Investigation of the global protein content from healthy human tears.

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 ±â€¯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.

EFFICACY OF INTRAVITREAL AFLIBERCEPT IN MACULAR TELANGIECTASIA TYPE 1 IS LINKED TO THE OCULAR ANGIOGENIC PROFILE.

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.

Measuring Serum Amyloid A for Infection Prediction in Aneurysmal Subarachnoid Hemorrhage.

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.

Human sweat metabolomics for lung cancer screening.

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.

Proteomic analysis of human substantia nigra identifies novel candidates involved in Parkinson's disease pathogenesis.

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).

A predictive clinical-genetic model of tissue plasminogen activator response in acute ischemic stroke.

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 clinical-genetic 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.

Discovery and verification of osteopontin and Beta-2-microglobulin as promising markers for staging human African trypanosomiasis.

Human African trypanosomiasis, or sleeping sickness, is a parasitic disease endemic in sub-Saharan Africa, transmitted to humans through the bite of a tsetse fly. The first or hemolymphatic stage of the disease is associated with presence of parasites in the bloodstream, lymphatic system, and body tissues. If patients are left untreated, parasites cross the blood-brain barrier and invade the cerebrospinal fluid and the brain parenchyma, giving rise to the second or meningoencephalitic stage. Stage determination is a crucial step in guiding the choice of treatment, as drugs used for S2 are potentially dangerous. Current staging methods, based on counting white blood cells and demonstrating trypanosomes in cerebrospinal fluid, lack specificity and/or sensitivity. In the present study, we used several proteomic strategies to discover new markers with potential for staging human African trypanosomiasis. Cerebrospinal fluid (CSF) samples were collected from patients infected with Trypanosoma brucei gambiense in the Democratic Republic of Congo. The stage was determined following the guidelines of the national control program. The proteome of the samples was analyzed by two-dimensional gel electrophoresis (n = 9), and by sixplex tandem mass tag (TMT) isobaric labeling (n = 6) quantitative mass spectrometry. Overall, 73 proteins were overexpressed in patients presenting the second stage of the disease. Two of these, osteopontin and ß-2-microglobulin, were confirmed to be potential markers for staging human African trypanosomiasis (HAT) by Western blot and ELISA. The two proteins significantly discriminated between S1 and S2 patients with high sensitivity (68% and 78%, respectively) for 100% specificity, and a combination of both improved the sensitivity to 91%. The levels of osteopontin and ß-2-microglobulin in CSF of S2 patients (µg/ml range), as well as the fold increased concentration in S2 compared with S1 (3.8 and 5.5 respectively) make the two markers good candidates for the development of a test for staging HAT patients.

pROC: an open-source package for R and S+ to analyze and compare ROC curves.

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.

Quantitative analysis of human cerebrospinal fluid proteins using a combination of cysteine tagging and amine-reactive isobaric labeling.

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.

Brain extracellular fluid protein changes in acute stroke patients.

In vivo human brain extracellular fluids (ECF) of acute stroke patients were investigated to assess the changes in protein levels associated with ischemic damages. Microdialysates (MDs) from the infarct core (IC), the penumbra (P), and the unaffected contralateral (CT) brain regions of patients suffering an ischemic stroke (n = 6) were compared using a shotgun proteomic approach based on isobaric tagging and mass spectrometry. Quantitative analysis showed 53 proteins with increased amounts in the IC or P with respect to the CT samples. Glutathione S-transferase P (GSTP1), peroxiredoxin-1 (PRDX1), and protein S100-B (S100B) were further assessed with ELISA on the blood of unrelated control (n = 14) and stroke (n = 14) patients. Significant increases of 8- (p = 0.0002), 20- (p = 0.0001), and 11-fold (p = 0.0093) were found, respectively. This study highlights the value of ECF as an efficient source to further discover blood stroke markers.

Matrix metalloproteinase-9 and intercellular adhesion molecule 1 are powerful staging markers for human African trypanosomiasis.

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.

Isobaric tagging-based selection and quantitation of cerebrospinal fluid tryptic peptides with reporter calibration curves.

In the past few years, mass spectrometry (MS) has emerged as an efficient tool for the multiplexed peptide and protein concentration determination by isotope dilution. Despite the growing use of isobaric tagging to perform relative quantitation for the discovery of potential biomarkers in biological fluids, no real application has so far been presented for their absolute quantitation. Isobaric tandem mass tags (TMTs) were used herein for the selection and quantitation of tryptic peptides derived from brain damage related proteins in cerebrospinal fluid (CSF). Proteotypic tryptic peptide analogues were synthesized, prepared in four reference amounts, differentially labeled with four isobaric TMTs with reporter-ions at m/z = 128.1, 129.1, 130.1, and 131.1, and mixed with CSF sample previously labeled with TMT 126.1. Off-gel electrophoresis (OGE) was used as first-dimension separation of the pooled sample. The resulting fractions were analyzed with reversed-phase liquid chromatography (RP-LC) tandem mass spectrometry (MS/MS), using tandem time-of-flight (TOF/TOF) and hybrid linear ion trap-orbitrap (LTQ-OT) instruments. Under collision-induced dissociation (CID) or higher-energy C-trap dissociation (HCD), the release of the reporter fragments from the TMT-labeled peptide standards provided an internal calibration curve to assess the concentration of these peptides in the CSF. This tool also allowed identifying selectively these peptides in CSF as only the targeted peptides showed specific fragmentation pattern in the TMT reporter-ion zone of the tandem mass spectra. Assays for the concentration measurements of peptides from PARK7, GSTP1, NDKA, and S100B proteins in CSF were further characterized using this novel, efficient, and straightforward approach.

From relative to absolute quantification of tryptic peptides with tandem mass tags: application to cerebrospinal fluid.

Quantification is a major task in proteomics. Among the different analytical strategies to enable peptide and protein quantification, tagging with isotopic labels has emerged as a practical, versatile, and efficient alternative. In particular, isobaric labels, such as TMT or iTRAQ, are now widely employed to make relative comparison of the protein amounts in separate biological samples with tandem mass spectrometry (MS/MS). We used herein a shotgun proteomic approach based on labelling with tandem mass tags (TMTs) for the relative quantification of proteins, and the absolute quantification of their tryptic peptides in human cerebrospinal fluid (CSF). First, the comparison of ante- and post-mortem CSF samples was carried out for the discovery of protein marker candidates of brain-damage disorders. Second, tryptic peptides representative of these candidates were measured in CSF using reporter-ion calibration curves. These works highlighted the advantages and limitations of such strategies for quantification purposes in proteomics.

Bioinformatics for protein biomarker panel classification: what is needed to bring biomarker panels into in vitro diagnostics?

A large number of biomarkers have been discovered over the past few years using proteomics techniques. Unfortunately, most of them are neither specific nor sensitive enough to be translated into in vitro diagnostics and routine clinical practice. From this observation, the idea of combining several markers into panels to improve clinical performances has emerged. In this article, we present a discussion of the bioinformatics aspects of biomarker panels and the concomitant challenges, including high dimensionality and low patient number and reproducibility.

Neopterin and CXCL-13 in Diagnosis and Follow-Up of Trypanosoma brucei gambiense Sleeping Sickness: Lessons from the Field in Angola.

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.

Iron is neurotoxic in retinal detachment and transferrin confers neuroprotection.

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.

Relative quantification of proteins in human cerebrospinal fluids by MS/MS using 6-plex isobaric tags.

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.

Anti-(apolipoprotein A-1) IgGs are associated with high levels of oxidized low-density lipoprotein in acute coronary syndrome.

ApoA-1 (apolipoprotein A-1) is the main component of HDL (high-density lipoprotein) and stabilizes PON-1 (paraoxonase-1), which prevents lipid peroxidation and oxLDL (oxidized low-density lipoprotein) formation. Autoantibodies against apoA-1 [anti-(apoA-1) IgG] have been found in antiphospholipid syndrome and systemic lupus erythematosous, two diseases with an increased risk of thrombotic events, as well as in ACS (acute coronary syndrome). OxLDL levels are also elevated in these diseases. Whether anti-(apoA-1) IgGs exist in other prothrombotic conditions, such as APE (acute pulmonary embolism) and stroke, has not been studied and their potential association with oxLDL and PON-1 activity is not known. In the present study, we determined prospectively the prevalence of anti-(apoA-1) IgG in patients with ACS (n=127), APE (n=58) and stroke (n=34), and, when present, we tested their association with oxLDL levels. The prevalance of anti-(apoA-1) IgG was 11% in the ACS group, 2% in the control group and 0% in the APE and stroke groups. The ACS group had significantly higher median anti-(apoA-1) IgG titres than the other groups of patients. Patients with ACS positive for anti-(apoA-1) IgG had significantly higher median oxLDL values than those who tested negative (226.5 compared with 47.7 units/l; P<0.00001) and controls. The Spearman ranked test revealed a significant correlation between anti-(apoA-1) IgG titres and serum oxLDL levels (r=0.28, P<0.05). No association was found between PON-1 activity and oxLDL or anti-(apoA-1) IgG levels. In conclusion, anti-(apoA-1) IgG levels are positive in ACS, but not in stroke or APE. In ACS, their presence is associated with higher levels of oxLDL and is directly proportional to the serum concentration of oxLDL. These results emphasize the role of humoral autoimmunity as a mediator of inflammation and coronary atherogenesis.

Correlation between cardiac biomarkers and right ventricular enlargement on chest CT in non massive pulmonary embolism.

BACKGROUND: Troponin I (cTnI), myoglobin, heart-type fatty acid binding protein (H-FABP), and natriuretic peptides (BNP, NTproBNP) were all reported to be elevated in patients with pulmonary embolism (PE). METHODS: To assess the correlation between the aforementioned markers and helical computed tomography (hCT) right ventricular dysfunction (RVD) in non massive PE, we performed this prospective pilot study on 50 patients. RESULTS: Patients with RVD had significant higher natriuretic peptides prevalence than cardiomyocytes damage-related markers (48% vs 20%, P=0.006). Significant prevalence differences were observed only for natriuretic peptides when patients with RVD and those without were compared (74% vs 33% for NT-pro BNP, P=0.005 and 65% vs 22% for BNP, P=0.003). Patients with RVD had significant higher biomarkers median plasmatic values than those without (BNP: 170 vs 36 pg/ml, P=0.0027; NT-proBNP: 1369 vs 170.7 pg/ml, P=0.0024; cTnI: 0.032 vs 0 ng/ml, P=0.0034; H-FABP: 4.32 vs 2.23 ng/ml, P=0.0032; myoglobin: 36.7 vs 28.2 ng/ml, P=0.03). Significant correlations were only obtained between RV/LV index and plasmatic natriuretic peptides (NT-proBNP: r=0.36, P=0.009; BNP r=0.28; P=0.047). CONCLUSIONS: Natriuretic peptides prevalence elevation and median values are significantly higher when RVD is present and significantly correlate with hCT RVD.

Thyroid-associated orbitopathy and tears: A proteomics study.

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.