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Background Osteoarthritis (OA) is characterised by cartilage degradation but also by other joint tissues modification like subchondral bone sclerosis Objectives In this study, we used a proteomic approach to compare secretome of osteoblasts isolated from sclerotic (SC) or non sclerotic (NSC) area of OA subchondral bone Methods Secretome was analysed using differential quantitative and relative label free analysis on nanoUPLC G2 HDMS system. mRNA of the more differentially secreted proteins were then quantified by RT-PCR and the most relevant proteins identified using western-blotting and immunoassays Results 175 proteins were identified in NSC osteoblasts secretome. Compared to NSC osteoblasts secretome, 13 proteins were significantly less secreted (Osteomodulin, CSF-1, IGFBP5, VCAM-1, IGF2, 78 kDa glucose-regulated protein, versican, calumenin, IGFBP2, thrombospondin-4, periostin, reticulocalbin 1 and osteonectin), and 12 proteins significantly more secreted by SC osteoblasts (CHI3L1, fibulin-3, SERPINE2, IGFBP6, SH3BGRL3, SERPINE1, reticulocalbin3, alpha-2-HS-glycoprotein, TIMP-2, IGFBP3, TIMP-1, SERPINF1). Similar changes in periostin, osteomodulin, SERPINE1, IGFBP6, fibulin-3 and CHI3L1 mRNA levels were observed. Finally, osteomodulin and fibulin-3 specific sequences were quantified by western blot and immunoassays in serum and osteoblasts conditioned culture supernatants. Conclusions We highlighted some proteins differentially secreted by NSC and SC osteoblasts of OA subchondral bone sclerosis. These changes contribute to explain some features observed in OA subchondral bone, like the increase of bone remodelling or abnormalities in bone matrix mineralization. Among identified proteins, osteomodulin was found decreased and fibulin-3 increased in serum of OA patients. These findings suggest that osteomodulin and fibulin-3 fragments could be biomarkers to monitor early changes in subchondral bone metabolism in OA. Acknowledgements The D-Board project has received funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 3 05 815. Disclosure of Interest None declared

Viral semaphorins are semaphorin 7A (sema7A) mimics found in pox- and herpesviruses. Among herpesviruses, semaphorins are encoded by gammaherpesviruses of the Macavirus genus only. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus that persistently infects wildebeest asymptomatically but induces malignant catarrhal fever (MCF) when transmitted to several species of susceptible ruminants and the rabbit model. MCF is caused by the activation/proliferation of latently infected T lymphocytes. Viral semaphorins have been suggested to mediate immune evasion mechanisms and/or directly alter host T cell function. We studied AlHV-sema, the viral semaphorin encoded by the A3 gene of AlHV-1. Phylogenetic analyses revealed independent acquisition of pox- and herpesvirus semaphorins, suggesting that these proteins might have distinct functions. AlHV-sema showed a predicted three-dimensional structure very similar to sema7A and conserved key residues in sema7A-plexinC1 interaction. Expression analyses revealed that AlHV-sema is a secreted 93-kDa glycoprotein expressed during the early phase of virus replication. Purified AlHV-sema was able to bind to fibroblasts and dendritic cells and induce F-actin condensation and cell retraction through a plexinC1 and Rho/cofilin-dependent mechanism. Cytoskeleton rearrangement was further associated with inhibition of phagocytosis by dendritic cells and migration to the draining lymph node. Next, we generated recombinant viruses and demonstrated that the lack of A3 did not significantly affect virus growth in vitro and did not impair MCF induction and associated lymphoproliferative lesions. In conclusion, AlHV-sema has immune evasion functions through mechanisms similar to poxvirus semaphorin but is not directly involved in host T cell activation during MCF. IMPORTANCE Whereas most poxviruses encode viral semaphorins, semaphorin-like genes have only been identified in few gammaherpesviruses belonging to the Macavirus genus. Alcelaphine herpesvirus 1 (AlHV-1) is a macavirus carried asymptomatically by wildebeest but induces a latency-associated lymphoproliferative disease of T lymphocytes in various ruminant species, namely, malignant catarrhal fever (MCF). Viral semaphorins have been hypothesized to have immune evasion functions and/or be involved in activating latently infected T cells. We present evidence that the viral semaphorin AlHV-sema inhibits dendritic cell phagocytosis and migration to the draining lymph node, both being indispensable mechanisms for protective antiviral responses. Next, we engineered recombinant viruses unable to express AlHV-sema and demonstrated that this protein is dispensable for the induction of MCF. In conclusion, this study suggests that herpesvirus and poxvirus semaphorins have independently evolved similar functions to thwart the immune system of the host while AlHV-sema is not directly involved in MCF-associated T-cell activation.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

A method for the high-throughput analysis of human serum for the 38 most prevalent polychlorinated biphenyls (PCBs) based on the use of fast gas chromatography-isotopic dilution time-of-flight mass spectrometry (GC-IDTOFMS) is presented. The chromatographic separation time was 8 min. The separation of the congeners was carried out either chromatographically or analytically using the mass spectral deconvolution capability of the TOFMS. The instrument and the method limits of detection (LODs) were 0.5 pg microL(-1) and 20 pg microL(-1), respectively, which is not as good as the one achieved using high resolution mass spectrometry (HRMS) but allows the detection and quantification of the prevalent PCBs present in real human serum samples. The dynamic range covered 3 orders of magnitude. The comparison with the high resolution mass spectrometry (HRMS) reference method (28 min) was good and some separation improvements have been observed. This method allows the analysis of 100 samples per day per instrument.

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10-20 μm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies.

Stable gadolinium(III) chelates are nowadays routinely used as contrast agents for magnetic resonance imaging (MRI). Their non-covalent binding to human serum albumin (HSA) has shown to improve their efficacy. Non-covalent interactions lead to complex formation that can be quantified by several techniques that are mostly tedious and time-consuming. In this study, electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interaction between HSA and several gadolinium(III) complexes. The results were compared with those obtained in the liquid phase. Four gadolinium complexes were investigated: Gd-DTPA 1, Gd-C4Me-DTPA 2, Gd-EOB-DTPA 3, and MP-2269 4. Relaxometry studies show that complexes 1 and 2 have no significant affinity for HSA, while complexes 3 and 4 have increasing affinities for the protein. 1:1 and 1:2 complexes between HSA and MP-2269 were detected by ESI-MS for a twofold excess of the contrast agent, whereas a ligand/protein molar ratio of 4:1 was necessary to observe a 1:1 stoichiometry for Gd-EOB-DTPA, an observation that is in good agreement with the known weaker affinity of the contrast agent for the protein. At a fourfold molar excess, no supramolecular complex was observed for Gd-DTPA 1 and Gd-C4Me-DTPA 2; a tenfold molar excess was necessary to detect a 1:1 complex, confirming the very weak affinity of these contrast agents for HSA. Copyright © 2004 John Wiley & Sons, Ltd.

Incinerators, dioxin crisis and health risks for the general population in Belgium. Bernard A., Fierens S., Mairesse H., C. Hermans, F. Broeckaert, J.-F. Focant, E. Depauw. Since a few years, there is some concern in Belgium about the potential health risks of dioxins emitted by municipal solid waste incinerators (MSWI) and more recently by the dioxins which were introduced in the food chain during the Belgian PCB incident. This communication reports studies which have been recently carried out to assess the possible health impact of dioxins in these two sit¬ uations. Dioxins (PCDDIFs ) were determined in the blood of 85 subjects living around two MSWI, one localized in an industrial area and the other in a rural area. By comparison with an age-matched control group of 27 subjects living in an unpolluted area, an increase of dioxin body burden (36,7 vs 24,3 pg TEQlg blood fat, 34 % on average) was found only in the vicinity of the MSWI in the rural area, most likely because of a higher consumption of locally produced foods around this incinerator. However, despite this increase, all subjects had dioxin levels still in the range of values reported in unpolluted areas in Europe. As to the dioxin crisis, the contamination was too limited in time and in the food chain to have caused adverse health effects in the general popula¬ tion of Belgium even if most contaminated foods (e.g. chicken meat) had dioxin levels more than 100 times above tolerance levels. Indeed, a doubling of the PCB and dioxin burden of the young adult population would have required the consumption of respectively 10 and 20 highly contaminated meals. In view of the very limited proportion of the food chain affected by the incident (less than 2% of poultry farms and of 0.1% of all farms in Bel¬ gium), such an extreme scenario was quite improbable at the pop¬ ulation level. It was conceivable only in farmers who could have consumed their own products. But even for these farmers, this would mean that their PCB and dioxin body burden would have been increased at levels of the 80s or of subjects regularly eating contaminated seafood., Bernard Alfred, Depauw Edwin, Fierens Sébastien, Mairesse Hélène, Hermans Cédric, Broeckaert Fabrice, Focant Jean-François. Incinérateurs, crise dioxine, et risques sanitaires pour la population belge. In: Bulletin de la Classe des sciences, tome 12, n°1-6, 2001. pp. 103-117.