Quantification of Autophagosomes in Human Fibroblasts Using Cyto-ID® Staining and Cytation Imaging.

As an essential process for the maintenance of cellular homeostasis and function, autophagy is responsible for the lysosome-mediated degradation of damaged proteins and organelles; therefore, dysregulation of autophagy in humans can lead to a variety of diseases. The link between impaired autophagy and disease highlights the need to investigate possible interventions to address dysregulations. One possible intervention is hyperthermia, which is described in this protocol. To investigate these interventions, a method for absolute quantification of autophagosomal compartments is required that allows comparison of autophagosomal activity under different conditions. Existing methods such as western blotting and immunohistochemistry for analysing the location and relative abundance of intracellular proteins associated with autophagy, or transmission electron microscopy (TEM), which are either very time-consuming, expensive, or both, are less suitable for this purpose. The method described in this protocol allows the absolute quantification of autophagosomes per cell in human fibroblasts using the CYTO-ID® Autophagy Detection Kit after heat therapy compared to a control. The Cyto-ID® assay is based on the use of a specific dye that selectively stains autophagic compartments, combined with an additional Hoechst 33342 dye for nuclear staining. The subsequent recognition of these stained compartments by the Cytation Imager enables the software to determine the number of autophagosomes per nucleus in living cells. Additionally, this absolute quantification uses an image-based method, and the protocol is easy to use and not time-consuming. Furthermore, the method is not only suitable for heat therapy but can also be adapted to any other desired therapy or substance. Key features • Absolute quantification of autophagic compartments in living cells. • Optimised protocol for the determination of autophagy in primary human skin fibroblasts. • Allows the testing of active substances and treatments concerning autophagy. • Imaging-based method for the determination of autophagy.

Heat treatment in health and disease: How water-filtered infrared-A (wIRA) irradiation affects key cellular mechanisms in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients compared to healthy donors.

Heat treatment or hyperthermia is a promising therapy for many diseases, especially cancer, and can be traced back thousands of years. Despite its long history, little is known about the cellular and molecular effects of heat on human cells. Therefore, we investigated the impact of water-filtered infrared-A (wIRA) irradiation (39 °C, 60 min) on key cellular mechanisms, namely autophagy, mitochondrial function and mRNA expression, in human fibroblasts and peripheral blood mononuclear cells (PBMCs) from healthy donors and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients. Our results show an induction of autophagy in healthy fibroblasts and PBMCs from healthy donors and ME/CFS patients. ME/CFS patients have higher mitochondrial function compared to healthy donors. The wIRA treatment leads to a slight reduction in mitochondrial function in PBMCs from ME/CFS patients, thereby approaching the level of mitochondrial function of healthy donors. Furthermore, an activation of the mRNA expression of the autophagy-related genes MAP1LC3B and SIRT1 as well as for HSPA1, which codes for a heat shock protein, can be observed. These results confirm an impact of heat treatment in human cells on key cellular mechanisms, namely autophagy and mitochondrial function, in health and disease, and provide hope for a potential treatment option for ME/CFS patients.

Ex vivo Assessment of Mitochondrial Function in Human Peripheral Blood Mononuclear Cells Using XF Analyzer.

Cellular health and function, as we know today, depend on a large extent on mitochondrial function. The essential function of mitochondria is the energy production, more precisely ATP production, via oxidative phosphorylation. Mitochondrial energy production parameters therefore represent important biomarkers. Studies on human cells have mainly been performed on in vitro cell cultures. However, peripheral blood mononuclear cells (PBMCs) are particularly suitable for such examinations. That's why this protocol describes a method to measure key parameters of mitochondrial function in freshly isolated PBMCs with the latest technology, the XF Analyzer. For this ex vivo approach PBMCs are first isolated out of human anticoagulated blood. Next, they are attached to the surface of special microplates pre-coated with Poly-D-Lysine. During the subsequent measurement of oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) the stress reagents oligomycin, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), rotenone and antimycin A are injected. Several mitochondrial parameters can be calculated from the results obtained. The application of this protocol allows the analysis of various influences, such as pharmaceuticals or environmental factors, on human cells.

mRNA expression of ageing-associated genes in calorie reduction is subject to donor variability and can be induced by calorie restriction mimetics.

BACKGROUND: Finding ways to a healthier ageing are increasingly becoming the focus of geriatric research. One way to accomplish this could be calorie restriction, as this is known to positively influence the ageing of model organisms. AIM: The aim of this study was to investigate the influence of calorie reduction (F. X. Mayr therapy) and of the calorie restriction mimetics resveratrol and spermidine on the expression of ageing-associated genes. METHODS: mRNA expression in peripheral blood mononuclear cells (PBMCs) of 18 participants taking part in an F. X. Mayr therapy was analysed. The PBMCs of one additional participant were treated ex vivo with spermidine or resveratrol. mRNA expression of SIRT1, SIRT3, FOXO3 and SOD2 was determined for these two calorie restriction mimetics. For the F. X. Mayr therapy samples, mRNA of XPA was analysed additionally. RESULTS: mRNA expression of the ageing-associated genes showed a distinct donor variation during F. X. Mayr therapy, with a significant increase in mRNA expression of SIRT1. Expression of XPA was similar to SIRT1, with a significant correlation at the last time point tested. Spermidine treatment of PBMCs resulted in a significantly increased expression of all genes tested, whereas resveratrol treatment caused a significant increase of SIRT3, FOXO3 and SOD2 mRNA expression. CONCLUSIONS: By increasing SIRT1 and XPA mRNA expression, calorie reduction in the form of F. X. Mayr therapy could contribute to a healthier ageing; however, the donor variability observed showed that not everyone benefited from this. Calorie restriction mimetics may be an option for promote healthier ageing for those who do not benefit from calorie reduction.

12 days of in vivo caloric reduction can improve important parameters of aging in humans.

Caloric reduction (CR) is considered as the most reasonable intervention to delay aging and age-related diseases. Numerous studies in various model organisms provide the main basis for this hypothesis. Human studies exist, but they differ widely in study design, characteristics of test persons and study outcome. In this study we investigated CR in humans on a molecular level to gain a better understanding in these processes. For that purpose, we analyzed human peripheral blood mononuclear cells of healthy people fasting according to F.X. Mayr. In a previous study our group could show a significantly improved DNA repair capacity after fasting. Here we were able to confirm these findings despite a slightly modified fasting therapy. Furthermore, the function of the mitochondrial respiratory chain and the mRNA levels of the mitochondria-associated genes SIRT3 and NDUFS1 were significantly affected by CR. However, these changes were only detectable in people who exhibited no improvement in DNA repair capacity. In contrast to that we could not observe any changes in ROS levels, mitochondrial DNA copy number and non-mitochondrial respiration. Altogether our results reveal that CR in form of F. X. Mayr therapy is able to positively influence several cellular parameters and especially mitochondrial function.