Tag: TORC1

Mitochondrial Measures in Primary Cells Isolated from Patients with ME/CFS

Abstract:

Fibroblasts and peripheral blood mononuclear cells (PBMCs) are commonly utilized cell types for the analysis of mitochondrial function. Fibroblasts, derived from connective tissue, provide a reliable model for studying mitochondrial metabolism due to their active role in energy production and their accessibility for experimental manipulations. PBMCs, on the other hand, are a heterogeneous population of immune cells that include lymphocytes and monocytes. They offer the advantage of reflecting mitochondrial function in circulating cells and providing insights into systemic aspects of mitochondrial biology. Both cell types can be cultured and treated with various substrates or stressors to assess parameters of mitochondrial function.

Here we describe the use of fibroblasts and PBMCs isolated from patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) to investigate mitochondrial abnormalities in the pathogenesis of this disease. Our techniques employ the use of fluorescent cellular dyes to measure mitochondrial mass, membrane potential and reactive oxygen species levels, luminescent measures of cellular NAD/NADH levels, and FRET-based measurements of the cellular and energy regulators, TORC1 and AMPK. These techniques are similarly useful for studying different physiological and pathological conditions.

Source: Allan CY, Katsaros T, Missailidis D, Fisher PR, Annesley SJ. Mitochondrial Measures in Primary Cells Isolated from Patients with ME/CFS. Methods Mol Biol. 2025;2920:203-223. doi: 10.1007/978-1-0716-4498-0_12. PMID: 40372685. https://link.springer.com/protocol/10.1007/978-1-0716-4498-0_12

Cell-Based Blood Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a devastating illness whose biomedical basis is now beginning to be elucidated. We reported previously that, after recovery from frozen storage, lymphocytes (peripheral blood mononuclear cells, PBMCs) from ME/CFS patients die faster in culture medium than those from healthy controls. We also found that lymphoblastoid cell lines (lymphoblasts) derived from these PBMCs exhibit multiple abnormalities in mitochondrial respiratory function and signalling activity by the cellular stress-sensing kinase Target Of Rapamycin Complex 1 (TORC1). These differences were correlated with disease severity, as measured by the Richardson and Lidbury weighted standing test.

The clarity of the differences between these cells derived from ME/CFS patient blood and those from healthy controls suggested that they may provide useful biomarkers for ME/CFS. Here, we report a preliminary investigation into that possibility using a variety of analytical classification tools, including linear discriminant analysis, logistic regression and receiver operating characteristic (ROC) curve analysis.

We found that results from three different tests-lymphocyte death rate, mitochondrial respiratory function and TORC1 activity-could each individually serve as a biomarker with better than 90% sensitivity but only modest specificity vís a vís healthy controls. However, in combination, they provided a cell-based biomarker with sensitivity and specificity approaching 100% in our sample.

This level of sensitivity and specificity was almost equalled by a suggested protocol in which the frozen lymphocyte death rate was used as a highly sensitive test to triage positive samples to the more time consuming and expensive tests measuring lymphoblast respiratory function and TORC1 activity. This protocol provides a promising biomarker that could assist in more rapid and accurate diagnosis of ME/CFS.

Source: Missailidis D, Sanislav O, Allan CY, Annesley SJ, Fisher PR. Cell-Based Blood Biomarkers for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Int J Mol Sci. 2020 Feb 8;21(3). pii: E1142. doi: 10.3390/ijms21031142. https://www.ncbi.nlm.nih.gov/pubmed/32046336