bluuebunny/crossref_metadata_2025 huggingface dataset:
Preassociation of IL-15 with IL-15Rα-IgG1-Fc Enhances Its Activity on Proliferation of NK and CD8+/CD44high T Cells and Its Antitumor Action
Authors: Dubois Sigrid , Patel Hiral J. , Zhang Meili , Waldmann Thomas A. , Müller Jürgen R.
Published: April 2014

In the induction of an immune response, IL-15Rα on APCs transpresents IL-15 to NK and CD8+/CD44high T cells that express the IL-2/15Rβ and γc subunits only. In this study, we show data mimicking this transpresentation by using IL-15 preassociated with a chimeric protein that is comprised of the extracellular domain of murine IL-15Rα and the Fc portion of human IgG1. When tested in vitro, IL-15Rα-IgG1-Fc strongly increased the IL-15-mediated proliferation of murine NK and CD8+/CD44high T cells. The effect of IL-15Rα-IgG1-Fc was dependent on the presence of both IgG1-Fc and IL-15Rα. When injected into mice, IL-15Rα-IgG1-Fc enhanced the capacity of IL-15 to expand the number of NK and CD8+/CD44high T cells. The effect on cell numbers in vivo also depended on Fc receptor binding because reduced expansion was observed in FcRγ−/− mice. NK cells cultured in IL-15/IL-15Rα-IgG1-Fc complex gained cytotoxic activity toward a number of NK-sensitive targets. When mice bearing the NK-sensitive syngeneic tumor B16 were treated, the presence of IL-15Rα-IgG1-Fc increased the antitumor activity of IL-15. Thus, a preassociation with IL-15Rα-IgG1-Fc enhances the activities of IL-15 in vivo and in vitro that may be useful in the treatment of tumors.
Prostaglandin E2 Suppresses Lipopolysaccharide-Stimulated IFN-β Production
Authors: Xu X. Julia , Reichner Jonathan S. , Mastrofrancesco Balduino , Henry William L. , Albina Jorge E.
Published: April 2014

Macrophages activate the production of cytokines and chemokines in response to LPS through signaling cascades downstream from TLR4. Lipid mediators such as PGE2, which are produced during inflammatory responses, have been shown to suppress MyD88-dependent gene expression upon TLR4 activation in macrophages. The study reported here investigated the effect of PGE2 on TLR3- and TLR4-dependent, MyD88-independent gene expression in murine J774A.1 macrophages, as well as the molecular mechanism underlying such an effect. We demonstrate that PGE2 strongly suppresses LPS-induced IFN-β production at the mRNA and protein levels. Poly (I:C)-induced IFN-β and LPS-induced CCL5 production were also suppressed by PGE2. The inhibitory effect of PGE2 on LPS-induced IFN-β expression is mediated through PGE2 receptor subtypes EP2 and EP4, and mimicked by the cAMP analog 8-Br-cAMP as well as by the adenylyl cyclase activator forskolin. The downstream effector molecule responsible for the cAMP-induced suppressive effect is exchange protein directly activated by cAMP (Epac) but not protein kinase A. Moreover, data demonstrate that Epac-mediated signaling proceeds through PI3K, Akt, and GSK3β. In contrast, PGE2 inhibits LPS-induced TNF-α production in these cells through a distinct pathway requiring protein kinase A activity and independent of Epac/PI3K/Akt. In vivo, administration of a cyclooxygenase inhibitor before LPS injection resulted in enhanced serum IFN-β concentration in mice. Collectively, data demonstrate that PGE2 is a negative regulator for IFN-β production in activated macrophages and during endotoxemia.
Ex Vivo Generated Regulatory T Cells Modulate Experimental Autoimmune Myasthenia Gravis
Authors: Aricha Revital , Feferman Tali , Fuchs Sara , Souroujon Miriam C.
Published: April 2014

Naturally occurring CD4+CD25+ regulatory T (Treg) cells are key players in immune tolerance and have therefore been suggested as potential therapeutic tools for autoimmune diseases. In myasthenia gravis (MG), reduced numbers or functionally impaired Treg cells have been reported. We have observed that PBL from myasthenic rats contain decreased numbers of CD4+CD25highFoxp3+ cells as compared with PBL from healthy controls, and we have tested whether Treg cells from healthy donors can suppress experimental autoimmune MG in rats. Because the number of naturally occurring Treg cells is low, we used an approach for a large-scale ex vivo generation of functional Treg cells from CD4+ splenocytes of healthy donor rats. Treg cells were generated ex vivo from CD4+ cells by stimulation with anti-CD3 and anti-CD28 Abs in the presence of TGF-β and IL-2. The obtained cells expressed high levels of CD25, CTLA-4, and Foxp3, and they were capable of suppressing in vitro proliferation of T cells from myasthenic rats in response to acetylcholine receptor, the major autoantigen in myasthenia. Administration of ex vivo-generated Treg cells to myasthenic rats inhibited the progression of experimental autoimmune MG and led to down-regulation of humoral acetylcholine receptor-specific responses, and to decreased IL-18 and IL-10 expression. The number of CD4+CD25+ cells in the spleen of treated rats remained unchanged, but the subpopulation of CD4+CD25+ cells expressing Foxp3 was significantly elevated. Our findings imply that Treg cells play a critical role in the control of myasthenia and could thus be considered as potential agents for the treatment of MG patients.
CD4+CD25+ Regulatory T Cell Repertoire Formation Shaped by Differential Presentation of Peptides from a Self-Antigen
Authors: Larkin Joseph , Rankin Andrew L. , Picca Cristina Cozzo , Riley Michael P. , Jenks Scott A. , Sant Andrea J. , Caton Andrew J.
Published: April 2014

We have used TCR transgenic mice directed to different MHC class II-restricted determinants from the influenza virus hemagglutinin (HA) to analyze how specificity for self-peptides can shape CD4+CD25+ regulatory T (Treg) cell formation. We show that substantial increases in the number of CD4+CD25+ Treg cells can occur when an autoreactive TCR directed to a major I-Ed-restricted determinant from HA develops in mice expressing HA as a self-Ag, and that the efficiency of this process is largely unaffected by the ability to coexpress additional TCR α-chains. This increased formation of CD4+CD25+ Treg cells in the presence of the self-peptide argues against models that postulate selective survival rather than induced formation as mechanisms of CD4+CD25+ Treg cell formation. In contrast, T cells bearing a TCR directed to a major I-Ad-restricted determinant from HA underwent little or no selection to become CD4+CD25+ Treg cells in mice expressing HA as a self-Ag, correlating with inefficient processing and presentation of the peptide from the neo-self-HA polypeptide. These findings show that interactions with a self-peptide can induce thymocytes to differentiate along a pathway to become CD4+CD25+ Treg cells, and that peptide editing by DM molecules may help bias the CD4+CD25+ Treg cell repertoire away from self-peptides that associate weakly with MHC class II molecules.
The Magnitude of the Antibody and Memory B Cell Responses during Priming with a Protein-Polysaccharide Conjugate Vaccine in Human Infants Is Associated with the Persistence of Antibody and the Intensity of Booster Response
Authors: Rohner Geraldine Blanchard , Snape Matthew D. , Kelly Dominic F. , John Tessa , Morant Anita , Yu Ly-Mee , Borkowski Astrid , Ceddia Francesca , Borrow Ray , Siegrist Claire-Anne , Pollard Andrew J.
Published: April 2014

Rapid waning of anti-polysaccharide bactericidal Ab and vaccine effectiveness is observed following infant immunization with the serogroup C meningococcal (MenC) glycoconjugate vaccine. This is despite the demonstrable presence of immunological memory. Persistence of functional Ab, therefore, appears to be the key determinant of MenC conjugate vaccine effectiveness. Ab persistence is thought to depend in the short term on the survival of plasma cells generated during priming and in the longer term on the production of new Ab secreting cells from memory B cells. In this study, we found a strong association between the level of MenC-specific Ab and the frequency of memory B cells measured at 5 mo of age (1 mo after 3-dose primary immunization with MenC conjugate vaccine), and the persistence of functional Ab at one year of age. These findings suggest that these two parameters are good markers of B cell responses to priming and can be used as predictors of long term humoral immunity induced by glycoconjugate vaccines received in early infancy.
Induction of Broad Cross-Subtype-Specific HIV-1 Immune Responses by a Novel Multivalent HIV-1 Peptide Vaccine in Cynomolgus Macaques
Authors: Azizi Ali , Anderson David E. , Torres José V. , Ogrel Andrei , Ghorbani Masoud , Soare Catalina , Sandstrom Paul , Fournier Jocelyne , Diaz-Mitoma Francisco
Published: April 2014

One of the major obstacles in the design of an effective vaccine against HIV-1 is its antigenic variation, which results in viral escape from the immune system. Through a bioinformatics approach, we developed an innovative multivalent HIV-1 vaccine comprised of a pool of 176 lipidated and nonlipidated peptides representing variable regions of Env and Gag proteins. The potency and breadth of the candidate vaccine against a panel of HIV-1 subtypes was evaluated in nonhuman primate (cynomolgus macaques) and humanized mouse (HLA-A2.1) models. The results demonstrate strong immunogenicity with both breadth (humoral and cellular immunity) and depth (immune recognition of widely divergent viral sequences) against heterologous HIV-1 subtypes A–F.
Bacterial Fimbriae Stimulate Proinflammatory Activation in the Endothelium through Distinct TLRs
Authors: Davey Michael , Liu Xinyan , Ukai Takashi , Jain Vishal , Gudino Cynthia , Gibson Frank C. , Golenbock Douglas , Visintin Alberto , Genco Caroline A.
Published: April 2014

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed Kd of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.
Differential Molecular and Anatomical Basis for B Cell Migration into the Peritoneal Cavity and Omental Milky Spots
Authors: Berberich Simon , Dähne Sabrina , Schippers Angela , Peters Thorsten , Müller Werner , Kremmer Elisabeth , Förster Reinhold , Pabst Oliver
Published: April 2014

The constitutive migration of B cells from the circulation into the peritoneal cavity and back is essential for peritoneal B cell homeostasis and function. However, the molecular machinery and the anatomical basis for these migratory processes have hardly been investigated. In this study, we analyze the role of integrins as well as the role of the omentum for B2 cell migration into and out of the peritoneal cavity of mice. We demonstrate that α4β7 integrin-mucosal addressin cell adhesion molecule 1 interaction enables B2 cell migration from the circulation into omental milky spots but not into the peritoneum. In contrast, α4β1 integrin mediates direct entry of B2 cells into the peritoneal cavity as well as their retention at that site, limiting B2 cell egress via the draining parathymic lymph nodes. Surgical removal of the omentum results in a 40% reduced immigration of B2 cells from the circulation into the peritoneum but does not impair B cell exit from this compartment. In conclusion, these data reveal the existence of alternative routes for B2 cell entry into the peritoneal cavity and identify integrins as key factors for peritoneal B2 cell homeostasis, mediating B2 cell migration into and out of the peritoneal cavity as well as their retention at this site.
Anti-MHC Class I Antibody Activation of Proliferation and Survival Signaling in Murine Cardiac Allografts
Authors: Jindra Peter T. , Hsueh Aileen , Hong Longshen , Gjertson David , Shen Xiu-Da , Gao Feng , Dang Julie , Mischel Paul S. , Baldwin William M. , Fishbein Michael C. , Kupiec-Weglinski Jerzy W. , Reed Elaine F.
Published: April 2014

Anti-MHC class I alloantibodies have been implicated in the process of acute and chronic rejection because these Abs can bind to endothelial cells and transduce signals leading to the activation of cell survival and proliferation pathways. To characterize the role of the MHC class I-signaling pathway in the pathogenesis of Ab-mediated rejection, we developed a mouse vascularized heterotopic cardiac allograft model in which B6.RAG1 KO hosts (H-2Kb/Db) received a fully MHC-incompatible BALB/c (H-2Kd/Dd) heart transplant and were passively transfused with anti-donor MHC class I Ab. We demonstrate that cardiac allografts of mice treated with anti-MHC class I Abs show characteristic features of Ab-mediated rejection including microvascular changes accompanied by C4d deposition. Phosphoproteomic analysis of signaling molecules involved in the MHC class I cell proliferation and survival pathways were elevated in anti-class I-treated mice compared with the isotype control-treated group. Pairwise correlations, hierarchical clustering, and multidimensional scaling algorithms were used to dissect the class I-signaling pathway in vivo. Treatment with anti-H-2Kd Ab was highly correlated with the activation of Akt and p70S6Kinase (S6K). When measuring distance as a marker of interrelatedness, multidimensional scaling analysis revealed a close association between members of the mammalian target of rapamycin pathway including mammalian target of rapamycin, S6K, and S6 ribosomal protein. These results provide the first analysis of the interrelationships between these signaling molecules in vivo that reflects our knowledge of the signaling pathway derived from in vitro experiments.
Canonical Transient Receptor Potential 5 Channel in Conjunction with Orai1 and STIM1 Allows Sr2+ Entry, Optimal Influx of Ca2+, and Degranulation in a Rat Mast Cell Line
Authors: Ma Hong-Tao , Peng Ze , Hiragun Takaaki , Iwaki Shoko , Gilfillan Alasdair M. , Beaven Michael A.
Published: April 2014

Degranulation of mast cells in response to Ag or the calcium mobilizing agent, thapsigargin, is dependent on emptying of intracellular stores of Ca2+ and the ensuing influx of external Ca2+, also referred to as store-operated calcium entry. However, it is unlikely that the calcium release-activated calcium channel is the sole mechanism for the entry of Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells. In this study we show that influx of Ca2+ and Sr2+ as well as degranulation are dependent on the presence of the canonical transient receptor potential (TRPC) channel protein TRPC5, in addition to STIM1 and Orai1, as demonstrated by knock down of each of these proteins by inhibitory RNAs in a rat mast cell (RBL-2H3) line. Overexpression of STIM1 and Orai1, which are known to be essential components of calcium release-activated calcium channel, allows entry of Ca2+ but not Sr2+, whereas overexpression of STIM1 and TRPC5 allows entry of both Ca2+ and Sr2+. These and other observations suggest that the Sr2+-permeable TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca2+ to generate a signal for degranulation.
Comprehensive Assessment and Mathematical Modeling of T Cell Population Dynamics and Homeostasis
Authors: Thomas-Vaslin Véronique , Altes Hester Korthals , de Boer Rob J. , Klatzmann David
Published: April 2014

Our current view of T cell differentiation and population dynamics is assembled from pieces of data obtained from separate experimental systems and is thus patchy. We reassessed homeostasis and dynamics of T cells 1) by generating a mathematical model describing the spatiotemporal features of T cell differentiation, and 2) by fitting this model to experimental data generated by disturbing T cell differentiation through transient depletion of dividing T cells in mice. This specific depletion was obtained by administration of ganciclovir to mice expressing the conditional thymidine kinase suicide gene in T cells. With this experimental approach, we could derive quantitative parameters describing the cell fluxes, residence times, and rates of import, export, proliferation, and death across cell compartments for thymocytes and recent thymic emigrants (RTEs). Among other parameters, we show that 93% of thymocytes produced before single-positive stages are eliminated through the selection process. Then, a postselection peripheral expansion of naive T cells contributes three times more to naive T cell production than the thymus, with half of the naive T cells consisting of dividing RTEs. Altogether, this work provides a quantitative population dynamical framework of thymocyte development, RTEs, and naive T cells.
Agonists of the Mas-Related Gene (Mrgs) Orphan Receptors as Novel Mediators of Mast Cell-Sensory Nerve Interactions
Authors: Lee Min-Goo , Dong Xinzhong , Liu Qin , Patel Kush N. , Choi Oksoon Hong , Vonakis Becky , Undem Bradley J.
Published: April 2014

IgE-dependent activation of mast cell activation is often associated with symptoms attributed to activation of sensory nerves. Depending on the tissues involved such symptoms include itching, sneezing, irritation, vasodilation, and reflex secretions. In the present study, we hypothesize that sensory neuroactive mediators released from mast cells may include agonists of recently discovered orphan receptors referred to as sensory nerve specific receptors or products of mas related genes. HEK-293 cells expressing MrgC11 receptors and wild-type HEK-293 cells were loaded with the calcium indicator Fura-2. A known stimulant of MrgC11 receptors the RF-amide, neuropeptide FF, evoked a rapid increase in cytosolic calcium in the MrgC11 expressing cells but not in the wild-type HEK-293 cells. IgE-dependent stimulation of either rat basophilic leukemia-2H3 cells (RBL-2H3 cells) or mouse bone marrow-derived mast cells, released a substance(s) that stimulated increases in cytosolic calcium in the MrgC11 expressing cells that far exceeded that seen in control cells. RT-PCR revealed that both mouse mast cells and RBL-2H3 cells express the RF-amide precursor gene proneuropeptide FF (A). Immunohistochemical analysis demonstrated RF-amide immunoreactivity in mouse skin mast cells in situ and in mast cells isolated from mouse skin. These data support the hypothesis that agonists of certain sensory nerve specific receptors or mas related genes may participate in mast cell sensory nerve interactions.
Maturing Dendritic Cells Depend on RAGE for In Vivo Homing to Lymph Nodes
Authors: Manfredi Angelo A. , Capobianco Annalisa , Esposito Antonio , De Cobelli Francesco , Canu Tamara , Monno Antonella , Raucci Angela , Sanvito Francesca , Doglioni Claudio , Nawroth Peter P. , Bierhaus Angelika , Bianchi Marco E. , Rovere-Querini Patrizia , Del Maschio Alessandro
Published: April 2014

The mobilization of dendritic cells (DCs) from peripheral tissues is critical for the establishment of T cell-dependent immune responses or tolerance, because the physical interaction of DCs with naive T cells takes place in the T cell areas of lymph nodes. The autocrine/paracrine release of the high mobility group box 1 (HMGB1) nuclear protein by DCs controls the outcome of the DC–T cell interaction, influencing the priming/Th1 polarization of naive T cells. We herein present evidence that the receptor for advanced glycation end products (RAGE), a multiligand member of the Ig superfamily of cell-surface molecules that acts as a receptor for HMGB1, plays a nonredundant role in DC homing to lymph nodes. We used noninvasive imaging by magnetic resonance and immunohistochemistry to track DCs after s.c. injection in the footpad of wild-type+/+ or RAGE−/− mice. Maturing DCs expressing RAGE effectively migrated in both conditions. In contrast, RAGE−/− DCs failed to reach the draining popliteal lymph nodes of +/+ and −/− mice, indicating that the integrity of RAGE is required for DC mobilization. Thus the HMGB1-RAGE pathway is a checkpoint in DC maturation and function and a candidate for targeted therapies.
A Critical Role of Costimulation during Intrathymic Development of Invariant NK T Cells
Authors: Chung Yeonseok , Nurieva Roza , Esashi Eiji , Wang Yi-Hong , Zhou Dapeng , Gapin Laurent , Dong Chen
Published: April 2014

CD1d-restricted Vα14+ invariant NK T (iNKT) cells are a specialized αβ T cell subset that regulates both innate and adaptive immunity. Although costimulatory molecules are required for the activation of conventional T cells and for the development of Foxp3+ T cells, their role in iNKT cell regulation is unclear. Here we report that mice deficient in CD80/CD86 and/or B7h exhibit severe defects in thymic iNKT cell maturation, associated with largely reduced iNKT cell number in the thymus and the periphery. We show that costimulation is necessary for the optimal expansion of postselected NK1.1− immature iNKT cells in the thymus and for the proper expression of the maturation markers T-bet and CD122. Surprisingly, costimulatory molecules on both hemopoietic and nonhematopoietic cells are required for iNKT cell development. Our results thus demonstrate a previously unknown function of costimulation in the intrathymic development of iNKT cells, distinct from that of conventional T cells and regulatory T cells.
Complement Activation Plays a Key Role in Antibody-Induced Infusion Toxicity in Monkeys and Rats
Authors: Tawara Tomonori , Hasegawa Kazumasa , Sugiura Yusuke , Harada Katsuhiko , Miura Toru , Hayashi Sunao , Tahara Tomoyuki , Ishikawa Masaharu , Yoshida Hideaki , Kubo Kinya , Ishida Isao , Kataoka Shiro
Published: April 2014

Infusion reactions are a major side effect of the administration of therapeutic Abs and are the result of a complex immune reaction. In this study, we report that substitutions of Fc amino acids in the anti-HLA-DR Ab HD8 reduce its ability to induce infusion reactions in rats and monkeys. We first showed that i.v. administration of IgG1- and IgG2-subclass HD8 Abs induces severe infusion reactions in monkeys. These Abs express strong complement-dependent cytotoxicity (CDC), and in vivo depletion of complement in rats by pretreatment with cobra venom factor abrogated the lethal infusion reactions generated by HD8-IgG1. Thus, the infusion reactions appear to be largely driven by the complement system. To reduce the CDC function of HD8-IgG1, its Fc region was modified by two amino acid substitutions at Pro331Ser and Lys322Ala. The modified Ab was incapable of expressing CDC in vitro and did not induce severe infusion reactions in rats and monkeys, even at extremely high doses. The modified Ab retained its Ab-dependent cellular cytotoxicity function as well as its antitumor activity in a tumor-bearing mouse model. In summary, complement appears to drive infusion reactions, and modifications that eliminate the CDC activity of an Ab also reduce its ability to induce infusion reactions.
Why T Cells of Thymic Versus Extrathymic Origin Are Functionally Different
Authors: Blais Marie-Ève , Brochu Sylvie , Giroux Martin , Bélanger Marie-Pier , Dulude Gaël , Sékaly Rafick-Pierre , Perreault Claude
Published: April 2014

Age-related thymic involution severely impairs immune responsiveness. Strategies to generate T cells extrathymically are therefore being explored with intense interest. We have demonstrated that T cells produced extrathymically were functionally deficient relative to thymus-derived T cells. The main limitation of extrathymic T cells is their undue susceptibility to apoptosis; they thus do not expand properly when confronted with pathogens. Using oncostatin M-transgenic mice, we found that in the absence of lymphopenia, T cells of extrathymic origin constitutively undergo excessive homeostatic proliferation that leads to overproduction of IL-2 and IFN-γ. IFN-γ up-regulates Fas and FasL on extrathymic CD8 T cells, thereby leading to their demise by Fas-mediated apoptosis. Moreover, IFN-γ and probably IL-2 curtail survival of extrathymic CD4 T cells by down-regulating IL-7Rα and Bcl-2, and they support a dramatic accumulation of FoxP3+ T regulatory cells. Additionally, we show that wild-type thymus-derived T cells undergoing homeostatic proliferation in a lymphopenic host shared key features of extrathymic T cells. Our work explains how excessive lymphopenia-independent homeostatic proliferation renders extrathymic T cells functionally defective. Based on previous work and data presented herein, we propose that extrathymic T cells undergo constitutive homeostatic proliferation because they are positively selected by lymph node hemopoietic cells rather than by thymic epithelial cells.
Three-Dimensional Structure of the Cross-Reactive Pollen Allergen Che a 3: Visualizing Cross-Reactivity on the Molecular Surfaces of Weed, Grass, and Tree Pollen Allergens
Authors: Verdino Petra , Barderas Rodrigo , Villalba Mayte , Westritschnig Kerstin , Valenta Rudolf , Rodriguez Rosalia , Keller Walter
Published: April 2014

Two EF-hand calcium-binding allergens (polcalcins) occur in the pollen of a wide variety of unrelated plants as highly cross-reactive allergenic molecules. We report the expression, purification, immunological characterization, and the 1.75-Å crystal structure of recombinant Che a 3 (rChe a 3), the polcalcin from the weed Chenopodium album. The three-dimensional structure of rChe a 3 resembles an α-helical fold that is essentially identical with that of the two EF-hand allergens from birch pollen, Bet v 4, and timothy grass pollen, Phl p 7. The extensive cross-reactivity between Che a 3 and Phl p 7 is demonstrated by competition experiments with IgE Abs from allergic patients as well as specific Ab probes. Amino acid residues that are conserved for the two EF-hand allergen family were identified in multiple sequence alignments of polcalcins from 15 different plants. Next, the three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 were used to identify conserved amino acids with high surface exposition to visualize surface patches as potential targets for the polyclonal IgE Ab response of allergic patients. The essentially identical three-dimensional structures of rChe a 3, rPhl p 7, and rBet v 4 explain the extensive cross-reactivity of allergic patients IgE Abs with two EF-hand allergens from unrelated plants. In addition, analyzing the three-dimensional structures of cross-reactive Ags for conserved and surface exposed amino acids may be a first approach to mapping the conformational epitopes on disease-related Ags that are recognized by polyclonal patient Abs.
Protective Effect of IL-18 on Kainate- and IL-1β-Induced Cerebellar Ataxia in Mice
Authors: Andoh Tsugunobu , Kishi Hiroyuki , Motoki Kazumi , Nakanishi Kenji , Kuraishi Yasushi , Muraguchi Atsushi
Published: April 2014

The pathogenesis of sporadic cerebellar ataxia remains unknown. In this study, we demonstrate that proinflammatory cytokines, IL-18 and IL-1β, reciprocally regulate kainate-induced cerebellar ataxia in mice. We show that systemic administration of kainate activated IL-1β and IL-18 predominantly in the cerebellum of mice, which was accompanied with ataxia. Mice deficient in caspase-1, IL-1R type I, or MyD88 were resistant to kainate-induced ataxia, while IL-18- or IL-18R α-deficient mice displayed significant delay of recovery from ataxia. A direct intracerebellar injection of IL-1β-induced ataxia and intracerebellar coinjection of IL-18 counteracted the effect of IL-1β. Our data firstly show that IL-18 and IL-1β display differential direct regulation in kainate-induced ataxia in mice. Our results might contribute toward the development of a new therapeutic strategy for cerebellar ataxia in humans.
C1q Binds Phosphatidylserine and Likely Acts as a Multiligand-Bridging Molecule in Apoptotic Cell Recognition
Authors: Païdassi Helena , Tacnet-Delorme Pascale , Garlatti Virginie , Darnault Claudine , Ghebrehiwet Berhane , Gaboriaud Christine , Arlaud Gérard J. , Frachet Philippe
Published: April 2014

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute “eat me” signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (KD = 3.7–7 × 10−8 M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.
HLA Class I Antibody-Mediated Endothelial Cell Proliferation via the mTOR Pathway
Authors: Jindra Peter T. , Jin Yi-Ping , Rozengurt Enquire , Reed Elaine F.
Published: April 2014

Anti-HLA Abs have been shown to contribute to the process of transplant vasculopathy by binding to HLA class I molecules expressed by the endothelial and smooth muscle cells of the graft and transducing intracellular signals that elicit cell proliferation. The aim of this study was to determine the role of mammalian target of rapamycin (mTOR) in HLA class I-induced endothelial cell proliferation and to explore in depth the relationship between mTOR complexes and their downstream targets following ligation of HLA class I molecules by anti-HLA Abs. We used small interfering RNA technology to abrogate mTOR, rapamycin-insensitive companion of mTOR (rictor), or regulatory associated protein of mTOR (raptor) to study the function of these gene products to activate proteins involved in MHC class I-induced cell proliferation and survival. Knockdown of mTOR inhibited class I-mediated phosphorylation of proteins downstream of mTOR complex 1 and mTOR complex 2. Furthermore, knockdown of mTOR, rictor, or raptor blocked HLA class I-induced endothelial cell proliferation. Long-term pretreatment with the mTOR inhibitor rapamycin significantly blocked both mTOR-raptor and mTOR-rictor complex formation. Interestingly, rapamycin also blocked class I-induced Akt phosphorylation at Ser473 and Bcl-2 expression. These results support the role of anti-HLA Abs in the process of transplant vasculopathy and suggest that exposure of the graft endothelium to anti-HLA Abs may promote proliferation through the mTOR pathway.
Human Trophoblasts Recruited T Lymphocytes and Monocytes into Decidua by Secretion of Chemokine CXCL16 and Interaction with CXCR6 in the First-Trimester Pregnancy
Authors: Huang Yu , Zhu Xiao-Yong , Du Mei-Rong , Li Da-Jin
Published: April 2014

During human early pregnancy, fetus-derived trophoblasts come into direct contact with maternal immune cells at the maternofetal interface. At sites of placental attachment, invasive extravillous trophoblasts encounter decidual leukocytes (DLC) that accumulate within the decidua. Because we first found chemokine CXCL16 was highly expressed in and secreted by the first-trimester human trophoblasts previously, in this study we tested the hypothesis of whether the fetal trophoblasts can direct migration of maternal T lymphocyte and monocytes into decidua by secreting CXCL16. We analyzed the transcription and translation of CXCL16 in the isolated first-trimester human trophoblast, and examined the kinetic secretion of CXCL16 in the supernatant of the primary-cultured trophoblasts. We demonstrated that the sole receptor of CXCL16, CXCR6, is preferentially expressed in T lymphocytes, NKT cells, and monocytes, hardly expressed in two subsets of NK cells from either the peripheral blood or decidua. We further demonstrated the chemotactic activity of CXCL16 in the supernatant of the primary trophoblast on the peripheral mononuclear cells and DLC. Moreover, the CXCL16/CXCR6 interaction is involved in the migration of the peripheral T lymphocytes, γδ T cells, and monocytes, but not NKT cells. In addition, the trophoblast-conditioned medium could enrich PBMC subsets selectively to constitute a leukocyte population with similar composition to that of DLC, which suggests that the fetus-derived trophoblasts can attract T cells, γδ T cells, and monocytes by producing CXCL16 and interaction with CXCR6 on these cells, leading to forming a specialized immune milieu at the maternofetal interface.
IL-13 Is Required for Eosinophil Entry into the Lung during Respiratory Syncytial Virus Vaccine-Enhanced Disease
Authors: Castilow Elaine M. , Meyerholz David K. , Varga Steven M.
Published: April 2014

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.
Pneumococcal Capsular Polysaccharide Is Immunogenic When Present on the Surface of Macrophages and Dendritic Cells: TLR4 Signaling Induced by a Conjugate Vaccine or by Lipopolysaccharide Is Conducive
Authors: Cohen Noam , Stolarsky-Bennun Michal , Amir-Kroll Hila , Margalit Raanan , Nussbaum Gabriel , Cohen-Sfady Michal , Pevsner-Fischer Meirav , Fridkin Mati , Bercovier Herve , Eisenbach Lea , Jung Steffen , Cohen Irun R.
Published: April 2014

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.
Integrin β1 Regulates Phagosome Maturation in Macrophages through Rac Expression
Authors: Wang Qing-Qing , Li Hong , Oliver Tim , Glogauer Michael , Guo Jian , He You-Wen
Published: April 2014

Phagocytosis and subsequent phagosome maturation by professional phagocytes are essential in the clearance of infectious microbial pathogens. The molecular regulation of phagosome maturation is largely unknown. We show that integrin β1 plays critical roles in the phagocytosis of microbial pathogens and phagosome maturation. Macrophages lacking integrin β1 expression exhibit reduced phagocytosis of bacteria, including group B streptococcus and Staphylococcus aureus. Furthermore, phagosomes from macrophages lacking integrin β1 show lowered maturation rate, defective acquisition of lysosome membrane markers, and reduced F-actin accumulation in the periphagosomal region. Integrin β1-deficient macrophages exhibit impaired bactericidal activity. We found that the expression of the Rho family GTPases Rac1, Rac2, and Cdc42 was reduced in integrin β1-deficient macrophages. Ectopic expression of Rac1, but not Cdc42, in integrin β1-deficient macrophages restored defective phagosome maturation and F-actin accumulation in the periphagosomal region. Importantly, macrophages lacking Rac1/2 also exhibit defective maturation of phagosomes derived from opsonized Escherichia coli or IgG beads. Taken together, these results suggest that integrin β1 regulates phagosome maturation in macrophages through Rac expression.
A238L Inhibits NF-ATc2, NF-κB, and c-Jun Activation through a Novel Mechanism Involving Protein Kinase C-θ-Mediated Up-Regulation of the Amino-Terminal Transactivation Domain of p300
Authors: Granja Aitor G. , Perkins Neil D. , Revilla Yolanda
Published: April 2014

The transcriptional coactivators CREB-binding protein and p300 regulate inducible transcription in multiple cellular processes and during the establishment of inflammatory and immune response. Several viruses have been shown to interfere with CREB-binding protein/p300 function, modulating their transcriptional activity. In this study, we report that the viral protein A238L interacts with the amino-terminal region of p300, inhibiting the acetylation and transcriptional activation of NF-ATc2, NF-κB, and c-Jun in stimulated human T cells. We demonstrate that A238L modulates the autoacetylation of p300 without altering its intrinsic histone acetyl transferase activity. Furthermore, we show that the molecular mechanism of the inhibition executed by the viral protein is conducted through blocking protein kinase C (PKC)-p300 interaction and further acetylation in the amino-terminal transactivation domain of the coactivator, and that Ser384, within the CH1 domain, is essential for the full transcriptional activation of the coactivator. Moreover, we show that overexpression of an active form of PKC-θ reverts the A238L-mediated inhibition of the transcriptional activity of p300, showing, for the first time, a PKC-θ-mediated up-regulation of the coactivator. These findings provide new strategies to develop therapies potentially useful in the control of disorders related to p300 deregulation.
Injury Enhances Resistance to Escherichia coli Infection by Boosting Innate Immune System Function
Authors: Maung Adrian A. , Fujimi Satoshi , MacConmara Malcolm P. , Tajima Goro , McKenna Ann M. , Delisle Adam J. , Stallwood Christopher , Onderdonk Andrew B. , Mannick John A. , Lederer James A.
Published: April 2014

Major injury is widely thought to predispose the injured host to opportunistic infections. This idea is supported by animal studies showing that major injury causes reduced resistance to polymicrobial sepsis induced by cecal ligation and puncture. Although cecal ligation and puncture represents a clinically relevant sepsis model, we wanted to test whether injury might also lead to greater susceptibility to peritoneal infection caused by a single common pathogen, Escherichia coli. Contrary to our expectation, we show herein that the LD50 for sham-injured mice was 103 CFU of E. coli, whereas the LD50 for burn-injured mice was 50 × 103 CFU at 7 days postinjury. This injury-associated enhanced resistance was apparent as early as 1 day after injury, and maximal resistance was observed at days 7 and 14. We found that burn-injured mice had higher numbers of circulating neutrophils and monocytes than did sham mice before infection and that injured mice were able to recruit greater numbers of neutrophils to the site of infection. Moreover, the peritoneal neutrophils in burn-injured mice were more highly activated than neutrophils from sham mice as determined by Mac-1 expression, superoxide generation, and bactericidal activity. Our findings suggest that the enhanced innate immune response that develops following injury, although it is commonly accepted as the mediator of the detrimental systemic inflammatory response syndrome, may also, in some cases, benefit the injured host by boosting innate immune antimicrobial defenses.
IL-23 Is Required for the Development of Severe Egg-Induced Immunopathology in Schistosomiasis and for Lesional Expression of IL-17
Authors: Rutitzky Laura I. , Bazzone Lindsey , Shainheit Mara G. , Joyce-Shaikh Barbara , Cua Daniel J. , Stadecker Miguel J.
Published: April 2014

In infection with the trematode helminth Schistosoma mansoni, the severity of CD4 T cell-mediated hepatic granulomatous and fibrosing inflammation against parasite eggs varies considerably in humans and among mouse strains. In mice, either the natural high pathology, or high pathology induced by concomitant immunization with schistosome egg Ags (SEA) in CFA (SEA/CFA), results from a failure to contain a net proinflammatory cytokine environment. We previously demonstrated that the induction of severe immunopathology was dependent on the IL-12/IL-23 common p40 subunit, and correlated with an increase in IL-17, thus implying IL-23 in the pathogenesis. We now show that mice lacking the IL-23-specific subunit p19 are impaired in developing severe immunopathology following immunization with SEA/CFA, which is associated with a marked drop of IL-17 in the granulomas, but not in the draining mesenteric lymph nodes, and with a markedly suppressed SEA-specific IFN-γ response regulated by a striking increase in IL-10. The granulomas are characterized by a significant reduction in Gr-1+ cell recruitment and by alternative macrophage activation. Taken together, these results demonstrate that IL-23 per se is not necessary for the generation of IL-17-producing T cells, but is essential for the development of severe schistosome egg-induced immunopathology, and its absence cannot be overcome with other possible compensatory mechanisms.
Malaria Parasites Require TLR9 Signaling for Immune Evasion by Activating Regulatory T Cells
Authors: Hisaeda Hajime , Tetsutani Kohhei , Imai Takashi , Moriya Chikako , Tu Liping , Hamano Shinjiro , Duan Xuefeng , Chou Bin , Ishida Hidekazu , Aramaki Akiko , Shen Jianying , Ishii Ken J. , Coban Cevayir , Akira Shizuo , Takeda Kiyoshi , Yasutomo Koji , Torii Motomi , Himeno Kunisuke
Published: April 2014

Malaria is still a life-threatening infectious disease that continues to produce 2 million deaths annually. Malaria parasites have acquired immune escape mechanisms and prevent the development of sterile immunity. Regulatory T cells (Tregs) have been reported to contribute to immune evasion during malaria in mice and humans, suggesting that activating Tregs is one of the mechanisms by which malaria parasites subvert host immune systems. However, little is known about how these parasites activate Tregs. We herein show that TLR9 signaling to dendritic cells (DCs) is crucial for activation of Tregs. Infection of mice with the rodent malaria parasite Plasmodium yoelii activates Tregs, leading to enhancement of their suppressive function. In vitro activation of Tregs requires the interaction of DCs with parasites in a TLR9-dependent manner. Furthermore, TLR9−/− mice are partially resistant to lethal infection, and this is associated with impaired activation of Tregs and subsequent development of effector T cells. Thus, malaria parasites require TLR9 to activate Tregs for immune escape.
Regulatory T Cells Prevent Control of Experimental African Trypanosomiasis
Authors: Wei Guojian , Tabel Henry
Published: April 2014

African trypanosomes are single-cell, extra-cellular blood parasites causing profound immunosuppression. Susceptible BALB/c mice infected s.c. into a footpad with 104 Trypanosoma congolense die with fulminating parasitemia within 10 days. We injected BALB/c mice 2 days before such an infection with different doses of a depleting mAb specific for CD25, a surface marker of regulatory T cells (Tregs). Pretreatment with a low, optimal dose of anti-CD25 resulted in a dramatic effect, in that the infected mice did not develop parasitemia, as well as eliminated all parasites and showed no signs of disease. Their spleens showed a 100% reduction of CD4+CD25high T cells and overall a 70% reduction of CD4+CD25+Foxp3+ T cells 7 days postinfection. The protective effect of treatment with an optimal dose of anti-CD25 could be reversed by administration of l-N6-(1-imminoethyl) lysine, a specific inhibitor of inducible NO synthase or administration of anti-CD8 Ab. Analysis of the cytokine patterns and cell surface marker in infected mice pretreated with anti-CD25 Abs pointed to a potential NKT cell response. We then conducted infections in CD1d−/− mice. From our observations, we conclude that CD4+CD25highFoxp3+ Tregs prevent, in normal infected susceptible mice, an early protective response mediated by CD8+ NKT cell-dependent activation of macrophages to kill parasites by production of NO. Our results also indicate that different populations of NKT cells have protective or suppressive effects. Our observations lead us to propose a hypothesis of cross-regulation of NKT cells and Tregs in trypanosome infections.