The Experts below are selected from a list of 1671 Experts worldwide ranked by ideXlab platform
Howard L Weiner - One of the best experts on this subject based on the ideXlab platform.
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TGF-β Induces Surface LAP Expression on Murine CD4 T Cells Independent of Foxp3 Induction
PloS one, 2010Co-Authors: Takatoku Oida, Howard L WeinerAbstract:Background It has been reported that human FOXP3+ CD4 Tregs express GARP-anchored surface Latency-Associated Peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3+ Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs.
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depletion of tgf β from fetal bovine serum
Journal of Immunological Methods, 2010Co-Authors: Takatoku Oida, Howard L WeinerAbstract:TGF-β is one of the key cytokines controlling immune responses. Measuring TGF-β from culture supernatants in vitro is an important index of immune function. However, fetal bovine serum (FBS) contains a high level of latent TGF-β that often hampers measuring T cell-derived TGF-β in culture using FBS-supplemented medium. In this report, we generated anti-Latency Associated Peptide (LAP) monoclonal antibodies which cross-react with bovine LAP, and developed a protocol to deplete TGF-β from FBS. This provides the ability to reliably quantify TGF-β in vitro without relying on serum-free media which do not support growth of murine T cells.
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overexpression of tgf β1 gene induces cell surface localized glucose regulated protein 78 Associated Latency Associated Peptide tgf β
Journal of Immunology, 2010Co-Authors: Takatoku Oida, Howard L WeinerAbstract:TGF-β plays a crucial role in immune regulation. It has been reported that pro–TGF-β, Latency-Associated Peptide (LAP), latent TGF-β and/or active TGF-β (LAP/TGF-β) is localized on the cell surface of Foxp3+ regulatory T cells. However, the molecular mechanism(s) of how LAP/TGF-β is anchored on the cell membrane is unknown. In this study, we show that forced expression of human TGF-β1 gene by retrovirus transduction into P3U1 mouse myeloma cells, and other cell types including murine CD4+CD25− T cells, makes these cells surface LAP/TGF-β-positive. The surface LAP/TGF-β contains high-glycosylated, furin-processed latent TGF-β, which is different from the low-glycosylated, furin-unprocessed intracellular form or the high-glycosylated, furin-unprocessed secreted form. Furthermore, surface LAP/TGF-β forms a complex with the molecular chaperone glucose-regulated protein 78 (GRP78, also known as BiP), and knockdown of GRP78 reduced the expression levels of surface LAP/TGF-β. GRP78, however, is not involved in GARP-mediated surface LAP/TGF-β. Our results suggest that GRP78 provides an additional surface localization mechanism for LAP/TGF-β, which may play an important role in controlling TGF-β activity.
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Cutting Edge: Human Latency-Associated Peptide+ T Cells: A Novel Regulatory T Cell Subset
Journal of immunology (Baltimore Md. : 1950), 2010Co-Authors: Roopali Gandhi, Mauricio F. Farez, Yue Wang, Deneen Kozoriz, Francisco J. Quintana, Howard L WeinerAbstract:Regulatory T cells (Tregs) play an important role in the maintenance of peripheral tolerance. Several molecules including TGF-β have been linked to the function and differentiation of Tregs. In this study, we describe a unique population of T cells expressing a membrane bound form of TGF-β, the Latency-Associated Peptide (LAP), and having regulatory properties in human peripheral blood. These CD4+LAP+ T cells lack Foxp3 but express TGF-βR type II and the activation marker CD69. CD4+LAP+ T cells are hypoproliferative compared with CD4+LAP− T cells, secrete IL-8, IL-9, IL-10, IFN-γ, and TGF-β upon activation, and exhibit TGF-β– and IL-10–dependent suppressive activity in vitro. The in vitro activation of CD4+LAP− T cells results in the generation of LAP+ Tregs, which is further amplified by IL-8. In conclusion, we have characterized a novel population of human LAP+ Tregs that is different from classic CD4+Foxp3+CD25high natural Tregs.
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Latency Associated Peptide identifies a novel cd4 cd25 regulatory t cell subset with tgfβ mediated function and enhanced suppression of experimental autoimmune encephalomyelitis
Journal of Immunology, 2008Co-Authors: Meiling Chen, Boshiun Yan, Yoshio Bando, Vijay K Kuchroo, Howard L WeinerAbstract:CD4+CD25+ regulatory T cells (Tregs) are essential for maintaining self-tolerance and immune homeostasis. Here we characterize a novel subset of CD4+CD25+ Tregs that express Latency-Associated Peptide (LAP) on their cell surface (CD4+CD25+LAP+ cells). CD4+CD25+LAP+ cells express elevated levels of Foxp3 and Treg-Associated molecules (CTLA4, glucocorticoid-induced TNFR-related gene), secrete TGFβ, and express both cell surface TGFβ and surface receptors for TGFβ. In vitro, the suppressive function of CD4+CD25+LAP+ cells is both cell contact and soluble factor dependent; this contrasts with CD4+CD25+LAP− cells, which are mainly cell contact dependent. In a model of experimental autoimmune encephalomyelitis, CD4+CD25+LAP+ cells exhibit more potent suppressive activity than CD4+CD25+LAP− cells, and the suppression is TGFβ dependent. We further show that CD4+CD25+LAP+ cells suppress myelin oligodendrocyte glycoprotein-specific immune responses by inducing Foxp3 and by inhibiting IL-17 production. Our findings demonstrate that CD4+CD25+ Tregs are a heterogeneous population and that the CD4+CD25+ subset that expresses LAP functions in a TGFβ-dependent manner and has greater in vivo suppressive properties. Our work helps elucidate the ambiguity concerning the role of TGFβ in CD4+CD25+ Treg-mediated suppression and indicates that LAP is an authentic marker able to identify a TGFβ-expressing CD4+CD25+ Treg subset.
Takatoku Oida - One of the best experts on this subject based on the ideXlab platform.
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TGF-β Induces Surface LAP Expression on Murine CD4 T Cells Independent of Foxp3 Induction
PloS one, 2010Co-Authors: Takatoku Oida, Howard L WeinerAbstract:Background It has been reported that human FOXP3+ CD4 Tregs express GARP-anchored surface Latency-Associated Peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3+ Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs.
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depletion of tgf β from fetal bovine serum
Journal of Immunological Methods, 2010Co-Authors: Takatoku Oida, Howard L WeinerAbstract:TGF-β is one of the key cytokines controlling immune responses. Measuring TGF-β from culture supernatants in vitro is an important index of immune function. However, fetal bovine serum (FBS) contains a high level of latent TGF-β that often hampers measuring T cell-derived TGF-β in culture using FBS-supplemented medium. In this report, we generated anti-Latency Associated Peptide (LAP) monoclonal antibodies which cross-react with bovine LAP, and developed a protocol to deplete TGF-β from FBS. This provides the ability to reliably quantify TGF-β in vitro without relying on serum-free media which do not support growth of murine T cells.
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overexpression of tgf β1 gene induces cell surface localized glucose regulated protein 78 Associated Latency Associated Peptide tgf β
Journal of Immunology, 2010Co-Authors: Takatoku Oida, Howard L WeinerAbstract:TGF-β plays a crucial role in immune regulation. It has been reported that pro–TGF-β, Latency-Associated Peptide (LAP), latent TGF-β and/or active TGF-β (LAP/TGF-β) is localized on the cell surface of Foxp3+ regulatory T cells. However, the molecular mechanism(s) of how LAP/TGF-β is anchored on the cell membrane is unknown. In this study, we show that forced expression of human TGF-β1 gene by retrovirus transduction into P3U1 mouse myeloma cells, and other cell types including murine CD4+CD25− T cells, makes these cells surface LAP/TGF-β-positive. The surface LAP/TGF-β contains high-glycosylated, furin-processed latent TGF-β, which is different from the low-glycosylated, furin-unprocessed intracellular form or the high-glycosylated, furin-unprocessed secreted form. Furthermore, surface LAP/TGF-β forms a complex with the molecular chaperone glucose-regulated protein 78 (GRP78, also known as BiP), and knockdown of GRP78 reduced the expression levels of surface LAP/TGF-β. GRP78, however, is not involved in GARP-mediated surface LAP/TGF-β. Our results suggest that GRP78 provides an additional surface localization mechanism for LAP/TGF-β, which may play an important role in controlling TGF-β activity.
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CD4+CD25− T Cells That Express Latency-Associated Peptide on the Surface Suppress CD4+CD45RBhigh-Induced Colitis by a TGF-β-Dependent Mechanism
Journal of immunology (Baltimore Md. : 1950), 2003Co-Authors: Takatoku Oida, Xingmin Zhang, Masao Goto, Satoshi Hachimura, Mamoru Totsuka, Shuichi Kaminogawa, Howard L WeinerAbstract:Murine CD4+CD25+ regulatory cells have been reported to express Latency-Associated Peptide (LAP) and TGF-β on the surface after activation, and exert regulatory function by the membrane-bound TGF-β in vitro. We have now found that a small population of CD4+ T cells, both CD25+ and CD25−, can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP+ cells are also positive for thrombospondin, which has the ability to convert latent TGF-β to the active form. In the CD4+CD45RBhigh-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25+LAP+ and CD25+LAP− cells, but also by CD25−LAP+ cells. CD4+CD25−LAP+ T cells were part of the CD45RBlow cell fraction. CD4+CD25−LAP−CD45RBlow cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25−LAP+ cells was abrogated in vivo by anti-TGF-β mAb. These results identify a new TGF-β-dependent regulatory CD4+ T cell phenotype that is CD25− and LAP+.
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cd4 cd25 t cells that express Latency Associated Peptide on the surface suppress cd4 cd45rbhigh induced colitis by a tgf β dependent mechanism
Journal of Immunology, 2003Co-Authors: Takatoku Oida, Xingmin Zhang, Masao Goto, Satoshi Hachimura, Mamoru Totsuka, Shuichi Kaminogawa, Howard L WeinerAbstract:Murine CD4+CD25+ regulatory cells have been reported to express Latency-Associated Peptide (LAP) and TGF-β on the surface after activation, and exert regulatory function by the membrane-bound TGF-β in vitro. We have now found that a small population of CD4+ T cells, both CD25+ and CD25−, can be stained with a goat anti-LAP polyclonal Ab without being stimulated. Virtually all these LAP+ cells are also positive for thrombospondin, which has the ability to convert latent TGF-β to the active form. In the CD4+CD45RBhigh-induced colitis model of SCID mice, regulatory activity was exhibited not only by CD25+LAP+ and CD25+LAP− cells, but also by CD25−LAP+ cells. CD4+CD25−LAP+ T cells were part of the CD45RBlow cell fraction. CD4+CD25−LAP−CD45RBlow cells had minimal, if any, regulatory activity in the colitis model. The regulatory function of CD25−LAP+ cells was abrogated in vivo by anti-TGF-β mAb. These results identify a new TGF-β-dependent regulatory CD4+ T cell phenotype that is CD25− and LAP+.
Nasreen Khalil - One of the best experts on this subject based on the ideXlab platform.
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Post translational activation of latent transforming growth factor beta (L-TGF-β): clinical implications
Histology and histopathology, 2001Co-Authors: Nasreen KhalilAbstract:Transforming growth factor-betas (TGF-βs) are multifunctional cytokines that exist in 3 isoforms in mammals. The TGF-Bs are ubiquitously expressed and al1 isoforms are secreted as biologically inactive precursors called latent TGF-β (L-TGF-β). LTGF-βs are generally not effective molecules because they are unable to interact with their receptors. However, the removal of or conformational change of the precursor protein called the Latency Associated Peptide (LAP) results in the generation of biologically active TGF-β. In vitro active TGF-β has many biological effects but from a clinical point of view one of the most recognized associations of aberrant TGF-β production is with diseases characterized by enhanced connective tissue synthesis. Recently a number of observations in the context of fibrotic disorders suggest mechanisms of activation of L-TGF-β1 in vivo . The recognition of mechanisms that activate L-TGF-β1 in vivo offers the possibiiity of interfering with the activation of L-TGFβ1 for therapeutic purposes.
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Activation of Rat Alveolar Macrophage-Derived Latent Transforming Growth Factor β-1 by Plasmin Requires Interaction with Thrombospondin-1 and its Cell Surface Receptor, CD36
The American journal of pathology, 1999Co-Authors: Teshome Yehualaeshet, Joanne E. Murphy-ullrich, Robert O'connor, Julia Green-johnson, Sabine Mai, Roy L. Silverstein, Nasreen KhalilAbstract:Transforming growth factor-β-1 (TGF-β1) is secreted by cells in a latent form (L-TGF-β1) noncovalently bound to a Latency-Associated Peptide. Activated alveolar macrophages obtained from rat lungs after bleomycin-induced pulmonary injury released increased amounts of active TGF-β1 as well as plasmin, a protease, and thrombospondin-1 (TSP-1), a trimeric glycoprotein. Previously we had demonstrated that plasmin was critical to the activation of L-TGF- β1. In the present study we demonstrated that TSP-1 is also important for the activation of L-TGF- β1 because the activation can be inhibited by anti-TSP-1 monoclonal antibody. Proteins obtained from alveolar macrophage cell lysates immunoprecipitated with antibodies specific for TSP-1 were identified on immunoblots as LAP and TGF-β1, indicating that TSP-1/L-TGF-β1 complexes are present on alveolar macrophages. However, in the presence of plasmin both Latency-Associated Peptide and TGF-β1 were decreased in the same cell lysates, indicating that L-TGF-β1 Associated with TSP-1 is released by plasmin. Using immunofluorescence and antibodies to TGF-β1 and CD36, a receptor for TSP-1, there was colocalization of TGF-β1 with CD36. Because TSP-1 but not TGF-β1 is a natural ligand for CD36, these findings suggest that the L-TGF-β1 in a complex with TSP-1 localizes to the macrophage cell surface when TSP-1 interacts with its receptor, CD36. Furthermore, the association of TSP-1/L-TGF-β1 complex with CD36 is necessary to the activation of L-TGF-β1 because antibodies to CD36 prevent the colocalization of TGF-β1 with CD36 as observed by immunofluorescence and inhibit activation of the L-TGF-β1 by explanted alveolar macrophages. These findings suggest that activation of L-TGF-β1 by plasmin occurs at the cell surface of activated alveolar macrophages and requires a TSP-1/CD36 interaction.
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plasmin regulates the activation of cell Associated latent tgf beta 1 secreted by rat alveolar macrophages after in vivo bleomycin injury
American Journal of Respiratory Cell and Molecular Biology, 1996Co-Authors: Nasreen Khalil, Steven Corne, Carol Whitman, Harold YacyshynAbstract:Transforming growth factor beta s (TGF-beta s) are 25-kD multifunctional proteins that regulate inflammation and connective tissue synthesis. With rare exception TGF-beta 1 is secreted noncovalently bound to a Latency-Associated Peptide (LAP) that renders the mature TGF-beta 1 biologically inactive. An important mechanism for the control of TGF-beta 1 action is the regulation of the post-translational processing that removes the LAP from the mature Peptide and renders it biologically active. In a model of pulmonary inflammation and fibrosis induced by the antineoplastic antibiotic, bleomycin, we have demonstrated that explanted alveolar macrophages secrete progressively increasing quantities of a biologically active form of TGF-beta 1, the secretion of which was maximal 7 days after bleomycin administration. Thereafter, there was a rapid decline in the secretion of the active form of TGF-beta 1, whereas the latent form continued to be secreted in elevated quantities. Plasmin, a serine protease, was transi...
Edward H Snell - One of the best experts on this subject based on the ideXlab platform.
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Structural insights into conformational switching in Latency-Associated Peptide between transforming growth factor β-1 bound and unbound states.
IUCrJ, 2020Co-Authors: Timothy R Stachowski, Mary E Snell, Edward H SnellAbstract:Transforming growth factor β-1 (TGFβ-1) is a secreted signalling protein that directs many cellular processes and is an attractive target for the treatment of several diseases. The primary endogenous activity regulatory mechanism for TGFβ-1 is sequestration by its pro-Peptide, Latency-Associated Peptide (LAP), which sterically prohibits receptor binding by caging TGFβ-1. As such, recombinant LAP is promising as a protein-based therapeutic for modulating TGFβ-1 activity; however, the mechanism of binding is incompletely understood. Comparison of the crystal structure of unbound LAP (solved here to 3.5 Å resolution) with that of the bound complex shows that LAP is in a more open and extended conformation when unbound to TGFβ-1. Analysis suggests a mechanism of binding TGFβ-1 through a large-scale conformational change that includes contraction of the inter-monomer interface and caging by the 'straight-jacket' domain that may occur in partnership through a loop-to-helix transition in the core jelly-roll fold. This conformational change does not appear to include a repositioning of the integrin-binding motif as previously proposed. X-ray scattering-based modelling supports this mechanism and reveals possible orientations and ensembles in solution. Although native LAP is heavily glycosylated, solution scattering experiments show that the overall folding and flexibility of unbound LAP are not influenced by glycan modification. The combination of crystallography, solution scattering and biochemical experiments reported here provide insight into the mechanism of LAP sequestration of TGFβ-1 that is of fundamental importance for therapeutic development.
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Structural consequences of transforming growth factor beta-1 activation from near-therapeutic X-ray doses
Journal of Synchrotron Radiation, 2019Co-Authors: Timothy R Stachowski, Thomas D. Grant, Edward H SnellAbstract:Dissociation of transforming growth factor beta-1 (TGFβ-1) from the inhibitory protein Latency-Associated Peptide (LAP) can occur from low doses of X-ray irradiation of the LAP-TGFβ-1 complex, resulting in the activation of TGFβ-1, and can have health-related consequences. Using the tools and knowledge developed in the study of radiation damage in the crystallographic setting, small-angle X-ray scattering (SAXS) and complementary techniques suggest an activation process that is initiated but not driven by the initial X-ray exposure. LAP is revealed to be extended when not bound to TGFβ-1 and has a different structural conformation compared to the bound state. These studies pave the way for the structural understanding of systems impacted at therapeutic X-ray doses and show the potential impact of radiation damage studies beyond their original intent.
Yuti Chernajovsky - One of the best experts on this subject based on the ideXlab platform.
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molecular engineering of short half life small Peptides vip αmsh and γ msh fused to Latency Associated Peptide results in improved anti inflammatory therapeutics
Annals of the Rheumatic Diseases, 2012Co-Authors: Sandrine Vessillier, Gill Adams, Rita Jones, M. P. Seed, Mauro Perretti, Trinidad Monteromelendez, Yuti ChernajovskyAbstract:Objective To facilitate the targeting to inflammation sites of small anti-inflammatory Peptides, with short half-lives, by fusion with the Latency-Associated Peptide (LAP) of transforming growth factor β1 through a cleavable matrix metalloproteinase (MMP) linker. This design improves efficacy, overcoming the limitations to their clinical use. Methods We generated latent forms of vasoactive intestinal Peptide (VIP), α-melanocyte-stimulating hormone (MSH) and γ 3 MSH by fusion to LAP through an MMP cleavage site using recombinant DNA technology. The biological activities of these latent therapeutics were studied in vivo using monosodium urate (MSU)-induced peritonitis and collagen-induced arthritis (CIA) models. We assessed gene therapy and purified protein therapy. Results The recruitment of the polymorphonuclear cells induced by MSU injection into mouse peritoneal cavity was reduced by 35% with γ 3 MSH (1 nmol), whereas administration of a much lower dose of purified latent LAP–MMP–γ 3 MSH (0.03 nmol) attenuated leucocyte influx by 50%. Intramuscular gene delivery of plasmids coding LAP–MMP–VIP and LAP–MMP–αMSH at disease onset reduced the development of CIA compared with LAP–MMP, which does not contain any therapeutic moiety. Histological analysis confirmed a significantly lower degree of inflammation, bone and cartilage erosion in groups treated with LAP–MMP–VIP or LAP–MMP–αMSH. Antibody titres to collagen type II and inflammatory cytokine production were also reduced in these two groups. Conclusion Incorporation of small anti-inflammatory Peptides within the LAP shell and delivered as recombinant protein or through gene therapy can control inflammatory and arthritic disease. This platform delivery can be developed to control human arthritides and other autoimmune diseases.
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Molecular engineering of short half-life small Peptides (VIP, αMSH and γ₃MSH) fused to Latency-Associated Peptide results in improved anti-inflammatory therapeutics.
Annals of the rheumatic diseases, 2011Co-Authors: Sandrine Vessillier, Gill Adams, Trinidad Montero-melendez, Rita Jones, M. P. Seed, Mauro Perretti, Yuti ChernajovskyAbstract:Objective To facilitate the targeting to inflammation sites of small anti-inflammatory Peptides, with short half-lives, by fusion with the Latency-Associated Peptide (LAP) of transforming growth factor β1 through a cleavable matrix metalloproteinase (MMP) linker. This design improves efficacy, overcoming the limitations to their clinical use. Methods We generated latent forms of vasoactive intestinal Peptide (VIP), α-melanocyte-stimulating hormone (MSH) and γ 3 MSH by fusion to LAP through an MMP cleavage site using recombinant DNA technology. The biological activities of these latent therapeutics were studied in vivo using monosodium urate (MSU)-induced peritonitis and collagen-induced arthritis (CIA) models. We assessed gene therapy and purified protein therapy. Results The recruitment of the polymorphonuclear cells induced by MSU injection into mouse peritoneal cavity was reduced by 35% with γ 3 MSH (1 nmol), whereas administration of a much lower dose of purified latent LAP–MMP–γ 3 MSH (0.03 nmol) attenuated leucocyte influx by 50%. Intramuscular gene delivery of plasmids coding LAP–MMP–VIP and LAP–MMP–αMSH at disease onset reduced the development of CIA compared with LAP–MMP, which does not contain any therapeutic moiety. Histological analysis confirmed a significantly lower degree of inflammation, bone and cartilage erosion in groups treated with LAP–MMP–VIP or LAP–MMP–αMSH. Antibody titres to collagen type II and inflammatory cytokine production were also reduced in these two groups. Conclusion Incorporation of small anti-inflammatory Peptides within the LAP shell and delivered as recombinant protein or through gene therapy can control inflammatory and arthritic disease. This platform delivery can be developed to control human arthritides and other autoimmune diseases.
