more pronounced for female than for male HIDs (47% vs. 65%, p = 0.001). RhIG preparations from seven manufacturers varied greatly in the level of Fc-fucosylation (56%-91%). The level of fucosylation slightly increased upon repeated immunization, although it remained fairly constant over time. The RhIG from the different manufacturers all demonstrated increased Fc-galactosylation (64%-82%) compared to total IgG (38%-51%). RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered. © 2014 AABB.

RI beam factory project at RIKEN

CERN Document Server

Motobayashi, T

2003-01-01

Construction of the RI beam factory project in the first phase has started. The aim of the project is to provide intense radio-isotopes (RI) beams at energies of several hundred MeV/nucleon in a wide range of atomic masses. These beams will be produced by the projectile fragmentation of primary beams accelerated by a cascade of the existing ring cyclotron and a series of new ring cyclotrons. Improvements of the existing facility made for the new cyclotron complex have extended the energy range of available beams, which already opened new domains of study.

Complexes prepared from protein A and human serum, IgG, or Fc gamma fragments: characterization by immunochemical analysis of ultracentrifugation fractions and studies on their interconversion.

Science.gov (United States)

Langone, J J; Das, C; Mainwaring, R; Shearer, W T

1985-01-01

Protein A of Staphylococcus aureus is an Fc receptor for IgG that has been used as a therapeutic reagent to treat cancer in humans and experimental animals. We used ultracentrifugation combined with analysis of isolated fractions by radioimmunoprecipitation and competitive radioimmunoassay with chicken antibodies that bind free protein A or protein A in complexes but do bind free immunoglobulin reagents to localize and characterize the types of complexes formed with different molar ratios of 125I-protein A and human 131I-IgG alone or in serum, and 131I-Fc gamma fragments. This approach offers a distinct advantage over direct counting of radioactivity in the fractions because resolution of complexes and free reagents is much improved. With excess 131I-IgG or 131I-Fc, all the 125I-protein A is present only in complexes that contained 4 molecules of immunoglobulin reagent and 2 molecules of protein A (4:2 complexes), whereas with excess 125I-protein A the stoichiometry of the complexes was 1:1. We have also shown the preformed 4:2 and 1:1 complexes will interconvert in the presence of added excess protein A or IgG, respectively, and that fresh IgG will exchange with IgG or Fc gamma in preformed complexes. Because protein A has been found to elute from an immobilized reagent used in serotherapy of human cancer and is present in a large excess of IgG, the 4:2 complexes may play an active role in the tumoricidal or toxic reactions observed.

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.

Science.gov (United States)

Park, Sol Ji; Cho, Bumrae; Koo, Ok Jae; Kim, Hwajung; Kang, Jung Taek; Hurh, Sunghoon; Kim, Su Jin; Yeom, Hye Jung; Moon, Joonho; Lee, Eun Mi; Choi, Ji Yei; Hong, Ju Ho; Jang, Goo; Hwang, Joing-Ik; Yang, Jaeseok; Lee, Byeong Chun; Ahn, Curie

2014-06-01

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury

RI beam factory and its expected pioneering work

International Nuclear Information System (INIS)

Yano, Yasushige

1996-01-01

The RARF (RIKEN Accelerator Research Facility) houses an intermediate-energy heavy-ion accelerator complex consisting of a K540-MeV ring cyclotron (RRC) and a couple of different types of the injectors: a variable-frequency heavy-ion linac and a K70-MeV AVF cyclotron. One of remarkable features of this facility is capability of supplying light-atomic-mass RI (radioactive isotope) beams with the world-highest level of intensities by the projectile-fragment separator, RIPS. In these several years nuclear physicists have opened up a quite new and fascinating heavy-ion science exploiting such RI beams. In order to further promote this new science, the RARF proposes 'RIKEN RI Beam Factory' as a next facility-expanding project. The factory takes the aim at providing RI beams covering over the whole atomic-mass range with the world-highest intensities in a wide energy range up to several hundreds MeV/nucleon. To realize the 'Factory' a K2500-MeV superconducting ring cyclotron will be built which boosts output energies of the RRC beams up to 400 MeV/nucleon for light ions and up to 100 MeV/nucleon for very heavy ions, preserving their beam intensities (typically 1 pμA). RI beams will be generated by the projectile fragmentation. A new type of experimental installation called 'MUSES' (Multi-USe Experimental Storage rings) will also be constructed. It consists of an accumulator-cooler ring, booster synchrotron ring and double storage rings. With MUSES, various types of unique colliding experiments will become possible: ion-ion merging or head-on collisions; collisions of either electrons or X-rays with ion (stable isotope or RI) beams; internal target experiments; and atomic and molecular physics with cooler electron beams. (author)

Association of C-Type Lectin Mincle with FcεRIβγ Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk.

Science.gov (United States)

Honjoh, Chisato; Chihara, Kazuyasu; Yoshiki, Hatsumi; Yamauchi, Shota; Takeuchi, Kenji; Kato, Yuji; Hida, Yukio; Ishizuka, Tamotsu; Sada, Kiyonao

2017-04-10

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6'-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

Domik Petra / Jüri Kuuskemaa

Index Scriptorium Estoniae

Kuuskemaa, Jüri, 1942-

2006-01-01

Kunstiajaloolane Jüri Kuuskemaa tutvustab Peeter I majamuuseumi Kadriorus. Maja ajaloost, sisustusest (mööbel, kunstiesemed). Keldriruumis eksponeeritud Salomon Zeltrechti ja tema abiliste poolt tehtud kivibüstidest

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.

Science.gov (United States)

Arora, Jayant; Hu, Yue; Esfandiary, Reza; Sathish, Hasige A; Bishop, Steven M; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B; Weis, David D

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C H 3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

Structure of RiVax: a recombinant ricin vaccine

International Nuclear Information System (INIS)

Legler, Patricia M.; Brey, Robert N.; Smallshaw, Joan E.; Vitetta, Ellen S.; Millard, Charles B.

2011-01-01

The X-ray crystal structure (at 2.1 Å resolution) of an immunogen under development as part of a ricin vaccine for humans is presented and structure-based analysis of the results was conducted with respect to related proteins and the known determinants for inducing or suppressing the protective immune response. RiVax is a recombinant protein that is currently under clinical development as part of a human vaccine to protect against ricin poisoning. RiVax includes ricin A-chain (RTA) residues 1–267 with two intentional amino-acid substitutions, V76M and Y80A, aimed at reducing toxicity. Here, the crystal structure of RiVax was solved to 2.1 Å resolution and it was shown that it is superposable with that of the ricin toxin A-chain from Ricinus communis with a root-mean-square deviation of 0.6 Å over 258 C α atoms. The RiVax structure is also compared with the recently determined structure of another potential ricin-vaccine immunogen, RTA 1–33/44–198 R48C/T77C. Finally, the locations and solvent-exposure of two toxin-neutralizing B-cell epitopes were examined and it was found that these epitopes are within or near regions predicted to be involved in catalysis. The results demonstrate the composition of the RiVax clinical material and will guide ongoing protein-engineering strategies to develop improved immunogens

Prediction of the Pharmacokinetics, Pharmacodynamics, and Efficacy of a Monoclonal Antibody, Using a Physiologically Based Pharmacokinetic FcRn Model

Science.gov (United States)

Chetty, Manoranjenni; Li, Linzhong; Rose, Rachel; Machavaram, Krishna; Jamei, Masoud; Rostami-Hodjegan, Amin; Gardner, Iain

2015-01-01

Although advantages of physiologically based pharmacokinetic models (PBPK) are now well established, PBPK models that are linked to pharmacodynamic (PD) models to predict pharmacokinetics (PK), PD, and efficacy of monoclonal antibodies (mAbs) in humans are uncommon. The aim of this study was to develop a PD model that could be linked to a physiologically based mechanistic FcRn model to predict PK, PD, and efficacy of efalizumab. The mechanistic FcRn model for mAbs with target-mediated drug disposition within the Simcyp population-based simulator was used to simulate the pharmacokinetic profiles for three different single doses and two multiple doses of efalizumab administered to virtual Caucasian healthy volunteers. The elimination of efalizumab was modeled with both a target-mediated component (specific) and catabolism in the endosome (non-specific). This model accounted for the binding between neonatal Fc receptor (FcRn) and efalizumab (protective against elimination) and for changes in CD11a target concentration. An integrated response model was then developed to predict the changes in mean Psoriasis Area and Severity Index (PASI) scores that were measured in a clinical study as an efficacy marker for efalizumab treatment. PASI scores were approximated as continuous and following a first-order asymptotic progression model. The reported steady state asymptote (Y ss) and baseline score [Y (0)] was applied and parameter estimation was used to determine the half-life of progression (Tp) of psoriasis. Results suggested that simulations using this model were able to recover the changes in PASI scores (indicating efficacy) observed during clinical studies. Simulations of both single dose and multiple doses of efalizumab concentration-time profiles as well as suppression of CD11a concentrations recovered clinical data reasonably well. It can be concluded that the developed PBPK FcRn model linked to a PD model adequately predicted PK, PD, and efficacy of efalizumab. PMID

The RiSE climbing robot: body and leg design

Science.gov (United States)

Saunders, A.; Goldman, D. I.; Full, R. J.; Buehler, M.

2006-05-01

The RiSE robot is a biologically inspired, six legged climbing robot, designed for general mobility in scansorial (vertical walls, horizontal ledges, ground level) environments. It exhibits ground reaction forces that are similar to animal climbers and does not rely on suction, magnets or other surface-dependent specializations to achieve adhesion and shear force. We describe RiSE's body and leg design as well as its electromechanical, communications and computational infrastructure. We review design iterations that enable RiSE to climb 90° carpeted, cork covered and (a growing range of) stucco surfaces in the quasi-static regime.

Verification of Representative Sampling in RI waste

International Nuclear Information System (INIS)

Ahn, Hong Joo; Song, Byung Cheul; Sohn, Se Cheul; Song, Kyu Seok; Jee, Kwang Yong; Choi, Kwang Seop

2009-01-01

For evaluating the radionuclide inventories for RI wastes, representative sampling is one of the most important parts in the process of radiochemical assay. Sampling to characterized RI waste conditions typically has been based on judgment or convenience sampling of individual or groups. However, it is difficult to get a sample representatively among the numerous drums. In addition, RI waste drums might be classified into heterogeneous wastes because they have a content of cotton, glass, vinyl, gloves, etc. In order to get the representative samples, the sample to be analyzed must be collected from selected every drum. Considering the expense and time of analysis, however, the number of sample has to be minimized. In this study, RI waste drums were classified by the various conditions of the half-life, surface dose, acceptance date, waste form, generator, etc. A sample for radiochemical assay was obtained through mixing samples of each drum. The sample has to be prepared for radiochemical assay and although the sample should be reasonably uniform, it is rare that a completely homogeneous material is received. Every sample is shredded by a 1 ∼ 2 cm 2 diameter and a representative aliquot taken for the required analysis. For verification of representative sampling, classified every group is tested for evaluation of 'selection of representative drum in a group' and 'representative sampling in a drum'

21 CFR 866.5530 - Immunoglobulin G (Fc fragment specific) immunological test system.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fc fragment specific... Test Systems § 866.5530 Immunoglobulin G (Fc fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fc fragment specific) immunological test system is a device that consists of...

IgE by itself affects mature rat mast cell preformed and de novo-synthesized mediator release and amplifies mast cell migratory response.

Directory of Open Access Journals (Sweden)

Aleksandra Słodka

Full Text Available BACKGROUND: Immunoglobulin E (IgE binds to high affinity receptor FcεRI numerously expressed on mast cells. Recent findings have revealed that IgE by itself may regulate various aspects of mast cell biology, however, detailed data is still limited. METHODOLOGY/FINDINGS: Here, we have examined the influence of IgE alone, used at different concentrations, on mast cell activity and releasability. For the study we have employed in vivo differentiated mature tissue mast cells isolated from rat peritoneal cavity. Mast cells were exposed to IgE alone and then the release of preformed and de novo-synthesized mediators, surface FcεRI expression and mast cell migratory response were assessed. IgE by itself was found to up-regulate FcεRI expression and activate mast cells to degranulation, as well as de novo synthesis and release of cysteinyl leukotrienes and TNF. We have provided evidence that IgE alone also amplified spontaneous and CCL5- or TNF-induced migration of mast cells. Importantly, IgE was effective only at concentrations ≥ 3 µg/mL. A molecular basis investigation using an array of specific inhibitors showed that Src kinases, PLC/PLA2, MAP kinases (ERK and p38 and PI3K were entirely or partially involved in IgE-induced mast cell response. Furthermore, IgE alone stimulated the phosphorylation of MAP kinases and PI3K in rat mast cells. CONCLUSION: Our results clearly demonstrated that IgE by itself, at higher concentrations, influences mast cell activity and releasability. As there are different conditions when the IgE level is raised it might be supposed that in vivo IgE is one of the important factors modulating mast cell biology within tissues.

Nucleic acid binding and other biomedical properties of artificial oligolysines

Directory of Open Access Journals (Sweden)

Roviello GN

2016-11-01

Full Text Available Giovanni N Roviello,1 Caterina Vicidomini,1 Vincenzo Costanzo,1 Valentina Roviello2 1CNR Istituto di Biostrutture e Bioimmagini, Via Mezzocannone site and Headquarters, 2Centro Regionale di Competenza (CRdC Tecnologie, Via Nuova Agnano, Napoli, Italy Abstract: In the present study, we report the interaction of an artificial oligolysine (referred to as AOL realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI and its complex with polycytidylic acid (poly I:C, RNAs with well-known interferon-inducing ability, and double-stranded (ds DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD and ultraviolet (UV studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine–protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL. Keywords: nucleic acid binding, polyinosinic acid, double-stranded nucleic acids, oligolysine, circular dichroism

[Söejoonistused] / Jüri Marran

Index Scriptorium Estoniae

Marran, Jüri, 1937-

2005-01-01

Jüri Marran on hariduselt füüsik ja kunstihariduse omandanud Melanie ja Erich Arraku ateljeekoolis. Lk. 1391 (Gravitatsioon), 1440 (Valgus), 1471 (Vastastikmõju), 1486 (Massenergia), 1519 (Valguse dualism), 1555 (Aja dilatatsioon)

Kas e-äri võrdub pankrotiga / Priit Vaikmaa

Index Scriptorium Estoniae

Vaikmaa, Priit

2001-01-01

E-äride pankrotilaine aastal 2000. Firmadevaheline äri internetis - B2B (business-to-business). B2B ärikeskkondade kodulehekülgede aadressid. B2B ja e-äri lahenduste pakkujad Eestis. Diagramm: B2B e-kommertsi prognoositud käive 2004.a.

Jüri Lina kui tegelikkusele osutav Patagoonia šamaan / Jüri Lina ; intervjueerinud Ingrid Peek

Index Scriptorium Estoniae

Lina, Jüri, 1949-

2011-01-01

2.-5. juunini Tallinnas kinos Sõprus toimuva festivali "Hallo, Kosmos!" raames 2. juunil esilinastuvast Jüri Lina filmist "Täitmatu Uroboros. Massoonide salakaval taktika", tema uuringute põhjusest. Festivali "Hallo, Kosmos!" kavast

Protein kinase activity associated with Fcγ/sub 2a/ receptor of a murine macrophage like cell line, P388D1

International Nuclear Information System (INIS)

Hirata, Y.; Suzuki, T.

1987-01-01

The properties of protein kinase activity associated with Fc receptor specific for IgG/sub 2a/(Fcγ/sub 2a/R) of a murine macrophage like cell line, P388D 1 , were investigated. IgG/sub 2a/-binding protein isolated from the detergent lysate of P388D 1 cells by affinity chromatography of IgG-Sepharose was found to contain four distinct proteins of M/sub r/ 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with [γ- 32 P]ATP. The autophosphorylation of Fcγ/sub 2a/ receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Phosphorylation of casein catalyzed by Fcγ/sub 2a/ receptor complex was dependent on casein concentration, increased with time or temperature, was dependent on the concentration of ATP and Mg 2+ , and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn 2+ or KCl or by a small amount of heparin and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fcγ/sub 2a/ receptor used ATP as substrate with an apparent K/sub m/ of 2 μM as well as GTP with an apparent K/sub m/ of 10 μM. Prior heating (60 0 C for 15 min) or treatment with protease (trypsin or Pronase) of Fcγ/sub 2a/ receptor complex almost totally abolished casein kinase activity. Thin-layer chromatography of a partial acid hydrolysate of the phosphorylated casein showed that the site of phosphorylation is at a seryl residue. These results suggest that Fcγ 2 /sub a/ receptor forms a molecule complex with protein kinase, whose characteristics resemble those of type II casein kinase but are different from those of cyclic nucleotide dependent protein kinase or from those of C protein kinase

Diagnostic Validity of RI Angiocardiography in Cardiac Diseases

Energy Technology Data Exchange (ETDEWEB)

Kim, Kwan Yop; Koh, Chang Soon; Lee, Mun Ho [Seoul National University College of Medicine, Seoul (Korea, Republic of)

1972-09-15

The employment of gamma-ray scintillation camera with the use of short-lived radioisotopic pharmaceuticals, such as {sup 99m}Tc sodium pertechnetate, have enabled us to perform RI (radioisotopic) angiocardiography. Although conventional cardiac catheterization or angiocardiography using contrast media have been an important diagnostic tool, they may carry some risks or serious complications. The author investigated on RI angiocardiography in twelve normal and twenty five patients with cardiovascular diseases in an effort to evaluate its diagnostic value. The results obtained with this study are as follows; l. In normal subjects, the scintillation camera transit time of arm-to-right heart was found to be 2.1+-0.67 seconds, right heart-to-lung 1.5+-0.40 seconds and lung-to-left heart 3.5+-0.86 seconds. 2. Transformation or displacement of the heart and the great vessel are easily discriminated by RI angiocardiography. Both in the cases with tricuspid atresia and tetralogy of Fallot, ventricular septal defects are well recognized by sequential RI angiocardiography. 3. It is also helpful in determining the site and extent of obstruction, and estimating the postoperative course both in the cases with superior vena cava syndrome and pulmonary stenosis. 4. Pericarditis with effusion is readily diagnosed by RI angiocardiogram showing characteristic 'dead space' between intracardiac and intrapulmonary radioactivity. 5. It was found that the diagnostic accuracy of this study was 78.4%. It is concluded that above results obtained are useful and accurate enough for the diagnostic screening method for clinical practice.

Diagnostic Validity of RI Angiocardiography in Cardiac Diseases

International Nuclear Information System (INIS)

Kim, Kwan Yop; Koh, Chang Soon; Lee, Mun Ho

1972-01-01

The employment of gamma-ray scintillation camera with the use of short-lived radioisotopic pharmaceuticals, such as 99m Tc sodium pertechnetate, have enabled us to perform RI (radioisotopic) angiocardiography. Although conventional cardiac catheterization or angiocardiography using contrast media have been an important diagnostic tool, they may carry some risks or serious complications. The author investigated on RI angiocardiography in twelve normal and twenty five patients with cardiovascular diseases in an effort to evaluate its diagnostic value. The results obtained with this study are as follows; l. In normal subjects, the scintillation camera transit time of arm-to-right heart was found to be 2.1±0.67 seconds, right heart-to-lung 1.5±0.40 seconds and lung-to-left heart 3.5±0.86 seconds. 2. Transformation or displacement of the heart and the great vessel are easily discriminated by RI angiocardiography. Both in the cases with tricuspid atresia and tetralogy of Fallot, ventricular septal defects are well recognized by sequential RI angiocardiography. 3. It is also helpful in determining the site and extent of obstruction, and estimating the postoperative course both in the cases with superior vena cava syndrome and pulmonary stenosis. 4. Pericarditis with effusion is readily diagnosed by RI angiocardiogram showing characteristic 'dead space' between intracardiac and intrapulmonary radioactivity. 5. It was found that the diagnostic accuracy of this study was 78.4%. It is concluded that above results obtained are useful and accurate enough for the diagnostic screening method for clinical practice.

Diagnostic Validity of RI Angiocardiography in Cardiac Diseases

Energy Technology Data Exchange (ETDEWEB)

Kim, Kwan Yop; Koh, Chang Soon; Lee, Mun Ho [Seoul National University College of Medicine, Seoul (Korea, Republic of)

1972-09-15

The employment of gamma-ray scintillation camera with the use of short-lived radioisotopic pharmaceuticals, such as {sup 99m}Tc sodium pertechnetate, have enabled us to perform RI (radioisotopic) angiocardiography. Although conventional cardiac catheterization or angiocardiography using contrast media have been an important diagnostic tool, they may carry some risks or serious complications. The author investigated on RI angiocardiography in twelve normal and twenty five patients with cardiovascular diseases in an effort to evaluate its diagnostic value. The results obtained with this study are as follows; l. In normal subjects, the scintillation camera transit time of arm-to-right heart was found to be 2.1+-0.67 seconds, right heart-to-lung 1.5+-0.40 seconds and lung-to-left heart 3.5+-0.86 seconds. 2. Transformation or displacement of the heart and the great vessel are easily discriminated by RI angiocardiography. Both in the cases with tricuspid atresia and tetralogy of Fallot, ventricular septal defects are well recognized by sequential RI angiocardiography. 3. It is also helpful in determining the site and extent of obstruction, and estimating the postoperative course both in the cases with superior vena cava syndrome and pulmonary stenosis. 4. Pericarditis with effusion is readily diagnosed by RI angiocardiogram showing characteristic 'dead space' between intracardiac and intrapulmonary radioactivity. 5. It was found that the diagnostic accuracy of this study was 78.4%. It is concluded that above results obtained are useful and accurate enough for the diagnostic screening method for clinical practice.

S1P₄ Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells.

Science.gov (United States)

Kulinski, Joseph M; Proia, Richard L; Larson, Elisabeth M; Metcalfe, Dean D; Olivera, Ana

2018-04-25

Mast cells are key players in the development of inflammatory allergic reactions. Cross-linking of the high-affinity receptor for IgE (FcεRI) on mast cells leads to the generation and secretion of the sphingolipid mediator, sphingosine-1-phosphate (S1P) which is able, in turn, to transactivate its receptors on mast cells. Previous reports have identified the expression of two of the five receptors for S1P on mast cells, S1P₁ and S1P₂, with functions in FcεRI-mediated chemotaxis and degranulation, respectively. Here, we show that cultured mouse mast cells also express abundant message for S1P₄. Genetic deletion of S1pr4 did not affect the differentiation of bone marrow progenitors into mast cells or the proliferation of mast cells in culture. A comprehensive characterization of IgE-mediated responses in S1P₄-deficient bone marrow-derived and peritoneal mouse mast cells indicated that this receptor is dispensable for mast cell degranulation, cytokine/chemokine production and FcεRI-mediated chemotaxis in vitro. However, interleukin-33 (IL-33)-mediated enhancement of IgE-induced degranulation was reduced in S1P₄-deficient peritoneal mast cells, revealing a potential negative regulatory role for S1P₄ in an IL-33-rich environment. Surprisingly, genetic deletion of S1pr4 resulted in exacerbation of passive systemic anaphylaxis to IgE/anti-IgE in mice, a phenotype likely related to mast cell-extrinsic influences, such as the high circulating levels of IgE in these mice which increases FcεRI expression and consequently the extent of the response to FcεRI engagement. Thus, we provide evidence that S1P₄ modulates anaphylaxis in an unexpected manner that does not involve regulation of mast cell responsiveness to IgE stimulation.

Fc receptor gamma subunit polymorphisms and systemic lupus erythematosus

International Nuclear Information System (INIS)

Al-Ansari, Aliya; Ollier, W.E.; Gonzalez-Gay, Miguel A.; Gul, Ahmet; Inanac, Murat; Ordi, Jose; Teh, Lee-Suan; Hajeer, Ali H.

2004-01-01

To investigate the possible association between Fc receptor gamma polymorphisms and systemic lupus erythematosus (SLE). We have investigated the full FcR gamma gene for polymorphisms using polymerase chain reaction (PCR)-single strand confirmational polymorphisms and DNA sequencing .The polymorphisms identified were genotype using PCR-restriction fragment length polymorphism. Systemic lupus erythematosus cases and controls were available from 3 ethnic groups: Turkish, Spanish and Caucasian. The study was conducted in the year 2001 at the Arthritis Research Campaign, Epidemiology Unit, Manchester University Medical School, Manchester, United Kingdom. Five single nucleotide polymorphisms were identified, 2 in the promoter, one in intron 4 and, 2 in the 3'UTR. Four of the 5 single nucleotide polymorphisms (SNPs) were relatively common and investigated in the 3 populations. Allele and genotype frequencies of all 4 investigated SNPs were not statistically different cases and controls. fc receptor gamma gene does not appear to contribute to SLE susceptibility. The identified polymorphisms may be useful in investigating other diseases where receptors containing the FcR gamma subunit contribute to the pathology. (author)

Direct evidence that FK506 inhibition of FcepsilonRI-mediated exocytosis from RBL mast cells involves calcineurin.

Science.gov (United States)

Hultsch, T; Brand, P; Lohmann, S; Saloga, J; Kincaid, R L; Knop, J

1998-05-01

FcepsilonRI-mediated exocytosis of preformed mediators from mast cells and basophils (e.g. histamine, serotonin, beta-hexosaminidase) is sensitive to the immunosuppressants cyclosporin A and FK506 (IC50 200 and 4 nM, respectively) but not rapamycin. The mechanism of inhibition does not appear to involve tyrosine phosphorylation, hydrolysis of inositol phosphates or calcium flux. Here we report experiments using a molecular approach to assess the role of calcineurin, a serine/threonine phosphatase thought to be the primary pharmacological target of these drugs. Calcineurin's activity requires association of its catalytic (A) subunit with an intrinsic regulatory (B) subunit. We hypothesized that calcineurin-sensitive signalling events should be affected by the depletion of calcineurin B subunits, thereby reducing the number of active A:B complexes. We therefore transfected rat basophilic leukemia (RBL) cells with an inhibitory (dominant negative) form of the calcineurin A subunit, which binds the calcineurin B subunit with high affinity but does not possess catalytic activity (B subunit knock-out, BKO). In these transfected cells, the dose-response curve for the inhibition of FcepsilonRI-mediated exocytosis by FK506 was shifted to the left, indicating an increased drug sensitivity of BKO-transfected cells. We conclude that FK506 inhibition of FcepsilonRI-mediated exocytosis in mast cells specifically targets calcineurin activity.

DNA Nucleotide Sequence Restricted by the RI Endonuclease

Science.gov (United States)

Hedgpeth, Joe; Goodman, Howard M.; Boyer, Herbert W.

1972-01-01

The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage λ has been determined. The 5′-terminal nucleotide labeled with 32P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in λ DNA was deduced from these data and from the 3′-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virus [Formula: see text] The RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5′-phosphoryls and short cohesive termini of four nucleotides, pApApTpT. The most striking feature of the sequence is its symmetry. PMID:4343974

Äriühingute vastastikune tunnustamine ja asukohavahetus Euroopa Liidus / Andres Vutt

Index Scriptorium Estoniae

Vutt, Andres, 1969-

2007-01-01

Äriühingute vastastikuse tunnustamise põhimõtetest ja võimalustest, kuidas saab Euroopa Liidus muuta äriühingu asukohta ühest riigist teise ning mida tuleb Eestil seoses sellega arvestada. Äriühingute asukohavahetusest Euroopa Kohtu lahendites. Artikkel on valminud ETF-i grandi 6747 abil

Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

International Nuclear Information System (INIS)

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z_B_p_a–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z_B_p_a–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z_B_p_a–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL"-"1, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL"-"1). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. - Highlights: • A photoactivable Z_B_p_a–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques. • A approach for Fc-specific photo-biotinylated IgG via Z_B_p_a–Biotin was proposed. • The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA. • It gave a LOD of 2 ng mL"-"1 CEA

Fc-specific biotinylation of antibody using an engineered photoactivatable Z–Biotin and its biosensing application

Energy Technology Data Exchange (ETDEWEB)

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao, E-mail: tangjb@wfmc.edu.cn

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody–biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z{sub Bpa}–Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG–biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z{sub Bpa}–Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z{sub Bpa}–Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL{sup -1}, is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL{sup -1}). Given that the (strept)avidin–biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. - Highlights: • A photoactivable Z{sub Bpa}–Biotin was fabricated by aaRS/tRNA and Avitag/BirA techniques. • A approach for Fc-specific photo-biotinylated IgG via Z{sub Bpa}–Biotin was proposed. • The photo-biotinylated IgG was used to fabricate an immunosensor for detecting CEA. • It gave a LOD

Remedial Investigation/Feasibility Study (RI/FS) process, elements and techniques guidance

Energy Technology Data Exchange (ETDEWEB)

1993-12-01

This manual provides detailed guidance on Remedial Investigation/Feasibility Studies (RI/FSs) conducted pursuant to the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) at Department of Energy (DOE) facilities. The purpose of the RI/FS, to assess the risk posed by a hazardous waste site and to determine the best way to reduce that risk, and its structure (site characterization, risk assessment, screening and detailed analysis of alternatives, etc.) is defined in the National Oil and Hazardous Substances Pollution Contingency Plan (NCP) and further explained in the Environmental Protection Agency`s (EPA`s) Guidance for Conducting Remedial Investigations and Feasibility Studies Under CERCLA (Interim Final) 540/G-89/004, OSWER Directive 9355.3-01, October 1988. Though issued in 1988, the EPA guidance remains an excellent source of information on the conduct and structure of an RI/FS. This document makes use of supplemental RI/FS-related guidance that EPA has developed since its initial document was issued in 1988, incorporates practical lessons learned in more than 12 years of experience in CERCLA hazardous site remediation, and drawing on those lessons, introduces the Streamlined Approach For Environmental Restoration (SAFER), developed by DOE as a way to proceed quickly and efficiently through the RI/FS process at DOE facilities. Thus as its title implies, this guidance is intended to describe in detail the process and component elements of an RI/FS, as well as techniques to manage the RI/FS effectively.

Omalizumab therapy in atopic dermatitis: depletion of IgE does not improve the clinical course - a randomized, placebo-controlled and double blind pilot study.

Science.gov (United States)

Heil, Peter Maximilian; Maurer, Dieter; Klein, Brigitte; Hultsch, Thomas; Stingl, Georg

2010-12-01

Our understanding of the pathogenic role of IgE in atopic dermatitis is incomplete. We asked whether blocking free IgE would alter the course of the disease. We administered either omalizumab, a humanized monoclonal mouse antibody against IgE, or placebo subcutaneously for 16 weeks to 20 atopic dermatitis patients and measured immunological and clinical disease parameters. Omalizumab (I) reduced free serum IgE, (II) lowered surface IgE and FcɛRI expression on different peripheral blood mononuclear cells, (III) reduced the saturation of FcɛRI with IgE, (IV) increased the number of free FcɛRI and (V) lowered the number of IgE+, but not of FcɛRI+ cells in skin. The in vivo relevance of these results is evidenced by the increase in the threshold allergen concentration required to give a type I hypersensitivity reaction in the titrated skin test. While not significantly altering the clinical disease parameters, omalizumab treatment led to an improvement of the atopy patch test results in single patients, i.e. an eczematous reaction upon epicutaneous allergen challenge. The interference with immediate and delayed type skin tests may imply that a therapeutic benefit of omalizumab treatment, if present at all, would be seen in patients with acute rather than chronic forms of the disease. © The Authors • Journal compilation © Blackwell Verlag GmbH, Berlin.

Chicken IgY Fc expressed by Eimeria mitis enhances the immunogenicity of E. mitis.

Science.gov (United States)

Qin, Mei; Tang, Xinming; Yin, Guangwen; Liu, Xianyong; Suo, Jingxia; Tao, Geru; Ei-Ashram, Saeed; Li, Yuan; Suo, Xun

2016-03-21

Eimeria species are obligate intracellular apicomplexan parasites, causing great economic losses in the poultry industry. Currently wild-and attenuated- type anticoccidial vaccines are used to control coccidiosis. However, their use in fast growing broilers is limited by vaccination side effects caused by medium and/or low immunogenic Eimeria spp. There is, therefore, a need for a vaccine with high immunogenicity for broilers. The avian yolk sac IgY Fc is the avian counterpart of the mammalian IgG Fc, which enhances immunogenicity of Fc-fusion proteins. Here, we developed a stable transgenic Eimeria mitis expressing IgY Fc (Emi.chFc) and investigated whether the avian IgY Fc fragment enhances the immunogenicity of E. mitis. Two-week-old broilers were immunized with either Emi.chFc or wild type Eimeria and challenged with wild type E. mitis to analyze the protective properties of transgenic Emi.chFc. Chickens immunized with Emi.chFc had significantly lower oocyst output, in comparison with PBS, mock control (transgenic E. mitis expressing HA1 from H9N2 avian influenza virus) and wildtype E. mitis immunized groups after challenge, indicating that IgY Fc enhanced the immunogenicity of E. mitis. Our findings suggest that IgY Fc-expressing Eimeria may be a better coccidiosis vaccine, and transgenic Eimeria expressing Fc-fused exogenous antigens may be used as a novel vaccine-delivery vehicle against a wide variety of pathogens.

Jüri Naela rahakotti prantsatas miljon krooni / Aigi Viira

Index Scriptorium Estoniae

Viira, Aigi

2009-01-01

Tallinna Ülikooli tantsukompositsiooni dotsent Jüri Nael sai Eesti haridus- ja teadusministeeriumilt miljonikroonise stipendiumi doktoriõpinguteks Londonis Royal Holloway University's. Jüri Nael pühendub Londonis õppides sellele, mis jääb teatri ja tantsu vahele ehk füüsilisele teatrile, täpsemalt – füüsilisele eksperimentaalteatrile

Statistical dynamic imaging of RI-labeled tracer from list-mode PET data

International Nuclear Information System (INIS)

Tanimoto, Michiaki; Kuroda, Yoshihiro; Oshiro, Osamu; Watabe, Hiroshi; Kuroda, Tomohiro

2009-01-01

Positron emission tomography (PET) can be used in physiological analysis to illustrate physiological states by visualizing the accumulation of radioisotope (RI)-labeled tracer in specific organs or tissues. PET obtains spatio-temporal statistics in the form of list-mode data. However, conventional imaging techniques, which sum up list-mode data over a given time period, cannot depict detailed temporal dynamics of the RI-labeled tracer. In this study, a spatio-temporal analysis approach was employed to visualize the temporal flow dynamics of RI-labeled tracer from the obtained list-mode data. Experiments to assess the visualization of simulated RI-labeled tracer dynamics as well as RI-labeled tracer dynamics in a vascular phantom showed that the proposed method successfully depicted detailed temporal flow dynamics that could not be visualized using conventional methods. (author)

Imaging of the bronchial blood flow using RI-angiography

International Nuclear Information System (INIS)

Fujii, Tadashige; Hirayama, Jiro; Kanai, Hisakata; Kobayashi, Toshio; Handa, Kenjiro

1979-01-01

RI-angiography with sup(99m)TcO 4 - was carried out using a scintillation camera with a digital minicomputer for the purpose of imaging of bronchial blood flow in various lung diseases, and as application of dual radioisotope techniques, other imagings such as tumor imaging with 197 HgCl 2 or 67 Ga-citrate and/or perfusion imaging with sup(99m)Tc-MAA, were performed simultaneously in patients remaining the same position, too. The image as a iso-count map extracted out of the image of 197 HgCl 2 , 67 Ga-citrate or sup(99m)Tc-MAA, was superimposed to the brightness image of RI-angiogram (aortic phase). By these procedures, the image of bronchial blood flow were obtained in some patients with lung cancer, pulmonary tuberculosis, lung abscess, and chronic bronchitis. The dual radioisotope techniques using RI-angiography and the other imaging were useful to make isotope diagnosis of lung diseases more reliable, and the image superimposition methods using RI-angiogram and the image of tumor of perfusion, were useful to improve anatomic orientation of the former. (author)

Analysis of concept and application or Risk-Informed Performed-Based Regulation (RI-PBR)

International Nuclear Information System (INIS)

Kim, W. S.; Sung, K. Y.; Lee, C. J.; Kim, H. J.

2002-01-01

For improving regulation of nuclear power plants, the USNRC is adopting the Risk-Informed Performance-Based Regulation (RI-PBR) as an alternative, in parallel with implementing current deterministic regulation. This paper introduces a research plan for 'Institutionalization of RI-PBR' that is being conducted by KINS as a national project for evaluating feasibility for application of the alternative to Korean regulation system. Analysis of regulation characteristics, case study and experience on RI-PBR were presented as interim research results. In addition, the future plan of development of RI-PBR concept as understandable to the public and evaluation of level of techniques needed for implementation of RI-PBR was introduced

40 CFR Table 25 to Subpart G of... - Effective Column Diameter (Fc)

Science.gov (United States)

2010-07-01

... 40 Protection of Environment 9 2010-07-01 2010-07-01 false Effective Column Diameter (Fc) 25 Table..., Table 25 Table 25 to Subpart G of Part 63—Effective Column Diameter (Fc) Column type Fc (feet) 9-inch by 7-inch built-up columns 1.1 8-inch-diameter pipe columns 0.7 No construction details known 1.0 ...

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge

Science.gov (United States)

Konduru, Krishnamurthy; Shurtleff, Amy C.; Bradfute, Steven B.; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105−106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Science.gov (United States)

Konduru, Krishnamurthy; Shurtleff, Amy C; Bradfute, Steven B; Nakamura, Siham; Bavari, Sina; Kaplan, Gerardo

2016-01-01

Ebola virus (EBOV), a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP) are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV) GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc) protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV) pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data further support

Ebolavirus Glycoprotein Fc Fusion Protein Protects Guinea Pigs against Lethal Challenge.

Directory of Open Access Journals (Sweden)

Krishnamurthy Konduru

Full Text Available Ebola virus (EBOV, a member of the Filoviridae that can cause severe hemorrhagic fever in humans and nonhuman primates, poses a significant threat to the public health. Currently, there are no licensed vaccines or therapeutics to prevent and treat EBOV infection. Several vaccines based on the EBOV glycoprotein (GP are under development, including vectored, virus-like particles, and protein-based subunit vaccines. We previously demonstrated that a subunit vaccine containing the extracellular domain of the Ebola ebolavirus (EBOV GP fused to the Fc fragment of human IgG1 (EBOVgp-Fc protected mice against EBOV lethal challenge. Here, we show that the EBOVgp-Fc vaccine formulated with QS-21, alum, or polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC adjuvants induced strong humoral immune responses in guinea pigs. The vaccinated animals developed anti-GP total antibody titers of approximately 105-106 and neutralizing antibody titers of approximately 103 as assessed by a BSL-2 neutralization assay based on vesicular stomatitis virus (VSV pseudotypes. The poly-ICLC formulated EBOVgp-Fc vaccine protected all the guinea pigs against EBOV lethal challenge performed under BSL-4 conditions whereas the same vaccine formulated with QS-21 or alum only induced partial protection. Vaccination with a mucin-deleted EBOVgp-Fc construct formulated with QS-21 adjuvant did not have a significant effect in anti-GP antibody levels and protection against EBOV lethal challenge compared to the full-length GP construct. The bulk of the humoral response induced by the EBOVgp-Fc vaccine was directed against epitopes outside the EBOV mucin region. Our findings indicate that different adjuvants can eliciting varying levels of protection against lethal EBOV challenge in guinea pigs vaccinated with EBOVgp-Fc, and suggest that levels of total anti-GP antibodies elicit by protein-based GP subunit vaccines do not correlate with protection. Our data

A strategy for bacterial production of a soluble functional human neonatal Fc receptor

DEFF Research Database (Denmark)

Andersen, Jan Terje; Justesen, Sune; Berntzen, Gøril

2008-01-01

The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus...

Fc-specific biotinylation of antibody using an engineered photoactivatable Z-Biotin and its biosensing application.

Science.gov (United States)

Yang, Hong-Ming; Bao, Ru-Meng; Yu, Chang-Mei; Lv, Yan-Na; Zhang, Wei-Fen; Tang, Jin-Bao

2017-01-01

The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (Z Bpa -Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz. photo-biotinylated IgG, was successfully achieved after UV exposure by combining the inherent Fc-binding capability of the Z-domain with the formation of covalent bond by the photo-crosslinker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis assay showed that more than 90% of IgGs conjugated with Z Bpa -Biotin molecules suffered 3 h UV irradiation. Further pepsin digestion analysis confirmed that the Z Bpa -Biotin was conjugated to the Fc fragment of IgG without interference. We took the tumor biomarker carcinoembryoic antigen (CEA) as model to evaluate the detection efficiency of the site-specific photo-biotinylated IgG in biosensing application using surface plasmon resonance (SPR) technology. The photo-biotinylated IgG coated surface gave a limit of detection (LOD) of 2 ng mL -1 , is 5-fold lower than that of the randomly NHS-biotinylated IgG (10 ng mL -1 ). Given that the (strept)avidin-biotin complex is extensively used in immunoassays, the proposed method for biotinylated IgG provides a powerful approach to further expand related applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults

OpenAIRE

Gaudinski, Martin R.; Coates, Emily E.; Houser, Katherine V.; Chen, Grace L.; Yamshchikov, Galina; Saunders, Jamie G.; Holman, LaSonji A.; Gordon, Ingelise; Plummer, Sarah; Hendel, Cynthia S.; Conan-Cibotti, Michelle; Lorenzo, Margarita Gomez; Sitar, Sandra; Carlton, Kevin; Laurencot, Carolyn

2018-01-01

Background VRC01 is a human broadly neutralizing monoclonal antibody (bnMAb) against the CD4-binding site of the HIV-1 envelope glycoprotein (Env) that is currently being evaluated in a Phase IIb adult HIV-1 prevention efficacy trial. VRC01LS is a modified version of VRC01, designed for extended serum half-life by increased binding affinity to the neonatal Fc receptor. Methods and findings This Phase I dose-escalation study of VRC01LS in HIV-negative healthy adults was conducted by the Vaccin...

Üksikud naljakad peaaegu kolmikud / Jüri Viikberg ; tlk. Jüri Viikberg

Index Scriptorium Estoniae

Viikberg, Jüri 1953-

2005-01-01

Arvustus: Punttila, Matti. Haaskannäköinen tyttö : kielikaskuista sanaleikkeihin. Helsinki, 1998 ; Punttila, Matti. Pilkettä silmäkulmaan : kielikaskuista sanaleikkeihin. Helsinki, 2001 ; Punttila, Matti. Poskettomia huulia : kielikaskuista sanaleikkeihin. Helsinki, 2004. Näiteid: Kui keel ei püsi hammaste taga ; Sõna keeles ja keel hammaste vahel / tõlkinud Jüri Viikberg // Oma Keel, 1995, nr. 1, lk. 55-56, 63-64

Live visualization of genomic loci with BiFC-TALE.

Science.gov (United States)

Hu, Huan; Zhang, Hongmin; Wang, Sheng; Ding, Miao; An, Hui; Hou, Yingping; Yang, Xiaojing; Wei, Wensheng; Sun, Yujie; Tang, Chao

2017-01-11

Tracking the dynamics of genomic loci is important for understanding the mechanisms of fundamental intracellular processes. However, fluorescent labeling and imaging of such loci in live cells have been challenging. One of the major reasons is the low signal-to-background ratio (SBR) of images mainly caused by the background fluorescence from diffuse full-length fluorescent proteins (FPs) in the living nucleus, hampering the application of live cell genomic labeling methods. Here, combining bimolecular fluorescence complementation (BiFC) and transcription activator-like effector (TALE) technologies, we developed a novel method for labeling genomic loci (BiFC-TALE), which largely reduces the background fluorescence level. Using BiFC-TALE, we demonstrated a significantly improved SBR by imaging telomeres and centromeres in living cells in comparison with the methods using full-length FP.

Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

International Nuclear Information System (INIS)

He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

2004-01-01

The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

RiTSE/KCGE: Application of artificial intelligence to improve plant availability

International Nuclear Information System (INIS)

Frank, M.V.; Epstein, S.A.

1986-01-01

Maintenance and other personnel related errors are a major cause of reactor trips in some U.S. plants. Software called RiTSE and KCGE, that can operate on a mini-computer work station, has been developed to dramatically reduce trips caused by clearance coordination problems. Such problems arise because of the enormous number of components that interrelate in ways that cannot always be anticipated during plant operation. Concurrent maintenance, test, surveillance and other operations activities contribute to the unanticipated interactions. RiTSE allows the appropriate plant personnel (i.e., those responsible for authorizing work during start-up and operation) to predict if their next authorizations would cause a reactor trip. With KCGE, RiTSE can warn these personnel of a condition of significantly reduced margin to trip. RiTSE also aids post-trip cause analysis by pointing out those components that could have contributed to the trip. It is believable that use of RiTSE is a quicker and more reliable way to authorize work during operation than reliance on memory and drawings. The software provides maximum benefit when used in conjunction with a configuration management program that assures accurate plant presentation and accurate updating of component status during operation. Application of RiTSE and KCGE to a client's PWR demonstrated part of their potential benefits. (Nogami, K.)

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D.

NARCIS (Netherlands)

Kapur, R.; Della Valle, L.; Verhagen, O.J.; Hipgrave Ederveen, A.; Ligthart, P.; de Haas, M.; Kumpel, B.; Wuhrer, M.; van der Schoot, C.E.; Vidarsson, G.

2015-01-01

Background RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions

Prophylactic anti-D preparations display variable decreases in Fc-fucosylation of anti-D

NARCIS (Netherlands)

Kapur, Rick; Della Valle, Luciana; Verhagen, Onno J. H. M.; Hipgrave Ederveen, Agnes; Ligthart, Peter; de Haas, Masja; Kumpel, Belinda; Wuhrer, Manfred; van der Schoot, C. Ellen; Vidarsson, Gestur

2015-01-01

RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with

Effects of microparticle size and Fc density on macrophage phagocytosis.

Directory of Open Access Journals (Sweden)

Patricia Pacheco

Full Text Available Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages.

Tailor-making a protein a-derived domain for efficient site-specific photocoupling to Fc of mouse IgG₁.

Directory of Open Access Journals (Sweden)

Feifan Yu

Full Text Available Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG₁ (mIgG₁. Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG₁ monoclonal antibodies (mAbs. The best variant, denoted Z(F5I corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP group as a probe for site-specific photoconjugation to Fc of mIgG₁, The best photocoupling efficiency to mIgG₁ Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG₁ mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG₁ antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG₁ mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly higher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP probe. This result indicates that the use of a site-specific and affinity probe

Genealogy of gas cells for low-energy RI-beam production

Energy Technology Data Exchange (ETDEWEB)

Wada, Michiharu, E-mail: mw@riken.jp

2013-12-15

Highlights: • In order to overcome serious limitations in the universality of the traditional isotope separator on-line technique, various endeavors have been made on gas catcher cells for converting relativistic RI-beams from in-flight separators to low-energy RI-beams. • The origin of the gas catcher is found in the IGISOL (Ion guide isotope separator on-line) technique. • Many developments have been made over the years to overcome the various difficulties and drawbacks found in the IGISOL technique. -- Abstract: In order to overcome serious limitations in the universality of the traditional isotope separator on-line technique, various endeavors have been made on gas catcher cells for converting relativistic RI-beams from in-flight separators to low-energy RI-beams. The origin of the gas catcher is found in the IGISOL (Ion guide isotope separator on-line) technique. Many developments have been made over the years to overcome the various difficulties and drawbacks found in the IGISOL technique.

Genealogy of gas cells for low-energy RI-beam production

International Nuclear Information System (INIS)

Wada, Michiharu

2013-01-01

Highlights: • In order to overcome serious limitations in the universality of the traditional isotope separator on-line technique, various endeavors have been made on gas catcher cells for converting relativistic RI-beams from in-flight separators to low-energy RI-beams. • The origin of the gas catcher is found in the IGISOL (Ion guide isotope separator on-line) technique. • Many developments have been made over the years to overcome the various difficulties and drawbacks found in the IGISOL technique. -- Abstract: In order to overcome serious limitations in the universality of the traditional isotope separator on-line technique, various endeavors have been made on gas catcher cells for converting relativistic RI-beams from in-flight separators to low-energy RI-beams. The origin of the gas catcher is found in the IGISOL (Ion guide isotope separator on-line) technique. Many developments have been made over the years to overcome the various difficulties and drawbacks found in the IGISOL technique

Tallinna linnapea Jüri Ratas : Keskerakond ei ole maffia! / Jüri Ratas ; interv. Urmo Soonvald

Index Scriptorium Estoniae

Ratas, Jüri, 1978-

2005-01-01

Tallinna linnapea Jüri Ratas vastab küsimustele, mis puudutavad Rein Ratase Tallinna Vee nõukogu liikmeks määramist, K-kohukse reklaami seost Keskerakonna valimiskampaaniaga, linnapeaks saamist, koostööd teiste poliitikutega, Edgar Savisaare osa Tallinna linna juhtimises

NRG1-Fc improves metabolic health via dual hepatic and central action.

Science.gov (United States)

Zhang, Peng; Kuang, Henry; He, Yanlin; Idiga, Sharon O; Li, Siming; Chen, Zhimin; Yang, Zhao; Cai, Xing; Zhang, Kezhong; Potthoff, Matthew J; Xu, Yong; Lin, Jiandie D

2018-03-08

Neuregulins (NRGs) are emerging as an important family of signaling ligands that regulate glucose and lipid homeostasis. NRG1 lowers blood glucose levels in obese mice, whereas the brown fat-enriched secreted factor NRG4 protects mice from high-fat diet-induced insulin resistance and hepatic steatosis. However, the therapeutic potential of NRGs remains elusive, given the poor plasma half-life of the native ligands. Here, we engineered a fusion protein using human NRG1 and the Fc domain of human IgG1 (NRG1-Fc) that exhibited extended half-life in circulation and improved potency in receptor signaling. We evaluated its efficacy in improving metabolic parameters and dissected the mechanisms of action. NRG1-Fc treatment triggered potent AKT activation in the liver, lowered blood glucose, improved insulin sensitivity, and suppressed food intake in obese mice. NRG1-Fc acted as a potent secretagogue for the metabolic hormone FGF21; however, the latter was largely dispensable for its metabolic effects. NRG1-Fc directly targeted the hypothalamic POMC neurons to promote membrane depolarization and increase firing rate. Together, NRG1-Fc exhibits improved pharmacokinetic properties and exerts metabolic benefits through dual inhibition of hepatic gluconeogenesis and caloric intake.

Herpes simplex virus immunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI

International Nuclear Information System (INIS)

Johnson, D.C.; Ligas, M.W.; Frame, M.C.; Cross, A.M.; Stow, N.D.

1988-01-01

Evidence was recently presented that herpes simplex virus type 1 (HSV-1) immunoglobulin G (IgG) Fc receptors are composed of a complex containing a previously described glycoprotein, gE, and a novel virus-induced polypeptide, provisionally named g70. Using a monoclonal antibody designated 3104, which recognizes g70, in conjunction with antipeptide sera and virus mutants unable to express g70 or gE, the authors have mapped the gene encoding g70 to the US7 open reading frame of HSV-1 adjacent to the gE gene. Therefore, g70 appears to be identical to a recently described polypeptide which was named gI. Under mildly denaturing conditions, monoclonal antibody 3104 precipitated both gI and gE from extracts of HSV-1-infected cells. In addition, rabbit IgG precipitated the gE-gI complex from extracts of cells transfected with a fragment of HSV-1 DNA containing the gI, gE, and US9 genes. Cells infected with mutant viruses which were unable to express gE or gI did not bind radiolabeled IgG; however, cells coinfected with two viruses, one unable to express gE and the other unable to express gI, bound levels of IgG approaching those observed with wild-type viruses. These results further support the hypothesis that gE and gI form a complex which binds IgG by the Fc domain and that neither polypeptide alone can bind IgG

Omalizumab in the management of patients with allergic (IgE-mediated asthma

Directory of Open Access Journals (Sweden)

Thomas Sandström

2009-05-01

Full Text Available Thomas SandströmDepartment of Respiratory Medicine and Allergy, University Hospital, Umeå, SwedenAbstract: Immunoglobulin E (IgE is central to the pathophysiology of allergic asthma. Omalizumab, an anti-IgE monoclonal antibody, binds to the FcεRI binding site on free IgE. As a result, circulating free IgE is reduced, IgE is prevented from attaching to mast cells and basophils, and FcεRI receptor expression is down-regulated. The inflammatory response to allergens and the acute and chronic effector phases of allergic inflammation are thereby attenuated. In clinical trials in adults and adolescents, omalizumab reduced asthma exacerbations, severe asthma exacerbations, inhaled corticosteroid requirements, and emergency visits, as well as significantly improving asthma-related quality of life, morning peak expiratory flow and asthma symptom scores in patients with severe allergic (IgE-mediated asthma. Results from clinical trials in children (< 12 years are consistent with those in the adult population. It is difficult to predict which patients will respond to omalizumab. Responders to omalizumab should be identified after a 16-week trial of therapy using the physician’s overall assessment. When treatment is targeted to these responders, omalizumab provides a cost-effective therapy for inadequately controlled severe allergic (IgE-mediated asthma. Long-term therapy with omalizumab shows the potential for disease-modification in asthma. Ongoing studies are also evaluating the use of omalizumab in other non-asthma IgE-mediated conditions.Keywords: omalizumab, IgE, allergic asthma

Construction and expression of an anti-VEGFR2 Nanobody-Fc fusionbody in NS0 host cell.

Science.gov (United States)

Qasemi, Maryam; Behdani, Mahdi; Shokrgozar, Mohammad Ali; Molla-Kazemiha, Vahid; Mohseni-Kuchesfahani, Homa; Habibi-Anbouhi, Mahdi

2016-07-01

Angiogenesis is the formation of new blood vessels which is involved in migration, growth and differentiation of endothelial cells. This process regularly occurs during growth and development in children however, in adults is usually part of a disease process such as cancer. The vascular endothelial growth factor (VEGF) is a vital player in the vascular development and angiogenesis in physiological and pathological processes. Camelid's immune system has unique antibodies which are composed of only a heavy chain homodimer and the variable domain (VHH, Nanobody). Nanobodies are small, around 15 kDa and stable. In this study, we engineered and constructed a new Nanobody-Fc fusion protein (fusionbody) composed of an anti-VEGFR2 Nanobody and an Fc fragment of human IgG1 antibody. The recombinant vector was transfected into NS0 host cells. Stable producer clones were developed and the recombinant fusionbody was expressed and purified. Functional assay showed the anti-VEGFR2 fusionbody could bind to VEGFR2 on cell surface via VHH part and could mediate killing the targeted cells through direct cell death and complement-dependent cytotoxicity (CDC). Copyright © 2016 Elsevier Inc. All rights reserved.

IgG receptor FcγRIIB plays a key role in obesity-induced hypertension.

Science.gov (United States)

Sundgren, Nathan C; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D; Tanigaki, Keiji; Yuhanna, Ivan S; Chambliss, Ken L; Mineo, Chieko; Shaul, Philip W

2015-02-01

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertension, FcγRIIB(-/-) mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet-fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. © 2014 American Heart Association, Inc.

IgG Receptor FcγRIIB Plays a Key Role in Obesity-Induced Hypertension

Science.gov (United States)

Sundgren, Nathan C.; Vongpatanasin, Wanpen; Boggan, Brigid-Meghan D.; Tanigaki, Keiji; Yuhanna, Ivan S.; Chambliss, Ken L.; Mineo, Chieko; Shaul, Philip W.

2015-01-01

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB+/+ mice developed obesity-induced hypertension, FcγRIIB−/− mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet–fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals. PMID:25368023

Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid.

Science.gov (United States)

Kiyokawa, Kazuya; Yamamoto, Shinji; Sato, Yukari; Momota, Naoto; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

2012-07-01

Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Näitus "Sajand eesti puugravüüri klotsidel" / Jüri Hain

Index Scriptorium Estoniae

Hain, Jüri, 1941-

2007-01-01

Eesti Rahvusraamatukogus 15. I-3. II eksponeeritud ja rahvusraamatukogu kollektsioonist koostatud näitusest "Sajand eesti puugravüüri klotsidel: 1840-1940". Järgmistest kunstnikest ja nende töödest: Aleksander Otto Gern, Bogdan Gottlieb Friedrich Kally, August Daugell, Hermann Eduard Hartmann, Eduard Magnus Jakobson, Mart Pukits, Andres Kuusk, Karl Friedrich Schnell, Anton Suurkask (1873-1965), Arkadio Laigo, Hando Mugasto, Eduard Järv, Richard Kaljo, Ott Kangilaski, Ants Puks (1918-?), Salome Trei

Binding Cellulose and Chitosan via Intermolecular Inclusion Interaction: Synthesis and Characterisation of Gel

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Jiufang Duan

2015-01-01

Full Text Available A novel cellulose-chitosan gel was successfully prepared in three steps: (1 ferrocene- (Fc- cellulose with degrees of substitution (DS of 0.5 wt% was synthesised by ferrocenecarboxylic acid and cellulose within dimethylacetamide/lithium chloride (DMAc/LiCl; (2 the β-cyclodextrin (β-CD groups were introduced onto the chitosan chains by reacting chitosan with epichlorohydrin in dimethyl sulphoxide and a DS of 0.35 wt%; (3 thus, the cellulose-chitosan gel was obtained via an intermolecular inclusion interaction of Fc-cellulose and β-CD-chitosan in DMA/LiCl, that is, by an intermolecular inclusion interaction, between the Fc groups of cellulose and the β-CD groups on the chitosan backbone at room temperature. The successful synthesis of Fc-cellulose and β-CD-chitosan was characterised by 13C-NMR spectroscopy. The gel based on β-CD-chitosan and Fc-cellulose was formed under mild conditions which can engender autonomous healing between cut surfaces after 24 hours: the gel cannot self-heal while the cut surfaces were coated with a solution of a competitive guest (adamantane acid. The cellulose-chitosan complex made by this method underwent self-healing. Therefore, this study provided a novel method of expanding the application of chitosan by binding it with another polymer.

Äri- ja ettevõtluskeeldude piiriülene mõju / Maarja Torga, Juhan Sarv

Index Scriptorium Estoniae

Torga, Maarja, 1984-

2017-01-01

Eestis kohaldatud äri- ja ettevõtluskeeldude piiriülesest mõjust ning välisriigis kohaldatud äri- ja ettevõtluskeelu mõjust Eestis. Äri- ja ettevõtluskeeldude piiriülesest tsiviilõiguslikust ja karistusõiguslikust mõjust. Sisaldab asjakohast kohtupraktikat

Fcγ receptor expression on splenic macrophages in adult immune thrombocytopenia

NARCIS (Netherlands)

Audia, S; Santegoets, K; Laarhoven, A G; Vidarsson, G; Facy, O; Ortega-Deballon, P; Samson, M; Janikashvili, N; Saas, P; Bonnotte, B; Radstake, T R

2017-01-01

Splenic macrophages play a key role in immune thrombocytopenia (ITP) pathogenesis by clearing opsonized platelets. Fcγ receptors (FcγR) participate in this phenomenon, but their expression on splenic macrophages and their modulation by treatment have scarcely been studied in human ITP. We aimed to

Mast cells & Company

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Friederike eJönsson

2012-02-01

Full Text Available Classically, allergy depends on IgE antibodies and on high-affinity IgE receptors expressed by mast cells and basophils. This long accepted IgE/FcεRI/mast cell paradigm, on which the definition of immediate hypersensitivity was based in the Gell and Coomb’s classification, appears too reductionist. Recently accumulated evidence indeed requires that not only IgE but also IgG antibodies, that not only FcεRI but also FcγR of the different types, that not only mast cells and basophils but also neutrophils, monocytes, macrophages, eosinophils, and other myeloid cells by considered as important players in allergy. This view markedly changes our understanding of allergic diseases and, possibly, their treatment.

Development of RI protein chip system for measurement of ADMA as risk factor of liver disease, cardiovascular disease and diabetes

Energy Technology Data Exchange (ETDEWEB)

Park, Gil Hong; An, Sin Ae; Choi, Hyun Mi; Cheong, Kyung Ah; Chang, Yeon Soo [Korea University, Seoul (Korea, Republic of)

2010-05-15

ADMA (asymmetric dimethylarginine) is an endogenous competitive NOS (nitric oxide synthase) inhibitor. Elevation of ADMA level in body fluid is related to various diseases including diabetes mellitus, atherosclerosis, hypercholesterolemia, chronic heart failure, and hypertension. Our goal was to elucidate the role for ADMA and protein arginine methylation in the pathogenesis of diabetes and develop RI protein chip system for easy determination of ADMA levels in blood or tissues. As a result, ADMA regulated by protein arginine methylation, DDAH1, and NOS was demonstrated to play some role in the pathogenesis of diabetes mellitus, and arginine methylation of some proteins indicated their possible involvement in the change in ADMA levels. The concept of the RI protein chip system was devised such that firstly, ADMA was acylated by radio-labelled succinic acid, and followed by binding of the complex to anti-acyl ADMA-specific antibody coated on chip. Acylation condition of ADMA with radio-labelled succinic acid was established

Development of RI protein chip system for measurement of ADMA as risk factor of liver disease, cardiovascular disease and diabetes

International Nuclear Information System (INIS)

Park, Gil Hong; An, Sin Ae; Choi, Hyun Mi; Cheong, Kyung Ah; Chang, Yeon Soo

2010-05-01

ADMA (asymmetric dimethylarginine) is an endogenous competitive NOS (nitric oxide synthase) inhibitor. Elevation of ADMA level in body fluid is related to various diseases including diabetes mellitus, atherosclerosis, hypercholesterolemia, chronic heart failure, and hypertension. Our goal was to elucidate the role for ADMA and protein arginine methylation in the pathogenesis of diabetes and develop RI protein chip system for easy determination of ADMA levels in blood or tissues. As a result, ADMA regulated by protein arginine methylation, DDAH1, and NOS was demonstrated to play some role in the pathogenesis of diabetes mellitus, and arginine methylation of some proteins indicated their possible involvement in the change in ADMA levels. The concept of the RI protein chip system was devised such that firstly, ADMA was acylated by radio-labelled succinic acid, and followed by binding of the complex to anti-acyl ADMA-specific antibody coated on chip. Acylation condition of ADMA with radio-labelled succinic acid was established

S1P4 Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells

Directory of Open Access Journals (Sweden)

Joseph M. Kulinski

2018-04-01

Full Text Available Mast cells are key players in the development of inflammatory allergic reactions. Cross-linking of the high-affinity receptor for IgE (FcεRI on mast cells leads to the generation and secretion of the sphingolipid mediator, sphingosine-1-phosphate (S1P which is able, in turn, to transactivate its receptors on mast cells. Previous reports have identified the expression of two of the five receptors for S1P on mast cells, S1P1 and S1P2, with functions in FcεRI-mediated chemotaxis and degranulation, respectively. Here, we show that cultured mouse mast cells also express abundant message for S1P4. Genetic deletion of S1pr4 did not affect the differentiation of bone marrow progenitors into mast cells or the proliferation of mast cells in culture. A comprehensive characterization of IgE-mediated responses in S1P4-deficient bone marrow-derived and peritoneal mouse mast cells indicated that this receptor is dispensable for mast cell degranulation, cytokine/chemokine production and FcεRI-mediated chemotaxis in vitro. However, interleukin-33 (IL-33-mediated enhancement of IgE-induced degranulation was reduced in S1P4-deficient peritoneal mast cells, revealing a potential negative regulatory role for S1P4 in an IL-33-rich environment. Surprisingly, genetic deletion of S1pr4 resulted in exacerbation of passive systemic anaphylaxis to IgE/anti-IgE in mice, a phenotype likely related to mast cell-extrinsic influences, such as the high circulating levels of IgE in these mice which increases FcεRI expression and consequently the extent of the response to FcεRI engagement. Thus, we provide evidence that S1P4 modulates anaphylaxis in an unexpected manner that does not involve regulation of mast cell responsiveness to IgE stimulation.

Reiu Tüüri maratonloeng kunstiturust / Reiu Tüür

Index Scriptorium Estoniae

Tüür, Reiu

2001-01-01

27. VI toimus Pärnus Kunstplatze Unobomberi galeriis Reiu Tüüri näitus-loeng kaasaegse kunsti galeriidest Londonis ja Eestis. Näitus on osa R. Tüüri sel sügisel Londoni linnaülikooli juures kaitstavast kunsti mänedzhmendi alasest magistritööst "Eesti kaasaegse kunsti turg". Juhendaja Jani Robertson Hollandist.

Generation of CMAHKO/GTKO/shTNFRI-Fc/HO-1 quadruple gene modified pigs.

Science.gov (United States)

Kim, Geon A; Lee, Eun Mi; Jin, Jun-Xue; Lee, Sanghoon; Taweechaipaisankul, Anukul; Hwang, Jong Ik; Alam, Zahid; Ahn, Curie; Lee, Byeong Chun

2017-08-01

As an alternative source of organs for transplantation into humans, attention has been directed to pigs due to their similarities in biological features and organ size. However, severe immune rejection has prevented successful xenotransplantation using pig organs and tissues. To overcome immune rejection, recently developed genetic engineering systems such as TALEN coupled with somatic cell nuclear transfer (SCNT) to make embryos could be used to produce pigs compatible with xenotransplantation. We used the TALEN system to target the non-Gal antigen cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene in pigs that is naturally deleted in humans. Gal-deleted cells expressing both soluble human tumor necrosis factor receptor I IgG 1 -Fc (shTNFRI-Fc) and human hemagglutinin -tagged-human heme oxygenase-1 (hHO-1) were transfected with a TALEN target for CMAH. Cells lacking CMAH were negatively selected using N-glyconeuraminic acid (Neu5Gc)/magnetic beads and the level of Neu5Gc expression of isolated cells were analyzed by FACS and DNA sequencing. Cloned embryos using 3 different genetically modified cell clones were respectively transferred into 3 recipients, with 55.6% (5/9) becoming pregnant and three cloned pigs were produced. Successful genetic disruption of the CMAH gene was confirmed by sequencing, showing lack of expression of CMAH in tail-derived fibroblasts of the cloned piglets. Besides decreased expression of Neu5Gc in piglets produced by SCNT, antibody-mediated complement-dependent cytotoxicity assays and natural antibody binding for examining immuno-reactivity of the quadruple gene modified pigs derived from endothelial cells and fibroblasts were reduced significantly compared to those of wild type animals. We conclude that by combining the TALEN system and transgenic cells, targeting of multiple genes could be useful for generating organs for xenotransplantation. We produced miniature pigs with quadruple modified genes CMAHKO/GTKO/shTNFRI-Fc

Competency remodelling and application plans for development of job competency in RI-biomics

Energy Technology Data Exchange (ETDEWEB)

Shin, Woo Ho; Park, Tai Jin [Korean Association for Radiation Application, Seoul (Korea, Republic of)

2017-03-15

RI-Biomics technology is advanced convergence technologies that can be measured in real time and track in vivo behavior and metabolism of substances using characteristics of the radioactive isotope. Its application fields are increasing such as drug development, agriculture, development of new materials and their utilization, etc. In addition, according to domestic and international developments and changes in the RI-Biomics environment, RI-Biomics professionals are needed to train continuously. To develop systematic human resources basement and competency-based curriculum, we perform competency modeling of pedagogical perspective to targeted at high-performance on RI-Biomics. Furthermore, we redefine the competency model and verified by industry experts with focus group interviews. In the result, two general competencies and three professional competencies were extracted by interview. Each competencies are organized six sub-competencies and nine sub-competencies. In the final steps, the same procedures were repeated to obtain the consensus of experts on derived competencies and behavioral objectives. The results of the study are applicable to enhance human resource management and to develop the curriculum for RI-Biomics expert training. It is expected to be used as reference material of long term-planning for RI-Biomics professional.

Competency remodelling and application plans for development of job competency in RI-biomics

International Nuclear Information System (INIS)

Shin, Woo Ho; Park, Tai Jin

2017-01-01

RI-Biomics technology is advanced convergence technologies that can be measured in real time and track in vivo behavior and metabolism of substances using characteristics of the radioactive isotope. Its application fields are increasing such as drug development, agriculture, development of new materials and their utilization, etc. In addition, according to domestic and international developments and changes in the RI-Biomics environment, RI-Biomics professionals are needed to train continuously. To develop systematic human resources basement and competency-based curriculum, we perform competency modeling of pedagogical perspective to targeted at high-performance on RI-Biomics. Furthermore, we redefine the competency model and verified by industry experts with focus group interviews. In the result, two general competencies and three professional competencies were extracted by interview. Each competencies are organized six sub-competencies and nine sub-competencies. In the final steps, the same procedures were repeated to obtain the consensus of experts on derived competencies and behavioral objectives. The results of the study are applicable to enhance human resource management and to develop the curriculum for RI-Biomics expert training. It is expected to be used as reference material of long term-planning for RI-Biomics professional

Teel kriisist õigusriiki / Jüri Raidla

Index Scriptorium Estoniae

Raidla, Jüri, 1957-

1990-01-01

Jüri Raidla, justiitsminister (kinnitati 17. 04. 1990). Sündinud 1957. a. Pärnus, 1980. a. lõpetas Tartu Ülikooli õigusteaduskonna, kaitses 12. 02. 1987. a. Leningradi Ülikoolis väitekirja "Mehaaniliste transpordivahenditega kahju tekitamisest tulenevad kohustused"

Fcγ receptor-mediated inflammation inhibits axon regeneration.

Directory of Open Access Journals (Sweden)

Gang Zhang

Full Text Available Anti-glycan/ganglioside antibodies are the most common immune effectors found in patients with Guillain-Barré Syndrome, which is a peripheral autoimmune neuropathy. We previously reported that disease-relevant anti-glycan autoantibodies inhibited axon regeneration, which echo the clinical association of these antibodies and poor recovery in Guillain-Barré Syndrome. However, the specific molecular and cellular elements involved in this antibody-mediated inhibition of axon regeneration are not previously defined. This study examined the role of Fcγ receptors and macrophages in the antibody-mediated inhibition of axon regeneration. A well characterized antibody passive transfer sciatic nerve crush and transplant models were used to study the anti-ganglioside antibody-mediated inhibition of axon regeneration in wild type and various mutant and transgenic mice with altered expression of specific Fcγ receptors and macrophage/microglia populations. Outcome measures included behavior, electrophysiology, morphometry, immunocytochemistry, quantitative real-time PCR, and western blotting. We demonstrate that the presence of autoantibodies, directed against neuronal/axonal cell surface gangliosides, in the injured mammalian peripheral nerves switch the proregenerative inflammatory environment to growth inhibitory milieu by engaging specific activating Fcγ receptors on recruited monocyte-derived macrophages to cause severe inhibition of axon regeneration. Our data demonstrate that the antibody orchestrated Fcγ receptor-mediated switch in inflammation is one mechanism underlying inhibition of axon regeneration. These findings have clinical implications for nerve repair and recovery in antibody-mediated immune neuropathies. Our results add to the complexity of axon regeneration in injured peripheral and central nervous systems as adverse effects of B cells and autoantibodies on neural injury and repair are increasingly recognized.

Pealinlased olid aktiivsemad valijad / Jüri Ratas

Index Scriptorium Estoniae

Ratas, Jüri, 1978-

2007-01-01

Ilmunud ka: Stolitsa, 14. märts 2007, lk. 2. Tallinna linnapea Jüri Ratas parlamendivalimiste tulemustest. Vt. samas: Kes Tallinnast riigikokku valiti?; Riigikogu valimise tulemused Tallinnas: diagramm

Reproduction of the FC/DFC units in nucleoli.

Science.gov (United States)

Smirnov, Evgeny; Hornáček, Matúš; Kováčik, Lubomír; Mazel, Tomáš; Schröfel, Adam; Svidenská, Silvie; Skalníková, Magdalena; Bartová, Eva; Cmarko, Dušan; Raška, Ivan

2016-04-25

The essential structural components of the nucleoli, Fibrillar Centers (FC) and Dense Fibrillar Components (DFC), together compose FC/DFC units, loci of rDNA transcription and early RNA processing. In the present study we followed cell cycle related changes of these units in 2 human sarcoma derived cell lines with stable expression of RFP-PCNA (the sliding clamp protein) and GFP-RPA43 (a subunit of RNA polymerase I, pol I) or GFP-fibrillarin. Correlative light and electron microscopy analysis showed that the pol I and fibrillarin positive nucleolar beads correspond to individual FC/DFC units. In vivo observations showed that at early S phase, when transcriptionally active ribosomal genes were replicated, the number of the units in each cell increased by 60-80%. During that period the units transiently lost pol I, but not fibrillarin. Then, until the end of interphase, number of the units did not change, and their duplication was completed only after the cell division, by mid G1 phase. This peculiar mode of reproduction suggests that a considerable subset of ribosomal genes remain transcriptionally silent from mid S phase to mitosis, but become again active in the postmitotic daughter cells.

Euroopa äriühingu mudel ja selle rakendamine finantssektori äriühingute poolt / Ingrid Ulst

Index Scriptorium Estoniae

Ulst, Ingrid, 1977-

2005-01-01

SE (Euroopa äriühingu) mudeli olemusest ja SE regulatsiooni olulisematest aspektidest, põgusalt Nordea Bank AB praegusest grupistruktuurist ja võimalikust restruktureerimisest. Lisatud tabel: Nordea Bank AB olulisemad grupiettevõtted ja järelevalve (01/01/2004)

Contribution of PIP-5 kinase Iα to raft-based FcγRIIA signaling

International Nuclear Information System (INIS)

Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

2009-01-01

Receptor FcγIIA (FcγRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P 2 and PI(4,5)P 2 -synthesizing PIP5-kinase Iα to rafts contributes to FcγRIIA signaling. A fraction of PIP5-kinase Iα was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P 2 . PIP5-kinase Iα bound PI(4,5)P 2 , and depletion of the lipid displaced PIP5-kinase Iα from the DRM. Activation of FcγRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P 2 . Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcγRIIA activation, PIP5-kinase Iα and PI(4,5)P 2 co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Iα and PI(4,5)P 2 were present at the edges of electron-dense assemblies containing activated FcγRIIA in their core. The data suggest that activation of FcγRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Iα and PI(4,5)P 2

Hubungan Polimorfisme Gen FcγRIIA dengan Densitas P. falciparum dan Efikasi Dihidroartemisinin-Piperakuin

Directory of Open Access Journals (Sweden)

Sylvia Sance Marantina

2016-06-01

Full Text Available Dimorfisme FcγRlla memiliki keterkaitan dengan kemampuan inang dalam mengeliminasi parasit malaria sehingga perlu dilakukan penelitian untuk mengetahui polimorfisme alel FcγRlla dari populasi di daerah endemis malaria di Indonesia agar dapat diketahui peran imunitas dalam mengeliminasi parasit malaria. Sebanyak 120 sampel dried blood spot (DBS malaria falsifarum yang diperoleh dari studi efikasi obat DHP di lima wilayah di Indonesia dianalisis dengan polymerase chain reaction (PCR dan sekuensing untuk melihat varian alel FcγRIIa-131 serta hubungannya dengan densitas parasit dan efikasi dihidroartemisinin-piperakuin. Analisis gen FcγRIIa menunjukkan bahwa genotip RH memiliki frekuensi paling tinggi (50,8% dibandingkan RR (17,5% dan HH (31,7%. Alel R131 gen FcγRIIa menunjukkan efek protektif terhadap high density parasitemia/HDP (>5000 parasit/μL; odds ratio [OR]= 0,133, 95% confidence interval [CI]= 0,053–0,334, p< 0,001 dan berhubungan dengan keberadaan gametosit yang lebih lama pada inang (>72 jam; relative risk [RR]= 1,571, 95% confidence interval [CI]= 1,005–2,456, p= 0,090. Kata Kunci: malaria falsiparum, dihidroartemisinin-piperakuin, K13, FcγRIIa, efikasi obat Polymorphism of Human FcγRIIa and Its Association with P. falciparum Density and Efficacy of Dihydroartemisinin- Piperaquine Abstract FcγRlla dimorphism has been related to the ability of the host to eliminate malaria parasite so it is necessary to investigate the allele polymorphism FcγRlla of population in malaria-endemic areas in Indonesia in order to know the role of immunity in eliminating malaria parasite. A total of 120 samples of Dried Blood Spot (DBS falciparum malaria acquired from DHP drug efficacy studies in 5 regions in Indonesia were analyzed by Polymerase Chain Reaction (PCR and sequencing, to look at variants of FcγRIIa-131 allele and its Association with Parasite DensityandEfficacy ofDihydroartemisinin- Piperaquine. The FcγRIIa gene analysis indicated

Preparation and Characterization of FC Films Coated on PET Substrates by RF Magnetron Sputtering

Directory of Open Access Journals (Sweden)

Huang Mei-lin

2018-01-01

Full Text Available Fluorocarbon (FC films were prepared on polyethylene terephthalate (PET plates and PET fabrics respectively by a radiofrequency (RF magnetron sputtering technique using polytetrafluoroethylene (PTFE as a target. Scanning electron microscope and X-ray photoelectron spectroscopy were used to investigate the morphology, structure and composition of the obtained FC films. The hydrophobicity and uvioresistant properties of the FC film coated fabric were studied. The results show that the FC films were successfully deposited on the PET substrates by a RF magnetron sputtering. The deposited films are made up of four components -CF3, -CF2-, CF- and -C-. The proportions of the four components and surface morphologies of the deposited films vary with the sputtering conditions. Compared with the original fabric samples, the hydrophobicity of the FC film coated fabrics is quite good and improved significantly.

[Mihai Eminescu luulefestivalil osales Jüri Talvet

Index Scriptorium Estoniae

2014-01-01

Mihai Eminescu luulefestivalil esitleti mahukat tõlkeantoloogiat "Antologia poeziei universal de astăzi" (Craiova: Editura Europa”, 2014), mille on koostanud Ion Deaconescu. Antoloogias on ka Jüri Talveti luulet

Unusual ZFC and FC magnetic behavior in thin Co multi-layered structure

Energy Technology Data Exchange (ETDEWEB)

Ben-Dor, Oren; Yochelis, Shira [Department of Applied Physics, Center of Nanoscience and Nanotechnology, Hebrew University, Jerusalem 91904 (Israel); Felner, Israel [Racah Institute of Physics, Hebrew University, Jerusalem 91904 (Israel); Paltiel, Yossi [Department of Applied Physics, Center of Nanoscience and Nanotechnology, Hebrew University, Jerusalem 91904 (Israel)

2017-04-15

The observation of unusual magnetic phenomena in a Ni -based magnetic memory device ( O. Ben-Dor et al., 2013) encouraged us to conduct a systematic research on Co based multi-layered structure which contains a α-helix L polyalanine (AHPA-L) organic compound. The constant Co thickness is 7 nm and AHPA-L was also replaced by non-chiral 1-Decanethiol organic molecules. Both organic compounds were chemisorbed on gold by a thiol group. The dc magnetic field (H) was applied parallel and perpendicular to the surface layers. The perpendicular direction is the easy magnetization axis and along this orientation only, the zero-field-cooled (ZFC) plots exhibit a pronounced peak around 55–58 K. This peak is suppressed in the second ZFC and field-cooled (FC) runs performed shortly after the virgin ZFC one. Thus, around the peak position ZFC>FC a phenomenon seldom observed. This peak reappears after measuring the same material six months later. This behavior appears in layers with the non-chiral 1-Decanethiol and it is very similar to that obtained in sulfur doped amorphous carbon. The peak origin and the peculiar ZFC>FC case are qualitatively explained. - Highlights: • FC curve crosses ZFC curve in a 7 nm Co and thiol-based organic molecules multi-layered structure. • The ZFC>FC phenomena occurs for H perpendicular along the easy axis. • This phenomenon disappears in the second FC-ZFC run performed shortly after. • The unusual behavior reappears after six months.

A novel activating chicken IgY FcR is related to leukocyte receptor complex (LRC)

NARCIS (Netherlands)

Viertlboeck, B.C.; Schmitt, R.; Hanczaruk, M.A.; Crooijmans, R.P.M.A.; Groenen, M.A.M.; Gobel, T.W.

2009-01-01

FcRs have multifaceted roles in the immune system. Chicken FcRs were demonstrated on macrophages decades ago; however, only recently the chicken Ig-like receptor AB1, encoded in the leukocyte receptor complex, was molecularly identified as a high-affinity FcR. The present study was initiated to

Laser spectroscopy of exotic RI atoms in superfluid helium-OROCHI experiment

International Nuclear Information System (INIS)

Furukawa, T.; Matsuo, Y.; Hatakeyama, A.; Fujikake, K.; Matsuura, Y.; Kobayashi, T.; Shimoda, T.

2010-01-01

We have been developing a new laser spectroscopic technique 'OROCHI,' which is based on the combination of superfluid helium as a stopper of radioactive isotope (RI) beam and in-situ laser spectroscopy of RI atoms, for determining spins and moments of exotic RIs. By using this unique technique, it is feasible to measure nuclear spins and electromagnetic moments of extremely low yield RI (estimated as less than 1 pps). Recently, we have demonstrated that nuclear spins and moments are obtained from Zeeman and hyperfine splittings of stable Rb isotopes measured using this OROCHI technique. Details of this laser spectroscopy method in He II 'OROCHI' and the summary of our development are presented.

Võrguäri Eestis kiratseb / Indrek Kald

Index Scriptorium Estoniae

Kald, Indrek, 1974-

2004-01-01

Autor otsib põhjusi, miks tänavuse Interneti-kaupluste edetabeli koostamiseks saatis oma äritulemused vaid viis võrgukauplust. Zebra Infosüsteemide tegevjuhi Peeter Tamme arvamus e-äri kiratsemise põhjuste kohta

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

Science.gov (United States)

Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

2016-01-01

Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

Directory of Open Access Journals (Sweden)

Omar Rafael Alemán

2016-01-01

Full Text Available Neutrophils (PMN are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

Study on the Contribution of Nuclear and RI Technology to the National Economy

International Nuclear Information System (INIS)

Lee, Man Ki; Moon, K. H.; Kim, S. S.

2004-12-01

This study evaluated the economic value addition of nuclear power generation technology and radio-isotope(RI) technology quantitatively by using modified Input-Output table. For the nuclear power generation technology, the economic effect of both power plant construction and operation was evaluated. On the other hand, the economic effect of the RI utilization on the national economy was evaluated for RI technology. Firstly, the total economic value addition from nuclear power generation(both construction and operation) was estimated as 1.4% of Gross Domestic Product(GDP) in 2000. Among them, 1.2% was from nuclear power plant operation and the rest was from construction. On the other hand, the economic value addition from RI technology has continuously increased. Its share of GDP increased from 0.30% in 1980 to 0.58 in 2000

77 FR 43514 - Anchorage Regulations; Narragansett Bay and Rhode Island Sound, RI

Science.gov (United States)

2012-07-25

...-AA01 Anchorage Regulations; Narragansett Bay and Rhode Island Sound, RI AGENCY: Coast Guard, DHS... Narragansett Bay, Rhode Island, and adding an offshore anchorage in Rhode Island Sound south of Brenton Point... rulemaking (NPRM) entitled ``Anchorage Regulations; Narragansett Bay and Rhode Island Sound, RI,'' in the...

Elagu eesti energia : Jaan ja Jüri Ojaver, Paul Rodgers / Reet Varblane

Index Scriptorium Estoniae

Varblane, Reet, 1952-

2001-01-01

Paul Rodgersi, Jüri ja Jaan Ojaveri ühisnäitusest "Koostöö" Rakvere Kesklinna galeriis. P. Rodgersilt ja Jüri Ojaverilt on väljas Viinist "Eesti energia" näituselt naasnud tööd, Jaan Ojaverilt minimalistlikud tööriistad-objektid

76 FR 18812 - Submission for Review: RI 30-10, Disabled Dependent Questionnaire

Science.gov (United States)

2011-04-05

... OFFICE OF PERSONNEL MANAGEMENT Submission for Review: RI 30-10, Disabled Dependent Questionnaire... Dependent Questionnaire. As required by the Paperwork Reduction Act of 1995 (Pub. L. 104-13, 44 U.S.C... (202) 606-0910. SUPPLEMENTARY INFORMATION: The RI 30-10, Disabled Dependent Questionnaire, is used to...

76 FR 36582 - Submission for Review: Disabled Dependent Questionnaire, RI 30-10

Science.gov (United States)

2011-06-22

... OFFICE OF PERSONNEL MANAGEMENT Submission for Review: Disabled Dependent Questionnaire, RI 30-10... Dependent Questionnaire. As required by the Paperwork Reduction Act of 1995 (Pub. L. 104-13, 44 U.S.C.... SUPPLEMENTARY INFORMATION: RI 30-10, Disabled Dependent Questionnaire, is used to collect sufficient information...

Helilooja Jüri Dmitrievi loominguline õhtu / Vladislav Horuzhenko

Index Scriptorium Estoniae

Horuzhenko, Vladislav

2006-01-01

Viiuldajast, pianistist ja heliloojast Jüri Dmitrievist. Loomingulisest õhtust 8. okt. Pärnu raekojas Pärnumaa Rahvakultuuri Keskseltsi korraldatava projekti "Inimesed Pärnumaa kultuurimaastikul" raames

Characterization of a second ligand binding site of the insulin receptor

International Nuclear Information System (INIS)

Hao Caili; Whittaker, Linda; Whittaker, Jonathan

2006-01-01

Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

Comprehensive evaluation of cesium removal by CuFC adsorption. The effects of initial concentration, CuFC dosage and co-existing ions in solution

International Nuclear Information System (INIS)

Yao Xu; Ping Gu; Guang-Hui Zhang; Jun Zhao; Lu Wang; Xiang-Zhu Xiao; Fei Han

2017-01-01

To use copper ferrocyanide (CuFC) more efficiently in wastewater treatment, the method of isotope carrying used in "1"3"7Cs removal was investigated. A calculation model based on Freundlich isotherm was established to determine the optimum initial cesium concentration, at which the highest decontamination factor (DF) could be obtained at a certain CuFC dosage. An accurate DF prediction model was developed to describe synergistic effects of sodium and potassium. A novel index called volumetric distribution coefficient (K_v_d) was proposed to evaluate adsorption performance in terms of DF and concentration factor. (author)

A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines.

Directory of Open Access Journals (Sweden)

Lanying Du

Full Text Available An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS, is caused by a novel coronavirus (MERS-CoV. It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588 in the truncated receptor-binding domain (RBD: residues 367-606 of MERS-CoV spike (S protein fused with human IgG Fc fragment (S377-588-Fc is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4, the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients' lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

Science.gov (United States)

Dumont, Jennifer A.; Liu, Tongyao; Low, Susan C.; Zhang, Xin; Kamphaus, George; Sakorafas, Paul; Fraley, Cara; Drager, Douglas; Reidy, Thomas; McCue, Justin; Franck, Helen W. G.; Merricks, Elizabeth P.; Nichols, Timothy C.; Bitonti, Alan J.; Pierce, Glenn F.

2012-01-01

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation. PMID:22246033

A research on the empowerment plan for specialist in RI-biomics field

Energy Technology Data Exchange (ETDEWEB)

Shin, Wo Ho; Park, Tai Jin; Yeom, Yu Sun [Korea Association for Radiation Application, Seoul (Korea, Republic of); Park, Sang Hyun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)

2014-12-15

Increasing utilization of radiation and RI (Radioisotope) in nuclear industry including non-power area has achieved sustainable development of radiation industry. Industries are no longer confined by a single technology or abilities but expanded for application gradually. RIBiomics fields are one of the convergence technology that is recognized on a high-tech industry. Unlike the conventional industry, RI-Biomics field needs to various specialists to perform related task. There is no domestic training program to educate the whole process. This study aims to suggest the plan for improvement of practical skills for specialists in RI-Biomics through development of our training program. For this purpose, we have first investigated the opinion about classification scheme from experts and then analyzed the results in order to reflecting our training program. Based on analyzed results, conformity assessment was executed to organize curriculum through status of constructed device and instructor in domestic. Our training program was performed jointly with KAERI (Korea Atomic Energy Research Institute). RI-Biomics center is prepared with facilities of overall experiment to improve quality of education. Due to the fact that specialists have routine task, we organized a five-day short course to reflect temporal difficulties. We performed a trial operation to 6 participants in RI-Biomics field. Through the survey for the specialists who participated in the program, we evaluated the efficiency of our training program. The results showed that participants were satisfied with the organized curriculum and educational materials. Therefore, our program is expected to be utilized as basic research data to develop feasible program for policy development and to improve practical skills in RI-Biomics.

A research on the empowerment plan for specialist in RI-biomics field

International Nuclear Information System (INIS)

Shin, Wo Ho; Park, Tai Jin; Yeom, Yu Sun; Park, Sang Hyun

2014-01-01

Increasing utilization of radiation and RI (Radioisotope) in nuclear industry including non-power area has achieved sustainable development of radiation industry. Industries are no longer confined by a single technology or abilities but expanded for application gradually. RIBiomics fields are one of the convergence technology that is recognized on a high-tech industry. Unlike the conventional industry, RI-Biomics field needs to various specialists to perform related task. There is no domestic training program to educate the whole process. This study aims to suggest the plan for improvement of practical skills for specialists in RI-Biomics through development of our training program. For this purpose, we have first investigated the opinion about classification scheme from experts and then analyzed the results in order to reflecting our training program. Based on analyzed results, conformity assessment was executed to organize curriculum through status of constructed device and instructor in domestic. Our training program was performed jointly with KAERI (Korea Atomic Energy Research Institute). RI-Biomics center is prepared with facilities of overall experiment to improve quality of education. Due to the fact that specialists have routine task, we organized a five-day short course to reflect temporal difficulties. We performed a trial operation to 6 participants in RI-Biomics field. Through the survey for the specialists who participated in the program, we evaluated the efficiency of our training program. The results showed that participants were satisfied with the organized curriculum and educational materials. Therefore, our program is expected to be utilized as basic research data to develop feasible program for policy development and to improve practical skills in RI-Biomics

Jüri Talvet maailmaluule tõlgendajana / Jüri Talvet’s Interpretations of World Poetry

OpenAIRE

Lauri Pilter

2016-01-01

Teesid: Tartu Ülikooli maailmakirjanduse professori, luuletaja, kirjandusteadlase ja hispaaniakeelse kirjanduse spetsialisti Jüri Talveti tõlketegevuse viljade hulka kuulub luulet ja proosat nii sajandeid vanast Hispaania klassikast kui ka 20. sajandil või tänapäeval romaani keeltes või inglise keeles loodud teostest. Käesolev artikkel keskendub sellele, kuidas professor Talvet on tõlgendanud luule ja poeetika, kuid ka kirjandusajaloo, iseäranis barokk-kirjanduse alaseid küsimusi oma kirjandu...

The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans

DEFF Research Database (Denmark)

Klasson, Lisa; Westberg, Joakim; Sapountzis, Panagiotis

2009-01-01

genome of W. pipientis strain wRi that induces very strong cytoplasmic incompatibility in its natural host Drosophila simulans. A comparison with the previously sequenced genome of W. pipientis strain wMel from Drosophila melanogaster identified 35 breakpoints associated with mobile elements and repeated...... sequences that are stable in Drosophila lines transinfected with wRi. Additionally, 450 genes with orthologs in wRi and wMel were sequenced from the W. pipientis strain wUni, responsible for the induction of parthenogenesis in the parasitoid wasp Muscidifurax uniraptor. The comparison of these A...

Eksjuhtide hoogne kinnisvaraäri / Koit Brinkmann

Index Scriptorium Estoniae

Brinkmann, Koit

2007-01-01

Endised Skanska EMV juhid Jaanus Otsa ja Olaf Herman ajasid põhitöökoha kõrvalt tulusat eraäri. Lisad: Skanska partnerlus Mati Tusoviga; Palju firmasid, kuid samad nimed; Väljavõte Skanska eetikakoodeksist. Kommenteerib Mika Toivola

FcγRIIb on myeloid cells rather than on B cells protects from collagen-induced arthritis.

Science.gov (United States)

Yilmaz-Elis, A Seda; Ramirez, Javier Martin; Asmawidjaja, Patrick; van der Kaa, Jos; Mus, Anne-Marie; Brem, Maarten D; Claassens, Jill W C; Breukel, Cor; Brouwers, Conny; Mangsbo, Sara M; Boross, Peter; Lubberts, Erik; Verbeek, J Sjef

2014-06-15

Extensive analysis of a variety of arthritis models in germline KO mice has revealed that all four receptors for the Fc part of IgG (FcγR) play a role in the disease process. However, their precise cell type-specific contribution is still unclear. In this study, we analyzed the specific role of the inhibiting FcγRIIb on B lymphocytes (using CD19Cre mice) and in the myeloid cell compartment (using C/EBPαCre mice) in the development of arthritis induced by immunization with either bovine or chicken collagen type II. Despite their comparable anti-mouse collagen autoantibody titers, full FcγRIIb knockout (KO), but not B cell-specific FcγRIIb KO, mice showed a significantly increased incidence and severity of disease compared with wild-type control mice when immunized with bovine collagen. When immunized with chicken collagen, disease incidence was significantly increased in pan-myeloid and full FcγRIIb KO mice, but not in B cell-specific KO mice, whereas disease severity was only significantly increased in full FcγRIIb KO mice compared with incidence and severity in wild-type control mice. We conclude that, although anti-mouse collagen autoantibodies are a prerequisite for the development of collagen-induced arthritis, their presence is insufficient for disease development. FcγRIIb on myeloid effector cells, as a modulator of the threshold for downstream Ab effector pathways, plays a dominant role in the susceptibility to collagen-induced arthritis, whereas FcγRIIb on B cells, as a regulator of Ab production, has a minor effect on disease susceptibility. Copyright © 2014 by The American Association of Immunologists, Inc.

Correlating the Impact of Well-Defined Oligosaccharide Structures on Physical Stability Profiles of IgG1-Fc Glycoforms.

Science.gov (United States)

More, Apurva S; Toprani, Vishal M; Okbazghi, Solomon Z; Kim, Jae H; Joshi, Sangeeta B; Middaugh, C Russell; Tolbert, Thomas J; Volkin, David B

2016-02-01

As part of a series of articles in this special issue describing 4 well-defined IgG1-Fc glycoforms as a model system for biosimilarity analysis (high mannose-Fc, Man5-Fc, GlcNAc-Fc and N297Q-Fc aglycosylated), the focus of this work is comparisons of their physical properties. A trend of decreasing apparent solubility (thermodynamic activity) by polyethylene glycol precipitation (pH 4.5, 6.0) and lower conformational stability by differential scanning calorimetry (pH 4.5) was observed with reducing size of the N297-linked oligosaccharide structures. Using multiple high-throughput biophysical techniques, the physical stability of the Fc glycoproteins was then measured in 2 formulations (NaCl and sucrose) across a wide range of temperatures (10°C-90°C) and pH (4.0-7.5) conditions. The data sets were used to construct 3-index empirical phase diagrams and radar charts to visualize the regions of protein structural stability. Each glycoform showed improved stability in the sucrose (vs. salt) formulation. The HM-Fc and Man5-Fc displayed the highest relative stability, followed by GlcNAc-Fc, with N297Q-Fc being the least stable. Thus, the overall physical stability profiles of the 4 IgG1-Fc glycoforms also show a correlation with oligosaccharide structure. These data sets are used to develop a mathematical model for biosimilarity analysis (as described in a companion article by Kim et al. in this issue). Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

fcGENE: a versatile tool for processing and transforming SNP datasets.

Directory of Open Access Journals (Sweden)

Nab Raj Roshyara

Full Text Available Modern analysis of high-dimensional SNP data requires a number of biometrical and statistical methods such as pre-processing, analysis of population structure, association analysis and genotype imputation. Software used for these purposes often rely on specific and incompatible input and output data formats. Therefore extensive data management including multiple format conversions is necessary during analyses.In order to support fast and efficient management and bio-statistical quality control of high-dimensional SNP data, we developed the publically available software fcGENE using C++ object-oriented programming language. This software simplifies and automates the use of different existing analysis packages, especially during the workflow of genotype imputations and corresponding analyses.fcGENE transforms SNP data and imputation results into different formats required for a large variety of analysis packages such as PLINK, SNPTEST, HAPLOVIEW, EIGENSOFT, GenABEL and tools used for genotype imputation such as MaCH, IMPUTE, BEAGLE and others. Data Management tasks like merging, splitting, extracting SNP and pedigree information can be performed. fcGENE also supports a number of bio-statistical quality control processes and quality based filtering processes at SNP- and sample-wise level. The tool also generates templates of commands required to run specific software packages, especially those required for genotype imputation. We demonstrate the functionality of fcGENE by example workflows of SNP data analyses and provide a comprehensive manual of commands, options and applications.We have developed a user-friendly open-source software fcGENE, which comprehensively supports SNP data management, quality control and analysis workflows. Download statistics and corresponding feedbacks indicate that software is highly recognised and extensively applied by the scientific community.

Hispaania kirjanduse elav keha / Jüri Talvet

Index Scriptorium Estoniae

Talvet, Jüri, 1945-

1996-01-01

Keha ja kehatus hispaania kirjanduses (Cervantese Dulcinea, Sancho Panza, don Quijote) ja maailmakirjanduses esinevate tegelaskujude näiteil : Ka Tõnu Õnnepalu Joonatanist romaanis 'Hind'. Ilmunud ka kogumikus: Talvet, Jüri. Tõrjumatu äär. Tartu : Ilmamaa, 2005, lk. 469-473

Syntaxin binding protein 1 is not required for allergic inflammation via IgE-mediated mast cell activation.

Directory of Open Access Journals (Sweden)

Zhengli Wu

Full Text Available Mast cells play a central role in both innate and acquired immunity. When activated by IgE-dependent FcεRI cross-linking, mast cells rapidly initiate a signaling cascade and undergo an extensive release of their granule contents, including inflammatory mediators. Some SNARE (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptor proteins and SM (Sec1/Munc18 family proteins are involved in mast cell degranulation. However, the function of syntaxin binding protein 1 (STXBP1, a member of SM family, in mast cell degranulation is currently unknown. In this study, we examined the role of STXBP1 in IgE-dependent mast cell activation. Liver-derived mast cells (LMCs from wild-type and STXBP1-deficient mice were cultured in vitro for the study of mast cell maturation, degranulation, cytokine and chemokine production, as well as MAPK, IκB-NFκB, and NFAT signaling pathways. In addition, in vivo models of passive cutaneous anaphylaxis and late-phase IgE-dependent inflammation were conducted in mast cell deficient W(sh mice that had been reconstituted with wild-type or STXBP1-deficient mast cells. Our findings indicate that STXBP1 is not required for any of these important functional mechanisms in mast cells both in vitro and in vivo. Our results demonstrate that STXBP1 is dispensable during IgE-mediated mast cell activation and in IgE-dependent allergic inflammatory reactions.

Preliminary Beam Irradiation Test for RI Production Targets at KOMAC

Energy Technology Data Exchange (ETDEWEB)

Yoon, Sang Pil; Kwon, Hyeok Jung; Kim, Han Sung; Cho, Yong Sub; Seol, Kyung Tae; Song, Young Gi; Kim, Dae Il; Jung, Myung Hwan; Kim, Kye Ryung; Min, Yi Sub [KAERI, Daejeon (Korea, Republic of)

2016-05-15

The new beamline and target irradiation facility has been constructed for the production of therapeutic radio-isotope. Sr-82 and Cu-67 were selected as the target isotope in this facility, they are promising isotope for the PET imaging and cancer therapy. For the facility commissioning, the irradiation test for the prototype-target was conducted to confirm the feasibility of radio-isotope production, the proto-type targets are made of RbCl pellet and the natural Zn metal for Sr-82 and Cu-67 production respectively, In this paper, an introduction to the RI production targetry system and the results of the preliminary beam irradiation test are discussed. the low-flux beam irradiation tests for proto-type RI target have been conducted. As a result of the beam irradiation tests, we could obtain the evidence of Sr-82 and Cu-67 production, have confirmed the feasibility of Sr-82 and Cu-67 production at KOMAC RI production facility.

Preliminary Beam Irradiation Test for RI Production Targets at KOMAC

International Nuclear Information System (INIS)

Yoon, Sang Pil; Kwon, Hyeok Jung; Kim, Han Sung; Cho, Yong Sub; Seol, Kyung Tae; Song, Young Gi; Kim, Dae Il; Jung, Myung Hwan; Kim, Kye Ryung; Min, Yi Sub

2016-01-01

The new beamline and target irradiation facility has been constructed for the production of therapeutic radio-isotope. Sr-82 and Cu-67 were selected as the target isotope in this facility, they are promising isotope for the PET imaging and cancer therapy. For the facility commissioning, the irradiation test for the prototype-target was conducted to confirm the feasibility of radio-isotope production, the proto-type targets are made of RbCl pellet and the natural Zn metal for Sr-82 and Cu-67 production respectively, In this paper, an introduction to the RI production targetry system and the results of the preliminary beam irradiation test are discussed. the low-flux beam irradiation tests for proto-type RI target have been conducted. As a result of the beam irradiation tests, we could obtain the evidence of Sr-82 and Cu-67 production, have confirmed the feasibility of Sr-82 and Cu-67 production at KOMAC RI production facility

HAL/S-FC compiler system functional specification

Science.gov (United States)

1974-01-01

Compiler organization is discussed, including overall compiler structure, internal data transfer, compiler development, and code optimization. The user, system, and SDL interfaces are described, along with compiler system requirements. Run-time software support package and restrictions and dependencies are also considered of the HAL/S-FC system.

Linnapea, keda rõõmustab titekisa / Jüri Ratas ; interv. Riina Luik

Index Scriptorium Estoniae

Ratas, Jüri, 1978-

2006-01-01

Tallinna linnapea vastab küsimustele, mis puudutavad tema suhtumist opositsiooni kriitikasse, linna juhtimist, perekonda, lapsepõlve, spordiga tegelemist, sõpruskonda, eraettevõtlusest loobumist ning suhteid kirikuga. Lisa: Jüri Ratas (27). Vt. samas lühiintervjuud linnapea abikaasa Karin Ratasega. Kommenteerib Jüri Ratase endine klassijuhataja Nõmme gümnaasiumist, võrkpallitreener Raimund Pundi

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

Science.gov (United States)

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

Evaluation of inventory change report (ICR) in 2008/2009 - 2012/2013 in MBA.RI-B

International Nuclear Information System (INIS)

Agustinus Suprijanto; Suratno; Sigit Pramana

2013-01-01

Evaluation has been performed for Inventory Change Report (ICR) in 2008/2009 - 2012/2013 in MBA.RI-B (MBA = Material Balance Area) (RI-B = Center for Accelerator and Material Process Technology). The evaluation performed on the inventory change report of nuclear material document. Changes in inventories occur when there is acceptance or shipment of nuclear materials outside nuclear facilities. Successive reports changes in inventory of nuclear material in 2008/2009 comprised 3 sheets ICR document numbers (153, 154 and 155), in 2009/2010 comprised 1 sheet ICR document number (159), in 2010/2011 comprised 3 sheets ICR document numbers (164, 165 and 166), and in 2012/2013 comprised 2 sheets ICR document numbers (175 and 176). From these data report changes in inventory of nuclear material can be concluded that in MBA.RI-B has been the displacement of nuclear material activities includes receipt of nuclear material from other nuclear facilities and shipments of nuclear materials out of facilities for research and development includes receipt 4 times from RI-E and 1 times from RI-A and 3 times shipment to RI-E. (author)

77 FR 5581 - Submission for Review: Financial Resources Questionnaire (RI 34-1, RI 34-17) and Notice of Amount...

Science.gov (United States)

2012-02-03

... OFFICE OF PERSONNEL MANAGEMENT Submission for Review: Financial Resources Questionnaire (RI 34- 1... opportunity to comment on a revised information collection request (ICR) 3206-0167, Financial Resources... collection techniques or other forms of information technology, e.g., permitting electronic submissions of...

Arved Viirlaid teises kaanonis / Jüri Talvet

Index Scriptorium Estoniae

Talvet, Jüri, 1945-

2002-01-01

Arved Viirlaid 80. Ka tema juubeliks ilmunud ingliskeelsest luulekogust "Selected poems" (tlk. Taimi Ene Moks ja R. W. Stedingh). Ilmunud ka kogumikus: Talvet, Jüri. Tõrjumatu äär. Tartu : Ilmamaa, 2005, lk. 417-420, pealk.: Teises Kaanonis: Viirlaiu luule inglise keeles

Investigating the function of F_c -specific binding of IgM to Plasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting

DEFF Research Database (Denmark)

Stevenson, Liz; Huda, Pie; Jeppesen, Anine

2015-01-01

of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via Fc to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect...

Application of RI power sources to cardiac pacemakers and aftercare in its implantation

International Nuclear Information System (INIS)

Hori, Motokazu

1974-01-01

RI power sources have long life when they are implanted into human bodies together with cardiac pacemakers, as compared with e.g. mercury batteries. Therefore, the frequency of their replacement can be by far less. However, there are the problems of radiation protection, high cost, availability, etc. The following matters are described: The cardiac pacemaker and its power supply, implantation into human body, problems with patients and conventional power sources; the current state of RI power sources for cardiac pacemakers, including plutonium-238 RTG and 147 Pm and 3 H batteries; and problems with the RI power sources. (Mori, K.)

Rembrandt Kadrioru lossis / Jüri Hain

Index Scriptorium Estoniae

Hain, Jüri, 1941-

2000-01-01

Kadrioru lossi kuppelsaali tundmatu meistri laemaali maalimise ajalugu. Maali aluseks on Rembrandti maal "Diana suplus". Laemaal on maalitud Magdalena de Passe (1600-1638) vasegravüüri põhjal, mis on tehtud Rembrandti maalist. Rembrandt on lähtunud Antonio Tempesta (1555-1630) kahest gravüürist, viimane on lähtunud Otto van Veeni (1556-1629) maalist

Thermal characteristic of insulation for optimum design of RI transport package

International Nuclear Information System (INIS)

Lee, J. C.; Bang, K. S.; Seo, K. S.

2002-01-01

A package to transport the high level radioactive materials in required to withstand the hypothetical accident conditions as well as normal transport conditions according to IAEA and domestic regulations. The regulations require that the package should maintain the shielding, thermal and structural integrities to release no radioactive material. Thermal characteristics of insulations were evaluated and optimum insulation thickness was deduced for RI transport package. The package has a maximum capacity of 600 Curies for Ir-192 sealed source. The insulation thickness was decided with 10 mm of polyurethane form to maintain the thermal safety under fire accident condition. Thermal analysis was carried out for RI transport package, and it was shown that the thermal integrity of the package was maintained. The results obtained this study will be applied to a basic data for design of RI transport cask

Endothelial Fcγ Receptor IIB Activation Blunts Insulin Delivery to Skeletal Muscle to Cause Insulin Resistance in Mice

Science.gov (United States)

Tanigaki, Keiji; Chambliss, Ken L.; Yuhanna, Ivan S.; Sacharidou, Anastasia; Ahmed, Mohamed; Atochin, Dmitriy N.; Huang, Paul L.

2016-01-01

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. FcγRIIB is expressed in numerous cell types including endothelium and B lymphocytes. Here we investigated how endothelial FcγRIIB influences glucose homeostasis, using mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated insulin delivery, and it was associated with blunted endothelial nitric oxide synthase (eNOS) activation in skeletal muscle. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and eNOS antagonism. Furthermore, in knock-in mice harboring constitutively active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin delivery to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin movement has been identified that may contribute to type 2 diabetes pathogenesis. PMID:27207525

Framework for applying RI-ISI methodology for Indian PHWRs

International Nuclear Information System (INIS)

Vinod, Gopika; Saraf, R.K.; Ghosh, A.K.; Kushwaha, H.S.

2006-01-01

Risk Informed In-Service Inspection (RI-ISI) aims at categorizing the components for In-Service inspection based on their contribution to Risk. For defining the contribution of risk from components, their failure probabilities and its subsequent effect on Core Damage Frequency (CDF) needs to be evaluated using Probabilistic Safety Assessment methodology. During the last several years, both the U.S. Nuclear Regulatory Commission (NRC) and the nuclear industry have recognized that Probabilistic Safety Assessment (PSA) has evolved to be more useful in supplementing traditional engineering approaches in reactor regulation. The paper highlights the various stages involved in applying RI-ISI and then compares the findings with existing ISI practices. (author)

Regulation of Monoclonal Antibody Immunotherapy by FcγRIIB.

Science.gov (United States)

Stopforth, Richard J; Cleary, Kirstie L S; Cragg, Mark S

2016-05-01

Monoclonal antibodies (mAb) are revolutionising the treatment of many different diseases. Given their differing mode of action compared to most conventional chemotherapeutics and small molecule inhibitors, they possess the potential to be independent of common modes of treatment resistance and can typically be combined readily with existing treatments without dose-limiting toxicity. However, treatments with mAb rarely result in cure and so a full understanding of how these reagents work and can be optimised is key for their subsequent improvement. Here we review how an understanding of the biology of the inhibitory Fc receptor, FcγRIIB (CD32B), is leading to the development of improved mAb treatments.

Mannan-Binding Lectin Is Involved in the Protection against Renal Ischemia/Reperfusion Injury by Dietary Restriction.

Directory of Open Access Journals (Sweden)

Shushimita Shushimita

Full Text Available Preoperative fasting and dietary restriction offer robust protection against renal ischemia/reperfusion injury (I/RI in mice. We recently showed that Mannan-binding lectin (MBL, the initiator of the lectin pathway of complement activation, plays a pivotal role in renal I/RI. Based on these findings, we investigated the effect of short-term DR (30% reduction of total food intake or three days of water only fasting on MBL in 10-12 weeks old male C57/Bl6 mice. Both dietary regimens significantly reduce the circulating levels of MBL as well as its mRNA expression in liver, the sole production site of MBL. Reconstitution of MBL abolished the protection afforded by dietary restriction, whereas in the fasting group the protection persisted. These data show that modulation of MBL is involved in the protection against renal I/RI induced by dietary restriction, and suggest that the mechanisms of protection induced by dietary restriction and fasting may be different.

Construction of a bimolecular fluorescence complementation (BiFC ...

African Journals Online (AJOL)

DR. NJ TONUKARI

2012-08-02

Aug 2, 2012 ... Accepted 8 June, 2012. Protein–protein interactions are essential for signal transduction in cells. ... BiFC is a novel technology that is used for identifying .... occasional fluorescence was observed, it was very weak ... Light field.

Data of evolutionary structure change: 1DEAA-2RI1A [Confc[Archive

Lifescience Database Archive (English)

Full Text Available 1DEAA-2RI1A 1DEA 2RI1 A A -MRLIPLTTAEQVGKWAARHIVNRINAFKPTADRPFVLG.../line> ASP CA 284 PHE CA 347 SER CA 228 1DEA... A 1DEAA ALVEMHKAGQVSFK 1DEA A 1DEAA GLU CA 160 ASP CA 224 VAL CA 284 1DEA

New Insights into the Functional Behavior of Antibodies as Revealed by Binding Studies on an Anti-Uranium Monoclonal Antibody

International Nuclear Information System (INIS)

Blake, Diane A.; Xia Li; Haini Yu; Blake, Robert C.

2004-01-01

As part of an ongoing effort to develop immunoassays for chelated uranium(VI) on a hand-held flow fluorimeter, an anti-uranium monoclonal antibody designated as 8A11 was fluorescently labeled using two different strategies. When 8A11 was coupled via reactive lysines to either ALEXATM 488 or Cy5TM, the resulting fluorescent antibody conjugate exhibited positive cooperativity in the presence of its antigen, U(VI) chelated with 2,9-dicarboxy-1,10-phenanthroline (U(VI)-DCP). That is, when one of the two binding sites on the covalently modified 8A11 was occupied with bound antigen, the affinity of the remaining site on the antibody for U(VI)-DCP appeared to increase. Unmodified 8A11 bound U(VI)-DCP with the expected hyperbolic dependence on the concentration of antigen, consistent with independent and equal binding of ligand at both sites. Proteolytic cleavage of the fluorescently conjugated 8A11 to produce the fluorescent monovalent Fab fragment yielded an active preparation that now bound U(VI)-DCP with no evidence of positive cooperativity. Although, in principle, any divalent antibody has the potential to exhibit positive cooperativity in its binding interactions with its antigen, very little literature precedent for this type of behavior exists. Native 8A11 was also noncovalently labeled with highly fluorescent ZENONTM reagents. These reagents are fluorescently-labeled Fab fragments of goat anti-mouse antibodies that bind to the Fc portion of 8A11. These high-affinity, monovalent fluorescent reagents permitted the intact 8A11 mouse antibody to be labeled in situ with no covalent modifications. Incubation of the 8A11 with ZENON 647 produced a fluorescent protein complex that showed an 8-fold higher affinity for U(VI)-DCP than did the free 8A11 alone. Again, very few literature precedents exist for this phenomenon, where agents that bind to the Fc portion of an intact antibody change the affinity of the antibody for the antigen at the structurally distant Fab portion

Ühest avastamata illustreeritud raamatust / Jüri Hain

Index Scriptorium Estoniae

Hain, Jüri, 1941-

2011-01-01

Erick, Françoise. Heute / ill. Sergei Slastnikov. Tallinn : Tallinna Eesti Kirjastus-Ühisus, 1926. S. Slastnikovi puugravüüris illustratsioonidest, tema kunstialastest kirjutistest ja puugravüüri kasutuselevõtust eesti raamatukunstis

Narragansett, RI Lab--Office of Research and Development

Science.gov (United States)

The EPA-ORD laboratory in Narragansett, RI is a recognized leader in scientific knowledge and expertise concerning the ecology of oceans, estuaries, and water-sheds, and the effects of human activities on that ecology.

Assessment of the binding properties of granuloszint

Energy Technology Data Exchange (ETDEWEB)

Schubiger, P.A.; Hasler, P.H.; Novak-Hofer, I.; Blaeuenstein, P.

1989-09-01

/sup 123/I-granuloszint (a murine monoclonal antibody - called AK-47 - against NCA-95 glycoprotein of granulocytes) has been proved to be a very convenient and successful radiopharmaceutical for visualizing infectious diseases. For a broad introduction in routine nuclear medicine it was necessary to optimize the labelling method and to determine in vitro exactly those biological and binding parameters which are relevant for an effective application in vivo. Binding to granulocytes has been shown to be specific and saturable (nonspecific binding about 10%) and is not via the Fc part of the antibody. The investigation of the binding properties of /sup 125/I-labelled AK-47 gave the following results: Affinity constant 5x10/sup 8/, 20,000-100,000 epitopes per granulocyte and an immunoreactivity of more than 90%. Labelling with /sup 123/I reduced the immunoreactivity to 40%. The Lindmo method and immunoblotting are used as quality control to check the likely in vivo behaviour of the labelled antibody. There is a good correspondence between the results from the two methods. With our special labelling method and the different in vitro checks we have found a reliable way to control the production and to assure an optimal binding behaviour of /sup 123/I-granuloszint. (orig.).

Assessment of the binding properties of granuloszint

International Nuclear Information System (INIS)

Schubiger, P.A.; Hasler, P.H.; Novak-Hofer, I.; Blaeuenstein, P.

1989-01-01

123 I-granuloszint (a murine monoclonal antibody - called AK-47 - against NCA-95 glycoprotein of granulocytes) has been proved to be a very convenient and successful radiopharmaceutical for visualizing infectious diseases. For a broad introduction in routine nuclear medicine it was necessary to optimize the labelling method and to determine in vitro exactly those biological and binding parameters which are relevant for an effective application in vivo. Binding to granulocytes has been shown to be specific and saturable (nonspecific binding about 10%) and is not via the Fc part of the antibody. The investigation of the binding properties of 125 I-labelled AK-47 gave the following results: Affinity constant 5x10 8 , 20,000-100,000 epitopes per granulocyte and an immunoreactivity of more than 90%. Labelling with 123 I reduced the immunoreactivity to 40%. The Lindmo method and immunoblotting are used as quality control to check the likely in vivo behaviour of the labelled antibody. There is a good correspondence between the results from the two methods. With our special labelling method and the different in vitro checks we have found a reliable way to control the production and to assure an optimal binding behaviour of 123 I-granuloszint. (orig.)

Comprehension of radiation for RI license

International Nuclear Information System (INIS)

Guk, Jin Sun

2005-08-01

This book starts with theoretical nuclear energy which explains technical terms such as nuclear physics and chemistry, RI and utilization of radiation, radiochemistry and radiation chemistry, radiation biology. It also explains radiation measurements technology including radiation detector and measurements radiation measurements circuit, measuring of energy spectrum. Radiation protection monitoring for radiation protection. It deals with nuclear law, enforcement regulation and ordinance.

Dual role of Fcγ receptors in host defense and disease in Borrelia burgdorferi-infected mice

Directory of Open Access Journals (Sweden)

Alexia Anne Belperron

2014-06-01

Full Text Available Arthritis in mice infected with the Lyme disease spirochete, Borrelia burgdorferi, results from the influx of innate immune cells responding to the pathogen in the joint and is influenced in part by mouse genetics. Production of inflammatory cytokines by innate immune cells in vitro is largely mediated by Toll-like receptor (TLR interaction with Borrelia lipoproteins, yet surprisingly mice deficient in TLR2 or the TLR signaling molecule MyD88 still develop arthritis comparable to that seen in wild type mice after B. burgdorferi infection. These findings suggest that other, MyD88-independent inflammatory pathways can contribute to arthritis expression. Clearance of B. burgdorferi is dependent on the production of specific antibody and phagocytosis of the organism. As Fc receptors (FcγR are important for IgG-mediated clearance of immune complexes and opsonized particles by phagocytes, we examined the role that FcγR play in host defense and disease in B. burgdorferi-infected mice. B. burgdorferi-infected mice deficient in the Fc receptor common gamma chain (FcεRγ-/- mice harbored ~10 fold more spirochetes than similarly infected wild type mice, and this was associated with a transient increase in arthritis severity. While the elevated pathogen burdens seen in B. burgdorferi-infected MyD88-/- mice were not affected by concomitant deficiency in FcγR, arthritis was reduced in FcεRγ-/-MyD88-/- mice in comparison to wild type or single knockout mice. Gene expression analysis from infected joints demonstrated that absence of both MyD88 and FcγR lowers mRNA levels of proteins involved in inflammation, including Cxcl1 (KC, Xcr1 (Gpr5, IL-1beta, and C reactive protein. Taken together, our results demonstrate a role for FcγR-mediated immunity in limiting pathogen burden and arthritis in mice during the acute phase of B. burgdorferi infection, and further suggest that this pathway contributes to the arthritis that develops in B. burgdorferi

Eduard Vääri : [Wiedemanni keeleauhinna laureaat 2001

Index Scriptorium Estoniae

2005-01-01

Sisu: Kogu elu eesti keele kaitsel : [F. J. Wiedemanni keeleauhinna vastuvõtmisel peetud kõne alusel] ; Laks, Meinhard. Liivi rannarahva lipp : [luuletus] = Livõ rńdarouv plaga / liivi keelde tõlkinud Eduard Vääri

The Ri chicken breed and livelihoods in North Vietnam: characterization and prospects

Directory of Open Access Journals (Sweden)

Pascal Leroy

2011-06-01

Full Text Available For the last twenty years, the consumption of poultry meat has boomed in Vietnam as in the rest of the developing world. Capital-intensive production has grown rapidly to satisfy this demand. Based on a few numbers of genetically uniform strains, these systems threaten biodiversity. In Vietnam, both rural and urban households still keep indigenous chickens as part of a diversified livelihood portfolio. In line with the national in situ conservation strategy, this study approached the context of local poultry keeping in two rural and one suburban districts of Northern Vietnam. It aimed at understanding households’ willingness, constraints and opportunities for practice improvement, including breeds’ management. As the Ri chicken constitutes the large majority of backyard flocks, two particular objectives of this study are the morpho-biometric characterisation of phenotypic diversity among individuals classified as Ri by farmers and an assessment of their productive potential. Chicken was found to hold a different place in livelihoods of the three districts with consequences on the management of genetic resources. The most favourable conditions for improvement of the Ri breed was found in the rural district of Luong-Son, due to market integration. In the more remote district of Ky-Son, living standards were lower and much would be gained from Ri conservation. Ri breed was the most threatened in the suburban Gia-Lam district, where poultry was a minor side-activity, lacking incentive for genetic management. From motives and constraints, tracks about breeding goals are suggested. Further considerations about conservation, improvement, market integration and livelihoods are proposed.

Antibody-mediated immunity to the obligate intracellular bacterial pathogen Coxiella burnetii is Fc receptor- and complement-independent

Directory of Open Access Journals (Sweden)

Heinzen Robert A

2009-05-01

Full Text Available Abstract Background The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonosis Q fever. The intracellular niche of C. burnetii has led to the assumption that cell-mediated immunity is the most important immune component for protection against this pathogen. However, passive immunization with immune serum can protect naïve animals from challenge with virulent C. burnetii, indicating a role for antibody (Ab in protection. The mechanism of this Ab-mediated protection is unknown. Therefore, we conducted a study to determine whether Fc receptors (FcR or complement contribute to Ab-mediated immunity (AMI to C. burnetii. Results Virulent C. burnetii infects and replicates within human dendritic cells (DC without inducing their maturation or activation. We investigated the effects of Ab opsonized C. burnetii on human monocyte-derived and murine bone marrow-derived DC. Infection of DC with Ab-opsonized C. burnetii resulted in increased expression of maturation markers and inflammatory cytokine production. Bacteria that had been incubated with naïve serum had minimal effect on DC, similar to virulent C. burnetii alone. The effect of Ab opsonized C. burnetii on DC was FcR dependent as evidenced by a reduced response of DC from FcR knockout (FcR k/o compared to C57Bl/6 (B6 mice. To address the potential role of FcR in Ab-mediated protection in vivo, we compared the response of passively immunized FcR k/o mice to the B6 controls. Interestingly, we found that FcR are not essential for AMI to C. burnetii in vivo. We subsequently examined the role of complement in AMI by passively immunizing and challenging several different strains of complement-deficient mice and found that AMI to C. burnetii is also complement-independent. Conclusion Despite our data showing FcR-dependent stimulation of DC in vitro, Ab-mediated immunity to C. burnetii in vivo is FcR-independent. We also found that passive immunity to this pathogen is independent of

Tüüri ava(sta)mas, kuid mitte ainult / Igor Garšnek

Index Scriptorium Estoniae

Garšnek, Igor, 1958-

2011-01-01

Kahest kontserdist Estonia kontserdisaalis - 4. märtsil ERSO ja Herbert Schuch (klaver) Olari Eltsi dirigeerimisel, kavas Tüüri Sümfoonia nr 8, Pärdi "Nekroloog" ja Ullmanni Klaverikontsert ning 10. märtsil Erkki-Sven Tüüri autorikontsert, kus esinesid Eesti Filharmoonia Kammerkoor ja Sinfonietta Riga Daniel Reussi juhatusel

Multiple regulatory roles of the mouse transmembrane adaptor protein NTAL in gene transcription and mast cell physiology.

Directory of Open Access Journals (Sweden)

Iva Polakovicova

Full Text Available Non-T cell activation linker (NTAL; also called LAB or LAT2 is a transmembrane adaptor protein that is expressed in a subset of hematopoietic cells, including mast cells. There are conflicting reports on the role of NTAL in the high affinity immunoglobulin E receptor (FcεRI signaling. Studies carried out on mast cells derived from mice with NTAL knock out (KO and wild type mice suggested that NTAL is a negative regulator of FcεRI signaling, while experiments with RNAi-mediated NTAL knockdown (KD in human mast cells and rat basophilic leukemia cells suggested its positive regulatory role. To determine whether different methodologies of NTAL ablation (KO vs KD have different physiological consequences, we compared under well defined conditions FcεRI-mediated signaling events in mouse bone marrow-derived mast cells (BMMCs with NTAL KO or KD. BMMCs with both NTAL KO and KD exhibited enhanced degranulation, calcium mobilization, chemotaxis, tyrosine phosphorylation of LAT and ERK, and depolymerization of filamentous actin. These data provide clear evidence that NTAL is a negative regulator of FcεRI activation events in murine BMMCs, independently of possible compensatory developmental alterations. To gain further insight into the role of NTAL in mast cells, we examined the transcriptome profiles of resting and antigen-activated NTAL KO, NTAL KD, and corresponding control BMMCs. Through this analysis we identified several genes that were differentially regulated in nonactivated and antigen-activated NTAL-deficient cells, when compared to the corresponding control cells. Some of the genes seem to be involved in regulation of cholesterol-dependent events in antigen-mediated chemotaxis. The combined data indicate multiple regulatory roles of NTAL in gene expression and mast cell physiology.

Sonya Smith ja telenovela-äri / Tiina Lepiste

Index Scriptorium Estoniae

Lepiste, Tiina

2006-01-01

Telenovelade ärist ja fenomenist. Ka Venetsueela päritolu näitlejannast Sonya Smithist seriaalis "Armastuse jõud". Sama: Teleleht, 17. Jul. 2006, nr. 28, lk. 6-7 : ill pealkirjaga "Seebiäri vohab ja õitseb"

Reward acts on the pFC to enhance distractor resistance of working memory representations.

Science.gov (United States)

Fallon, Sean James; Cools, Roshan

2014-12-01

Working memory and reward processing are often thought to be separate, unrelated processes. However, most daily activities involve integrating these two types of information, and the two processes rarely, if ever, occur in isolation. Here, we show that working memory and reward interact in a task-dependent manner and that this task-dependent interaction involves modulation of the pFC by the ventral striatum. Specifically, BOLD signal during gains relative to losses in the ventral striatum and pFC was associated not only with enhanced distractor resistance but also with impairment in the ability to update working memory representations. Furthermore, the effect of reward on working memory was accompanied by differential coupling between the ventral striatum and ignore-related regions in the pFC. Together, these data demonstrate that reward-related signals modulate the balance between cognitive stability and cognitive flexibility by altering functional coupling between the ventral striatum and the pFC.

Mida me Tallinnas teinud oleme / Jüri Ratas

Index Scriptorium Estoniae

Ratas, Jüri, 1978-

2006-01-01

Tallinna linnapea Jüri Ratas annab ülevaate linnavalitsuse tegevusest esimese saja päeva jooksul. Linnavalitsuse tegevust hindavad Tallinna volikogu Isamaaliidu fraktsiooni esimees Toomas Tõniste, volikogu Reformierakonna esimees Keit Pentus ja volikogu sotsiaaldemokraatide fraktsiooni aseesimees Hannes Rumm

The role of the observational approach in RI planning: WAG 5 case study

International Nuclear Information System (INIS)

Brill, A.K.; Kuhaida, A.J. Jr.

1992-10-01

A Remedial Investigation (RI) Plan was developed for the Oak Ridge National Laboratory (ORNL) Waste Area Grouping (WAG) 5 in March 1988 and submitted to the Environmental Protection Agency (EPA) Region IV and the State of Tennessee for review and approval. The observational approach was evaluated by the ORNL Environmental Restoration (ER) Program and accepted by EPA as an accelerated and cost-effective approach to the RI/Feasibility Study (FS) process for remediation of WAG 5. The traditional approach used in preparing the 1988 RI Plan focused on data completeness, included a typical range of RI data-gathering activities for determining the nature and extent of contamination at WAG 5, and used multiple iterations of sampling activities to deal with uncertainties without consideration of potential deviations. In fall 1991, a revised Field Sampling Plan (FSP) was developed that used the observational approach to integrate site characterization with site remediation needs. This approach recognized the uncertainties of site characterization/remedial planning and developed contingency plans for dealing with them. The observational approach emphasizes data sufficiency to support remedial planning decisions for WAG 5

President vabastas Jüri Sakkarti kohtuniku ametist / Tanel Mazur

Index Scriptorium Estoniae

Mazur, Tanel, 1971-

2010-01-01

President Toomas Hendrik Ilves kirjutas 9. veebruaril 2010 alla otsusele vabastada Jüri Sakkart Viru Maakohtu kohtuniku ametist tema enda soovil. Kaitsepolitsei on J. Sakkarti kuriteokahtlustusega kinni pidanud

FC-TLBO: fully constrained meta-heuristic algorithm for abundance ...

Indian Academy of Sciences (India)

Omprakash Tembhurne

hyperspectral unmixing; meta-heuristic approach; teaching-learning-based optimisation (TLBO). 1. ... area of research due to its real-time applications. Satellite .... describes the detailed methodology of proposed FC-TLBO. Section 4 contains ...

Human Endogenous Retrovirus HERV-Fc1 Association with Multiple Sclerosis Susceptibility: A Meta-Analysis

Science.gov (United States)

García-Montojo, Marta; Alcina, Antonio; Fedetz, María; Alloza, Iraide; Astobiza, Ianire; Leyva, Laura; Fernández, Oscar; Izquierdo, Guillermo; Antigüedad, Alfredo; Arroyo, Rafael; Álvarez-Lafuente, Roberto; Vandenbroeck, Koen; Matesanz, Fuencisla; Urcelay, Elena

2014-01-01

Background Human endogenous retroviruses (HERVs) are repetitive sequences derived from ancestral germ-line infections by exogenous retroviruses and different HERV families have been integrated in the genome. HERV-Fc1 in chromosome X has been previously associated with multiple sclerosis (MS) in Northern European populations. Additionally, HERV-Fc1 RNA levels of expression have been found increased in plasma of MS patients with active disease. Considering the North-South latitude gradient in MS prevalence, we aimed to evaluate the role of HERV-Fc1on MS risk in three independent Spanish cohorts. Methods A single nucleotide polymorphism near HERV-Fc1, rs391745, was genotyped by Taqman chemistry in a total of 2473 MS patients and 3031 ethnically matched controls, consecutively recruited from: Northern (569 patients and 980 controls), Central (883 patients and 692 controls) and Southern (1021 patients and 1359 controls) Spain. Our results were pooled in a meta-analysis with previously published data. Results Significant associations of the HERV-Fc1 polymorphism with MS were observed in two Spanish cohorts and the combined meta-analysis with previous data yielded a significant association [rs391745 C-allele carriers: pM-H = 0.0005; ORM-H (95% CI) = 1.27 (1.11–1.45)]. Concordantly to previous findings, when the analysis was restricted to relapsing remitting and secondary progressive MS samples, a slight enhancement in the strength of the association was observed [pM-H = 0.0003, ORM-H (95% CI) = 1.32 (1.14–1.53)]. Conclusion Association of the HERV-Fc1 polymorphism rs391745 with bout-onset MS susceptibility was confirmed in Southern European cohorts. PMID:24594754

Downmodulation of Vaccine-Induced Immunity and Protection against the Intracellular Bacterium Francisella tularensis by the Inhibitory Receptor FcγRIIB

Directory of Open Access Journals (Sweden)

Brian J. Franz

2015-01-01

Full Text Available Fc gamma receptor IIB (FcγRIIB is the only Fc gamma receptor (FcγR which negatively regulates the immune response, when engaged by antigen- (Ag- antibody (Ab complexes. Thus, the generation of Ag-specific IgG in response to infection or immunization has the potential to downmodulate immune protection against infection. Therefore, we sought to determine the impact of FcγRIIB on immune protection against Francisella tularensis (Ft, a Category A biothreat agent. We utilized inactivated Ft (iFt as an immunogen. Naïve and iFt-immunized FcγRIIB knockout (KO or wildtype (WT mice were challenged with Ft-live vaccine strain (LVS. While no significant difference in survival between naïve FcγRIIB KO versus WT mice was observed, iFt-immunized FcγRIIB KO mice were significantly better protected than iFt-immunized WT mice. Ft-specific IgA in serum and bronchial alveolar lavage, as well as IFN-γ, IL-10, and TNF-α production by splenocytes harvested from iFt-immunized FcγRIIB KO, were also significantly elevated. In addition, iFt-immunized FcγRIIB KO mice exhibited a reduction in proinflammatory cytokine levels in vivo at 5 days after challenge, which correlates with increased survival following Ft-LVS challenge in published studies. Thus, these studies demonstrate for the first time the ability of FcγRIIB to regulate vaccine-induced IgA production and downmodulate immunity and protection. The immune mechanisms behind the above observations and their potential impact on vaccine development are discussed.

Tartu kui märk / Jüri Talvet

Index Scriptorium Estoniae

Talvet, Jüri, 1945-

1996-01-01

Ilmus algkujul hispaania keeles Sevillas Andaluusia Semiootikaassotsiatsiooni ajakirja Discurso Tartu semiootikakoolkonnale pühendatud erinumbris (nr. 8, 1993). Ilmunud ka kogumikus: Talvet, Jüri. Ameerika märkmeid, ehk, Kaemusi Eestist. Tartu : Ilmamaa, 2000, lk. 62-71, pealk.: Tartu vaim

77 FR 28253 - Safety Zone; America's Cup World Series, East Passage, Narragansett Bay, RI

Science.gov (United States)

2012-05-14

...-AA00 Safety Zone; America's Cup World Series, East Passage, Narragansett Bay, RI AGENCY: Coast Guard... navigable waters of the East Passage, Narragansett Bay, Rhode Island, during the America's Cup World Series... rulemaking (NPRM) entitled ``Safety Zones; America's Cup World Series, East Passage, Narragansett Bay, RI...

Current situation of RI's and radiation utilization in Japan

International Nuclear Information System (INIS)

1976-01-01

Usage of RIs and radiation is now extensive, for the improvement of industrial production techniques and also in medicine and agriculture. The current situation in Japan is described as follows: enterprises using RIs, circulation of RIs, the equipments provided with RIs in private enterprises, commercial RI, and radiation utilization, expenditures and sales related to RIs and radiation in mining and industries, and personnel. The number of places using RIs and radiation totaled about 3,200 (in 1975). The expenditures for RIs and radiation in private enterprises are about 17,500 million yen while sales are 20,800 million yen (both in fiscal 1974). The total number of personnel engaged in RI and radiation works is about 62,000. (Mori, K.)

Comparison of D2 receptor binding (123I-IBZM) and rCBF (99mTc-HMPAO) in extrapyramidal disorders

International Nuclear Information System (INIS)

Saur, H.B.; Bartenstein, P.; Schober, O.; Oberwittler, C.; Lerch, H.; Masur, H.

1994-01-01

The aim of this SPECT study was to determine whether there is a correlation between rCBF ( 99m Tc-HMPAO) and D2 receptor binding ( 123 I-IBZM) in disorders of the extrapyramidal system and in which situation the 99 MTc-HMPAO scan could predict the outcome of the 123 I-IBZM study. 13 patients with Parkinson's syndrome and 13 patients with hyperkinetic extrapyramidal disorders were studied. In all patients the two SPECT studies were performed within 2-7 days. ROIs were placed over the basal ganglia (BG), the frontal cortex (FC) and the cerebellum (CE). The ratios BG/FC and BG/CE were calculated. In both groups the scatter was lower when the frontal cortex was used as reference region. Among the patients with hyperkinetic extrapyramidal hyperkinetic extrapyramidal disorders the two patients with Huntington's chorea had lower rCBF and D2 receptor binding compared to other hyperkinetic extrapyramidal disorders. There was no correlation between D2 receptor binding and rCBF in the basal ganglia. The 99 MTc-HMPAO studies did not provide clinically useful information, except in Huntington's chorea. (orig.) [de

Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen

OpenAIRE

Jarvis, Gavin E.; Best, Denise; Watson, Steve P.

2004-01-01

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induc...

Studying astrophysical reactions with low-energy RI beams at CRIB

Directory of Open Access Journals (Sweden)

Yamaguchi H.

2016-01-01

Full Text Available Studies on nuclear astrophysics, nuclear structure, and other interests have been performed using the radioactive-isotope (RI beams at the low-energy RI beam separator CRIB, operated by Center for Nuclear Study (CNS, the University of Tokyo. A typical measurement performed at CRIB is the elastic resonant scattering with the inverse kinematics. One recent experiment was on the α resonant scattering with 7Li and 7Be beams. This study is related to the astrophysical 7Li/7Be(α,γ reactions, important at hot p-p chain and νp-process in supernovae. There have also been measurements based on other experimental methods. The first THM measurement using an RI beam has been performed at CRIB, to study the 18F(p, α15O reaction at astrophysical energies via the three body reaction 2H(18F, α15On. The 18F(p, α 15O reaction rate is crucial to understand the 511-keV γ-ray production in nova explosion phenomena, and we successfully evaluated the reaction cross section at novae temperature and below experimentally for the first time.

Endurance test for IR rig for RI production assembly (test procedure)

International Nuclear Information System (INIS)

Chung, Heung June; Ryu, Jeong Soo

2000-08-01

This test procedure details the test loop, test method, and test procedure for pressure drop, vibration and endurance test of IR Rig for RI production. From the pressure drop test, the hydraulic design requirements of the capsule are verified. HANARO limit condition is checked and the compatibility with HANARO core is verified. From flow induced vibration test vibration frequency and displacement are investigated. The wear of IR Rig is investigated through endurance test, and these data are used to evaluate the expected wear at maximum resident time of the IR Rig for RI production

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

Science.gov (United States)

Bhatti, L; Behle, K; Stevens, R H

1992-10-01

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

Energy Technology Data Exchange (ETDEWEB)

Barb, Adam W., E-mail: abarb@iastate.edu; Subedi, Ganesh P. [Iowa State University, Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology (United States)

2016-01-15

Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb{sup 3+} with high affinity (0.70 and 0.13 μM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (−0.221 to 0.081 ppm) and residual dipolar couplings (−7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb{sup 3+}){sub 2} complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems.

An encodable lanthanide binding tag with reduced size and flexibility for measuring residual dipolar couplings and pseudocontact shifts in large proteins

International Nuclear Information System (INIS)

Barb, Adam W.; Subedi, Ganesh P.

2016-01-01

Metal ions serve important roles in structural biology applications from long-range perturbations seen in magnetic resonance experiments to electron-dense signatures in X-ray crystallography data; however, the metal ion must be secured in a molecular framework to achieve the maximum benefit. Polypeptide-based lanthanide-binding tags (LBTs) represent one option that can be directly encoded within a recombinant protein expression construct. However, LBTs often exhibit significant mobility relative to the target molecule. Here we report the characterization of improved LBTs sequences for insertion into a protein loop. These LBTs were inserted to connect two parallel alpha helices of an immunoglobulin G (IgG)-binding Z domain platform. Variants A and B bound Tb 3+ with high affinity (0.70 and 0.13 μM, respectively) and displayed restricted LBT motion. Compared to the parent construct, the metal-bound A experienced a 2.5-fold reduction in tag motion as measured by magnetic field-induced residual dipolar couplings and was further studied in a 72.2 kDa complex with the human IgG1 fragment crystallizable (IgG1 Fc) glycoprotein. The appearance of both pseudo-contact shifts (−0.221 to 0.081 ppm) and residual dipolar couplings (−7.6 to 14.3 Hz) of IgG1 Fc resonances in the IgG1 Fc:(variant A:Tb 3+ ) 2 complex indicated structural restriction of the LBT with respect to the Fc. These studies highlight the applicability of improved LBT sequences with reduced mobility to probe the structure of macromolecular systems

Ethanol Inhibits High-Affinity Immunoglobulin E Receptor (Fc epsilon RI) Signaling in Mast Cells by Suppressing the Function of Fc epsilon RI-Cholesterol Signalosome

Czech Academy of Sciences Publication Activity Database

Dráberová, Lubica; Paulenda, Tomáš; Hálová, Ivana; Potůčková, Lucie; Bugajev, Viktor; Bambousková, Monika; Tůmová, Magda; Dráber, Petr

2015-01-01

Roč. 10, č. 12 (2015), e0144596-e0144596 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GBP302/12/G101; GA MŠk LD12073 Institutional support: RVO:68378050 Keywords : KAPPA-B Activation * Lipid rafts * Nadph oxidase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.057, year: 2015

Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

Science.gov (United States)

Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

2015-12-01

Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

Tüüri autoriplaat on MIDEM-i auhinna nominent

Index Scriptorium Estoniae

2008-01-01

Muusikamessi MIDEM klassikalise muusika plaatide auhinnaõhtul on üheks preemia nominendiks Luciano Berio ja Krzysztof Penderecki kõrval Erkki-Sven Tüüri autoriplaat "Magma", messist 27.-31. jaan. Cannes'is Prantsusmaal