Proteomic analysis of inflammatory protein expression patterns in cell culture and transgenic animal models for Alzheimer's disease

Dementia is a syndrome characterized by failure of recent memory and other cognitive functions that is usually insidious in onset but steadily progresses with age. Alzheimer’s disease (AD) is the most common form of senile dementia. It is neuropathologically characterized by extracellular and perivascular aggregation of amyloid β (Aβ) peptide, by the generation of intracellular neurofibrillary tangles due to a hyperphosphorylation of tau protein and by an increased rate of neuronal degeneration. The degenerative process starts 20-30 years before the clinical onset of the disease. Clinical diagnosis of AD is difficult but possible, but can only be confirmed by biopsy or autopsy. At present, no biological marker exists for early diagnosis of AD during life. Therefore, identification of biomarkers for AD would be of great value for clinical diagnosis of incipient AD. Recent studies have proven the involvement of inflammatory processes in the neurodegenerative events in AD. Inflammation may not be the first event in the progression of the disease, but it involves activation of glia cells including microglia and astrocytes and subsequent release of proinflammatory mediators. Cytokines released such as IL-1, TNF-α and IL-6 are the main proinflammatory cytokines that can modulate inflammatory responses as well as glial proliferation and activation. Oxidative stress triggered by inflammatory processes causes changes in proteins such as tyrosine nitration or lipid peroxidation. Aβ deposits, tau hyperphosphorylation, inflammation and oxidative stress may finally lead to changes in synaptic connectivity and efficacy including perturbation of long-term potentiation (LTP), important in the formation of memory. Proteomic technology used in these studies is a recent technology which is a two step process: separation of proteins and their subsequent analysis by mass spectrometry. Moreover, this technology can provide new information concerning the expression level, post-translational modification of specific proteins as well as their conformational changes during disease progression. In our study, this technology was modified and improved, e.g by the miniaturization of the complete process. Proteomic technology was also used in parallel with other methods such as chromatography in order to increase the sensitivity of detection by mass spectrometry. This study aimed: 1) To establish that cytokine treatment of human microglia cells is an efficient method to study certain aspects of AD pathogenesis. For this analysis, a map of protein expression in normal and in treated microglia cells was made. 2) To map protein expression in APP/PS2 transgenic mice, a model for human AD, in order to compare human AD brain with murine models. 3) To identify highly nitrated proteins in brains of transgenic animals. Several proteins were found to be modified after injury. 4) To provide evidence for instability of synapses in AD brains. To start with this study, the technologies used to map mouse brain cytosolic proteins were improved. 5) To isolate synaptosomal membranes from the whole brain and to analyse it by massspectrometry. For mapping synaptic membrane protein expression in controls or transgenic mouse models, the technology was miniaturizated and optimized. This study is still in progress

Vestibular Schwann cells are a distinct subpopulation of peripheral glia with specific sensitivity to growth factors and extracellular matrix components

International audienceVestibular nerve Schwann cells are predisposed to develop schwannoma. While knowledge concerning this condition has greatly improved, little is known about properties of normal vestibular Schwann cells. In an attempt to understand this predisposition, we evaluated cell density regulation and proliferative features of these cells taken from 6-day-old rats. Data were compared to those obtained with sciatic Schwann cells. In both vestibular and sciatic 7-day-old cultures, Schwann cells appear as bipolar or flattened cells. However , sciatic and vestibular cells greatly differ in other aspects: on poly-L-lysine coating, sciatic cells specifically synthesize myelin basic protein, while expression of P0 mRNAs is restricted to some vestibular cells. Laminin increases sciatic cell density but not that of vestibular cells. Fibronectin selectively enhances the proliferation of vestibular Schwann cells and lacks an effect on sciatic ones. Comparison of cell density changes between sciatic and vestibular cells shows that they are sensitive to two different sets of growth factors. Progesterone and FGF-2 combined with forskolin selectively enhance the cell density of sciatic glia, while IGF-1 and GDNF specifically increase vestibular cell density. Furthermore, BrdU incorporation assays indicate that GDNF is also a mitogen for vestibular cells. Altogether, vestibular Schwann cells display phenotypic features and responsiveness to exog-enous signals that are significantly different from sciatic Schwann cells, suggesting that vestibular glia form a subpopulation of Schwann cells

MAO-A-induced mitogenic signaling is mediated by reactive oxygen species, MMP-2, and the sphingolipid pathway.

International audienceThe degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall

A signaling cascade mediated by ceramide, src and PDGFRβ coordinates the activation of the redox-sensitive neutral sphingomyelinase-2 and sphingosine kinase-1.

International audienceStress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H2O2. In mesenchymal cells, H2O2 elicits a dose-dependent biphasic effect, mitogenic at low concentration (5μM), and anti-proliferative and toxic at high concentration (100μM). Low H2O2 concentration triggered activation of nSMase2 and SK1 through a nSMase2/Cer-dependent signaling pathway that acted upstream of activation of SK1. Further results implicated src and the trans-activation of PDGFRβ, as supported by the blocking effect of specific siRNAs, pharmacological inhibitors, and genetically deficient cells for nSMase2, src and SK1. The H2O2-induced src/PDGFRβ/SK1 signaling cascade was impaired in nSMase2-deficient fro/fro cells and was rescued by exogenous C2Cer that activated src/PDGFRβ/SK1. Thus, the results define a nSMase2/SK1 signaling pathway implicated in the mitogenic response to low oxidative stress. On the other hand, high oxidative stress induced inhibition of SK1. The results also showed that the toxicity of high H2O2 concentration was comparable in control and nSMase2-deficient cells. Taken together the results identify a tightly coordinated nSMase2/SK1 pathway that mediates the mitogenic effects of H2O2 and may sense the degree of oxidative stress

Annexin II-dependent actin remodelling evoked by hydrogen peroxide requires the metalloproteinase/sphingolipid pathway

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Influenza Vaccination Coverage in the 2004/05, 2005/06, and 2006/07 Seasons: A Secondary Data Analysis Based on Billing Data of the German Associations of Statutory Health Insurance Physicians

Hintergrund: Zur Senkung der Krankheitslast von saisonaler Influenza empfiehlt die Ständige Impfkommission eine jährliche Impfung für Risikogruppen. Ziel ist das Erreichen einer Impfquote von 75 % bei über 60-Jährigen bis zum Jahr 2010. Die Autoren stellen Influenzaimpfquoten der Saisons 2004/05 bis 2006/07 vor, die anhand von Abrechnungsdaten der Kassenärztlichen Vereinigungen (KVen) bestimmt wurden. Methode: Pseudonymisierte Daten von 14 der 17 KVen wurden analysiert. Studienpopulation sind alle gesetzlich Krankenversicherten der untersuchten KV-Gebiete (n = 61,5 Millionen; 86 % der Bevölkerung der 14 KV-Gebiete). Die Impfquoten werden berechnet durch die Anzahl geimpfter Personen bezogen auf die jeweilige Population. Ergebnisse: Die Influenzaimpfquote der Bevölkerung beziehungsweise der über 60-Jährigen in den untersuchten KV-Gebieten beträgt 19 % beziehungsweise 45 % (Saison 2004/05), 22 % beziehungsweise 50 % (Saison 2005/06) und 21 % beziehungsweise 49 % (Saison 2006/07) und liegt in den neuen Bundesländern höher als in den alten Bundesländern. Gut ein Drittel aller geimpften Personen wurde in allen drei Saisons geimpft, bei den über 60-Jährigen fast die Hälfte. Schlussfolgerung: Seit der Saison 2005/06 ist die Impfquote nicht weiter angestiegen, so dass es eine besondere Herausforderung für alle Akteure darstellt, das anvisierte Ziel einer Impfquote der über 60-Jährigen von 75 % bis zum Jahr 2010 zu erreichen. Die Auswertung der KV-Daten kann ein kontinuierliches Monitoring der Influenzaimpfquoten ermöglichen.Background: The German Standing Committee on Vaccination recommends annual vaccination for persons in high-risk groups in order to lower the disease burden associated with seasonal influenza. The stated target is 75% vaccination coverage of people over age 60 by the year 2010. We present statistics based on billing data of the german associations of statutory health insurance physicians regarding vaccination coverage for influenza in the three seasons from 2004/05 to 2006/07. Methods: We analyzed anonymous data from 14 of the 17 associations of statutory health insurance physicians in Germany. The study population consisted of all persons covered by statutory health insurance in the geographical areas under study (61.5 million persons, or 86% of the total population of these areas). Vaccination coverage was calculated as the number of vaccinated persons divided by the number of persons covered by statutory health insurance. Results: The influenza vaccination coverage of the overall study population was 19% in 2004/05, 22% in 2005/06, and 21% in 2006/07. The coverage of persons over age 60 was 45% in 2004/05, 50% in 2005/06, and 49% in 2006/07 and was higher in areas that were formerly part of East Germany than in the rest of the country. More than a third of all vaccinated persons were vaccinated in all three seasons, as were almost half of the vaccinated persons over age 60. Conclusion: There was no secular increase in influenza vaccination coverage over the period 2005/06 to 2006/07. The stated target of 75% vaccination coverage for persons over age 60 by the year 2010 would thus seem to represent a major challenge for all persons involved. The analysis of data of the associations of statutory health insurance physicians enables continuous monitoring of influenza vaccination coverage

Safety of rituximab in rheumatoid arthritis patients with a history of severe or recurrent bacterial infection: observational study of 30 cases in everyday practice.

International audienceOBJECTIVES: To report our experience with rituximab therapy in patients with rheumatoid arthritis (RA) and a history of severe or recurrent bacterial infections. PATIENTS AND METHODS: Retrospective observational study in five rheumatology departments experienced in the use of biotherapies. Patients were included if they had RA and a history of severe or recurrent bacterial infection (requiring admission and/or intravenous antimicrobial therapy) that contraindicated the introduction or continuation of TNFalpha antagonist therapy. RESULTS: Of 161 RA patients given rituximab in the five study centers, 30 met the inclusion criteria, 23 females and seven males with a mean age of 58.4+/-11.8 years and a mean disease duration of 11.4+/-13.9 years. Among them, 22 had rheumatoid factors and 21 had received TNFalpha antagonist therapy (one agent in 15 patients, two in five patients and three in one patient). Prior infections were as follows: septicemia, n=2; lower respiratory tract infection or lung abscess, n=12; prosthesis infection, n=3; septic arthritis, n=3; endocarditis, n=1; pyelonephritis, n=2; osteitis, n=4; and various skin infections (erysipelas, cellulitis or skin abscess), n=6. Of these 33 infections, 21 occurred during TNFalpha antagonist therapy. During rituximab therapy, all patients received concomitant glucocorticoid therapy (mean dosage, 12+/-7.9 mg/day). The number of rituximab cycles was one in 13 patients, two in seven patients and three or more in 10 patients. Mean time from the single or last serious infection and the first rituximab infusion was 20.1+/-18.7 months. Mean follow-up since the first rituximab infusion was 19.3+/-7.4 months. During follow-up, six (20%) patients experienced one infection each. Immunoglobulin levels after rituximab therapy were within the normal range. CONCLUSION: Rituximab therapy was well tolerated in 24 (80%) of 30 patients with RA and a history of severe or recurrent bacterial infection. In everyday practice, rituximab therapy seems safe with regard to the recurrence of infectious episodes. However, longer follow-ups are needed

Structural basis for substrate selectivity and nucleophilic substitution mechanisms in human adenine phosphoribosyltransferase catalyzed reaction

International audienceThe reversible adenine phosphoribosyltransferase enzyme (APRT) is essential for purine homeostasis in prokaryotes and eukaryotes. In humans, APRT (hAPRT) is the only enzyme known to produce AMP in cells from dietary adenine. APRT can also process adenine analogs, which are involved in plant development or neuronal homeostasis. However, the molecular mechanism underlying substrate specificity of APRT and catalysis in both directions of the reaction remains poorly understood. Here we present the crystal structures of hAPRT complexed to three cellular nucleotide analogs (hypoxanthine, IMP, and GMP) that we compare with the phosphate-bound enzyme. We established that binding to hAPRT is substrate shape-specific in the forward reaction, whereas it is base-specific in the reverse reaction. Furthermore , a quantum mechanics/molecular mechanics (QM/ MM) analysis suggests that the forward reaction is mainly a nucleophilic substitution of type 2 (S N 2) with a mix of S N 1-type molecular mechanism. Based on our structural analysis, a magnesium-assisted S N 2-type mechanism would be involved in the reverse reaction. These results provide a framework for understanding the molecular mechanism and substrate discrimination in both directions by APRTs. This knowledge can play an instrumental role in the design of inhibitors, such as antiparasitic agents, or adenine-based substrates

NGS developed in AQUAGENET. Designs and main results

AQUAGENET network comprises six partners from France, Spain and Portugal that cooperate for the development and application of biotechnology in the aquaculture industry. This network has implemented the most recent NGS technologies for the study genome and transcriptome of several species of fish, molluscs and pathogens. &nbsp; This study has been co-funded by project AQUAGENET (SOE2/P1/ E287) program INTERREG IVB SUDOE, and by project RTA2009-00066-00-00 from the Instituto Nacional de Investigaci&oacute;n y Tecnolog&iacute;a Agraria y Alimentaria (INIA, Spain), and FEDER (EU).www.juntadeandalucia.es/agriculturaypesca/ifapa/aquagenetLa red AQUAGENET incluye seis beneficiarios de Francia, Espa&ntilde;a y Portugal que cooperan para el desarrollo y aplicaci&oacute;n de biotecnolog&iacute;a en la industria acu&iacute;cola. Esta red ha implementado las m&aacute;s recientes tecnolog&iacute;as NGS para el estudio del genoma y transcriptoma de diferentes especies de peces, moluscos y pat&oacute;genos. Este trabajo ha sido cofinanciado por el proyecto AQUAGENET (SOE2/P1/ E287) programa INTERREG IVB SUDOE, y por el proyecto RTA2009-00066-00-00 para el Instituto Nacional de Investigaci&oacute;n y Tecnolog&iacute;a Agraria y Alimentaria (INIA, Spain), and FEDER (EU). www.juntadeandalucia.es/agriculturaypesca/ifapa/aquagenet</p