In the search for a noninvasive and reliable rapid screening method to detect biomarkers, a metabolomics fingerprinting approach was developed and applied to rat serum samples using capillary electrophoresis coupled to an electrospray ionization-time of flight-mass spectrometer (CE-TOF-MS). An ultrafiltration method was used for sample pretreatment. To evaluate performance the method was validated with carnitine, choline, ornithine, alanine, acetylcarnitine, betaine, and citrulline, covering the entire electropherogram of pool of rat serum. The linearity for all metabolites was >0.99, with good recovery and precision. Approximately 34 compounds were also confirmed in the pool of rat serum. The method was successfully applied to real serum samples from rats with ventilator-induced lung injury, an experimental rat model for acute lung injury (ALI), giving a total of 1163 molecular features. By use of univariate and multivariate statistics 18 significant compounds were found, of which five were confirmed. The involvement of arginase and nitric oxide synthase has been proved for other lung diseases, meaning the increase of asymmetric dimethyl arginine (ADMA) and ornithine and the decrease of arginine found were in accordance with published literature. Ultimately this fingerprinting approach offers the possibility of identifying biomarkers that could be regularly screened for as part of routine disease control. In this way it might be possible to prevent the development of ALI in patients in critical care units.
The aim of this study was to evaluate platelet activation in gastric cancer patients with regard to histopathological classification and the presence of distant metastases, by using platelet morphological parameters: MPV, L-PLT, MPC, as well as quantitative evaluation of surface receptor expression: CD41a, CD61, CD42b, CD62P, by flow cytometry at the resting state and after TRAP activation. In gastric cancer patients higher values of MPV and LP, as well as decreased MPC values were determined. Quantitative evaluation of surface antigen expression also revealed higher number of CD41a, CD61 and CD62P molecules, as compared with the platelets in the control group. Significant decrease of CD42b molecules' number after TRAP incubation, and the increased CD41a, CD61 and CD62P expression also point to the retained reactivation capacity of platelets. Good correlation between morphological parameters and the number of CD62P molecules indicates the usefulness of routine tests in evaluation of platelet activation.
PAH is characterised by upregulated tryptophan metabolism and enhanced biosynthesis of kynurenine. Elevated kynurenine concentration is associated with an adverse clinical course. Dysregulated immunity, delineated by Treg-Th17 imbalance, is directly related to diverse activation of the kynurenine pathway, indicating the potential interplay between kynurenines and the immune system in PAH.
We investigated whether the choice of anticoagulant or the application of density gradient mononuclear cell isolation may account for conflicting published data regarding the levels of the scavenger receptors' expression in healthy individuals. We demonstrate that the detection of CD163, but not CD36, differs dramatically among the methods.CD163 is a member of the scavenger receptor cysteine-rich family of proteins, accounting for the clearance of hemoglobinhaptoglobin complexes, which in turn fuel an anti-inflammatory response mediated by heme metabolites (6, 10). CD163 is also an attractive candidate for potential diagnostic use as a marker of monocyte/macrophage activity in inflammatory diseases (1,3,8,11). However, it is still unclear how many circulating monocytes in normal subjects express the CD163 molecule on their surface. Previous studies with Mac 2-48, RM3/1, Ber-Mac3, and other monoclonal antibodies indicated that 0 to 100% of CD14 ϩ monocytes were positive for CD163, as detected by flow cytometry (2,4,11,12,13). Possibly due to different protocols used for CD163 identification in various studies, some discrepancies were found even when the same clone of monoclonal antibody was used for different studies.In the current study, we analyzed the influence of three commonly used anticoagulants and mononuclear cell isolation on the level of CD163 expression evaluated with the use of commercially available, fluorochrome-conjugated GHI/61 monoclonal antibody. For comparison, the same kind of analysis was performed for another scavenger receptor, CD36, which is also expressed on monocytes and is involved mainly in phagocytosis of apoptotic neutrophils and uptake of modified low-density lipoproteins.Forty-eight individuals (20 female and 28 male) had blood drawn between 7 and 9 a.m. The donors were healthy volunteers aged 18 to 56 years and selected in accordance with the guidelines of the Bialystok Local Ethics Committee (no. 8-I-003/132/2004). Blood samples were divided into tubes with EDTA, citrate, or heparin and immediately processed.Whole-blood samples were incubated with CD14 fluorescein isothiocyanate (clone MP9), CD163 phycoerythrin (clone GHI/61), CD36 allophycocyanin (clone CB38) (all obtained from Becton Dickinson, San Jose, CA), and fluorochromeconjugated isotype controls for 30 min at room temperature. Red blood cells were lysed for 10 min with fluorescence-activated cell sorter lysing solution (Becton Dickinson, San Jose, CA). The remaining white blood cells were washed twice with phosphate-buffered saline and fixed with 400 l of 2% paraformaldehyde (5).Portions of each whole-blood sample were also used for isolation of mononuclear cells. Mononuclear cells were isolated using Histopaque (density, 1.077 g/ml; Sigma-Aldrich) density gradient centrifugation as described previously (9). The isolated cells were washed twice with phosphate-buffered saline, and the cell concentration was adjusted to 1 ϫ 10 6 cells/ ml. Mononuclear cells were stained with 20 l of fluorochrome-conjugated monoclonal antib...
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