Impact of long-term cryopreservation on blood immune cell markers in ME/CFS: implications for biomarker discovery

Impact of long-term cryopreservation on blood immune cell markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: implications for biomarker discovery, by Elisabet Gómez-Mora,  Jorge Carrillo,  Víctor Urrea,  Josepa Rigau,  José Alegre,  Cecilia Cabrera,  Elisa Oltra,  Jesús Castro-Marrero and  Julià Blanco in Front. Immunol., 17 November 2020 [doi.org/10.3389/fimmu.2020.582330]

 

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a complex neuroimmune disorder characterized by numerous symptoms of unknown etiology. The ME/CFS immune markers reported so far have failed to generate a clinical consensus, perhaps partly due to the limitations of biospecimen biobank.

To address this issue, we performed a comparative analysis of the impact of long-term biobanking on previously identified immune markers and also explored additional potential immune markers linked to infection in ME/CFS. A correlation analysis of marker cryostability across immune cell subsets based on flow cytometry immunophenotyping of fresh blood and frozen PBMC samples collected from individuals with ME/CFS (n = 18) and matched healthy controls (n = 18) was performed.

The functionality of biobanked samples was assessed on the basis of cytokine production assay after stimulation of frozen PBMCs. T cell markers defining Treg subsets and the expression of surface glycoprotein CD56 in T cells and the frequency of the effector CD8 T cells, together with CD57 expression in NK cells, appeared unaltered by biobanking. By contrast, NK cell markers CD25 and CD69 were notably increased, and NKp46 expression markedly reduced, by long-term cryopreservation and thawing. Further exploration of Treg and NK cell subsets failed to identify significant differences between ME/CFS patients and healthy controls in terms of biobanked PBMCs.

Our findings show that some of the previously identified immune markers in T and NK cell subsets become unstable after cell biobanking, thus limiting their use in further immunophenotyping studies for ME/CFS. These data are potentially relevant for future multisite intervention studies and cooperative projects for biomarker discovery using ME/CFS biobanked samples. Further studies are needed to develop novel tools for the assessment of biomarker stability in cryopreserved immune cells from people with ME/CFS.

Posted in News | Tagged , , , , , , , , , , , , , , | Comments Off on Impact of long-term cryopreservation on blood immune cell markers in ME/CFS: implications for biomarker discovery

High prevalence of perineural cysts in patients with FM & CFS

High prevalence of perineural cysts in patients with fibromyalgia and Chronic Fatigue Syndrome, by Mieke Hulens, Frans Bruyninckx, Wim Dankaerts, Ricky Rasschaert, Peter De Mulder, Ingeborg Stalmans, Greet Vansant, Chris Bervoets in Pain Medicine, 1 Dec 2020 pnaa410 [doi.org/10.1093/pm/pnaa410]

 

Research abstract

Objective:
Pain in fibromyalgia (FM) and chronic fatigue syndrome (CFS) is assumed to originate from central sensitization. Perineural cysts or Tarlov cysts (TCs) are nerve root dilations resulting from pathologically increased cerebrospinal fluid pressure. These cysts initially affect sensory neurons and axons in dorsal root ganglia and produce sensory symptoms (pain and paresthesia). Symptomatic TC (STC) patients often complain about widespread pain and fatigue. Consequently, STC patients may initially be diagnosed with FM, CFS, or both. The objective of this study was to document the prevalence of TCs in patients diagnosed with FM or CFS.

Design:
A retrospective study.

Setting:
An outpatient clinic for musculoskeletal disorders.

Subjects:
Patients diagnosed with FM according to the 1990 American College of Rheumatology criteria or with CFS according to the 1994 Centers for Disease Control criteria were selected.

Methods:
Review of lumbar and sacral magnetic resonance imaging scans including TCs ≥5 mm in size.

Results:
In total, 197 patients with FM, CFS, or both underwent magnetic resonance imaging. Ninety-one percent were women. The mean age was 48.1 (±11.9) years. TCs were observed in 39% of patients, with a mean size of 11.8 (±5.2) mm. In males, the prevalence was 12%, vs. 42% in females.

Conclusions:
In patients diagnosed with FM or CFS, the prevalence of TCs was three times higher than that in the general population. This observation supports the hypothesis that STCs, FM, and CFS may share the same pathophysiological mechanism, i.e., moderately increased cerebrospinal fluid pressure, causing irritation of neurons and axons in dorsal root ganglia.

 

Read full article

Posted in News | Tagged , , , , , , , , , , , , , | Comments Off on High prevalence of perineural cysts in patients with FM & CFS

Role of mitochondria, oxidative stress & the response to antioxidants ME/CFS: a possible approach to SARS-CoV-2 ‘long-haulers’?

Role of mitochondria, oxidative stress and the response to antioxidants in myalgic encephalomyelitis/chronic fatigue syndrome: a possible approach to SARS-CoV-2 ‘long-haulers’?, by , Warren Tate in Chronic Dis Transl Med. 2020 Nov 21 [doi: 10.1016/j.cdtm.2020.11.002]

 

Review article abstract:

A significant number of SARS-CoV-2 (COVID-19) pandemic patients have developed chronic symptoms lasting weeks or months which are very similar to those described for myalgic encephalomyelitis/chronic fatigue syndrome.

This paper reviews the current literature and understanding of the role that mitochondria, oxidative stress and antioxidants may play in the understanding of the pathophysiology and treatment of chronic fatigue. It describes what is known about the dysfunctional pathways which can develop in mitochondria and their relationship to chronic fatigue.

It also reviews what is known about oxidative stress and how this can be related to the pathophysiology of fatigue, as well as examining the potential for specific therapy directed at mitochondria for the treatment of chronic fatigue in the form of antioxidants. This review identifies areas which require urgent, further research in order to fully elucidate the clinical and therapeutic potential of these approaches.

Conclusion

Research into mitochondrial function of ME/CFS has been increasing in recent years and appears to hold potential for better understanding this enigmatic disease that may be increasing its prevalence as a result of the SARS-CoV-2 pandemic. So far, however, the results have proved somewhat contradictory. There are several reasons for this.

An accurate diagnosis for ME/CFS is difficult in the absence a molecular biomarker diagnostic test, individuals in the ME/CFS group are often heterogeneous and have differing disease severity.

There are multiple clinical case definitions that are being used for diagnosis. A vicious circle exists: without a biochemical diagnostic test it is difficult to be certain which patients have ME/CFS and not another fatigue illness with overlapping symptoms, but determining a valid test requires identifying an homogenous group of test subjects.

If ME/CFS is indeed a state of CoQ10 deficiency – particularly in high energy-demand organs like the brain – then an effective way of assessing CoQ10 is needed. This could then be a cellular biomarker that is monitored after oral supplementation in treatment of ME/CFS. Changes in mitochondrial function and markers of oxidative stress may be the biological component most impacted upon by CoQ10 supplementation, due to CoQ10’s vital role in the electron transport chain.

There is also clear need to establish whether MitoQ does indeed improve mitochondrial function and oxidative stress and lessens symptoms in both ME/CFS and chronic symptoms from SARS-CoV-19. There is a biologically plausible mechanism for expected improvement with MitoQ’s superior bioavailability to cells and to mitochondria within them, and by how it can restore oxidative balance and therefore improve mitochondrial function.

A respiratory index, like the BHI, also has the potential to predict and monitor pathophysiology in both ME/CFS and ‘long-haulers’ from SARS-CoV-2. Further research into ME/CFS and SARS-CoV-2, two perplexing and seriously debilitating diseases, is urgently required.

Posted in News | Tagged , , , , , , , , , , , | Comments Off on Role of mitochondria, oxidative stress & the response to antioxidants ME/CFS: a possible approach to SARS-CoV-2 ‘long-haulers’?

Systematic review of Primary Outcome Measurements for CFS/ME in randomized controlled trials

Systematic review of primary Outcome Measurements for Chronic Fatigue Syndrome/ Myalgic Encephalomyelitis (CFS/ME) in randomized controlled trials, by  Do-Young Kim, Jin-Seok Lee and Chang-Gue Son in J. Clin. Med. 2020, 9(11) 3463 [doi.org/10.3390/jcm9113463] (This article belongs to the Special Issue Chronic Fatigue Syndrome/Myalgic Encephalomyelitis: Diagnosis and Treatment)

 

Research abstract:

Background:

Due to its unknown etiology, the objective diagnosis and therapeutics of chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) are still challenging.

Generally, the patient-reported outcome (PRO) is the major strategy driving treatment response because the patient is the most important judge of whether changes are meaningful.

Methods:

In order to determine the overall characteristics of the main outcome measurement applied in clinical trials for CFS/ME, we systematically surveyed the literature using two electronic databases, PubMed and the Cochrane Library, throughout June 2020. We analyzed randomized controlled trials (RCTs) for CFS/ME focusing especially on main measurements.

Results:

Fifty-two RCTs out of a total 540 searched were selected according to eligibility criteria. Thirty-one RCTs (59.6%) used single primary outcome and others adapted ≥2 kinds of measurements. In total, 15 PRO-derived tools were adapted (50 RCTs; 96.2%) along with two behavioral measurements for adolescents (4 RCTs; 7.7%). The 36-item Short Form Health Survey (SF-36; 16 RCTs), Checklist Individual Strength (CIS; 14 RCTs), and Chalder Fatigue Questionnaire (CFQ; 11 RCTs) were most frequently used as the main outcomes. Since the first RCT in 1996, Clinical Global Impression (CGI) and SF-36 have been dominantly used each in the first and following decade (26.1% and 28.6%, respectively), while both CIS and Multidimensional Fatigue Inventory (MFI) have been the preferred instruments (21.4% each) in recent years (2016 to 2020).

Conclusions:

This review comprehensively provides the choice pattern of the assessment tools for interventions in RCTs for CFS/ME. Our data would be helpful practically in the design of clinical studies for CFS/ME-related therapeutic development.

Posted in News | Tagged , , , , , , | Comments Off on Systematic review of Primary Outcome Measurements for CFS/ME in randomized controlled trials

Paradigm shift to disequilibrium in the genesis of orthostatic intolerance

Paradigme shift to disequilibrium in the genesis of orthostatic intolerance in patients with CFS, by K Miwa in European Heart Journal Vol 40, Supp 1, Oct 2019

 

Research abstract

Background
Chronic fatigue syndrome (CFS) characterized by severe disabling fatigue and prolonging post-exertional malaise. The dysfunction of the central nervous system associated with myalgic encephalomyelitis (ME) has been postulated as the main cause of CFS. Orthostatic intolerance (OI) causes a marked reduction in the activities of daily living and impairs the quality of life in patients with ME/CFS. OI has been surmised to be a cardiovascular symptom with cerebral hypo-perfusion and exaggerated sympathetic nervous activation.

Purpose
Postural instability or disequilibrium may be involved in the etiology of OI because postural stability is an essential element for static balance.

Methods
The study comprised 72 patients with ME/CFS (18 men and 54 women; mean age, 37±10 years), who underwent neurological examinations and the active 10-min standing test.

Results
Disequilibrium defined as instability on standing with their feet together and eyes shut, was detected in 23 (32%) patients while postural orthostatic tachycardia in 16 (22%). Compared with 49 patients without disequilibrium, patients with disequilibrium more prevalently failed to complete the 10-min standing test (74% vs. 4%, p<0.01) and body sway was significantly more prevalently observed during the test (100% vs. 12%, p<0.01).

The performance status score was significantly higher in patients with disequilibrium than those without it (median: 7 vs. 5, p<0.01), suggesting more severely restricted activity of daily living in the former. The prevalence of postural orthostatic tachycardia during the standing test was comparable between the patients with disequilibrium (23%) and those without it (22%, p=1.00). The 19 (26%) patients who failed to complete the 10-min standing test had disequilibrium more prevalently than those who completed it (89% vs. 11%, p<0.01).

Performance status score was significantly higher in patients who failed to complete it than those who completed it (median: 6 vs. 5, p<0.01), suggesting more severe restriction of activity of daily living in the former. Significantly higher rates of disequilibrium (89% vs. 11%, p<0.01), unstable standing on one leg (84% vs. 17%, p<0.01) as well as abnormal tandem gait (79% vs. 11%, p<0.01) were noted in patients who failed to complete it than those who completed it. Body sway during the standing test was significantly more prevalently observed in the patients who failed to complete it than those who completed it (89% vs. 23%, p<0.01).

The prevalence of postural orthostatic tachycardia during the standing test was comparable between the patients who failed to complete it and those who completed it (21% vs. 23%, p=1.00). Among the patients who failed to complete it 8 had the previous study records which revealed that 6 of them had completed it 6–24 months before when all the 6 patients had had no disequilibrium.

Conclusion
Disequilibrium should be recognized as an important cause of OI in patients with ME/CFS.

Posted in News | Tagged , , | Comments Off on Paradigm shift to disequilibrium in the genesis of orthostatic intolerance

Bias caused by reliance on patient-reported outcome measures in non-blinded randomized trials

Bias caused by reliance on patient-reported outcome measures in non-blinded randomized trials: an in-depth look at exercise therapy for chronic fatigue syndrome
by Michiel Tack, David M Tuller & Caroline Struthers in Fatigue: Biomedicine, Health & Behavior Vol 8, 2020 Issue 4, 25 Nov 2020 [doi.org/10.1080/21641846.2020.1848262]

 

Research abstract: 

Background
Several randomized trials have reported that graded exercise therapy (GET) is an effective treatment for chronic fatigue syndrome (CFS). These trials were not blinded and relied on patient-reported outcome measures (PROMs). We investigate whether bias introduced by this study design influenced the results.

Methods
We extracted standardized mean differences from the most recent meta-analysis on exercise therapy for CFS to analyze their size, consistency over time, and congruence with objective measurements. A narrative review methodology was used to examine mediation analyses, plausible mechanisms of improvement, and risk of response bias.

Results
Patient-reported improvements in exercise trials for CFS tend to be small, transient, and poorly supported by objective measurements. The risk of expectancy effects and response bias was high as patients were actively encouraged to adopt a positive attitude towards exercise therapy. Mediation analyses suggest that self-reported improvements in fatigue and physical function are not mediated by objective measures of fitness.

Conclusions
Treatment effects seen in exercise trials for CFS could be the result of bias associated with the use of PROMs in non-blinded trials. This might explain the discrepancy between positive results reported in randomized trials and views on exercise therapy expressed by patient organizations. We hope that this case study furthers critical assessment of patient-reported improvements in areas of medicine where blinding of therapists and trial participants faces practical limitations.

Read full paper

A summary of our paper on exercise therapy and bias caused by lack of blinding

Posted in News | Tagged , , , , , , , | Comments Off on Bias caused by reliance on patient-reported outcome measures in non-blinded randomized trials

Podcast – Long COVID & ME/CFS

Long-term COVID & ME/CFS – a podcast

 

A TWiV podcast hosted by Prof Vincent Racaniello with Prof Mady Hornig, Fiona Lowenstein, Prof David Tuller and 2 patients to discuss long-term COVID & the similarities & differences with ME/CFS”. [1h 52min]

 

 

Posted in News | Tagged , , , , , | Comments Off on Podcast – Long COVID & ME/CFS

Changes in DNA methylation profiles of ME/CFS patients reflect systemic dysfunctions

Changes in DNA methylation profiles of myalgic encephalomyelitis/chronic fatigue syndrome patients reflect systemic dysfunctions, by AM Helliwell, EC Sweetman,
PA Stockwell, CD Edgar, A Chatterjee & WP Tate in Clinical Epigenetics vol 12, no: 167 (2020)

 

Research abstract:

Background:
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a lifelong debilitating disease with a complex pathology not yet clearly defined. Susceptibility to ME/CFS involves genetic predisposition and exposure to environmental factors, suggesting an epigenetic association. Epigenetic studies with other ME/CFS cohorts have used array- based technology to identify differentially methylated individual sites. Changes in RNA quantities and protein abundance have been documented in our previous investigations with the same ME/CFS cohort used for this study.

Results:
DNA from a well-characterised New Zealand cohort of 10 ME/CFS patients and 10 age-/sex-matched healthy controls was isolated from peripheral blood mononuclear (PBMC) cells, and used to generate reduced genome-scale DNA methylation maps using reduced representation bisulphite sequencing (RRBS). The sequencing data were analysed utilising the DMAP analysis pipeline to identify differentially methylated fragments, and the MethylKit pipeline was used to quantify methylation differences at individual CpG sites.

DMAP identified 76 differentially methylated fragments and Methylkit identified 394 differentially methylated cytosines that included both hyper- and hypo-methylation. Four clusters were identified where differentially methylated DNA fragments overlapped with or were within close proximity to multiple differentially methylated individual cytosines. These clusters identified regulatory regions for 17 protein encoding genes related to metabolic and immune activity.

Analysis of differentially methylated gene bodies (exons/introns) identified 122 unique genes. Comparison with other studies on PBMCs from ME/CFS patients and controls with array technology showed 59% of the genes identified in this study were also found in one or more of these studies. Functional pathway enrichment analysis identified 30 associated pathways. These included immune, metabolic and neurological-related functions differentially regulated in ME/CFS patients compared to the matched healthy controls.

Conclusions:
Major differences were identified in the DNA methylation patterns of ME/CFS patients that clearly distinguished them from the healthy controls. Over half found in gene bodies with RRBS in this study had been identified in other ME/CFS studies using the same cells but with array technology. Within the enriched functional immune, metabolic and neurological pathways, a number of enriched neurotransmitter and neuropeptide reactome pathways highlighted a disturbed neurological pathophysiology within the patient group.

Posted in News | Tagged , , , , , , , , , , | Comments Off on Changes in DNA methylation profiles of ME/CFS patients reflect systemic dysfunctions

A hierarchical logistic regression predicting rapid respiratory rates from post-exertional malaise

A hierarchical logistic regression predicting rapid respiratory rates from post-exertional malaise, by Joseph Cotler, Ben Z Katz, Corine Reurts-Post, Ruud Vermeulen & Leonard A Jason in Scientific Reports vol 10, no. 19933 (2020) Published online: 16 Nov 2020 [doi.org/10.1080/21641846.2020.1845287]

 

Research abstract: 

Background:
Past research has found high rates of hyperventilation in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), but hyperventilation can be influenced by psychological factors. Clinical respiratory rates have been less frequently assessed.

Aim
This study aimed to identify the predictors of rapid respiratory rates in patients referred an outpatient clinic specializing in ME/CFS.

Methods
Adults (n = 216) referred to an outpatient clinic specializing in Myalgic Encephalomyelitis/ Chronic Fatigue Syndrome (ME/CFS) participated in a two-day cardiopulmonary exercise test. As part of that evaluation, subjects had resting respiratory rates measured on two consecutive days. The current study used questionnaires to assess the relationship between tachypnea (rapid respiratory rates) and a variety of domains including post-exertional malaise (PEM), a common complaint in patients with ME/CFS, and psychiatric/somatic symptoms, using hierarchical logistic regression analysis.

Results
PEM was a significant predictor of tachypnea, while psychological/somatic assessments and sedentary behaviors were not significantly predictive of tachypnea.

Conclusions
These findings suggest that respiratory rate may be useful as an objective clinical metric of PEM, and potentially ME/CFS.

Read full article

Posted in News | Tagged , , , , , , , , , , , | Comments Off on A hierarchical logistic regression predicting rapid respiratory rates from post-exertional malaise

The profile of circulating microRNAs in ME & their relation to symptom severity, & disease pathophysiology

Profile of circulating microRNAs in myalgic encephalomyelitis and their relation to symptom severity, and disease pathophysiology, by Evguenia Nepotchatykh, Wesam Elremaly, Iurie Caraus, Christian Godbout, Corinne Leveau, Lynda Chalder, Catherine Beaudin, Emi Kanamaru, Renata Kosovskaia, Shawn Lauzon, Yanick Maillet, Anita Franco, Viorica Lascau-Coman, Saadallah Bouhanik, Yaned Patricia Gaitan, Dawei Li & Alain Moreau in Scientific Reports volume 10, no. 19620 (2020) 12 Nov 2020

 

Abstract:

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex chronic disease, rooted in multi-system dysfunctions characterized by unexplained debilitating fatigue. Post-exertional malaise (PEM), defined as the exacerbation of the patient’s symptoms following minimal physical or mental stress, is a hallmark of ME/CFS. While multiple case definitions exist, there is currently no well-established biomarkers or laboratory tests to diagnose ME/CFS.

Our study aimed to investigate circulating microRNA expression in severely ill ME/CFS patients before and after an innovative stress challenge that stimulates PEM.

Our findings highlight the differential expression of eleven microRNAs associated with a physiological response to PEM. The present study uncovers specific microRNA expression signatures associated with ME/CFS in response to PEM induction and reports microRNA expression patterns associated to specific symptom severities.

The identification of distinctive microRNA expression signatures for ME/CFS through a provocation challenge is essential for the elucidation of the ME/CFS pathophysiology, and lead to accurate diagnoses, prevention measures, and effective treatment options.

 

CTV News: Montreal researchers develop new test to diagnose chronic fatigue syndrome

“We based our test on the cardinal symptom of the disease, which is discomfort after exertion,” said Moreau. “This is really what is most specific to myalgic encephalomyelitis.”

The test could also allow patients to be grouped into subgroups, in order not only to better understand the molecular mechanisms involved in certain symptoms but also to better select patients who could benefit from certain treatments.

“It becomes more interesting for both trying to work with more homogeneous subgroups to understand the disease and see what explains the severity or all of the symptoms,” said Moreau. “It may speed up research to better understand the disease.”

Finally, the test could allow early detection of patients in whom ME is developing, so that they can start treating them as quickly as possible.

OMF: Montréal ME/CFS Research Center Paper Published in Scientific Reports

MicroRNAs can represent potential indicators for diseases such as ME/CFS, and changes in microRNA expression could indicate cellular dysfunction and degeneration. Using a test to induce mild-but-reproducible Post Exertional Malaise (PEM), our team has so far uncovered and validated 11 different microRNAs associated with ME/CFS that are capable of differentiating ME/CFS patients from healthy patients — with 90 percent accuracy! 

This post-exertional stress challenge provoking PEM in ME/CFS patients has helped us gain unprecedented insight into the pathophysiology of ME/CFS. Based on the 11 different microRNA signatures discovered in ME/CFS, machine learning algorithms have also led to the classification of ME/CFS patients into four clusters associated with symptom severity.

These exciting results could lead to the development of a new, non-invasive diagnostic test for ME/CFS, a prognostic tool used to predict future cases, and identification of effective treatment options.

The Scientist: Blood MicroRNA Patterns Linked to Chronic Fatigue Syndrome

To Williams, the major value of the study lies in an analysis Moreau’s team conducted with the microRNA data that untangles the molecular pathways the 11 sequences are involved in.

This revealed that 7 out of the 11 microRNAs were involved in regulating immune functions, which “certainly fits with one arm of the research that suggests that immune activation is very important in leading to chronic fatigue,” she says.

An additional network analysis flagged the key genes each microRNA is associated with and other diseases they’ve been linked to, which included viral infection, sleep disorders, and cognitive impairments. “Using a network approach, you can start to shed light on which cellular processes are important. And then if that ties in with what we know already about the cellular processes in ME/CFS, then that all begins to paint a bit of a picture.”

Posted in News | Tagged , , , , , , , , , , , , , , , , , , , , | Comments Off on The profile of circulating microRNAs in ME & their relation to symptom severity, & disease pathophysiology