Lobar lung resection in elderly patients with non-small cell lung carcinoma: impact of cardiac comorbidity on surgical outcome.

PRINCIPLES: The aim of this study was to evaluate the impact of cardiac comorbidity on the perioperative morbidity and mortality after lobar lung resection for lung cancer in patients aged 70 years and older. METHODS: The medical records of all 68 patients ≥70 years, who underwent lobar lung resection for non-small cell lung cancer (NSCLC) from 2003 to 2011 at our department, were reviewed retrospectively. Twenty-two patients with a mean age of 76.3 years had cardiac comorbidities (Group A) including previous cardiac operations in 4 patients, previous myocardial infarction in 5 patients, previous coronary stent insertion in 3 patients, medically treated coronary artery disease in 10 patients and medically treated valvular heart disease in 2 patients whereas 46 patients (mean age = 74.5 years) had no previous cardiac history (Group B). RESULTS: There were no significant differences in postoperative morbidity (13.6% in Group A vs. 17.4% in Group B) between both groups. No in-hospital mortality was observed in both groups. CONCLUSION: In our experience lobar lung resections for NSCLC in elderly patients with cardiac comorbidity seem to be a safe therapy option for this increasing subpopulation. Though, our retrospective data with the small number of study objects require further confirmation in larger prospective trials.

Value and pitfalls of neurophysiological monitoring in thoracic and thoracoabdominal aortic replacement and endovascular repair.

BACKGROUND: The aim of our study was to analyze the neurophysiological monitoring method with regard to its potential problems during thoracic and thoracoabdominal aortic open or endovascular repair. Furthermore, preventive strategies to the main pitfalls with this method were developed. METHODS: Between 11/2000 and 05/2007 in 97 cases open surgery or endovascular stentgraft-implantation was performed on the thoracic or thoracoabdominal aorta. Intraoperatively, neurophysiologic motor- and somatosensory-evoked potentials were monitored. RESULTS: Our cases were divided into four groups: event-free patients with normal potentials (A, 63 cases), with correlation of modified evoked potentials and neurological outcome (B, 14 cases), false-positive or false-negative results (C, 4 cases), and medication interaction or technical issues (D, 16 cases). We observed a sensitivity of 93 % and a specificity of 96 % for the neurophysiological monitoring. CONCLUSIONS: Monitoring spinal cord function during surgical and endovascular interventions on the thoracic and thoracoabdominal aorta is necessary. It can be made more effective by precisely analyzing the interference factors of the neurophysiological monitoring method itself. Successful strategies of immediate troubleshooting could be identified.

Biological actions of curcumin on articular chondrocytes.

OBJECTIVES: Curcumin (diferuloylmethane) is the principal biochemical component of the spice turmeric and has been shown to possess potent anti-catabolic, anti-inflammatory and antioxidant, properties. This article aims to provide a summary of the actions of curcumin on articular chondrocytes from the available literature with the use of a text-mining tool. We highlight both the potential benefits and drawbacks of using this chemopreventive agent for treating osteoarthritis (OA). We also explore the recent literature on the molecular mechanisms of curcumin mediated alterations in gene expression mediated via activator protein 1 (AP-1)/nuclear factor-kappa B (NF-kappaB) signalling in chondrocytes, osteoblasts and synovial fibroblasts. METHODS: A computer-aided search of the PubMed/Medline database aided by a text-mining tool to interrogate the ResNet Mammalian database 6.0. RESULTS: Recent work has shown that curcumin protects human chondrocytes from the catabolic actions of interleukin-1 beta (IL-1beta) including matrix metalloproteinase (MMP)-3 up-regulation, inhibition of collagen type II and down-regulation of beta1-integrin expression. Curcumin blocks IL-1beta-induced proteoglycan degradation, AP-1/NF-kappaB signalling, chondrocyte apoptosis and activation of caspase-3. CONCLUSIONS: The available data from published in vitro and in vivo studies suggest that curcumin may be a beneficial complementary treatment for OA in humans and companion animals. Nevertheless, before initiating extensive clinical trials, more basic research is required to improve its solubility, absorption and bioavailability and gain additional information about its safety and efficacy in different species. Once these obstacles have been overcome, curcumin and structurally related biochemicals may become safer and more suitable nutraceutical alternatives to the non-steroidal anti-inflammatory drugs that are currently used for the treatment of OA.

Biochemical biomarkers of oxidative collagen damage.

Collagens are major constituents of connective tissues in the animal kingdom. During aging and inflammatory-related diseases, the collagen network undergoes oxidation that leads to structural and biochemical alterations within the collagen molecule. Collagen oxidation appears to be a key determinant of aging and a critical physiopathologic mechanism of numerous diseases. Further, the detection of oxidized-collagen peptides seems to be a promising approach for the diagnosis and the prognosis of inflammatory diseases. This chapter reviews the structural and biochemical changes to collagen induced by reactive oxygen and nitrogen species and discusses recent data on the use of collagen-derived biomarkers for measuring oxidative damage.

Curcumin inhibits pro-inflammatory mediators and metalloproteinase-3 production by chondrocytes.

OBJECTIVE AND DESIGN: This study aims to investigate the effects of curcumin (Cur) on the extracellular matrix protein metabolism of articular chondrocytes and on their production of inflammatory mediators. METHODS: Human chondrocytes in alginate beads and human cartilage explants were cultured in the absence or in the presence of interleukin (IL)-1beta (10(-11) M) and with or without Cur (5-20 microM). Nitric oxide (NO) synthesis was measured by the Griess spectrophotometric method; prostaglandin (PG) E(2) by a specific radioimmunoassay; and IL-6, IL-8, aggrecan (Agg), matrix metalloproteinase (MMP)-3, and tissue inhibitor of metalloproteinase (TIMP)-1 by specific enzyme-amplified immunoassays. Proteoglycan degradation was evaluated by the release of (35)S-glycosaminoglycans (GAG) from human cartilage explants. RESULTS: In alginate beads and cartilage explant models, Cur inhibited the basal and the IL-1beta-stimulated NO, PGE(2), IL-6, IL-8, and MMP-3 production by human chondrocytes in a concentration-dependent manner. The TIMP-1 and the Agg productions were not modified. In the basal condition, (35)S-GAG release from cartilage explants was decreased by Cur. CONCLUSIONS: Curcumin was a potent inhibitor of the production of inflammatory and catabolic mediators by chondrocytes, suggesting that this natural compound could be efficient in the treatment of osteoarthritis.

Interleukin-1beta and interleukin-6 disturb the antioxidant enzyme system in bovine chondrocytes: a possible explanation for oxidative stress generation.

OBJECTIVE: Beside matrix metalloproteinases, reactive oxygen species (ROS) are the main biochemical factors of cartilage degradation. To prevent ROS toxicity, chondrocytes possess a well-coordinated enzymatic antioxidant system formed principally by superoxide dismutases (SODs), catalase (CAT) and glutathione peroxidase (GPX). This work was designed to assess the effects of interleukin (IL)-1beta and IL-6 on the enzymatic activity and gene expression of SODs, CAT and GPX in bovine chondrocytes. METHODS: Bovine chondrocytes were cultured in monolayer for 4-96 h in the absence or in the presence of IL-1beta (0.018-1.8ng/ml) or IL-6 (10-100 ng/ml). To study signal transduction pathway, inhibitors of mitogen-activated protein kinases (MAPK) (PD98059, SB203580 and SP600125) (5-20 microM) and nuclear factor (NF)-kappaB inhibitors [BAY11-7082 (1-10 microM) and MG132 (0.1-10 microM)] were used. SODs, CAT and GPX enzymatic activities were evaluated in cellular extract by using colorimetric enzymatic assays. Mn SODs, Cu/Zn SOD, extracellular SOD (EC SOD), CAT and GPX gene expressions were quantified by real-time and quantitative polymerase chain reaction (PCR). RESULTS: Mn SOD and GPX activities were dose and time-dependently increased by IL-1beta. In parallel, IL-1beta markedly enhanced Mn SOD and GPX gene expressions, but decreased Cu/Zn SOD, EC SOD and CAT gene expressions. Induction of SOD enzymatic activity and Mn SOD mRNA expression were inhibited by NF-kappaB inhibitors but not by MAPK inhibitors. IL-6 effects were similar but weaker than those of IL-1beta. CONCLUSIONS: In conclusion, IL-1beta, and to a lesser extend IL-6, dysregulates enzymatic antioxidant defenses in chondrocyte. These changes could lead to a transient accumulation of H(2)O(2) in mitochondria, and consequently to mitochondria damage. These changes contribute to explain the mitochondrial dysfunction observed in osteoarthritis chondrocytes.

Efficacy and frequency of cerebrospinal fluid drainage in operative management of thoracoabdominal aortic aneurysms.

BACKGROUND: Paraplegia remains the most dreaded complication following thoracoabdominal aortic repair. We investigated the efficacy of cerebrospinal fluid drainage as a spinal cord-protecting modality. We also evaluated the correlation between the frequency of cerebrospinal fluid drainage and the Crawford classification. METHODS: Spinal cord function was monitored during 20 open surgical procedures (group I) and 27 stent-graft implantations (group II). Evoked potentials and intracranial pressure were monitored in each operation. If intracranial pressure exceeded 15 mmHg, cerebrospinal fluid was drained. RESULTS: Cerebrospinal fluid drainage was necessary in 75 % of patients in group I (Crawford type I: 33 %, type II: 40 %, type III: 20 %, type IV: 7 %) and in 22 % of patients in group II (Crawford type I: 33 %, type II: 66 %). Evoked potential alterations correlated with an increase in intracranial pressure. Timely cerebrospinal fluid drainage reversed these changes in 72 %. Three patients remained paraplegic. CONCLUSION: Cerebrospinal fluid drainage is a valuable neuroprotective interventional tool to lower the risk of spinal cord ischemia. The combination of neurophysiological monitoring and cerebrospinal fluid drainage optimizes the prevention of paraplegia during aortic repair.

Development of a special balloon occlusion device to prevent adverse events in high-risk patients during open aortic surgery.

OBJECTIVE: To prevent clamp injury that may occur during aortic surgery, we aimed to develop a special balloon occlusion (BO) device to lower the thromboembolic risk in patients with severe atherosclerosis during aortic aneurysm repair. METHODS: The study comprised two test phases: a laboratory-testing series focussing on flexible artificial aortas, and an experimental series conducted on 10 pigs. RESULTS: The device proved to be effective during the laboratory tests and the experiments on pigs. No complications such as intraoperative balloon rupture, dislocation, or occlusion leaks occurred. No damage to the aortic vessels was observed in further histological examinations. CONCLUSIONS: This BO device has the potential to become an alternative to cross-clamping for vascular surgeons in patients with severely atherosclerotic vessels.

Nitric oxide-related products and myeloperoxidase in bronchoalveolar lavage fluids from patients with ALI activate NF-kappa B in alveolar cells and monocytes.

An increased production of NO* and peroxynitrite in lungs has been suspected during acute lung injury (ALI) in humans, and recent studies provided evidence for an alveolar production of nitrated compounds. We observed increased concentrations of nitrites/nitrates, nitrated proteins and markers of neutrophil degranulation (myeloperoxidase, elastase and lactoferrine) in the fluids recovered from bronchoalveolar lavage fluids (BALF) of patients with ALI and correlated these changes to the number of neutrophils and the severity of the ALI. We also observed that BALFs stimulated the DNA-binding activity of the nuclear transcription factor kappa B (NF-kappaB) as detected by electrophoretic mobility shift assay in human alveolar cells (A549) and monocytes (THP1). The level of activation of the NF-kappaB-binding activity was correlated to the concentration of nitrated proteins and myeloperoxidase. Furthermore, in vitro studies confirmed that NO*-derived species (peroxynitrite and nitrites) and the neutrophil enzyme myeloperoxidase by themselves increased the activation of NF-kappaB, thereby arguing for an in vivo pathogenetic role of NO*-related products and neutrophil enzymes to human ALI.

Thoracoabdominal aortic aneurysm repair: interplay of spinal cord protecting modalities.

BACKGROUND: The purpose of this study was to assess the complementary use of different methods of measuring spinal cord perfusion during thoracoabdominal aortic surgery. METHODS: The spinal cords of 28 patients undergoing surgery on the thoracoabdominal aorta were monitored with transcranial electrical stimulation (tcMEP) and somatosensory-evoked potentials (SSEP). Available approaches of spinal cord-protection included: Moderate systemic hypothermia, constant cerebrospinal fluid (CSF) drainage and pressure monitoring, reimplantation of segmental arteries, cardiopulmonary bypass (CPB), and staged clamping. RESULTS: Fourteen of 19 patients (75%) undergoing open surgical treatment (Group I) exhibited loss of tcMEP after proximal aortic clamping. In nine cases (47%), we observed recovery of tcMEP after intraoperative interventions, while two patients subsequently developed paraplegia and three died. Seventeen of 19 patients showed loss of SSEP, with recovery in 12 cases (63%). During stent-graft implantation (Group II), one of nine patients (11%) demonstrated tcMEP loss with intraoperative, intervention-related recovery. The SSEP-recording course remained stable. CONCLUSIONS: tcMEP/SSEP monitoring has proved to be an excellent means of detecting spinal cord ischaemia during surgery on thoracoabdominal aortic aneurysms. The prognostic value of tcMEP monitoring should be considered superior to that of SSEP measurements, because of its direct and rapid response to spinal malperfusion. Through combined neurophysiological monitoring, vital parameter balancing and intraoperative interventions, spinal cord perfusion improves and recovery of tcMEP and SSEP is achievable, reducing the prevalence of postoperative paraplegia.

Comparative effects of IL-1beta and hydrogen peroxide (H2O2) on catabolic and anabolic gene expression in juvenile bovine chondrocytes.

OBJECTIVE: To compare the effects of hydrogen peroxide (H(2)O(2)) to those of interleukin-1beta (IL-1beta) on gene expression in juvenile bovine articular chondrocytes (BAC). The study analyses the activation of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) transcription factors, and the mRNA steady-state levels of the type II collagen, aggrecan core protein matrix, metalloproteinases (MMP-1, -3), and transforming growth factor-beta1 (TGF-beta1) genes. METHODS: Confluent BAC cultures were treated for 3 and 24h with IL-1beta and/or different concentrations of H(2)O(2) (Protocol 1). Following initial treatment, a part of the cells was further subjected to another 24h with medium, in the presence of IL-1beta, to determine the effect of the cytokine on H(2)O(2) pre-treated cells (Protocol 2). Total RNA and nuclear protein extractions were performed to study mRNA steady-state levels (real-time polymerase chain reaction) and AP-1/NF-kappaB DNA binding (Electrophoretic Mobility Shift Assays), respectively. RESULTS: IL-1beta enhanced both AP-1 and NF-kappaB binding, whereas H(2)O(2) only activated AP-1. H(2)O(2) pre-treatment decreased the IL-1beta activation of NF-kappaB. Both H(2)O(2) and IL-1beta down-regulated type II collagen and aggrecan expression and increased that of MMP-1 and -3. When cells were pre-treated with H(2)O(2), followed by IL-1beta, the effects were the same as those observed with H(2)O(2) alone. However, although H(2)O(2) and IL-1beta were capable of increasing TGF-beta1 expression separately, subsequent incubation with both factors led to a partial or total abolition of TGF-beta1 up-regulation. CONCLUSION: The different regulation of NF-kappaB and AP-1 by H(2)O(2) and IL-1beta underlines the distinct roles played by the two transcription factors in the regulation of gene expression. H(2)O(2) and IL-1beta exert similar effects on matrix, MMPs and TGF-beta1 gene expression. However, the association of H(2)O(2) and IL-1beta does not cause synergic effect, and rather leads, in some cases, to an opposite effect. These data provide further insights into the respective roles of reactive oxygen species and cytokine in the pathophysiology of joint diseases.

Improved spinal cord perfusion during thoracoabdominal aortic repair.

OBJECTIVES: The study's aim is to evaluate whether intraoperative neurophysiological monitoring with transcranial motor-evoked potentials (tcMEP) permits early recognition of neuronal lesions, thus making interventions to prevent irreversible spinal cord damage possible. MATERIAL AND METHODS: TcMEP monitoring was carried out in twelve patients (mean age 60 years) during open surgical thoracoabdominal aortic replacement. Current approaches for corrective, spinal cord-protecting interventions consist of: raising distal perfusion by increasing cardiopulmonary bypass (CPB) flow, catecholamine application, reducing central venous pressure, reimplantation of segmental arteries, and cerebrospinal fluid (CSF) drainage. RESULTS: Nine patients exhibited loss of tcMEP after segmental aorta clamping. In five patients we observed a recovery of tcMEP through counteractive measures. Three patients died intraoperatively, one patient presented with postoperative paraplegia and loss of tcMEP. CONCLUSION: TcMEP loss is associated with spinal cord ischaemia, causing postoperative paraplegia. TcMEP monitoring is an excellent method to detect spinal cord ischaemia at an early stage.

Setup of neurophysiological monitoring with tcMEP/SSEP during thoracoabdominal aneurysm repair.

OBJECTIVES: The article describes a procedure for the intraoperative neurophysiological placement of electrodes to control the spinal cord function during thoracoabdominal aortic aneurysm repair. MATERIAL AND METHODS: Intraoperative monitoring is performed by motor-evoked myogenic potentials after transcranial electric stimulation (tcMEP) and somatosensory-evoked potentials (SSEP). In tcMEP, the stimulating percutaneous needle electrodes are placed at C3 and C4 according to the 10 - 20 system for EEG recordings. TcMEP are recorded from the anterior tibial and gastrocnemius muscles on both sides. The SSEP electrodes are placed laterally and caudally onto the malleolus medialis in order to stimulate the tibial nerve. The stimulus is documented via electrodes attached to the scalp within the sensory cortex region. RESULTS: The application of the electrodes is both easy to learn and can be performed without further difficulties. Once attached, the electrodes provide a quick assessment and interpretation of spinal cord function. The identification of external sources of disturbance during the monitoring (e. g. insufficient impedance, unfavourable electrode positioning, and technical interference caused by medical equipment) enables the supervisor to differentiate between normal and abnormal neurological responses. CONCLUSIONS: TcMEP and SSEP allow an adequate, direct, and reliable intraoperative assessment of spinal cord function, enabling the surgeon to diagnose an impending ischaemia and act accordingly. This measurement technique provides the surgical team with a means of integrating neurological aspects during thoracoabdominal aneurysm repair.

Influence of oxygen tension on nitric oxide and prostaglandin E2 synthesis by bovine chondrocytes.

OBJECTIVES: To determine the in vitro effects of oxygen tension on interleukin (IL)-1beta induced nitric oxide (*NO) and prostaglandin E(2) (PGE(2)) production by bovine chondrocytes. DESIGN: Enzymatically isolated bovine chondrocytes were cultured for different periods in suspension in 21 (atmospheric), 5 or 1% (low) oxygen tension and in the absence or in the presence of increased amounts (0.01 to 1nM) of IL-1beta. Nitrite and nitrate concentrations in the culture supernatants were determined by a spectrophotometric method based upon the Griess reaction. PGE(2) production was quantified by a specific radioimmunoassay (RIA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) mRNA steady state levels were also quantified by real-time polymerase chain reaction (PCR). RESULTS: In the absence of IL-1beta, ()NO production remained stable whatever the oxygen tension used. IL-1beta dose-dependently increased *NO production in both atmospheric and low oxygen conditions but the effect was more pronounced in low (1 and 5%) than in atmospheric (21%) oxygen tension (P<0.001). Under low and atmospheric oxygen tension, iNOS gene expression was increased by IL-1beta, but to a lesser extent in 21% than in 1 or 5% oxygen (P<0.01). In the basal condition, bovine chondrocytes spontaneously produced PGE(2) whatever the oxygen tension used. At 21% oxygen, IL-1beta dose-dependently increased PGE(2) production while no significant effect was observed at 1 or 5% oxygen. COX-2 gene expression was significantly upregulated by IL-1beta in both low and atmospheric oxygen tension. No significant difference between oxygen tension conditions was observed. CONCLUSIONS: This study demonstrates that a hypoxic environment fully blocks COX-2 activity but favours iNOS gene expression in chondrocytes culture. These findings indicate that O(2) tension modulates cellular behaviour in culture and supports the concept of chondrocyte culture in low oxygen tension to reproduce in vitro the life conditions of chondrocytes.

Effect of hypoxia and reoxygenation on gene expression and response to interleukin-1 in cultured articular chondrocytes.

OBJECTIVE: To determine the effects of hypoxia and reoxygenation on the metabolism of chondrocytes and their response to interleukin-1beta (IL-1beta). The study included activation of hypoxia-inducible factor 1 (HIF-1), NF-kappaB, and activator protein 1 (AP-1) transcription factors, expression of matrix components and metalloproteases and transforming growth factor beta (TGFbeta) and TGFbeta receptors, and production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). METHODS: Bovine articular chondrocytes (BACs) were cultured to confluency in either 5% O(2) (hypoxia) or 21% O(2) (normoxia) in media supplemented with 10% fetal calf serum (FCS). BACs were preincubated for 18 hours in media with 1% FCS only and then incubated for 24 hours in the presence of IL-1beta. For reoxygenation experiments, cells were treated in the same way in 5% O(2), except that cultures were transferred to normal atmospheric conditions and used after 4 hours for RNA extraction or after 30 minutes for cytoplasmic or nuclear protein extraction. RESULTS: In hypoxic and reoxygenated chondrocytes, we observed strong DNA binding of HIF-1. IL-1beta-induced DNA binding of NF-kappaB and AP-1 was significantly higher in hypoxic and reoxygenated cultures than in normoxia. Greater activation of the MAPKs was also observed with IL-1beta treatment in hypoxia compared with normoxia. Steady-state levels of type II collagen and aggrecan core protein messenger RNA (mRNA) were decreased by IL-1beta in all instances. Matrix metalloprotease 1 (MMP-1) and MMP-3 mRNA were increased by IL-1beta in normoxia and hypoxia, whereas only MMP-3 mRNA was enhanced in reoxygenated cultures. The MMP-2 mRNA level was not significantly affected by IL-1beta in normoxia or hypoxia, whereas it was enhanced in reoxygenated cultures. MMP-9 mRNA was dramatically decreased by IL-1beta only in low oxygen tension. Tissue inhibitor of metalloproteinases 1 (TIMP-1) message was significantly enhanced by the cytokine in most instances, whereas TIMP-2 message was markedly decreased by IL-1beta in reoxygenated cultures. Stimulation of TGFbeta1 expression by IL-1beta was observed only in normal atmospheric conditions. One of the more striking findings of the study was the greater stimulating effect of IL-1beta on NO production observed in hypoxia, which was much higher than in normoxia, whereas the reverse was observed for IL-1beta-induced PGE(2) production. CONCLUSION: Oxygen level and reoxygenation stress significantly modulate gene expression and the response of articular chondrocytes to cytokines such as IL-1beta. In hypoxic conditions, which mimic the in vivo condition of cartilage, the effects of IL-1beta on both synthesis and degradative processes are significantly different from those in normoxia, conditions that are unlikely encountered by chondrocytes in a normal state. In low oxygen tension, high IL-1beta-induced NO production is associated with a significant decrease in PGE(2) synthesis. These data should influence our concept of the role of oxygen in the pathophysiology of joint disease and may help define the best conditions in which to develop bioartificial cartilage.

Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes.

OBJECTIVES: To determine the regulatory effects of reactive oxygen species (ROS) on the expression by human osteoarthritic chondrocytes of interleukin (IL)-1beta, -6 and -8, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene in response to interleukin (IL)-1beta or lipopolysaccharide (LPS). METHODS: Human chondrocytes in monolayer culture were incubated for 3 h with ROS generating molecules such as S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 microM), 3-morpholinosydnonimine (SIN-1, 100 microM), with chemically synthesised peroxynitrite (ONOO-, 10 microM) or hydrogen peroxide (H2O2, 100 microM). After treatment by ROS, chondrocytes were washed and then cultured for the next 24 h with or without lipopolysaccharide LPS (10 microg/ml) or IL-1beta (1.10(-11) M). IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression was analysed by real time and quantitative RT PCR. IL-6, IL-8 and prostaglandin (PG) E2 productions were assayed by specific immunoassays. Nitrite was measured in the culture supernatants by the Griess procedure. RESULTS: LPS and IL-1beta stimulated IL-1beta, IL-6, IL-8, iNOS and COX-2 gene expression. SNAP significantly downregulated LPS induced overall gene expressions, whereas SIN-1 had no effect. ONOO- inhibited iNOS and COX-2 gene expression but not that of the cytokine genes. When chondrocytes were incubated with IL-1beta, SIN-1 and ONOO dramatically decreased all gene expressions while SNAP was inefficient. H2O2 treatment inhibited both LPS and IL-1beta induced gene expressions. CONCLUSIONS: These data provide an evidence that ROS may have anti-inflammatory properties by depressing inflammatory gene expression. Further, we demonstrate that ROS effects are dependent on the nature of radical species and the signalling pathway that is activated. These findings should be taken into consideration for the management of antioxidant therapy in treatment of inflammatory joint diseases.

Regulation by reactive oxygen species of interleukin-1beta, nitric oxide and prostaglandin E(2) production by human chondrocytes.

OBJECTIVES: To determine the effects of two drugs, N-monomethyl-L-arginine (L-NMMA) and N-acetylcysteine (NAC), on interleukin-1beta (IL-1beta), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production by human chondrocytes. The effect of aceclofenac (ACECLO), a non-steroidal antiinflammatory drug (NSAID), was also examined. METHODS: Human chondrocytes were enzymatically isolated from osteoarthritic knee cartilage and then maintained in culture in suspension for 48h in the absence or in the presence of lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5mM), NAC (1mM) or ACECLO (6.10(-6)M). IL-1beta and PGE(2) productions were quantified by specific immunoassays. Nitrite was measured in the culture supernatants by a spectrophotometric method based upon the Griess reaction. Cyclooxygenase-2 (COX-2), inducible NO synthase (iNOS) and IL-1beta gene expressions were quantified by transcription of mRNA followed by real time and quantitative polymerase chain reaction. COX-2 protein expression was analysed by Western blot. RESULTS: LPS markedly increased the expression of IL-1beta, iNOS and COX-2 genes. In parallel, NO(2) and PGE(2) amounts found in the culture supernatants were significantly enhanced whereas IL-1beta was immunologically undetectable. The addition of L-NMMA (0.5mM) fully blocked LPS-induced NO production but greatly increased PGE(2) production, suggesting a negative effect of NO on PGE(2) synthesis. Inversely, NO production was stimulated by NAC while PGE(2) production was not affected. Interestingly, NAC increased the IL-1beta and iNOS mRNA levels but did not significantly modify COX-2 mRNA expression. L-NMMA did not significantly affect the expression of IL-1beta, iNOS and COX-2. The amount of COX-2 protein did not change in the presence of the antioxidants. Finally, ACECLO fully blocked the production of PGE(2) by chondrocytes without affecting the levels of COX-2 mRNA. CONCLUSIONS: The stimulation of IL-1beta, NO and PGE(2) production by LPS is differentially controlled by reactive oxygen species (ROS). In fact, L-NMMA and NAC have different mechanisms of action on the regulation of NO and PGE(2) productions. L-NMMA fully inhibits NO but increases PGE(2) production whereas NAC up-regulates NO but does not modify PGE(2) synthesis. The stimulating effect of L-NMMA on PGE(2) production is not controlled at the transcriptional level. These findings suggest that antioxidant therapy could have different effects according to the oxygen radical species targeted.

Expression of TGF-betas and their receptors is differentially modulated by reactive oxygen species and nitric oxide in human articular chondrocytes.

OBJECTIVES: To study the effects exerted by two antioxidants, N-monomethyl-L-arginine (L-NMMA), as an inhibitor of nitric oxide (NO) synthesis, and N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, on the expression of the major growth factor involved in cartilage repair, TGF-beta, under the three isoforms beta1, beta2 and beta3, and the receptors I and II of this factor, using lipopolysaccharide (LPS)-treated human chondrocytes in culture. METHODS: Suspension cultures of human chondrocytes derived from the knee of osteoarthritic patients were treated for 48 h with lipopolysaccharide (LPS) (10 microg/ml), L-NMMA (0.5 mM) or NAC (1 mM). Nitrite levels were assayed on the culture media using the Griess spectrophotometric method. After total RNA extraction, the expression of inducible NO synthase (iNOS), TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta receptors I and II, was determined by semi-quantitative polymerase chain-reaction (RT-PCR). RESULTS: LPS induced a dramatic increase of both NO production and iNOS mRNA level. The addition of L-NMMA (0.5 mM) abolished NO production without affecting iNOS mRNA levels. In contrast NAC (1 mM) strongly synergized with LPS to stimulate NO synthesis. LPS treatment did not significantly alter TGF-beta1 expression whereas L-NMMA inhibited its production. TGF-beta2 mRNA level was decreased by LPS and was not changed in the presence of L-NMMA. On the other hand, NAC was capable of counteracting the LPS-induced inhibition of TGF-beta2 expression. TGFbeta3 mRNA level was markedly reduced by LPS alone, or with both L-NMMA and NAC. Finally, the expression of TGF-betaRI was slightly increased in the presence of combined LPS and L-NMMA or NAC whereas that of TGFbeta-RII was reduced in the same conditions. CONCLUSIONS: The modulation of TGF-beta system was found to be differentially controlled by NO and ROS productions. Indeed, the control exerted on TGF-beta expression varied according to the isoform: TGF-beta1 mRNA level depends on NO whereas that of TGF-beta2 is regulated by ROS and TGF-beta3 seems to be unaffected by both of them. The expression of TGF-beta receptors appeared to be modulated by NO and ROS levels. The relevance of the present findings to osteoarthritis (OA) physiopathology and the potential use of antioxidant therapy to treat this disease are discussed.

Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN-1).

We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO-) donor 3-morpholino sydnonimine (SIN-1). Cells were incubated overnight with 0.5, 1.0 or 2.0 mM SIN-1, with or without 10-3 M propofol (Diprivan). Cytotoxicity, assessed by measuring the release of pre-incorporated 51Cr, increased when the concentration of SIN-1 increased, and was significantly decreased by 10-3 M propofol (90%, 78% and 28% of protection against 0.5, 1.0 and 2.0 mM SIN-1, respectively). Cell protection against 1 mM SIN-1 was tested with 0.03-1.0 mM propofol and this was compared to tyrosine, a target molecule for peroxynitrite. Propofol protected cells in a dose-dependent manner (r = 0.98; p < 0.001) and was as effective as tyrosine. Finally, using high-performance liquid chromatography, we demonstrated that propofol reacted with ONOO- more rapidly than did tyrosine, inhibiting nitrotyrosine formation. In the absence of propofol, 3.5 mM ONOO- with 1 mM tyrosine yielded 39.6% nitrotyrosine, but nitrotyrosine was not produced when 5 mM propofol was added. We conclude that propofol protects endothelial cells against the toxicity of ONOO-. The anti-oxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite, a property that might be relevant in pathological situations involving a significant contribution of peroxynitrite to tissue damage.

Nitrated proteins in bronchoalveolar lavage fluid of patients at risk of ventilator-associated bronchopneumonia.

The study was designed to identify markers of oxidative injury, related to the nitric oxide derived cascade, in bronchoalveolar lavage (BAL) fluid from intensive care patients suspected of ventilator-associated pneumonia (VAP) and/or acute respiratory distress syndrome (ARDS). Thirty-eight patients developing VAP and/or ARDS (VAP/ARDS group) were compared to 20 ventilated patients without VAP/ARDS (control group). Myeloperoxidase (MPO) and elastase, taken as markers of neutrophil activation were measured by enzymatic techniques, and nitrated proteins (NTPs) by an immunological method. The cytotoxicity of the BAL fluid was tested using cultured human epithelial alveolar cells by the release of pre-incorporated 51Cr. Mean NTP concentration and, MPO and elastase activities were different between the VAP/ARDS and control groups (p<0.05 for NTPs; p<0.005 for MPO; p<0.005 for elastase). NTP concentration correlated with MPO and elastase activity and neutrophil number (r=0.93, 0.91 and 0.87, respectively), but not to protein concentration and arterial oxygen tension/inspiratory oxygen fraction. The cytotoxicity of BAL correlated with NTP concentration (r=0.92) and MPO activity (r=0.89). It was concluded that the concentrations of nitrated proteins in bronchoalveolar lavage fluid correlated with the oxidant activity of neutrophils and that, bronchoalveolar lavage fluid cytotoxicity was correlated with the nitrated protein concentration and may be mediated by oxidants.