Relevance of grade 1 gray-scale ultrasound findings in wrists and small joints to the assessment of subclinical synovitis in rheumatoid arthritis.

OBJECTIVE: To investigate the clinical relevance of grade 1 findings on gray-scale ultrasound (GSUS) of the joints in patients with rheumatoid arthritis (RA). METHODS: We examined the wrists and small joints of 100 patients with early or established RA and 30 healthy controls, using GSUS and power Doppler ultrasound (PDUS). Independent clinical assessment of all joints for tenderness and swelling according to the European League Against Rheumatism examination technique was performed. Joints with grade 1 findings on GSUS were identified, and associations with swelling, pain, and findings on PDUS were assessed. Grade 1 findings on GSUS in patients with early RA were reassessed after 6 months of antirheumatic treatment. RESULTS: Grade 1 results represented the majority of all GSUS findings in patients with RA and were also frequently recorded in healthy controls. Grade 1 GSUS findings were not associated with tenderness, swelling, or positive results on PDUS. In comparison to joints with grade 2 and grade 3 findings on GSUS, joints with grade 1 findings were less likely to respond to treatment. CONCLUSION: The present results indicate that grade 1 findings on GSUS have limited clinical relevance.

The ABC transporter family efflux pump PvdRT-OpmQ of Pseudomonas putida KT2440: purification and initial characterization.

Tripartite efflux systems of the ABC-type family transport a variety of substrates and contribute to the antimicrobial resistance of Gram-negative bacteria. PvdRT-OpmQ, a member of this family, is thought to be involved in the secretion of the newly synthesized and recycled siderophore pyoverdine in Pseudomonas species. Here, we purified and characterized the inner membrane component PvdT and the periplasmic adapter protein PvdR of the plant growth-promoting soil bacterium Pseudomonas putida KT2440. We show that PvdT possesses an ATPase activity that is stimulated by the addition of PvdR. In addition, we provide the first biochemical evidence for direct interactions between pyoverdine and PvdRT.

The RND efflux system ParXY affects siderophore secretion in Pseudomonas putida KT2440.

IMPORTANCE: Gram-negative bacteria from the Pseudomonas group are survivors in various environmental niches. For example, the bacteria secrete siderophores to capture ferric ions under deficiency conditions. Tripartite efflux systems are involved in the secretion of siderophores, which are also important for antibiotic resistance. For one of these efflux systems, the resistance-nodulation-cell division transporter ParXY from the model organism Pseudomonas putida KT2440, we show that it influences the secretion of the siderophore pyoverdine in addition to its already known involvement in antibiotic resistance. Phenotypically, its role in pyoverdine secretion is only apparent when other pyoverdine secretion systems are inactive. The results confirm that the different tripartite efflux systems have overlapping substrate specificities and can at least partially functionally substitute for each other, especially in important physiological activities such as supplying the cell with iron ions. This fact must be taken into account when developing specific inhibitors for tripartite efflux systems.

Resistance to Bipyridyls Mediated by the TtgABC Efflux System in Pseudomonas putida KT2440.

Resistance-nodulation-division (RND) transporters are involved in antibiotic resistance and have a broad substrate specificity. However, the physiological significance of these efflux pumps is not fully understood. Here, we have investigated the role of the RND system TtgABC in resistance to metal ion chelators in the soil bacterium Pseudomonas putida KT2440. We observed that the combined action of an RND inhibitor and the chelator 2,2'-bipyridyl inhibited bacterial growth. In addition, the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl and natural bipyridyl derivatives such as caerulomycin A, indicating that TtgABC is required for detoxification of compounds of the bipyridyl family. Searching for the basis of growth inhibition by bipyridyls, we found reduced adenosine triphosphate (ATP) levels in the ttgB mutant compared to the wild type. Furthermore, the expression of genes related to iron acquisition and the synthesis of the siderophore pyoverdine were reduced in the mutant compared to the wild type. Investigating the possibility that 2,2'-bipyridyl in the ttgB mutant mediates iron accumulation in cells (which would cause the upregulation of genes involved in oxidative stress via the Fenton reaction), we measured the expression of genes coding for proteins involved in intracellular iron storage and the response to oxidative stress. However, none of the genes was significantly upregulated. In a further search for a possible link between 2,2'-bipyridyl and the observed phenotypes, we considered the possibility that the ion chelator limits the intracellular availability of metabolically important metal ions. In this context, we found that the addition of copper restores the growth of the ttgB mutant and the production of pyoverdine, suggesting a relationship between copper availability and iron acquisition. Taken together, the results suggest that detoxification of metal chelating compounds of the bipyridyl family produced by other bacteria or higher ordered organisms is one of the native functions of the RND efflux pump TtgABC. Without the efflux pump, these compounds may interfere with cell ion homeostasis with adverse effects on cell metabolism, including siderophore production. Finally, our results suggest that TtgABC is involved in resistance to bile salts and deoxycholate.

PvdRT-OpmQ and MdtABC-OpmB efflux systems are involved in pyoverdine secretion in Pseudomonas putida KT2440.

Fluorescent pseudomonads produce and secrete a siderophore termed pyoverdine to capture iron when it becomes scarce. The molecular basis of pyoverdine secretion is only partially understood. Here, we investigate the role of the putative PvdRT-OpmQ and MdtABC-OpmB efflux systems in pyoverdine secretion in the soil bacterium Pseudomonas putida KT2440. Expression from the respective promoters is stimulated by iron limitation albeit to varying degrees. Deletion of pvdRT-opmQ leads to reduced amounts of pyoverdine in the medium and decreased growth under iron limitation. Deletion of mdtABC-opmB does not affect growth. However, when both systems are deleted, strong effects on growth and pyoverdine secretion (yellow colony phenotype, less pyoverdine in medium, more pyoverdine in the periplasm) are observed. Overexpression of pvdRT-opmQ causes the opposite effect. These results provide first evidence for an involvement of the multidrug efflux system MdtABC-OpmB in pyoverdine secretion. In addition, the PvdRT-OpmQ system was shown to contribute to pyoverdine secretion in P. putida KT2440, extending previous investigations on its role in Pseudomonas species. Since the double deletion mutant still secrets pyoverdine, at least one additional efflux system participates in the transport of the siderophore. Furthermore, our results suggest a contribution of both efflux systems to ampicillin resistance.

WNT5A is induced by inflammatory mediators in bone marrow stromal cells and regulates cytokine and chemokine production.

WNT5A has recently been implicated in inflammatory processes, but its role as a bone marrow stromal cell (BMSC)-derived mediator of joint inflammation in arthritis is unclear. Here, we investigated whether inflammatory stimuli induce WNT5A in BMSC to control inflammatory responses. WNT5A levels were determined in human BMSC after stimulation with lipopolysaccharide (LPS) or tumor necrosis factor α (TNF-α,) and in synovial cells and tissue of patients with rheumatoid arthritis (RA) and human TNF-α transgenic (hTNFtg) mice. A microarray analysis of WNT5A-treated murine osteoblasts was performed using Affymetrix gene chips. The regulation of cytokine/chemokine expression was confirmed by qPCR, ELISA, and Luminex technology in BMSC after stimulation with WNT5A or WNT5A knockdown. Relevant signaling pathways were identified using specific inhibitors. Migration of MACS-purified T lymphocytes and monocytes was assessed using the FluoroBlok system. WNT5A expression was increased threefold in BMSC after stimulation with LPS or TNF-α. Synovial fibroblasts from patients with RA showed a twofold increase of WNT5A expression compared with control cells, and its expression was highly induced in the synovial tissue of patients with RA and hTNFtg mice. Microarray analysis of WNT5A-treated osteoblasts identified cytokines and chemokines as targets. The induction of IL-1ß, IL-6, CCL2, CCL5, CXCL1, and CXCL5 by WNT5A was confirmed in BMSC and depended on the activation of the NF-κB, mitogen-activated protein (MAPK), and Akt pathways. Accordingly, knockdown of WNT5A markedly reduced the basal and LPS-induced cytokine/chemokine production. Finally, migration of monocytes and T cells toward the supernatant of WNT5A-treated BMSC was increased by 25% and 20%, respectively. This study underlines the critical role of BMSC-derived WNT5A in the regulation of inflammatory processes and suggests its participation in the pathogenesis of RA.

Dissociation of osteogenic and immunological effects by the selective glucocorticoid receptor agonist, compound A, in human bone marrow stromal cells.

Glucocorticoids (GCs) regulate various physiological processes, including bone remodeling. Whereas physiological amounts of GCs are required for proper human osteoblast differentiation, prolonged exposure to GCs leads to substantial bone loss in vivo predominantly by inhibiting osteoblast functions. Compound A (CpdA) is a novel GC receptor modulator with the potential of an improved benefit/risk profile. Here we tested the osteoimmunological effects of CpdA on primary human osteoblasts and their paracrine interactions with osteoclasts. To assess the antiinflammatory potential of CpdA in human bone marrow stromal cell (BMSC)-derived osteoblasts, cells were stimulated with lipopolysaccharide and cytokine expression was determined. Similar to dexamethasone (DEX), CpdA profoundly suppressed lipopolysaccharide-induced TNF-α (-63%), IL-1ß (-38%), and IL-6 (-36%) (P < 0.05) mRNA levels. Of note, CpdA failed to induce osteogenic differentiation of BMSCs, whereas DEX and budesonide enhanced matrix mineralization an d increased runt-related transcription factor 2 and alkaline phosphatase mRNA levels up to 5-fold in a dose-dependent manner. Interestingly, each substance promoted cell proliferation by 7-10% and suppressed apoptosis by 25-30% at low concentrations and early differentiation stages, whereas high concentrations (1 µm) suppressed proliferation and stimulated apoptosis in mature osteoblasts. Finally, CpdA did not increase the receptor activator of nuclear factor-κB ligand to osteoprotegerin mRNA ratio as compared with DEX and did not stimulate the formation of osteoclasts in coculture with BMSCs. In summary, CpdA displays dissociated osteogenic and immunological effects in human BMSCs that are distinct from those of conventional GCs. Whether the specific osteoimmunological profile of CpdA translates into a relevant in vivo effect needs to be further explored.

Interleukin-4 and interleukin-13 stimulate the osteoclast inhibitor osteoprotegerin by human endothelial cells through the STAT6 pathway.

INTRODUCTION: Endothelial cells of the bone vasculature modulate development, remodeling, and repair of bone by secreting osteotropic cytokines and hormones, which can act on osteoblastic and osteoclastic lineage cells. RANKL is the essential factor for differentiation, activation, and survival of osteoclasts, whereas osteoprotegerin (OPG) is a soluble decoy receptor and inhibitor for RANKL. MATERIALS AND METHODS: In this study, we analyzed the regulation of OPG by T helper 2 (Th2) cytokines interleukin (IL)-4 and the closely related IL-13 in human umbilical vein endothelial cells (HUVECs), the underlying signaling pathway, and its functional relevance on osteoclastic resorption. RESULTS: IL-4 and IL-13 induced OPG mRNA levels and protein secretion in HUVEC by up to 4-fold in a dose- and time-dependent fashion (maximum effect after 48 h and at 10 ng/ml). Activation of the transcription factor STAT6 preceded IL-4-induced OPG expression, and blockade of IL-4-induced STAT6 activation by the phospholipase C-specific inhibitor D609 decreased OPG expression. Soluble IL-4 receptor (sIL-4R) dose-dependently abolished both IL-4-induced STAT6 phosphorylation and OPG expression. RANKL stimulated the activity of osteoclasts, which was antagonized by HUVEC-derived supernatant containing OPG. The inhibitory effect on osteoclastogenesis was completely and specifically abrogated by a neutralizing OPG antibody in unstimulated HUVEC supernatant and partially in IL-4-stimulated HUVEC supernatant. CONCLUSIONS: In summary, IL-4 and IL-13 induced OPG expression through activation of STAT6 in endothelial cells, and HUVEC-derived OPG is an IL-4/IL-13-induced inhibitor of osteoclastic resorption. These data underline the impact of Th2 cytokines on bone resorption through modulation of endothelial cell-derived cytokines.