Tag: pathophysiology

Circulating extracellular vesicles-microRNAs as potential biomarkers for the identification of ME/CFS: differentiating fatigue-related conditions

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating, multi-systemic condition that has gained renewed global attention due to its clinical overlap with the growing population of long COVID patients. Despite ongoing efforts to characterize the disease, definitive diagnostic molecular markers are yet to be fully established, posing challenges in clinically differentiating from idiopathic chronic fatigue (ICF) and depression (Dep). This study aimed to identify circulating extracellular vesicles (EVs)-associated microRNAs (miRNAs) that serve as both diagnostic signatures and windows into the disease’s underlying pathophysiology.

Methods: Circulating EVs from ME/CFS (n = 6), ICF (n = 6), and depression (n = 8) patients were analyzed using flow cytometry, nano-tracking analysis, and comprehensive miRNA analysis. Differentially expressed miRNAs were analyzed using KEGG pathway enrichment to identify ME/CFS-specific signatures. Key candidate biomarkers were further validated in an additional healthy control (HC) cohort (n = 4).

Results: ME/CFS-EVs exhibited a unique subpopulation with high calcein intensity and larger diameters. Initial global miRNA profiling (Volcano plot) identified miR-21-5p, let-7f-5p, miR-26b-5p, and miR-20a-5p as significantly dysregulated EV-miRNAs in ME/CFS compared to ICF and Dep. To explore systemic pathophysiology, we identified a 114 EV-miRNA signature that achieved 87.0 ± 4.8% sensitivity and 93.7 ± 2.4% specificity within repeated cross-validation of the discovery cohort. After adjusting for covariates, 91 miRNAs remained significant; pathway analysis of the 62 up-regulated EV-miRNAs revealed significant enrichment in neuro-systemic axes, encompassing cellular structural integrity (focal adhesion), core signaling hubs (PI3K-Akt), and systemic homeostasis (such as insulin signaling and endocrine functions). Preliminary evaluation confirmed that these target EV-miRNAs remained at minimal or undetectable levels in the HC group.

Conclusions: A 62 EV-miRNA signature provides insight into the interconnected neuro-systemic pathways disrupted in ME/CFS, particularly those governing neuronal connectivity and cellular scaffolding. Within this candidate EV-miRNA signature, the top-ranked miRNAs-miR-21-5p, let-7f-5p, miR-26b-5p, and miR-20a-5p-emerge as potential candidate biomarkers whose specific elevation was not shared by HC. These findings establish a valuable framework for targeted diagnosis and enhance our understanding of the molecular pathways involved in synaptic and structural alterations in ME/CFS.

Source: Eguchi A, Kuratsune H, Nakatomi Y, Yasui T, Nakagawa R, Watanabe Y, Fukuda S. Circulating extracellular vesicles-microRNAs as potential biomarkers for the identification of ME/CFS: differentiating fatigue-related conditions. J Transl Med. 2026 Jul 27;24(1):979. doi: 10.1186/s12967-026-08695-w. PMID: 42533331. https://link.springer.com/article/10.1186/s12967-026-08695-w (Full text)

Metabolomic Classification of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome via Explainable Ensemble Learning and Pareto-Guided Feature Selection

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating multisystem illness characterised by post-exertional malaise, non-restorative sleep, and cognitive impairment, yet no objective diagnostic biomarkers have been established. Untargeted plasma metabolomics provides a broad view of the biochemical disturbances underlying ME/CFS; however, the high dimensionality of omics datasets and the limited interpretability of conventional classifiers nevertheless hinder translation into clinical practice. This study evaluates three ensemble classifiers-Explainable Boosting Machine (EBM), XGBoost, and LightGBM-for binary ME/CFS classification using plasma metabolomic and lipidomic profiles from 197 participants (106 ME/CFS; 91 healthy controls; 888 features).

Feature dimensionality was reduced using a Pareto-Guided Recursive Neural Network (PRNN) pipeline. Model performance was assessed via 50-repeat stratified hold-out validation. EBM achieved the highest accuracy (0.909; 95% CI: 0.868-0.949) and area under the receiver operating characteristic curve (AUC: 0.940; 95% CI: 0.909-0.983), with XGBoost and LightGBM performing comparably. Interpretability analyses revealed that pairwise metabolite interaction terms-particularly proline & indole-3-lactate, tyrosine & N-acetylornithine, and maleic acid & arachidic acid-contributed the greatest discriminative signal.

An ablation analysis comparing the full interaction-augmented EBM (AUC = 0.940) with a main-effects-only EBM (AUC = 0.882) confirmed that pairwise metabolite co-variation contributes additional discriminative value beyond individual metabolite levels, implicating amino acid catabolism, tryptophan-kynurenine pathway dysregulation, mitochondrial energy impairment, and lipid remodelling as central pathophysiological features. Global and instance-level explanations jointly demonstrated population-level metabolic signatures alongside individual heterogeneity, highlighting the added clinical value of explainable artificial intelligence (XAI) in metabolomics.

These findings support EBM-based metabolomic profiling as an internally validated approach for ME/CFS classification, subject to external validation, calibration assessment, and prospective testing.

Source: Yagin FH, Korkmaz Y, Colak C, Alzakari SA, Alkhalifa AK, Al-Hashem F, Aghaei M. Metabolomic Classification of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome via Explainable Ensemble Learning and Pareto-Guided Feature Selection. Int J Mol Sci. 2026 Jun 30;27(13):5920. doi: 10.3390/ijms27135920. PMID: 42450188. https://www.mdpi.com/1422-0067/27/13/5920 (Full text)

Disrupted glymphatic function and its relationship with sleep and cognitive impairment in ME/CFS assessed via DTI-ALPS

Abstract:

The glymphatic system is a recently discovered brain waste clearance system that is mostly active during sleep and disengaged during wakefulness. Impaired glymphatic function leads to the deposition of metabolic waste products in the brain potentially causing inflammation leading to various symptoms in ME/CFS. While the glymphatic function has been assessed in other neurodegenerative diseases using ‘diffusion tensor imaging along the perivascular space’ (DTI-ALPS), it has not been studied in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

This preliminary study investigates glymphatic function in 58 participants (ME/CFS = 31 and healthy controls = 27) using the DTI-ALPS index derived from DTI data acquired with 3 T MRI. The bilateral hemispheric DTI-ALPS index was estimated to assess glymphatic function, and an asymmetry index was calculated to determine interhemispheric asymmetry in glymphatic function.

We found that the global DTI-ALPS index was significantly lower in ME/CFS patients compared to healthy controls (ME/CFS: 1.44 ± 0.086; healthy controls: 1.51 ± 0.11, p = 0.014), indicating reduced glymphatic function in ME/CFS. Examining the hemispheres separately, showed the right hemisphere DTI-ALPS index was lower in ME/CFS than healthy controls (ME/CFS = 1.41 ± 0.097; healthy controls = 1.49 ± 0.12; p = 0.009) but not different on the left. Additionally, we did not find any significant difference in asymmetry index between ME/CFS and healthy controls. We observed an association between the global DTI-ALPS index and severity of ‘sleep disturbance’ (p = 0.013, r = -0.47) and “impaired concentration” (p = 0.026, r = -0.43).

This study demonstrated impaired glymphatic function in ME/CFS which may lead to symptoms such as cognitive dysfunction and sleep disturbance experienced by ME/CFS.

Source: Thapaliya K, Marshall-Gradisnik S, Inderyas M, Barnden L. Disrupted glymphatic function and its relationship with sleep and cognitive impairment in ME/CFS assessed via DTI-ALPS. Front Neurosci. 2026 Jun 19;20:1875420. doi: 10.3389/fnins.2026.1875420. PMID: 42403482; PMCID: PMC13329448. https://pmc.ncbi.nlm.nih.gov/articles/PMC13329448/ (Full text)

Exploring differences in protein cargo of extracellular vesicles from ME/CFS patient plasma compared to healthy controls

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic and debilitating disease characterized by post-exertional malaise, fatigue and pain. Yet, its underlying biological mechanisms remain poorly understood. Extracellular vesicles (EVs) are nanoparticles carrying biological cargo and are involved in cell-cell communication. Plasma EVs reflect several disease states and may serve as minimally invasive biomarkers. In this exploratory study, we characterized the plasma EV profiles of ME/CFS patients (N = 49) and healthy controls (N = 50), by enriching for EVs by size-exclusion chromatography coupled to high-resolution quantitative proteomics.

The ME/CFS patients had significantly higher concentrations of EVs than healthy controls. Among the 424 detected proteins included for analyses, 11 had different levels in EVs from ME/CFS patients. The ME/CFS associated EV proteins appear to mainly originate from erythroid cells, hepatocytes and plasma B cells, based on their tissue expression. Albeit differences in EV protein levels did not withstand correction for multiple testing, our study is the largest to date, thereby encouraging future investigations on the role of EV and its cargo in ME/CFS.

Source: Rydland A, Yran ES, Nyman TA, Strand EB, Trøseid AS, Øvstebø R, Heinicke F, Lie BA, Viken MK. Exploring differences in protein cargo of extracellular vesicles from ME/CFS patient plasma compared to healthy controls. Biochem Biophys Rep. 2026 Jun 20;47:102679. doi: 10.1016/j.bbrep.2026.102679. PMID: 42375682; PMCID: PMC13312568. https://pmc.ncbi.nlm.nih.gov/articles/PMC13312568/ (Full text)

Tryptophan Metabolism and Aryl-Hydrocarbon Receptor Agonists in the Gut Microbiome of People With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic disease with unknown biological basis and no cure. Microbiome dysbiosis has been reported in people with ME/CFS but its relevance to pathophysiology is unknown. Gut microbes are an important source of tryptophan metabolites that activate the aryl hydrocarbon receptor (AHR), a regulator of homeostatic and inflammatory genes. Dysregulated activation of AHR contributes to pathophysiology of several neuroimmune and chronic diseases but its role in ME/CFS has not been investigated. The purpose of this study was to investigate the production of tryptophan metabolites and AHR agonists by gut microbes of people with ME/CFS.

We found lower diversity and altered microbiome community structure in people with ME/CFS and changes in the subcommunity of microbes that correlated with tryptophan metabolites. Using targeted metabolomics we identified nine metabolites elevated in the stool of people with ME/CFS, including three AHR agonists. Stool ex vivo cultures were tested for their capacity to activate AHR in a reporter cell line and by qPCR. AHR activation did not differ between people with ME/CFS and controls, however, we detected elevated agonist activity in people with neurocognitive symptoms, regardless of underlying disease.

These findings are consistent with previous work revealing changes in the gut microbiome of people with ME/CFS and adds further support to alterations in tryptophan metabolism associated with the disease. Altered AHR activity by gut microbial metabolites may be a common mechanism contributing to neurocognitive symptoms in diseases including ME/CFS.

Source: Esteban DJ, Conrad B, Cullinan A, Luong S, Albaum J, Wilk V. Tryptophan Metabolism and Aryl-Hydrocarbon Receptor Agonists in the Gut Microbiome of People With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Microbiologyopen. 2026 Jun;15(3):e70333. doi: 10.1002/mbo3.70333. PMID: 42325052. https://onlinelibrary.wiley.com/doi/10.1002/mbo3.70333 (Full text)

Systems neuroendocrinology in ME/CFS and long COVID: a chronobiological framework for hormone-based research

Abstract:

Hormonal dysregulation is increasingly reported in ME/CFS and Long COVID, yet the broader role of neuroendocrine disruption in these conditions remains underexplored. While changes in steroid, peptide, and neuropeptide hormones have been identified, these findings are often considered in isolation and without attention to their timing or integration within broader physiological systems. The hypothalamic-pituitary axes regulate endocrine, immune, autonomic, nervous, and metabolic functions, systems commonly affected in both conditions, yet their circadian and menstrual dynamics are rarely investigated.

In this review, we examine the evidence for neuroendocrine dysfunction in ME/CFS and Long COVID, focusing on hormone output, functional assays, receptor expression, and the coordination of endocrine biorhythms. Sex hormone signalling emerges as a key area of vulnerability, particularly given the female predominance in both conditions and the complexity of reproductive hormone regulation.

We argue that accurate hormone measurement and time-structured sampling, including circadian and menstrual rhythms, are essential for detecting meaningful biological differences. By embedding chronobiology-aware, dense-sampling strategies and integrating multi-omic analyses into multi-system study designs, we outline a framework for investigating dynamic endocrine mechanisms underlying symptom variability and multisystem dysfunction, which may ultimately support the development of more targeted, personalised interventions.

Source: Thomas N, Huang K, Schneider-Futschik EK, Pollack B, Tal MC, Fineberg D, Wang X, Gurvich C, Pretorius R, Bergquist J, Armstrong CW. Systems neuroendocrinology in ME/CFS and long COVID: a chronobiological framework for hormone-based research. Front Neuroendocrinol. 2026 Jun 19:101268. doi: 10.1016/j.yfrne.2026.101268. Epub ahead of print. PMID: 42320559. https://www.sciencedirect.com/science/article/abs/pii/S0091302226000385 (Full text)

Irisin Signaling Resistance in Myalgic Encephalomyelitis: A Proposed Mechanistic Framework for Post-Exertional Malaise Involving the TSP-1-HSP90α-αvβ5 Axis

Abstract:

Myalgic Encephalomyelitis (ME) is a chronic, multisystem disease characterized by systemic metabolic dysfunction and post-exertional malaise (PEM). In this study, we investigated the dysregulation of irisin, an exercise-induced myokine, and its potential antagonism by thrombospondin-1 (TSP-1).

In a cross-sectional study (92 ME patients vs. 44 sedentary healthy controls), plasma irisin and TSP-1 levels were measured at baseline and after a 90 min mechanical stress challenge applied to induce PEM. ME patients exhibited significantly lower baseline irisin (p < 0.05) and a blunted exertional response (p < 0.05). Paradoxically, baseline irisin was an independent predictor of fatigue severity (β = 0.728, p = 0.018), with moderate-to-severe patients showing elevated levels of both irisin and TSP-1 (p < 0.05), suggesting a compensatory but ineffective response. Functional cellular dielectric spectroscopy indicated that TSP-1 inhibits irisin signaling in a concentration-dependent manner. Irisin signaling was markedly reduced by both αvβ5 blockade and HSP90α inhibition in this experimental system, consistent with a diminished ability to counteract TSP-1.

Collectively, these findings support a model in which dysregulation of the irisin-TSP-1 axis contributes to metabolic dysfunction in ME. Elevated circulating TSP-1 levels are associated with symptom severity and are linked to impaired irisin signaling in an HSP90α- and αvβ5-dependent context. This interaction is consistent with defective metabolic adaptation and highlights a potential therapeutic target that warrants further validation to restore energy homeostasis.

Source: Souma B, Elremaly W, Akoume MY, Elbakry M, Godbout C, Moreau A. Irisin Signaling Resistance in Myalgic Encephalomyelitis: A Proposed Mechanistic Framework for Post-Exertional Malaise Involving the TSP-1-HSP90α-αvβ5 Axis. Int J Mol Sci. 2026 May 26;27(11):4770. doi: 10.3390/ijms27114770. PMID: 42278300. https://www.mdpi.com/1422-0067/27/11/4770 (Full text)

Dynamic microclot profiling: thromboelastography advances precision management in long COVID and myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Long COVID and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) share overlapping symptoms, and emerging evidence implicates persistent fibrinoid microclots in their pathophysiology, contributing to impaired microcirculation. This review explores the role of microclots and evaluates thromboelastography (TEG) as a potential diagnostic tool.

A comprehensive literature review was conducted using major biomedical databases. Studies indicate microclots are prevalent in both conditions. Long COVID patients demonstrate a TEG profile of increased clot strength (maximum amplitude) and reduced fibrinolysis (LY30), suggesting a persistent hypercoagulable state. Despite its advantages in real-time assessment, TEG interpretation faces challenges from preanalytical variability and a lack of standardized protocols. Promising therapeutic trials, including anticoagulants (e.g., apixaban) and fibrinolytics (e.g., lumbrokinase), require further validation. Technological advancements like AI-driven TEG analysis and portable devices could improve diagnostic precision.

In conclusion, persistent microclots are a key pathophysiological feature. TEG provides a promising, novel approach for detecting coagulation abnormalities and could guide treatment, but requires standardization in future clinical trials. Future research should integrate multiomics biomarkers for precision therapeutics to improve patient outcomes.

Source: Saleem S, Hussain A, Haroon M, Raza A, Afzal U, Anwar MF, Imran S, Iqbal MU, Hajj F. Dynamic microclot profiling: thromboelastography advances precision management in long COVID and myalgic encephalomyelitis/chronic fatigue syndrome. Blood Coagul Fibrinolysis. 2026 Jun 11. doi: 10.1097/MBC.0000000000001439. Epub ahead of print. PMID: 42274123. https://pubmed.ncbi.nlm.nih.gov/42274123/

Deficient TRPM3-linked mitochondrial Ca2+ influx in natural killer cells associated with myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

Introduction: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic illness, commonly associated with dysregulation of the immune system including reduced cytotoxicity of natural killer (NK) cells and post-exertional neuroimmune exhaustion. Previously, transient receptor potential melastatin 3 (TRPM3) ion channel impairment associated with reduced Ca2+ mobilisation in NK cells from ME/CFS patients was reported. To further explore the pathomechanisms involved in ME/CFS, we investigated the downstream impact of TRPM3 ion channel dysfunction on mitochondrial Ca2+ mobilisation in NK cells.

Method: Fluorescence live-cell imaging was used to investigate Ca2+ mobilisation in NK cells of (N = 10) ME/CFS, classified using Canadian Consensus Criteria, and (N = 10) healthy control (HC) participants. Cytoplasmic and mitochondrial Ca2+ entry was determined using Fluo-8 AM and Rhod-2 AM Ca2+ indicators, respectively. The effect of TRPM3 modulation on Ca2+ mobilisation ex vivo, was examined using pregnenolone sulfate and ononetin to activate and inhibit the channel, respectively.

Results: Cytosolic Ca2+ influx amplitude and slope were significantly reduced (p < 0.001), with a significantly shorter T1/2 response (p = 0.001) in ME/CFS compared to HC. Ca2+ influx amplitude (p < 0.001) and slope (p < 0.041) into the mitochondria were significantly higher in ME/CFS compared to HC. TRPM3 activation triggered pronounced cytosolic response (P < 0.001) accompanied by mitochondrial Ca2+ increase in HC. TRPM3-dependent cytosolic and mitochondrial Ca2+ mobilisation (P < 0.015) were significantly reduced with a shorter T1/2 response (p < 0.02) in ME/CFS compared to HC.

Conclusion: The results demonstrate that altered TRPM3-mediated cytosolic Ca2+ influx may significantly impact Ca2+ mobilisation into the mitochondria of people with ME/CFS. Alterations that interfere with the optimal function of Ca2+ permeable channels may cumulatively impact downstream signalling, leading to detrimental cellular consequences. Collectively these findings provide an avenue for further studies on the physiological functions of TRPM3 ion channel and its role in ME/CFS.

Source: Magawa CT, Eaton-Fitch N, Muraki K, Marshall-Gradisnik S. Deficient TRPM3-linked mitochondrial Ca2+ influx in natural killer cells associated with myalgic encephalomyelitis/chronic fatigue syndrome. BMC Immunol. 2026 May 23. doi: 10.1186/s12865-026-00849-1. Epub ahead of print. PMID: 42177403. https://link.springer.com/article/10.1186/s12865-026-00849-1 (Full text)

3D Virtual Reality Performance Metrics as a Future Fatigue Biomarker in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS)

Abstract:

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disorder, characterized by symptoms such as post-exertional malaise (PEM) and cognitive impairments. This study assessed reaction time (RT) metrics in three-dimensional (3D) visual tasks with the aim of objectively quantifying the cognitive impairments in ME/CFS patients compared to controls.

Methods: A total of 120 participants (60 ME/CFS patients and 60 controls) were recruited at the Department of Ophthalmology, Universität of Erlangen-Nürnberg. RT was assessed using a virtual reality-oculomotor test system, presenting 3D stimuli at three disparity levels (275″, 550″, and 1100″) within three gaming repetitions (R1, R2, and R3). Mixed-effects models were used to evaluate group differences, with age and gender as covariates. Pairwise contrasts were calculated to assess changes across repetitions. Fatigue self-assessments were recorded by validated questionnaires, (FACIT Fatigue Scale, Chalder Fatigue Scale, Bell Score and Health Assessment Questionnaire), and their correlation with RT metrics was portrayed using a Spearman correlation matrix.

Results: Estimated means (EM-means) for RT were significantly prolonged in ME/CFS patients compared to controls at disparity 275″ (1969 ms vs. 1384 ms; p = 0.0001), 550″ (1409 vs. 1071 ms; p = 0.0012) and 1100″ (1126 ms vs. 891 ms; p = 0.00223). Age was a significant covariate (p < 0.001), while gender showed no effect. Both groups demonstrated improvements in RT over repetitions; however, ME/CFS patients showed a significantly lower improvement compared to controls, reaching significance in R3 (p = 0.0042). RT metrics did not correlate with patients’ self-assessment scores.

Conclusions: ME/CFS patients showed consistently slower RTs compared to controls, particularly in later, easier gaming repetitions, potentially reflecting the impact of fatigue.

Source: Ladek AM, Priebe L, Harrer T, Harrer E, Michelson G, Knauer TS, Dias-Nunes DX, Mardin CY, Bergua A, Hohberger B. 3D Virtual Reality Performance Metrics as a Future Fatigue Biomarker in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS). Biomedicines. 2026 Apr 9;14(4):855. doi: 10.3390/biomedicines14040855. PMID: 42072397. https://www.mdpi.com/2227-9059/14/4/855 (Full text)