Identification of a Novel Serum Proteomic Signature for Primary Sjögren's Syndrome.

Context: Primary Sjögren's syndrome (pSS) is a complex heterogeneous autoimmune disease (AID) which can mimic rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Our exploratory study investigated serum biomarkers that may discriminate pSS from RA and SLE. Methods: Serum concentrations of 63 biomarkers involved in immune cell trafficking, inflammatory response, cellular movement, and cell-to-cell signaling were measured in AID patients, included prospectively into the study at the Montpellier University Hospital. A multivariate analysis by multiple logistic regression was performed, and discriminative power assessed using logistic regression adjusted on significant demographic factors. Results: Among the 95 patients enrolled, 42 suffered from pSS, 28 from RA, and 25 from SLE. Statistical analysis showed that concentrations of BDNF (OR = 0.493 with 95% CI [0.273-0.891]; p = 0.0193) and I-TAC/CXCL11 (OR = 1.344 with 95% CI [1.027-1.76]; p = 0.0314) can significantly discriminate pSS from RA. Similarly, greater concentrations of sCD163 (OR = 0.803 with 95% CI [0.649-0.994]; p = 0.0436), Fractalkine/CX3CL1 (OR = 0.534 with 95% CI [0.287-0. 991]; p = 0.0466), MCP-1/CCL2 (OR = 0.839 with 95% CI [0.732-0.962]; p = 0.0121), and TNFa (OR = 0.479 with 95% CI [0.247-0.928]; p = 0.0292) were associated with SLE diagnosis compared to pSS. In addition, the combination of low concentrations of BDNF and Fractalkine/CX3CL1 was highly specific for pSS (specificity 96.2%; positive predictive value 80%) compared to RA and SLE, as well as the combination of high concentrations of I-TAC/CXCL11 and low concentrations of sCD163 (specificity 98.1%; positive predictive value 75%). Conclusion: Our study highlights biomarkers potentially involved in pSS, RA, and SLE pathophysiology that could be useful for developing a pSS-specific diagnostic tool.

Combining culture and microbead-based immunoassay for the early and generic detection of bacteria in platelet concentrates.

BACKGROUND: Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS: A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS: Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION: In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

Tissue-type plasminogen activator controls neuronal death by raising surface dynamics of extrasynaptic NMDA receptors.

N-methyl-d-aspartate receptors (NMDARs) are ion channels whose synaptic versus extrasynaptic localization critically influences their functions. This distribution of NMDARs is highly dependent on their lateral diffusion at the cell membrane. Each obligatory subunit of NMDARs (GluN1 and GluN2) contains two extracellular clamshell-like domains with an agonist-binding domain and a distal N-terminal domain (NTD). To date, the roles and dynamics of the NTD of the GluN1 subunit in NMDAR allosteric signaling remain poorly understood. Using single nanoparticle tracking in mouse neurons, we demonstrate that the extracellular neuronal protease tissue-type plasminogen activator (tPA), well known to have a role in the synaptic plasticity and neuronal survival, leads to a selective increase of the surface dynamics and subsequent diffusion of extrasynaptic NMDARs. This process explains the previously reported ability of tPA to promote NMDAR-mediated calcium influx. In parallel, we developed a monoclonal antibody capable of specifically blocking the interaction of tPA with the NTD of the GluN1 subunit of NMDAR. Using this original approach, we demonstrate that the tPA binds the NTD of the GluN1 subunit at a lysine in position 178. Accordingly, when applied to mouse neurons, our selected antibody (named Glunomab) leads to a selective reduction of the tPA-mediated surface dynamics of extrasynaptic NMDARs, subsequent signaling and neurotoxicity, both in vitro and in vivo. Altogether, we demonstrate that the tPA is a ligand of the NTD of the obligatory GluN1 subunit of NMDAR acting as a modulator of their dynamic distribution at the neuronal surface and subsequent signaling.

Use of a synthetic biosensor for neutralizing activity-biased selection of monoclonal antibodies against atroxlysin-I, an hemorrhagic metalloproteinase from Bothrops atrox snake venom.

BACKGROUND: The snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy. Little is known about the immunochemistry of the P-I SVMPs since few monoclonal antibodies (mAbs) against these molecules have been obtained. In addition, producing toxin-neutralizing mAbs remains very challenging. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report on the set-up of a functional screening based on a synthetic peptide used as a biosensor to select neutralizing mAbs against SVMPs and the successful production of neutralizing mAbs against Atroxlysin-I (Atr-I), a P-I SVMP from B. atrox. Hybridomas producing supernatants with inhibitory effect against the proteolytic activity of Atr-I towards the FRET peptide Abz-LVEALYQ-EDDnp were selected. Six IgG1 Mabs were obtained (named mAbatr1 to mAbatr6) and also two IgM. mAbatrs1, 2, 3 and 6 were purified. All showed a high specific reactivity, recognizing only Atr-I and B. atrox venom in ELISA and a high affinity, showing equilibrium constants in the nM range for Atr-I. These mAbatrs were not able to bind to Atr-I overlapping peptides, suggesting that they recognize conformational epitopes. CONCLUSIONS/SIGNIFICANCE: For the first time a functional screening based on a synthetic biosensor was successfully used for the selection of neutralizing mAbs against SVMPs.

Circulating osteoglycin and NGAL/MMP9 complex concentrations predict 1-year major adverse cardiovascular events after coronary angiography.

OBJECTIVE: Previous proteomics experiments have demonstrated that several proteins are differentially expressed in vulnerable human carotid plaques compared with stable plaques. This study aims to investigate the prognostic value of 13 such circulating biomarkers in patients with coronary artery disease. APPROACH AND RESULTS: Between 2008 and 2011, 768 patients who underwent coronary angiography for acute coronary syndrome or stable angina pectoris were included in a prospective biomarker study. Plasma concentrations of 13 biomarkers were measured in 88 patients who experienced a major adverse cardiovascular event (MACE) within 1 year and 176 control patients without MACE who were matched on age, sex, and number of diseased coronary vessels. MACE comprised all-cause mortality, acute coronary syndrome, unplanned coronary revascularization, and stroke. After adjustment for established cardiovascular risk factors, osteoglycin (OGN; odds ratio per SD increase in ln-transformed OGN, 1.53; 95% confidence interval, 1.11-2.11; P=0.010) and neutrophil gelatinase-associated lipocalin/matrix metalloproteinase 9 (NGAL/MMP9; odds ratio per SD increase in ln-transformed NGAL/MMP9, 1.37; 95% confidence interval, 1.01-1.85; P=0.042) complex were independently associated with MACE during follow-up. These associations were independent of C-reactive protein levels. Adding OGN or NGAL/MMP9 to a model containing conventional risk factors did not significantly improve discriminatory power (OGN: area under receiver operating characteristic curve, 0.75 versus 0.67; NGAL/MMP9: 0.73 versus 0.67) but did significantly improve risk reclassification (OGN: net reclassification index=0.29; 95% confidence interval, 0.05-0.53; P<0.019; NGAL/MMP9: net reclassification index=0.44; 95% confidence interval, 0.20-0.69; P<0.001). CONCLUSIONS: Circulating OGN and NGAL/MMP9 complex are promising biomarkers that are expressed in vulnerable atherosclerotic plaques and may have incremental value for prediction of MACE within 1 year after coronary angiography.

Local carotid atherosclerotic plaque proteins for the identification of circulating biomarkers in coronary patients.

OBJECTIVE: To identify circulating biomarkers that originate from atherosclerotic vulnerable plaques and that could predict future cardiovascular events. METHODS: After a protein enrichment step (combinatorial peptide ligand library approach), we performed a two-dimensional electrophoresis comparative analysis on human carotid plaque protein extracts (fibrotic and hemorrhagic atherosclerotic plaques). In silico analysis of the biological processes was applied on proteomic data. Luminex xMAP assays were used to quantify inflammatory components in carotid plaques. The systemic quantification of proteins originating from vulnerable plaques in blood samples from patients with stable and unstable coronary disease was evaluated. RESULTS: A total of 118 proteins are differentially expressed in fibrotic and hemorrhagic plaques, and allowed the identification of three biological processes related to atherosclerosis (platelet degranulation, vascular autophagy and negative regulation of fibrinolysis). The multiplex assays revealed an increasing expression of VEGF, IL-6, IL-8, IP-10 and RANTES in hemorrhagic as compared to fibrotic plaques (p<0.05). Measurement of protein expressions in plasmas from patients with stable and unstable coronary disease identified a combination of biomarkers, including proteins of the smooth muscle cell integrity (Calponin-1), oxidative stress (DJ-1) and inflammation (IL-8), that allows the accurate classification of patients at risk (p=0.0006). CONCLUSION: Using tissue protein enrichment technology, we validated proteins that are differentially expressed in hemorrhagic plaques as potential circulating biomarkers of coronary patients. Combinations of such circulating biomarkers could be used to stratify coronary patients.

N-truncated Aß peptides in complex fluids unraveled by new specific immunoassays.

Previous studies have highlighted the potential physiopathological and diagnostic role of N- and C-terminally truncated amyloid-ß (Aß) peptides in Alzheimer's disease. However, our knowledge about their production remains incomplete, in part due to the lack of very specific and sensitive tools for their detection. We thus developed specific monoclonal antibodies that target either Aß11-x or Aß17-x species, which result from the combined cleavages by ß/γ- or α/γ-secretases, respectively. The presence of Aß peptides truncated at residue 11 and 17 peptides was qualitatively and quantitatively assessed, using surface enhanced laser desorption ionization-time of flight mass spectrometry and xMAP (Multi-Analyte Profiling) immunoassays, in the supernatant of HEK293 cells that overexpress wild type or mutant Aß protein precursor or in which α- and ß-secretase activities had been modulated. Our results show a differential secretion of Aß11-40 and Aß17-40 species by these HEK293 cell lines. Finally, Aß11-40 concentration in human cerebrospinal fluid (measured with the new xMAP immunoassays) from a first pilot study was higher in cerebrospinal fluid samples from patients with Alzheimer's disease than in samples from patients with other types of dementia.

Identification and characterization of cholest-4-en-3-one, oxime (TRO19622), a novel drug candidate for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive death of cortical and spinal motor neurons, for which there is no effective treatment. Using a cell-based assay for compounds capable of preventing motor neuron cell death in vitro, a collection of approximately 40,000 low-molecular-weight compounds was screened to identify potential small-molecule therapeutics. We report the identification of cholest-4-en-3-one, oxime (TRO19622) as a potential drug candidate for the treatment of ALS. In vitro, TRO19622 promoted motor neuron survival in the absence of trophic support in a dose-dependent manner. In vivo, TRO19622 rescued motor neurons from axotomy-induced cell death in neonatal rats and promoted nerve regeneration following sciatic nerve crush in mice. In SOD1(G93A) transgenic mice, a model of familial ALS, TRO19622 treatment improved motor performance, delayed the onset of the clinical disease, and extended survival. TRO19622 bound directly to two components of the mitochondrial permeability transition pore: the voltage-dependent anion channel and the translocator protein 18 kDa (or peripheral benzodiazepine receptor), suggesting a potential mechanism for its neuroprotective activity. TRO19622 may have therapeutic potential for ALS and other motor neuron and neurodegenerative diseases.