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Background. Thyroid-associated orbitopathy (TAO) is the most common autoimmune disease of the orbit. It occurs more often in patients presenting with hyperthyroidism, characteristic of Graves’ disease, but may be associated with hypothyroidism or euthyroidism. The diagnosis of TAO is based on clinical orbital features, radiological criteria, and the potential association with thyroid disease. To date, there is no specific marker of the orbital disease, making the early diagnosis difficult, especially if the orbital involvement precedes the thyroid dysfunction. Summary. The goal of this review is to present the disease and combine the available data in the literature concerning investigation of TAO biomarkers. Conclusions. Despite the progress done in the understanding of TAO disease, some important pieces are still missing. Typically, for the future, major efforts have to be done in the discovery of new biomarkers, validation of the suspected candidates on multicenter cohorts with standardized methodologies, and establishment of their clinical performances on the specific clinical application fields in order to improve not only the management of the TAO patients but also the therapeutic options and follow-up.

Supplemental Digital Content is Available in the Text. In this interventional case series, 8 patients presenting macular telangiectasia Type 1 with macular edema were treated by intravitreal aflibercept. The favorable clinical response observed with this treatment is consistent with the profile of angiogenic factors analyzed in their aqueous humors., Purpose: To evaluate intravitreal aflibercept in macular telangiectasia Type 1 (MacTel 1) patients and measure their ocular angiogenic profile. Methods: Eight subjects with MacTel 1 refractory to bevacizumab, ranibizumab, or laser therapy and switched to aflibercept were included. Best-corrected visual acuity, central macular thickness, and cystic areas quantified on optical coherence tomography B-scans were assessed during 12 months. Perifoveal capillary densities were measured on optical coherence tomography angiography. Aqueous humor was sampled from six patients and eight control subjects undergoing cataract extraction. Growth factors were quantified using a multiarray immunoassay. Results: Over 12 months, patients received 6.6 ± 1.4 (range, 5–8) intravitreal aflibercept injections. Twelve months after switching to aflibercept, best-corrected visual acuity increased by ≥5 letters in 5 of 8 patients, compared with preaflibercept levels. Mean best-corrected visual acuity improved from 79.6 (∼20/50) to 88.0 (∼20/35) Early Treatment Diabetic Retinopathy Study letters (P = 0.042), and central macular thickness decreased from 434 ± 98 μm to 293 ± 59 μm (P = 0.014). Compared with control subjects, the profile of angiogenic factors in MacTel 1 eyes revealed no difference in vascular endothelial growth factor-A levels but significantly higher levels of placental growth factor (P = 0.029), soluble vascular endothelial growth factor receptor-1 (sFlt-1; P = 0.013), vascular endothelial growth factor-D (P = 0.050), and Tie-2 (P = 0.019). Placental growth factor levels inversely correlated with both superficial and deep capillary plexus densities on optical coherence tomography angiography (P = 0.03). Conclusion: The clinical response to aflibercept coupled to the angiogenic profile of MacTel 1 eyes support the implication of the placental growth factor/Flt-1 pathway in MacTel 1.

Human African Trypanosomiasis may become manageable in the next decade with fexinidazole. However, currently stage diagnosis remains difficult to implement in the field and requires a lumbar puncture. Our study of an Angolan cohort of T. b. gambiense-infected patients used other staging criteria than those recommended by the WHO. We compared WHO criteria (cell count and parasite identification in the CSF) with two biomarkers (neopterin and CXCL-13) which have proven potential to diagnose disease stage or relapse. Biological, clinical, and neurological data were analysed from a cohort of 83 patients. A neopterin concentration below 15.5 nmol/L in the CSF denoted patients with stage 1 disease, and a concentration above 60.31 nmol/L characterized patients with advanced stage 2 (trypanosomes in CSF and/or cytorachia higher than 20 cells) disease. CXCL-13 levels below 91.208 pg/mL denoted patients with stage 1 disease, and levels of CXCL-13 above 395.45 pg/mL denoted patients with advanced stage 2 disease. Values between these cut-offs may represent patients with intermediate stage disease. Our work supports the existence of an intermediate stage in HAT, and CXCL-13 and neopterin levels may help to characterize it.

OBJECT Aneurysmal subarachnoid hemorrhage (aSAH) is associated with high rates of mortality and morbidity. The main predictor for the poor outcome is the World Federation of Neurosurgical Societies (WFNS) scale. However, this scale does not take into account proinflammatory events, such as infection occurring after the aSAH, which could modify the long-term status of patients. The aim of this study was to evaluate neopterin as an inflammatory biomarker for outcome and infection prediction in aSAH patients. METHODS Plasma concentrations of neopterin were measured in 61 aSAH patients (22 male and 39 female; mean age [± SD] 52.8 ± 11.8 years) using a commercial ELISA kit. Samples were collected daily for 10 days. Outcome at 12 months was determined using the Glasgow Outcome Scale (GOS) and dichotomized as poor (GOS score 1, 2, or 3) or good (GOS score 4 or 5). Infection was determined by the presence of a positive bacterial culture. RESULTS Patients with poor outcome at 12 months had higher concentrations of neopterin than patients with good outcome. In the same way, patients who had an infection during the hospitalization had significantly higher concentrations of neopterin than patients without infection (p = 0.001). Moreover, neopterin concentrations were significantly (p < 0.008) elevated in infected patients 2 days before infection detection and antibiotic therapy. CONCLUSIONS Neopterin is an efficient outcome predictor after aSAH. Furthermore, it is able to differentiate between infected and uninfected patients as early as 2 days before clinical signs of infection, facilitating earlier antibiotic therapy and better management.

International audience; In retinal detachment (RD), photoreceptor death and permanent vision loss are caused by neurosensory retina separating from the retinal pigment epithelium because of subretinal fluid (SRF), and successful surgical reattachment is not predictive of total visual recovery. As retinal iron overload exacerbates cell death in retinal diseases, we assessed iron as a predictive marker and therapeutic target for RD. In the vitreous and SRF from patients with RD, we measured increased iron and transferrin (TF) saturation that is correlated with poor visual recovery. In ex vivo and in vivo RD models, iron induces immediate necrosis and delayed apoptosis. We demonstrate that TF decreases both apoptosis and necroptosis induced by RD, and using RNA sequencing, pathways mediating the neuroprotective effects of TF are identified. Since toxic iron accumulates in RD, we propose TF supplementation as an adjunctive therapy to surgery for improving the visual outcomes of patients with RD.

Considering absence of invasiveness and side effects, tears emerge as a particularly attractive fluid for biomarker discovery and therefore for daily clinical use. However, to date this fluid remains poorly studied in healthy condition. Here, we present an updated in-depth characterisation of the human healthy tear protein composition using proteomics approach. Both eyes of 8 healthy controls (4 men and 4 women, average age: 38 ± 18) were collected using the Schirmer's strip method. After liquid digestion and off-gel electrophoresis fractionation, three independent proteomics analyses were performed on a LTQ-Orbitrap Velos Pro. Globally, 1351 proteins were identified with 2 unique peptides and 1% FDR. Gene ontology analyses showed up that 39% of the tear proteins were enzymes, with high numbers of dehydrogenases, phosphatases, kinases and ligases. Immunoglobulins, serpins and 14-3-3 domains proteins also emerged as the main tear protein families. The glycolysis and the coagulation and complement cascades, which were already shown in tears as involved in ocular and systemic diseases, were highlighted performing pathway analyses. Our study therefore complements the existing data on healthy tears proteome. Nevertheless, extensive studies for deeply and definitively characterise this promising fluid are required in the near future in order to be able to routinely use this fluid in clinics. A better understanding of its protein content will probably open new avenues in the biomarker discovery and clinical practice in the near future.

To date, Thyroid-Associated Orbitopathy (TAO), an autoimmune inflammatory disease affecting the eye, remains poorly characterised and its diagnosis challenging. The aim of this study was to investigate the tears of the TAO patients in order to identify potential biomarkers. Two independent quantitative Tandem Mass Tag™ 6-plex experiments were done. After in-solution digestion and isoelectric fractionation, the 12 fractions were analysed with a LTQ Orbitrap Velos coupled to a liquid chromatography. Raw files were searched against Swiss-Prot-AC database using Proteome Discoverer software, with a false discovery rate of 1% at peptide and protein levels. The differential proteins were then verified using orthogonal approaches in independent patients. Globally, 712 tear proteins were quantified with 2 unique peptides. Interestingly, cystatin c (TAO/controls ratio: 1.53), alpha-1 antichymotrypsin (ratio: 1.70) and retinal dehydrogenase (ratio: 0.68), displaying differential levels in the tears of TAO patients using proteomics experiments emerged as highly promising biomarkers after verification. In conclusion, this proteomics study supports the idea that tears reflect biological modifications occurring in a disease context and can therefore be a promising fluid for biomarker discovery. Moreover, our study identified three candidates that could in the future open new avenues in the diagnosis of TAO disease. Significance Thyroid associated orbitopathy (TAO) is the most common disease affecting the orbit. Moreover, the later, severe stages of the disease can be sight threating [1] . On the other hand, the early sign and symptoms can be mistaken with other ocular pathologies [2] . Here we explore the modification of the tear content of the TAO patients using proteomics strategies and we proposed three new biomarker candidates, which could allow the early diagnosis of the disease and prompt action to prevent more severe stages. Moreover, our findings could also help to better understand the pathophysiology of the disease.

Sweat is one of the less employed biofluids for discovery of markers in spite of its increased application in medicine for detection of drugs or for diagnostic of cystic fibrosis. In this research, human sweat was used as clinical sample to develop a screening tool for lung cancer, which is the carcinogenic disease with the highest mortality rate owing to the advanced stage at which it is usually detected. In this context, a method based on the metabolite analysis of sweat to discriminate between patients with lung cancer versus smokers as control individuals is proposed. The capability of the metabolites identified in sweat to discriminate between both groups of individuals was studied and, among them, a trisaccharide phosphate presented the best independent performance in terms of the specificity/sensitivity pair (80 and 72.7%, respectively). Additionally, two panels of metabolites were configured using the PanelomiX tool as an attempt to reduce false negatives (at least 80% specificity) and false positives (at least 80% sensitivity). The first panel (80% specificity and 69% sensitivity) was composed by suberic acid, a tetrahexose, and a trihexose, while the second panel (69% specificity and 80% sensitivity) included nonanedioic acid, a trihexose, and the monoglyceride MG(22:2). Thus, the combination of the five metabolites led to a single panel providing 80% specificity and 79% sensitivity, reducing the false positive and negative rates to almost 20%. The method was validated by estimation of within-day and between-days variability of the quantitative analysis of the five metabolites.

The majority of patients with mild traumatic brain injury (mTBI) will have normal Glasgow coma scale (GCS) of 15. Furthermore, only 5%–8% of them will be CT-positive for an mTBI. Having a useful biomarker would help clinicians evaluate a patient’s risk of developing intracranial lesions. The S100B protein is currently the most studied and promising biomarker for this purpose. Heart fatty-acid binding protein (H-FABP) has been highlighted in brain injury models and investigated as a biomarker for stroke and severe TBI, for example. Here, we evaluate the performances of S100B and H-FABP for differentiating between CT-positive and CT-negative patients. A total of 261 patients with a GCS score of 15 and at least one clinical symptom of mTBI were recruited at three different European sites. Blood samples from 172 of them were collected ≤ 6 h after trauma. Patients underwent a CT scan and were dichotomised into CT-positive and CT-negative groups for statistical analyses. H-FABP and S100B levels were measured using commercial kits, and their capacities to detect all CT-positive scans were evaluated, with sensitivity set to 100%. For patients recruited ≤ 6 h after trauma, the CT-positive group demonstrated significantly higher levels of both H-FABP (p = 0.004) and S100B (p = 0.003) than the CT-negative group. At 100% sensitivity, specificity reached 6% (95% CI 2.8–10.7) for S100B and 29% (95% CI 21.4–37.1) for H-FABP. Similar results were obtained when including all the patients recruited, i.e. hospital arrival within 24 h of trauma onset. H-FABP out-performed S100B and thus seems to be an interesting protein for detecting all CT-positive mTBI patients with a GCS score of 15 and at least one clinical symptom.

Type 2 diabetes is a worldwide disease reaching epidemic dimensions. The rapid progression of the disease urgently calls for both a broader and deeper understanding of its pathophysiology. In line with this statement, the Human Diabetes Proteome Project (HDPP) was officially launched at the 11th HUPO meeting in Boston, 2012. A special session was dedicated to this new initiative, gathering experts in the main topics related to diabetes and its associated complications. Key issues were debated with a focus on how deranged circulating glucose and free fatty acids induce dysfunction. It has been decided that HDPP will therefore focus on studying the early stages of diabetes that lead to abnormal glucose and lipid levels. The initiative will initially focused on islets of Langerhans, insulin-producing cell lines, and blood human samples from diabetes-related cohorts. In subsequent stages HDPP will investigate target tissues in which glucose and lipids could promote protein dysfunctions. Omics-rooted systems approaches enhanced by bioinformatics will be deployed to unravel effects of lipids and glucose triggering diabetes initiation and progression. A first milestone has been defined for the 12th HUPO meeting in Yokohama, 2013: the 1000 diabetes-associated protein (the 1000-HDPP) database, i.e. a freely available internet resource ( www.HDPP.info ) of more than 1000 proteins with links to their corresponding proteotypic peptides, affinity reagents and protein-specific biological/biomedical information.

Accurately determining time-of-onset of cerebral infarction is important to clearly identify patients who could benefit from reperfusion therapies. We assessed the kinetics of peroxiredoxin 1 (PRDX1), a protein involved in oxidative stress during the acute phase of ischemia, and its ability to determine stroke onset in a population of patients with known onset of less than 24 hours and in a control group. Median PRDX1 levels were significantly higher in stroke patients compared to controls. PRDX1 levels were also higher from blood samples withdrawn before vs. after 3 hours following stroke onset, and before vs. after 6 hours. ROC analysis with area under the curve (AUC), sensitivity (Se) and specificity (Sp) determined from the Youden index was performed to assess the ability of PRDX1 levels to determine onset. Diagnostic performances of PRDX1 levels were defined by an AUC of 69%, Se of 53% and Sp of 86% for identifying cerebral infarction occurring

Despite decades of intensive investigations, the precise sequence of molecular events and the specific proteins mediating the degenerative process underlying Parkinson's disease (PD) remain unraveled. Proteomic strategies may provide unbiased tools to identify novel candidates and explore original mechanisms involved in PD. Substantia nigra pars compacta (SN) tissue, whose degeneration is the hallmark of PD, was dissected from neuropathologically confirmed PD patients (n=3) and control subjects (n=3), before being submitted to a comparative 2-DE analysis. The present study revealed a subset of neuronal and/or glial proteins that appears to be deregulated in PD and likely to contribute to neurodegeneration. Observed alterations not only consolidate well accepted concepts surrounding PD pathogenesis such as oxidative stress and mitochondrial dysfunction but also point out to novel pathways. Among the latter, cytosolic non specific dipeptidase 2 (CNDP2), a relatively unknown protein not yet reported to be associated with PD pathogenesis, was shown to be increased in the SN of PD patients, as confirmed by Western blot. Immunohistochemical analyses demonstrated the presence of CNDP2 within the cytoplasm of SN dopaminergic neurons. Altogether, our findings support a key role of CNDP2 in PD neurodegeneration, by mechanisms that could involve oxidative stress, protein aggregation or inflammation. This article is part of a Special Issue entitled: Translational Proteomics.

The pathogenesis of delayed cerebral ischemia (DCI) in aneurysmal subarachnoid hemorrhage (aSAH) is multi-factorial and not completely elucidated. Matrix metalloproteinase-9 (MMP-9) might participate in wall remodeling leading to luminal narrowing. The authors investigated MMP-9 concentration in brain extracellular fluid of aSAH patients and assessed whether this enzyme could have a predictive value for the risk of DCI.Patients were classified according to the grading of the World Federation of Neurological Surgeons (WFNS) in low- and high-grade patients (WFNS = 1-3, n = 9 and WFNS = 4-5, n = 12, respectively). Cerebral microdialysis probes were placed in brain parenchyma of the respective vascular territory of the aneurysm in 21 consecutive aSAH patients, enrolled in a prospective study on inflammation. Microdialysis samples, collected daily from day 0 until day 8 after aSAH, were retrospectively analyzed for MMP-9 by zymography.Initial concentration of the MMP-9 proform (pro-MMP-9) was significantly higher in high-grade patients as compared to low-grade patients. Furthermore, initial pro-MMP-9 concentration in patients with DCI was seven-fold higher than in asymptomatic patients. Pro-MMP-9 values greater than or equal to 0·27 pg/μl showed 83% sensitivity and 63% specificity in predicting vasospasm. The mature form of the MMP-9 could be preferentially detected in patients with DCI but was usually low.The pro and mature forms of MMP-9 were released locally in the brain after aSAH. Pro-MMP-9 release was related to WFNS grade severity. This protease, considered as playing a critical role in endothelial basal membrane damage, may contribute to the inflammatory processes leading to arterial narrowing.

Objective: Wide interindividual variability exists in response to tissue plasminogen activator (t-PA) treatment in the acute phase of ischemic stroke. We aimed to find genetic variations associated with hemorrhagic transformation (HT) and mortality rates after t-PA. We then generated a clinicalgenetic model for predicting t-PA response. Methods: Our prospective study used SNPlex to genotype 140 single nucleotide polymorphisms (SNPs) from 97 candidate genes in 3 patient cohorts. The cohorts included 1,172 patients who were treated with t-PA; 20.9% of them developed HT as evaluated by systematic brain computed tomography scan, and 10.6% died. A predictive model was generated by logistic regression (LR). Functional studies included real time quantitative polymerase chain reaction, nephelometry, and Western blot for alpha-2-macroglobulin (A2M) and activated partial thromboplastin time measurement for coagulation factor XII (FXII). Results: Replication analysis revealed that the SNP rs669 (Val1000Ile) in A2M was associated with HT, and rs1801020 (-4C>T) of F12 was associated with in-hospital death. The rs669 SNP withstood Bonferroni correction for HT (p < 3.57E-4). LR-based scores predicted HT occurrence (p = 9.13E-15) and in-hospital mortality (p = 8.7E-9) and were validated in an independent cohort. Val1000Ile modified A2M serum levels at baseline and after t-PA infusion, but not mRNA expression or protein structure; -4C>T affected FXII activity both prior to and after t-PA treatment. Interpretation: Two functional polymorphisms were consistently associated with t-PA safety. Our validated LR-based score predicts t-PA safety in the Spanish population. ANN NEUROL 2012;72:716729

The lectin pathway of the complement system has been implicated in secondary ischemic/inflammatory injury after traumatic brain injury (TBI). However, previous experimental studies have yielded conflicting results, and human studies are scarce. In this exploratory study, we investigated associations of several lectin pathway proteins early after injury and single-nucleotide polymorphisms (SNP) with outcomes after severe TBI (mortality at 14 days [primary outcome] and consciousness assessed with the Glasgow Coma Scale [GCS] at 14 days, disability assessed with the Glasgow Outcome Scale Extended [GOSE] at 90 days). Forty-four patients with severe TBI were included. Plasma levels of lectin pathway proteins were sampled at 6, 12, 24, and 48 h after injury and eight mannose-binding lectin (MBL) and ficolin (FCN)2 SNPs were analyzed by enzyme-linked immunosorbent assay (ELISA) and genotyping, respectively. Plasma protein levels were stable with only a slight increase in mannose-binding protein-associated serine protease (MASP)-2 and FCN2 levels after 48 h (p 0.05), respectively. Neither lectin protein plasma levels (6 h or mean levels) nor MBL2 genotypes or FCN2 variant alleles were associated with 14 day mortality or 14 day consciousness. However, FCN2, FCN3, and MASP-2 levels were higher in patients with an unfavorable outcome (GOSE 1-4) at 90 days (p 0.05), whereas there was no difference in MBL2 genotypes or FCN2 variant alleles. In particular, higher mean MASP-2 levels over 48 h were independently associated with a GOSE score4 at 90 days after adjustment (odds ratio 3.46 [95% confidence interval 1.12-10.68] per 100 ng/mL increase, p = 0.03). No association was observed between the lectin pathway of the complement system and 14 day mortality or 14 day consciousness. However, higher plasma FCN2, FCN3, and, in particular, MASP-2 levels early after injury were associated with an unfavorable outcome at 90 days (death, vegetative state, and severe disability) which may be related to an increased activation of the lectin pathway.

OBJECTIVES A critical step before treatment of human African trypanosomiasis (HAT) is the correct staging of the disease. As late stage is established when trypanosomes cross the blood-brain barrier and invade the central nervous system, we hypothesized that matrix metalloproteinases and cell adhesion molecules could indicate, alone or in combination, the disease progression from the first to the second stage of HAT. METHODS We measured the levels of MMP-2, MMP-9, ICAM-1, VCAM-1 and E-selectin in the cerebrospinal fluid (CSF) of 63 Trypanosoma brucei gambiense-infected patients (15 stage 1 and 48 stage 2). Staging was based on counting of white blood cells (WBC) and/or parasite detection in CSF. Concentrations were obtained either by ELISA or multiplex bead suspension assays, and results were compared with three known HAT staging markers (CXCL10, CXCL8 and H-FABP). RESULTS ICAM-1 and MMP-9 accurately discriminated between stage 1 and stage 2 patients with HAT with 95% sensitivity (SE) for 100% specificity (SP), which was better than CXCL10 (93% SE for 100% SP), one of the most promising known markers. Combination of ICAM-1 and MMP-9 with H-FABP provided a panel that resulted in 100% of SE and SP for staging HAT. CONCLUSIONS ICAM-1 and MMP-9, alone or in combination, appeared as powerful CSF staging markers of HAT. Final validation of all newly discovered staging markers on a large multi-centric cohort including both forms of the disease as well as patients with others infections should be performed.

Purpose To investigate the molecular composition of subretinal fluid (SRF) in central serous chorioretinopathy (CSCR) and rhegmatogenous retinal detachment (RRD) using proteomics and metabolomics. Methods SRF was obtained from one patient with severe nonresolving bullous CSCR requiring surgical subretinal fibrin removal, and two patients with long-standing RRD. Proteins were trypsin-digested, labeled with Tandem-Mass-Tag and fractionated according to their isoelectric point for identification and quantification by tandem mass spectrometry. Independently, metabolites were extracted on cold methanol/ethanol, and identified by untargeted ultra-high performance liquid chromatography and high-resolution mass spectrometry. Bioinformatics analyses were conducted. Results In total, 291 proteins and 651 metabolites were identified in SRF samples. Compared with RRD, 128 proteins (77 downregulated; 51 upregulated) and 76 metabolites (43 downregulated; 33 upregulated) differed in the SRF from CSCR. Protein and metabolites notably deregulated in CSCR were related to glycolysis/gluconeogenesis, inflammation (including serum amyloid P component, versican), alternative complement pathway (complement factor H and complement factor H–related protein), cellular adhesion, biliary acid metabolism (farnesoid X receptor/retinoid X receptor), and gluco- and mineralocorticoid systems (aldosterone, angiotensin, and corticosteroid-binding globulin). Conclusions Proteomics and metabolomics can be performed on SRF. A unique SRF sample from CSCR exhibited a distinct molecular profile compared with RRD. Translational Relevance This first comparative multiomics analysis of SRF improved the understanding of CSCR and RRD pathophysiology. It identified pathways potentially involved in the better photoreceptor preservation in CSCR, suggesting neuroprotective targets that will require additional confirmation.

PURPOSE: Accurate early anticipation of long-term irreversible brain damage during the acute phase of patients with aneurysmal subarachnoid hemorrhage (aSAH) remains difficult. Using a combination of clinical scores together with brain injury-related biomarkers (H-FABP, NDKA, UFD1 and S100beta), this study aimed at developing a multiparameter prognostic panel to facilitate early outcome prediction following aSAH. METHODS: Blood samples of 141 aSAH patients from two separated cohorts (sets of 28 and 113 patients) were prospectively enrolled and analyzed with 14 months of delay. Patients were admitted within 48 h following aSAH onset. A venous blood sample was withdrawn within 12 h after admission. H-FABP, NDKA, UFD1, S100beta and troponin I levels were determined using classical immunoassays. The World Federation of Neurological Surgeons (WFNS) at admission and the Glasgow Outcome Score (GOS) at 6 months were evaluated. RESULTS: In the two cohorts, blood concentration of H-FABP, S100beta and troponin I at admission significantly predicted unfavorable outcome (GOS 1-2-3). A multivariate analysis identified a six-parameter panel, including WFNS, H-FABP, S100beta, troponin I, NDKA and UFD-1; when at least three of these parameters were simultaneously above cutoff values, prediction of unfavorable outcome reached around 70% sensitivity in both cohorts for 100% specificity. CONCLUSION: The use of this panel, including four brain injury-related proteins, one cardiac marker and a clinical score, could be a valuable tool to identify aSAH patients at risk of poor outcome.

A new 6-plex isobaric mass tagging technology is presented, and proof of principle studies are carried out using standard protein mixtures and human cerebrospinal fluid (CSF) samples. The Tandem Mass Tags (TMT) comprise a set of structurally identical tags which label peptides on free amino-terminus and epsilon-amino functions of lysine residues. During MS/MS fragmentation, quantification information is obtained through the losses of the reporter ions. After evaluation of the relative quantification with the 6-plex version of the TMT on a model protein mixture at various concentrations, the quantification of proteins in CSF samples was performed using shotgun methods. Human postmortem (PM) CSF was taken as a model of massive brain injury and comparison was carried out with antemortem (AM) CSF. After immunoaffinity depletion, triplicates of AM and PM CSF pooled samples were reduced, alkylated, digested by trypsin, and labeled, respectively, with the six isobaric variants of the TMT (with reporter ions from m/z = 126.1 to 131.1 Th). The samples were pooled and fractionated by SCX chromatography. After RP-LC separation, peptides were identified and quantified by MS/MS analysis with MALDI TOF/TOF and ESI-Q-TOF. The concentration of 78 identified proteins was shown to be clearly increased in PM CSF samples compared to AM. Some of these proteins, like GFAP, protein S100B, and PARK7, have been previously described as brain damage biomarkers, supporting the PM CSF as a valid model of brain insult. ELISA for these proteins confirmed their elevated concentration in PM CSF. This work demonstrates the validity and robustness of the tandem mass tag (TMT) approach for quantitative MS-based proteomics.

Background Inflammation is known to worsen cerebral damage at the acute phase of stroke. In this setting, cell adhesion molecules (CAMs) play a crucial role mediating migration of immune cells into the infarcted area. However, their value in long-term outcome prediction for patients with cerebrovascular diseases (CVD) is less described. Methods Levels of four CAMs (E-selectin, P-selectin glycoprotein ligand-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 (VCAM-1)) and six other known biomarkers (C-reactive protein (CRP), interleukin-6 (IL-6), N-terminal pro-brain natriuretic peptide (NT-proBNP), troponin I, vasopressin-neurophysin 2-copeptin, and S100 calcium-binding protein B) were measured in a population of patients presenting CVD. Blood collections for analysis were performed within different time windows after stroke onset: 0–6 h, 6–36 h, 2–3 days, 5–7 days, and 2–3 weeks. Independent associations with poor outcome at 3 months (modified Rankin Scale score > 2) were sought using univariate and multivariate analysis after adjustments for age and National Institute of Health Stroke Scale score. Predictive ability of each biomarker has also been assessed with ROC analysis. Results One hundred patients were prospectively included whom 75 presented with ischemic strokes, nine with hemorrhagic strokes and 16 with transient ischemic attacks. During the first 6 h after stroke onset, E-selectin was found to be an independent predictor of 3-month outcome (odds ratio (OR) =24; 95 % confidence interval (95 % CI), 2–354; p = 0.022) (area under the curve (AUC) =78 %), as was VCAM-1 during the third week after onset (OR = 8; 95 % CI, 2–37; p = 0.01) (AUC = 73 %). Associations remained after the exclusion of patients with hemorrhagic strokes and transient ischemic attacks. Independent associations with outcome were also found for CRP (OR = 5; 95 % CI, 1–22; p = 0.023) and IL-6 (OR = 5; 95 % CI, 1–17; p = 0.021) at 2–3 days and for NT-proBNP at 6–36 h (OR = 20; 95 % CI, 1–337; p = 0.04). Conclusions E-selectin and VCAM-1 were independent predictors of outcome in a population of patients with CVD. The predictive capability of other biomarkers known to be indicators for prognosis also emerged, confirming the study’s robustness. CAMs levels could be considered as objective biological criteria for prognosis in CVD. Electronic supplementary material The online version of this article (doi:10.1186/s12950-015-0106-z) contains supplementary material, which is available to authorized users.

Aneurysmal subarachnoid hemorrhage (aSAH) is associated with high rates of mortality and morbidity. Nosocomial infections, such as pneumonia or urinary tract infections, are among the main causes of worsening outcomes and death. The aim of this study was to discover a biomarker to predict infection in aSAH patients. For this purpose, the plasma of infected and noninfected patients was compared using quantitative mass spectrometry. The most interesting differentially expressed proteins were selected for validation by immunoassays on plasma samples taken from patients (n = 81) over 10 days of hospitalization. Predictive performances were established using Mann-Whitney U tests and receiver operating characteristic curves. Quantitative proteomics identified 17 significantly regulated proteins. Of these, levels of serum amyloid A (SAA) were significantly higher in infected patients (p < 0.007). ELISA confirmed that the concentrations were significantly higher (p < 0.002) already at hospital admission in patients who subsequently developed an infection during their hospitalization, (AUC of 76%) for a cutoff value of 90.9 μg/mL. Our data suggested that measuring SAA could be an efficient means of detecting patients susceptible of developing an infection during hospitalization after an aSAH. Its predictive capacity could lead to earlier antibiotherapy, improved patient management, and potentially better long-term outcomes.

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.

Intestinal epithelial cells are characterized by continuous renewal and differentiation events, which may be influenced by the basement membrane, and in particular laminins, which are major components of this specialized extracellular matrix. The function and signaling pathways of laminins in these processes are still poorly documented. In this study, we investigated the possible role and the subcellular localization of nucleolin, a nuclear shuttling protein, in relation to differentiation of human intestinal epithelial Caco2/TC7 cells triggered by exogenous laminin-1. Immunofluorescence and Western blot analysis indicated that laminin-1 induced early differentiation of the cells concomitantly to a decrease in nuclear nucleolin and its a cell surface location. We also showed that both effects of laminin-1 on Caco2/TC7 cells—induction of the differentiation marker sucrase-isomaltase and redistribution of nucleolin—could be mediated by a β1-integrin dependent cascade that implicated activation of the p38 MAPK pathway. In addition, knock-down of nucleolin expression by the small interfering RNA strategy mimicked the effect of laminin-1 as it resulted in the induction of cell polarization and differentiation. Thus, our study suggests that changes in the subcellular distribution and expression level of nucleolin play an important role in intestinal cell differentiation and relay the signaling pathway induced by laminin-1. J. Cell. Physiol. 206: 545–555, 2006. © 2005 Wiley-Liss, Inc.

Self-renewing tissues such as the intestine contain progenitor proliferating cells which subsequently differentiate. Cell proliferation and differentiation involve gene regulation processes which take place in the nucleus. A human intestinal epithelial cell line model (Caco2/TC7) which reproduces these dynamic processes has been used to perform proteomic studies on nuclear proteins. Nuclei from Caco2/TC7 cells at proliferative and differentiated stages were purified by subcellular fractionation. After two-dimensional gel electrophoresis separation and ruthenium staining, 400 protein spots were detected by image analysis. Eighty-five spots corresponding to 60 different proteins were identified by matrix-assisted laser desorption/ionization mass spectrometry in nuclei from proliferative cells. Comparison of nuclear proteomes from proliferative or differentiated cells by differential display resulted in the identification of differentially expressed proteins such as nucleolin, hnRNP A2/B1 and hnRNP A1. By using Western blot analysis, we found that the expression and number of specific isoforms of these nuclear proteins decreased in differentiated cells. Immunocytochemistry experiments also showed that in proliferative cells nucleolin was distributed in nucleoli-like bodies. In contrast, hnRNPs A2/B1 and A1 were dispersed throughout the nucleus. This study of the nuclear proteome from intestinal epithelial cells represents the first step towards the establishment of a protein database which will be a valuable resource in future studies on the differential expression of nuclear proteins in response to physiological, pharmacological and pathological modulations.

Introduction L’hemorragie meningee est une maladie associee a des taux eleves de mortalite et de morbidite. Environ 40 % des patients meurent dans les premieres 24 heures apres l’hemorragie et parmi ceux qui survivent beaucoup d’entre eux restent hospitalises dans le service de reanimation. Les infections nosocomiales, telles que la pneumonie ou l’infection des voies urinaires sont une des principales causes de mauvais pronostique. Jusqu’a present, aucun biomarqueur sanguin efficace n’est disponible pour la detection precoce et la prevention de ces infections. Le but de cette etude est d’utiliser des strategies de proteomique afin de decouvrir de nouveaux biomarqueurs pour la prediction de l’infection chez des patients souffrant d’hemorragie meningee. Materiel et methodes Cette etude a ete effectuee apres accord du CPP local. Les echantillons plasmatiques de 4 patients infectes et 4 patients non infectes ont ete compares en utilisant la spectrometrie de masse (TMT-10 plex). Parmi les proteines significativement exprimees entre les deux groups, la plus interessante a ete selectionnee pour une verification ulterieure dans le plasma de 54 patients infectes et 27 patients non infectes. Les performances predictives ont ete etablies en utilisant des tests de Mann-Whitney U et courbes ROC. Resultats Les resultats proteomiques ont montre 17 proteines significativement regulees entre les patients infectes et non infectes. Parmi celles-ci, la SAA a ete choisie pour des etudes de verification. A l’admission a l’hopital, les concentrations de SAA etaient deja nettement plus elevee chez les patients qui developpent une infection lors d’hospitalisation (p Discussion Nos donnees suggerent que la combinaison de la SAA, une molecule associee a la phase aigue d’une infection, avec les leucocytes, le WFNS et l’âge pourrait etre un outil efficace pour la determination de l’infection chez les patients affectes par une hemorragie meningee. Leur capacite de prediction pourrait conduire a une antibiotherapie plus precoce ainsi qu’a l’amelioration du pronostique. Afin d’evaluer son utilite clinique, ces resultats doivent etre valides dans une etude multicentrique plus grande.

Parkinson's disease (PD) pathology spreads throughout the brain following a region-specific process predominantly affecting the substantia nigra (SN) pars compacta. SN exhibits a progressive loss of dopaminergic neurons responsible for the major cardinal motor symptoms, along with the occurrence of Lewy bodies in the surviving neurons. To gain new insights into the underlying pathogenic mechanisms in PD, we studied postmortem nigral tissues dissected from pathologically confirmed PD cases (n = 5) and neurologically intact controls (n = 8). Using a high-throughput shotgun proteomic strategy, we simultaneously identified 1795 proteins with concomitant quantitative data. To date, this represents the most extensive catalog of nigral proteins. Of them, 204 proteins displayed significant expression level changes in PD patients versus controls. These were involved in novel or known pathogenic processes including mitochondrial dysfunction, oxidative stress, or cytoskeleton impairment. We further characterized four candidates that might be relevant to PD pathogenesis. We confirmed the differential expression of ferritin-L and seipin by Western blot and demonstrated the neuronal localization of gamma glutamyl hydrolase and nebulette by immunohistochemistry. Our preliminary findings suggest a role for nebulette overexpression in PD neurodegeneration, through mechanisms that may involve cytoskeleton dynamics disruption. All MS data have been deposited in the ProteomeXchange with identifier PXD000427 (http://proteomecentral.proteomexchange.org/dataset/PXD000427).

Multiple sclerosis is the first cause of progressive neurological disability among young adults living in Western countries. Its diagnosis is mostly based on clinical evaluation, neuroimaging, and in some cases cerebrospinal fluid (CSF) analysis, but no definitive diagnostic test exists. We proposed here that the exploration of tears from multiple sclerosis patients could lead to the discovery of new biomarkers.Thirty multiple sclerosis patients (20% men) recruited to the Geneva University Hospitals were included in our study (mean age ± SD [years]: 42.4 ± 15.9). Twenty-five control patients (32% men) were also enrolled (mean age ± SD [years]: 42.7±15.1). Tears, CSF or blood was collected for each patient. Three independent quantitative (tandem mass tag) experiments were carried out between tears from multiple sclerosis and control patients. Protein verification was performed by Western blot on tears and CSF and by ELISA on serum samples.Combined proteomics analyses provided 185 identified tear proteins. Among the differential proteins, alpha-1 antichymotrypsin was the only one to be significantly increased in the three experiments with similar ratios (ratios 1.6 to 2.5, p0.05). Its tear, CSF and serum elevation were further confirmed by Western blot and ELISA, respectively.This study supports the concept that modifications of the tear proteome can reflect biological abnormalities associated with multiple sclerosis and perhaps other inflammatory conditions affecting the CNS. In addition, alpha-1 antichymotrypsin elevation in tear fluid emerges as a promising biomarker for the diagnosis of multiple sclerosis.

The outcome after severe traumatic brain injury (TBI) is largely unfavorable, with approximately two thirds of patients suffering from severe disabilities or dying during the first 6 months. Existing predictive models displayed only limited utility for outcome prediction in individual patients. Time courses of heart-fatty acidic binding protein (H-FABP) and their association with outcome were investigated and compared with S100b. Forty-nine consecutive patients with severe TBI (sTBI; Head component of the Abbreviated Injury Scale [HAIS]3) with mono and multiple trauma were enrolled in this study. Enzyme-linked immunosorbent assay measured blood concentrations of H-FABP and S100b at 6, 12, 24, and 48 h after TBI. Outcome measures were conscious state at 14 days (Glasgow Coma Scale), disability (Glasgow Outcome Scale Extended; GOSE), and mortality at 3 months. Univariate logistic regression analysis and receiver operating characteristic curves analysis were carried out. Maximal H-FABP and S100b concentrations were observed at 6 h after TBI (34.4±34.0 and 0.64±0.99 ng/mL, respectively). Patients with multi-trauma had significantly higher H-FABP concentrations at 24 and 48 h (22.6±25.6 and 12.4±18.2 ng/mL, respectively), compared to patients with mono trauma (6.9±5.1 and 3.7±4.2 ng/mL, respectively). In the first 48 h, H-FABP and S100b were inversely correlated with the GOSE at 3 months; H-FABP at 48 h predicted mortality with 75% sensitivity and 93% specificity. Early blood levels of H-FABP after sTBI have prognostic significance for survival and disability.

International audience; Accurate stage determination is crucial in the choice of treatment for patients suffering from sleeping sickness, also known as human African trypanosomiasis (HAT). Current staging methods, based on the counting of white blood cells (WBC) and the detection of parasites in the cerebrospinal fluid (CSF) have limited accuracy. We hypothesized that immune mediators reliable for staging T. b. gambiense HAT could also be used to stratify T. b. rhodesiense patients, the less common form of HAT.A population comprising 85 T. b. rhodesiense patients, 14 stage 1 (S1) and 71 stage 2 (S2) enrolled in Malawi and Uganda, was investigated. The CSF levels of IgM, MMP-9, CXCL13, CXCL10, ICAM-1, VCAM-1, neopterin and B2MG were measured and their staging performances evaluated using receiver operating characteristic (ROC) analyses.IgM, MMP-9 and CXCL13 were the most accurate markers for stage determination (partial AUC 88%, 86% and 85%, respectively). The combination in panels of three molecules comprising CXCL13-CXCL10-MMP-9 or CXCL13-CXCL10-IgM significantly increased their staging ability to partial AUC 94% (p value < 0.01).The present study highlighted new potential markers for stage determination of T. b. rhodesiense patients. Further investigations are needed to better evaluate these molecules, alone or in panels, as alternatives to WBC to make reliable choice of treatment.

Background Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome. Methodology/Principal findings Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers. Conclusions/Significance In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment., Author Summary The reduction of the number of lumbar punctures performed during the follow-up of patients affected by sleeping sickness (HAT) is considered a research priority. Follow-up, consisting of the examination of cerebrospinal fluid (CSF) for presence of parasites and for the number of leukocytes, is necessary to assess treatment outcome. However, diagnosis of treatment failure is still imperfect and WHO encourages improvements in defining criteria. Many studies have attempted to standardize actual methods and to define a cut-off for the number of white blood cells in CSF to define relapses, while only few have proposed alternatives to current practice. Here we show that neopterin, already proven to be a powerful marker for staging T. b. gambiense HAT, is also useful in evaluating post-therapeutic outcome. The measurement of neopterin concentration in CSF during the follow-up may allow reduction in the number of lumbar punctures from five to three for the majority of cured patients.

International audience; BACKGROUND: Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients. METHODS AND FINDINGS: Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging. CONCLUSIONS: This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.

BACKGROUND: Ability to accurately determine time of stroke onset remains challenging. We hypothesized that an early biomarker characterized by a rapid increase in blood after stroke onset may help defining better the time window during which an acute stroke patient may be candidate for intravenous thrombolysis or other intravascular procedures. METHODS: The blood level of 29 proteins was measured by immunoassays on a prospective cohort of stroke patients (N = 103) and controls (N = 132). Mann-Whitney U tests, ROC curves and diagnostic odds ratios were applied to evaluate their clinical performances. RESULTS: Among the 29 molecules tested, GST-π concentration was the most significantly elevated marker in the blood of stroke patients (p3 h after onset). According to goal-oriented distinct cut-offs (sensitivity(Se)-oriented: 17.7 or specificity(Sp)-oriented: 65.2 ug/L), the GST-π test obtained 91%Se/50%Sp and 50%Se/91%Sp, respectively. Moreover, GST-π showed also the highest AUC (0.83) and performances for detecting patients treated with tPA (N = 12) compared to ineligible patients (N = 103). CONCLUSIONS: This study demonstrates that GST-π can accurately predict the time of stroke onset in over 50% of early stroke patients. The GST-π test could therefore complement current guidelines for tPA administration and potentially increase the number of patients accessing thrombolysis.

Highly complex and dynamic protein mixtures are hardly comprehensively resolved by direct shotgun proteomic analysis. As many proteins of biological interest are of low abundance, numerous analytical methodologies have been developed to reduce sample complexity and go deeper into proteomes. The present work describes an analytical strategy to perform cysteinyl-peptide subset enrichment and relative quantification through successive cysteine and amine-isobaric tagging. A cysteine-reactive covalent capture tag (C³T) allowed derivatization of cysteines and specific isolation on a covalent capture (CC) resin. The 6-plex amine-reactive tandem mass tags (TMT) served for relative quantification of the targeted peptides. The strategy was first evaluated on a model protein mixture with increasing concentrations to assess the specificity of the enrichment and the quantitative performances of the workflow. It was then applied to human cerebrospinal fluid (CSF) from post-mortem and ante-mortem samples. These studies confirmed the specificity of the C³T and the CC technique to cysteine-containing peptides. The model protein mixture analysis showed high precision and accuracy of the quantification with coefficients of variation and mean absolute errors of less than 10% on average. The CSF experiments demonstrated the potential of the strategy to study complex biological samples and identify differential brain-related proteins. In addition, the quantification data were highly correlated with a classical TMT experiment (i.e., without C³T cysteine-tagging and enrichment steps). Altogether, these results legitimate the use of this quantitative C³T strategy to enrich and relatively quantify cysteine-containing peptides in complex mixtures.

Background Receiver operating characteristic (ROC) curves are useful tools to evaluate classifiers in biomedical and bioinformatics applications. However, conclusions are often reached through inconsistent use or insufficient statistical analysis. To support researchers in their ROC curves analysis we developed pROC, a package for R and S+ that contains a set of tools displaying, analyzing, smoothing and comparing ROC curves in a user-friendly, object-oriented and flexible interface. Results With data previously imported into the R or S+ environment, the pROC package builds ROC curves and includes functions for computing confidence intervals, statistical tests for comparing total or partial area under the curve or the operating points of different classifiers, and methods for smoothing ROC curves. Intermediary and final results are visualised in user-friendly interfaces. A case study based on published clinical and biomarker data shows how to perform a typical ROC analysis with pROC. Conclusions pROC is a package for R and S+ specifically dedicated to ROC analysis. It proposes multiple statistical tests to compare ROC curves, and in particular partial areas under the curve, allowing proper ROC interpretation. pROC is available in two versions: in the R programming language or with a graphical user interface in the S+ statistical software. It is accessible at http://expasy.org/tools/pROC/ under the GNU General Public License. It is also distributed through the CRAN and CSAN public repositories, facilitating its installation.