Explore the vast range of titles available.

ObjectiveWe 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.MethodsSynovial 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.ResultsFHL2 levels were higher in RA than in OA and in hTNFtg than in wild-type mice. Surprisingly, while transforming growth factor (TGF)β-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.ConclusionsThese 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.

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-1β 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 100μg 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.

Novel molecules that specifically target human TNF[alpha] in rheumatoid arthritis pose problems for preclinical assessment of efficacy. In this study collagen antibody-induced arthritis (CAIA) has been induced in human TNF[alpha] transgenic mice to provide a novel model that has been optimised for the evaluation of molecules targeting human TNF[alpha]. Tg1278TNFko mice lack murine TNF[alpha] and are heterozygous for multiple copies of the human TNF[alpha] 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 [mu]g) i.p. on Day 1 or Day 4. Animals were assessed for arthritis symptoms using a clinical score, cytokine levels (human TNF[alpha], IL-1[beta] and IL-6) in sera and joints, and histopathology. The dependence of the model on human TNF[alpha] was determined by dosing animals with etanercept. Tg1278TNFko animals treated with 2, 4 or 8 mg CAb on Day 0, with 100[mu]g 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 [mu]g) 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. The features of CAIA in Tg1278TNFko animals make the model well-suited to testing the next generation of therapeutics that will target human TNF[alpha] in rheumatoid arthritis.

Chemokines are an important family of molecules involved in various pathophysiological manifestations. The B-chemokines RANTES, MIP-1α and MIP-1β were also implicated to participate in T cell activation. This study demonstrates that freshly purified T cell proliferation and RANTES secretion did not always correlate. CD3/CD28 costimulation or CD2/CD28 costimulation both caused comparable proliferation, but only CD3/CD28 supported modest RANTES secretion. Meanwhile, the phorbol ester PMA caused a robust RANTES secretion without promoting IL-2 production and T cell proliferation. Additions of various α- or β-chemokines, or neutralising anti-chemokine antibodies failed to affect T cell proliferation in response to various costimuli. The effects of PMA on RANTES secretion appeared to be chemokine-specific as MEP-lawas not equally affected, and depended on protein kinase C (PKC) isoenzyme activation. Signals causing calcium mobilisation caused a reduction in RANTES secretion compared to the PMA response. Ligation of CTLA-4, expression of which depends on sustained calcium elevation, inhibited RANTES secretion induced by CD3/CD28, but had no effect on PMA-stimulated RANTES secretion. The use of inhibitors indicated that PKC-, Ras- and PTK-dependent pathways were crucial for any RANTES secretion, whereas PI3K-, Calcineurin-, mTOR-, ERKl/2 and p38-dependent pathways were only important in CD3/CD28-costimulated RANTES secretion. Analysis of CXCR4 signalling indicated that calcium mobilisation, unlike Gi-protein- dependent PI3K activation, was not necessary for chemotaxis to SDF-1 and its peptide analogues. CXCR4-induced chemotaxis, D-3 phosphoinositide lipid accumulation, PKB and ERKl/2 activation was sensitive to PI3K or Gi-protein inhibition. In contrast, SDF- 1-induced actin polymerisation was partially dependent on PI3K, whereas CXCR4 internalisation was only sensitive to PKC inhibition. Despite the observed PKB activation, SDF-1 did not rescue or promote basal T cell apoptosis, or apoptosis in response to Fas ligation. Finally, T-tropic HIV-1 gpl20s did not cause significant chemotaxis, CXCR4 internalisation or PtdIns(3,4,5)P3 generation and antagonised SDF-1 responses on calcium mobilisation assays