bluuebunny/crossref_metadata_2025_split huggingface dataset:
Syk- and Lyn-Dependent Phosphorylation of Syk on Multiple Tyrosines Following B Cell Activation Includes a Site That Negatively Regulates Signaling
Authors: Keshvara Lakhu M. , Isaacson Christina C. , Yankee Thomas M. , Sarac Radmila , Harrison Marietta L. , Geahlen Robert L. 
Published: December 2022

Abstract
The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-SH2 domain tyrosines is catalyzed primarily by Syk itself, but only occurs to an appreciable extent in cells that express Lyn. We propose that these sites are phosphorylated following the binding of Syk to immunoreceptor tyrosine-based activation motif.
Impaired Cutaneous Immune Responses in Thy-1-Deficient Mice
Authors: Beissert Stefan , He Hai-Tao , Hueber Anne-Odile , Lellouch Annemarie C. , Metze Dieter , Mehling Annette , Luger Thomas A. , Schwarz Thomas , Grabbe Stephan 
Published: December 2022

AbstractThy-1 is a cell surface glycoprotein expressed mainly on brain and lymphoid tissue. Although the functions of Thy-1 are incompletely understood, evidence exists that Thy-1 participates in T cell activation. To examine the functional role of Thy-1 in cutaneous immune responses in vivo, Thy-1 gene-targeted mice (Thy-1−/−) and wild-type mice (Thy-1+/+) were immunized with the hapten oxazolone. After challenge with oxazolone, contact hypersensitivity responses in Thy-1−/− mice were reduced by 25% compared with Thy-1+/+ mice. Likewise, irritant dermatitis induced by croton oil was also decreased. In addition, Thy-1−/− mice showed a significantly reduced delayed-type hypersensitivity response after injection of allogeneic spleen cells into the hind footpads of allosensitized animals when compared with Thy-1+/+ mice. Moreover, proliferative responses to immobilized anti-CD3 were decreased in peripheral Thy-1−/− lymphocytes; this decrease was associated with a significantly reduced intracellular Ca2+ influx and protein tyrosine phosphorylation, indicating impairment of early lymphocyte activation. In contrast, the T cell proliferation induced by mitogens was normal, suggesting that Thy-1 expression weakly contributes to TCR-mediated T cell activation. Epidermal Langerhans cells and bone marrow-derived dendritic cells from Thy-1−/− mice exhibited a normal expression of costimulatory surface molecules as well as an unaltered ability to stimulate allogeneic T cells. Taken together, these findings demonstrate that a lack of Thy-1 expression does not generally compromise the immune system; however, Thy-1 expression may be involved in the fine-tuning of T cell-mediated immune responses.
Induction and Inhibition of CD40-CD40 Ligand Interactions: A New Strategy Underlying Host-Virus Relationships
Authors: Sharma Madhav D. , Leite de Moraes Maria , Zavala Flora , Pontoux Christiane , Papiernik Martine 
Published: December 2022

AbstractInteraction between CD40 and the CD40 ligand (CD40L) is required for mouse mammary tumor virus (MMTV) propagation. We found that Fas was expressed on B cells and CD40L on a small subset of viral superantigen-cognate T cells 12 h after MMTV(SW) infection. CD40L and Fas were down-regulated after 24 h. All CD4 T cells then became resistant to anti-CD3-induced CD40L induction in vitro for 2 wk. Initiation of CD40L expression and its rapid shut-off was associated with IL-12 production and was controlled by IFN-γ and shedding of soluble CD40. These results suggest that a rapid, transient CD40-CD40L interaction involving a small number of cells is sufficient for MMTV propagation. Modulation of CD40L expression may be a major mechanism regulating the balance between viral propagation and host defenses, allowing mutual survival.
Attenuation of Inducible Th2 Immunity with Autoimmune Disease Progression
Authors: Tian Jide , Kaufman Daniel L. 
Published: December 2022

Abstract
Autoantigen-based immunotherapeutics have been shown to activate regulatory responses capable of inhibiting T cell-mediated autoimmune disease in animal models. However, their efficacy generally declines, as treatment occurs later in the disease process, and their mechanism of action is a matter of intense debate. Here, we report that the early administration of β cell autoantigens (βCAAs) to nonobese diabetic (NOD) mice broadly diverts the natural development of potentially pathogenic Th1-biased autoimmune responses toward the Th2 phenotype through Th2 spreading. With disease progression, there was a steady decline in the ability of βCAA treatment to promote Th2-type cellular and humoral autoimmunity. Late in the disease process, some βCAAs were still able to induce Th2 responses and Th2 spreading (although to a much lesser extent), while other autoantigens were not. This attenuation of inducible Th2 immunity with disease progression is likely to reflect a reduction in the availability of uncommitted autoantigen-reactive precursor T cells. These findings suggest that there are inherent differences in the frequency of βCAA-reactive T cells and that, in advanced stages of autoimmune disease, regulatory responses may be best elicited with target tissue Ags against which large uncommitted T cell pools are still available. Since individuals presenting the first signs of autoimmune disease are likely to already have an advanced disease process, these findings may be useful for the rational design of Ag-based immunotherapeutics.
Phosphatidylinositol 3-Kinase Is Required for CD28 But Not CD3 Regulation of the TEC Family Tyrosine Kinase EMT/ITK/TSK: Functional and Physical Interaction of EMT with Phosphatidylinositol 3-Kinase
Authors: Lu Yiling , Cuevas Bruce , Gibson Spencer , Khan Humera , LaPushin Ruth , Imboden John , Mills Gordon B. 
Published: December 2022

AbstractLigation of the TCR or CD28 induces activation of phosphatidylinositol 3-kinase (PI3K), the TEC family protein tyrosine kinase, EMT/ITK/TSK (EMT), and the SRC family tyrosine kinase, LCK. LCK is required for the activation and phosphorylation of EMT induced by ligation of the TCR or CD28 placing LCK upstream of EMT in T cell signaling cascades. We report herein that inhibition of PI3K activity with the specific inhibitors LY294002 and wortmannin markedly decreased EMT activation induced by CD28 cross-linking but not by CD3 cross-linking. Further, inhibition of PI3K markedly decreased EMT in vitro autokinase activity induced by activated LCK. In contrast, PI3K inhibitors did not alter CD28 or CD3 cross-linking or LCK-induced EMT phosphorylation. Consistent with the requirement of PI3K activity for CD28 but not CD3-induced stimulation of the EMT in vitro autokinase activity, a small but significant portion of cellular EMT associates with PI3K following CD28 cross-linking but not following CD3 cross-linking. CD28-induced association of EMT with PI3K also requires functional expression of LCK. Fusion proteins containing the SRC homology 2 domain of EMT interact with PI3K or a PI3K-associated molecule in a tyrosine phosphorylation-dependent manner. Taken together, the data suggest that EMT is differentially regulated and recruited to different signaling complexes following ligation of CD28 or the TCR complex, perhaps contributing to the disparate roles that EMT appears to play downstream of CD28 and the TCR.
Secretory Component Delays the Conversion of Secretory IgA into Antigen-Binding Competent F(ab′)2: A Possible Implication for Mucosal Defense
Authors: Crottet Pascal , Corthésy Blaise 
Published: December 2022

AbstractSecretory component (SC) represents the soluble ectodomain of the polymeric Ig receptor, a membrane protein that transports mucosal Abs across epithelial cells. In the protease-rich environment of the intestine, SC is thought to stabilize the associated IgA by unestablished molecular mechanisms. To address this question, we reconstituted SC-IgA complexes in vitro by incubating dimeric IgA (IgAd) with either recombinant human SC (rSC) or SC isolated from human colostral milk (SCm). Both complexes exhibited an identical degree of covalency when exposed to redox agents, peptidyl disulfide isomerase, and temperature changes. In cross-competition experiments, 50% inhibition of binding to IgAd was achieved at ∼10 nM SC competitor. Western blot analysis of IgAd digested with intestinal washes indicated that the α-chain in IgAd was primarily split into a 40-kDa species, a phenomenon delayed in rSC- or SCm-IgAd complexes. In the same assay, either of the SCs was resistant to degradation only if complexed with IgAd. In contrast, the κ light chain was not digested at all, suggesting that the F(ab′)2 region was left intact. Accordingly, IgAd and SC-IgAd digestion products retained functionality as indicated by Ag reactivity in ELISA. Size exclusion chromatography under native conditions of digested IgAd and rSC-IgAd demonstrates that SC exerts its protective role in secretory IgA by delaying cleavage in the hinge/Fc region of the α-chain, not by holding together degraded fragments. The function of integral secretory IgA and F(ab′)2 is discussed in terms of mucosal immune defenses.
Alterations in TCR-MHC Contacts Subsequent to Cross-Recognition of Class I MHC and Singly Substituted Peptide Variants
Authors: Ono Toshiro , DiLorenzo Teresa P. , Wang Fuming , Kalergis Alexis M. , Nathenson Stanley G. 
Published: December 2022

AbstractVesicular stomatitis virus (VSV) elicits H-2Kb-restricted CTLs specific for the immunodominant VSV octapeptide RGYVYQGL. To study the structural features important for interaction between the TCR β-chain and the peptide/MHC complex, we immunized TCR α-chain transgenic mice with the VSV peptide and raised a panel of anti-VSV CTL clones with identical TCR α-chains. Consistent with our previous analysis of uncloned populations of primary CTLs, the anti-VSV CTL clones were all Vβ13+ and expressed TCR β-chains with highly homologous complementarity-determining region 3 (CDR3) loops. Although the clones expressed similar TCRs, they differed in their ability to cross-react with VSV peptide variants singly substituted at TCR contact positions 4 and 6. These findings allowed us to identify short stretches of amino acids in the C-terminal region of the CDR3β loop that, when altered, modify the cross-reaction capability of the TCR to position 4 and position 6 variant peptides. To further probe the structural correlates of biologic cross-reactivity, we used cross-reactive CTL clones and cell lines expressing point mutations in H-2Kb to investigate the effect of single amino acid changes in the peptide on the pattern of recognition of the TCR for the peptide/MHC complex. Single conservative substitutions in the peptide were sufficient to alter the recognition contacts between a cross-reactive TCR and the MHC molecule, supporting the idea that the TCR can make overall structural adjustments in MHC contacts to accommodate single amino acid changes in the peptide.
The Charge of Endotoxin Molecules Influences Their Conformation and IL-6-Inducing Capacity
Authors: Schromm Andra B. , Brandenburg Klaus , Loppnow Harald , Zähringer Ulrich , Rietschel Ernst Th. , Carroll Stephen F. , Koch Michel H. J. , Kusumoto Shoichi , Seydel Ulrich 
Published: December 2022

AbstractThe activation of cells by endotoxin (LPS) is one of the early host responses to infections with Gram-negative bacteria. The lipid A part of LPS molecules is known to represent the endotoxic principle; however, the specific requirements for the expression of biologic activity are still not fully understood. We previously found that a specific molecular conformation (endotoxic conformation) is a prerequisite for lipid A to be biologically active. In this study, we have investigated the interdependence of molecular charge and conformation of natural and chemically modified LPS and lipid A and its transport and intercalation into phospholipid membranes mediated by human LPS-binding protein, as well as IL-6 production after stimulation of whole blood or PBMCs. We found that the number, nature, and location of negative charges strongly modulate the molecular conformation of endotoxin. In addition, the LPS-binding protein-mediated transport of LPS into phospholipid membranes depends on the presence of net negative charge, yet charge is only a necessary, but not a sufficient, prerequisite for transport and intercalation. The biologic activity is determined mainly by the molecular conformation: only conical molecules are highly biologically active, whereas cylindrical ones are largely inactive. We could demonstrate that the net negative charge of the lipid A component and its distribution within the hydrophilic headgroup strongly influence the molecular conformation and, therefore, also the biologic activity.
A Site for CD4 Binding in the β1 Domain of the MHC Class II Protein HLA-DR1
Authors: Brogdon Jennifer , Eckels David D. , Davies Christopher , White Stephen , Doyle Carolyn 
Published: December 2022

AbstractUsing a lymphocyte binding assay, we have previously demonstrated that the CD4 protein can mediate cell adhesion by direct interaction with MHC class II molecules. In this report, we have used this assay to test whether synthetic peptides, corresponding to DRβ sequences, could inhibit CD4-class II adhesion. A peptide derived from sequences within the β1 domain (DRβ41–55), as well as two peptides derived from sequences within the β2 domain (DRβ121–135 and DRβ141–155), were shown to inhibit CD4-class II adhesion. Inasmuch as a site for CD4 binding in the β2 domain had been previously documented, these studies were designed to investigate the role of the β1 domain as an additional site of interaction with CD4. Sixteen site-specific mutations were engineered within the β1 domain of DRβ1*0101. Several mutations were shown to disrupt CD4-dependent T cell activation. Based on these results, we propose a model for the molecular interaction of CD4 with MHC class II proteins in which both the β1 and β2 domains of class II interact with the two amino-terminal Ig-like domains of CD4.
IL-12 Enhances Antibody Responses to T-Independent Polysaccharide Vaccines in the Absence of T and NK Cells
Authors: Buchanan Renee M. , Arulanandam Bernard P. , Metzger Dennis W. 
Published: December 2022

AbstractPolysaccharide vaccines to encapsulated bacteria such as Neisseria meningitidis and Streptococcus pneumoniae are weakly immunogenic due to their T-independent (TI) nature. Even when converted to T-dependent forms through conjugation to foreign proteins, polysaccharides induce responses that are deficient in many respects, such as induction of murine IgG2a Ab, the isotype that mediates optimal complement fixation and opsonization. We now show that IL-12 treatment of mice induces significantly increased levels of IgG2a Ab to the model TI-2 Ag, DNP-Ficoll, and to vaccines composed of polysaccharides from pneumococci and meningococci. Use of immunodeficient mice lacking T cells and/or NK cells demonstrated that such cells were not responsible for the observed Ab enhancement. Furthermore, the use of IFN-γ knockout mice showed that stimulation of TI-2 Ab responses by IL-12 was only partially dependent on IFN-γ. The ability of IL-12 to dramatically enhance TI Ab responses suggests that IL-12 will be useful as a powerful vaccine adjuvant to induce protective immune responses against encapsulated pathogens.
Bacterial Lipopolysaccharide Can Enter Monocytes Via Two CD14-Dependent Pathways
Authors: Kitchens Richard L. , Wang Ping-yuan , Munford Robert S. 
Published: December 2022

AbstractHost recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.
Regulation of Hepatitis B Virus mRNA Expression in a Hepatitis B Surface Antigen Transgenic Mouse Model by IFN-γ-Secreting T Cells After DNA-Based Immunization
Authors: Mancini Maryline , Hadchouel Michelle , Tiollais Pierre , Michel Marie-Louise 
Published: December 2022

AbstractThe immunotherapeutic effect of DNA-mediated immunization against chronic hepatitis B virus (HBV) infection has been evaluated in transgenic mice expressing the sequences that code for the envelope proteins of HBV in the liver. In this model of HBV chronic carriers, a single i.m. injection of plasmid DNA encoding HBV envelope proteins is sufficient to generate specific immune responses leading to the clearance of the transgene expression product and the control of HBV mRNA. The relative contributions of the T cell subpopulations induced by DNA immunization were examined using adoptive transfer experiments. It was shown that either CD8+ or CD4+ T lymphocytes from immunocompetent DNA-immunized animals were sufficient to control viral gene expression in the livers of the recipient transgenic mice. This effect was mediated by a cytokine-dependent mechanism common to both T cell subpopulations; this mechanism did not require cell lysis, but did involve the production of IFN-γ by the activated T cells.
Evaluation of IL-12 in Immunotherapy and Vaccine Design in ExperimentalMycobacterium aviumInfections
Authors: Silva Regina A. , Pais Teresa F. , Appelberg Rui 
Published: December 2022

AbstractIL-12 is a pivotal cytokine in the induction of IFN-γ-mediated protective immune responses. We tested the effects of rIL-12 administration to Mycobacterium avium-infected mice and found a limited ability to induce protection against the infection; this ability varied according to the mycobacterial strain studied. IL-12 accelerated the expression and production of IFN-γ in both immunocompetent and immunodeficient SCID or CD4-depleted mice. Evidence of NK cell activation was found as well as an enhancement of the ability to adoptively transfer resistance with T cell-enriched spleen cell populations and an increase in inflammatory cell recruitment in the liver. The protective ability of IL-12 was dependent upon the endogenous production of IFN-γ as evaluated by the use of specific neutralizing Abs or IFN-γ gene-disrupted mice. IL-12 potentiated the protective immunity conferred by a subunit vaccine containing M. avium culture filtrate proteins and dimethyl dioctadecyl ammonium chloride as an adjuvant. Thus, we show limited immunotherapeutic benefits from IL-12 administration in M. avium infections and promising results in its use as a coadjuvant in vaccine design.
Staphylococcal Enterotoxin A-Induced Injury of Human Lung Endothelial Cells and IL-8 Accumulation Are Mediated by TNF-α
Authors: Fujisawa Nobumitsu , Hayashi Shinichiro , Kurdowska Anna , Noble James M. , Naitoh Keiko , Miller Edmund J. 
Published: December 2022

AbstractStaphylococcal enterotoxin A (SEA), a superantigen produced by some strains of Staphylococcus aureus, causes a variety of clinical manifestations ranging from food poisoning to shock. S. aureus can also be associated with the development of acute respiratory distress syndrome, and SEA has been shown to cause an inflammatory reaction in the lung. Therefore, we examined possible interactions between SEA, PBMCs, polymorphonuclear cells (PMNs), and normal human lung microvascular endothelial cells (HMVEC-L), as well as the role of these interactions on the secretion of IL-8. Injury to HMVEC-L, as measured by the release of 51Cr, increased significantly when HMVEC-L were incubated with SEA and PBMCs. IL-8 was secreted by both PBMCs and HMVEC-L. The accumulation of IL-8 in the culture medium of HMVEC-L was increased by SEA in a dose-dependent manner and was directly related to the number of PBMCs present. Although neither anti-human IL-8 nor IL-1 mAb inhibited HMVEC-L cytotoxicity, anti-human TNF-α mAb inhibited both the cytotoxicity and IL-8 accumulation completely. When HMVEC-L were incubated with supernatants from SEA-treated PBMCs, HMVEC-L cytotoxicity was comparable with HMVEC-L incubated with SEA and PBMCs at the same time. Although high concentrations of purified PMNs induced HMVEC-L lysis in a dose-dependent manner, the effect of PMNs was not changed in the presence of SEA. These findings suggest that TNF-α secreted by SEA-stimulated PBMCs plays a leading role in HMVEC-L injury.