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Choulaki C.,University of Crete | Papadaki G.,University of Crete | Repa A.,University of Crete | Kampouraki E.,University of Crete | And 7 more authors.
Arthritis Research and Therapy | Year: 2015

Introduction: Interleukin-1β (IL-1β) is a major inflammatory cytokine, produced predominantly by innate immune cells through NLRP3-inflammasome activation. Both intrinsic and extrinsic danger signals may activate NLRP3. Genetic variations in NLRP3-inflammasome components have been reported to influence rheumatoid arthritis (RA) susceptibility and severity. We sought to assess the activity of NLRP3-inflammasome in patients with active RA compared to healthy individuals. Method: Intracellular protein expression of NLRP3, ASC, pro- and active caspase-1, pro- and active IL-1β was assessed by immunoblotting both at baseline and upon inflammasome activation. NLRP3 function (IL-1β secretion) was assessed upon priming of TLR2 (Pam(3)CysSK(4), TLR3 (poly(I:C)) or TLR4 (LPS) and ATP sequential treatment. We used caspase inhibitors (casp-1, 3/7 and 8) to assess their contribution to IL-1β maturation. All experiments were performed in whole blood cells. Results: Active RA patients (n = 11) expressed higher basal intracellular levels of NLRP3 (p < 0.008), ASC (p < 0.003), active caspase-1 (p < 0.02) and pro-IL-1β (p < 0.001). Upon priming with TLR4 (LPS) and ATP, RA-derived cell extracts (n = 7) displayed increased expression of NLRP3 (p < 0.01) and active caspase-1 (p < 0.001). Secreted IL-1β in culture supernatants from whole blood cells activated with TLR4 (LPS) or TLR3 agonist (poly(I:C)) plus ATP was higher in RA patients (n = 20) versus controls (n = 18) (p < 0.02 for both). Caspase-1 inhibition significantly reduced IL-1β secretion induced by all stimuli, whereas caspase-8 inhibition affected only TLR4 and TLR3 cell priming. Conclusion: Patients with active RA have increased expression of NLRP3 and NLRP3-mediated IL-1β secretion in whole blood cells upon stimulation via TLR3 and TLR4 but not TLR2. In these patients, IL-1β secretion seems to be predominately driven by caspase-1 and caspase-8. Targeting NLRP3 or downstream caspases may be of benefit in suppressing IL-1β production in RA. © 2015 Choulaki et al. Source

Fanouriakis A.,University of Crete | Krasoudaki E.,Venizeleion Hospital of Heraklion | Tzanakakis M.,University of Crete | Boumpas D.T.,University of Crete | Boumpas D.T.,Institute of Molecular Biology and Biotechnology FORTH
Modern Rheumatology | Year: 2012

The treatment of lupus nephritis has seen significant advances during the past decade mainly due to the publication of well-designed randomized clinical trials (RCTs). The choice of treatment is guided by the histopathologic classification but is also influenced by demographic, clinical, and laboratory characteristics that allow for the identification of patients at risk for more aggressive disease. For the induction arm, low-dose cyclophosphamide regimens and mycophenolate mofetil have been validated as alternatives to the established National Institutes of Health regimen of high-dose cyclophosphamide; for the maintenance phase, azathioprine and mycophenolate compete for treatment of first choice. Rituximab is efficacious in real-life clinical practice but ineffective in clinical trials. The role of recently approved belimumab in lupus nephritis eagerly awaits further documentation. Aggressive management of comorbid conditions, such as hypertension and dyslipidemia, is of utmost importance. Here, we review the latest advances in lupus nephritis therapy with a focus on recent RCTs as well as new biologic agents under development. Furthermore, we propose a therapeutic algorithm in an effort to facilitate clinical decision-making in this gradually changing landscape. Upcoming European and American recommendations should provide further clarification. © Japan College of Rheumatology 2012. Source

Papadakis G.,Advanced Materials and Processes | Gizeli E.,Institute of Molecular Biology and Biotechnology FORTH | Gizeli E.,University of Crete
Analytical Methods | Year: 2014

Screening for mutations in the tumor-suppressor genes BRCA1 and BRCA2 is of great importance for breast and ovarian cancer prevention. We describe a methodology for mutation screening and detection based on acoustic wave devices. In particular, we detect four mutations located in BRCA1 and BRCA2 genes using the quartz crystal microbalance technique. The detection is based on measurements of the acoustic ratio of dissipation versus frequency change (ΔD/ΔF) of double-stranded DNA molecules bound to the device surface that are produced after PCR amplification and restriction digestion; the acoustic ratio has been shown to be a measure of the intrinsic viscosity of the attached molecules, which, in turn, depends on the size of the dsDNAs. Novel features of this approach are the lack of a hybridization step, the label free sensing of the length, rather than mass, of the DNA molecules and the direct detection of the digested DNA products without prior purification. The method is generic, simple and capable of detecting single base mutations to long genomic rearrangements; it is also suitable and applicable to a Lab-on-a-chip concept. This journal is © 2014 The Royal Society of Chemistry. Source

Tilstra J.S.,University of Pittsburgh | Robinson A.R.,University of Pittsburgh | Wang J.,University of California at Riverside | Gregg S.Q.,University of Pittsburgh | And 17 more authors.
Journal of Clinical Investigation | Year: 2012

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB - activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging. Source

Papanastasiou M.,Institute of Molecular Biology and Biotechnology FORTH | Orfanoudaki G.,Institute of Molecular Biology and Biotechnology FORTH | Orfanoudaki G.,University of Crete | Koukaki M.,Institute of Molecular Biology and Biotechnology FORTH | And 7 more authors.
Molecular and Cellular Proteomics | Year: 2013

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected +19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies +25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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