Proteome profiling of interleukin-12 treated human T helper cells.

Selective activation of T helper subsets 1 (Th1) and 2 (Th2) plays a crucial role in different pathological conditions. Th1 cell response is involved in pathogenesis of autoimmune diseases, such as type II diabetes and multiple sclerosis, and Th2 cell response in pathogenesis of allergy and asthma. Cytokine interleukin-12 (IL-12) is one of the key factors in the differentiation of naïve CD4(+) T cells into Th1 cells. In this study we used 2-DE and MS to find and identify IL-12 regulated proteins in human CD4(+) T cells. In total, 42 protein spots were found to be differentially expressed following IL-12 stimulation, of which 22 were up- and 20 down-regulated. Among the upregulated proteins there are a multifunctional cytokine macrophage migration inhibitory factor and a known IL-12 target gene Programmed cell death 4. Downregulated proteins include p21-activated kinase 2 and its upstream GTPase Cdc42. Compared to previous reports our analysis provides a new view on the IL-12 induced changes on CD4(+) T cells underscoring the importance of creating and combining the data generated at various levels to build a comprehensive view of a given biological process of the cell.

Proteomic and transcriptomic characterization of interferon-alpha-induced human primary T helper cells.

Interferon-alpha (IFN-alpha) is a multifunctional cytokine that modulates immune response. In spite of the numerous comprehensive studies on the effects of IFN-alpha on various cell types, novel characteristics of this versatile agent emerge continuously. In the present study a differential proteomic approach was used to identify new IFN-alpha-regulated proteins in human primary CD4(+) T cells. Two IFN-alpha-inducible proteins, soluble N-ethylmaleimide-sensitive factor attachment protein alpha (alpha-SNAP) and cleavage stimulation factor-64 (CstF-64) previously not described in this context, were identified. Additionally, several proteins already known as IFN-stimulated genes were observed. The results of proteomics experiments were further studied at the mRNA level using real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Both peripheral blood and cord blood CD4(+) T cells were used in order to see if there are differences in IFN-alpha response between these populations. Differences were observed between the IFN-alpha-induced expression kinetics in peripheral blood and cord blood transcripts. The induction was more rapid in peripheral blood than in cord blood cells. CstF-64 expression was upregulated by IFN-alpha at the protein, but not at the mRNA level.

Comparison of PDQuest and Progenesis software packages in the analysis of two-dimensional electrophoresis gels.

Efficient analysis of protein expression by using two-dimensional electrophoresis (2-DE) data relies on the use of automated image processing techniques. The overall success of this research depends critically on the accuracy and the reliability of the analysis software. In addition, the software has a profound effect on the interpretation of the results obtained, and the amount of user intervention demanded during the analysis. The choice of analysis software that best meets specific needs is therefore of interest to the research laboratory. In this paper we compare two advanced analysis software packages, PDQuest and Progenesis. Their evaluation is based on quantitative tests at three different levels of standard 2-DE analysis: spot detection, gel matching and spot quantitation. As test materials we use three gel sets previously used in a similar comparison of Z3 and Melanie, and three sets of gels from our own research. It was observed that the quality of the test gels critically influences the spot detection and gel matching results. Both packages were sensitive to the parameter or filter settings with respect to the tendency of finding true positive and false positive spots. Quantitation results were very accurate for both analysis software packages.