Candice Trocmé

Circulating Matrix Metalloproteinases 1, 2, 9 and Their Inhibitors TIMP-1 and TIMP-2 as Serum Markers of Liver Fibrosis in Patients With Chronic Hepatitis C: Comparison With PIIINP and Hyaluronic Acid

OBJECTIVES:Histological examination of liver biopsy is currently required in the management of patients with chronic hepatitis C. Our aim was to evaluate the diagnostic utility of a panel of circulating markers in detecting the stage of fibrosis.METHODS:One hundred and ninety four-patients who had undergone a percutaneous liver biopsy before antiviral treatment, and 194 age- and sex-matched healthy subjects were studied. Serum levels of hyaluronate, procollagen type III N-terminal peptide (PIIINP), matrix metalloproteinases (MMP)-1, MMP-2, MMP-9 and their tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 were determined by RIA and ELISA. Histological lesions were staged according to the METAVIR score.RESULTS:Hyaluronate, PIIINP, TIMP-1, and TIMP-2 serum levels were significantly higher in patients than in controls. Six markers were significantly correlated with fibrosis: MMP-2 (r = 0.28; p< 0.01), TIMP-1 (r = 0.42; p< 0.001), HA (r = 0.50; p< 0.001), PIIINP (r = 0.62; p< 0.0001), MMP-1 (r =−0.32; p< 0.01), and MMP-9 (r =−0.22; p< 0.05). By multivariate analysis, only PIIINP and MMP-1 were independently associated with fibrosis, and were combined using the equation of the logistic regression model. Using receiver-operating characteristics analysis, the area under the curve of the score to discriminate mild (F0/F1) from significant fibrosis (F2/F3/F4) was 0.82, with a sensitivity of 60% for a specificity of 92%.CONCLUSIONS:Our results suggest that combining two serum markers reflecting fibrogenesis (PIIINP) and fibrolysis (MMP-1) may provide a useful tool for evaluating liver fibrosis.

Comparison of nine blood tests and transient elastography for liver fibrosis in chronic hepatitis C: the ANRS HCEP-23 study.

BACKGROUND & AIMS Blood tests and transient elastography (Fibroscan™) have been developed as alternatives to liver biopsy. This ANRS HCEP-23 study compared the diagnostic accuracy of nine blood tests and transient elastography (Fibroscan™) to assess liver fibrosis, vs. liver biopsy, in untreated patients with chronic hepatitis C (CHC). METHODS This was a multicentre prospective independent study in 19 French University hospitals of consecutive adult patients having simultaneous liver biopsy, biochemical blood tests (performed in a centralized laboratory) and Fibroscan™. Two experienced pathologists independently reviewed the liver biopsies (mean length=25±8.4 mm). Performance was assessed using ROC curves corrected by Obuchowskis method. RESULTS Fibroscan™ was not interpretable in 113 (22%) patients. In the 382 patients having both blood tests and interpretable Fibroscan™, Fibroscan™ performed similarly to the best blood tests for the diagnosis of significant fibrosis and cirrhosis. Obuchowskis measure showed Fibrometer® (0.86), Fibrotest® (0.84), Hepascore® (0.84), and interpretable Fibroscan™ (0.84) to be the most accurate tests. The combination of Fibrotest®, Fibrometer®, or Hepascore® with Fibroscan™ or Apri increases the percentage of well classified patients from 70-73% to 80-83% for significant fibrosis, but for cirrhosis a combination offers no improvement. For the 436 patients having all the blood tests, AUROCs ranged from 0.82 (Fibrometer®) to 0.75 (Hyaluronate) for significant fibrosis, and from 0.89 (Fibrometer® and Hepascore®) to 0.83 (FIB-4) for cirrhosis. CONCLUSIONS Contrarily to blood tests, performance of Fibroscan™ was reduced due to uninterpretable results. Fibrotest®, interpretable Fibroscan™, Fibrometer®, and Hepascore® perform best and similarly for diagnosis of significant fibrosis and cirrhosis.

Human B lymphocytes synthesize the 92-kDa gelatinase, matrix metalloproteinase-9

Matrix metalloproteinases (MMPs) are involved in the remodeling of connective tissue as well as in disease states associated with acute and chronic inflammation or tumoral metastatic processes. Despite detailed and extensive studies of the mechanisms of lymphocyte extravasation, remarkably little is known about the expression and regulation of metalloproteinases involved in the migratory process. By using zymography and reverse transcription-polymerase chain reaction experiments, we have demonstrated that Epstein-Barr virus-immortalized B lymphocytes are able to secrete a 92-kDa metalloproteinase with gelatinolytic activity which has been purified and identified as being MMP-9. Moreover, the tissue inhibitor of metalloproteinase was shown to be constitutively expressed by the B cells. The expression of 92-kDa gelatinase is mediated by cytokines, growth factors, lipopolysaccharide, concanavalin A, and the tumor promotor phorbol 12-myristate 13-acetate. Time dependence activity increased rapidly up to 24 h of incubation with lipopolysaccharide or concanavalin A stimulation while it requires a delay and more time to have an optimum effect when cytokines were the stimulating agents; transforming growth factor-β abolished 92-kDa gelatinase production. Both staurosporine and wortmannin are inductive stimuli, and the level of MMP-9 secreted into the media is greater than that observed with other agents except concanavalin A. Elicitation of the chemotactic migration of B cells through a model basement membrane by lipopolysaccharide was shown to be correlated with gelatinase expression and inhibited by 7 mm captopril. Our study indicates that Epstein-Barr virus-B lymphocytes express 92-kDa gelatinase, the production of which can be modified by a variety of physiological and pharmacological signals which have been shown to differ according to the cell type.

The Role of Oxidative Stress in Amyotrophic Lateral Sclerosis and Parkinson’s Disease

We examined oxidative stress markers of 31 patients suffering from ALS, 24 patients suffering from PD and 30 healthy subjects were included. We determined the plasma levels of lipid peroxidation (malondialdehyde, MDA), of protein oxidative lesions (plasma glutathione, carbonyls and thiols) and the activity of antioxidant enzymes i.e. erythrocyte Cu,Zn-Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px) and catalase. MDA and thiols were significantly different in both neurodegenerative diseases versus control population. A trend for an enhancement of oxidized glutathione was noted in ALS patients. Univariate analysis showed that SOD activity was significantly decreased in ALS and GSH-Px activity was decreased in PD. After adjusting for demographic parameters and enzyme cofactors, we could emphasize a compensatory increase of SOD activity in PD. Different antioxidant systems were not involved in the same way in ALS and PD, suggesting that oxidative stress may be a cause rather than a consequence of the neuronal death.

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

OBJECTIVE 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.

Diagnostic accuracy, reproducibility and robustness of fibrosis blood tests in chronic hepatitis C: A meta-analysis with individual data

OBJECTIVES To evaluate the diagnostic accuracy of liver fibrosis tests and its influencing factors in a meta-analysis with individual data. DESIGN AND METHODS Four independent centers provided four blood tests and Metavir staging from 825 patients with chronic hepatitis C. RESULTS FibroMeter AUROC (0.840) for significant fibrosis was superior to those of Fibrotest (0.803, p=0.049), APRI (0.789, p=0.001) and Hepascore (0.781, p<0.001). The misclassification rate was lower for FibroMeter (23%) than for Fibrotest and Hepascore (both 28%, p<0.001). The variation in the diagnostic cut-offs of tests among centers, reflecting the overall reproducibility, was: FibroMeter: 4.2%, APRI: 24.0%, Fibrotest: 24.2%, Hepascore: 35.0%. Accordingly, the proportion of patients diagnosed with significant fibrosis changed: FibroMeter: 0.8%, Hepascore: 2.4% (p=0.02 vs FibroMeter), Fibrotest: 5.8% (p<10(-3)), APRI: 18.2% (p<10(-3)). CONCLUSIONS This study on clinical applicability shows significant differences in diagnostic accuracy, inter-center reproducibility, and robustness of biomarkers to changes in population characteristics between blood tests.

Nonalcoholic Fatty Liver Disease, Nocturnal Hypoxia, and Endothelial Function in Patients With Sleep Apnea

BACKGROUND Nocturnal hypoxia, the hallmark of OSA, is a potential contributing factor for nonalcoholic fatty liver disease (NAFLD). NAFLD severity and its implication in OSA-related endothelial dysfunction have not been investigated in a large, unselected OSA population, including nonobese subjects. METHODS Noninvasive blood tests (SteatoTest, NashTest, and FibroTest) were used to evaluate steatosis, nonalcoholic steatohepatitis (NASH), and fibrosis in a large cohort of patients with OSA. In the same group, endothelial function and its links with NAFLD severity were assessed. RESULTS Of the 226 subjects included who were referred for suspicion of OSA (men, 55%; median age, 56 years; median BMI, 34.2 kg/m2 [33% with BMI<30 kg/m2]), 61.5% exhibited moderate or severe steatosis. By multivariate analysis, independent factors for liver steatosis were, as expected, triglyceride levels (P<.0001) and insulin resistance (P=.0004) as well as nocturnal cumulative time spent<90% of oxygen saturation (CT90) (P=.01). Thirty-eight percent had borderline or possible NASH (N1 or N2 with NashTest). CT90 was significantly associated with borderline or possible NASH (P=.035) in univariate but not in multivariate analysis. The dose-response relationship between the severity of nocturnal hypoxia and liver injury was established only in morbid obesity and not in lean. Multivariate models showed that steatosis was independently associated with endothelial dysfunction after adjustment for confounders. CONCLUSIONS In a large, unselected OSA population, the severity of nocturnal hypoxia was independently associated with steatosis. Preexisting obesity exacerbated the effects of nocturnal hypoxemia. NAFLD is a potential mechanism of endothelial dysfunction in OSA.

Intervertebral disk degeneration and herniation: the role of metalloproteinases and cytokines.

This article reviews the role of metabolic factors, including metalloproteinases and cytokines, in the occurrence of degenerative disk disease and disk herniation. Given that mechanical factors alone cannot cause disk degeneration, studies must explore metabolic, genetic, nutritional, and age-related factors. Zinc metalloproteinases exert particularly important effects, not only directly, but also indirectly through promotion of neovascularization. The production of these enzymes is dependent on a number of cytokines and on the cell changes they induce. This complex effect acts both on disk matrix degeneration and on the pain generated by contact between the protruding disk and the nerve roots. However, it can have a favorable effect by promoting resorption of the herniated disk. Available data on the role for mechanical factors on the disk chondrocyte metabolism and on metalloproteinase production show that mechanical and metabolic factors interact closely to produce disk disorders.

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

Abstract Background : 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. a The ANRS HC EP 23 Fibrostar Study Group Sponsor: French National Agency for Research on aids and viral hepatitis (ANRS), Paris. Scientific Committee: Coordinators: J.-P. Zarski, Grenoble; M. Vaubourdolle, Paris; Hepatologists: J.-P. Bronowicki, Lille; P. Calès, Angers; A. Mallat, Créteil; P. Mathurin, Lille; Pathologists: N. Sturm, Grenoble; E.S. Zafrani, Créteil; Biologists: B. Poggi, Lyon; J. Guéchot, Paris; Methodologists and administrators: J.L. Bosson; A. Paris; ANRS: L. Allain, Paris. Methodologists and administrators: A. Bechet, J.-L. Bosson, A. Paris, A. Plages, S. Royannais, Centre Hospitalier Universitaire de Grenoble Biologists: R.-C. Boisson, Centre Hospitalier Lyon Sud, Hospices Civils de Lyon M.-C. Gelineau, B. Poggi, Hôtel Dieu, Hospices Civils de Lyon J.-C. Renversez, C. Trocmé, Centre Hospitalier Universitaire de Grenoble J. Guéchot, E. Lasnier, M. Vaubourdolle, Hôpital Saint-Antoine, AP-HP, Paris H. Voitot, Hôpital Beaujon, AP-HP, Paris A. Vassault, Hôpital Necker, AP-HP, Paris A. Rosenthal-Allieri, Centre Hospitalier Universitaire de Nice A. Lavoinne, F. Ziegler, Centre Hospitalier Universitaire de Rouen M. Bartoli, C. Dorche, C. Lebrun, Centre Hospitalier de Chambéry A. Myara, Growwupe Hospitalier Paris Saint-Joseph, Paris F. Guerber, A. Pottier, Laboratoire Elibio-Groupe Oriade, Vizille, La Mure Hepatologists: V. Leroy, J.-P. Zarski, Centre Hospitalier Universitaire de Grenoble A. Poujol-Robert, R. Poupon, Hôpital Saint-Antoine, AP-HP, Paris A. Abergel, Centre Hospitalier Universitaire de Clermont-Ferrand J.P. Bronowicki, H ô pital de Brabois, Centre Hospitalier Universitaire de Nancy S. Metivier, J.P. Vinel, Hôpital Purpan, Centre Hospitalier Universitaire de Toulouse V. De Ledinghen, Hôpital Haut Levêque, Centre Hospitalier Universitaire de Bordeaux O. Goria, Centre Hospitalier Universitaire de Rouen M. Maynard-Muet, C. Trepo, Hôtel Dieu, Hospices Civils de Lyon Ph. Mathurin, Centre Hospitalier Universitaire de Lille H. Danielou, D. Guyader, Hôpital Pontchaillou, Centre Hospitalier Universitaire de Rennes O. Rogeaux, Centre Hospitalier de Chambéry S. Pol, Ph. Sogni, Hôpital Cochin, AP-HP, Paris A. Tran, Hôpital De l’Archet, Centre Hospitalier Universitaire de Nice P. Calès, Centre Hospitalier Universitaire d’Angers T. Asselah, P. Marcellin, Hôpital Beaujon, AP-HP, Clichy M. Bourlière, V. Oulès, Hôpital Saint Joseph, Assistance Publique- Hôpitaux de Marseille D. Larrey, Centre Hospitalier Universitaire de Montpellier F. Habersetzer, Centre Hospitalier Universitaire de Strasbourg M. Beaugrand, Hôpital Jean Verdier, AP-HP, Bondy Pathologists: N. Sturm, Centre Hospitalier Universitaire de Grenoble E.-S. Zafrani, Hôpital Henri Mondor, AP-HP, Créteil

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

Tissue 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)-1beta 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.