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1.
Ann Rheum Dis ; 74(12): 2216-23, 2015 Dec.
Article in English | MEDLINE | ID: mdl-25125695

ABSTRACT

OBJECTIVE: We analysed the role of the adaptor molecule four-and-a-half Lin11, Isl-1 & Mec-3 (LIM) domain protein 2 (FHL2) in the activation of fibroblast-like synoviocytes in human rheumatoid arthritis (RA) and tumour necrosis factor α (TNFα)-dependent animal models of the disease. METHODS: Synovial tissues of patients with RA and osteoarthritis (OA) as well as hind paw sections from arthritic human TNFα transgenic (hTNFtg) mice and synovial fibroblasts from these were analysed. The effects of cytokines on the expression of FHL2 and disease-relevant matrixmetalloproteases (MMPs) were determined. Analyses of human tissue specimens from patients treated with anti-TNFα as well as anti-TNFα treatment of hTNFtg mice were performed to substantiate the TNFα effects on FHL2 levels. FHL2(-/-) mice and hTNFtg mice (with constitutive or inducible transgene expression) were crossbred to generate TNFα overexpressing FHL2-deficient animals. Signalling pathways were analysed in cells from these mice and in human cells after knock down of FHL2 by western blot. RESULTS: FHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)ß-induced FHL2 expression, TNFα suppressed FHL2. In vivo, anti-TNFα treatment led to higher FHL2 levels both in RA patients and hTNFtg mice. The loss of FHL2 increased joint destruction in hTNFtg mice, which was accompanied by elevated MMP-13. In vitro, TNFα-mediated MMP-13 was significantly higher in FHL2(-/-) cells and after knock down of FHL2, which was caused by prolonged p38 MAPK activation. CONCLUSIONS: These data suggest that FHL2 serves as a protective factor and that, rather than promoting the pathology, the upregulation of FHL2 in RA occurs in frame of a regenerative attempt.


Subject(s)
DNA/genetics , Gene Expression Regulation , LIM-Homeodomain Proteins/genetics , Muscle Proteins/genetics , Osteoarthritis/genetics , Synovial Membrane/metabolism , Transcription Factors/genetics , Animals , Cells, Cultured , Chronic Disease , Humans , Immunoblotting , LIM-Homeodomain Proteins/biosynthesis , Mice , Mice, Transgenic , Muscle Proteins/biosynthesis , Osteoarthritis/metabolism , Osteoarthritis/pathology , Real-Time Polymerase Chain Reaction , Signal Transduction , Synovial Membrane/pathology , Transcription Factors/biosynthesis
2.
J Transl Med ; 12: 285, 2014 Oct 25.
Article in English | MEDLINE | ID: mdl-25344414

ABSTRACT

BACKGROUND: Novel molecules that specifically target human TNFα in rheumatoid arthritis pose problems for preclinical assessment of efficacy. In this study collagen antibody-induced arthritis (CAIA) has been induced in human TNFα transgenic mice to provide a novel model that has been optimised for the evaluation of molecules targeting human TNFα. METHODS: Tg1278TNFko mice lack murine TNFα and are heterozygous for multiple copies of the human TNFα transgene that is expressed under normal physiological control. To establish CAIA, a collagen II monoclonal antibody cocktail (CAb) at 2, 4 or 8 mg was injected i.p. on Day 0 followed by a lipopolysaccharide (LPS) boost (10 or 100 µg) i.p. on Day 1 or Day 4. Animals were assessed for arthritis symptoms using a clinical score, cytokine levels (human TNFα, IL-1ß and IL-6) in sera and joints, and histopathology. The dependence of the model on human TNFα was determined by dosing animals with etanercept. RESULTS: Tg1278TNFko animals treated with 2, 4 or 8 mg CAb on Day 0, with 100 µg LPS on Day 4, had more severe arthritis and earlier symptoms than wild type animals at all doses of CAb tested. Subsequently it was found that the transgenic model did not require LPS at all for arthritis development but a lower dose of LPS (10 µg) was found necessary for reproducible and robust disease (close to 100% incidence, well-synchronised, with high arthritis scores). Furthermore the LPS challenge could be brought forward to Day 1 so that its' actions to facilitate disease could be separated temporally from the arthritis phase (beginning about Day 4). Etanercept, administered immediately after the serum spike of cytokines associated with LPS had subsided, was able to dose-dependently inhibit arthritis development and this was associated with a marked protection of the joints histologically on Day 14. Etanercept was also able to reverse the signs of arthritis when given therapeutically allowing animals to be matched for disease burden before dosing begins. CONCLUSIONS: The features of CAIA in Tg1278TNFko animals make the model well-suited to testing the next generation of therapeutics that will target human TNFα in rheumatoid arthritis.


Subject(s)
Antibodies/administration & dosage , Arthritis, Experimental/drug therapy , Arthritis, Rheumatoid/drug therapy , Collagen Type II/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Animals , Arthritis, Rheumatoid/metabolism , Etanercept/therapeutic use , Humans , Mice , Mice, Knockout , Mice, Transgenic
3.
Biologicals ; 39(6): 384-95, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21996051

ABSTRACT

Etanercept is a soluble tumor necrosis factor (TNF) receptor originally approved for treatment of moderate-to-severe rheumatoid arthritis, juvenile rheumatoid arthritis, and psoriatic arthritis. We have developed a non-innovator version of the recombinant protein etanercept, with the investigational name AVG01 (trade name AVENT™), using a novel expression vector-based technology. Here we show, by extensive analytical characterization, that AVG01 is highly similar to the reference product Enbrel® and demonstrates similar efficacy in pre-clinical studies.


Subject(s)
Arthritis/drug therapy , Immunoglobulin G/pharmacology , Recombinant Proteins/pharmacology , Animals , Antirheumatic Agents/pharmacology , Blotting, Western , CHO Cells , Cell Line, Tumor , Chromatography, High Pressure Liquid , Cricetinae , Cricetulus , Disease Models, Animal , Drug Evaluation, Preclinical , Etanercept , Female , Humans , Immunoglobulin G/analysis , Immunoglobulin G/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Receptors, Tumor Necrosis Factor/analysis , Receptors, Tumor Necrosis Factor/genetics , Recombinant Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Treatment Outcome
5.
Chem Biol ; 10(3): 207-13, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12670534

ABSTRACT

At least one Holy Grail for many academic researchers and pharmaceutical research divisions alike has been to identify therapeutically useful selective PI3K inhibitors. There are several different but closely related PI3Ks which are thought to have distinct biological roles. Until now, however, researchers have been frustrated by poor selectivity of the available pharmacological inhibitors, which are unable to distinguish the different isoforms of PI3K adequately. Fortunately, recently published work gives cause for optimism; there are now several patent specifications published that describe new PI3K inhibitors, including some that are more selective for the delta isoform of PI3K. Given the involvement of PI3Ks in a plethora of biological settings, such isoform-selective inhibitors may have immense potential use for the treatment of patients with inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases.


Subject(s)
Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Autoimmune Diseases/drug therapy , Autoimmune Diseases/enzymology , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/enzymology , Enzyme Inhibitors/chemistry , Humans , Inflammation/drug therapy , Inflammation/enzymology , Isoenzymes/antagonists & inhibitors , Neoplasms/drug therapy , Neoplasms/enzymology , Structure-Activity Relationship
6.
J Immunol ; 170(8): 4021-30, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12682230

ABSTRACT

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kgamma (KD-PI3Kgamma) or Deltap85 (a construct unable to bind class I(A) p110alpha, -beta, or -delta) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kgamma markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40-60% inhibition. Interestingly, the expression of Deltap85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kgamma mutant, typically in the range of 20-40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.


Subject(s)
Chemokines, CXC/physiology , Chemotaxis, Leukocyte/immunology , Isoenzymes/physiology , Phosphatidylinositol 3-Kinases/physiology , Protein Serine-Threonine Kinases , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/immunology , Animals , Cattle , Cell Migration Inhibition , Chemokine CXCL12 , Chemokines, CXC/antagonists & inhibitors , Chromones/pharmacology , Class Ib Phosphatidylinositol 3-Kinase , Clone Cells , Drug Synergism , Enzyme Activation/genetics , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation/immunology , Genetic Vectors , Humans , Insulin-Like Growth Factor I/antagonists & inhibitors , Insulin-Like Growth Factor I/physiology , Isoenzymes/antagonists & inhibitors , Isoenzymes/biosynthesis , Isoenzymes/genetics , Jurkat Cells , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Morpholines/pharmacology , Phosphatidylinositol 3-Kinases/biosynthesis , Phosphatidylinositol 3-Kinases/genetics , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Swine , T-Lymphocyte Subsets/metabolism , Tetracycline/metabolism
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