Urine Proteomics Differentiate Primary Thrombotic Antiphospholipid Syndrome From Obstetric Antiphospholipid Syndrome

Antiphospholipid syndrome (APS) is a multisystem disorder characterized by thrombosis and/or recurrent fetal loss. This clinical phenotype heterogeneity may result in differences in response to treatment and prognosis. In this study, we aimed to identify primary thrombotic APS (TAPS) from primary obstetric APS (OAPS) using urine proteomics as a non-invasive method. Only patients with primary APS were enrolled in this study from 2016 to 2018 at a single clinical center in Shanghai. Urine samples from 15 patients with TAPS, 9 patients with OAPS, and 15 healthy controls (HCs) were collected and analyzed using isobaric tags for relative and absolute quantification (iTRAQ) labeling combined with liquid chromatography-tandem mass spectrometry analysis to identify differentially expressed proteins. Cluster analysis of urine proteomics identified differentiated proteins among the TAPS, OAPS, and HC groups. Urinary proteins were enriched in cytokine and cytokine receptor pathways. Representative secreted cytokines screened out (fold change >1.20, or <0.83, p<0.05) in these differentiated proteins were measured by enzyme-linked immunosorbent assay in a validation cohort. The results showed that the levels of C-X-C motif chemokine ligand 12 (CXCL12) were higher in the urine of patients with TAPS than in those with OAPS (p=0.035), while the levels of platelet-derived growth factor subunit B (PDGFB) were lower in patients with TAPS than in those with OAPS (p=0.041). In addition, correlation analysis showed that CXCL12 levels were positively correlated with immunoglobulin G anti-β2-glycoprotein I antibody (r=0.617, p=0.016). Our results demonstrated that urinary CXCL12 and PDGFB might serve as potential non-invasive markers to differentiate primary TAPS from primary OAPS.

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Urinary Ceruloplasmin Concentration Predicts Development of Kidney Disease in Sickle Cell Disease Patients

Blood ◽

10.1182/blood.v128.22.4865.4865 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4865-4865 ◽

Cited By ~ 1

Author(s):

Santosh L. Saraf ◽

Xionghao Lin ◽

Gillian Lee ◽

Elena Afia Adjei ◽

Namita Kumari ◽

...

Keyword(s):

Mass Spectrometry ◽

Sickle Cell Disease ◽

Kidney Disease ◽

Renal Disease ◽

Mass Spectrometry Analysis ◽

Cell Disease ◽

Free Hemoglobin ◽

Spectrometry Analysis ◽

Non Invasive ◽

Ckd Stage

Abstract BACKGROUND: Chronic kidney disease (CKD) is a prevalent complication of sickle cell disease (SCD) and associated with early mortality. Hemoglobinuria is a risk factor for the development and progression of CKD. Discovery and validation of non-invasive biomarkers for early stage renal disease are needed to facilitate optimal CKD treatment. Mass-spectrometry analysis of patient urine is a modern method for biomarker discovery. Urine from patients with late stages of glomerular disease contains a large number of abundant plasma proteins that overwhelm and complicate mass-spectrometry analysis. Thus, analysis of samples collected before the onset of kidney disease is a promising approach. Abnormal renal iron metabolism including cortical iron deposition is characteristic of SCD nephropathy. Ceruloplasmin is a ferrioxidase that is important facilitator of cellular iron export by ferroportin and of iron binding by transferrin. OBJECTIVES: We aimed to use mass-spectrometry to determine urinary biomarkers in SCD patients without renal disease that may predict the development of CKD and to validate the biomarkers by ELISA. METHODS: Mass-spectrometry analysis was performed on urine samples from eight University of Illinois at Chicago (UIC) SCD patients without CKD. Proteins were identified using Proteome Discoverer 1.4 and quantified using SIEVE 2.0 program. Ceruloplasmin concentrations were determined by ELISA in these eight subjects for the validation of our mass spectrometry analysis findings plus an additional 12 UIC SCD patients without CKD. Urine ceruloplasmin and free hemoglobin concentrations were determined by ELISA in an additional 34 UIC SCD patients with CKD stage ranging from 0-5. RESULTS: Label-free quantitative proteomic analysis of urine samples collected from SCD patients without CKD showed greater ceruloplasmin levels in 2 samples with hemoglobinuria versus 6 samples without hemoglboinuria (37.4-fold, p=2.7x10-8). Analysis of all twenty non-CKD samples by ELISA showed 2.5-fold higher levels of ceruloplasmin in hemoglobinuria samples (p=0.003). To determine whether urine ceruloplasmin correlated with CKD stage, we analyzed an additional 34 samples with and without CKD. Samples with CKD stages 2-5 (N=12) demonstrated higher levels of ceruloplasmin than stages 0-1 (N=22) (1.7-fold increase, p=0.008) (Figure 1A). In an additional analysis of these results, individual CKD disease stage correlated with urinary concentrations of ceruloplasmin (N=34, r=0.49, p=0.0035) (Figure 1B) and cell free hemoglobin (N=34, r=0.45, p=0.007) (Figure 1C). The correlation of ceruloplasmin concentration with CKD stage in the SCD patients showed high sensitivity and specificity (area under curve 90.7±6.8; p-value=0.018) by ROCK analysis. Finally, urinary ceruloplasmin concentration demonstrated a strong correlation with free hemoglobin concentration (r=0.79). CONCLUSIONS: Urinary ceruloplasmin may complement urinary free hemoglobin as a non-invasive biomarker of risk for CKD in SCD patients. ACKNOWLEDGMENTS: This work was supported by NIH Research Grants 1P50HL118006, 1R01HL125005, 5G12MD007597 and K23HL125984. The content is solely the responsibility of the authors and does not necessarily represent the official view of NHLBI, NIMHD or NIH. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Role of lgtC in Resistance of Nontypeable Haemophilus influenzae Strain R2866 to Human Serum

Infection and Immunity ◽

10.1128/iai.00722-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6226-6235 ◽

Cited By ~ 35

Author(s):

Alice L. Erwin ◽

Simon Allen ◽

Derek K. Ho ◽

Paul J. Bonthius ◽

Justin Jarisch ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Human Serum ◽

Mass Spectrometry Analysis ◽

Start Codon ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Resistance ◽

Nontypeable Haemophilus Influenzae ◽

Spectrometry Analysis ◽

Nthi Strain ◽

High Level

ABSTRACT We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame (“off”) in most colonies of R3392 but in frame with its start codon (“on”) in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galα1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

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Streptococcus mutans Surface α-Enolase Binds Salivary Mucin MG2 and Human Plasminogen

Infection and Immunity ◽

10.1128/iai.72.11.6748-6752.2004 ◽

2004 ◽

Vol 72 (11) ◽

pp. 6748-6752 ◽

Cited By ~ 64

Author(s):

Jingping Ge ◽

Diana M. Catt ◽

Richard L. Gregory

Keyword(s):

Streptococcus Mutans ◽

Mass Spectrometry Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Ionization Time ◽

Spectrometry Analysis ◽

Flight Mass Spectrometry ◽

Salivary Mucin ◽

Transmission Electron ◽

A Cell ◽

Human Plasminogen

ABSTRACT Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis identified enolase as a cell surface component of Streptococcus mutans, which was confirmed by enzyme-linked immunosorbent assay, Western blotting, and transmission electron microscopy. Surface enolase was demonstrated to bind to human plasminogen and salivary mucin MG2. The results suggested a role for enolase in S. mutans attachment, clearance, or breach of the bloodstream barrier.

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Urinary vitamin D-binding protein as a marker of ovarian reserve

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00762-9 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Sanglin Li ◽

Lina Hu ◽

Chanyu Zhang

Keyword(s):

Vitamin D ◽

Ovarian Reserve ◽

Polycystic Ovary ◽

Binding Protein ◽

Expression Patterns ◽

Mass Spectrometry Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Vitamin D Binding Protein ◽

Characteristic Analysis ◽

Spectrometry Analysis

Abstract Background Ovarian reserve reflects the quality and quantity of available oocytes and has become an indispensable measure for the better understanding of reproductive potential. Proteomic approaches are especially helpful in discerning differential protein expression patterns associated with normal and diseased states and, thus, proteomic analyses are increasingly used to identify clinically useful biomarkers. The aim of this study was to investigate proteins secreted in the urine of patients with different ovarian reserve by proteomic techniques to identify potential markers for assessing ovarian reserve. Methods Urine samples were obtained from patients with polycystic ovary syndrome (PCOS) and diminished ovarian reserve (DOR), and from normal control (NC)participants. We used isobaric tags for relative and absolute quantification (iTRAQ) technology combined with mass spectrometry analysis to identify candidate urinary proteins in the three groups. The selected proteins were confirmed using western blot analysis and enzyme-linked immunosorbent assay (ELISA). Diagnostic performance of the selected proteins was assessed using receiver operating characteristic analysis. Results When Compared with NC samples, 285 differentially expressed proteins (DEPs) were identified in the DOR samples and 372 in the PCOS samples. By analyzing the intersection of the two groups of DEPs, we found 26 proteins with different expression trends in the DOR and PCOS groups. Vitamin D-binding protein (VDBP) was the key protein for the protein-protein interaction network. ELISA quantification of urinary VDBP revealed the highest levels in the PCOS group, followed by the NC group and the lowest levels in the DOR group (115.90 ± 26.02, 81.86 ± 23.92 and 52.84 ± 21.37 ng/ml, respectively; P < 0.05). As a diagnostic marker, VDBP had a sensitivity of 67.4% and a specificity of 91.8% for DOR, and a sensitivity of 93.8% and a specificity of 77.6% for PCOS. Conclusions Urinary VDBP is closely associated with ovarian reserve and can be considered as a novel noninvasive biomarker of ovarian reserve. However, studies including large sample sizes are needed to validate these results.

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Assessing the feasibility and acceptability of online measurements of exhaled volatile organic compounds (VOCs) in children with preschool wheeze: a pilot study

BMJ Paediatrics Open ◽

10.1136/bmjpo-2020-001003 ◽

2021 ◽

Vol 5 (1) ◽

pp. e001003

Author(s):

Karl Holden ◽

Misty Makinde ◽

Michael Wilde ◽

Matthew Richardson ◽

Tim Coats ◽

...

Keyword(s):

Mass Spectrometry ◽

Preschool Children ◽

Volatile Organic Compounds ◽

Real Time ◽

Organic Compounds ◽

Mass Spectrometry Analysis ◽

Breathing Patterns ◽

Spectrometry Analysis ◽

Non Invasive ◽

Volatile Organic

BackgroundInvestigating airway inflammation and pathology in wheezy preschool children is both technically and ethically challenging. Identifying and validating non-invasive tests would be a huge clinical advance. Real-time analysis of exhaled volatile organic compounds (VOCs) in adults is established, however, the feasibility of this non-invasive method in young children remains undetermined.AimTo determine the feasibility and acceptability of obtaining breath samples from preschool children by means of real-time mass spectrometry analysis of exhaled VOCs.MethodsBreath samples from preschool children were collected and analysed in real time by proton transfer reaction–time of flight–mass spectrometry (PTR–TOF–MS) capturing unique breath profiles. Acetone (mass channel m/z 59) was used as a reference profile to investigate the breath cycle in more detail. Dynamic time warping (DTW) was used to compare VOC profiles from adult breath to those we obtained in preschool children.Results16 children were recruited in the study, of which eight had acute doctor-diagnosed wheeze (mean (range) age 3.2 (1.9–4.5) years) and eight had no history of wheezing (age 3.3 (2.2–4.1) years). Fully analysable samples were obtained in 11 (68%). DTW was used to ascertain the distance between the time series of mass channel m/z 59 (acetone) and the other 193 channels. Commonality of 12 channels (15, 31, 33, 41, 43, 51, 53, 55, 57, 60, 63 and 77) was established between adult and preschool child samples despite differences in the breathing patterns.ConclusionReal-time measurement of exhaled VOCs by means of PTR–MS is feasible and acceptable in preschool children. Commonality in VOC profiles was found between adult and preschool children.

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Deep proteomic analysis of chicken erythropoiesis

10.1101/289728 ◽

2018 ◽

Cited By ~ 1

Author(s):

Marjorie Leduc ◽

Emilie-Fleur Gautier ◽

Anissa Guillemin ◽

Cédric Broussard ◽

Virginie Salnot ◽

...

Keyword(s):

Expression Patterns ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Absolute Quantification ◽

Mass Spectrometry Analysis ◽

Differentiation Process ◽

Erythroid Cells ◽

Spectrometry Analysis ◽

Gata Transcription Factors ◽

Chicken Erythrocytes

AbstractIn contrast to mammalian erythroid cells that lost their nucleus at the end of the differentiation process, circulating chicken erythrocytes, like erythrocytes of most other non-mammalian vertebrates, are nucleated although their nucleus is believed to be transcriptionally silent. This major difference suggests that the erythroid differentiation process is likely to present both similarities and differences in mammals compared to other vertebrates. Since proteins are the major cellular effectors, analysis of the proteome is more prone to reflect true differences than analysis of the pattern of mRNA expression. We have previously reported the evolution of the proteome of human erythroid cells throughout their differentiation process. Here we report the analysis of the proteome of chicken erythroblasts during their terminal differentiation. We used the T2EC cellular model that allows to obtain homogenous populations of immature erythroblasts. Induction of their terminal differentiation led to their maturation and the possibility to obtain cells at different differentiation stages. Mass spectrometry analysis of these cell populations allowed the absolute quantification of 6167 proteins throughout the terminal differentiation process. Beside many proteins with similar expression patterns between chicken and human erythroblasts, like SLC4A1 (Band3), GATA1 or CD44, this analysis also revealed that other important proteins like Kit or other GATA transcription factors exhibit fully different patterns of expression.

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Detection of Antiphospholipid Antibodies by Flow Cytometry: Rapid Detection of Antibody Isotype and Phospholipid Specificity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649636 ◽

1993 ◽

Vol 70 (04) ◽

pp. 603-607 ◽

Cited By ~ 18

Author(s):

M W Stewart ◽

W S Etches ◽

A S Russell ◽

J S Percy ◽

C A Johnston ◽

...

Keyword(s):

Flow Cytometry ◽

Antiphospholipid Syndrome ◽

Antiphospholipid Antibodies ◽

Fetal Loss ◽

Laboratory Diagnosis ◽

Enzyme Linked Immunosorbent Assay ◽

Antibody Isotype ◽

Anionic Phospholipids ◽

Flow Cytometric Method ◽

Antibody Isotypes

SummaryLaboratory diagnosis of antiphospholipid antibodies is important in patients with clinical features of the antiphospholipid syndrome, such as thrombosis and fetal loss. We have developed a novel method for the detection of antiphospholipid antibodies using flow cytometry. Anionic phospholipids cardiolipin, phosphatidylserine and phosphatidylinositol are coated onto polystyrene beads of different sizes, allowing detection and semiquantitation of their respective phospholipid antibody isotypes. The results of the flow cytometric method closely correlate those of the standardised anticardiolipin enzyme-linked immunosorbent assay (ELISA), but the method is quicker and is versatile in its ability to detect IgG, IgM and IgA antibody isotypes at the same time. The method promises to be useful in evaluating the significance of phospholipid specificity and antibody isotypes in patients with the antiphospholipid syndrome.

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Rapid and Sensitive Screening of Sulfamethazine in Porcine Urine with an Enzyme-Linked Immunosorbent Assay and a Field-Portable Immunofiltration Assay

Journal of Food Protection ◽

10.4315/0362-028x-65.5.820 ◽

2002 ◽

Vol 65 (5) ◽

pp. 820-827 ◽

Cited By ~ 10

Author(s):

PATRICIA CRABBE ◽

CARLOS VAN PETEGHEM

Keyword(s):

Detection Limit ◽

Immunosorbent Assay ◽

Visual Detection ◽

Close Contact ◽

Negative Control ◽

Mass Spectrometry Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Blue Color ◽

Nylon Membrane ◽

Spectrometry Analysis

An enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunofiltration assay (ELIFA) were developed for the screening of sulfamethazine (SMZ) in porcine urine. Incurred urine samples were measured by ELISA with a working range of 0 to 10 ng of SMZ per ml. The assay showed good accuracy and precision, with recoveries above 99.8% and intra-and interassay coefficients of variation (CVs) ranging from 2.6 to 5.6% and from 5.9 to 12.7%, respectively. Good agreement was observed when the results of the immunoassay were compared with those of liquid chromatography/tandem mass spectrometry analysis. For the ELIFA, a nylon membrane is placed on top of an absorbent material and covalently coated with rabbit anti-rat immunoglobulins. Free binding sites are blocked, and monoclonal anti-SMZ antibodies, SMZ standard or urine, and SMZ–horse radish peroxidase conjugate are subsequently dropped onto the membrane. During the assay, the reactants are drawn through the membrane because of its close contact with the absorbent pad. Finally, a substrate solution is added for blue color development. The blue spot produced can be visually evaluated or instrumentally measured (numeric value), and the intensity of its color is inversely proportional to the analyte concentration. When a blue dot appears on the membrane, even if its color is less intense than that of the negative control, the sample is considered “negative,” i.e., it is thought to contain a concentration of SMZ that is below the visual detection limit. If no color appears on the test membrane, the sample is considered “positive,” i.e., it is thought to contain a concentration of SMZ that is equal to or above the visual detection limit. Validation of the assay showed good inter- and intra-assay precision (CV < 10%). Because samples can be analyzed after a simple dilution in <30 min with this assay format, it has strong potential for application in the field.

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Genes Encoding Bacteriocins and Their Expression and Potential Virulence Factors of Enterococci Isolated from Wood Pigeons (Columba palumbus)

Journal of Food Protection ◽

10.4315/0362-028x-69.3.520 ◽

2006 ◽

Vol 69 (3) ◽

pp. 520-531 ◽

Cited By ~ 31

Author(s):

MARÍA MARTÍN ◽

JORGE GUTIÉRREZ ◽

RAQUEL CRIADO ◽

CARMEN HERRANZ ◽

LUIS M. CINTAS ◽

...

Keyword(s):

Antimicrobial Activity ◽

Virulence Factors ◽

Virulence Genes ◽

Pcr Amplification ◽

Virulence Gene ◽

Antagonistic Activity ◽

Mass Spectrometry Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Virulence Determinants ◽

Spectrometry Analysis

Samples of the intestinal content and carcasses of wood pigeons (Columba palumbus) were evaluated for enterococci with antimicrobial activity. Enterococcus faecium comprised the largest enterococcal species with antagonistic activity, followed by Enterococcus faecalis and Enterococcus columbae. PCR amplification of genes coding bacteriocins and determination of their nucleotide sequence, and the use of specific antipeptide bacteriocin antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay, permitted characterization of enterococci coding that described bacteriocins and their expression. The efaAfm determinant was the only virulence gene detected in E. faecium, whereas E. faecalis showed a larger number of virulence determinants, and E. columbae did not carry any of the virulence genes examined. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of producer cells. Purification of the antagonistic activity of E. columbae PLCH2 showed multiple chromatographic fragments after matrix-assisted laser desorption–ionization time-of-flight mass spectrometry analysis, suggesting the active peptide(s) had not yet purified to homogeneity. Bacteriocinogenic E. faecium and E. columbae isolates may be considered hygienic for production of enterocins and potentially safe due to their low incidence of potential virulence genes and susceptibility of most relevant clinical antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors is an issue that must be considered further.

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