Biomarker Discovery in Subclinical Mycobacterial Infections of Cattle

BACKGROUND: Bovine tuberculosis is a highly prevalent infectious disease of cattle worldwide; however, infection in the United States is limited to 0.01% of dairy herds. Thus detection of bovine TB is confounded by high background infection with M. avium subsp. paratuberculosis. The present study addresses variations in the circulating peptidome based on the pathogenesis of two biologically similar mycobacterial diseases of cattle. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that serum proteomes of animals in response to either M. bovis or M. paratuberculosis infection will display several commonalities and differences. Sera prospectively collected from animals experimentally infected with either M. bovis or M. paratuberculosis were analyzed using high-resolution proteomics approaches. iTRAQ, a liquid chromatography and tandem mass spectrometry approach, was used to simultaneously identify and quantify peptides from multiple infections and contemporaneous uninfected control groups. Four comparisons were performed: 1) M. bovis infection versus uninfected controls, 2) M. bovis versus M. paratuberculosis infection, 3) early, and 4) advanced M. paratuberculosis infection versus uninfected controls. One hundred and ten differentially elevated proteins (P < or = 0.05) were identified. Vitamin D binding protein precursor (DBP), alpha-1 acid glycoprotein, alpha-1B glycoprotein, fetuin, and serine proteinase inhibitor were identified in both infections. Transthyretin, retinol binding proteins, and cathelicidin were identified exclusively in M. paratuberculosis infection, while the serum levels of alpha-1-microglobulin/bikunin precursor (AMBP) protein, alpha-1 acid glycoprotein, fetuin, and alpha-1B glycoprotein were elevated exclusively in M. bovis infected animals. CONCLUSIONS/SIGNIFICANCE: The discovery of these biomarkers has significant impact on the elucidation of pathogenesis of two mycobacterial diseases at the cellular and the molecular level and can be applied in the development of mycobacterium-specific diagnostic tools for the monitoring progression of disease, response to therapy, and/or vaccine based interventions

Plasma proteome changes in cardiovascular disease patients: novel isoforms of apolipoprotein A1

<p>Abstract</p> <p>Background</p> <p>The aim of this proteomic study was to look for changes taking place in plasma proteomes of patients with acute myocardial infarction (AMI), unstable angina pectoris (UAP), and stable angina pectoris (SAP).</p> <p>Methods</p> <p>Depleted plasma proteins were separated by 2D SDS-PAGE (pI 4-7), and proteomes were compared using Progenesis SameSpots statistical software. Proteins were identified by nanoLC-MS/MS. Proteins were quantified using commercial kits. Apolipoprotein A1 was studied using 1D and 2D SDS-PAGE, together with western blotting.</p> <p>Results</p> <p>Reciprocal comparison revealed 46 unique, significantly different spots; proteins in 34 spots were successfully identified and corresponded to 38 different proteins. Discrete comparisons of patient groups showed 45, 41, and 8 significantly different spots when AMI, UAP, and SAP were compared with the control group. On the basis of our proteomic data, plasma levels of two of them, alpha-1 microglobulin and vitamin D-binding protein, were determined. The data, however, failed to prove the proteins to be suitable markers or risk factors in the studied groups. The plasma level and isoform representation of apolipoprotein A1 were also estimated. Using 1D and 2D SDS-PAGE, together with western blotting, we observed extra high-molecular weight apolipoprotein A1 fractions presented only in the patient groups, indicating that the novel high-molecular weight isoforms of apolipoprotein A1 may be potential new markers or possible risk factors of cardiovascular disease.</p> <p>Conclusion</p> <p>The reported data show plasma proteome changes in patients with AMI, UAP, and SAP. We propose some apolipoprotein A1 fractions as a possible new disease-associated marker of cardiovascular disorders.</p

Proteomics Comparison of Cerebrospinal Fluid of Relapsing Remitting and Primary Progressive Multiple Sclerosis

Background: Based on clinical representation of disease symptoms multiple sclerosis (MScl) patients can be divided into two major subtypes; relapsing remitting (RR) MScl (85-90%) and primary progressive (PP) MScl (10-15%). Proteomics analysis of cerebrospinal fluid (CSF) has detected a number of proteins that were elevated in MScl patients. Here we specifically aimed to differentiate between the PP and RR subtypes of MScl by comparing CSF proteins. Methodology/Principal Findings: CSF samples (n = 31) were handled according to the same protocol for quantitative mass spectrometry measurements we reported previously. In the comparison of PP MScl versus RR MScl we observed a number of differentially abundant proteins, such as protein jagged-1 and vitamin D-binding protein. Protein jagged-1 was over three times less abundant in PP MScl compared to RR MScl. Vitamin D-binding protein was only detected in the RR MScl samples. These two proteins were validated by independent techniques (western blot and ELISA) as differentially abundant in the comparison between both MScl types. Conclusions/Significance: The main finding of this comparative study is the observation that the proteome profiles of CSF in PP and RR MScl patients overlap to a large extent. Still, a number of differences could be observed. Protein jagged-1 is a ligand for multiple Notch receptors and involved in the mediation of Notch signaling. It is suggested in literature that the Notch pathway is involved in the remyelination of MScl lesions. Aberration of normal homeostasis of Vitamin D, of which approximately 90% is bound to vitamin D-binding protein, has been widely implicated in MScl for some years now. Vitamin D directly and indirectly regulates the differentiation, activation of CD4+ T-lymphocytes and can prevent the development of autoimmune processes, and so it may be involved in neuroprotective elements in MScl

Proteomics Mapping of Cord Blood Identifies Haptoglobin “Switch-On” Pattern as Biomarker of Early-Onset Neonatal Sepsis in Preterm Newborns

Intra-amniotic infection and/or inflammation (IAI) are important causes of preterm birth and early-onset neonatal sepsis (EONS). A prompt and accurate diagnosis of EONS is critical for improved neonatal outcomes. We sought to explore the cord blood proteome and identify biomarkers and functional protein networks characterizing EONS in preterm newborns.We studied a prospective cohort of 180 premature newborns delivered May 2004-September 2009. A proteomics discovery phase employing two-dimensional differential gel electrophoresis (2D-DIGE) and mass spectrometry identified 19 differentially-expressed proteins in cord blood of newborns with culture-confirmed EONS (n = 3) versus GA-matched controls (n = 3). Ontological classifications of the proteins included transfer/carrier, immunity/defense, protease/extracellular matrix. The 1(st)-level external validation conducted in the remaining 174 samples confirmed elevated haptoglobin and haptoglobin-related protein immunoreactivity (Hp&HpRP) in newborns with EONS (presumed and culture-confirmed) independent of GA at birth and birthweight (P<0.001). Western blot concurred in determining that EONS babies had conspicuous Hp&HpRP bands in cord blood ("switch-on pattern") as opposed to non-EONS newborns who had near-absent "switch-off pattern" (P<0.001). Fetal Hp phenotype independently impacted Hp&HpRP. A bayesian latent-class analysis (LCA) was further used for unbiased classification of all 180 cases based on probability of "antenatal IAI exposure" as latent variable. This was then subjected to 2(nd)-level validation against indicators of adverse short-term neonatal outcome. The optimal LCA algorithm combined Hp&HpRP switch pattern (most input), interleukin-6 and neonatal hematological indices yielding two non-overlapping newborn clusters with low (≤20%) versus high (≥70%) probability of IAI exposure. This approach reclassified ∼30% of clinical EONS diagnoses lowering the number needed to harm and increasing the odds ratios for several adverse outcomes including intra-ventricular hemorrhage.Antenatal exposure to IAI results in precocious switch-on of Hp&HpRP expression. As EONS biomarker, cord blood Hp&HpRP has potential to improve the selection of newborns for prompt and targeted treatment at birth

Vitamin D Binding Protein Genotype Is Associated with Serum 25-Hydroxyvitamin D and PTH Concentrations, as Well as Bone Health in Children and Adolescents in Finland

Peer reviewe

Genetic variation in vitamin D-related genes and risk of colorectal cancer in African Americans

PurposeDisparities in both colorectal cancer (CRC) incidence and survival impact African Americans (AAs) more than other US ethnic groups. Because vitamin D is thought to protect against CRC and AAs have lower serum vitamin D levels, genetic variants that modulate the levels of active hormone in the tissues could explain some of the cancer health disparity. Consequently, we hypothesized that genetic variants in vitamin D-related genes are associated with CRC risk.MethodsTo test this hypothesis, we studied 39 potentially functional single-nucleotide polymorphisms (SNPs) in eight genes (CYP2R1, CYP3A4, CYP24A1, CYP27A1, CYP27B1, GC, DHCR7, and VDR) in 961 AA CRC cases and 838 healthy AA controls from Chicago and North Carolina. We tested whether SNPs are associated with CRC incidence using logistic regression models to calculate p values, odds ratios, and 95% confidence intervals. In the logistic regression, we used a log-additive genetic model and used age, gender, and percent West African ancestry, which we estimated with the program STRUCTURE, as covariates in the models.ResultsA nominally significant association was detected between CRC and the SNP rs12794714 in the vitamin D 25-hydroxylase gene CYP2R1 (p=0.019), a SNP that has previously been associated with serum vitamin D levels. Two SNPs, rs16847024 in the GC gene and rs6022990 in the CYP24A1 gene, were nominally associated with left-sided CRC (p=0.015 and p=0.018, respectively).ConclusionsOur results strongly suggest that genetic variation in vitamin D-related genes could affect CRC susceptibility in AAs. Electronic supplementary materialThe online version of this article (doi:10.1007/s10552-014-0361-y) contains supplementary material, which is available to authorized users