Synovial fluid levels of bradykinin correlate with biochemical markers for cartilage degradation and inflammation in knee osteoarthritis.

OBJECTIVE: To determine the content of bradykinin (BK) and markers of cartilage degradation and inflammation in the synovial fluid (SF) of patients with knee osteoarthritis (OA), and to evaluate correlations with biomarkers or clinical parameters. METHODS: SFs were obtained from 30 patients with knee OA. Levels of basal and generated BK, cartilage oligomeric matrix protein (COMP), interleukin (IL) 1, IL-6, IL-8 and matrix metalloprotease (MMP) 1, MMP-3, MMP-13 and sulfated glycosaminoglycans (GAGs) were measured by enzyme-linked immunosorbent assay (ELISA) or colorimetric assays. RESULTS: The mean concentration of basal BK (in the presence of peptidase and protease inhibitors to avoid degradation and de novo formation of BK) was 422 pg/ml (95% confidence interval, CI, 281-563) whereas that of in vitro generated BK (in the presence of peptidase inhibitors SFs were incubated 60 min at 37°C to measure the potential capability to generate BK) was 3427 pg/ml (2591-4264). The content of MMP-13, IL-1α, and IL-1ß was under assay sensitivity. Basal BK levels positively correlated (Spearman's rank correlation) with GAGs (40 µg/ml, 26-54, r = 0.4834, P = 0.0308) and IL-6 (553 pg/ml, 171-935, r = 0.3946, P = 0.0377) similarly to the generated BK (GAGs, r = 0.4563, P = 0.0431; IL-6, r = 0.5605, P = 0.0019). Statistical analysis of basal BK and biomarkers was significant (P = 0.0483). When applying a stepwise logistic regression analysis considering biomarkers together with clinical parameters, results indicated that K/L radiographic OA grade and COMP improved the model (P = 0.0032). CONCLUSION: The presence of BK in the knee OA SF and its correlations with cartilage degradation and inflammation markers of OA support its participation in OA pathology.

Fasitibant prevents the bradykinin and interleukin 1ß synergism on prostaglandin E2 release and cyclooxygenase 2 expression in human fibroblast-like synoviocytes.

This study investigates the effect of the selective and potent B(2) receptor antagonist fasitibant (MEN16132) on the proinflammatory effect of bradykinin (BK) and its interaction with interleukin 1ß (IL-1ß) in human synoviocytes. PGE(2) content was detected in the surnatants and COX-2 and COX-1 gene and protein expression determined in the cells. Radioligand binding ([(3) H]BK) and BK-induced inositolphosphate experiments were performed. Incubation of synoviocytes with BK induced a sustained production of PGE(2) and transient COX-2 gene expression that were prevented by pretreatment with fasitibant (1 µM, 30 min preincubation). IL-1ß increased PGE(2) release and COX-2 expression more than BK alone. The combined treatment of cells with BK and IL-1ß induced an even increase of released PGE(2) and COX-2 gene and protein expression indicating a synergistic rather than an additive effect, not related to an increase of B(2) receptors density or its coupling. These potentiating effects of BK on PGE(2) production and increased COX-2 expression produced by IL-1ß were B(2)-receptor-mediated as fasitibant could prevent them. None of the treatments induced changes in the COX-1 expression. The synergistic PGE(2) production was abolished by the specific NF-kappaB inhibitor (BAY-117085), whereas specific inhibitors for the p38 (SB203580), JNK (SP600125), and ERK1/2 (PD98059) mitogen-activated protein kinases could prevent the prostanoid release. BK can potentiate the COX-2 gene expression and consequent prostanoid production induced by IL-1ß. The prevention of this synergism by fasitibant indicates BK B(2) receptor blockade as an alternative symptomatic therapy for osteoarthritis.

Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B2 receptor.

BACKGROUND AND PURPOSE: Icatibant is a well-known kinin B2 receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B2 receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists. EXPERIMENTAL APPROACH: Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B2 receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B2 receptors. KEY RESULTS: Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B2 receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B2 receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B2 receptor complex is proposed. CONCLUSIONS AND IMPLICATIONS: MEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

Bradykinin and B2 receptor antagonism in rat and human articular chondrocytes.

BACKGROUND AND PURPOSE: In osteoarthritis (OA), bradykinin (BK) is known to contribute to pain and synovitis, but not to cartilage degradation. Here, we investigated effects of BK and its antagonists on chondrocytes, cells involved in cartilage homeostasis. EXPERIMENTAL APPROACH: BK receptor density and affinities of BK, its analogues and antagonists were measured in cultured human and rat chondrocytes by radioligand binding. Effects of BK were assessed by accumulation of inositol phosphates (IP) and release of interleukin (IL)-6 and IL-8. KEY RESULTS: Density of [³H]-BK binding sites was higher (13-30-fold) and BK evoked a greater (48-fold) IP production, in human than in rat chondrocytes. The BK B2 receptor antagonists MEN16132 and icatibant displayed similar binding affinity. MEN16132 was 40-fold more potent than icatibant in the IP assay. In human chondrocytes, BK increased release (over 24 h) of IL-6 and IL-8, effects blocked by MEN16132 but not by the B1 receptor antagonist Lys-[Leu8][desArg9]BK. BK-induced release of IL-6, but not of IL-8, was partially inhibited by indomethacin (10 µM) and nordihydroguaiaretic acid (10 µM). Antagonists for the prostanoid EP receptors (AH6809 10 µM; L-798,196, 200 nM; L-161,982, 1 µM) were ineffective. Dexamethasone (100 nM) partially inhibited release of both IL-6 and IL-8. Inhibitors of intracellular downstream signalling pathways (SB203580 10 µM; PD98059, 30 µM; SP600125, 30 µM; BAY-117085, 5 µM) indicated the involvement of p38 MAPK and the activation of NF-κB. CONCLUSION AND IMPLICATIONS: BK mediated inflammatory changes and cartilage degradation and B2 receptor blockade would, therefore, be a potential treatment for OA.

Novel effects mediated by bradykinin and pharmacological characterization of bradykinin B2 receptor antagonism in human synovial fibroblasts.

BACKGROUND AND PURPOSE: Bradykinin (BK) and B2 receptors have been implicated in the pathophysiology of osteoarthritis (OA), and synovitis is one of its hallmarks. Here, the selective B2 receptor antagonists MEN16132 and icatibant have been pharmacologically characterized in human synovial cells. EXPERIMENTAL APPROACH: Radioligand and functional studies (inositol phosphate (IP) accumulation, interleukin (IL)-6 and IL-8 release) were performed in cultured synoviocytes. KEY RESULTS: [3H]-BK saturation studies indicated receptor density (Bmax) and K(d) values of 121,550 sites per cell and 1.14 nM respectively. In synoviocytes, MEN16132 (pK(I) 8.9) was threefold more potent than icatibant (pK(I) 8.4). Both antagonists showed competitive antagonism in the BK-induced IP assay (control EC50 0.45 nM), with pK(B) values of 9.9 (MEN16132) and 8.1 (icatibant). 24h incubation with BK induced IL-6 (EC50 216 nM) and IL-8 (EC50 53 nM) release. Both MEN16132 (IL-6: pIC50 8.1; IL-8: pIC50 8.4) and icatibant (IL-6: pIC50 6.6; IL-8: pIC50 6.7) completely prevented this BK-induced release. Indomethacin did not affect the basal or the IL-6/IL-8 release induced by BK, whereas nordihydroguaiaretic acid decreased the basal release, although BK still increased IL-6 and IL-8 production. BK-induced IL-8 release was attenuated by inhibitors of phospholipase C (U73122), p38 (SB203580), JNK (SP600125), ERK 1/2 (PD98059) MAPKs, phosphoinositide 3-kinase (LY294002), NF-kappaB (BAY-117085) and by the glucocorticoid dexamethasone. CONCLUSIONS AND IMPLICATIONS: Bradykinin via B2 receptors can participate in inflammatory events in synovitis. MEN16132 is a highly potent B2 receptor antagonist capable of blocking pro-inflammatory responses to BK evoked in human synoviocytes.

Comparative antagonist pharmacology at the native mouse bradykinin B2 receptor: radioligand binding and smooth muscle contractility studies.

BACKGROUND AND PURPOSE: The aim was to characterize the recently discovered non-peptide antagonist MEN16132 at the mouse B2 receptor, relative to other antagonists. EXPERIMENTAL APPROACH: [3H]-BK binding experiments used mouse lung and ileum tissue membranes and antagonist potency was measured in the isolated ileum contractility assay. KEY RESULTS: Two BK binding sites resulted from saturation and homologous competition experiments. A role for the B1 receptor was excluded because of the poor affinity of B1 receptor ligands (pIC50<5). MEN16132, and the other reference antagonists, inhibited only one portion of BK specific binding, and the rank order of potency was (pIC50): Icatibant (lung 10.7; ileum 10.2)=MEN11270 (lung 10.4; ileum 9.9)=MEN16132 (lung 10.5; ileum 9.9).>LF16-0687 (lung 8.9; ileum 8.8)>FR173657 (lung 8.6; ileum 8.2). BK homologous curves performed with lung membranes after treatment with the antagonist MEN16132 or Icatibant (10 nM) displayed only the low affinity site. The functional antagonism by MEN16132 (pA2 9.4) and Icatibant (pA2 9.1), towards BK (control EC50 6.1 nM) induced ileum contractions, was concentration-dependent and surmountable, but the Schild plot slope was less than unity. CONCLUSIONS AND IMPLICATIONS: In mouse tissue, radiolabelled BK recognizes two binding sites and B2 receptor antagonists can compete only for the higher affinity one. The pharmacological profile of the novel non-peptide antagonist MEN16132 indicates that it exhibits subnanomolar affinity and potency for the mouse B2 receptor and is suitable for further characterization in in vivo pathophysiological models.

A survey on the use of antibacterial in orthopaedic surgery.

The aim of this study is the comprehensive (pre-operative, operative and post-operative) evaluation of the use of antibacterial drugs on a group of patients subjected to orthopaedical surgery. The data regarding 1000 orthopaedic surgical operations, corresponding to 1000 non-urgent patients, have been examined. This study reveals the incongruity in the use of antibacterial drugs in the orthopaedic surgical activity mainly in operative prophylaxis, but also the administration during the intra-operative period, and the use of the same drugs in the post operative period.

Prevalence of HCV infection among health care workers in a hospital in central Italy.

Health care workers are at risk of acquiring transmissible diseases. Controversial results have been reported about HCV. The aims of the present study are to assess the prevalence of HCV in health care workers in Pistoia General Hospital (central Italy) and to compare prevalence with other groups, particularly with a sample of the general population. Serum samples collected from 511 health care employees engaged in direct clinical task and 222 clerical and nurse school attendees have been tested by ELISA and confirmed by RIBA. Total seroprevalence was 3.8%:4.7% in the first group; 1.8% in the second group. The data showed a slight increase in prevalence by age and not by length of stay in the health care workers' group. Comparison of HCV prevalence with blood donors from the province of Pistoia and the general population from Sersale (Catanzaro, southern Italy), restricted to under-40 subjects, indicates an increasing trend in this order: Blood donors, general population, clerical and nursing school attendees, health care workers' group with the highest value (3.4%). A cohort study is needed to evaluate pattern of HCV seroconversion.

Encephalitis caused directly by Mycoplasma pneumoniae.

A case of non-fatal encephalitis in a 21-y-old immunocompetent woman is described. High titre serum antibodies against Mycoplasma pneumoniae were found. In addition, Mycoplasma pneumoniae DNA was detected in the cerebrospinal fluid by polymerase chain reaction. Neuroimaging findings by magnetic resonance and computed tomographic scanning of the brain, and laboratory investigations, including a search for serum antibodies to gangliosides, did not support an immune-mediated mechanism. No other pathogens were found. These results strongly suggest that the encephalitis was caused directly by Mycoplasma pneumoniae invasion of the central nervous system. They also indicate that such pathogenetic mechanism may sometimes be sufficient to explain neurological manifestations occurring during the course of Mycoplasma pneumoniae infection. The consequences for therapy are discussed.

Serum NSE, CEA, CT, CA 15-3 levels in human lung cancer.

The significance of neuron specific enolase (NSE) was investigated in comparison with other tumor markers (CEA, CT, CA 15-3) used in the diagnosis and treatment monitoring of lung cancer. As previously described, the calcitonin assay proved to have very low sensitivity for small cell lung cancer (SCLC). The serum NSE assay was, however, shown to be a useful diagnostic aid for discrimination between histologically different lung cancers, and therefore this assay may be a valuable tool for treatment monitoring in SCLC patients. CA 15-3, also an unspecific marker, showed similar sensitivity to the NSE assay in SCLC patients, the sensitivity being higher than CEA in non small cell lung cancer (NSCLC).

[Glycosaminoglycans in pleural effusions. Preliminary results]. / I glicosaminoglicani nei versamenti pleurici. Risultati preliminari.

Distinction between benign and malignant pleural effusions is often a very difficult problem. Glycosaminoglycans play an important role in cellular proliferation and differentiation. The authors examined glycosaminoglycans in 17 samples, 11 malignant and 7 benign. Separation of glycosaminoglycans has been achieved by centrifugation-addition of trichloracetic acid-centrifugation-addition of cold ethanol. Glycosaminoglycans have been analyzed by electrophoresis on cellulose acetate sheets. 6 malignant samples had higher levels of HA and CS than standard. The authors distinguished benign effusions in two subgroups: inflammatory and cardiac. Among inflammatory, 3 had higher levels of HS and 1 of HA and CS. Further investigations are necessary to elucidate possible different patterns.