Quinone compounds regulate the level of ROS production by the NADPH oxidase Nox4

NADPH oxidase Nox4 is expressed in a wide range of tissues and plays a role in cellular signaling by providing reactive oxygen species (ROS) as intracellular messengers. Nox4 oxidase activity is thought to be constitutive and regulated at the transcriptional level; however, we challenge this point of view and suggest that specific quinone derivatives could modulate this activity. In fact, we demonstrated a significant stimulation of Nox4 activity by 4 quinone derivatives (AA-861, tBuBHQ, tBuBQ, and duroquinone) observed in 3 different cellular models, HEK293E, T-REx™, and chondrocyte cell lines. Our results indicate that the effect is specific toward Nox4 versus Nox2. Furthermore, we showed that NAD(P)H:quinone oxidoreductase (NQO1) may participate in this stimulation. Interestingly, Nox4 activity is also stimulated by reducing agents that possibly act by reducing the disulfide bridge (Cys226, Cys270) located in the extracellular E-loop of Nox4. Such model of Nox4 activity regulation could provide new insight into the understanding of the molecular mechanism of the electron transfer through the enzyme, i.e., its potential redox regulation, and could also define new therapeutic targets in diseases in which quinones and Nox4 are implicated. © 2013 Elsevier Inc. All rights reserved

Independent validation of the Enhanced Liver Fibrosis (ELF) score in the ANRS HC EP 23 Fibrostar cohort of patients with chronic hepatitis C

International audienceBackground : The Enhanced Liver Fibrosis (ELF) score combining serum hyaluronan, N-terminal peptide of type III procollagen and tissue inhibitor of metalloproteinase-1, was reported as relevant in predicting liver fibrosis in chronic liver disease and proposed as an alternative to liver biopsy.Methods : We evaluated the ELF score in a cohort of chronic hepatitis C (CHC) patients included in a multicenter prospective study (ANRS HC EP 23 Fibrostar) using commercial reagents, different from those developed by the manufacturer of the Siemens ELF™ test.Results : In 512 CHC, the ELF score, using ROC curves, showed good predictive performances for severe fibrosis [AUROC=0.82; 95% confidence interval (CI) 0.78–0.86]and for cirrhosis (AUROC=0.85; 95% CI 0.81–0.90), but slightly lower for significant fibrosis (AUROC=0.78; 95% CI 0.74–0.82). The Obuchowski measure (0.81) showed that the ELF score globally performed as a marker of liver fibrosis. The ELF score predicted significant fibrosis (cut-off=9.0) with a sensitivity of 0.86, a specificity of 0.62, a positive predictive value (PPV) of 0.80 and a negative predictive value (NPV) of 0.70. For extensive fibrosis (cut-off=9.33), sensitivity was 0.90, specificity was 0.63, PPV was 0.73 and NPV was 0.85. For cirrhosis (cut-off=9.35), sensitivity was 0.83, specificity was 0.75, PPV was 0.44 and NPV was 0.95.Conclusions : This study confirms the ELF score performance as an index to predict liver fibrosis or cirrhosis in CHC. The ELF test, using validated reagents, could be added to the health authorities approved non-invasive tests in assessing fibrosis as surrogate to liver biopsy

Predictive diagnostic of chronic allograft dysfunction using urinary proteomics analysis.

International audienceBACKGROUND: Kidney transplant Chronic Allograft Dysfunction (CAD), a major cause of long-term graft failure, is currently diagnosed at a late and irreversible stage by graft biopsies. Our goal was to identify predictive urinary biomarkers of CAD before renal lesions appeared by analysis of the urine proteomic profile. METHODS/METHODS: Twenty-nine urinary samples withdrawn three months post-transplant were analyzed by SELDI-TOF technology. CAD development was evaluated by serum creatinine level and confirmed by allograft biopsy one year after transplantation. Comparison of protein profile of both groups revealed 18 biomarkers predictive of CAD occurrence. RESULTS: The biomarker demonstrating the highest diagnostic performance was a protein of 8860 Da that predicted CAD with a sensitivity of 93% and a specificity of 65%. Moreover combination of these biomarkers in two multivariate analyses improved the diagnostic potential of CAD. Relevance of these individual biomarkers and a decisional algorithm constituted of 3 proteins was confirmed in an independent cohort of patients with undetermined CAD status one year post-transplant. CONCLUSIONS: These non invasive biomarkers, detected as soon as three months post-grafting, allowed identification of patients who would develop CAD as late as 4 years after graft. Systematic measurement of these biomarkers would greatly improve the management of immunosuppressive therapy of kidney grafted patients

Investigating metalloproteinases MMP-2 and MMP-9 mechanosensitivity to feedback loops involved in the regulation of in vitro angiogenesis by endogenous mechanical stresses.

International audienceAngiogenesis is a complex morphogenetic process regulated by growth factors, but also by the force balance between endothelial cells (EC) traction stresses and extracellular matrix (ECM) viscoelastic resistance. Studies conducted with in vitro angiogenesis assays demonstrated that decreasing ECM stiffness triggers an angiogenic switch that promotes organization of EC into tubular cords or pseudo-capillaries. Thus, mechano-sensitivity of EC with regard to proteases secretion, and notably matrix metalloproteinases (MMPs), should likely play a pivotal role in this switching mechanism. While most studies analysing strain regulation of MMPs used cell cultured on stretched membranes, this work focuses on MMP expression during self-assembly of EC into capillary-like structures within fibrin gels, i.e. on conditions that mimics more closely the in vivo cellular mechanical microenvironment. The activity of MMP-2 and MMP-9, two MMPs that have a pivotal role in capillaries formation, has been monitored in pace with the progressive elongation of EAhy926 cells that takes place during the emergence of cellular cords. We found an increase of the zymogen proMMP-2 that correlates with the initial stages of EC cords formation. However, MMP-2 was not detected. ProMMP-9 secretion decreased, with levels of MMP-9 kept at a rather low value. In order to analyse more precisely the observed differences of EAhy926 response on fibrin and plastic substrates, we proposed a theoretical model of the mechano-regulation of proMMP-2 activation in the presence of type 2 tissue inhibitor of MMPs (TIMP-2). Using association/dissociation rates experimentally reported for this enzymatic network, the model adequately describes the synergism of proMMP-2 and TIMP-2 strain activation during pseudo-capillary morphogenesis. All together, these results provide a first step toward a systems biology approach of angiogenesis mechano-regulation by cell-generated extracellular stresses and strains

TIMP-1/MMP-9 imbalance in an EBV-immortalized B lymphocyte cellular model: evidence for TIMP-1 multifunctional properties

AbstractTissue inhibitors of metalloproteinases (TIMPs) were initially described as agents controlling metalloproteinase activity. The purpose of this study was to investigate the expression and the roles of TIMP-1 secreted by Epstein–Barr-virus (EBV)-immortalized B lymphocytes. TIMP-1 was isolated from conditioned medium of interleukin (IL)-1β stimulated EBV-B lymphocytes; purified TIMP-1 was identified by mass spectrometry and immunochemistry. TIMP-1-free MMP-9 was quantified after purification by zymography and enzyme-linked immunosorbent assay. EBV-B lymphocyte-secreted TIMP-1 inhibited MMP-9 gelatinolytic activity resulting in decreased B-cell transmigration as measured in vitro. The release of huge amounts of TIMP-1 in proportion to MMP-9 from B lymphocytes after EBV transformation was shown to be correlated with secretion of IL-10 and dependent on culture time. In contrast, there was little TIMP-1 and almost no IL-10 released from native B cells, suggesting a possible IL-10 mediated autocrine regulation mechanism of TIMP-1 synthesis. The MMP-9/TIMP-1 imbalance observed in the culture medium of EBV-B lymphocytes (TIMP-1>MMP-9) and of native B cells (MMP-9>TIMP-1) is suggestive of a new function for TIMP-1. We propose that TIMP-1 acts as a survival factor controlling B-cell growth and apoptosis through an autocrine regulation process involving IL-10 secreted by EBV-B lymphocytes

A 12‐month pilot study outcomes of vagus nerve stimulation in Crohn's disease

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Synovial fluid proteomic fingerprint: S100A8, S100A9 and S100A12 proteins discriminate rheumatoid arthritis from other inflammatory joint diseases.

International audienceOBJECTIVE: We investigated SF and serum proteomic fingerprints of patients suffering from RA, OA and other miscellaneous inflammatory arthritides (MIAs) in order to identify RA-specific biomarkers. METHODS: SF profiles of 65 patients and serum profiles of 31 patients were studied by surface-enhanced laser desorption and ionization-time-of-flight-mass spectrometry technology. The most discriminating RA biomarkers were identified by matrix-assisted laser desorption ionization-time of flight and their overexpression was confirmed by western blotting and ELISA. RESULTS: Three biomarkers of 10 839, 10 445 and 13 338 Da, characterized as S100A8, S100A12 and S100A9 proteins, were the most up-regulated proteins in RA SF. Their expression was about 10-fold higher in RA SF vs OA SF. S100A8 exhibited a sensitivity of 82% and a specificity of 69% in discriminating RA from other MIAs, whereas S100A12 displayed a sensitivity of 79% and a specificity of 64%. Three peptides of 3351, 3423 and 3465 Da, corresponding to the alpha-defensins-1, -2 and -3, were also shown to differentiate RA from other MIAs with weaker sensitivity and specificity. Levels of S100A12, S100A8 and S100A9 were statistically correlated with the neutrophil count in MIA SF but not in the SF of RA patients. S100A8, S100A9, S100A12 and alpha-defensin expression in serum was not different in the three populations. CONCLUSION: The most enhanced proteins in RA SF, the S100A8, S100A9 and S00A12 proteins, distinguished RA from MIA with high accuracy. Possible implication of resident cells in this increase may play a role in RA physiopathology

Unfolded protein response gene GADD34 is overexpressed in rheumatoid arthritis and related to the presence of circulating anti-citrullinated protein antibodies

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Unfolded protein response gene GADD34 is overexpressed in rheumatoid arthritis and related to the presence of circulating anti-citrullinated protein antibodies

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