Category: Methodology

Induced pluripotent stem cells as suitable sensors for fibromyalgia and myalgic encephalomyelitis/chronic fatigue syndrome Monzón-Nomdedeu MB, Morten KJ, Oltra E. Induced pluripotent stem cells as suitable sensors for fibromyalgia and myalgic encephalomyelitis/chronic fatigue syndrome

Abstract:

BACKGROUND: Fibromyalgia (FM) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) are devastating metabolic neuroimmune diseases that are difficult to diagnose because of the presence of numerous symptoms and a lack of specific biomarkers. Despite patient heterogeneity linked to patient subgroups and variation in disease severity, anomalies are found in the blood and plasma of these patients when compared with healthy control groups. The seeming specificity of these “plasma factors”, as recently reported by Ron Davis and his group at Stanford University, CA, United States, and observations by our group, have led to the proposal that induced pluripotent stem cells (iPSCs) may be used as metabolic sensors for FM and ME/CFS, a hypothesis that is the basis for this in-depth review.

AIM: To identify metabolic signatures in FM and/or ME/CFS supporting the existence of disease-associated plasma factors to be sensed by iPSCs.

METHODS: A PRISMA (Preferred Reported Items for Systematic Reviews and Meta-analysis)-based systematic review of the literature was used to select original studies evaluating the metabolite profiles of FM and ME/CFS body fluids. The MeSH terms “metabolomic” or “metabolites” in combination with FM and ME/CFS disease terms were screened against the PubMed database. Only original studies applying omics technologies, published in English, were included. The data obtained were tabulated according to the disease and type of body fluid analyzed. Coincidences across studies were searched and P-values reported by the original studies were gathered to document significant differences found in the disease groups.

RESULTS: Eighteen previous studies show that some metabolites are commonly altered in ME/CFS and FM body fluids. In vitro cell-based assays have the potential to be developed as screening platforms, providing evidence for the existence of factors in patient body fluids capable of altering morphology, differentiation state and/or growth patterns. Moreover, they can be further developed using approaches aimed at blocking or reversing the effects of specific plasma/serum factors seen in patients. The documented high sensitivity and effective responses of iPSCs to environmental cues suggests that these pluripotent cells could form robust, reproducible reporter systems of metabolic diseases, including ME/CFS and FM. Furthermore, culturing iPSCs, or their mesenchymal stem cell counterparts, in patient-conditioned medium may provide valuable information to predict individual outcomes to stem-cell therapy in the context of precision medicine studies.

CONCLUSION: This opinion review explains our hypothesis that iPSCs could be developed as a screening platform to provide evidence of a metabolic imbalance in FM and ME/CFS.

Source: Monzón-Nomdedeu MB, Morten KJ, Oltra E. Induced pluripotent stem cells as suitable sensors for fibromyalgia and myalgic encephalomyelitis/chronic fatigue syndrome. World J Stem Cells 2021; 13(8): 1134-1150 [DOI: 10.4252/wjsc.v13.i8.1134]  https://www.wjgnet.com/1948-0210/full/v13/i8/1134.htm (Full study)

Medical Care Situation of People with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in Germany

Abstract:

Background and Objective: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a severe illness with the hallmark symptom of Post-Exertional Malaise (PEM). Currently, no biomarkers or established diagnostic tests for ME/CFS exist. In Germany, it is estimated that over 300,000 people are affected by ME/CFS. Research from the United States and the UK shows that patients with ME/CFS are medically underserved, as they face barriers to medical care access and are dissatisfied with medical care. The first aim of the current research was to investigate whether patients with ME/CFS are medically underserved in Germany in terms of access to and satisfaction with medical care. Second, we aimed at providing a German-language version of the DePaul Symptom Questionnaire Short Form (DSQ-SF) as a tool for ME/CFS diagnostics and research in German-speaking countries.

Materials and Methods: The current research conducted an online questionnaire study in Germany investigating the medical care situation of patients with ME/CFS. The questionnaire was completed by 499 participants who fulfilled the Canadian Consensus Criteria and reported PEM of 14 h or longer.

Results: Participants frequently reported geographic and financial reasons for not using the available medical services. Furthermore, they reported low satisfaction with medical care by the physician they most frequently visited due to ME/CFS. The German version of the DSQ-SF showed good reliability, a one-factorial structure and construct validity, demonstrated by correlations with the SF-36 as a measure of functional status.

Conclusions: Findings provide evidence that patients with ME/CFS in Germany are medically underserved. The German-language translation of the DSQ-SF provides a brief, reliable and valid instrument to assess ME/CFS symptoms to be used for research and clinical practice in German-speaking countries. Pathways to improve the medical care of patients with ME/CFS are discussed.

Source: Froehlich L, Hattesohl DBR, Jason LA, Scheibenbogen C, Behrends U, Thoma M. Medical Care Situation of People with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in Germany. Medicina (Kaunas). 2021 Jun 23;57(7):646. doi: 10.3390/medicina57070646. PMID: 34201825. https://pubmed.ncbi.nlm.nih.gov/34201825/

Broadband electrical impedance as a novel characterization of oxidative stress in single L6 skeletal muscle cells

Abstract:

Oxidative stress (OS) is one of the leading causes of cytotoxicity and is linked to many human physio-pathological conditions. In particular, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) induced by OS is debilitating to quality of life, while no clear biological markers have been identified for diagnostic measures. Recently, impedance measurements of peripheral blood cells of ME/CFS patients have been shown as a promising approach to diagnose the disease. Inspired by this study and aiming to interrogate muscle cells directly, we investigated if broadband measurements of single muscle cells could differentiate normal and oxidatively stressed cell populations.

We first optimized a protocol through H2O2 treatment to introduce oxidative stress to cultured rat L6 skeletal muscle cells. The treated cells were further characterized through broadband impedance spectroscopy of single cells using a microfluidic lab-on-a-chip system. The resulting dielectric properties of cytoplasm permittivity and conductivity are electrically distinct from normally cultured cells. The reflection and transmission coefficients, ΔS11 and ΔS21, of the normal cells are tightly clustered and closely resemble those of the cell-free solution across the frequency range of 9 kHz to 9 GHz. On the other hand, dielectric properties of the oxidized cells have a wide distribution in the GHz range, deviating both in the positive and negative directions from the normally cultured cells.

Simulation results guide our hypothesis that the dielectric differences could be linked to ion alterations, while calcium imaging directly supports the contribution of calcium flux to the observed deviation of S parameters. The unique electrical profile associated with oxidized cells in the GHz frequencies provide a framework for future development of technologies to diagnose oxidative-stress related diseases such as ME/CFS.

Source: Ferguson C, Pini N, Du X, Farina M, Hwang JMC, Pietrangelo T, Cheng X. Broadband electrical impedance as a novel characterization of oxidative stress in single L6 skeletal muscle cells. Anal Chim Acta. 2021 Aug 15;1173:338678. doi: 10.1016/j.aca.2021.338678. Epub 2021 May 25. PMID: 34172152. https://pubmed.ncbi.nlm.nih.gov/34172152/

Evaluation of natural killer cell assay performance on shipped blood specimens

Abstract:

Documenting the importance of NK cell function as a biomarker for diseases and physiologic conditions including myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), will require assays amenable to clinical implementation and standardization. Research studies typically perform NK functional assays on the day of sample collection. This pilot study was conducted to compare assay formats and specimen processing to identify those that are most tolerant of conditions required for shipping and amenable to standardization as shown by inter-assay and inter-laboratory correlation of results.

We compared performance within and between assays that measure NK cell function using direct cytotoxicity [chromium-51 release (CRCA) or fluorescence (Flow Cytometry Cytotoxicity Assay, FCCA)] or an indirect surrogate marker (CD107a surface expression)]. Additional variables for within/between assay comparisons included time of testing (same day as specimen collection or next day within 24 h), specimen types [whole blood or isolated peripheral blood mononuclear cells (PBMCs)], and processing method (fresh or cryopreserved). Statistical measures included number of samples tested in assay conditions (n), medians (x͂), interquartile range (IQR), Pearson correlation coefficient (R2), and correlation p-value (p).

Samples came from 3 clinics and included 31 participants. Same day testing was only available for the subset of participants enrolled from the site of the laboratory performing CRCA. Results from same day CRCA testing of whole blood were considered the gold standard [n = 10, x͂=10.0%, IQR = 7.2%], and correlated well with PBMCs isolated next day [n = 26, x͂= 15.6%, IQR = 13.1%] [R2 = 0.59, p = 0.03]. Next day CRCA results were compromised using whole blood or frozen PBMCs. Next day FCCA cytotoxicity in PBMC [n = 30, x͂=34.1%, IQR = 15.5%] correlated with same day CRCA PMBC [R2 = 0.8, p = 0.001] and next day CRCA PMBC [R2 = 0.5, p < 0.0001]. CD107a expression after induction by PMA and ionomycin did not correlate with other cytotoxicity measures. NK function can be measured in PBMCs isolated after overnight shipping/storage at ambient temperature and CRCA and FCCA results on this sample type are well correlated.

Source: Querec TD, Abrams J, Stewart JJ, Barnes Z, Balbin E, Klimas N, Fletcher MA, Brown L, Bertolli J, Unger ER. Evaluation of natural killer cell assay performance on shipped blood specimens. J Immunol Methods. 2021 Apr 2:113049. doi: 10.1016/j.jim.2021.113049. Epub ahead of print. PMID: 33819446. https://pubmed.ncbi.nlm.nih.gov/33819446/

An exploratory study of discrepancies between objective and subjective measurement of the physical activity level in female patients with chronic fatigue syndrome

Abstract:

Objective: To explore the ability of a self-report activity diary to measure the physical activity level (PAL) in female patients with chronic fatigue syndrome (CFS) and whether illness-related complaints, health-related quality of life domains (HRQOL) or demographic factors are associated with discrepancies between self-reported and objectively measured PAL.

Methods: Sixty-six patients with CFS, recruited from the chronic fatigue clinic of a university hospital, and twenty matched healthy controls wore an accelerometer (Actical) for six consecutive days and registered their activities in an activity diary in the same period. Participants’ demographic data was collected and all subjects completed the CFS Symptom List (illness-related complaints) daily and Short-Form-36 (HRQOL domains) during the first and second appointment.

Results: A significant, but weak association between the activity diary and Actical was present in patients with CFS (rs = 0.376 and rs = 0.352; p < 0.001) and a moderately strong association in healthy controls (rs = 0.605; and rs = 0.644; p < 0.001) between week and weekend days, respectively. A linear mixed model identified a negative association between age and the discrepancy between the self-reported and objective measure of PA in both patients with CFS and healthy controls.

Conclusion: The activity diary showed limited ability to register the PAL in female patients with CFS. The discrepancy between measures was not explained by illness-related complaints, HRQOL domains or demographic factors. The activity diary cannot replace objective activity monitoring measured with an accelerometer, but may provide additional information about the perceived activity.

Source: Vergauwen K, Huijnen IPJ, Smeets RJEM, Kos D, van Eupen I, Nijs J, Meeus M. An exploratory study of discrepancies between objective and subjective measurement of the physical activity level in female patients with chronic fatigue syndrome. J Psychosom Res. 2021 Mar 10;144:110417. doi: 10.1016/j.jpsychores.2021.110417. Epub ahead of print. PMID: 33773330. https://pubmed.ncbi.nlm.nih.gov/33773330/

Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery

Abstract:

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 biobanking. 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.

Source: Gómez-Mora E, Carrillo J, Urrea V, Rigau J, Alegre J, Cabrera C, Oltra E, Castro-Marrero J, Blanco J. Impact of Long-Term Cryopreservation on Blood Immune Cell Markers in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Implications for Biomarker Discovery. Front Immunol. 2020 Nov 17;11:582330. doi: 10.3389/fimmu.2020.582330. PMID: 33329554; PMCID: PMC7732598. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7732598/  (Full text)

The Prospects of the Two-Day Cardiopulmonary Exercise Test (CPET) in ME/CFS Patients: A Meta-Analysis

Abstract:

Background: The diagnosis of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is problematic due to the lack of established objective measurements. Postexertional malaise (PEM) is a hallmark of ME/CFS, and the two-day cardiopulmonary exercise test (CPET) has been tested as a tool to assess functional impairment in ME/CFS patients. This study aimed to estimate the potential of the CPET.

Methods: We reviewed studies of the two-day CPET and meta-analyzed the differences between ME/CFS patients and controls regarding four parameters: volume of oxygen consumption and level of workload at peak (VO2peak, Workloadpeak) and at ventilatory threshold (VO2@VT, Workload@VT).

Results: The overall mean values of all parameters were lower on the 2nd day of the CPET than the 1st in ME/CFS patients, while it increased in the controls. From the meta-analysis, the difference between patients and controls was highly significant at Workload@VT (overall mean: -10.8 at Test 1 vs. -33.0 at Test 2, p < 0.05), which may reflect present the functional impairment associated with PEM.

Conclusions: Our results show the potential of the two-day CPET to serve as an objective assessment of PEM in ME/CFS patients. Further clinical trials are required to validate this tool compared to other fatigue-inducing disorders, including depression, using well-designed large-scale studies.

Source: Lim EJ, Kang EB, Jang ES, Son CG. The Prospects of the Two-Day Cardiopulmonary Exercise Test (CPET) in ME/CFS Patients: A Meta-Analysis. J Clin Med. 2020 Dec 14;9(12):E4040. doi: 10.3390/jcm9124040. PMID: 33327624. https://pubmed.ncbi.nlm.nih.gov/33327624/

Measuring improvement and deterioration in myalgic encephalomyelitis/chronic fatigue syndrome: the pitfalls of the Chalder Fatigue Questionnaire

Adamson et al. considered a 2-point decrease in Chalder Fatigue Questionnaire score to indicate improvement in fatigue and a 2-point increase in Chalder Fatigue Questionnaire score to indicate deterioration in fatigue.1 While intuitively appealing, data exist that suggest a more complex relationship between changes in Chalder Fatigue Questionnaire scores and clinical change.

Collin and Crawley studied treatment outcomes at 11 specialist myalgic encephalomyelitis/chronic fatigue syndrome clinics in England.2 The authors tabulated mean change in Chalder Fatigue Questionnaire score at one year against Clinical Global Impression scores (see additional file 1, table S3). A 2-point decrease in Chalder Fatigue Questionnaire score was reported patients who deemed their health as follows: ‘no change’, ‘a little worse’, ‘much worse’ and ‘very much worse’. The mean changes in Chalder Fatigue Questionnaire score in those categories were similar, with overlapping 95% confidence intervals within the range [4.77, 2.29]. This suggests that a 2-point decrease in Chalder Fatigue Questionnaire score indicates deterioration or no change in the health of a person with myalgic encephalomyelitis/chronic fatigue syndrome, not improvement.

Read the rest of this letter here: https://sci-hub.se/10.1177/0141076820977843

Source: Kirke KD. Measuring improvement and deterioration in myalgic encephalomyelitis/chronic fatigue syndrome: the pitfalls of the Chalder Fatigue Questionnaire. J R Soc Med. 2020 Dec 15:141076820977843. doi: 10.1177/0141076820977843. Epub ahead of print. PMID: 33319615.

Systematic Review of Primary Outcome Measurements for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) in Randomized Controlled Trials

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.

Source: Kim DY, Lee JS, Son CG. Systematic Review of Primary Outcome Measurements for Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME) in Randomized Controlled Trials. J Clin Med. 2020 Oct 28;9(11):E3463. doi: 10.3390/jcm9113463. PMID: 33126460. https://pubmed.ncbi.nlm.nih.gov/33126460/

Reliability and Validity of the Modified Korean Version of the Chalder Fatigue Scale (mKCFQ11)

Abstract:

Fatigue can accompany various diseases; however, fatigue itself is a key symptom for patients with chronic fatigue syndrome (CFS). Due to the absence of biological parameters for the diagnosis and severity of CFS, the assessment tool for the degree of fatigue is very important. This study aims to verify the reliability and validity of the modified Korean version of the Chalder Fatigue Scale (mKCFQ11).

This study was performed using data from 97 participants (Male: 37, Female: 60) enrolled in a clinical trial for an intervention of CFS. The analyses of the coefficient between the mKCFQ11 score and the Fatigue Severity Scale (FSS), the Visual Analogue Scale (VAS) or the 36-item Short-Form Health Survey (SF-36) at two time points (baseline and 12 weeks) as well as their changed values were conducted. The mKCFQ11 showed strong reliability, as evidenced by the Cronbach’s alpha coefficient of 0.967 for the whole item and two subclasses (0.963 for physical and 0.958 for mental fatigue) along with the suitable validity of the mKCFQ11 structure shown by the principal component analysis. The mKCFQ11 scores also strongly correlated (higher than 0.7) with the VAS, FSS and SF-36 on all data from baseline and 12 weeks and changed values. This study demonstrated the clinical usefulness of the mKCFQ11 instrument, particularly in assessing the severity of fatigue and the evaluation of treatments for patients suffering from CFS.

Source: Ahn YC, Lee JS, Son CG. Reliability and Validity of the Modified Korean Version of the Chalder Fatigue Scale (mKCFQ11). Healthcare (Basel). 2020 Oct 24;8(4):E427. doi: 10.3390/healthcare8040427. PMID: 33114401. https://www.mdpi.com/2227-9032/8/4/427  (Full text)