The profile of 122 metabolites in the cerebrospinal fluid (CSF) of patients suffering from Alzheimer’s disease (AD) and controls was studied. Among the 122 metabolites analyzed, 61 could be detected. Statistically significant differences between the AD and control group were only detected for metabolites of the glycolysis. Thus, accurate quantification of 11 glycolytic metabolites was done. We detected a significant reduction of five of them, namely phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, pyruvate and dihydroxyacetone phosphate in the AD CSF compared to controls. These results correlate with the known reduction of glucose metabolism in the brain of patients with AD and indicate that metabolic analysis of the central carbon metabolism can be a potential tool in AD diagnostic. Although the Receiver operating characteristic (ROC) analyses of the metabolites do not reach the level of the diagnostic informativity of AD biomarkers, the combination of specific glycolysis metabolites with the established biomarkers may lead to an improvement in sensitivity and specificity.

Analysis of the white blood cell differential as part of a flow cytometry-based approach is a common routine diagnostic tool used in clinics and research. For human blood, the methodological approach, suitable markers, and gating strategies are well-established. However, there is a lack of information regarding the mouse blood count. In this article, we deliver a fast and easy protocol for reprocessing mouse blood for the purpose of flow cytometric analysis, as well as suitable markers and gating strategies. We also present two possible applications: for the analysis of the whole blood count, with blood from a cardiac puncture, and for the analysis of a certain leukocyte subset at multiple time points in the framework of a mouse experiment, using blood from the facial vein. Additionally, we provide orientation values by applying the method to 3-month-old and 24-month-old male and female C57BL/6J mice. Our analyses demonstrate differences in the leukocyte fractions depending on age and sex. We discuss the influencing factors and limitations that can affect the results and that, therefore, need to be considered when applying this method. The present study fills the gap in the knowledge related to the rare information on flow cytometric analysis of mouse blood and, thus, lays the foundation for further investigations in this area.

(1) Background: Hemoadsorption is a method of blood purification with a wide spectrum of indications. Pre-emptive use of hemoadsorption in patients undergoing heart surgery with cardiopulmonary bypass is considered to reduce the risk of postoperative systemic inflammatory response syndrome. The current study aimed to identify the spectrum of blood proteins adsorbed on the polymer matrix of the CytoSorb hemoadsorption system and to investigate their influence on cultured endothelial cells in vitro. (2) Methods: Adsorbers used for intraoperative hemoadsorption were obtained from patients undergoing on-pump valve surgery in acute endocarditis. Proteins were extracted from the adsorbers, purified, identified with mass-spectrometry and applied to cultured human aortic endothelial cells. (3) Results: A broad range of blood proteins were identified in the material eluted from the CytoSorb adsorber. When added to cultured ECs, these protein extracts caused severe reduction in cell viability and migration. After 24 h exposure, transcriptional changes with up-regulation of multiple metabolic regulators were observed and verified on the protein level. Genes responsible for control of mitosis were significantly down-regulated. (4) Conclusions: In summary, our data reveal that intraoperative hemoadsorption allows broad spectrum removal of a wide range of molecules eliciting endothelial damage.

Background: Vitamin C is one of the major extracellular nonenzymatic antioxidants involved in the biosynthesis of collagen. It promotes the growth of fibroblasts, wound healing processes, and enhances the survival and differentiation of osteoblasts. The potential effects of ascorbic acid on human dental pulp cells (DPC) and the cells of the apical papilla (CAP) used in actual regenerative endodontic procedures remain largely unknown. In this study, we investigated the possible employment of ascorbic acid in the differentiation and regenerative therapies of DPC and CAP. Methods: Nine extracted human wisdom teeth were selected for this study. Subpopulations of stem cells within DPC and CAP were sorted with the mesenchymal stem cell marker STRO-1, followed by treatments with different concentrations (0 mM, 0.1 mM, 0.5 mM, and 1.0 mM) of ascorbic acid (AA), RT-PCR, and Western blot analysis. Results: FACS analysis revealed the presence of cell subpopulations characterized by a strong expression of mesenchymal stem cell marker STRO-1 and dental stem cell markers CD105, CD44, CD146, CD90, and CD29. Treatment of the cells with defined amounts of AA revealed a markedly increased expression of proliferation marker Ki-67, especially in the concentration range between 0.1 mM and 0.5 mM. Further investigations demonstrated that treatment with AA led to significantly increased expression of common stem cell markers OCT4, Nanog, and Sox2. The most potent proliferative and expressional effects of AA were observed in the concentration of 0.1 mM. Conclusions: AA might be a novel and potent growth promoter of human dental cells. Increasing the properties of human dental pulp cells and the cells of the apical papilla using AA could be a useful factor for further clinical developments of regenerative endodontic procedures.

Advanced glycation end products (AGEs) result from a non-enzymatic reaction of proteins with reactive carbohydrates. Heat-processed food, such as bread, contains high amounts of AGEs. The activation of the NF-κB signaling pathway by bread crust extract (BCE) is well understood. However, it is largely unknown whether NRF2, the master regulator of oxidative stress resistance in mammalian cells, is affected by BCE. We have investigated the molecular mechanisms by which BCE induces antioxidant gene expression in cellular models. Our data showed that soluble extracts from bread crust are capable of stimulating the NRF2 signaling pathway. Furthermore, NRF2 pathway activation was confirmed by microarray and reporter-cell analyses. QRT-PCR measurements and Western blot analyses indicated an induction of antioxidative genes such as HMOX1, GCLM and NQO1 upon BCE treatment. Moreover, BCE pretreated cells had a survival advantage compared to control cells when exposed to oxidative stress. BCE induces phosphorylation of AKT and ERK kinase in EA.hy926 cells. By mass spectrometry, several new, potentially active modifications in BCE were identified. Our findings indicate that BCE activates NRF2-dependent antioxidant gene expression, thus provoking a protection mechanism against oxidative stress-mediated tissue injury. Hence, BCE can be considered as functional food with antioxidative and cardioprotective potential.

Tissue concentrations of advanced glycation end product (AGE) and peripheral soluble receptor of AGE (sRAGE) levels may be associated with periodontitis severity. Both parameters and periodontitis might serve as outcome predictors for patients undergoing coronary artery bypass grafting (CABG). This study aimed to investigate possible associations between periodontitis and AGE/sRAGE. Ultimately, we wanted to examine whether AGE, sRAGE, and severe periodontitis are associated with the incidence of new cardiovascular events within 3 years of follow-up after CABG. Ninety-five patients with coronary vascular disease (CVD) (age 69 years, 88.3% males) needing CABG surgery were included. Periodontal diagnosis was made according to the guidelines of the “Centers for Disease Control and Prevention (CDC)” (2007) and staged according to the new classification of periodontal diseases (2018). AGE tissue concentrations were assessed as skin autofluorescence (sAF). sRAGE levels were determined by using a commercially available enzyme-linked immunoabsorbance assay (ELISA) kit. Univariate and multivariate baseline and survival analyses were carried out with Mann–Whitney U test, Chi² test, Kaplan–Meier curves with Log-Rank test, and logistic and Cox regression. sAF was identified as an independent risk indicator for severe periodontitis with respect to the cofactors age, gender, plaque index, and diabetes (adjusted odds ratio [OR] = 2.9, p = 0.028). The degree of subgingival inflammation assessed as a percentage of sites with bleeding on probing (BOP) was inversely correlated with sRAGE concentration (r = −0.189, p = 0.034). Both sAF (Hazard Ratio [HR] = 2.4, p = 0.004) and sRAGE (HR = 1.9, p = 0.031) increased the crude risk for new adverse events after CABG. The occurrence of severe periodontitis trends towards a higher risk for new cardiovascular events (HR = 1.8, p = 0.115). Applying multivariate Cox regression, only peripheral arterial disease (adjusted HR = 2.7, p = 0.006) and history of myocardial infarction (adjusted HR = 2.8, p = 0.010) proved to be independent risk factors for cardiovascular outcome. We conclude that sAF may represent a new, independent risk indicator for severe periodontitis. In contrast, sAF, sRAGE, and severe periodontitis were not independent prognostic factors for postoperative outcome in patients undergoing CABG.