bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pasteurella multocida Toxin Increases Endothelial Permeability via Rho Kinase and Myosin Light Chain Phosphatase
Authors: Essler Markus , Hermann Karin , Amano Mutsuki , Kaibuchi Kozo , Heesemann Jürgen , Weber Peter C. , Aepfelbacher Martin 
Published: December 2022

Abstract
Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho. Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells. Stimulation of endothelial layers with PMT enhanced transendothelial permeability &gt;10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum. The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation. PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase. Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase. The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.
High Titer, Prostate Specific Antigen-Specific Human IgG Production byhu-PBL-SCID Mice Immunized with Antigen-Mouse IgG2a Complex-Pulsed Autologous Dendritic Cells
Authors: Coccia Marco A. , Brams Peter 
Published: December 2022

AbstractWe report here that immunization of human PBMC reconstituted SCID mice (hu-PBL-SCID mice) with in vitro cultured autologous dendritic cells (DC) pulsed with prostate specific antigen (PSA) complexed to a PSA-specific mouse IgG2a (PSA-IgG2a) consistently and reproducibly stimulates PSA-specific human IgG production. On day 0, female PBMC were used to reconstitute SCID mice and to generate DC in vitro. DC cultures were pulsed with PSA or PSA-IgG2a on day 6. The previously reconstituted hu-PBL-SCID mice were immunized with either PSA-pulsed DC and PSA, PSA-IgG2a-pulsed DC and PSA-IgG2a, or additional PBMC and PSA-IgG2a on day 7. Mice immunized with PSA-IgG2a-pulsed DC had, on the average, up to 31.5 times greater PSA-specific IgG serum concentrations than control mice. Competition ELISA confirmed the PSA specificity of serum IgG. Immunoblot analysis suggested that sera IgG preferentially recognized conformational epitopes on PSA. Therefore, our results represent a major step toward cloning human tumor-associated Ag-specific human mAbs from hu-PBL-SCID mice. In addition, flow cytometry showed that PSA-pulsed DC express significantly more B7.1, B7.2, CD40, and MHC class II surface molecules than mock-treated DC, but PSA-IgG2a-pulsed DC only had significantly enhanced B7.2 surface expression. Interestingly, PSA-specific IgG responses were reproducibly stimulated by DC expressing more B7.2, a molecule associated with Th2-type immune deviation, but not by those expressing more B7.1 and CD40, molecules associated with Th1-type immune deviation. Thus, our results show that stimulation with either Ag or Ag complexed to mAb yields DC with different phenotypes and APC effector functions.
TCR Transgenic Mice in Which Usage of Transgenic α- and β-Chains Is Highly Dependent on the Level of Selecting Ligand
Authors: Kersh Gilbert J. , Donermeyer David L. , Frederick Katherine E. , White J. Michael , Hsu Benjamin L. , Allen Paul M. 
Published: December 2022

AbstractWe have produced a TCR transgenic mouse that uses a TCR derived from a Th1 clone that is specific for residues 64 to 76 of the d allele of murine hemoglobin presented by I-Ek. Examination of these TCR transgenic mice on an H-2k/k background that expressed the nonstimulatory s allele of murine hemoglobin revealed that these mice express many endogenous TCR chains from both α and β loci. We found that this transgenic TCR is also very inefficient at mediating β selection, thereby showing a direct linkage between β selection and allelic exclusion of TCR β. We have also examined these mice on MHC backgrounds that have reduced levels of I-Ek and found that positive selection of cells with high levels of the transgenic TCR depends greatly on the ligand density. Decreasing the selecting ligand density is a means of reducing the number of available selecting niches, and the data reveal that the 3.L2 TCR is used sparingly for positive selection under conditions where the number of niches becomes limiting. The results, therefore, show a way that T cells may get to the periphery with two self-restricted TCRs: one that efficiently mediates positive selection, and another that is inefficient at positive selection with the available niches.
Isolation and Analysis of a T Cell Clone Variant Exhibiting Constitutively Phosphorylated Ser133 cAMP Response Element-Binding Protein
Authors: Belkowski Stanley M. , Rubin Charles S. , Prystowsky Michael B. 
Published: December 2022

Abstract
In driving T cell proliferation, IL-2 stimulates a new program of gene expression that includes proliferating cell nuclear antigen (PCNA), a requisite processivity factor for DNA polymerase δ. PCNA transcription is regulated in part through tandem CRE sequences in the promoter and CRE binding proteins; IL-2 stimulates CREB phosphorylation in the resting cloned T lymphocyte, L2. After culturing L2 cells for greater than 91 days, we consistently isolate a stable variant that exhibits constitutive CREB phosphorylation. L2 and L2 variant cells were tested for IL-2 responsiveness and rapamycin sensitivity with respect to specific kinase activity, PCNA expression and proliferation. In L2 cells, IL-2 stimulated and rapamycin inhibited the following: cAMP-independent CREB kinase activity, PCNA expression and proliferation. In L2 variant cells, CREB kinase activity was constitutively high; IL-2 stimulated and rapamycin blocked PCNA expression and proliferation. These results indicate that IL-2 induces a rapamycin-sensitive, cAMP-independent CREB kinase activity in L2 cells. However, phosphorylation of CREB alone is not sufficient to drive PCNA expression and L2 cell proliferation in the absence of IL-2.
The IL-1β-Converting Enzyme (Caspase-1) Inhibits Apoptosis of Inflammatory Neutrophils Through Activation of IL-1β
Authors: William R. , Watson G. , Rotstein Ori D. , Parodo Jean , Bitar Richard , Marshall John C. 
Published: December 2022

AbstractIL-1β-converting enzyme (ICE), also known as caspase-1, subserves two dichotomous biologic roles. It processes newly synthesized pro-IL-1β to yield the active cytokine and, as the human homologue of the Caenorhabditis elegans gene product, ced-3, it also induces cellular apoptosis through the cleavage of key intracellular structural and regulatory proteins and through the catalytic activation of other caspase family members. We show here that two different proinflammatory stimuli, LPS and granulocyte-macrophage-CSF, up-regulate the expression of both ICE and IL-1β in human polymorphonuclear neutrophils, and that the ICE-dependent cleavage of pro-IL-1β results in delayed expression of the constitutive cell death program. The apoptotic delay can be blocked by inhibiting tyrosine kinases or NF-κB activation and by inhibiting protein synthesis. Since an antisense oligonucleotide for IL-1β, a blocking Ab to IL-1β, and preincubation with the IL-1R antagonist all prevent the delay in apoptosis, we conclude that IL-1β acts in an autocrine manner to inhibit granulocyte programmed cell death. We conclude that caspase-1 (ICE) subserves both pro- and antiapoptotic roles; the latter role is evident during inflammation as an inhibition of spontaneous neutrophil apoptosis through the processing of IL-1β. The ICE-dependent activation of IL-1β may represent a common autocrine pathway for the divergent stimuli that inhibit the constitutive expression of neutrophil programmed cell death during inflammation.
Cutting Edge: A Newly Identified Response Element in the CD95 Ligand Promoter Contributes to Optimal Inducibility in Activated T Lymphocytes
Authors: Norian Lyse A. , Latinis Kevin M. , Koretzky Gary A. 
Published: December 2022

Abstract
Inducible expression of CD95 ligand on activated T lymphocytes contributes to both cytotoxic effector mechanisms and peripheral T cell homeostasis. To understand better the transcriptional events that regulate this expression, we have examined the CD95 ligand promoter to determine which regions are required for its induced activity following T cell stimulation. We report here the identification of a new response element within the promoter that is required for its optimal function in activated Jurkat T cells. This region is bound by proteins contained in nuclear extracts of activated, but not resting, T cells. Multimerization of this sequence independently drives transcription in response to T cell activation, while mutation of it substantially decreases inducible promoter activity. Finally, we provide evidence that T cell activation-induced transcription of the CD95 ligand gene is regulated coordinately by this response element together with two previously defined sites for nuclear factor of activated T cells (NFAT).
Termination of Peripheral Tolerance to a T Cell Epitope by Heteroclitic Antigen Analogues
Authors: Zügel Ulrich , Wang Rongfang , Shih Grace , Sette Alessandro , Alexander Jeff , Grey Howard M. 
Published: December 2022

AbstractTreating mice with an immunodominant T cell epitope from moth cytochrome c (MCC88–103) can induce T cell unresponsiveness under certain conditions of administration. In this report, we determined whether T cell tolerance to MCC88–103 in adult animals can be overcome by immunization with cross-reactive analogues of the tolerizing Ag. A panel of analogues of the tolerogen were tested for their capacity to terminate the tolerant state following in vivo immunization. As analyzed by their stimulatory capacity for a representative MCC88–103-specific T cell clone, this panel covered a wide range of cross-reactivity, including nonantigenic, antagonistic, weakly, and strongly antigenic peptides. Interestingly, only heteroclitic analogues, as measured in vitro by their enhanced antigenicity for the T cell clone that was specific for MCC88–103, were capable of breaking tolerance. Thus, an immune response to the cross-reactive, heteroclitic analogues of tolerized self Ags may represent a mechanism by which Ag molecular mimicry operates.
Multiple Signaling Pathways for the Activation of JNK in Mast Cells: Involvement of Bruton’s Tyrosine Kinase, Protein Kinase C, and JNK Kinases, SEK1 and MKK7
Authors: Kawakami Yuko , Hartman Stephen E. , Holland Pamela M. , Cooper Jonathan A. , Kawakami Toshiaki 
Published: December 2022

AbstractStimulation of the high affinity IgE receptor (FcεRI) as well as a variety of stresses induce activation of c-Jun N-terminal protein kinases (JNKs) stress-activated protein kinases in mast cells. At least three distinct signaling pathways leading to JNK activation have been delineated based on the involvements of Bruton’s tyrosine kinase (Btk), protein kinase C (PKC), and the JNK-activating cascades composed of multiple protein kinases. The PKC-dependent pathway, which is inhibited by a PKC inhibitor Ro31-8425 and can be activated by PMA, functions as a major route in FcεRI-stimulated mast cells derived from btk gene knockout mice. On the other hand, wild-type mouse-derived mast cells use both PKC-dependent and PKC-independent pathways for JNK activation. A PKC-independent pathway is regulated by Btk and SEK1 via the PAK→MEKK1→SEK1→JNK cascade, and is sensitive to phosphatidylinositol 3-kinase inhibitors, wortmannin and LY-294002, while the PKC-dependent pathway is affected to a lesser extent by both wortmannin treatment and overexpression of wild-type and dominant negative mutant SEK1 proteins. Another PKC-independent pathway involves Btk and MKK7, a recently cloned direct activator of JNK. Among the stresses tested, UV irradiation seems to activate Btk and JNK via the PKC-independent pathways.
Characterization of a Low Molecular Weight Isoform of IL-1 Receptor Antagonist
Authors: Malyak Mark , Guthridge Joel M. , Hance Kenneth R. , Dower Steven K. , Freed John H. , Arend William P. 
Published: December 2022

AbstractIL-1R antagonist (IL-1Ra) exists in two well-characterized forms, 17-kDa secretory IL-1Ra (sIL-1Ra) and 18-kDa intracellular IL-1Ra (icIL-1Ra), that arise by alternative transcription of the same IL-1Ra gene. A third, lower molecular mass form (∼16 kDa) was detected by immunoblot within lysates of a variety of cells, including human monocytes and myelomonocytic cell lines. The 16-kDa isoform was designated icIL-1RaII, and the previously established 18-kDa form was designated icIL-1RaI. Intracellular IL-1RaII bound type I IL-1R up to fivefold less avidly than did sIL-1Ra and icIL-1RaI. Microsequencing of cyanogen bromide fragments of purified icIL-1RaII provided evidence consistent with initiation of protein translation at the second start site in either IL-1Ra mRNA. The results of site-directed mutation experiments established that icIL-1RaII could be derived by alternative translation initiation. In vitro transcription and translation of intact sIL-1Ra cDNA in rabbit reticulocyte lysates led to both pro-sIL-1Ra and icIL-1RaII proteins, whereas transcription and translation of icIL-1RaI cDNA produced both icIL-1RaI and icIL-1RaII proteins. Mutation of the first 5′ ATG in sIL-1Ra cDNA led to translation of only icIL-1RaII, while only sIL-1Ra was observed after mutation of the second ATG. These results indicate that icIL-1RaII is a third member of the IL-1Ra family and is a 16-kDa, 143-amino acid intracellular protein derived by alternative translation initiation from either sIL-1Ra mRNA or icIL-1Ra mRNA. The role in biology of either intracellular form of IL-1Ra remains unknown.
Fcε Receptor I on Dendritic Cells Delivers IgE-Bound Multivalent Antigens into a Cathepsin S-Dependent Pathway of MHC Class II Presentation
Authors: Maurer Dieter , Fiebiger Edda , Reininger Bärbel , Ebner Christof , Petzelbauer Peter , Shi Guo-Ping , Chapman Harold A. , Stingl Georg 
Published: December 2022

AbstractIn this study, we elucidate the FcεRI-mediated Ag uptake and presentation mechanisms of dendritic cells (DC). We found that FcεRI-bound IgE, after polyvalent but not after monovalent ligation, is efficiently internalized into acidic, proteolytic compartments, degraded, and delivered into organelles containing MHC class II, HLA-DM, and lysosomal proteins. To follow the fate of the fragmented ligand, we sought to interfere with invariant chain (Ii) degradation, a process critical for peptide loading of nascent MHC class II molecules. We found DC to express cathepsin (Cat) S, a cysteine protease involved in li processing by B cells. Exposure of DC to a specific, active-site inhibitor of Cat S resulted in the loss of anti-Cat S immunoreactivity, led to the appearance of an N-terminal Ii remnant, and decreased the export of newly synthesized MHC class II to the DC surface. Furthermore, inactivation of Cat S as well as blockade of protein neosynthesis by cycloheximide strongly reduced IgE/FcεRI-mediated Ag presentation by DC. Thus, multimeric ligands of FcεRI, instead of being delivered into a recycling MHC class II pathway, are channeled efficiently into MIIC (MHC class II compartment)-like organelles of DC, in which Cat S-dependent li processing and peptide loading of newly synthesized MHC class II molecules occur. This IgE/FcεRI-dependent signaling pathway in DC may be a particularly effective route for immunization and a promising target for interfering with the early steps of allergen presentation.
Two Distinct Phospholipases C ofListeria monocytogenesInduce Ceramide Generation, Nuclear Factor-κB Activation, and E-Selectin Expression in Human Endothelial Cells
Authors: Schwarzer Nicole , Nöst Ralph , Seybold Joachim , Parida Shreemanta K. , Fuhrmann Oliver , Krüll Matthias , Schmidt Reinhold , Newton Robert , Hippenstiel Stefan , Domann Eugen , Chakraborty Trinad , Suttorp Norbert 
Published: December 2022

AbstractInfection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGDΔplcA and EGDΔplcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGDΔplcA or EGDΔplcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-κB activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-κB activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.
High Resolution Mapping of the B Cell Epitopes of Staphylokinase in Humans Using Negative Selection of a Phage-Displayed Antigen Library
Authors: Jenné Stéphane , Brepoels Katleen , Collen Désiré , Jespers Laurent 
Published: December 2022

AbstractStaphylokinase (Sak), a 16-kDa protein secreted by Staphylococcus aureus, induces fibrin-specific thrombolysis in patients with thrombotic disorders. However, Sak also elicits high titers of neutralizing Abs that persist for several months and preclude its repeated use in humans. To identify the antigenic determinants of Sak recognized by humans, a phage-displayed library of Sak variants was selected for mutants that escape binding to an affinity matrix derivatized with patient-specific polyclonal anti-Sak Abs. Fifty-six escape Sak variants were identified after three selection cycles using human polyclonal anti-Sak IgGs obtained from four different patients. DNA sequencing revealed 213 amino acid substitutions, of which 73% were found at 25 positions clustered in eight discontinuous Sak antigenic segments. Although each antigenic segment was recognized to a variable extent by each patient antiserum, the main epitopes of Sak in all patients were roughly targeted to two large discontinuous areas covering 35% of the solvent-accessible surface of Sak. The antigenic area I comprises three segments centered on residues 66, 73, and 135, while the antigenic area II consists of four segments centered on positions 20, 95, 102, and 121. These results suggest that a secondary immune response against Sak can occur in patients, and confirm an initial site-directed mutagenesis study wherein amino acid Lys74 was shown to play a prominent antigenic role. Comprehensive mapping of the most relevant sites of Sak that are antigenic for humans will guide efforts to modulate the immunogenicity of this therapeutically important molecule.
Cyclosporin A Enhances IL-12 Production by CpG Motifs in Bacterial DNA and Synthetic Oligodeoxynucleotides
Authors: Redford Thomas W. , Yi Ae-Kyung , Ward Courtney T. , Krieg Arthur M. 
Published: December 2022

AbstractCertain sequences of nucleotides (CpG motifs) in bacterial DNA or synthetic oligonucleotides (CpG DNA) promote the production of proinflammatory cytokines, including TNF-α, IFN-γ, IL-6, and IL-12. Here we demonstrate that the immunosuppressant cyclosporin A (CsA) unexpectedly enhanced CpG DNA-induced IL-12 production in murine splenocytes. CsA did not inhibit CpG DNA-induced TNF-α or IL-6 production, but decreased the production of IFN-γ by CpG DNA. Upon examining mechanisms by which CsA increases IL-12 production, we found that CpG DNA can also induce IL-10 production in B cells and that this production was sensitive to CsA. IL-10 has anti-inflammatory effects and can reduce the production of IL-12. To determine the possible role of CsA-modulated IL-10 production in mediating the increased IL-12 levels, splenocytes from IL-10 gene-disrupted mice (IL-10 −/−) and splenocytes cultured in anti-IL-10 Ab were studied. CpG DNA-stimulated IL-10 (−/−) splenocytes demonstrated no increase in IL-12 levels in the presence of CsA. Anti-IL-10 Ab treatment of normal splenocytes increased the magnitude of CpG DNA-induced IL-12 production to that seen with CsA. These results suggest that CpG DNA induces CsA-sensitive IL-10 production in B cells and that IL-10 acts as a negative feedback regulator of CpG DNA-induced IL-12 production.
Factor Xa Induces Cytokine Production and Expression of Adhesion Molecules by Human Umbilical Vein Endothelial Cells
Authors: Senden Nicole H. M. , Jeunhomme Trudi M. A. A. , Heemskerk Johan W. M. , Wagenvoord Rob , van’t Veer Cornelis , Hemker H. Coenraad , Buurman Wim A. 
Published: December 2022

AbstractProinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5–80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1α and TNF-α were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from γ-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and γ-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.