Rheumatoid arthritis is a complex systemic disease that ultimately leads to the progressive destruction of articular and periarticular structures. Novel data indicate that the innate immune system ...(through activation of Toll-like receptors) is involved in articular pathophysiology, including the recruitment of inflammatory cells, and that periarticular factors such as adipocytokines contribute to the perpetuation of joint inflammation. The deleterious process of joint destruction is mediated by intracellular signaling pathways involving transcription factors, such as nuclear factor kappaB, cytokines, chemokines, growth factors, cellular ligands, and adhesion molecules. Advances in molecular biology techniques have identified T-cell-independent and B-cell-independent pathways that operate at different stages of the disease. Cytokine-independent pathways appear to be responsible for maintaining basic disease activity that is not affected by currently available therapies. Using this knowledge in combination with gene-transfer and gene-silencing approaches, bench-to-bedside strategies will be developed, thus enabling the creation of novel treatments for rheumatoid arthritis.
Objective To analyse the expression of SIRT1 in synovial tissues and cells of patients with rheumatoid arthritis (RA) and to study the function of SIRT1 in inflammation and apoptosis in RA. Methods ...Levels of SIRT1 expression were analysed in synovial tissues and cells from patients with RA by real-time PCR and western blotting before and after stimulation with toll-like receptor ligands, tumour necrosis factor α (TNFα) and interleukin 1β (IL-1β). Immunohistochemistry was used to study the localisation of SIRT1. Fluorescence activated cell sorting analysis was performed to investigate the effect of SIRT1 on apoptosis. Peripheral blood monocytes and rheumatoid arthritis synovial fibroblasts (RASFs) were transfected with wild-type or enzymatically inactive SIRT1 expression vectors or with siRNA targeting SIRT1. Cytokine analysis of IL-6, IL-8 and TNFα were performed by ELISA to study the role of SIRT1 on proinflammatory mediators of RA. Results SIRT1 was found to be constitutively upregulated in synovial tissues and cells from patients with RA compared to osteoarthritis. TNFα stimulation of RASFs and monocytes resulted in further induced expression levels of SIRT1. Silencing of SIRT1 promoted apoptosis in RASFs, whereas SIRT1 overexpression protected cells from apoptosis. Inhibition of SIRT1 enzymatic activity by inhibitors, siRNA and overexpression of an enzymatically inactive form of SIRT1 reduced lipopolysaccharide-induced levels of TNFα in monocytes. Similarly, knockdown of SIRT1 resulted in a reduction of proinflammatory IL-6 and IL-8 in RASFs. Conclusion The TNFα-induced overexpression of SIRT1 in RA synovial cells contributes to chronic inflammation by promoting proinflammatory cytokine production and inhibiting apoptosis.
ObjectivesTo investigate the role of microRNA-193b-3p (miR-193b) in the vascular pathophysiology of systemic sclerosis (SSc).MethodsExpression of miR-193b in skin biopsies and fibroblasts from ...patients with SSc and normal healthy (NH) controls were determined by real-time PCR. Transfection with miR-193b precursor and inhibitor were used to confirm targets of miR-193b. Proliferative effects of urokinase-type plasminogen activator (uPA) were determined by water-soluble tetrazolium salt-1 assay and by analysis of proliferating cell nuclear antigen expression. Fluorescence activated cell sorting analysis was performed to investigate the effect of uPA on apoptosis. For inhibition of the uPA-cellular receptor for uPA (uPAR) pathway, uPAR neutralising antibodies and low molecular weight uPA were used.ResultsWe found that miR-193b was downregulated in SSc fibroblasts and skin sections as compared with NH controls. The expression of miR-193b was not affected by major profibrotic cytokines and hypoxia. Induction of miR-193b in SSc fibroblasts suppressed, and accordingly, knockdown of miR-193b increased the levels of messenger RNA and protein for uPA. uPA was found to be upregulated in SSc as compared with NH controls in a transforming growth factor-β dependent manner, and uPA was strongly expressed in vascular smooth muscle cells in SSc skin section. Interestingly, uPA induced cell proliferation and inhibited apoptosis of human pulmonary artery smooth muscle cells, and these effects were independent of uPAR signalling.ConclusionsIn SSc, the downregulation of miR-193b induces the expression of uPA, which increases the number of vascular smooth muscle cells in an uPAR-independent manner and thereby contributes to the proliferative vasculopathy with intimal hyperplasia characteristic for SSc.
In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its ...promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction.
The development of antimicrobial resistance has made it necessary to measure antimicrobial usage in animal production sectors. France is a major European producer of white veal calves, but few data ...were previously available for that sector, even though these young animals are particularly susceptible to infection and considered as a reservoir of antimicrobial resistance. A cross-sectional study was conducted on 186 batches of French calves to estimate the exposure of white veal calves to antimicrobials and identify the potential risk factors related to antimicrobial usage. An indicator of calf exposure was calculated as a count of the number of antimicrobial treatments per calf. The indicator was based on veterinary prescriptions (products, quantity dispensed and dosage prescribed) and the estimated weight of calves at treatment, using the dates of treatment collected from farm registers.
The study showed that calves were exposed to an average of 8.55 antimicrobial treatments (SD: 2.21, range: 2.75–15.86) over the five to six months of the fattening process. Group treatments were predominant (95.8%) and administered by the oral route. The “starting treatments”, given during the first two weeks of the fattening period, were administered systematically (to all the calves in all the farms) and accounted for a third of all treatments. Tetracyclines, polypeptides and macrolides were the most widely used antimicrobials, with respectively 4.32, 1.59 and 1.01 treatments per calf. Only rare uses of 3rd and 4th generation cephalosporins and fluoroquinolones, considered as critically important in human medicine, were reported.
Despite low variability of exposure between farms, a linear mixed-effects model highlighted a higher variability between farmers (ICC=0.14) or veterinarians (ICC=0.12), than between integrators (ICC=0.06). The number of calves per pen, introduced as a fixed effect in the model, was also significant: calves housed in pens of 6–10 and fed in buckets had on average 2.55 more antimicrobial treatments per calf than calves housed in pairs with the same feeding system. The model also highlighted an increase of 1.48 treatments per calf for farms with more than five percent of mortality, versus those with two percent or less.
The present study showed that antimicrobial treatments are numerous in veal calf fattening farms, particularly at the arrival of the animals. Taking into account the development of resistance to antimicrobials, the necessity and the effectiveness of some of these treatments should be re-evaluated.
In this study, we analyzed the methylation status of human promoters in rheumatoid arthritis synovial fibroblasts (RASF). Differentially methylated genes between RASF and osteoarthritis synovial ...fibroblasts (OASF) were identified by methylated DNA immunoprecipitation and hybridization to human promoter tiling arrays. The methylation status was confirmed by pyrosequencing. Gene and protein expression of differentially methylated genes was evaluated with real-time PCR, Western blot, and immunohistochemistry. Chromatin immunoprecipitation was used to measure the gene promoter-associated acetylation and methylation of histones. Transcription factor-specific targets were identified with microarray and luciferase assays. We found that the transcription factor T-box transcription factor 5 (TBX5) was less methylated in rheumatoid arthritis (RA) synovium and RASF than in osteoarthritis (OA) samples. Demethylation of the TBX5 promoter in RASF and RA synovium was accompanied by higher TBX5 expression than in OASF and OA synovium. In RA synovium, TBX5 expression was primarily localized to the synovial lining. In addition, the TBX5 locus was enriched in activating chromatin marks, such as histone 4 lysine 4 trimethylation and histone acetylation, in RASF. In our functional studies, we observed that 790 genes were differentially expressed by 2-6-fold after overexpression of TBX5 in OASF. Bioinformatic analysis of these genes revealed that the chemokines IL-8, CXCL12, and CCL20 were common targets of TBX5 in OASF. Taken together, our data show that TBX5 is a novel inducer of important chemokines in RASF. Thus, we conclude that RASF contribute to the inflammatory processes operating in the pathogenesis of RA via epigenetic control of TBX5.
To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand ...bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.
Bonobos (Pan paniscus) inhabit regions south of the Congo River including all areas between its southerly tributaries. To investigate the genetic diversity and evolutionary relationship among bonobo ...populations, we sequenced mitochondrial DNA from 376 fecal samples collected in seven study populations located within the eastern and western limits of the species' range. In 136 effective samples from different individuals (range: 7-37 per population), we distinguished 54 haplotypes in six clades (A1, A2, B1, B2, C, D), which included a newly identified clade (D). MtDNA haplotypes were regionally clustered; 83 percent of haplotypes were locality-specific. The distribution of haplotypes across populations and the genetic diversity within populations thus showed highly geographical patterns. Using population distance measures, seven populations were categorized in three clusters: the east, central, and west cohorts. Although further elucidation of historical changes in the geological setting is required, the geographical patterns of genetic diversity seem to be shaped by paleoenvironmental changes during the Pleistocene. The present day riverine barriers appeared to have a weak effect on gene flow among populations, except for the Lomami River, which separates the TL2 population from the others. The central cohort preserves a high genetic diversity, and two unique clades of haplotypes were found in the Wamba/Iyondji populations in the central cohort and in the TL2 population in the eastern cohort respectively. This knowledge may contribute to the planning of bonobo conservation.
There is an ongoing debate as to whether the gastrointestinal safety of COX-2 inhibition compared with nonsteroidal antiinflammatory drugs (NSAIDs) may come at the cost of increased cardiovascular ...events. In view of the large number of patients at cardiovascular risk requiring chronic analgesic therapy with COX-2 inhibitors for arthritic and other inflammatory conditions, the effects of selective COX-2 inhibition on clinically useful surrogates for cardiovascular disease, particularly endothelial function, need to be determined.
Fourteen male patients (mean age, 66+/-3 years) with severe coronary artery disease (average of 2.6 vessels with stenosis >75%) undergoing stable background therapy with aspirin and statins were included. The patients received celecoxib (200 mg BID) or placebo for a duration of 2 weeks in a double-blind, placebo-controlled, crossover fashion. After each treatment period, flow-mediated dilation of the brachial artery, high-sensitivity C-reactive protein, oxidized LDL, and prostaglandins were measured. Celecoxib significantly improved endothelium-dependent vasodilation compared with placebo (3.3+/-0.4% versus 2.0+/-0.5%, P=0.026), whereas endothelium-independent vasodilation, as assessed by nitroglycerin, remained unchanged (9.0+/-1.6% versus 9.5+/-1.3%, P=0.75). High-sensitivity C-reactive protein was significantly lower after celecoxib (1.3+/-0.4 mg/L) than after placebo (1.8+/-0.5 mg/L, P=0.019), as was oxidized LDL (43.6+/-2.4 versus 47.6+/-2.6 U/L, P=0.028), whereas prostaglandins did not change.
This is the first study to demonstrate that selective COX-2 inhibition improves endothelium-dependent vasodilation and reduces low-grade chronic inflammation and oxidative stress in coronary artery disease. Thus, selective COX-2 inhibition holds the potential to beneficially impact outcome in patients with cardiovascular disease.
The acute-phase response is an inflammatory process triggered mainly by the cytokine IL-6. Signaling of IL-6 is transduced by activation of STAT3 (signal transducer and activator of transcription 3), ...which rapidly induces the production of acute-phase proteins such as haptoglobin and fibrinogen. Another target of the IL-6/STAT3 signal transduction pathway is the microRNA cluster miR-17/92. Here, we investigated the interplay of miR-17/92 and STAT3 signaling and its impact on the acute-phase response in primary human hepatocytes and hepatoma (HepG2) cells. Employing a reporter gene system consisting of STAT3-sensitive promoter sequences, we show that the miR-17/92 cluster member miR-18a enhanced the transcriptional activity of STAT3. IL-6 stimulation experiments in miR-18a-overexpressing hepatocytes and HepG2 cells revealed an augmented acute-phase response indicated by increased expression and secretion of haptoglobin and fibrinogen. This effect was due, at least in part, to repression of PIAS3 (protein inhibitor of activated STAT, 3), a repressor of STAT3 activity, which we identified as a novel direct target of miR-18a. Finally, we demonstrate that the expression of miR-17/92 in primary hepatocytes and HepG2 cells is modulated by IL-6. Our data reveal, for the first time, a microRNA-mediated positive feedback loop of IL-6 signal transduction leading to an enhanced acute-phase response in human hepatocytes.
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