Recommendations about structuring proteomic biomarker studies should increase the probability that such markers will be clinically useful.

Purpose: Urine proteomics is emerging as a powerful tool for biomarker discovery. The purpose of this study is the development of a well-characterized ''real life'' sample that can be used as reference standard in urine clinical proteomics studies. Experimental design: We report on the generation of male and female urine samples that are extensively characterized by different platforms and methods (CE-MS, LC-MS, LC-MS/MS, 1-D gel analysis in combination with nano-LC MS/MS (using LTQ-FT ultra), and 2-DE-MS) for their proteome and peptidome. In several cases analysis involved a definition of the actual biochemical entities, i.e. proteins/peptides associated with molecular mass and detected PTMs and the relative abundance of these compounds. Results: The combination of different technologies allowed coverage of a wide mass range revealing the advantages and complementarities of the different technologies. Application of these samples in ''inter-laboratory'' and ''inter-platform'' data comparison is also demonstrated. Conclusions and clinical relevance: These well-characterized urine samples are freely available upon request to enable data comparison especially in the context of biomarker discovery and validation studies. It is also expected that they will provide the basis for the comprehensive characterization of the urinary proteome.

Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are unable to respond to IFN-γ due to disturbed chromatin remodelling, but can be rescued using histone deacetylase inhibitors.

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Background: Identification of markers for prediction of the clinical course of diabetic nephropathy remains a major challenge in disease management. We established a proteomics approach for identification of diabetic nephropathy-related biomarkers in urine. Methods: We used SELDI-TOF mass spectrometry and SAX2 protein arrays to compare protein profiles from urine of 4 defined patient groups. Samples from patients with type 2 diabetes (DM; n ‫؍‬ 45) without nephropathy and without microalbuminuria (DM-WNP), patients with DM with macro-or microalbuminuria (DM-NP; n ‫؍‬ 38), patients with proteinuria due to nondiabetic renal disease (n ‫؍‬ 34), and healthy controls (n ‫؍‬ 45) were analyzed. Anionic exchange, reversedphase fractionation, gel electrophoresis, and mass spectrometry were used to isolate and identify proteins with high discriminatory power. Results: A protein with m/z 6188 (P <0.0000004) was strongly released in the urine of healthy controls, patients with proteinuria due to nondiabetic disease, and DM-WNP in contrast to DM-NP patients. An m/z 14 766 protein (P <0.00008) was selectively excreted in the urine of DM-NP patients, whereas the protein with m/z 11 774 (P <0.000004) was significantly excreted by pa-