cytochrome-c-t and Acute-Phase-Reaction

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.

Topics: Acute-Phase Reaction; alpha-2-HS-Glycoprotein; Animals; Cattle; Chromatography, Liquid; Computational Biology; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Filtration; Haptoglobins; Hemopexin; Inflammation; Orosomucoid; Proteins; Proteolysis; Sepsis; Software; Sorption Detoxification; Swine; Tandem Mass Spectrometry

Tissue injury induces the acute phase response, aimed at minimizing damage and starting the healing process. Polymorphonuclear leukocytes (PMNs) respond to the presence of specific chemoattractants and begin to appear in large numbers. The aim of this study was to investigate the influence of reactive oxygen species (ROS) produced by PMNs on the interaction between colon carcinoma cells and mesothelial cells. An experimental human in vitro model was designed using Caco-2 colon carcinoma cells and primary cultures of mesothelial cells. Tumor cell adhesion to a mesothelial monolayer was assessed after preincubation of the mesothelium with stimulated PMNs and unstimulated PMNs. Mesothelial cells were also incubated with xanthine/xanthine oxidase (X/XO) complex producing ROS after which adhesion of Caco-2 cells was investigated and the expression of adhesion molecules (ICAM-1, VCAM-1, and CD44) by means of enzyme immunoassay. In the control situation the average adhesion of Caco-2 cells to the mesothelial monolayers was 23%. Mesothelial monolayers incubated with unstimulated PMNs showed a 25% increase of tumor cell adhesion (P < 0.05). The adhesion of tumor to the monolayers incubated with the N-formyl-methionyl-leucyl-phenylalanine-stimulated PMNs increased with 40% (P < 0.01). Incubation of the mesothelium with X/XO resulted in an enhancement of adhesion of Caco-2 cells of 70% and an up-regulation of expression of ICAM-1, VCAM-1, and CD44. This study reveals an increase of tumor cell adhesion to the mesothelium induced by incubating the mesothelial monolayers with PMNs. PMNs are producing a number of products, like proteolytic enzymes, cytokines, and ROS. These factors up-regulate the expression of adhesion molecules and in that way stimulate the adhesion of tumor to the mesothelium.

Topics: Acute-Phase Reaction; Caco-2 Cells; Cell Adhesion; Colonic Neoplasms; Cytochromes c; Epithelium; Humans; Immunoenzyme Techniques; In Vitro Techniques; Neoplasm Recurrence, Local; Neutrophils; Reactive Oxygen Species; Reproducibility of Results; Xanthine; Xanthine Oxidase