Effect of hypoxia/reoxygenation on the cytokine-induced production of nitric oxide and superoxide anion in cultured osteoarthritic synoviocytes.

OBJECTIVE: Hypoxia/reoxygenation (H/R) is an important feature in the osteoarthritis (OA) physiopathology. Nitric oxide (NO) is a significant proinflammatory mediator in the inflamed synovium. The purpose of this study was to investigate the effects of H/R on inducible NO synthase (iNOS) activity and expression in OA synoviocytes. In addition we studied the relationship between nitrosative stress and NADPH oxidase (NOX) in such conditions. METHODS: Human cultured synoviocytes from OA patients were treated for 24 h with interleukin 1-ß (IL-1ß), tumour necrosis factor α (TNF-α) or neither; for the last 6 h, they were submitted to either normoxia or three periods of 1-h of hypoxia followed by 1-h of reoxygenation. ·NO metabolism (iNOS expression, nitrite and peroxynitrite measurements) was investigated. Furthermore, superoxide anion O2(·-) production, NOX subunit expression and nitrosylation were also assessed. RESULTS: iNOS expression and nitrite (but not peroxynitrite) production were ~0.20 to ~0.12 nmol min(-1) mg proteins(-1) (P < 0.05), while NOXs' subunit expression and p47-phox phosphorylation were increased. NOXs and p47-phox were dramatically nitrosylated under H/R conditions (P < 0.05 vs normoxia). Using NOS inhibitors under H/R conditions, p47-phox nitrosylation was prevented and O2(·-) production was restored at normoxic levels (0.21 nmol min(-1) mg of proteins(-1)). CONCLUSIONS: Our results provide evidence for an up-regulation of iNOS activity in OA synoviocytes under H/R conditions, associated to a down-regulation of NOX activity through nitrosylation. These findings highlight the importance of radical production to OA pathogenesis, and appraise the metabolic modifications of synovial cells under hypoxia.

[Evaluation of analytical and diagnostic performances of the anti-cyclic citrullinated peptides (anti-CCP) automated assay on Elecsys analyzer]. / Dosage automatisé des anticorps anti-peptides cycliques citrullinés (anti-CCP) sur l'analyseur Elecsys: validation analytique et valeur diagnostique.

Anti-cyclic citrullinated peptides (anti-CCP) are highly characteristics of rheumatoid arthritis (RA). Since 2006, anti-CCP assays have been included in both French and European recommendations. We have evaluated the analytical and clinical performances of the anti-CCP assay on the Elecsys analyzer (Roche Diagnostics). Two plasma pools (target values: 17.2 and 363.0 U/mL) and two quality controls (target values: 24.5 and 157.0 U/mL) were tested; we also analyzed three hundred plasma samples from healthy subjects (n = 86) and diseased patients (presenting with RA, non rheumatoid disorders, or undifferentiated arthritis: n = 214). Analytical performances (intra- and inter-assay precisions) and clinical performances (ROC analysis and method comparison) were evaluated. Elecsys assay was compared to Immunoscan RA(R) assay using contingency tables. Intra- and inter-assay precisions showed coefficients of variation less than 5%. ROC analysis showed an area under the curve at 0.886. Considering the value of 17 U/mL as the optimal cut-off, we found sensibility and specificity at 75% and 95%, respectively. Comparison of the Elecsys anti-CCP assay with the Immunoscan RA(R) assay showed an overall agreement of 98,3%. We conclude that the the Elecsys anti-CCP assay displayed a high precision and clinical performances comparable to that of the efficient anti-CCP assay Immunoscan RA(R).

N-Terminal pro B-type natriuretic peptide testing for short-term prognosis in breathless older adults.

BACKGROUND: Amino-terminal pro-brain natriuretic peptide (NT-proBNP) is useful for the triage of patients with dyspnea. Our aim was to determine whether NT-proBNP levels could predict in-hospital outcome in breathless elderly patients. METHODS: At admission, NT-proBNP plasma concentrations were determined in 324 dyspneic patients aged 75 years and older. The association between NT-proBNP values and in-hospital mortality was assessed. RESULTS: Median NT-proBNP concentrations were not different in deceased patients (n = 43, 13%) compared to that of survivors (n = 281, 87%) (4354 vs 2499 pg/mL, respectively; P = .06). To predict in-hospital mortality, the optimum threshold of NT-proBNP was 3855 pg/mL, as defined by the receiver operating characteristic (ROC) curve, with a nonsignificant area under the ROC curve of 0.59. Mortality was significantly higher in patients (n = 139) with NT-proBNP levels 3855 pg/mL or higher (17.9% vs 9.7%, P = .045). After multivariate analysis, NT-proBNP level 3855 pg/mL or higher at admission was predictive of mortality (odds ratio, 2.41; 95% confidence interval, 1.02-5.68; P = .04). CONCLUSION: NT-proBNP higher than 3855 pg/mL is associated with in-hospital mortality in patients aged 75 years and older admitted for dyspnea.

High N-terminal pro-brain natriuretic peptide levels and low diffusing capacity for carbon monoxide as independent predictors of the occurrence of precapillary pulmonary arterial hypertension in patients with systemic sclerosis.

OBJECTIVE: To evaluate predictors of pulmonary arterial hypertension (PAH) in a prospective cohort of patients with systemic sclerosis (SSc). METHODS: Routine clinical assessments as well as measurements of the diffusing capacity for carbon monoxide/alveolar volume (DLCO/VA) ratio and N-terminal pro-brain natriuretic peptide (NT-proBNP) level were performed in a prospective cohort of 101 SSc patients who did not have PAH or severe comorbidities. After a planned 36-month followup, we evaluated the predictive value of these parameters for the development of precapillary PAH, as demonstrated by cardiac catheterization, disease progression, and death. Criteria for cardiac catheterization were a systolic pulmonary artery pressure (PAP) of >40 mm Hg on echocardiography, a DLCO value of <50% without pulmonary fibrosis, and unexplained dyspnea. RESULTS: Eight patients developed PAH, 29 had disease progression, and 10 died during a median followup of 29 months. Kaplan-Meier analysis identified the following baseline parameters as being predictors of PAH: DLCO/VA ratio <70% or <60% (P<0.01 for each comparison), elevated plasma NT-proBNP level (>97th percentile of normal; P = 0.005), echocardiographically estimated systolic PAP >40 mm Hg (P=0.08), and erythrocyte sedimentation rate >28 mm/hour (P=0.015). In multivariate analyses, an elevated baseline NT-proBNP level (hazard ratio [HR] 9.97 [95% confidence interval (95% CI) 1.69-62.42]) and a DLCO/VA ratio <60% (HR 36.66 [95% CI 3.45-387.6]) were predictors of the occurrence of PAH during followup. An increased NT-proBNP level together with a decreased DLCO/VA ratio of <70% was highly predictive of the occurrence of PAH during followup (HR 47.20 [95% CI 4.90-450.33]). CONCLUSION: This prospective study identified a decreased DLCO/VA ratio and an increased NT-proBNP as predictors of PAH in SSc. Use of these markers should result in improved PAH risk stratification and allow earlier initiation of therapy.

[The N-terminal pro-brain natriuretic peptide (NT-proBNP) assay with the Stratus CS analyzer]. / Evaluation des performances analytiques du dosage du NT-proBNP sur analyseur semi-automatisé Stratus CS.

BACKGROUND: NT-proBNP is an efficient biomarker for the evaluation, management and prognosis of patients with heart failure. METHODS: We evaluated the analytical performance of the NT-proBNP immunoenzymatic assay with the Stratus CS semi-automated analyzer in two hospital laboratories. The characteristics assessed included imprecision, functional sensitivity, linearity/recovery, interferences study, high-dose hook effect and a comparison of Acute Care(TM) pPBNP (on Stratus)CS) versus PBNP (on Dimension HM) results on patient heparinized plasma samples. RESULTS: Total imprecision reached < 5% coefficient of variation at NT-proBNP concentrations of 186-19,649 ng/L; recovery values for diluted samples were between 89.0 and 110.0 %; functional sensitivity reached 21 ng/L; there was no high-dose hook effect at concentrations up to 400,000 ng/L; hemaoglobin affected negatively but <10% the NT-proBNP assay, while bilirubin and triglycerides did not affected it more than 5%; Stratus CS results were strongly correlated with Dimension HM results (R(2)=1,00). CONCLUSION: The Stratus CS Acute Care pPBNP assay demonstrated excellent analytical performance which agreed with the Dimension HM PBNP assay. This analyzer is therefore suitable for use by low NT-proBNP test volume laboratories, and also by Emergency departments and Intensive care units.

Lack of association between three vascular endothelial growth factor gene polymorphisms and systemic sclerosis: results from a multicenter EUSTAR study of European Caucasian patients.

INTRODUCTION: Systemic sclerosis (SSc) is characterised by disturbed vessel morphology and an overproduction of vascular endothelial growth factor (VEGF). The VEGF gene located on chromosome 6p21.3 has several polymorphisms. OBJECTIVE: To test the hypothesis that disturbed angiogenesis may be related to the genetic background of the VEGF gene. MATERIALS AND METHODS: EUSTAR centres included European Caucasian patients with SSc and matched controls with osteoarthritis. The VEGF gene was genotyped by polymerase chain reaction, followed by restriction enzyme analysis. The 634 C/T and 936 C/G mutations and an 18-base pair insertion/deletion at -2549 of the VEGF promoter region were tested. RESULTS: 416 patients with SSc and 249 controls were included in the study population. Of the patients with SSc, 42% had a diffuse cutaneous subtype, 16% had increased pulmonary arterial pressure and 61% had decreased carbon monoxide diffusion capacity. The genotype frequencies in the patients with SSc and in controls were in Hardy-Weinberg equilibrium. The allele and genotype frequencies of the polymorphisms did not differ between patients with SSc and controls. No association was found between these polymorphisms and disease phenotypes. CONCLUSION: This study shows that there is no association between the three selected functional VEGF polymorphisms and SSc.

Superoxide production and NADPH oxidase expression in human rheumatoid synovial cells: regulation by interleukin-1beta and tumour necrosis factor-alpha.

OBJECTIVES: to evaluate the rheumatoid synovial cell capacity to produce superoxide anion in response to interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha), and to study the NADPH oxidase involvement in this production. MATERIAL AND METHODS: Synovial cells obtained from 7 rheumatoid arthritis (RA), 5 osteoarthritic (OA) patients, and dermal fibroblasts, were stimulated (i) with IL-1beta and TNF-alpha, or (ii) with specific oxidase activators and inhibitors, before studying superoxide production; we also studied NADPH oxidase mRNAs and protein expression, and p47-phox phosphorylation. RESULTS: Constitutive superoxide production by RA cells was increased in comparison to OA cells and dermal fibroblasts, and was stimulated by PMA and ionomycin. This production was increased after cytokine treatment of RA synovial cells. Cytokine-induced superoxide production by RA cells was inhibited by iodonium diphenyl or apocynin, suggesting the involvement of NADPH oxidase. RT-PCR and western blot analysis revealed the presence of p47-phox, gp91-phox and Nox4 in RA and OA cells, and in dermal fibroblasts. P47-phox phosphorylation was enhanced after cytokine-treatment in RA and OA cells, suggesting a PKC-mediated up-regulation of NADPH oxidase. CONCLUSIONS: NADPH oxidase is involved in the superoxide release by RA synovial cells, constitutively and after cytokine up-regulation. These cells express two different homologues (gp91-phox and Nox4).

Serum protein oxidation in patients with rheumatoid arthritis and effects of infliximab therapy.

OBJECTIVE: To examine protein oxidation in rheumatoid arthritis (RA) and evaluate its evolution after infliximab therapy in a subgroup of patients. METHODS: Seventy-one consecutive patients with RA were included. Among them, 30 patients refractory to conventional therapy were treated with infliximab. Serum markers of oxidative stress were determined at baseline and before the infusions of infliximab at weeks 6 and 30. Baseline values were compared with those in 30 healthy volunteers. RESULTS: Mean levels of serum carbonyl groups were significantly higher in RA patients than in controls (1.29+/-0.76 versus 0.58+/-0.39 nmol/mg of protein, p<0.0001), whereas thiol levels were found to be lower (238.3+/-61.6 versus 316.5+/-54.8 micromol/L, p<0.0001). Thiol levels inversely correlated with the disease activity score (r=-0.42, p=0.004), and with CRP values (r=-0.45, p=0.001). Immunoblots showed that albumin and heavy chain immunoglobulin were oxidized more markedly than in healthy volunteers. Significantly lower levels of thiol groups were detected in patients with refractory RA disease (208.9+/-66.8 versus 264.2+/-43.0 micromol/L, p<0.0004) but concentrations of carbonyl groups were similar. Short-term treatment with infliximab significantly decreased carbonyl groups (0.97+/-0.47 nmol/mg protein, p=0.02) and increased thiol (231.2+/-48.7 micromol/L, p=0.02) levels. CONCLUSION: Our results highlight free radical protein damage in RA and a link with inflammation, as underlined by the beneficial effects of infliximab.

Influence of renal function on N-terminal pro-brain natriuretic peptide (NT-proBNP) in patients admitted for dyspnoea in the Emergency Department: comparison with brain natriuretic peptide (BNP).

BACKGROUND: Renal dysfunction influences the optimum brain natriuretic peptide (BNP) threshold for a diagnosis of cardiac-related dyspnoea, but this has not been demonstrated for N-terminal pro-brain natriuretic peptide (NT-proBNP). We studied the influence of renal function on NT proBNP and BNP concentrations in dyspnoeic patients admitted by night to the Emergency Department (ED). METHODS: NT-proBNP, BNP, and creatinine levels were measured in blood samples collected routinely from 381 patients; estimated glomerular filtration rate (eGFR) was calculated. RESULTS: Cardiac-related dyspnoea was found in 115 patients (30.2%). NT-proBNP and BNP values were elevated in patients with cardiac-related dyspnoea (6823+/-6569 vs. 2716+/-4838 pg/ml, and 642+/-329 vs. 243+/-267 pg/ml, p<0.0001, respectively). Log-transformed NT-proBNP and BNP values were correlated to eGFR values. Mean NT-proBNP and BNP values stratified by ED diagnosis increased in line with eGFR categories, but in each category both peptide concentrations remained elevated in cardiac-related dyspnoea when compared with non-cardiac-related dyspnoea (p<0.05). NT-proBNP (and BNP) cut-off points rose as a function of eGFR categories: from 1360 (and 290) pg/ml in patients with eGFR 60-89 ml/min/1.73 m2, to 6550 (and 515) pg/ml in patients with eGFR 15-29 ml/min/1.73 m2. CONCLUSION: Renal function influences the optimal cut-off points of NT-proBNP and BNP for the diagnosis of cardiac-related dyspnoea.

Increased plasma soluble CD40 ligand concentrations in systemic sclerosis and association with pulmonary arterial hypertension and digital ulcers.

BACKGROUND: Increased expression of CD40L has been reported on activated CD4+ T lymphocytes in systemic sclerosis. CD40L can be expressed in soluble form (sCD40L). OBJECTIVE: To compare sCD40L concentrations in patients with systemic sclerosis and healthy controls. METHODS: Quantitative sandwich ELISA was used to measure plasma sCD40L in systemic sclerosis (n = 50) and matched healthy controls (n = 20). Patients with systemic sclerosis had limited cutaneous disease (29), digital ulcers (14), pulmonary arterial hypertension (PAH) (10), pulmonary fibrosis on CT (23), positive anti-Scl70 (14), and anti-centromere antibodies (10). Calcium channel blockers were discontinued 72 hours before measurements. RESULTS: Median (range) sCD40L concentration (pg/ml) was higher in systemic sclerosis than in controls (495 (10 to 7720) v 79 (50 to 118); p = 0.003), in limited cutaneous disease v diffuse disease (620 (20 to 7720) v 250 (10 to 2690); p = 0.005), in patients with digital ulcers v those without (1430 (36 to 7720) v 370 (10 to 2320); p = 0.002), and in those with PAH v those without (995 (15 to 3850) v 400 (10 to 7720); p = 0.048). sCD40L correlated with pulmonary arterial pressure estimated by Doppler echocardiography (r = 0.41; p = 0.005). CONCLUSIONS: The soluble form of CD40L is increased in plasma in systemic sclerosis and may be associated with vascular complications of the disease.

N-terminal pro-brain natriuretic peptide as a diagnostic marker of early pulmonary artery hypertension in patients with systemic sclerosis and effects of calcium-channel blockers.

OBJECTIVE: To evaluate N-terminal pro-brain natriuretic peptide (NT-proBNP) as a marker of early pulmonary artery hypertension (PAH) and to study changes in the levels of this marker following treatment with dihydropyridine-type calcium-channel blocker (DTCCB) in patients with systemic sclerosis (SSc). METHODS: We evaluated 40 consecutive SSc patients who had been hospitalized for followup care (mean +/- SD age 56 +/- 11 years and mean +/- SD duration of cutaneous disease 9 +/- 9 years; 27 with limited cutaneous and 13 with diffuse cutaneous disease) but who had no clinical symptoms of heart failure and had a normal left ventricular ejection fraction. At baseline, 10 patients had PAH, defined as a systolic pulmonary artery pressure (sPAP) >40 mm Hg, as measured by echocardiography. Levels of NT-proBNP were determined at baseline (after discontinuation of DTCCB treatment for 72 hours), after taking 3 doses of DTCCB following treatment reinitiation (assessment 1), and after 6-9 months of continuous DTCCB treatment (assessment 2) in the 20 patients who attended regular appointments (including the 10 patients with PAH at baseline). RESULTS: At baseline, 13 patients had high NT-proBNP values for their ages. High NT-proBNP levels identified patients with PAH with a sensitivity of 90%, a specificity of 90.3%, a positive predictive value of 69.2%, and a negative predictive value of 96%. The NT-proBNP level correlated with the sPAP (r = 0.44; P = 0.006). By assessment 1, the number of patients with PAH and high levels of NT-proBNP had decreased from 9 of 10 to 2 of 10 (P = 0.02). This decrease was partially sustained at assessment 2 (4 of 10 patients; P = 0.06). CONCLUSION: NT-proBNP is a useful biologic marker that can be used to diagnose early PAH in SSc patients without clinical heart failure. Measurement of NT-proBNP may be valuable for the evaluation of treatment with DTCCB and vasodilators in patients with PAH.

Variability in urinary excretion of bone resorption markers: limitations of a single determination in clinical practice.

In this study we assessed the within and between-subject variability of the concentrations of two urinary markers, free deoxypyridinoline (DPD) and C telopeptide (CTX-I), in healthy patients with the aim of setting reliable thresholds to enable physicians to take decisions about individual patients with confidence.Between-subject variability for the women was 25.4% for DPD and 38.2% for CTX-I, and for the men was 12.9% for DPD and 23.8% for CTX-I. The coefficients of variation were similar for daily, weekly and monthly determinations, giving means of 13.8 and 28.1% for DPD and CTX-I respectively. Critical difference (CD) was lower for DPD than for CTX-I (about 44 and 80% respectively). The number of samples required to determine the true mean with a CD at the 5% level was 29 for DPD and more than 113 for CTX-I.DPD was the least biologically variable. One determination was not sufficient to determine bone resorption status and a 44% decrease in DPD levels and an 80% decrease in CTX-I levels were required to demonstrate the efficacy of antiresorptive therapy in individual patients.

Low levels of nitric oxide (NO) in systemic sclerosis: inducible NO synthase production is decreased in cultured peripheral blood monocyte/macrophage cells.

OBJECTIVE: To investigate nitric oxide (NO) production and inducible NO synthase expression by cultured peripheral blood mononuclear cells (PBMC) in patients with systemic sclerosis (SSc). METHODS: Eighteen patients with SSc were compared with two control groups: 16 patients with rheumatoid arthritis (RA) and 23 patients with mechanical sciatica. Nitrate was determined by fluorimetry in plasma and by spectrophotometry in supernatants. Inducible NO synthase (iNOS) was detected in cultured PBMC by immunofluorescence, immunoblotting and flow cytometry with or without treatment of the cells with interleukin (IL) 1beta+ tumour necrosis factor alpha (TNF-alpha), IL-4 or interferon gamma (IFN-gamma) from day 1 to day 5. RESULTS: NO metabolite concentrations were lower in SSc patients (mean+/-s.e.m. 34.3+/-2.63 micromol/l) than in RA (48.3+/-2.82 micromol/l; P<0.02) and sciatica (43.3+/-5.24 micromol/l; P<0.03) patients. iNOS was detected in cultured monocytes in all three groups but induction occurred on day 1 in RA, day 2 in sciatica and only on day 3 in SSc, whatever the stimulus. CONCLUSIONS: The concentrations of NO metabolites are decreased in SSc patients and the metabolism of these compounds in PBMC is altered. Low levels of NO, a vasodilator, may be involved in vasospasm, which is critical in SSc. This may have therapeutic implications.

Tetracyclines inhibit nitrosothiol production by cytokine-stimulated osteoarthritic synovial cells.

OBJECTIVE AND DESIGN: To evaluate the capacity of doxycycline and minocycline to inhibit NO production and N-nitrosation reactions in vitro. METHODS: Synovial cells obtained from 6 patients with osteoarthritic joint disease were incubated for 24 hours with (i) or without (ii) IL-1beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(4) U/ml) plus minocycline or doxycycline (10(-4) to 10(-6) M), diclofenac (10(-5) M), or cortisol (10(-5) M). Nitrosothiols were determined by fluorimetry, nitrite by the Griess reaction, nitrate by a spectrophotometric assay using oxidation by nitrate reductase and iNOS by immunoblotting. RESULTS: After 24 hours of stimulation, the level of NO production was much higher than that in untreated cells: about 5.5 times higher for nitrosothiols, 5.2 times higher for nitrate and about 3.5 times higher for nitrite. Doxycycline and minocycline induced a dose-dependent decrease in the production of nitrosothiols, nitrate and nitrite, and inhibited the synthesis of the iNOS protein. Doxycycline and minocycline inhibited the N-nitrosation reaction of DAN effectively, with IC50 values close to 100 microM. Diclofenac and cortisol had no effect. CONCLUSION: This study provides new information on the mechanism by which tetracyclines exert anti-inflammatory effects, via inhibiting nitrosothiols.

[Inducible nitric oxide synthase expression and nitric oxide production by monocytes in systemic sclerosis]. / Expression de la NO synthase inductible et production du monoxyde d'azote par les cellules mononucléées sanguines dans la sclérodermie systémique.

We investigated nitric oxide (NO) production and inducible NO synthase (iNOS) expression by cultured peripheral blood mononuclear cells (PBMC) in systemic sclerosis (SSc). Eighteen patients with SSc were compared to two control groups: 16 rheumatoid arthritis patients (RA) and 23 mechanical sciatica patients. The sum of nitrites and nitrates was determined by fluorimetry in sera and spectrophotometry in supernatants. Inducible iNOS was detected in cultured PBMC by immunofluorescence, immunoblot and flow cytometry with or without IL-1 beta + TNF alpha, IL-4 or IFN gamma from day 1 to day 5. NO metabolite concentrations in the plasma were lower in SSc (34.3 mumol/l +/- 2.63 SEM) than in RA (48.3 mumol/l +/- 2.2; p < 0.02) and sciatica (43.3 mumol/l +/- 5.24; p < 0.03) patients. iNOS was detected in cultured monocytes in the 3 groups but induction occurred on day 1 in RA, day 2 in sciatica and only on day 3 in SSc, whatever the stimulus. The concentrations of NO metabolites are decreased in SSc patients and the induction of iNOS in PBMC is delayed. Low levels of NO, a vasodilator, may be involved in vasospasm, which is critical in SSc. This may suggest therapeutic implications.

Inhibition of the nitrosothiol production of cultured osteoarthritic chondrocytes by rhein, cortisol and diclofenac.

OBJECTIVE: Nitric oxide (NO degrees ) is a free molecule produced by NO synthases which acts as a mediator in inflammatory processes. NO degrees can react with thiol groups of proteins to produce nitrosothiols. Increased concentrations of these bioactive compounds have been found in sera and synovial fluids from patients with osteoarthritis (OA). The aim of this study was to assess the ability of human osteoarthritic chondrocytes to synthesize nitrosothiols and to compare the in vitro effects of rhein, cortisol and diclofenac on nitrosothiol and nitrite production. METHODS: Osteoarthritic chondrocytes were incubated for 24 h with 1 ng/ml of recombinant human interleukin-1beta (IL-1beta) in the presence or absence of rhein (1.3x10(-5) M, 6.5x10(-6) M, or 1.3x10(-6) M), cortisol (10(-5) M) or diclofenac (10(-5) M or 10(-6) M). Nitrite levels were measured in cell supernatants by the Griess method; nitrosothiol levels were determined in supernatants and cellular lysates by fluorimetry. RESULTS: At the basal level, nitrosothiols represented 80% of the total of nitrite and nitrosothiol production. After IL-1beta stimulation, NO degrees production was highly increased in the supernatants (45-fold increase in nitrite, 60-fold increase in nitrosothiols) as well as in cell lysates (35-fold increase in nitrosothiols). Rhein caused a dose-dependent decrease in nitrosothiol and nitrite production. In comparison, diclofenac (10(-5) M) moderately decreased nitrite and nitrosothiol levels in the supernatants but had no effect on lysate nitrosothiol. Cortisol had no significant effect on NO degrees production. CONCLUSIONS: The IL-1beta stimulation increased nitrosothiol production by osteoarthritic chondrocytes. These results demonstrate the need to measure nitrosothiol as well as nitrite production. Rhein inhibited the IL-1beta induced NO degrees production, and may be a suitable treatment for osteoarthritis.

Nitric oxide modifies glycolytic pathways in cultured human synoviocytes.

Nitric oxide (NO) is a free radical produced during inflammation following activation of an inducible NO synthase by pro-inflammatory cytokines such as IL-1beta. Since both NO and IL-1beta are involved in the physiopathology of inflammatory arthropathies, we investigated the effects of exogenous NO on glycolytic pathways in cultured human osteoarthritic synovial cells. NO generated from S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP) inhibited glucose uptake (by 50% after 1 h of incubation) and lactate production by 16% (SNAP) and 8.5% (SNP) after 3 h. Both NO donors also reduced production of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme of the glycolytic pathway. This effect was reversed by haemoglobin, a NO scavenger with higher affinity for the radical. In contrast, the effect on glucose uptake appeared to be irreversible.

System A neutral amino acid transporter regulation by interleukin-1beta in human osteoarthritic synovial cells: evidence for involvement of prostaglandin E(2) as a second messenger.

We studied the long-terms effects of interleukin-1beta (IL-1beta; 3 to 6 h) on alpha-(methylamino) isobutyric acid (MeAIB), a nonmetabolizable amino acid transported by system A. We found that IL-1beta induced a large decrease in MeAIB uptake by human osteoarthritic synovial cells and a concomitant increase in prostaglandin E(2) (PGE(2)) synthesis. Therefore, we investigated whether PGE(2) acts as a mediator for the long-term action of IL-1beta. We found that exogenous PGE(2) inhibited MeAIB uptake, and that AH6809, a PGE(2) receptor antagonist, inhibited IL-1beta-mediated MeAIB uptake. To identify the enzymes involved in the IL-1beta-mediated synthesis of PGE(2) that inhibits MeAIB uptake, we studied the expression of secreted (s) and cytosolic (c) phospholipase A(2) (PLA(2)). Because both were expressed, we selected a broad spectrum of inhibitors to determine which of the two PLA(2)s was involved. We used AACOCF3, a cPLA(2) inhibitor, and dithiothreitol (DTT) and bromophenacyl bromide (BPB), which are sPLA(2) inhibitors. Our results suggest that the PLA(2) involved in the IL-1beta-mediated synthesis of PGE(2) was sPLA(2). We also showed the expression of cyclooxygenase (COX)-2 and its partial involvement using a potent selective COX-2 inhibitor, L-745337. These findings provide insight into the mechanisms underlying the IL-1beta-mediated regulation of transport system A. The Il-1beta-induced inhibition of MeAIB uptake in human osteoarthritic synovial cells thus seems to be essentially mediated by PGE(2) production via the activation of sPLA(2) and the partial activation of COX-2.

Nitric oxide synthase is expressed in the lymphomononuclear cells of synovial fluid in patients with rheumatoid arthritis.

OBJECTIVE: To investigate the expression of inducible nitric oxide synthase (iNOS) in subpopulations of peripheral blood and synovial fluid (SF) leukocytes in patients with rheumatoid arthritis (RA). METHODS: iNOS was detected in peripheral blood and SF samples after cell permeabilization, by 2 color immunofluorescence flow cytometry. Samples from 14 patients with RA and 8 with osteoarthritis (OA) were studied. Nitrite concentration was determined by Griess reaction, interleukin 1beta and tumor necrosis factor alpha by an immunoenzymatic assay, and C-reactive protein (CRP) by an immunonephelometric method. RESULTS: In SF, iNOS was detected in 11 of 14 patients with RA and 2 of 8 with OA. In blood cells, iNOS was detected in 8 of 14 patients with RA and none of the OA group. iNOS was consistently detected in monocytes and was not detected in granular cells. In RA, there was no correlation between the number of iNOS positive mononuclear cells and cytokine concentrations. CRP concentration was correlated with the number of iNOS positive mononuclear cells in RA SF samples. CONCLUSION: SF mononuclear cells from patients with RA express iNOS and are involved in NO production in the joint. The number of positive cells is correlated with CRP concentration, suggesting the implication of NO production in the inflammatory process.

Is alpha-ketoisocaproyl-glutamine a suitable glutamine precursor to sustain fibroblast growth?

BACKGROUND: Glutamine is considered an essential nutrient for cellular growth. AIM: To test the suitability of alpha-ketoisocaproyl-Gln (Kic-Gln) as a new glutamine (Gln) precursor to sustain human fibroblast growth. METHODS: [3H] thymidine uptake into cellular DNA of human fibroblasts. Extracellular and intracellular amino acid patterns were determined with peptides and acylated compounds. RESULTS: L-alanyl-L-glutamine (used here as a recognized Gln precursor) promoted DNA synthesis, while N-acetyl-L-glutamine (used here as a negative control since it is known to be a poor Gln precursor) and alpha-ketoisocaproyl-glutamine had no effect. Alanyl-glutamine progressively gave rise to free glutamine in the growth medium. In contrast, glutamine supplied in acylated form was poorly available and did not appear in free form in the medium. In addition, only alanyl-glutamine increased intracellular glutamine and glutamate levels. In contrast, Kic-Gln was able to sustain net protein synthesis as judged by total protein content and reduced intracellular levels of most essential amino acids. CONCLUSION: Kic-Gln appears to be a poor extra-cellular precursor of Gln to sustain cell growth.