Tag: Pretorius

Virus-induced endothelial senescence as a cause and driving factor for ME/CFS and long COVID: mediated by a dysfunctional immune system

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are two post-viral diseases, which share many common symptoms and pathophysiological alterations. Yet a mechanistic explanation of disease induction and maintenance is lacking. This hinders the discovery and implementation of biomarkers and treatment options, and ultimately the establishment of effective clinical resolution. Here, we propose that acute viral infection results in (in)direct endothelial dysfunction and senescence, which at the blood-brain barrier, cerebral arteries, gastrointestinal tract, and skeletal muscle can explain symptoms.

The endothelial senescence-associated secretory phenotype (SASP) is proinflammatory, pro-oxidative, procoagulant, primed for vasoconstriction, and characterized by impaired regulation of tissue repair, but also leads to dysregulated inflammatory processes. Immune abnormalities in ME/CFS and long COVID can account for the persistence of endothelial senescence long past the acute infection by preventing their clearance, thereby providing a mechanism for the chronic nature of ME/CFS and long COVID.

The systemic and tissue-specific effects of endothelial senescence can thus explain the multisystem involvement in and subtypes of ME/CFS and long COVID, including dysregulated blood flow and perfusion deficits. This can occur in all tissues, but especially the brain as evidenced by findings of reduced cerebral blood flow and impaired perfusion of various brain regions, post-exertional malaise (PEM), gastrointestinal disturbances, and fatigue.

Paramount to this theory is the affected endothelium, and the bidirectional sustainment of immune abnormalities and endothelial senescence. The recognition of endothelial cell dysfunction and senescence as a core element in the aetiology of both ME/CFS and Long COVID should aid in the establishment of effective biomarkers and treatment regimens.

Source: Nunes M, Kell L, Slaghekke A, Wüst RC, Fielding BC, Kell DB, Pretorius E. Virus-induced endothelial senescence as a cause and driving factor for ME/CFS and long COVID: mediated by a dysfunctional immune system. Cell Death Dis. 2026 Jan 9;17(1):16. doi: 10.1038/s41419-025-08162-2. PMID: 41513611; PMCID: PMC12789617. https://pmc.ncbi.nlm.nih.gov/articles/PMC12789617/ (Full text)

Post-translational modifications within fibrinaloid microclot complexes distinguish Pre-COVID-19 Postural Orthostatic Tachycardia Syndrome, Long COVID, and Long COVID-POTS and reveal disease-specific molecular pathways

Abstract:

Background: Pre-COVID-19 Postural orthostatic tachycardia syndrome (PC-POTS), Long COVID, and their overlap (LC-POTS) are chronic post-viral conditions marked by debilitating symptoms despite frequently normal routine laboratory results. We have previously identified insoluble fibrinaloid microclot complexes (FMCs) in Long COVID. It is not known whether FMCs are also present in PC-POTS, or whether post-translational modifications (PTMs) within FMC-entrapped proteins contribute to disease mechanisms.

Methods: Platelet-poor plasma from healthy controls, PC-POTS patients (collected prior to the COVID-19 pandemic), Long COVID (without POTS) and LC-POTS patients underwent fluorescence imaging flow cytometry to quantify FMCs. Proteomic analyses were performed on insoluble protein fractions using a double trypsin digestion strategy and data-independent liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential protein abundance, PTMs, and amyloidogenicity were compared across groups.

Results: Measured with imaging flow cytometry in objects/mL, higher levels of FMCs were present in disease groups compared to controls, although not statistically significant. Statistically significant differences potentially lay within FMC sizes and composition. Furthermore, despite only a few dysregulated proteins, FMC proteomics revealed extensive and disease-specific peptides with PTM dysregulation across coagulation, immune, and metabolic pathways. Long COVID displayed FMCs with PTMs of coagulation proteins including prominent advanced glycation end-products (AGE)- and oxidation-based modifications of fibrinogen subunits, particularly fibrinogen subunit A (FIBA), resembling diabetic glycation profiles. FMCs in PC-POTS showed fewer fibrinogen PTMs but markedly increased modifications in metabolic proteins, including oxidised apoA1 and apoB, and immune patterns with complement-related proteins (C3, C4A/B, IC1), immunoglobulin G1 (IGG1) and alpha 2 macroglobulin (A2MG). LC-POTS shared coagulation pathology with Long COVID and immune pathology with PC-POTS. Many dysregulated peptides were determined by in silco methods to be highly amyloidogenic, consistent with FMCs as beta-sheet-rich aggregates. Protein-level differences were minimal compared with PTM changes.

Conclusions: This study provides the first evidence that post-translational modifications (PTMs) within fibrinaloid microclots complexes (FMCs) uniquely distinguish pre-COVID-19 POTS, Long COVID, and Long COVID-POTS. Because PC-POTS samples were collected before SARS-CoV-2, their PTM patterns reflect intrinsic disease biology, allowing a clear separation from Long COVID-related changes. PTM profiling revealed pro-coagulant fibrinogen modifications in Long COVID and LC-POTS, metabolic-oxidative disruptions in Long COVID and PC-POTS, and immune dysregulation in PC-POTS and LC-POTS. None of these is detectable with routine assays, and all are independent of protein abundance. The consistent presence of amyloidogenic peptides suggests a contribution to microvascular dysfunction. These findings define disease-specific PTM landscapes and support new diagnostic and therapeutic avenues across autonomic and post-viral disorders.

Source: Renata Madre Booyens, Mare Vlok, Cecile Bester, Rashmin Hira, M Asad Khan, Douglas B Kell, Satish R Raj, Etheresia Pretorius. Post-translational modifications within fibrinaloid microclot complexes distinguish Pre-COVID-19 Postural Orthostatic Tachycardia Syndrome, Long COVID, and Long COVID-POTS and reveal disease-specific molecular pathways.
bioRxiv 2025.12.29.696828; doi: https://doi.org/10.64898/2025.12.29.696828 https://www.biorxiv.org/content/10.64898/2025.12.29.696828v1 (Full text available as PDF file)

Dysregulation of lipid metabolism, energy production, and oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome, Gulf War Syndrome and fibromyalgia

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), Gulf War Syndrome (GWS), and Fibromyalgia (FM) are complex, chronic illnesses with overlapping clinical features. Symptoms that are reported across these conditions include post-exertional malaise (PEM), fatigue, and pain, yet the etiology of these illnesses remains largely unknown. Diagnosis is challenging in patients with these conditions as definitive biomarkers are lacking; patients are required to meet clinical criteria and often undergo lengthy testing to exclude other conditions, a process that is often prolonged, costly, and burdensome for patients.

The identification of reliable validated biomarkers could facilitate earlier and more accurate diagnosis and drive the development of targeted pharmacological therapies that might address the underlying pathophysiology of these diseases. Major driving forces for biomarker identification are the advancing fields of metabolomics and proteomics that allow for comprehensive characterization of metabolites and proteins in biological specimens. Recent technological developments in these areas enable high-throughput analysis of thousands of metabolites and proteins from a variety of biological samples and model systems, that provides a powerful approach to unraveling the metabolic phenotypes associated with these complex diseases.

Emerging evidence suggests that ME/CFS, GWS, and FM are all characterized by disturbances in metabolic pathways, particularly those related to energy production, lipid metabolism, and oxidative stress. Altered levels of key metabolites in these pathways have been reported in studies highlighting potential common biochemical abnormalities. The precise mechanisms driving altered metabolic pathways in ME/CFS, GWS, and FM remain to be elucidated; however, the elevated oxidative stress observed across these illnesses may contribute to symptoms and offer a potential target for therapeutic intervention.

Investigating the mechanisms, and their role in the disease process, could provide insights into disease pathogenesis and reveal novel treatment targets. As such, comprehensive metabolomic and proteomic analyses are crucial for advancing the understanding of these conditions in-order to identify both common, and unique, metabolic alterations that could serve as diagnostic markers or therapeutic targets.

Source: Davis L, Higgs M, Snaith A, Lodge TA, Strong J, Espejo-Oltra JA, Kujawski S, Zalewski P, Pretorius E, Hoerger M, Morten KJ. Dysregulation of lipid metabolism, energy production, and oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome, Gulf War Syndrome and fibromyalgia. Front Neurosci. 2025 Mar 10;19:1498981. doi: 10.3389/fnins.2025.1498981. PMID: 40129725; PMCID: PMC11931034. https://pmc.ncbi.nlm.nih.gov/articles/PMC11931034/ (Full text)

Data-independent LC-MS/MS analysis of ME/CFS plasma reveals a dysregulated coagulation system, endothelial dysfunction, downregulation of complement machinery

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating chronic condition that is characterized by unresolved fatigue, post-exertion symptom exacerbation (PESE), cognitive dysfunction, orthostatic intolerance, and other symptoms. ME/CFS lacks established clinical biomarkers and requires further elucidation of disease mechanisms.

A growing number of studies demonstrate signs of hematological and cardiovascular pathology in ME/CFS cohorts, including hyperactivated platelets, endothelial dysfunction, vascular dysregulation, and anomalous clotting processes. To build on these findings, and to identify potential biomarkers that can be related to pathophysiology, we measured differences in protein expression in platelet-poor plasma (PPP) samples from 15 ME/CFS study participants and 10 controls not previously infected with SARS-CoV-2, using DIA LC-MS/MS.

We identified 24 proteins that are significantly increased in the ME/CFS group compared to the controls, and 21 proteins that are significantly downregulated. Proteins related to clotting processes – thrombospondin-1 (important in platelet activation), platelet factor 4, and protein S – were differentially expressed in the ME/CFS group, suggestive of a dysregulated coagulation system and abnormal endothelial function. Complement machinery was also significantly downregulated, including C9 which forms part of the membrane attack complex. Additionally, we identified a significant upregulation of lactotransferrin, protein S100-A9, and an immunoglobulin variant.

The findings from this experiment further implicate the coagulation and immune system in ME/CFS, and bring to attention the pathology of or imposed on the endothelium. This study highlights potential systems and proteins that require further research with regards to their contribution to the pathogenesis of ME/CFS, symptom manifestation, and biomarker potential, and also gives insight into the hematological and cardiovascular risk for ME/CFS individuals affected by diabetes mellitus.

Source: Nunes, M., Vlok, M., Proal, A. et al. Data-independent LC-MS/MS analysis of ME/CFS plasma reveals a dysregulated coagulation system, endothelial dysfunction, downregulation of complement machinery. Cardiovasc Diabetol 23, 254 (2024). https://doi.org/10.1186/s12933-024-02315-x https://cardiab.biomedcentral.com/articles/10.1186/s12933-024-02315-x (Full text)

Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) has received more attention since the characterization of Long COVID (LC), a condition somewhat similar in symptom presentation and, to some extent, pathophysiological mechanisms. A prominent feature of LC pathology is amyloid, fibrinolysis-resistant fibrin(ogen) fragments, termed microclots. Despite prior identification of microclots in ME/CFS, quantitative analysis has remained challenging due to the reliance on representative micrographs and software processing for estimations.

Addressing this gap, the present study uses a cell-free imaging flow cytometry approach, optimized for the quantitative analysis of Thioflavin T-stained microclots, to precisely measure microclot concentration and size distribution across ME/CFS, LC, and healthy cohorts. We refer to our cell-free flow cytometry technique for detecting microclots as ‘flow clotometry’.

We demonstrate significant microclot prevalence in ME/CFS and LC, with LC patients exhibiting the highest concentration (18- and 3-fold greater than the healthy and ME/CFS groups, respectively). This finding underscores a common pathology across both conditions, emphasizing a dysregulated coagulation system. Moreover, relating to microclot size distribution, the ME/CFS group exhibited a significantly higher prevalence across all area ranges when compared to the controls, but demonstrated a significant difference for only a single area range when compared to the LC group.

This suggests a partially overlapping microclot profile in ME/CFS relative to LC, despite the overall higher concentration in the latter. The present study paves the way for prospective clinical application that aims to efficiently detect, measure and treat microclots.

Source: Etheresia Pretorius, Massimo Nunes, Jan pretorius et al. Flow Clotometry: Measuring Amyloid Microclots in ME/CFS, Long COVID, and Healthy Samples with Imaging Flow Cytometry, 24 June 2024, PREPRINT (Version 1) available at Research Square [https://doi.org/10.21203/rs.3.rs-4507472/v1] https://www.researchsquare.com/article/rs-4507472/v1 (Full text)

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: the biology of a neglected disease

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic, debilitating disease characterised by a wide range of symptoms that severely impact all aspects of life. Despite its significant prevalence, ME/CFS remains one of the most understudied and misunderstood conditions in modern medicine. ME/CFS lacks standardised diagnostic criteria owing to variations in both inclusion and exclusion criteria across different diagnostic guidelines, and furthermore, there are currently no effective treatments available.

Moving beyond the traditional fragmented perspectives that have limited our understanding and management of the disease, our analysis of current information on ME/CFS represents a significant paradigm shift by synthesising the disease’s multifactorial origins into a cohesive model. We discuss how ME/CFS emerges from an intricate web of genetic vulnerabilities and environmental triggers, notably viral infections, leading to a complex series of pathological responses including immune dysregulation, chronic inflammation, gut dysbiosis, and metabolic disturbances.

This comprehensive model not only advances our understanding of ME/CFS’s pathophysiology but also opens new avenues for research and potential therapeutic strategies. By integrating these disparate elements, our work emphasises the necessity of a holistic approach to diagnosing, researching, and treating ME/CFS, urging the scientific community to reconsider the disease’s complexity and the multifaceted approach required for its study and management.

Source: Arron HE, Marsh BD, Kell DB, Khan MA, Jaeger BR, Pretorius E. Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: the biology of a neglected disease. Front Immunol. 2024 Jun 3;15:1386607. doi: 10.3389/fimmu.2024.1386607. PMID: 38887284; PMCID: PMC11180809. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11180809/ (Full text)

Possible Role of Fibrinaloid Microclots in Postural Orthostatic Tachycardia Syndrome (POTS): Focus on Long COVID

Abstract:

Postural orthostatic tachycardia syndrome (POTS) is a common accompaniment of a variety of chronic, inflammatory diseases, including long COVID, as are small, insoluble, ‘fibrinaloid’ microclots.
We here develop the argument, with accompanying evidence, that fibrinaloid microclots, through their ability to block the flow of blood through microcapillaries and thus cause tissue hypoxia, are not simply correlated with but in fact, by preceding it, may be a chief intermediary cause of POTS, in which tachycardia is simply the body’s exaggerated ‘physiological’ response to hypoxia. Similar reasoning accounts for the symptoms bundled under the term ‘fatigue’.
Amyloids are known to be membrane disruptors, and when their targets are nerve membranes, this can explain neurotoxicity and hence the autonomic nervous system dysfunction that contributes to POTS. Taken together as a system view, we indicate that fibrinaloid microclots can serve to link POTS and fatigue in long COVID in a manner that is at once both mechanistic and explanatory. This has clear implications for the treatment of such diseases.
Source: Kell DB, Khan MA, Kane B, Lip GYH, Pretorius E. Possible Role of Fibrinaloid Microclots in Postural Orthostatic Tachycardia Syndrome (POTS): Focus on Long COVID. Journal of Personalized Medicine. 2024; 14(2):170. https://doi.org/10.3390/jpm14020170 https://www.mdpi.com/2075-4426/14/2/170 (Full text)

Herpesvirus Infection as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome

Abstract:

Understanding the pathophysiology of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is critical for advancing treatment options. This review explores the novel hypothesis that herpesviruses’ infection of endothelial cells (ECs) may underlie ME/CFS symptomatology.
We review evidence linking herpesviruses to persistent EC infection and the implications for endothelial dysfunction, encompassing blood flow regulation, coagulation, and cognitive impairment – symptoms consistent with ME/CFS and Long COVID. The paper provides a synthesis of current research on herpesvirus latency and reactivation, detailing the impact on ECs and subsequent systemic complications, including latent modulation and long-term maladaptation.
We suggest that the chronicity of ME/CFS symptoms and the multisystemic nature of the disease may be partly attributable to herpesvirus-induced endothelial maladaptation. Our conclusions underscore the necessity for further investigation into the prevalence and load of herpesvirus infection within ECs of ME/CFS patients.
This review offers a conceptual advance by proposing an endothelial infection model as a systemic mechanism contributing to ME/CFS, steering future research towards potentially unexplored avenues in understanding and treating this complex syndrome.
Source: Nunes, J.M.; Kell, D.B.; Pretorius, E. Herpesvirus Infection as a Systemic Pathological Axis in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Preprints 2024, 2024011486. https://doi.org/10.20944/preprints202401.1486.v1 https://www.preprints.org/manuscript/202401.1486/v1 (Full text available as PDF file)

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (Me/Cfs): The Biology of a Neglected Disease

Abstract:

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic disease with debilitating symptoms that impact all aspects of life. The diverse symptom presentation indicates that ME/CFS is likely to have a multifactorial origin. However, it is an extremely understudied disease with no standardised diagnostic criteria or proven treatment avenues. It is hypothesised that environmental insults (such as acute infection, mainly viral) or stress in genetically susceptible individuals may trigger the development of ME/CFS.

These insults result in acute inflammatory responses, along with aberrant immune activation. A spiralling disruption of homeostasis promotes subsequent patho-mechanisms including gut dysbiosis and systemic inflammation, and eventually a pathological clotting system, chronic endothelialitis, vasoconstriction, and hypoxia. Additionally, dysfunctional energy metabolism including oxidative stress is also present in the development of ME/CFS. Since the exact pathophysiology of ME/CFS remains unclear, additional research is required to reveal further insight into this “neglected” disease.

Source: Arron, Hayley and Marsh, Benamin and Khan, M. Asad and Jaeger, Beate and Kell, Douglas and Pretorius, Etheresia, Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (Me/Cfs): The Biology of a Neglected Disease. Available at SSRN: https://ssrn.com/abstract=4622074 or http://dx.doi.org/10.2139/ssrn.4622074 (Full text available as PDF file)

Accelerating discovery: A novel flow cytometric method for detecting fibrin(ogen) amyloid microclots using long COVID as a model

Abstract:

Long COVID has become a significant global health and economic burden, yet there are currently no established methods or diagnostic tools to identify which patients might benefit from specific treatments. One of the major pathophysiological factors contributing to Long COVID is the presence of hypercoagulability; this results in insoluble amyloid microclots that are resistant to fibrinolysis. Our previous research using fluorescence microscopy has demonstrated a significant amyloid microclot load in Long COVID patients. However, this approach lacked the elements of statistical robustness, objectivity, and rapid throughput.

In the current study, we have used imaging flow cytometry for the first time to show a significantly increased concentration and size of these microclots. We identified notable variations in size and fluorescence between microclots in Long COVID and those of controls even using a 20× objective. By combining cell imaging and the high-event-rate and full-sample analysis nature of a conventional flow cytometer, imaging flow cytometry can eliminate erroneous results and increase accuracy in gating and analysis beyond what pure quantitative measurements from conventional flow cytometry can provide.

Although imaging flow cytometry was used in our study, our results suggest that the signals indicating the presence of microclots should be easily detectable using a conventional flow cytometer. Flow cytometry is a more widely available technique than fluorescence microscopy and has been used in pathology laboratories for decades, rendering it a potentially more suitable and accessible method for detecting microclots in individuals suffering from Long COVID or conditions with similar pathology, such as myalgic encephalomyelitis.

Source: Turner S, Laubscher GJ, Khan MA, Kell DB, Pretorius E. Accelerating discovery: A novel flow cytometric method for detecting fibrin(ogen) amyloid microclots using long COVID as a model. Heliyon. 2023 Aug 29;9(9):e19605. doi: 10.1016/j.heliyon.2023.e19605. PMID: 37809592; PMCID: PMC10558872. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10558872/ (Full text)