Oxygen tension modulates the effects of TNFα in compressed chondrocytes.

OBJECTIVE AND DESIGN: Oxygen tension and biomechanical signals are factors that regulate inflammatory mechanisms in chondrocytes. We examined whether low oxygen tension influenced the cells response to TNFα and dynamic compression. MATERIALS AND METHODS: Chondrocyte/agarose constructs were treated with varying concentrations of TNFα (0.1-100 ng/ml) and cultured at 5 and 21 % oxygen tension for 48 h. In separate experiments, constructs were subjected to dynamic compression (15 %) and treated with TNFα (10 ng/ml) and/or L-NIO (1 mM) at 5 and 21 % oxygen tension using an ex vivo bioreactor for 48 h. Markers for catabolic activity (NO, PGE2) and tissue remodelling (GAG, MMPs) were quantified by biochemical assay. ADAMTS-5 and MMP-13 expression were examined by real-time qPCR. 2-way ANOVA and a post hoc Bonferroni-corrected t test were used to analyse data. RESULTS: TNFα dose-dependently increased NO, PGE2 and MMP activity (all p < 0.001) and induced MMP-13 (p < 0.05) and ADAMTS-5 gene expression (pp < 0.01) with values greater at 5 % oxygen tension than 21 %. The induction of catabolic mediators by TNFα was reduced by dynamic compression and/or L-NIO (all p < 0.001), with a greater inhibition observed at 5% than 21 %. The stimulation of GAG synthesis by dynamic compression was greater at 21 % than 5 % oxygen tension and this response was reduced with TNFα or reversed with L-NIO. CONCLUSIONS: The present findings revealed that TNFα increased production of NO, PGE2 and MMP activity at 5 % oxygen tension. The effects induced by TNFα were reduced by dynamic compression and/or the NOS inhibitor, linking both types of stimuli to reparative activities. Future therapeutics should develop oxygen-sensitive antagonists which are directed to interfering with the TNFα-induced pathways.

C-type natriuretic peptide signalling drives homeostatic effects in human chondrocytes.

Signals induced by mechanical loading and C-type natriuretic peptide (CNP) represent chondroprotective routes that may potentially prevent osteoarthritis (OA). We examined whether CNP will reduce hyaluronan production and export via members of the multidrug resistance protein (MRP) and diminish pro-inflammatory effects in human chondrocytes. The presence of interleukin-1ß (IL-1ß) increased HA production and export via MRP5 that was reduced with CNP and/or loading. Treatment with IL-1ß conditioned medium increased production of catabolic mediators and the response was reduced with the hyaluronan inhibitor, Pep-1. The induction of pro-inflammatory cytokines by the conditioned medium was reduced by CNP and/or Pep-1, αCD44 or αTLR4 in a cytokine-dependent manner, suggesting that the CNP pathway is protective and should be exploited further.

Cytokine release assays for the prediction of therapeutic mAb safety in first-in man trials--Whole blood cytokine release assays are poorly predictive for TGN1412 cytokine storm.

The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs.

Meropenem stability to beta-lactamase hydrolysis and comparative in vitro activity against several beta-lactamase-producing Gram-negative strains.

The interaction between meropenem and class A, B, C and D beta-lactamases was studied by a spectrophotometric method. Class A, C and D beta-lactamases were unable to confer in vitro resistance to carbapenems. Surprisingly, several class B metallo-beta-lactamases expressed in Escherichia coli failed to confer resistance when a conventional inoculum (105 cfu/mL) was used.

Hydrolysis of glycine-containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference for glycine-rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 +/- 9 min(-1) x mmol(-1) x L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation-mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG(cleavage site)A being especially sensitive to hydrolysis. Both positions P2 and P'2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P'3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.

Capillary electrophoresis in the assay of the hydrolysis of glycine-containing peptides by a protease from Pseudomonas aeruginosa.

A rapid and simple capillary zone electrophoresis (CZE) method for measuring the activity of a minor protease from Pseudomonas aeruginosa is described. When glycine-containing oligopeptides were used as substrates, it was possible to separate and quantify substrate and products. Moreover oligopeptide hydrolysates were analysed by fast atom bombardment mass spectrometry providing the sites of splitting of the substrates. By comparison with CZE calibration curves constructed with reference peptides, the initial rates of hydrolysis were calculated. The method, validated for pentaglycine hydrolysis, was also used for the analysis of reaction mixtures and for monitoring the enzymic hydrolysis of various peptides in order to investigate enzyme specificity.