bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prolonged Exposure to Lipopolysaccharide Inhibits Macrophage 5-Lipoxygenase Metabolism Via Induction of Nitric Oxide Synthesis
Authors: Coffey Michael J. , Phare Susan M. , Peters-Golden Marc 
Published: April 2014

Abstract
LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.
CD4 Help-Independent Induction of Cytotoxic CD8 Cells to Allogeneic P815 Tumor Cells Is Absolutely Dependent on Costimulation
Authors: Zhan Yifan , Corbett Alexandra J. , Brady Jamie L. , Sutherland Robyn M. , Lew Andrew M. 
Published: April 2014

AbstractMice made transgenic (Tg) for a rat anti-mouse CD4 Ab (GK mice) represent a novel CD4-deficient model. They not only lack canonical CD4 cells in the periphery, but also lack the residual aberrant Th cells that are found in CD4−/− mice and MHC class II−/− mice. To analyze the role of CD4 help and costimulation for CTL induction against alloantigens, we have assessed the surface and functional phenotype of CD8 cells in vivo (e.g., clearance of allogeneic P815 cells) and in vitro. In our CD4-deficient GK mice, CTL responses to allogeneic P815 cells were induced, albeit delayed, and were sufficient to eliminate P815 cells. Induction of CTL and elimination of allogeneic P815 cells were inhibited both in the presence and absence of CD4 cells by temporary CD40 ligand blockade. This indicated that direct interaction of CD40/CD40L between APCs and CD8 cells may be an accessory signal in CTL induction (as well as the indirect pathway via APC/CD4 interaction). Furthermore, whereas in CTLA4Ig single Tg mice P815 cells were rejected promptly, in the double Tg GK/CTLA4Ig mice CTL were not induced and allogeneic P815 cells were not rejected. These findings suggest that CD40/CD40L is involved in both CD4-dependent and CD4-independent pathways, and that B7/CD28 is pivotal in the CD4-independent pathway of CTL induction against allogeneic P815 cells.
Conventional, Naive CD4+ T Cells Provide an Initial Source of IL-4 During Th2 Differentiation
Authors: Noben-Trauth Nancy , Hu-Li Jane , Paul William E. 
Published: April 2014

AbstractIL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor α-chain (IL-4Rα−/−) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Rα−/−mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Rα−/− DO11.10/scid mice secreted ∼50 pg of IL-4 per106 cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-γ and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Rα−/− mice gave rise to a minor proportion (∼2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-γ and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-γ and IL-12, this IL-4 can induce Th2 polarization.
Differential Survival of Naive CD4 and CD8 T Cells
Authors: Ferreira Cristina , Barthlott Thomas , Garcia Sylvie , Zamoyska Rose , Stockinger Brigitta 
Published: April 2014

AbstractIn this paper we compare survival characteristics of transgenic and polyclonal CD4 and CD8 T cells. Transgenic CD4 T cells have an intrinsically lower capacity for survival, reflected in their gradual disappearance in thymectomized hosts, their increased sensitivity to apoptosis in vitro, and fewer divisions during homeostatic proliferation upon transfer into syngeneic lymphopenic hosts compared with CD8 T cells. Homeostatic proliferation, however, does not generally result in phenotypic conversion of activation markers unless cognate or cross-reactive Ag is present. T cells from the A18 TCR transgenic strain normally selected into the CD4 lineage are fragile as CD4 T cells, yet display the typical robust survival pattern of CD8 T cells when diverted into the CD8 lineage in a CD4-deficient host. Polyclonal CD4 and CD8 T cells also show distinctive patterns of survival, emphasizing that survival signals are relayed differently in the two lymphocyte subpopulations. However, expression levels of Bcl-2 in either transgenic or polyclonal naive CD4 and CD8 T cells are similar, excluding a role for this molecule as a key factor in differential survival of CD4 vs CD8 T cells.
Development of Dendritic Epidermal T Cells with a Skewed Diversity of γδTCRs in Vδ1-Deficient Mice
Authors: Hara Hiromitsu , Kishihara Kenji , Matsuzaki Goro , Takimoto Hiroaki , Tsukiyama Tadasuke , Tigelaar Robert E. , Nomoto Kikuo 
Published: April 2014

AbstractOne of the most intriguing features of γδ T cells that reside in murine epithelia is the association of a specific Vγ/Vδ usage with each epithelial tissue. Dendritic epidermal T cells (DETCs) in the murine epidermis, are predominantly derived from the “first wave” Vγ5+ fetal thymocytes and overwhelmingly express the canonical Vγ5/Vδ1-TCRs lacking junctional diversity. Targeted disruption of the Vδ1 gene resulted in a markedly impaired development of Vγ5+ fetal thymocytes as precursors of DETCs; however, γδTCR+ DETCs with a typical dendritic morphology were observed in Vδ1−/− mice and their cell densities in the epidermis were slightly lower than those in Vδ1+/− epidermis. Moreover, the Vδ1-deficient DETCs were functionally competent in their ability to up-regulate cytokines and keratinocyte growth factor-expression in response to keratinocytes. Vγ5+ DETCs were predominant in the Vδ1−/− epidermis, though Vγ5− γδTCR+ DETCs were also detected. The Vγ5+ DETCs showed a typical dendritic shape, γδTCRhigh, and age-associated expansion in epidermis as observed in conventional DETCs of normal mice, whereas the Vγ5− γδTCR+ DETCs showed a less dendritic shape, γδTCRlow, and no expansion in the epidermis, consistent with their immaturity. These results suggest that optimal DETC development does not require a particular Vγ/Vδ-chain usage but requires expression of a limited diversity of γδTCRs, which allow DETC precursors to mature and expand within the epidermal microenvironment.
Thymocyte Maturation: Selection for In-Frame TCR α-Chain Rearrangement Is Followed by Selection for Shorter TCR β-Chain Complementarity-Determining Region 3
Authors: Yassai Maryam , Gorski Jack 
Published: April 2014

AbstractThymocyte maturation consists of a number of stages, the goal of which is the production of functioning T cells that respond to foreign antigenic peptides using their clonotypic receptors. Selection of a productively rearranged TCR β-chain is the first stage in the process and occurs at the double-negative to double-positive (DP) transition. Later maturation stages are based on changes in markers such as CD5, CD69, or IL-7R. A stage in which α-chains are selected has also been identified using β-chain transgenic mice. Here we identify two additional selection stages in human thymocytes based on characteristics of the TCR. α selection is measured directly by identification of in-frame rearrangements and is associated with the appearance of CD3 on the DP thymocyte surface. The next stage has not yet been described and involves selection of thymocytes that express shorter TCR β-chain complementarity-determining region 3 (CDR3). This stage is associated with the acquisition of high levels of CDR3 by DP cells and the transition to SP thymocytes. The extent of CDR3 length selection observed is a function of the TCR V and J genes. We propose that CDR3 length selection is based on recognition of the MHC. Thus, there exist limitations on the allowable length of that portion of the TCR most intimately in contact with MHC and peptide. This may be a physical representation of positive selection.
Activation of Toll-Like Receptor 2 on Human Dendritic Cells Triggers Induction of IL-12, But Not IL-10
Authors: Thoma-Uszynski Sybille , Kiertscher Sylvia M. , Ochoa Maria Teresa , Bouis Deborah A. , Norgard Michael V. , Miyake Kensuke , Godowski Paul J. , Roth Michael D. , Modlin Robert L. 
Published: April 2014

AbstractMammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1a+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.
CD28 Utilizes Vav-1 to Enhance TCR-Proximal Signaling and NF-AT Activation
Authors: Michel Frédérique , Mangino Giorgio , Attal-Bonnefoy Géraldine , Tuosto Loretta , Alcover Andrés , Roumier Anne , Olive Daniel , Acuto Oreste 
Published: April 2014

AbstractThe mechanism through which CD28 costimulation potentiates TCR-driven gene expression is still not clearly defined. Vav-1, an exchange factor for Rho GTPases thought to regulate, mainly through Rac-1, various signaling components leading to cytokine gene expression, is tyrosine phosphorylated upon CD28 engagement. Here, we provide evidence for a key role of Vav-1 in CD28-mediated signaling. Overexpression of Vav-1 in Jurkat cells in combination with CD28 ligation strongly reduced the concentration of staphylococcus enterotoxin E/MHC required for TCR-induced NF-AT activation. Surprisingly, upon Vav-1 overexpression CD28 ligation sufficed to activate NF-AT in the absence of TCR engagement. This effect was not mediated by overexpression of ZAP-70 nor of SLP-76 but necessitated the intracellular tail of CD28, the intactness of the TCR-proximal signaling cascade, the Src-homology domain 2 (SH2) domain of Vav-1, and SLP-76 phosphorylation, an event which was favored by Vav-1 itself. Cells overexpressing Vav-1 formed lamellipodia and microspikes reminiscent of Rac-1 and Cdc42 activation, respectively, for which the SH2 domain of Vav-1 was dispensable. Together, these data suggest that CD28 engagement activates Vav-1 to boost TCR signals through a synergistic cooperation between Vav-1 and SLP-76 and probably via cortical actin changes to facilitate the organization of a signaling zone.
Conformational Epitopes on the Diabetes Autoantigen GAD65 Identified by Peptide Phage Display and Molecular Modeling
Authors: Myers Mark A. , Davies Janet M. , Tong Jonathan C. , Whisstock James , Scealy Marita , Mackay Ian R. , Rowley Merrill J. 
Published: April 2014

AbstractThe major diabetes autoantigen, glutamic acid decarboxylase (GAD65), contains a region of sequence similarity, including six identical residues PEVKEK, to the P2C protein of coxsackie B virus, suggesting that cross-reactivity between coxsackie B virus and GAD65 can initiate autoimmune diabetes. We used the human islet cell mAbs MICA3 and MICA4 to identify the Ab epitopes of GAD65 by screening phage-displayed random peptide libraries. The identified peptide sequences could be mapped to a homology model of the pyridoxal phosphate (PLP) binding domain of GAD65. For MICA3, a surface loop containing the sequence PEVKEK and two adjacent exposed helixes were identified in the PLP binding domain as well as a region of the C terminus of GAD65 that has previously been identified as critical for MICA3 binding. To confirm that the loop containing the PEVKEK sequence contributes to the MICA3 epitope, this loop was deleted by mutagenesis. This reduced binding of MICA3 by 70%. Peptide sequences selected using MICA4 were rich in basic or hydroxyl-containing amino acids, and the surface of the GAD65 PLP-binding domain surrounding Lys358, which is known to be critical for MICA4 binding, was likewise rich in these amino acids. Also, the two phage most reactive with MICA4 encoded the motif VALxG, and the reverse of this sequence, LAV, was located in this same region. Thus, we have defined the MICA3 and MICA4 epitopes on GAD65 using the combination of phage display, molecular modeling, and mutagenesis and have provided compelling evidence for the involvement of the PEVKEK loop in the MICA3 epitope.
Mechanism of IL-4-Mediated Up-Regulation of the Polymeric Ig Receptor: Role of STAT6 in Cell Type-Specific Delayed Transcriptional Response
Authors: Schjerven Hilde , Brandtzaeg Per , Johansen Finn-Eirik 
Published: April 2014

AbstractThe polymeric IgR (pIgR) mediates transport of dimeric IgA and pentameric IgM across mucosal epithelia, thereby generating secretory Abs. Its expression is up-regulated at the transcriptional level by IL-4 in HT-29 cells. In this study, we demonstrate that IL-4 mediates up-regulation of human pIgR through a 554-bp IL-4-responsive enhancer in intron 1. Mutation of a binding site for STAT-6 within this region abolished IL-4-induced enhancement, while an adjacent putative C/EBP site was dispensable. IL-4 treatment induced binding of STAT6 to the intronic STAT6 site, but cooperation with nearby upstream and downstream DNA elements was required for IL-4 responsiveness. Furthermore, IL-4-mediated increased transcription of the pIgR-derived enhancer, like the endogenous pIgR gene, required de novo protein synthesis. Interestingly, a conditionally active form of STAT6 sufficed to activate a pIgR-derived enhancer in HT-29 cells, but not in Cos-1 cells, suggesting a requirement for cell type-specific factors. Thus, STAT6 activation mediates a delayed transcriptional enhancement of pIgR by induction of a de novo synthesized protein that cooperates with STAT6 itself bound to its cognate DNA element in intron 1. This mechanism may represent a general strategy for how pleiotropic cytokines elicit cell type-specific transcriptional responses.
Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-α
Authors: Giroux Mélanie , Descoteaux Albert 
Published: April 2014

AbstractCyclooxygenase-2 (COX-2) is an inducible enzyme responsible for high levels of PG production during inflammation and immune responses. Previous studies with pharmacological inhibitors suggested a role for protein kinase C (PKC) in PG production possibly by regulating COX-2 expression. In this study, we addressed the role of PKC-α in the modulation of COX-2 expression and PGE2 synthesis by the overexpressing of a dominant-negative (DN) mutant of this isoenzyme in the mouse macrophage cell line RAW 264.7. We investigated the effect of various stimuli on COX-2 expression, namely, LPS, IFN-γ, and the intracellular parasite Leishmania donovani. Whereas LPS-induced COX-2 mRNA and protein expression were down-regulated in DN PKC-α-overexpressing clones, IFN-γ-induced COX-2 expression was up-regulated in DN PKC-α-overexpressing clones with respect to normal RAW 264.7 cells. Measurements of PGE2 levels revealed a strong correlation between PGE2 secretion and IFN-γ-induced COX-2 mRNA and protein levels in DN PKC-α-overexpressing clones. Taken together, these results suggest a role for PKC-α in the modulation of LPS- and IFN-γ-induced COX-2 expression, as well as in IFN-γ-induced PGE2 secretion.
Activated Murine Endothelial Cells Have Reduced Immunogenicity for CD8+ T Cells: A Mechanism of Immunoregulation?
Authors: Marelli-Berg Federica M. , Scott Diane , Bartok Istvan , Peek Emma , Dyson Julian , Lechler Robert I. 
Published: April 2014

AbstractThe immunogenic properties of primary cultures of murine lung microvascular endothelial cells (EC) were analyzed. Resting endothelial cells were found to constitutively express low levels of MHC class I and CD80 molecules. IFN-γ treatment of EC resulted in a marked up-regulation of MHC class I, but no change was observed in the level of CD80 expression. No CD86 molecules were detectable under either condition. The ability of peptide-pulsed EC to induce the proliferation of either the HY-specific, H2-Kk-restricted CD8+ T cell clone (C6) or C6 TCR-transgenic naive CD8+ T cells was analyzed. Resting T cells were stimulated to divide by quiescent peptide-prepulsed EC, while peptide-pulsed, cytokine-activated EC lost the ability to induce T cell division. Furthermore, Ag presentation by cytokine-activated EC induced CD8+ T cell hyporesponsiveness. The immunogenicity of activated EC could be restored by adding nonsaturating concentrations of anti-H2-Kk Ab in the presence of an optimal concentration of cognate peptide. This is consistent with the suggestion that the ratio of TCR engagement to costimulation determines the outcome of T cell recognition. In contrast, activated peptide-pulsed EC were killed more efficiently by fully differentiated effector CD8+ T cells. Finally, evidence is provided that Ag recognition of EC can profoundly affect the transendothelial migration of CD8+ T cells. Taken together, these results suggest that EC immunogenicity is regulated in a manner that contributes to peripheral tolerance.
A Physiological Ligand of Positive Selection Is Recognized as a Weak Agonist
Authors: Berg Rance E. , Irion Stefan , Kattman Steve , Princiotta Michael F. , Staerz Uwe D. 
Published: April 2014

AbstractPositive selection is a process that ensures that peripheral T cells express TCR that are self-MHC restricted. This process occurs in the thymus and requires both self-MHC and self-peptides. We have recently established a TCR transgenic (TCRtrans+) mouse model using the C10.4 TCR restricted to the MHC class Ib molecule, H2-M3. Having defined H2-M3 as the positively selecting MHC molecule, the severely limited number of H2-M3 binding peptides allowed us to characterize a mitochondrial NADH dehydrogenase subunit 1-derived 9-mer peptide as the physiological ligand of positive selection. Here, we demonstrate that the NADH dehydrogenase subunit 1 self-peptide is seen by mature C10.4 TCRtrans+ T cells as a weak agonist and induces positive selection at a defined concentration range. We also found that the full-length cognate peptide, a strong agonist for mature C10.4 TCRtrans+ T cells, initiated positive selection, albeit at significantly lower concentrations. At increased peptide concentrations, and thus increased epitope densities, either peptide only induced the development of partially functional T cells. We conclude that successful positive selection only proceeded at a defined, yet fairly narrow window of avidity.
Depletion of CD4 and CD8 T Lymphocytes in Mice In Vivo Enhances 1,25-Dihydroxyvitamin D3-Stimulated Osteoclast-Like Cell Formation In Vitro by a Mechanism That Is Dependent on Prostaglandin Synthesis
Authors: Grčević Danka , Lee Sun-Kyeong , Marušić Ana , Lorenzo Joseph A. 
Published: April 2014

AbstractTo investigate the role of T lymphocytes in osteoclastogenesis, we performed in vivo depletion of CD4 and/or CD8 T lymphocyte subsets and evaluated in vitro osteoclast-like cell (OCL) formation. T lymphocyte depletion (TLD) with mAbs was confirmed 24 h later by flow cytometry. OCL formation was stimulated with 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in bone marrow and with recombinant mouse (rm) receptor activator of NF-κB ligand (RANK-L) and rmM-CSF in bone marrow and spleen cell cultures. OCL formation was up to 2-fold greater in 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice than in those from intact mice. In contrast, TLD did not alter OCL formation in bone marrow or spleen cell cultures that were stimulated with rmRANK-L and rmM-CSF. The effects of TLD seemed to be mediated by enhanced PG synthesis, because the PGE2 concentration in the medium of 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice was 5-fold higher than that in cultures from intact mice, and indomethacin treatment abolished the stimulatory effect of TLD on OCL formation. There was a 2-fold increase in RANK-L expression and an almost complete suppression of osteoprotegerin expression in 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice compared with those from intact mice. Although there was a small (20%) increase in IL-1α expression in 1,25-(OH)2D3-stimulated bone marrow cultures from TLD mice, TLD in mice lacking type I IL-1R and wild-type mice produced similar effects on OCL formation. Our data demonstrate that TLD up-regulates OCL formation in vitro by increasing PG production, which, in turn, produces reciprocal changes in RANK-L and osteoprotegerin expression. These results suggest that T lymphocytes influence osteoclastogenesis by altering bone marrow stromal cell function.