bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1
Authors: Sendide Khalid , Reiner Neil E. , Lee Jimmy S. I. , Bourgoin Sylvain , Talal Amina , Hmama Zakaria 
Published: April 2014

Abstract
The glycosylphosphatidyl anchored molecule CD14 to the monocyte membrane plays a prominent role in innate immunity, and the paradigms for CD14 selective signaling are beginning to be elucidated. In this study, transfected human monocytic cell line THP-1 and Chinese hamster ovary (CHO) fibroblastic cells were used to examine phagocytosis of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Flow cytometry was combined with molecular and biochemical approaches to demonstrate a dual mechanism for BCG internalization involving either CD14 alone or a CD14-regulated complement receptor (CR)3-dependent pathway. Phagocytosis by CD14-positive THP-1 cells was attenuated by phosphatidylinositol-3 inhibitors LY294002 and wortmannin and experiments using transfected CHO cells showed substantial accumulation of phosphatidylinositol-3,4,5-trisphosphate at the BCG attachment site in CHO cells expressing CD14 and TLR2 suggesting that bacteria bind to CD14 and use TLR2 to initiate a PI3K signaling pathway. Additional experiments using blocking Abs showed that anti-TLR2 Abs inhibit phagocytosis of BCG by THP-1 cells. Furthermore, knockdown of cytohesin-1, a PI3K-regulated adaptor molecule for β2 integrin activation, specifically abrogated CD14-regulated CR3 ingestion of BCG consistent with the observation of physical association between CR3 and cytohesin-1 in cells stimulated with mycobacterial surface components. These findings reveal that mycobacteria promote their uptake through a process of “inside-out” signaling involving CD14, TLR2, PI3K, and cytohesin-1. This converts low avidity CR3 into an active receptor leading to increased bacterial internalization.
Uptake of Granulysin via Lipid Rafts Leads to Lysis of Intracellular Listeria innocua
Authors: Walch Michael , Eppler Elisabeth , Dumrese Claudia , Barman Hanna , Groscurth Peter , Ziegler Urs 
Published: April 2014

Abstract
The bacteriolytic activity of CTL is mediated by granulysin, which has been reported to kill intracellular Mycobacterium tuberculosis in dendritic cells (DC) with high efficiency. Despite that crucial effector function, the killing mechanism and uptake of granulysin into target cells have not been well investigated. To this end we analyzed granulysin binding, uptake, and the subsequent lysis of intracellular Listeria innocua in human DC. Recombinant granulysin was found to be actively taken up by DC into early endosomal Ag 1-labeled endosomes, as detected by immunofluorescence. Further transfer to L. innocua-containing phagosomes was indicated by colocalization of bacterial DNA with granulysin. After uptake of granulysin by DC, lysis of L. innocua was found in a dose-dependent manner. Uptake as well as lysis of Listeria were inhibited after blocking endocytosis by lowering the temperature and by cholesterol depletion of DC. Colocalization of granulysin with cholera toxin during uptake showed binding to and internalization via lipid rafts. In contrast to cholera toxin, which was targeted to the perinuclear compartment, granulysin was found exclusively in endosomal-phagosomal vesicles. Lipid raft microdomains, enriched in the immunological synapse, may thus enhance uptake and transfer of granulysin into bacterial infected host cells.
Early Role of CD4+ Th1 Cells and Antibodies in HER-2 Adenovirus Vaccine Protection against Autochthonous Mammary Carcinomas
Authors: Park Jong Myun , Terabe Masaki , Sakai Yoshio , Munasinghe Jeeva , Forni Guido , Morris John C. , Berzofsky Jay A. 
Published: April 2014

Abstract
HER-2 is an oncogenic tumor-associated Ag that is overexpressed in several human tumors including breast and ovarian cancer. The efficacy and mechanism of a HER-2-expressing recombinant adenoviral vaccine to protect against tumorigenesis was examined using HER-2 transgenic (BALB-neuT) mice, which develop spontaneous breast tumors in all 10 mammary glands, and also using a transplantable mouse tumor model. Vaccination beginning at 6–8 wk of age (through 19 wk of age) prevented development of spontaneous mammary tumors even after 50 wk, whereas the animals in the control groups had tumors in all mammary glands by 25 wk. Such long-term protection after the last boost has not been achieved previously in this transgenic mouse in which the oncogene is continuously spawning tumorigenesis. Using β2-microglobulin-knockout, IFN-γ-knockout, and B cell-deficient mice, CD4+ and CD8+ cell depletion, and Ab transfer studies, we show that induction of anti-HER-2/neu Abs are both necessary and sufficient for protection, and the IgG2a isotype is most effective. In contrast, CD8+ T cells are not necessary at all, and CD4+ T cells are necessary for only 36–48 h after immunization to provide help for B cells but not as effector cells. Equal protection in immunized mice deficient in FcγRI/III excluded an FcR-mediated mechanism. Anti-HER-2 serum not only inhibited growth of mammary tumor cell lines expressing HER-2 in vitro but also protected mice from tumors in vivo, suggesting a direct action of Ab on the tumor cells. Such a vaccine may provide Ab-mediated protection against HER-2-expressing breast cancers in humans.
Infection Biology of a Novel α-Crystallin of Mycobacterium tuberculosis: Acr2
Authors: Wilkinson Katalin A. , Stewart Graham R. , Newton Sandra M. , Vordermeier H. Martin , Wain John R. , Murphy Helen N. , Horner Katherine , Young Douglas B. , Wilkinson Robert J. 
Published: April 2014

Abstract
Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the α-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-γ-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.
Mastoparan, a G Protein Agonist Peptide, Differentially Modulates TLR4- and TLR2-Mediated Signaling in Human Endothelial Cells and Murine Macrophages
Authors: Lentschat Arnd , Karahashi Hisae , Michelsen Kathrin S. , Thomas Lisa S. , Zhang Wenxuan , Vogel Stefanie N. , Arditi Moshe 
Published: April 2014

Abstract
Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-κB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-α, or IL-1β was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.
Structure-Function Relationships among Human Cathelicidin Peptides: Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities
Authors: Braff Marissa H. , Hawkins Mi‘i A. , Nardo Anna Di , Lopez-Garcia Belen , Howell Michael D. , Wong Cathy , Lin Kenneth , Streib Joanne E. , Dorschner Robert , Leung Donald Y. M. , Gallo Richard L. 
Published: April 2014

Abstract
Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.
Requirement for TLR9 in the Immunomodulatory Activity of Propionibacterium acnes
Authors: Kalis Christoph , Gumenscheimer Marina , Freudenberg Nikolaus , Tchaptchet Sandrine , Fejer György , Heit Antje , Akira Shizuo , Galanos Chris , Freudenberg Marina A. 
Published: April 2014

Abstract
Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-α and IFN-γ in response to LPS, lipid A, synthetic lipopeptide Pam3CysK4, or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9−/−, mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9−/− mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-γ is a key mediator of these effects, and enhanced IFN-γ mRNA expression was absent in spleen and liver of P. acnes-primed TLR9−/− mice, we conclude that TLR9 is required for the induction of IFN-γ by P. acnes.
Regulation of p38 MAPK by MAPK Kinases 3 and 6 in Fibroblast-Like Synoviocytes
Authors: Inoue Tomoyuki , Hammaker Deepa , Boyle David L. , Firestein Gary S. 
Published: April 2014

Abstract
The p38 MAPK signal transduction pathway is a key regulator of IL-1 and TNF-α production in rheumatoid arthritis. Previous studies demonstrated that upstream MAPK kinases (MKK3 and MKK6) that regulate p38 are activated in rheumatoid arthritis synovium. However, their functional relevance in fibroblast-like synoviocytes (FLS) has not been determined. To investigate the relative contribution of MKK3 and MKK6 to p38 activation, the effect of dominant-negative (DN) MKK3 and MKK6 constructs on cultured FLS was evaluated. Cultured FLS were stimulated with medium or IL-1β, and immunoblotting was performed. In some experiments, cells were lysed and immunoprecipitated with anti-p38 Ab, followed by in vitro kinase assay with [γ-32P]ATP and GST-activating transcription factor-2 as substrate. IL-1β rapidly induced p38 phosphorylation in cells transfected with empty vector (pcDNA3.1), but was inhibited by 25% in cells expressing DN MKK3 or DN MKK6. Cotransfection with both DN plasmids decreased phospho-p38 by almost 75%. In vitro kinase assays on IL-1-stimulated FLS also showed that the combination of DN MKK3 and DN MKK6 markedly decreased kinase activity compared with empty vector or the individual DN plasmids. Furthermore, IL-1β-induced IL-8, IL-6, and matrix metalloproteinase-3 protein production was significantly inhibited in DN MKK3/DN MKK6-transfected cells. The constructs had no effect on the respective mediator mRNA levels. These data demonstrate that MKK3 and MKK6 make individual contributions to p38 activation in FLS after cytokine stimulation, but that both must be blocked for maximum inhibition.
Activation Pathways Implicate Anti-HLA-DP and Anti-LFA-1 Antibodies as Lead Candidates for Intervention in Chronic Berylliosis
Authors: Chou Yuan K. , Edwards David M. , Weinberg Andrew D. , Vandenbark Arthur A. , Kotzin Brian L. , Fontenot Andrew P. , Burrows Gregory G. 
Published: April 2014

Abstract
CD4+ T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4+ T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the presence of APCs, beryllium induced strong proliferation responses of BAL CD4+ T cells, production of superoptimal concentrations of secreted proinflammatory cytokines, IFN-γ, TNF-α,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4+ T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4+ T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4+ T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease.
Induction of Hyper Th1 Cell-Type Immune Responses by Dendritic Cells Lacking the Suppressor of Cytokine Signaling-1 Gene
Authors: Hanada Toshikatsu , Tanaka Kentaro , Matsumura Yumiko , Yamauchi Moriyasu , Nishinakamura Hitomi , Aburatani Hiroyuki , Mashima Ryuichi , Kubo Masato , Kobayashi Takashi , Yoshimura Akihiko 
Published: April 2014

AbstractSuppressor of cytokine signaling (SOCS1/JAB) has been shown to play an important role in regulating dendritic cell (DC) function and suppressing inflammatory diseases and systemic autoimmunity. However, role of SOCS1 in DCs for the initiation of Th cell response has not been clarified. Here we demonstrate that SOCS1-deficient DCs induce stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher IFN-γ production from naive T cells than wild-type (WT) DCs in vitro. Lymph node T cells also produced a higher amount of IFN-γ when SOCS1-deficient bone marrow-derived DCs (BMDCs) were transferred in vivo. Moreover, SOCS1−/− BMDCs raised more effective anti-tumor immunity than WT BMDCs. Microarray analysis revealed that IFN-inducible genes were highly expressed in SOCS1-deficient DCs without IFN stimulation, suggesting hyper STAT1 activation in SOCS1−/− DCs. These phenotypes of SOCS1-deficient DCs were similar to those of CD8α+ DCs, and in the WT spleen, SOCS1 is expressed at higher levels in the Th2-inducing CD4+ DC subset, relative to the Th1-inducing CD8α+ DC subset. We propose that reduction of the SOCS1 gene expression in DCs leads to CD8α+ DC-like phenotype which promotes Th1-type hyperresponses.
HIV-1 Transactivator of Transcription Protein Induces Mitochondrial Hyperpolarization and Synaptic Stress Leading to Apoptosis
Authors: Perry Seth W. , Norman John P. , Litzburg Angela , Zhang Dabao , Dewhurst Stephen , Gelbard Harris A. 
Published: April 2014

Abstract
Despite the efficacy of highly active antiretroviral therapy in reducing viral burden, neurologic disease associated with HIV-1 infection of the CNS has not decreased in prevalence. HIV-1 does not induce disease by direct infection of neurons, although extensive data suggest that intra-CNS viral burden correlates with both the severity of virally induced neurologic disease, and with the generation of neurotoxic metabolites. Many of these molecules are capable of inducing neuronal apoptosis in vitro, but neuronal apoptosis in vivo does not correlate with CNS dysfunction, thus prompting us to investigate cellular and synaptic events occurring before cell death that may contribute to HIV-1-associated neurologic disease. We now report that the HIV-1 regulatory protein transactivator of transcription protein (Tat) increased oxidative stress, ATP levels, and mitochondrial membrane potential in primary rodent cortical neurons. Additionally, a proinflammatory cellular metabolite up-regulated by Tat, platelet-activating factor, also induced oxidative stress and mitochondrial hyperpolarization in neurons, suggesting that this type of metabolic dysfunction may occur on a chronic basis during HIV-1 infection of the CNS. Tat-induced mitochondrial hyperpolarization could be blocked with a low dose of the protonophore FCCP, or the mitochondrial KATP channel antagonist, tolbutamide. Importantly, blocking the mitochondrial hyperpolarization attenuated Tat-induced neuronal apoptosis, suggesting that increased mitochondrial membrane potential may be a causal event in precipitating neuronal apoptosis in cell culture. Finally, Tat and platelet-activating factor also increased neuronal vesicular release, which may be related to increased mitochondrial bioenergetics and serve as a biomarker for early damage to neurons.
Protective CD8 T Cell Immunity Triggered by CpG-Protein Conjugates Competes with the Efficacy of Live Vaccines
Authors: Heit Antje , Schmitz Frank , O’Keeffe Meredith , Staib Caroline , Busch Dirk H. , Wagner Hermann , Huster Katharina M. 
Published: April 2014

Abstract
In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 μg of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.
In Vitro and In Vivo Characterization of a Novel Antibody-Like Single-Chain TCR Human IgG1 Fusion Protein
Authors: Mosquera Luis A. , Card Kimberlyn F. , Price-Schiavi Shari A. , Belmont Heather J. , Liu Bai , Builes Janette , Zhu Xiaoyun , Chavaillaz Pierre-Andre , Lee Hyung-il , Jiao Jin-an , Francis John L. , Amirkhosravi Ali , Wong Richard L. , Wong Hing C. 
Published: April 2014

Abstract
We have constructed a protein composed of a soluble single-chain TCR genetically linked to the constant domain of an IgG1 H chain. The Ag recognition portion of the protein binds to an unmutated peptide derived from human p53 (aa 264–272) presented in the context of HLA-A2.1, whereas the IgG1 H chain provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells and promoting target and effector cell conjugation. The protein also has potent antitumor effects in an in vivo tumor model and can mediate cell killing by Ab-dependent cellular cytotoxicity. Therefore, single-chain TCRs linked to IgG1 H chains behave like Abs but possess the ability to recognize Ags derived from intracellular targets. These fusion proteins represent a novel group of immunotherapeutics that have the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by the conventional Ab-based approach.