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  • Wagner, Hermann  (11)
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  • 1
    Language: English
    In: BMC Biotechnology, April 13, 2010, Vol.10, p.31
    Description: Background Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody ([alpha]T2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. [alpha]T2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly.sub.4 Ser).sub.3 amino acid sequence. Results Coexpression of [alpha]T2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with [alpha]T2ib indicated interaction of [alpha]T2ib with its cognate antigen within cells. [alpha]T2ib inhibited NF-[kappa]B driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding [alpha]T2ib into HEK293 cells demonstrated high efficiency of the TLR2-[alpha]T2ib interaction. The [alpha]T2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-[alpha]T2ib. Transduction with AdV[alpha]T2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNF[alpha] mRNA accumulation, as well as TNF[alpha] translation and release by macrophages were largely abrogated upon transduction of [alpha]T2ib. [alpha]T2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdV[alpha]T2ib. Systemic transduction applying AdV[alpha]T2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. Conclusion The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of [alpha]T2ib in microbial or viral infections.
    Keywords: Macrophages -- Properties ; Gene Expression -- Physiological Aspects ; Natural Immunity -- Genetic Aspects ; Antibodies -- Properties ; Cell Receptors -- Properties ; Dna Sequencing -- Methods
    ISSN: 1472-6750
    Source: Cengage Learning, Inc.
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  • 2
    Language: English
    In: BMC Biotechnology, 01 April 2010, Vol.10(1), p.31
    Description: Abstract Background Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (αT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. αT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence. Results Coexpression of αT2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with αT2ib indicated interaction of αT2ib with its cognate antigen within cells. αT2ib inhibited NF-κB driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding αT2ib into HEK293 cells demonstrated high efficiency of the TLR2-αT2ib interaction. The αT2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-αT2ib. Transduction with AdVαT2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNFα mRNA accumulation, as well as TNFα translation and release by macrophages were largely abrogated upon transduction of αT2ib. αT2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdVαT2ib. Systemic transduction applying AdVαT2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. Conclusion The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of αT2ib in microbial or viral infections.
    Keywords: Engineering
    ISSN: 1472-6750
    E-ISSN: 1472-6750
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  • 3
    Language: English
    In: The Journal of biological chemistry, 12 November 2004, Vol.279(46), pp.48004-12
    Description: Toll-like receptors (TLRs) mediate activation of the immune system upon challenge with microbial agonists, components of disintegrating cells of the body, or metabolic intermediates of lipidic nature. Comparison of murine (m) and human (h) TLR2 primary sequences revealed 65% of identical residues within the extracellular domains in contrast to 84% in the intracellular domains. Comparative analysis of TLR2-driven cell activation by various TLR2 agonists showed that the tri-lauroylated lipopeptide analog (Lau(3)CSK(4)) is recognized efficiently through mTLR2 but not hTLR2. Genetically complemented human embryonic kidney 293 cells and murine TLR2(-/-) embryonic fibroblasts, as well as human and murine macrophage cells, were used for this analysis. In contrast to cellular activation, which depended on blockable access of the TLR2-ligand to TLR2, cellular uptake of Lau(3)CSK(4) and tri-palmitoylated peptide (P(3)CSK(4)) was independent of TLR2. A low-conserved region spanning from leucine-rich repeat (LRR) motif 7 to 10 was found to control TLR2 species-specific cell activation. Exchange of mLRR8 for hLRR8 in mTLR2 abrogated mTLR2-typical cell activation upon cellular challenge with Lau(3)CSK(4) but not P(3)CSK(4), implicating mLRR8 as a central element of Lau(3)CSK(4) recognition. The point mutation L112P within LRR3 abrogated hTLR2-dependent recognition of lipopeptides but merely attenuated mTLR2 function, whereas deletion of the N-terminal third of each LRR-rich domain (LRRs 1 to 7) had the opposite effect on P(3)CSK(4) recognition. Despite similar domain structure of both TLR2 molecules, species-specific properties thus exist. Our results imply distinct susceptibilities of humans and mice to challenge with specific TLR2 ligands.
    Keywords: Fatty Acids -- Chemistry ; Membrane Glycoproteins -- Metabolism ; Peptides -- Metabolism ; Receptors, Cell Surface -- Metabolism
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 4
    Language: English
    In: BMC Biotechnology, April 13, 2010, Vol.10, p.31
    Description: Background Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody ([alpha]T2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. [alpha]T2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly.sub.4 Ser).sub.3 amino acid sequence. Results Coexpression of [alpha]T2ib and mouse TLR2 in HEK293 cells led to efficient retention and accumulation of TLR2 inside the ER compartment. Co-immunoprecipitation of human TLR2 with [alpha]T2ib indicated interaction of [alpha]T2ib with its cognate antigen within cells. [alpha]T2ib inhibited NF-[kappa]B driven reporter gene activation via TLR2 but not through TLR3, TLR4, or TLR9 if coexpressed in HEK293 cells. Co-transfection of human TLR2 with increasing amounts of the expression plasmid encoding [alpha]T2ib into HEK293 cells demonstrated high efficiency of the TLR2-[alpha]T2ib interaction. The [alpha]T2ib open reading frame was integrated into an adenoviral cosmid vector for production of recombinant adenovirus (AdV)-[alpha]T2ib. Transduction with AdV[alpha]T2ib specifically inhibited TLR2 surface expression of murine RAW264.7 and primary macrophages derived from bone marrow (BMM). Furthermore, TLR2 activation dependent TNF[alpha] mRNA accumulation, as well as TNF[alpha] translation and release by macrophages were largely abrogated upon transduction of [alpha]T2ib. [alpha]T2ib was expressed in BMM and splenocytes over 6 days upon systemic infection with AdV[alpha]T2ib. Systemic transduction applying AdV[alpha]T2ib rendered immune cells largely non-responsive to tripalmitoyl-peptide challenge. Our results show persistent paralysis of TLR2 activity and thus inhibition of immune activation. Conclusion The generated anti-TLR2 scFv intrabody inhibits specifically and very efficiently TLR2 ligand-driven cell activation in vitro and ex vivo. This indicates a therapeutic potential of [alpha]T2ib in microbial or viral infections.
    Keywords: Macrophages -- Properties ; Gene Expression -- Physiological Aspects ; Natural Immunity -- Genetic Aspects ; Antibodies -- Properties ; Cell Receptors -- Properties ; Dna Sequencing -- Methods
    ISSN: 1472-6750
    Source: Cengage Learning, Inc.
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  • 5
    Language: English
    In: The Journal of biological chemistry, 07 June 2002, Vol.277(23), pp.20847-53
    Description: The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor kappaB-driven reporter genes and mitogen- and stress-activated protein kinases and induces IkappaBalpha degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/TLR2(-/-) mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.
    Keywords: Drosophila Proteins ; Dendritic Cells -- Immunology ; Endoplasmic Reticulum -- Metabolism ; Heat-Shock Proteins -- Metabolism ; Membrane Glycoproteins -- Metabolism ; Receptors, Cell Surface -- Metabolism
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 6
    Language: English
    In: International Immunology, 1993, Vol.5(8), pp.929-937
    Description: The continuous presence of antigen and powerful immune responses (exhaustive cell proliferation) of ligand reactive T cells are currently thought to condition clonal deletion and/or induction of unresponsiveness to endogenous or exogenous superantigens (SAg). Here we report that in vivo induction of unresponsiveness to the SAg staphylococcal enterotoxin B (SEB) can be an immediate process. Within ours a large portion of ligand reactive V beta 8+ T cells becomes clonally deleted by apoptosis. In parallel, the remaining V beta 8+ T cells are unresponsive to SEB, yet at the same time express functional IL-2 receptors (IL-2R) and thus are highly responsive to the growth promoting effects of IL-2. In a subsequent step refractory IL-2R+V beta 8+ T cells undergo a wave of cell proliferation for 48 h, presumably driven by IL-2. Thereafter a large proportion of V beta 8+ T cells succumb to apoptosis, the remaining cells display the hallmarks of split unresponsiveness, i.e. they display a selective failure to produce IL-2 upon SEB stimulation in vitro combined with a preserved capability to express functional IL-2R. Early deletion and induction of unresponsiveness to SEB are cyclosporin A (CsA) resistant, while clonal expansion with subsequent cell deletion is blocked by CsA, yet the development of split unresponsiveness is not impaired by CsA. The results suggest that IL-2 driven growth of refractory T cells may mimic powerful immune responses of ligand reactive V beta 8+ T cells. Since unresponsiveness to SEB precedes in vivo expansion, the results as such question the concept of 'exhaustive cell proliferation' as a prerequisite for induction of unresponsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)
    Keywords: Lymphocyte Activation ; Cyclosporine -- Pharmacology ; Enterotoxins -- Immunology ; Staphylococcus Aureus -- Immunology ; Superantigens -- Immunology ; T-Lymphocytes -- Immunology;
    ISSN: 0953-8178
    E-ISSN: 1460-2377
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  • 7
    Language: English
    In: European Journal of Immunology, April 1999, Vol.29(4), pp.1209-1218
    Description: CpG‐containing oligodeoxynucleotides (CpG‐ODN) act as powerful adjuvant during induction of T cell responses. While CpG‐ODN directly activate antigen‐presenting cells (APC) and thus exert an extrinsic activity on T cells, it is unclear whether they directly affect T cells (intrinsic activity). Here we analyze the effects of CpG‐ODN on T cells in an APC‐free cell culture. We report that CpG‐ODN co‐stimulate T cells provided they were triggered via their TCR. CpG‐ODN induced IL‐2 production, IL‐2 receptor expression and thus proliferation. Proliferation was blocked by cyclosporin A or anti‐IL‐2 monoclonal antibodies (mAb) but not by anti‐IL‐4 mAb. Moreover, CpG‐co‐stimulated T cells differentiated into cytolytic T lymphocytes . Of note, IL‐2‐driven growth of primed T cells was not affected by CpG‐ODN. Co‐stimulation was also operative in T cells from CD28 mice and in TCR‐transgenic T cells stimulated with peptide. CpG‐ODN‐mediated co‐stimulation of T cells may thus explain part of the potent adjuvant effects of CpG‐ODN .
    Keywords: Cpg‐Oligodeoxynucleotide ; T Cell ; Co‐Stimulation ; Immunostimulatory Dna ; Cd28
    ISSN: 0014-2980
    E-ISSN: 1521-4141
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  • 8
    Language: English
    In: International Immunology, 1995, Vol.7(1), pp.105-114
    Description: Clonal deletion and/or inactivation establishes tolerance to self antigens. Endogenous and exogenous (bacterial) superantigens, like the staphylococcal enterotoxins, induce ligand-specific clonal anergy in vivo and thus are believed to mirror aspects of post-thymic tolerance mechanisms in mature peripheral T cells. Here we analyzed the level of anergy of ligand-responsive V sub( beta )8 super(+) T cells from staphylococcal enterotoxin B (SEB)-primed mice in vivo and in vitro. Upon in vitro restimulation with SEB, CD4 super(+)V sub( beta )8 super(+) and CD8 super(+)V sub( beta )8 super(+) T cells failed to produce IL-2. However, functional IL-2 receptors were triggered, since supplementation with IL-2 induced clonal growth in virtually all CD4 super(+)V sub( beta )8 super(+) and CD8 super(+)V sub( beta )8 super(+) T cells as determined by limiting dilution analyses. Thus in vitro unresponsiveness of lymphocytes from SEB-primed mice reflects the inability of SEB-reactive V sub( beta )8 super(+) T cells to produce IL-2. Surprisingly, anergy as defined in vitro was at variance with that in vivo. Following further challenge with SEB, systemic and acute lymphokine production (including IL-2 and tumor necrosis factor) occurred with almost identical peak values and kinetics to primary in vivo responses, and D-galactosamine-sensitized mice succumbed to lethal shock. Polymerase chain reaction analyses revealed that CD4 super(+)V sub( beta )8 super(+) expressed IL-2-specific mRNA in vivo upon restimulation with SEB. While lymphokine production and expression of the IL-2 receptor was similar to the response to in vivo primary stimulation, only CD8 super(+)V sub( beta )8 super(+) T cells expanded clonally upon reintroduction of SEB in vivo. Hence primed V sub( beta )8 super(+) T cells challenged with SEB display in vitro anergy yet in vivo responsiveness, at least in part. We conclude that the state of anergy is reversible, dependent upon the quality of activation signals provided in in vivo rather than in in vitro culture conditions.
    Keywords: Staphylococcus Aureus ; Staphylococcus Aureus ; Enterotoxins ; Superantigens ; Lymphocytes T ; Anergy ; in Vitro ; in Vivo ; Enterotoxins ; Superantigens ; Lymphocytes T ; Anergy ; in Vitro ; in Vivo ; Function ; Antigenic Properties and Virulence ; Mice ; Mice ; Mice;
    ISSN: 0953-8178
    E-ISSN: 1460-2377
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  • 9
    Language: English
    In: European Journal of Immunology, April 1997, Vol.27(4), pp.825-833
    Description: Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD 14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro‐inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100‐fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin‐6, and interferon‐γ (IFN‐γ) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with ‐galactosamine (‐GalN). Opposed to ‐GalN‐pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti‐IFN‐γ monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell‐derived IFN‐γ is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB‐induced T cell deletion and anergy induction. Since Gram‐positive and Gram‐negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.
    Keywords: Lipopolysaccharide ; Endotoxin ; Staphylococcal Enterotoxin B ; Superantigen ; Lethal Shock
    ISSN: 0014-2980
    E-ISSN: 1521-4141
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  • 10
    Language: English
    In: Zentralblatt für Bakteriologie : medical microbiology, virology, parasitology, infectious diseases, 1991, Vol.275(2), pp.264-268
    Description: Resting CDS and CD4 murine T cells become efficiently activated by the superantigen (SA) staphylococcal enterotoxin B (SEB). Limiting dilution experiments reveal that roughly every third CDS or CD4 T cell could be induced to proliferate. Surprisingly, besides CDS T cells, also CD4 cells acquire a high cytolytic activity upon activation with SEB. Both subpopulations only lyse MHC class II positive target cells in the presence of SEB. However the recognition of SEB by CTL is independent of the haplotype of the MHC, i.e. is MHC-unrestricted. In addition the recognition of SEB is independent of the coreceptor molecules CD4 and CDS, respectively. Ruhende CDS und CD4 T Zellen der Maus werden durch das Superantigen (SA) Staphylokokken Enterotoxin B (SEB) aktiviert. Grenzverdiinnungsanalysen zeigen, daß ungefähr jede dritte CDS oder CD4 T Zelle zur Proliferation induziert wird. Überraschenderweise entwickeln neben den CDS T Zellen auch die CD4 Zellen eine hohe zytolytische Aktivitat nach Aktivierung durch SEB. Beide Subpopulationen lysieren nur MHC II positive Targetzellen in Anwesenheit von SEB. Jedoch ist die Erkennung von SEB unabhängig vom Haplotyp des MHC, d.h. MHC unrestringiert. Außerdem wird SEB unabhängig von den Korezeptoren CD4 und CDS erkannt.
    Keywords: Biology
    ISSN: 0934-8840
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