Preterm delivery and selected biomarkers : phosphorylated insulin-like growth factor-binding protein-1 and matrix metalloproteinase-8 – in cervical fluid

Premature delivery is a major cause of neonatal morbidity and mortality. The incidence of premature deliveries has increased around the world. In Finland 5.3%, or about 3,000 children per year are born prematurely, before 37 weeks of gestation. The corresponding figure in the United States is about 13%. The morbidity and mortality are highest among infants delivered before 32 weeks of gestation - about 600 children each year in Finland. Approximately 70% of premature deliveries are unexplained. Preterm delivery can be caused by an asympto-matic infection between uterus and the fetal membranes, such can begin already in early pregnancy. It is difficult to predict preterm delivery, and many patients are therefore unnecessarily admitted to hospital for observation and exposed to medical treatments. On the other hand, the high risk women should be identified early for the best treatment of the mother and preterm infant. --- In the prospective study conducted at the Department of Obstetric and Gynecology, Helsinki University Central Hospital two biochemical inflammation related markers were measured in the lower genital tract fluids of asymp-tomatic women in early and mid pregnancy in an order to see whether these markers could identify women with an increased risk of preterm delivery. These biomarkers were phosphorylated insulin-like growth factor binding protein-1 (phIGFBP-1) and matrix metalloproteinase-8 (MMP-8). The study involved 5180 asymptomatic pregnant women, examined during the first and second ultrasound screening visits. The study samples were taken from the vagina and cervicix. In addition, 246 symptomatic women were studied (pregnancy weeks 22 – 34). The study showed that increased phIGFBP-1 concentration in cervical canal fluid in early pregnancy increased the risk for preterm delivery. The risk for very premature birth (before 32 weeks of gestation) was nearly four-fold. Low MMP-8 concentration in mid pregnancy increased the risk of subsequent premature preterm rupture of fetal membranes (PPROM). Significantly high MMP-8 concentrations in the cervical fluid increased the risk for prema-ture delivery initiated by preterm labour with intact membranes. Among women with preterm contractions the shortened cervical length measured by ultrasound and elevated cervical fluid phIGFBP-1 both predicted premature delivery. In summary, because of the relatively low sensitivity of cervical fluid phIGFBP-1 this biomarker is not suitable for routine screening, but provides an additional tool in assessing the risk of preterm delivery. Cervical fluid MMP-8 is not useful in early or mid pregnancy in predicting premature delivery because of its dual role. Further studies on the role of MMP-8 are therefore needed. Our study confirms that phIGFBP-1 testing is useful in predicting pre-term delivery.

Proteomics of Trypanosoma evansi Infection in Rodents

Background: Trypanosoma evansi infections, commonly called 'surra', cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS).Methodology/Principal Findings: Using shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Conclusions/Significance: Previous proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.

Serum proteomics of hepatitis C virus infection reveals retinol-binding protein 4 as a novel regulator

Persistent infection of hepatitis C virus (HCV) can lead to liver cirrhosis and hepatocellular carcinoma, which are currently diagnosed by invasive liver biopsy. Approximately 15-20% of cases of chronic liver diseases in India are caused by HCV infection. In North India, genotype 3 is predominant, whereas genotype 1 is predominant in southern parts of India. The aim of this study was to identify differentially regulated serum proteins in HCV-infected Indian patients (genotypes 1 and 3) using a two-dimensional electrophoresis approach. We identified eight differentially expressed proteins by MS. Expression levels of one of the highly upregulated proteins, retinol-binding protein 4 (RBP4), was validated by ELISA and Western blotting in two independent cohorts. We also confirmed our observation in the JFH1 infectious cell culture system. Interestingly, the HCV core protein enhanced RBP4 levels and partial knockdown of RBP4 had a positive impact on HCV replication, suggesting a possible role for this cellular protein in regulating HCV infection. Analysis of RBP4-interacting partners using a bioinformatic approach revealed novel insights into the possible involvement of RBP4 in HCV-induced pathogenesis. Taken together, this study provided information on the proteome profile of the HCV-infected Indian population, and revealed a link between HCV infection, RBP4 and insulin resistance.

High resolution mass spectrometric alveolar proteomics: Identification of surfactant protein SP-A and SP-D modifications in proteinosis and cystic fibrosis patients

In the present study, one- and two-dimensional gel electrophoresis combined with high resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) have been applied as powerful approaches for the proteome analysis of surfactant proteins SP-A and SP-D, including identification of structurally modified and truncation forms, in bronchoalveolar lavage fluid from patients with cystic fibrosis, chronic bronchitis and pulmonary alveolar proteinosis. Highly sensitive micro preparation techniques were developed for matrix-assisted laser desorption/ionization (MALDI) FT-ICR MS analysis which provided the identification of surfactant proteins at very low levels. Owing to the high resolution, FT-ICR MS was found to provide substantial advantages for the structural identification of surfactant proteins from complex biological matrices with high mass determination accuracy. Several protein bands corresponding to SP-A and SP-D were identified by MALDI-FT-ICR MS after electrophoretic separation by one- and two-dimensional gel electrophoresis, and provided the identification of structural modifications (hydroxy-proline) and degradation products.

Fucoidan inhibits the development of proteinuria in active Heymann nephritis

Fucoidan, the sulphated polysaccharide extracted from brown seaweed, has various biological activities. The effect of fucoidan on the formation of proteinuria and renal functions in active Heymann nephritis was investigated in this study. Active Heymann nephritis was induced by administering brush border protein of rat proximal uriniferous tubules (FX1A). Fucoidan was administered by oral intubation to Heymann nephritis rats at three doses (50, 100 and 200 mg/kg) once daily for 4 weeks. The elevated urinary protein excretion and plasma creatinine due to the induction of Heymann nephritis were significantly reduced by fucoidan at doses of 100 and 200 mg/kg. The results indicated that fucoidan has a renoprotective effect on active Heymann nephritis and is a promising therapeutic agent for nephritis. Copyright (c) 2005 John Wiley F Sons, Ltd.

Elucidating the Molecular Composition of Cartilage by Proteomics.

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers.

Protein expression profiling during chick retinal maturation: a proteomics-based approach

Background: The underlying pathways that drive retinal neurogenesis and synaptogenesis are still relatively poorly understood. Protein expression analysis can provide direct insight into these complex developmental processes. The aim of this study was therefore to employ proteomic analysis to study the developing chick retina throughout embryonic (E) development commencing at day 12 through 13, 17, 19 and post-hatch (P) 1 and 33 days.

Results: 2D proteomic and mass spectrometric analysis detected an average of 1514 spots per gel with 15 spots demonstrating either modulation or constitutive expression identified via MS. Proteins identified included alpha and beta-tubulin, alpha enolase, B-creatine kinase, gamma-actin, platelet-activating factor (PAF), PREDICTED: similar to TGF-beta interacting protein 1, capping protein (actin filament muscle Z line), nucleophosmin 1 (NPM1), dimethylarginine dimethylaminohydrolase, triosphoaphate isomerase, DJ1, stathmin, fatty acid binding protein 7 (FABP7/B-FABP), beta-synuclein and enhancer of rudimentary homologue.

Conclusion: This study builds upon previous proteomic investigations of retinal development and represents the addition of a unique data set to those previously reported. Based on reported bioactivity some of the identified proteins are most likely to be important to normal retinal development in the chick. Continued analysis of the dynamic protein populations present at the early stages and throughout retinal development will increase our understanding of the molecular events underpinning retinogenesis.

A pilot survey for Fusarium mycotoxin biomarkers in women from Golestan, northern Iran

Fusarium mycotoxins are frequent contaminants of cereals in many world regions, and are suggested risk factors for various acute and chronic human diseases. To date a lack of exposure tools has restricted epidemiological studies of the potential health effects. Recently established exposure biomarkers for deoxynivalenol (DON) and fumonisins are now available and here a pilot biomarker survey of 110 women (aged 39 to 72 years) from Golestan, northern Iran was conducted on samples collected at one time point during August-September 2007. Urinary DON and DON-glucuronide combined were detected frequently (79/110, 72%), mean 1.3 ng DON/ml urine, range not detected (nd)-6.5 ng/ml; mean creatinine adjusted levels were 1.5 ng DON/mg creatinine, range nd-7.1 ng/mg). Neither urinary de-epoxy DON (DOM-1) and DOM-1 glucuronide combined, nor urinary fumonisin B-1 were detected. This study is the first reported biomarker based exposure assessment of DON and fumonisins in this region. Overall DON exposure at this time point appears modest compared to other world regions where data are available.

Determining mycotoxins and mycotoxigenic fungi in food and feed:Developing biomarkers of human exposure to mycotoxins

Exposure assessment is a critical part of epidemiological studies into the effect of mycotoxins on human health. Whilst exposure assessment can be made by estimating the quantity of ingested toxins from food analysis and questionnaire data, the use of biological markers (biomarkers) of exposure can provide a more accurate measure of individual level of exposure in reflecting the internal dose. Biomarkers of exposure can include the excreted toxin or its metabolites, as well as the products of interaction between the toxin and macromolecules such as protein and DNA. Samples in which biomarkers may be analysed include urine, blood, other body fluids and tissues, with urine and blood being the most accessible for human studies. Here we describe the development of biomarkers of exposure for the assessment of three important mycotoxins; aflatoxin, fumonisin and deoxynivalenol. A number of different biomarkers and methods have been developed that can be applied to human population studies, and these approaches are reviewed in the context of their application to molecular epidemiology research.

Biomarkers in diabetes:hemoglobin A1c, vascular and tissue markers

Biomarkers are conventionally defined as "biological molecules that represent health and disease states." They typically are measured in readily available body fluids (blood or urine), lie outside the causal pathway, are able to detect subclinical disease, and are used to monitor clinical and subclinical disease burden and response to treatments. Biomarkers can be "direct" endpoints of the disease itself, or "indirect" or surrogate endpoints. New technologies (such as metabolomics, proteomics, genomics) bring a wealth of opportunity to develop new biomarkers. Other new technologies enable the development of nonmolecular, functional, or biophysical tissue-based biomarkers. Diabetes mellitus is a complex disease affecting almost every tissue and organ system, with metabolic ramifications extending far beyond impaired glucose metabolism. Biomarkers may reflect the presence and severity of hyperglycemia (ie, diabetes itself) or the presence and severity of the vascular complications of diabetes. Illustrative examples are considered in this brief review. In blood, hemoglobin A1c (HbA1c) may be considered as a biomarker for the presence and severity of hyperglycemia, implying diabetes or prediabetes, or, over time, as a "biomarker for a risk factor," ie, hyperglycemia as a risk factor for diabetic retinopathy, nephropathy, and other vascular complications of diabetes. In tissues, glycation and oxidative stress resulting from hyperglycemia and dyslipidemia lead to widespread modification of biomolecules by advanced glycation end products (AGEs). Some of these altered species may serve as biomarkers, whereas others may lie in the causal pathway for vascular damage. New noninvasive technologies can detect tissue damage mediated by AGE formation: these include indirect measures such as pulse wave analysis (a marker of vascular dysfunction) and more direct markers such as skin autofluorescence (a marker of long-term accumulation of AGEs). In the future, we can be optimistic that new blood and tissue-based biomarkers will enable the detection, prevention, and treatment of diabetes and its complications long before overt disease develops.

3-Deoxyfructose concentrations are increased in human plasma and urine in diabetes

3-Deoxyglucosone (3-DG) is a reactive dicarbonyl sugar thought to be a key intermediate in the nonenzymatic polymerization and browning of proteins by glucose. 3-DG may be formed in vivo from fructose, fructose 3-phosphate, or Amadori adducts to protein, such as N epsilon-fructoselysine (FL), all of which are known to be elevated in body fluids or tissues in diabetes. Modification of proteins by 3-DG formed in vivo is thought to be limited by enzymatic reduction of 3-DG to less reactive species, such as 3-deoxyfructose (3-DF). In this study, we have measured 3-DF, as a metabolic fingerprint of 3-DG, in plasma and urine from a group of diabetic patients and control subjects. Plasma and urinary 3-DF concentrations were significantly increased in the diabetic compared with the control population (0.853 +/- 0.189 vs. 0.494 +/- 0.072 microM, P <0.001, and 69.9 +/- 44.2 vs. 38.7 +/- 16.1 nmol/mg creatinine, P <0.001, respectively). Plasma and urinary 3-DF concentrations correlated strongly with one another, with HbA1c (P <0.005 in all cases), and with urinary FL (P <0.02 and P = 0.005, respectively). The overall increase in 3-DF concentrations in plasma and urine in diabetes and their correlation with other indexes of glycemic control suggest that increased amounts of 3-DG are formed in the body during hyperglycemia in diabetes and then metabolized to 3-DF. These observations are consistent with a role for increased formation of the dicarbonyl sugar 3-DG in the accelerated browning of tissue proteins in diabetes.

Effect of diabetes and aging on carboxymethyllysine levels in human urine

Carboxymethyllysine (CML) has been identified as a modified amino acid that accumulates with age in human lens proteins and collagen. CML may be formed by oxidation of fructoselysine (FL), the Amadori adduct formed on nonenzymatic glycosylation of lysine residues in protein, or by reaction of ascorbate with protein under autoxidizing conditions. We proposed that measurements of tissue and urinary CML may be useful as indices of oxidative stress or damage to proteins in vivo. To determine the extent to which oxidation of nonenzymatically glycosylated proteins contributes to urinary CML, we measured the urinary concentrations of FL and CML in diabetic (n = 26) and control (n = 28) patients. The urinary concentration of FL correlated strongly with HbA1 measurements and was significantly higher in diabetic compared with control samples (9.2 +/- 6.5 and 4.0 +/- 2.8 micrograms/mg creatinine, respectively; P less than 0.0001). There was also a strong correlation between the concentrations of CML and FL in both diabetic and control urine (r = 0.67, P less than 0.0001) but only a weakly significant increase in the CML concentration in diabetic compared with control urine (1.2 +/- 0.5 and 1.0 +/- 0.3 micrograms/mg creatinine, respectively; P = 0.05). The molar ratio of CML to FL was significantly lower in diabetic compared with control patients (0.25 +/- 0.12 and 0.43 +/- 0.16, respectively; P less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Proteomics on porcine haptoglobin and IgG/IgA show protein species distribution and glycosylation pattern to remain similar in PCV2-SD infection

Haptoglobin (Hp) and immunoglobulins are plasma glycoproteins involved in the immune reaction of the organism after infection and/or inflammation. Porcine circovirus type 2-systemic disease (PCV2-SD), formerly known as postweaning multisystemic wasting syndrome (PMWS), is a globally spread pig disease of great economic impact. PCV2-SD affects the immunological system of pigs causing immunosuppression. The aim of this work was to characterize the Hp protein species of healthy and PCV2-SD affected pigs, as well as the protein backbone and the glycan chain composition of porcine Hp. PCV2-SD affected pigs had an increased overall Hp level, but it did not affect the ratio between Hp species. Glycoproteomic analysis of the Hp β subunits confirmed that porcine Hp is N-glycosylated and, unexpectedly, O-glycosylated, a PTM that is not found on Hp from healthy humans. The glyco-profile of porcine IgG and IgA heavy chains was also characterized; decreased levels of both proteins were found in the investigated group of PCV2-SD affected pigs. Obtained results indicate that no significant changes in the N- and O-glycosylation patterns of these major porcine plasma glycoproteins were detectable between healthy and PCV2-SD affected animals.