Dinucleoside polyphosphates : newly detected uraemic compounds with an impact on leucocyte oxidative burst

Background. Dinucleoside polyphosphates (NpnN) have pathophysiologic roles in cardiovascular disease and are newly detected uraemic retention solutes. They were retrieved in human plasma, tissues and cells. Although their impact on several cell systems involved in vascular damage (endothelium, smooth muscle cells and thrombocytes) has been evaluated, their effect on different types of leucocytes has never been studied. Methods. This study evaluates, for the first time, the impact of NpnN on monocyte, granulocyte and lymphocyte oxidative burst activity at baseline and after stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA) in whole blood. Diadenosine triphosphate (Ap(3)A) to diadenosine hexaphosphate (Ap(6)A) were tested to investigate the effect of the number of phosphate groups on reactive oxygen species (ROS) production. The effect of the type of nucleoside was evaluated by comparing adenosine guanosine tetraphosphate, diguanosine tetraphosphate, uridine adenosine tetraphosphate (Up(4)A) and diadenosine tetraphosphate (Ap(4)A). Results. This study demonstrated that lymphocytes are especially susceptible to intracellular diadenosine polyphosphates. Depending on the phosphate chain length, different effects were observed. At baseline and with fMLP Ap(4)A, Ap(5)A and Ap(6)A enhanced lymphocyted-free radical production. In addition, Ap(3)A, Ap(4)A and Ap5A increased PMA-stimulated ROS production in lymphocytes. Monocytes and granulocytes parallel the lymphocyte response albeit with an inhibition of Ap(6)A on granulocytes. Considering NpnN with four phosphate groups, Up(4)A showed the most important stimulatory effects on monocytes and Ap(4)A on lymphocytes. Conclusions. NpnN mainly have a leucocyte-activating impact, most significant for Ap(4)A, considering phosphate chain length, and for Up(4)A, considering the type of nucleosides. These results suggest that the pro-inflammatory effects of NpnN can contribute to the development of atherosclerosis, probably in the early stages of chronic kidney disease, but their chemical composition affects their activity

Isolation, identification and characterization of vasoactive substances from endothelial cells, platelets and mononuclear leukocytes

The cause of essential hypertension is unknown and the pathogenesis is far from being under-stood completely. In the framework of this thesis, some unknown substances with strong ef-fects on the vasoregulatory system were isolated, identified and characterised. The basis of the thesis were the changes of the chromatographic and mass-spectrometric methods during the last ten years, which offered the possibility to isolate and to identify un-known biomolecules, and to clarify hereby unknown pathogenetic mechanisms. In the first part of the thesis, diadenosine polyphosphates (with 3-6 phosphates) were isolated, identified and quantified from different tissues and body fluids like adrenal glands, heart and platelets and plasma by using these innovative chromatographic and mass-spectrometric methods. Moreover, a potent vasoconstrictive dinucleoside polyphosphate with a purine and pyrimidine base was isolated from supernatants of stimulated endothelial cells. The concen-tration of this uridine adenosine tetraphosphate (Up4A) in plasma is sufficient to affect the vascular tone. The second part of the thesis deals with the isolation and characterisation of hypertensive agents in chronic renal failure (CRF) patients. In the secretome of mononuclear leukocytes, both Angiotensin II (Ang II) and des[Asp1]-[Ala1]-Ang II (named as “Ang A”) were isolated. The ratio Ang A / Ang II was significantly increased in plasma of CRF patients. Furthermore, p-hydroxy-hippuric acid was identified as a potent inhibitor of the Ca2+-ATPase, and phenylacetic acid was described as an inhibitor of the inducible NO synthase (iNOS). Finally, the effects of different hemofiltration membranes on the filtration rate of some identified sub-stances were analysed. In summary, some unknown substances with strong effects on the vasoregulatory system were isolated, identified and characterised. The thesis corroborates the hypothesis that hypertension is a disease caused by many yet unknown different factors and regulatory systems. It is very likely that there are still many other unknown factors. The identification of each unknown fac-tor offers the possibility to develop new and more appropriate therapeutic approaches

BcCluster: a bladder cancer database at the molecular level

Background: Bladder Cancer (BC) has two clearly distinct phenotypes. Non-muscle invasive BC has good prognosis and is treated with tumor resection and intravesical therapy whereas muscle invasive BC has poor prognosis and requires usually systemic cisplatin based chemotherapy either prior to or after radical cystectomy. Neoadjuvant chemotherapy is not often used for patients undergoing cystectomy. High-throughput analytical omics techniques are now available that allow the identification of individual molecular signatures to characterize the invasive phenotype. However, a large amount of data produced by omics experiments is not easily accessible since it is often scattered over many publications or stored in supplementary files. Objective: To develop a novel open-source database, BcCluster (http://www.bccluster.org/), dedicated to the comprehensive molecular characterization of muscle invasive bladder carcinoma. Materials: A database was created containing all reported molecular features significant in invasive BC. The query interface was developed in Ruby programming language (version 1.9.3) using the web-framework Rails (version 4.1.5) (http://rubyonrails.org/). Results: BcCluster contains the data from 112 published references, providing 1,559 statistically significant features relative to BC invasion. The database also holds 435 protein-protein interaction data and 92 molecular pathways significant in BC invasion. The database can be used to retrieve binding partners and pathways for any protein of interest. We illustrate this possibility using survivin, a known BC biomarker. Conclusions: BcCluster is an online database for retrieving molecular signatures relative to BC invasion. This application offers a comprehensive view of BC invasiveness at the molecular level and allows formulation of research hypotheses relevant to this phenotype

Urinary CE-MS peptide marker pattern for detection of solid tumors

Urinary profiling datasets, previously acquired by capillary electrophoresis coupled to mass-spectrometry were investigated to identify a general urinary marker pattern for detection of solid tumors by targeting common systemic events associated with tumor-related inflammation. A total of 2,055 urinary profiles were analyzed, derived from a) a cancer group of patients (n = 969) with bladder, prostate, and pancreatic cancers, renal cell carcinoma, and cholangiocarcinoma and b) a control group of patients with benign diseases (n = 556), inflammatory diseases (n = 199) and healthy individuals (n = 331). Statistical analysis was conducted in a discovery set of 676 cancer cases and 744 controls. 193 peptides differing at statistically significant levels between cases and controls were selected and combined to a multi-dimensional marker pattern using support vector machine algorithms. Independent validation in a set of 635 patients (293 cancer cases and 342 controls) showed an AUC of 0.82. Inclusion of age as independent variable, significantly increased the AUC value to 0.85. Among the identified peptides were mucins, fibrinogen and collagen fragments. Further studies are planned to assess the pattern value to monitor patients for tumor recurrence. In this proof-of-concept study, a general tumor marker pattern was developed to detect cancer based on shared biomarkers, likely indicative of cancer-related features

Proteomic-biostatistic integrated approach for finding the underlying molecular determinants of hypertension in human plasma

Despite advancements in lowering blood pressure, the best approach to lower it remains controversial because of the lack of information on the molecular basis of hypertension. We, therefore, performed plasma proteomics of plasma from patients with hypertension to identify molecular determinants detectable in these subjects but not in controls and vice versa. Plasma samples from hypertensive subjects (cases; n=118) and controls (n=85) from the InGenious HyperCare cohort were used for this study and performed mass spectrometric analysis. Using biostatistical methods, plasma peptides specific for hypertension were identified, and a model was developed using least absolute shrinkage and selection operator logistic regression. The underlying peptides were identified and sequenced off-line using matrix-assisted laser desorption ionization orbitrap mass spectrometry. By comparison of the molecular composition of the plasma samples, 27 molecular determinants were identified differently expressed in cases from controls. Seventy percent of the molecular determinants selected were found to occur less likely in hypertensive patients. In cross-validation, the overall R(2) was 0.434, and the area under the curve was 0.891 with 95% confidence interval 0.8482 to 0.9349, P<0.0001. The mean values of the cross-validated proteomic score of normotensive and hypertensive patients were found to be -2.007±0.3568 and 3.383±0.2643, respectively, P<0.0001. The molecular determinants were successfully identified, and the proteomic model developed shows an excellent discriminatory ability between hypertensives and normotensives. The identified molecular determinants may be the starting point for further studies to clarify the molecular causes of hypertension

One More Bottleneck towards Biomarker Validation and Clinical Implementation

ELISA is the main approach for the sensitive quantification of protein biomarkers in body fluids and is currently employed in clinical laboratories for the measurement of clinical markers. As such, it also constitutes the main methodological approach for biomarker validation and further qualification. For the latter, specific assay performance requirements have to be met, as described in respective guidelines of regulatory agencies. Even though many clinical ELISA assays in serum are regularly used, ELISA clinical applications in urine are significantly less. The scope of our study was to evaluate ELISA assay analytical performance in urine for a series of potential biomarkers for bladder cancer, as a first step towards their large scale clinical validation. Seven biomarkers (Secreted protein acidic and rich in cysteine, Survivin, Slit homolog 2 protein, NRC-Interacting Factor 1, Histone 2B, Proteinase-3 and Profilin-1) previously described in the literature as having differential expression in bladder cancer were included in the study. A total of 11 commercially available ELISA tests for these markers were tested by standard curve analysis, assay reproducibility, linearity and spiking experiments. The results show disappointing performance with coefficients of variation>20% for the vast majority of the tests performed. Only 3 assays (for Secreted protein acidic and rich in cysteine, Survivin and Slit homolog 2 protein) passed the accuracy thresholds and were found suitable for further application in marker quantification. These results collectively reflect the difficulties in developing urine-based ELISA assays of sufficient analytical performance for clinical application, presumably attributed to the urine matrix itself and/or presence of markers in various isoforms

Integrative analysis of extracellular and intracellular bladder cancer cell line proteome with transcriptome: improving coverage and validity of -omics findings

Characterization of disease-associated proteins improves our understanding of disease pathophysiology. Obtaining a comprehensive coverage of the proteome is challenging, mainly due to limited statistical power and an inability to verify hundreds of putative biomarkers. In an effort to address these issues, we investigated the value of parallel analysis of compartment-specific proteomes with an assessment of findings by cross-strategy and cross-omics (proteomics-transcriptomics) agreement. The validity of the individual datasets and of a “verified” dataset based on crossstrategy/omics agreement was defined following their comparison with published literature. The proteomic analysis of the cell extract, Endoplasmic Reticulum/Golgi apparatus and conditioned medium of T24 vs. its metastatic subclone T24M bladder cancer cells allowed the identification of 253, 217 and 256 significant changes, respectively. Integration of these findings with transcriptomics resulted in 253 “verified” proteins based on the agreement of at least 2 strategies. This approach revealed findings of higher validity, as supported by a higher level of agreement in the literature data than those of individual datasets. As an example, the coverage and shortlisting of targets in the IL-8 signalling pathway are discussed. Collectively, an integrative analysis appears a safer way to evaluate -omics datasets and ultimately generate models from valid observations

Threonine 150 phosphorylation of keratin 5 is linked to EBS and regulates filament assembly, cell cycle and oxidative stress response

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII–/–) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis