Reproducibility of pulmonary function tests in patients with systemic sclerosis.

Systemic sclerosis (SSc) is a rare autoimmune disease in which interstitial lung disease (ILD) is the leading cause of morbidity and mortality. Clinical management of the lung disease is mainly based on pulmonary function testing (PFT) and their changes over time. Little is known about the reproducibility of PFT testing in SSc patients. The aim of this study was to assess the test-retest reliability and reproducibility of PFTs in SSc patients with or without ILD over 30 days in order determine the potential physiologic variation over the time. We performed prospective observational study of SSc patients. The FVC, FEV1/FVC ratio, DLCO and KCO parameters were assessed in this population at four different timepoints; T0 (time 0) and H3 (T0 + 3 h) defined test-retest reliability, D15 (T0 + 15 days) and D30 (T0 + 30 days) for reproducibility. A mixed linear model was used to test the effect of time (and therefore reproducibility) on patients and we looked for an interaction. We included 25 SSc patients divided in two groups, 14 with ILD and 11 non-ILD. Interactions between time and group were not significant and were not reported. Time and group did not significantly influence the different measures of the PFT: FVC [p values time and group effect respectively (0.33; 0.34)], FEV1/FVC ratio (0.093; 0.056) and DLCO (0.99; 0.13) in the ILD and non ILD group (Table S2). The analyse with interactions between time and group were not significant and are not reported. We also used a Bland Altman test to assess reproducibility for FVC (L) and DLCO (mMKpa/min/L), Figs. 1 and 2 respectively. The measurements were therefore reproducible over time and in each group. PFT parameters are reproducible over time in a clinically stable population of SSc (no significant effect of the time T0, H3, D15 and D30) and there is no significant distinction between patients with ILD and no ILD. These respiratory functional data can further underline their use in clinical practice.

Inflammatory profile of induced sputum composition in systemic sclerosis and comparison with healthy volunteers.

Systemic sclerosis (SSc) is a potentially serious and disabling connective tissue disease specially in case of interstitial lung disease (SSc-ILD). The aim of our study was to evaluate the potential utility of dosing in the induced sputum (IS) and to compare their levels in SSc-ILD and SSc-nonILD patients, as well as in healthy volunteers (HV). IS and sera values were also compared. In a prospective cross-sectional analysis, we studied the IS and serum provided from 25 SSc patients, 15 SSc-nonILD and 10 SSc-ILD, compared to 25 HV. We analyzed sputum cell composition and quantified in the supernatant and corresponding serum by commercially available immunoassays: IGFBP-1, IGFBP-2, IGFBP-3, TGF-ß, IL-8, TNF-α, YKL-40, MMP-7 and MMP-9. Lung function was studied by the determination of FEV-1 (%), FVC (%), DLCO (%) and KCO (%). The IS of SSc patients had a lower weight than HV (p<0.05, p<0.01) without any significant difference with regard to the cellularity. IGFBP-1 (p < 0.0001), TGF-ß (p < 0.05), IL-8 (p < 0.05), YKL-40 (p < 0.0001) and MMP-7 (p < 0.01) levels were increased in the IS of SSc patients compared to HV. Only IL-8 serum levels (p < 0.001) were increased in SSc patients compared to HV. Neither in IS nor in serum were observed differences between SSc-ILD and SSc-nonILD patients. Correlations were observed between IS IL-8 levels and FEV-1 (%) (r = = - 0.53, p < 0.01), FVC (%) (r = - 0.51, p < 0.01) and annualized ∆KCO (%) (r = 0.57, p < 0.05), between IS TGF-ß levels and annualized ∆FEV-1 (%) (r = = - 0.57, p < 0.05), between IS IGFBP-2 levels and annualized ∆KCO (%) (r = 0.56, p < 0.05). Our study showed that SSc patients exhibit raised IS levels of IGFBP-1, TGF-ß, IL-8, YKL-40 and MMP-7, molecules known to be involved in lung remodeling and fibrotic process, without any significant difference between SSc-ILD and SSc-nonILD patients. IL-8, TGF-ß and IGFBP-2 are correlated with lung function in SSc patients which emphasize clinical relevance. IS analysis represents a new approach to understand lung inflammatory process in SSc patients. A longitudinal study is needed to evaluate their pathophysiological relevance.

Restriction of spontaneous and prednisolone-induced leptin production to dedifferentiated state in human hip OA chondrocytes: role of Smad1 and ß-catenin activation.

OBJECTIVE: The aetiology of OA is not fully understood although several adipokines such as leptin are known mediators of disease progression. Since leptin levels were increased in synovial fluid compared to serum in OA patients, it was suggested that joint cells themselves could produce leptin. However, exact mechanisms underlying leptin production by chondrocytes are poorly understood. Nevertheless, prednisolone, although displaying powerful anti-inflammatory properties has been recently reported to be potent stimulator of leptin and its receptor in OA synovial fibroblasts. Therefore, we investigated, in vitro, spontaneous and prednisolone-induced leptin production in OA chondrocytes, focusing on transforming growth factor-ß (TGFß) and Wnt/ß-catenin pathways. DESIGN: We used an in vitro dedifferentiation model, comparing human freshly isolated hip OA chondrocytes cultivated in monolayer during 1 day (type II, COL2A1 +; type X, COL10A1 + and type I collagen, COL1A1 -) or 14 days (COL2A1 -; COL10A1 - and COL1A1+). RESULTS: Leptin expression was not detected in day1 OA chondrocytes whereas day14 OA chondrocytes produced leptin, significantly increased with prednisolone. Activin receptor-like kinase 1 (ALK1)/ALK5 ratio was shifted during dedifferentiation, from high ALK5 and phospho (p)-Smad2 expression at day1 to high ALK1, endoglin and p-Smad1/5 expression at day14. Moreover, inactive glycogen synthase kinase 3 (GSK3) and active ß-catenin were only found in dedifferentiated OA chondrocytes. Smad1 and ß-catenin but not endoglin stable lentiviral silencing led to a significant decrease in leptin production by dedifferentiated OA chondrocytes. CONCLUSIONS: Only dedifferentiated OA chondrocytes produced leptin. Prednisolone markedly enhanced leptin production, which involved Smad1 and ß-catenin activation.

[Proteomics studies on arthritis by SELDI-TOF-MS: identification of the S100 proteins family as proteins of interest]. / Protéomique par SELDI-TOF-MS des maladies inflammatoires articulaires: identification des protéines S100 comme protéines d'intérêt.

Clinical proteomics is a technical approach studying the entire proteome expressed by cells, tissues or organs. It describes the dynamics of cell regulation by detecting molecular events related to diseases development. Proteomic techniques focus mainly on identification of new biomarkers or new therapeutic targets. It is a multidisciplinary approach using medical, biological, bioanalytical and bioinformatics knowledges. A strong collaboration between these fields allowed SELDI-TOF-MS proteomics studies to be performed at the CHU and the University of Liège, in GIGA-Research facilities. The aim of these studies was driven along three main axes of research related to the identification of biomarkers specific to a studied pathology, to a common biological pathway and, finally, to a treatment response. This work was presented in the setting of the "Synthèse CHU 2009" meeting.

Active-site mutants of class B beta-lactamases: substrate binding and mechanistic study.

Increased resistance to beta-lactam antibiotics is mainly due to beta-lactamases. X-ray structures of zinc beta-lactamases unraveled the coordination of the metal ions, but their mode of action remains unclear. Recently, enzymes in which one of the zinc ligands was mutated have been characterized and their catalytic activity against several beta-lactam antibiotics measured. A molecular modeling study of these enzymes was performed here to explain the catalytic activity of the mutants. Coordination around the zinc ions influences the way the tetrahedral intermediate is bound; any modification influences the first recognition of the substrate by the enzyme. For all the studied mutants, at least one of the interactions fails, inducing a loss of catalytic efficiency compared to the wild type. The present studies show that the enzyme cavity is a structure of high plasticity both structurally and mechanistically and that local modifications may propagate its effects far from the mutated amino acid.

Metal ion binding and coordination geometry for wild type and mutants of metallo-beta -lactamase from Bacillus cereus 569/H/9 (BcII): a combined thermodynamic, kinetic, and spectroscopic approach.

One high affinity (nm) and one low affinity (microM) macroscopic dissociation constant for the binding of metal ions were found for the wild-type metallo-beta-lactamase from Bacillus cereus as well as six single-site mutants in which all ligands in the two metal binding sites were altered. Surprisingly, the mutations did not cause a specific alteration of the affinity of metal ions for the sole modified binding site as determined by extended x-ray absorption fine structure (EXAFS) and perturbed angular correlation of gamma-rays spectroscopy, respectively. Also UV-visible absorption spectra for the mono-cobalt enzymes clearly contain contributions from both metal sites. The observations of the very similar microscopic dissociation constants of both binding sites in contrast to the significantly differing macroscopic dissociation constants inevitably led to the conclusion that binding to the two metal sites exhibits negative cooperativity. The slow association rates for forming the binuclear enzyme determined by stopped-flow fluorescence measurements suggested that fast metal exchange between the two sites for the mononuclear enzyme hinders the binding of a second metal ion. EXAFS spectroscopy of the mono- and di-zinc wild type enzymes and two di-zinc mutants provide a definition of the metal ion environments, which is compared with the available x-ray crystallographic data.

Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved cysteine in the catalytic mechanism.

When expressed by pathogenic bacteria, Zn2+-beta-lactamases induce resistance to most beta-lactam antibiotics. A possible strategy to fight these bacteria would be a combined therapy with non-toxic inhibitors of Zn2+-beta-lactamases together with standard antibiotics. For this purpose, it is important to verify that the inhibitor is effective under all clinical conditions. We have investigated the correlation between the number of zinc ions bound to the Zn2+-beta-lactamase from Bacillus cereus and hydrolysis of benzylpenicillin and nitrocefin for the wild type and a mutant where cysteine 168 is replaced by alanine. It is shown that both the mono-Zn2+ (mononuclear) and di-Zn2+ (binuclear) Zn2+-beta-lactamases are catalytically active but with different kinetic properties. The mono-Zn2+-beta-lactamase requires the conserved cysteine residue for hydrolysis of the beta-lactam ring in contrast to the binuclear enzyme where the cysteine residue is not essential. Substrate affinity is not significantly affected by the mutation for the mononuclear enzyme but is decreased for the binuclear enzyme. These results were derived from kinetic studies on two wild types and the mutant enzyme with benzylpenicillin and nitrocefin as substrates. Thus, targeting drug design to modify this residue might represent an efficient strategy, the more so if it also interferes with the formation of the binuclear enzyme.

Interaction between class B beta-lactamases and suicide substrates of active-site serine beta-lactamases.

The most widely used inactivators of active-site serine beta-lactamases behave as substrates of four class B metallo-beta-lactamases, but the efficiency of the catalytic process can vary by several orders of magnitude. A comparison of the kinetic parameters for the alpha and beta isomers of 6-iodopenicillanic acid shows that there is no general preference for the alpha isomer and that the efficient hydrolysis of imipenem by these enzymes must rest on other factors.