A proteome signature for intrauterine growth restriction derived from multifactorial analysis of mass spectrometry-based cord blood serum profiling

Intrauterine growth restriction (IUGR) is defined as a condition in which the fetus does not reach its genetically given growth potential, resulting in low birth weight. IUGR is an important cause of perinatal morbidity and mortality, thus contributing substantially to medically indicated preterm birth in order to prevent fetal death. We subjected umbilical cord blood serum samples either belonging to the IUGR group (n = 15) or to the control group (n = 15) to fractionation by affinity chromatography using a bead system with hydrophobic interaction capabilities. So prepared protein mixtures were analyzed by MALDI-TOF mass spectrometric profiling. The six best differentiating ion signals at m/z 8205, m/z 8766, m/z 13 945, m/z 15 129, m/z 15 308, and m/z 16 001 were collectively assigned as IUGR proteome signature. Separation confidence of our IUGR proteome signature reached a sensitivity of 0.87 and a specificity of 0.93. Assignment of ion signals in the mass spectra to specific proteins was substantiated by SDS-PAGE in conjunction with peptide mass fingerprint analysis of cord blood serum proteins. One constituent of this proteome signature, apolipoprotein C-III(0) , a derivative lacking glycosylation, has been found more abundant in the IUGR cord blood serum samples, irrespective of gestational age. Hence, we suggest apolipoprotein C-III(0) as potential key-marker of the here proposed IUGR proteome signature, as it is a very low-density lipoprotein (VLDL) and high-density lipoprotein (HDL) member and as such involved in triglyceride metabolism that itself is discussed as being of importance in IUGR pathogenesis. Our results indicate that subtle alterations in protein glycosylation need to be considered for improving our understanding of the pathomechanisms in IUGR.

Comparative stability studies of poly(2-methyl-2-oxazoline) and poly(ethylene glycol) brush coatings

Non-fouling surfaces that resist non-specific adsorption of proteins, bacteria, and higher organisms are of particular interest in diverse applications ranging from marine coatings to diagnostic devices and biomedical implants. Poly(ethylene glycol) (PEG) is the most frequently used polymer to impart surfaces with such non-fouling properties. Nevertheless, limitations in PEG stability have stimulated research on alternative polymers that are potentially more stable than PEG. Among them, we previously investigated poly(2-methyl-2-oxazoline) (PMOXA), a peptidomimetic polymer, and found that PMOXA shows excellent anti-fouling properties. Here, we compare the stability of films self-assembled from graft copolymers exposing a dense brush layer of PEG and PMOXA side chains, respectively, in physiological and oxidative media. Before media exposure both film types prevented the adsorption of full serum proteins to below the detection limit of optical waveguide in situ measurements. Before and after media exposure for up to 2 weeks, the total film thickness, chemical composition, and total adsorbed mass of the films were quantified using variable angle spectroscopic ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), and optical waveguide lightmode spectroscopy (OWLS), respectively. We found (i) that PMOXA graft copolymer films were significantly more stable than PEG graft copolymer films and kept their protein-repellent properties under all investigated conditions and (ii) that film degradation was due to side chain degradation rather than due to copolymer desorption.

Dysferlin is a new marker for leaky brain blood vessels in multiple sclerosis

Dysferlin is a muscle protein involved in cell membrane repair and its deficiency is associated with muscular dystrophy. We describe that dysferlin is also expressed in leaky endothelial cells. In the normal central nervous system (CNS), dysferlin is only present in endothelial cells of circumventricular organs. In the inflamed CNS of patients with multiple sclerosis (MS) or in animals with experimental autoimmune encephalomyelitis, dysferlin reactivity is induced in endothelial cells and the expression is associated with vascular leakage of serum proteins. In MS, dysferlin expression in endothelial cells is not restricted to vessels with inflammatory cuffs but is also present in noninflamed vessels. In addition, many blood vessels with perivascular inflammatory infiltrates lack dysferlin expression in inactive lesions or in the normal-appearing white matter. In vitro, dysferlin can be induced in endothelial cells by stimulation with tumor necrosis factor-alpha. Hence, dysferlin is not only a marker for leaky brain vessels, but also reveals dissociation of perivascular inflammatory infiltrates and blood-brain barrier disturbance in multiple sclerosis.

Pathogenesis of bacterial meningitis: contributions by experimental models in rabbits

Rabbits models of bacterial meningitis have contributed substantially to our understanding of the disease, although the technical characteristics of these models only allow the study of specific aspects of the disease. Bacterial multiplication in the subarachnoidal space is not substantially influenced by host defense mechanisms, mainly because of the lack of sufficient amounts of specific antibodies and functional complement in infected CSF. The multiplying bacteria induce profound changes in the blood-brain barrier, an influx of serum proteins into the CSF and the invasion of polymorphonuclear leukocytes at the site of the infection. The presence of polymorphonuclear leukocytes in CSF not only appears to be of limited value in combating the infection, but also seems to produce deleterious effects on the central nervous system. Components of the leukocytes, such as unsaturated fatty acids, arachidonic metabolites and free oxygen radicals, may contribute to the profound hydrodynamic, structural and metabolic changes that are currently under study in experimental models of the disease. A better understanding of the pathophysiology of bacterial meningitis may allow us to design more effective therapeutic strategies and improve the outcome of this disease.

In vitro activity of photoactivated disinfection using a diode laser in infected root canals

OBJECTIVE To investigate the lethal activity of photoactivated disinfection (PAD) on Enterococcus faecalis (ATCC 29212) and mixed populations of aerobic or anaerobic bacteria in infected root canals using a diode laser after the application of a photosensitizer (PS). MATERIALS AND METHODS First, the bactericidal activity of a low power diode laser (200 mW) against E. faecalis ATCC 29212 pre-treated with a PS (toluidine blue) for 2 min were examined after different irradiation times (30 s, 60 s and 90 s). The bactericidal activity in the presence of human serum or human serum albumin (HSA) was also examined. Second, root canals were infected with E. faecalis or with mixed aerobic or anaerobic microbial populations for 3 days and then irrigated with 1.5% sodium hypochlorite and exposed to PAD for 60 s. RESULTS Photosensitization followed by laser irradiation for 60 s was sufficient to kill E. faecalis. Bacteria suspended in human serum (25% v/v) were totally eradicated after 30 s of irradiation. The addition of HSA (25 mg/ml or 50 mg/ml) to bacterial suspensions increased the antimicrobial efficacy of PAD after an irradiation time of 30 s, but no longer. The bactericidal effect of sodium hypochlorite was only enhanced by PAD during the early stages of treatment. PAD did not enhance the activity of sodium hypochlorite against a mixture of anaerobic bacteria. CONCLUSIONS The bactericidal activity of PAD appears to be enhanced by serum proteins in vitro, but is limited to bacteria present within the root canal.

Irisin – a myth rather than an exercise-inducible myokine

The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20 kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.

Angiogenic factors and acute-phase proteins in serum samples of preeclampsia and HELLP patients: a matched-pair analysis

To investigate the serum level distribution of angiogenic markers (PlGF, endoglin, sFlt-1) and acute-phase proteins (SAA, CRP) in patients with HELLP syndrome or preeclampsia (PE) including matched controls.

Physiologic cold shock of Moraxella catarrhalis affects the expression of genes involved in the iron acquisition, serum resistance and immune evasion

Background Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. It was previously shown that the prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis are greatest in winter. The aim of this study was to investigate how M. catarrhalis uses the physiologic exposure to cold air to upregulate pivotal survival systems in the pharynx that may contribute to M. catarrhalis virulence. Results A 26°C cold shock induces the expression of genes involved in transferrin and lactoferrin acquisition, and enhances binding of these proteins on the surface of M. catarrhalis. Exposure of M. catarrhalis to 26°C upregulates the expression of UspA2, a major outer membrane protein involved in serum resistance, leading to improved binding of vitronectin which neutralizes the lethal effect of human complement. In contrast, cold shock decreases the expression of Hemagglutinin, a major adhesin, which mediates B cell response, and reduces immunoglobulin D-binding on the surface of M. catarrhalis. Conclusion Cold shock of M. catarrhalis induces the expression of genes involved in iron acquisition, serum resistance and immune evasion. Thus, cold shock at a physiologically relevant temperature of 26°C induces in M. catarrhalis a complex of adaptive mechanisms that enables the bacterium to target their host cellular receptors or soluble effectors and may contribute to enhanced growth, colonization and virulence.

Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.

iTRAQ identification of candidate serum biomarkers associated with metastatic progression of human prostate cancer

A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer 'BPH', (ii) localised cancer with no evidence of progression, 'non-progressing' (iii) localised cancer with evidence of biochemical progression, 'progressing', and (iv) bone metastasis at presentation 'metastatic'. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p<0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and 'panels' of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation.

Clonal analysis of Inquilinus limosus isolates from six cystic fibrosis patients and specific serum antibody response

Inquilinus limosus is a novel Gram-negative bacterium of the subdivision alpha-Proteobacteria recently found in the airways of patients with cystic fibrosis (CF). Here, the authors report on the clinical courses of six CF patients colonized with I. limosus. Five patients suffered from either an acute respiratory exacerbation or a progressive loss of pulmonary function, whereas one patient was in a stable clinical situation. This study focused on two aims: (i) the clonal analysis of I. limosus isolates by random amplified polymorphic DNA (RAPD)-PCR, and (ii) the clarification of whether the presence of I. limosus in the respiratory tract is associated with a specific serum antibody response. Serum IgG was detected by immunoblotting using I. limosus whole-cell-lysate proteins as antigens. Sera from healthy blood donors (n=10) and from CF patients colonized with Pseudomonas aeruginosa (n=10) were found to be immunoblot negative. All six Inquilinus-positive patients raised serum IgG antibodies against various I. limosus antigens. Surprisingly, in one patient, a specific I. limosus serum antibody response was already detected 1 year prior to Inquilinus-positive sputum cultures. Two prominent antigens were characterized by MALDI-MS: a 23 kDa protein revealed homology to the outer membrane lipoprotein OmlA of Actinobacillus pleuropneumoniae, and an 18 kDa protein to a protein-tyrosine phosphatase of Burkholderia cepacia. In conclusion, detection of I. limosus is accompanied by a specific serum antibody response and may reflect the infectious/pathogenic potential of I. limosus. Moreover, IgG immunoblotting may be useful to detect early infection with I. limosus and may support the selective cultivation of this novel emerging pathogen.

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

Evaluation of reproducibility of protein identification results after multidimensional human serum protein separation

We developed a gel- and label-free proteomics platform for comparative studies of human serum. The method involves the depletion of the six most abundant proteins, protein fractionation by Off-Gel IEF and RP-HPLC, followed by tryptic digestion, LC-MS/MS, protein identification, and relative quantification using probabilistic peptide match score summation (PMSS). We evaluated performance and reproducibility of the complete platform and the individual dimensions, by using chromatograms of the RP-HPLC runs, PMSS based abundance scores and abundance distributions as objective endpoints. We were interested if a relationship exists between the quantity ratio and the PMSS score ratio. The complete analysis was performed four times with two sets of serum samples containing different concentrations of spiked bovine beta-lactoglobulin (0.1 and 0.3%, w/w). The two concentrations resulted in significantly differing PMSS scores when compared to the variability in PMSS scores of all other protein identifications. We identified 196 proteins, of which 116 were identified four times in corresponding fractions whereof 73 qualified for relative quantification. Finally, we characterized the PMSS based protein abundance distributions with respect to the two dimensions of fractionation and discussed some interesting patterns representing discrete isoforms. We conclude that combination of Off-Gel electrophoresis (OGE) and HPLC is a reproducible protein fractionation technique, that PMSS is applicable for relative quantification, that the number of quantifiable proteins is always smaller than the number of identified proteins and that reproducibility of protein identifications should supplement probabilistic acceptance criteria.

Determination of ethyl glucuronide in human serum by capillary zone electrophoresis and an immunoassay

Ethyl glucuronide (EtG) is a marker of recent alcohol consumption. For the optimization of the analysis of EtG by CZE with indirect absorbance detection, the use of capillaries with permanent and dynamic wall coatings, the composition of the BGE, and various sample preparation procedures, including dilution with water, ultrafiltration, protein precipitation, and SPE, were investigated. Two validated screening assays for the determination of EtG in human serum, a CZE-based approach and an enzyme immunoassay (EIA), are described. The CZE assay uses a coated capillary, 2,4-dimethylglutaric acid as an internal standard, and a pH 4.65 BGE comprising 9 mM nicotinic acid, epsilon-aminocaproic acid and 10% v/v ACN. Proteins are removed via precipitation with ACN prior to analysis and the LOQ is 0.50 mg/L. The EIA is based upon commercial reagents which are promoted for the determination of urinary EtG. Krebs-Ringer solution containing 5% BSA is used as a calibration matrix. All samples are ultrafiltered prior to analysis of the ultrafiltrate on a Mira Plus analyzer. Assay calibration ranged between 0 and 2 mg/L and the upper reference limit was determined to be 0.05 mg/L. Both assays proved to be suitable for the analysis of samples from different individuals. For EtG levels above 0.50 mg/L, good agreement was observed for the comparison of the results of the two methods.