Tear-Derived Exosome Proteins Are Increased in Patients with Thyroid Eye Disease.

Exosomes contain proteins, lipids, RNA, and DNA that mediate intercellular signaling. Exosomes can contribute to the pathological processes of various diseases, although their roles in ocular diseases are unclear. We aimed to isolate exosomes from tear fluids (TF) of patients with Thyroid eye disease (TED) and analyze the exosomal proteins. TFs were collected from eight patients with TED and eight control subjects. The number of TF exosomes were measured using nanoparticle-tracking analysis. The expression of specific proteins in the purified exosome pellets were analyzed using a Proteome Profiler Array Kit. Cultured normal orbital fibroblasts were incubated with TF exosomes from patients with TED and control subjects, and changes in inflammatory cytokine levels were compared. TF exosomes from TED patients showed more exosomes than the control subjects. The expression levels of exosomal proteins vitamin D-binding (VDB) protein, C-reactive protein (CRP), chitinase 3-like 1 (CHI3L1), matrix metalloproteinase-9 (MMP-9), and vascular adhesion molecule-1 (VCAM-1) were significantly increased in patients with TED, compared to those of controls. Orbital fibroblasts exposed to TF exosomes from patients with TED showed significantly higher levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1) production than those treated with control TF exosomes. Specific proteins showed higher expression in exosomes from TED patients, implying that they may play keys roles in TED pathogenesis.

Maternal biomarker patterns for metabolism and inflammation in pregnancy are influenced by multiple micronutrient supplementation and associated with child biomarker patterns and nutritional status at 9-12 years of age.

Maternal nutritional status influences fetal development and long-term risk for adult non-communicable diseases. However, the underlying mechanisms remain poorly understood. We examined whether biomarkers for metabolism and inflammation during pregnancy were associated with maternal health and with child biomarkers and health at 9-12 years of age in 44 maternal-child dyads from the Supplementation with Multiple Micronutrients Intervention Trial (SUMMIT, ISRCTN34151616) in Lombok, Indonesia. Archived blood for each dyad from maternal enrollment, later in pregnancy, postpartum, and from children at 9-12 years comprised 132 specimens. Multiplex microbead immunoassays were used to quantify vitamin D-binding protein (D), adiponectin (A), retinol-binding protein 4 (R), C-reactive protein (C), and leptin (L). Principal component analysis (PCA) revealed distinct variance patterns, i.e. principal components (PC), for baseline pregnancy, bp.pc1.D↓A↓R↓ and bp.pc2.C↓L↑; combined follow-up during pregnancy and postpartum, dp-pp.pc1.D↑↓A↑R↑↓L↓ and dp-pp.pc2.A↑C↑L↑; and children, ch.pc1.D↑R↑C↑ and ch.pc2.D↓A↑L↑. Maternal multiple micronutrient (MMN) supplementation led to an association of baseline maternal bp.pc2.C↓L↑ with decreased post-supplementation maternal dp-pp.pc2.A↑C↑L↑ (p = 0.022), which was in turn associated with both increased child ch.pc1.D↑R↑C↑ (p = 0.036) and decreased child BMI z-score (BMIZ) (p = 0.022). Further analyses revealed an association between maternal dp-pp.pc1.D↑↓A↑R↑↓L↓ and increased child BMIZ (p = 0.036). Child ch.pc1.D↑R↑C↑ was associated with decreased birth weight (p = 0.036) and increased child BMIZ (p = 0.002). Child ch.pc2.D↓A↑L↑ was associated with increased child BMIZ (p = 0.005), decreased maternal height (p = 0.030) and girls (p = 0.002). A pattern of elevated maternal adiponectin and leptin in pregnancy was associated with increased C-reactive protein, vitamin A, and D binding proteins pattern in children, suggesting biomarkers acting in concert may have qualitative as well as quantitative influence beyond single biomarker effects. Patterns in pregnancy proximal to birth were more associated with child status. In addition, child patterns were more associated with child status, particularly child BMI. MMN supplementation affects maternal biomarker patterns of metabolism and inflammation in pregnancy, and potentially in the child. However, child nutrition conditions after birth may have a greater impact on metabolism and inflammation.

Pathophysiological implications of actin-free Gc-globulin concentration changes in blood plasma and cerebrospinal fluid collected from patients with Alzheimer's disease and other neurological disorders.

BACKGROUND: The extracellular actin scavenging system (EASS) is composed of plasma Gc-globulin and gelsolin, and is responsible for the elimination of toxic actin from the bloodstream. OBJECTIVES: In this study, we assessed the actin-free Gc-globulin concentrations in blood plasma and cerebrospinal fluid (CSF) obtained from subjects with neurodegenerative and inflammatory diseases of the central nervous system (CNS) as well as in a control group. MATERIAL AND METHODS: Using an enzyme-linked immunosorbent assay (ELISA), we measured the actinfree Gc-globulin concentrations in blood plasma and CSF obtained from subjects diagnosed with Alzheimer's disease (AD) (n = 20), amyotrophic lateral sclerosis (ALS) (n = 12), multiple sclerosis (MS) (n = 42), tick-borne encephalitis (TBE) (n = 12), and from a control group (n = 20). RESULTS: The concentrations of free Gc-globulin in plasma collected from patients diagnosed with AD (509.6 ±87.6 mg/L) and ALS (455.5 ±99.8 mg/L) did not differ significantly between each other, but were significantly higher compared to the reference group (311.7 ±87.5 mg/L) (p < 0.001 and p < 0.006, respectively) as well as to MS (310.8 ±66.6 mg/L) (p < 0.001 and p < 0.001, respectively) and TBE (256.7 ±76 mg/L) (p < 0.001 and p < 0.003, respectively). In CSF collected from patients diagnosed with AD and ALS, the concentrations of free Gc-globulin were 2.6 ±1.1 mg/L and 2.7 ±1.9 mg/L, respectively. They did not differ significantly between each other and were significantly higher compared to the reference group (1.5 ±0.9 mg/L) (p < 0.005 and p < 0.041, respectively). Interestingly, in patients with AD, significantly higher values of Gcglobulin were detected compared to multiple sclerosis patients (1.7 ±0.9 mg/L) (p < 0.013). CONCLUSIONS: Higher concentrations of free Gc-globulin in blood plasma and CSF collected from patients suffering from neurodegenerative diseases may indicate a potential role of this protein in their pathogenesis, and represent a potential tool for the diagnosis of CNS diseases.

A monoclonal antibody sandwich ELISA for vitamin D-binding protein (VDBP) is unaffected by Gc-globulin phenotype peptides and actin and demonstrates reduced levels in sepsis and non-sepsis intensive care patients.

The measurement of vitamin D-binding protein (VDBP) by immunoassay has been confounded by variable antibody recognition of the Gc1s, Gc1F and Gc2 phenotypes. This has led to spurious conclusions regarding vitamin D status in different ethnic groups. In order to overcome these problems there is a requirement for VDBP antibodies that are unaffected by phenotype status. Here we report the generation and testing of three monoclonal antibodies to VDBP which recognise linear epitopes and are unaffected by vast molar excesses of synthetic peptides spanning these phenotypic domains. These IgG1 kappa antibodies were purified and biotinylated to allow suitable pairings to develop a sandwich ELISA for circulating VDBP. The VDBP ELISA is unaffected by actin and confirms that VDBP levels are significantly reduced in sepsis patients and non-sepsis intensive care patients compared to normal healthy subjects. Levels of VDBP along with total 25OH vitamin D3 can be used to calculate free 25OH vitamin D3 levels and these compare well with consensus values determined independently. The VDBP ELISA meets acceptable performance criteria and as such can be used in conjunction with total 25OH vitamin D3 to determine the free 25OH vitamin D3 status in various cohorts.

Quantification of Total Vitamin-D-Binding Protein and the Glycosylated Isoforms by Liquid Chromatography-Isotope Dilution Mass Spectrometry.

Vitamin-D-binding protein (VDBP), a transporter of 25-hydroxyvitamin D metabolites, has three common isoforms. The relationship of the isoforms and their glycosylation state with various diseases has been under recent examination. In this work, liquid chromatography coupled to isotope dilution mass spectrometry was evaluated for quantification of VDBP, the three common isoforms, and total glycosylation. Protocols using guanidine, urea, RapiGest, trifluoroethanol, or tris buffer were also evaluated for optimal tryptic digestion. Differences in peptide release were detected between purified and plasma VDBP; however, for both protein sources, ELPEHTVK, TSALSAK, and VLEPTLK concentrations were reproducible between most protocols tested. The isoform-specific peptides, LPDATPK, LPDATPTELAK, and LPEATPTELAK, were optimally released when TFE was added to plasma. The total VDBP concentration calculated from the three shared peptides resulted in 97.6% accuracy compared with the concentration from amino acid analysis. Glycosylation of VDBP was also calculated for purified protein and donor samples using the ratio of the isoform-specific peptide(s) to the total protein concentration. Glycosylation of purified VDBP was found to be 99.5-111.1% the value determined by semiquantitative analysis of the intact protein by LC-MS. This approach may be used to quantify other samples containing a mixture of isoforms and post-translational modifications.

Optimizing High-Resolution Mass Spectrometry for the Identification of Low-Abundance Post-Translational Modifications of Intact Proteins.

Intact protein analysis by liquid chromatography-mass spectrometry (LC-MS) is now possible due to the improved capabilities of mass spectrometers yielding greater resolution, mass accuracy, and extended mass ranges. Concurrent measurement of post-translational modifications (PTMs) during LC-MS of intact proteins is advantageous while monitoring critical proteoform status, such as for clinical samples or during production of reference materials. However, difficulties exist for PTM identification when the protein is large or contains multiple modification sites. In this work, analyses of low-abundance proteoforms of proteins of clinical or therapeutic interest, including C-reactive protein, vitamin D-binding protein, transferrin, and immunoglobulin G (NISTmAb), were performed on an Orbitrap Elite mass spectrometer. This work investigated the effect of various instrument parameters including source temperatures, in-source CID, microscan type and quantity, resolution, and automatic gain control on spectral quality. The signal-to-noise ratio was found to be a suitable spectral attribute which facilitated identification of low abundance PTMs. Source temperature and CID voltage were found to require specific optimization for each protein. This study identifies key instrumental parameters requiring optimization for improved detection of a variety of PTMs by LC-MS and establishes a methodological framework to ensure robust proteoform identifications, the first step in their ultimate quantification.

Current Assays to Determine Free 25-Hydroxyvitamin D in Serum.

The 25-hydroxylated metabolite of vitamin D is the best clinical indicator of vitamin D status. For many years, emphasis has been on measuring total levels of 25-hydroxyvitamin D [25(OH)D], but recently, interest in measuring free 25(OH)D as a potentially better marker of vitamin D status has arisen. Since the 1980s when the first measurements of free 25(OH)D were made, little progress has been made in the development of rapid, reliable methods to determine the levels of free 25(OH)D. For many years, assessment of free 25(OH)D relied on calculations using levels of total 25(OH)D, albumin, and vitamin D binding protein (VDBP), for which many assays exist. However, because of vagaries in the measurement of VDBP in particular and the assumption of a constant affinity of VDBP for the vitamin D metabolites (which has been shown to be problematic), calculated values have proved suspect. This changed a few years ago when a new immunoassay was developed to measure free 25(OH)D directly. This review examines methods for determining free 25(OH)D, the different methods used in clinical studies, and the relationships between free 25(OH)D and other vitamin D metabolites and the physiologic functions affected by vitamin D metabolites, such as bone cell activity and turnover. The review also comments on the value of assessing free 25(OH)D and the efforts to standardize the assays.

The rat vomeronasal organ is a vitamin D target.

We studied the expression of vitamin D receptor and of vitamin D binding protein in the rat vomeronasal organ. With immunofluorescence, in situ hybridization and with reverse transcriptase PCR we found both proteins in sensory as well as in non-sensory cells. Sensory neurons contained immunoreactivity for vitamin D3 receptor in nuclei, in portions of the cytoplasm, and in apical dendrites and their microvilli. Vitamin D binding protein was observed in sensory neuron axons and cytoplasm, mostly confined to dendrites. Colocalization appeared in the contact zone of supporting cells and sensory dendrites. Both proteins were also found in single ciliated cells within the non-sensory epithelium. Vitamin D binding protein was also localized in secretory vesicles in a portion of the vomeronasal glands. Our findings suggest that the rat vomeronasal organ is a vitamin D target.

VITAMIN D BINDING PROTEIN AND 25-HYDROXYVITAMIN D LEVELS: EMERGING CLINICAL APPLICATIONS.

The precursor of the active form of vitamin D, 25-hydroxyvitamin D (25(OH)D), is recognized as the optimal indicator of vitamin D status. Vitamin D3 undergoes conversion through a multitude of enzymatic reactions described within the paper, and vitamin D levels are dependent on many factors including the vitamin D binding protein (DBP). The free hormone hypothesis postulates that protein-bound hormones are not biologically available and that unbound hormones are biologically active. The majority of circulating 25(OH)D and 1,25-dihydroxyvitamin D is tightly bound to DBP and albumin, with less than 1% circulating in an unbound form. As a result, factors affecting DBP alter the interpretation of 25(OH)D levels. The aim of this review is to assess the current methodology used to measure total and free 25(OH)D, and DBP. Additionally, we analyze the effects of other endocrine hormones and disease processes on DBP levels and subsequently, the interpretation of 25(OH)D levels. ABBREVIATIONS: CF = cystic fibrosis DBP = vitamin D binding protein ELISA = enzyme-linked immunosorbent assay ESLD = end-stage liver disease HC = hormone contraceptives iPTH = intact parathyroid hormone LC-MS = liquid chromatography-mass spectrometry MS = multiple sclerosis 25(OH)D = 25-hydroxyvitamin D PHPT = primary hyperparathyroidism RIA = radioimmunoassay.

GC and VDR SNPs and Vitamin D Levels in Parkinson's Disease: The Relevance to Clinical Features.

Vitamin D deficiency is suggested to be associated with Parkinson's disease (PD). Our aim was to investigate the serum 25-hydroxyvitamin D3 (25OHD) levels of PD patients in Turkish cohort, to investigate any association of vitamin D binding protein (GC) genotypes with PD due to the significant role of GC in vitamin D transport, to determine whether vitamin D receptor (VDR) haplotype that we previously demonstrated to be a risk haplotype for AD is also a common haplotype for PD and to investigate any relevant consequence of serum 25OHD levels, GC or VDR genotypes on clinical features of PD. Three hundred eighty-two PD patients and 242 healthy subjects were included in this study. The serum 25OHD levels were investigated by CLIA, and GC and VDR SNPs were evaluated with LightSnip. Our results indicated a strong relationship between low serum 25OHD levels and PD (p < 0.001). rs7041 of GC and ApaI of VDR were associated with the PD risk (p < 0.05). Minor allele carriers for BsmI of VDR gene in both PD patients and healthy subjects had significantly higher levels of serum 25OHD (p < 0.05). The homozygous major allele carriers for rs2282679, rs3755967 and rs2298850 of GC gene in PD patients with slower progression had significantly higher levels of serum 25OHD (p  < 0.05). Minor allele carriers for FokI of VDR gene were more frequent in patients with advanced-stage PD (p < 0.05). Consequently, this is the first study demonstrating GC gene as a risk factor for PD. The relationship between PD's clinical features and low 25OHD or risk genotypes might have effects on PD independently.

Vitamin D binding protein: a multifunctional protein of clinical importance.

Since the discovery of group-specific component and its polymorphism by Hirschfeld in 1959, research has put spotlight on this multifunctional transport protein (vitamin D binding protein, DBP). Besides the transport of vitamin D metabolites, DBP is a plasma glycoprotein with many important functions, including sequestration of actin, modulation of immune and inflammatory responses, binding of fatty acids, and control of bone development. A considerable DBP polymorphism has been described with a specific allele distribution in different geographic area. Multiple studies have shed light on the interesting relationship between polymorphisms of the DBP gene and the susceptibility to diseases. In this review, we give an overview of the multifunctional character of DBP and describe the clinical importance of DBP and its polymorphisms. Finally, we discuss the possibilities to use DBP as a novel therapeutic agent.

Vitamin D binding protein isoforms as candidate predictors of disease extension in childhood arthritis.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic autoimmune diseases with variable clinical outcomes. We investigated whether the synovial fluid (SF) proteome could distinguish a subset of patients in whom disease extends to affect a large number of joints. METHODS: SF samples from 57 patients were obtained around time of initial diagnosis of JIA, labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with expression verified by immunochemical methods. Protein glycosylation status was confirmed by hydrophilic interaction liquid chromatography. RESULTS: A truncated isoform of vitamin D binding protein (VDBP) is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Furthermore, sialylated forms of immunopurified synovial VDBP were significantly reduced in extended oligoarticular patients (p<0.005). CONCLUSION: Reduced conversion of VDBP to a macrophage activation factor may be used to stratify patients to determine risk of disease extension in JIA patients.

Urinary and dialysate losses of vitamin D-binding protein in children on chronic peritoneal dialysis.

BACKGROUND: Vitamin D deficiency is widely prevalent in chronic kidney disease [CKD] patients. The aim of our study was to determine whether losses of vitamin D binding protein [VDBP] in urine and dialysate contribute to circulating 25-hydroxyvitamin D [25OHD] levels in chronic peritoneal dialysis [PD] patients. METHODS: Dialysate, serum, and urine VDBP levels were measured in 16 children on PD and compared with serum and urine VDBP in ten CKD4-5 patients. Serum VDBP levels were correlated with total circulating 25OHD and peritoneal VDBP losses. RESULTS: The mean age of the study population was 9.4 ± 3.8 years and the median time on dialysis 7.5 (1-18) months. In CKD4-5 patients, urinary VDBP losses were >300-fold higher than seen in age-matched healthy children and correlated with urinary albumin loss (p = 0.0008). There was a significant correlation between serum VDBP and total dialysate and urine losses of VDBP (p = 0.03, r = -0.53). Dialysate VDBP losses correlate with dialysate albumin loss (p = 0.01). VDBP losses in the long daytime dwell were higher than in the overnight drain (p = 0.04). Serum VDBP levels were lower in children with a longer dialysis vintage (p = 0.0004, r = -0.77). In PD patients, the mean total loss of VDBP in dialysate and urine was 1.91 ± 1.6 µmol/day, equivalent to ~7% of the total circulating level of VDBP in healthy controls. There was no correlation between 25(OH)D and VDBP. CONCLUSIONS: Peritoneal VDBP losses mirror both dialysate and urinary albumin losses, and are associated with a longer dialysis vintage but do not contribute to vitamin D deficiency in children on PD.

Polymorphism in vitamin D-binding protein as a genetic risk factor in the pathogenesis of endometriosis.

CONTEXT: Previous studies have implicated a deficiency in the inflammatory response in women who develop endometriosis. The specific immunological deficits have not been completely elucidated. OBJECTIVE: Our objective was to identify differences in protein expression in serum that might shed light on the pathophysiology of endometriosis. DESIGN AND SETTING: This cross-sectional study of women undergoing laparoscopy between 2003 and 2005 took place at a university medical center. PATIENTS: Patients included consenting women age 18-49 yr undergoing surgery for pain and/or infertility or elective tubal ligation. Women with acute or chronic medical conditions were excluded. INTERVENTION: Blood was collected preoperatively. MAIN OUTCOME MEASURE: Proteomic analysis of serum was done using two-dimensional difference gel electrophoresis. RESULTS: We found 25 protein spots with a significant difference in abundance between women with endometriosis and controls, including acute-phase proteins and complement components. The abundance of vitamin D-binding protein was higher in all endometriosis pools by a factor of approximately 3 compared with the control pool (P < 0.02). Analysis of specific allele products using nano-scale liquid chromatography-electrospray ionization-mass spectrometry indicated that it was the GC*2 allele product that was in greater concentration in serum pools, as well as in single validation samples, in women with endometriosis (P = 0.006). In contrast to the GC*1 allele product, which is readily converted to a potent macrophage factor (Gc protein-derived macrophage-activating factor), the GC*2 allele product undergoes practically no such conversion. CONCLUSIONS: We speculate that the inability to sufficiently activate macrophages' phagocytotic function in those carrying the GC*2 polymorphism (more prevalent in endometriosis) may allow endometriotic tissues to implant in the peritoneal cavity. Future studies evaluating specific vitamin D-binding protein polymorphisms as a risk factor for endometriosis in larger populations of women are warranted.

Hypogelsolinemia, a disorder of the extracellular actin scavenger system, in patients with multiple sclerosis.

BACKGROUND: Extracellular gelsolin (GSN) and GC-globulin/Vitamin D-binding protein (DBP) appear to play an important role in clearing the actin from extracellular fluids and in modulating cellular responses to anionic bioactive lipids. In this study we hypothesized that cellular actin release and/or increase in bioactive lipids associated with multiple sclerosis (MS) development will translate into alteration of the actin scavenger system protein concentrations in blood and cerebrospinal fluid (CSF) of patients with MS. METHODS: We measured GSN and DBP concentrations in blood and CSF obtained from patients diagnosed with MS (n = 56) in comparison to a control group (n = 20) that includes patients diagnosed with conditions such as idiopathic cephalgia (n = 11), idiopathic (Bell's) facial nerve palsy (n = 7) and ischialgia due to discopathy (n = 2). GSN and DBP levels were measured by Western blot and ELISA, respectively. RESULTS: We found that the GSN concentration in the blood of the MS group (115 ± 78 µg/ml) was significantly lower (p < 0.001) compared to the control group (244 ± 96 µg/ml). In contrast, there was no statistically significant difference between blood DBP concentrations in patients with MS (310 ± 68 µg/ml) and the control group (314 ± 82 µg/ml). GSN and DBP concentrations in CSF also did not significantly differ between those two groups. CONCLUSIONS: The decrease of GSN concentration in blood and CSF of MS subjects suggests that this protein may be involved in chronic inflammation associated with neurodegeneration. Additionally, the results presented here suggest the possible utility of GSN evaluation for diagnostic purposes. Reversing plasma GSN deficiency might represent a new strategy in MS treatment.

Analysis of the Albumin/alpha-Fetoprotein/Afamin/Group specific component gene family in the context of zebrafish liver differentiation.

Members of the Albumin/alpha-Fetoprotein/Afamin/Group specific component (Alb/Afp/Afm/Gc) multi-gene family perform physiological functions essential for body homeostasis and are among the earliest genes to be expressed in the fetal liver in mammals. A comprehensive search of the zebrafish genome has led to the isolation of a single member of this gene family, exhibiting close homology to group specific component (gc; also described as vitamin D binding protein (dbp)). Our phylogenetic analyses did not uncover albumin in the genome, indicating its likely absence in zebrafish, whereas the absence of afp and afm is in agreement with previous findings that both genes arose at a later stage of vertebrate evolution. gc mRNA expression is initiated weakly around 55 hours post fertilisation (hpf) in the developing liver, and increases until it reaches a continuously high level from about 72 hpf onwards. Investigation of gc mRNA in hdac1 mutants revealed a severe delay of expression, indicating a defect in progression of hepatic differentiation. This provides further evidence for Hdac1 regulating the precise timely execution of hepatic gene expression programmes. Conversely, onset of gc expression was unaltered in cloche mutant embryos, which lack hepatic vasculature, suggesting that this particular step of hepatic differentiation occurs independently from endothelial cells. Our studies identify gc as the likely only member of the Alb/Afp/Afm/Gc gene family in zebrafish, providing important insights into the evolution of this multi-gene family in vertebrates. Furthermore, the identification of gc adds a valuable temporal marker for investigating progressive hepatic differentiation in zebrafish.

Differential proteomic characterization between normal peritoneal fluid and diabetic peritoneal dialysate.

BACKGROUND: Since the mechanism of comorbidity and mortality in peritoneal dialysis is unclear, a comparison of peritoneal dialysate and normal peritoneal fluid may provide clues to the biological and pathological processes involved in peritoneal damage. METHODS: Peritoneal dialysate and control samples were collected from five diabetes mellitus (DM) patients and two patients receiving laparoscopic cholecystectomy. Proteins were separated by two-dimensional gel electrophoresis (2D-GE). After image analysis, altered gel spots between these two sample groups were subjected to tryptic digestion and mass spectrometry analysis. The results were searched against the NCBI database. RESULTS: A total of 26 protein spots were considered altered in 2D-GE between the two sample groups. After western blotting confirmation, vitamin D-binding protein, haptoglobin and alpha-2-microglobulin were at higher levels in the DM samples, while complement C4-A and IGK@ protein were at lower levels compared to the control samples. CONCLUSION: The loss of vitamin D-binding protein, haptoglobin and alpha-2-microglobulin may be due to a change in the permeability of the peritoneal membrane to middle-sized proteins or leakage from peritoneal inflammation. Lower levels of complement C4-A in dialysate may shed light on the beginning of peritoneal membrane scleroses.

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.

Initial comparison of proteomic profiles of whole unstimulated saliva obtained from generalized aggressive periodontitis patients and healthy control subjects.

BACKGROUND AND OBJECTIVE: Salivary proteomics technology can be used to evaluate the disease progression of periodontitis and the systemic screening of proteomes of saliva from subjects with aggressive periodontitis has not been available. The objective of this preliminary study was to compare the proteomic profile of whole unstimulated saliva of subjects with generalized aggressive periodontitis (GAgP) with that of healthy volunteers to identify proteins, the levels of which were significantly altered between the two groups. MATERIAL AND METHODS: Whole unstimulated saliva was obtained from five subjects with GAgP and five healthy subjects, and proteins were separated using two-dimensional gel electrophoresis. Proteins, the levels of which were significantly different between the two groups, were identified by computer image analyses and subsequent electrospray ionization tandem mass spectrometry. RESULTS: Eleven proteins that exhibited a different level in the GAgP group vs. the control group were identified. Compared with whole saliva of healthy control subjects, the levels of serum albumin, immunoglobulin (Ig) gamma2 chain C region, Ig alpha2 chain C region, vitamin D-binding protein, salivary alpha-amylase and zinc-alpha2 glycoprotein were increased in whole unstimulated saliva of GAgP subjects, while those of lactotransferrin, elongation factor 2, 14-3-3 sigma, short palate, lung and nasal epithelium carcinoma-associated protein 2 precursor and carbonic anhydrase 6 were decreased. CONCLUSION: Comparison of the proteomic profile of whole unstimulated saliva of GAgP subjects with that of healthy control subjects revealed at least 11 differential proteins. The approach applied herein might be helpful to aid understanding of the etiology of GAgP.

The relationship between concentrations of vitamin D-binding protein (DBP) in serum and colostrum of mares and in serum of their foals in the neonatal period.

Vitamin D-binding protein (DBP) participates in the actin scavenger system, it is a carrier of vitamin D and its derivatives, it manifests the capacity to bind mainly monounsaturated and saturated fatty acids, it binds to the surface of several cells and enhances chemotactic activity of C5a of the complement. The present study was aimed at answering the question whether serum DBP level in mares is related to levels of this protein in colostrum and in serum of its progeny. For this purpose, sera from 77 mares, colostra from 72 mares and sera from 69 Thoroughbred foals were collected. Mother's age, number of deliveries experienced in the past, month of delivery, feeding of foals with colostra were recorded. Blood of the foals was sampled from the umbilical vein during delivery (0h) and 36-48 h after delivery from the external jugular vein, colostra of the mares were obtained after delivery and blood of the mares was sampled 36-48 h after delivery. Concentration of DBP was estimated by a self-designed ELISA. In the present study, DBP concentrations in newborn's serum were found independent of their concentrations in mother's serum, her age and number of parities experienced in the past. Colostrum DBP level was found to be lower than that in the mare's serum and was not correlated to the concentration of this protein in mare's serum. There was no effect of colostrum feeding on DBP level in the foal serum. These results indicate that serum DBP concentration in newborn foals depends on factors which act directly on the foal. Because of the lack of correlation between plasma and colostrum concentrations of DBP, it can be assumed that DBP is synthesised in the mammary gland and/or specific transport mechanisms exist in the mammary gland.