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)
Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious and complex physical illness that affects all body systems with a multiplicity of symptoms, but key hallmarks of the disease are pervasive fatigue and ‘post-exertional malaise’, exacerbation after physical and/or mental activity of the intrinsic fatigue and other symptoms that can be highly debilitating and last from days to months. Although the disease can vary widely between individuals, common symptoms also include pain, cognitive deficits, sleep dysfunction, as well as immune, neurological and autonomic symptoms. Typically, it is a very isolating illness socially, carrying a stigma because of the lack of understanding of the cause and pathophysiology.
Methods: To gain insight into the pathophysiology of ME/CFS, we examined the proteomes of peripheral blood mononuclear cells (PBMCs) by SWATH-MS analysis in a small well-characterised group of patients and matched controls. A principal component analysis (PCA) was used to stratify groups based on protein abundance patterns, which clearly segregated the majority of the ME/CFS patients (9/11) from the controls. This majority subgroup of ME/CFS patients was then further compared to the control group.
Results: A total of 60 proteins in the ME/CFS patients were differentially expressed (P < 0.01, Log10 (Fold Change) > 0.2 and < -0.2). Comparison of the PCA selected subgroup of ME/CFS patients (9/11) with controls increased the number of proteins differentially expressed to 99. Of particular relevance to the core symptoms of fatigue and post-exertional malaise experienced in ME/CFS, a proportion of the identified proteins in the ME/CFS groups were involved in mitochondrial function, oxidative phosphorylation, electron transport chain complexes, and redox regulation. A significant number were also involved in previously implicated disturbances in ME/CFS, such as the immune inflammatory response, DNA methylation, apoptosis and proteasome activation.
Conclusions: The results from this study support a model of deficient ATP production in ME/CFS, compensated for by upregulation of immediate pathways upstream of Complex V that would suggest an elevation of oxidative stress. This study and others have found evidence of a distinct pathology in ME/CFS that holds promise for developing diagnostic biomarkers.
Source: Sweetman E, Kleffmann T, Edgar C, de Lange M, Vallings R, Tate W. A SWATH-MS analysis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome peripheral blood mononuclear cell proteomes reveals mitochondrial dysfunction. J Transl Med. 2020 Sep 24;18(1):365. doi: 10.1186/s12967-020-02533-3. PMID: 32972442. https://translational-medicine.biomedcentral.com/articles/10.1186/s12967-020-02533-3 (Full text)
BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterised by fatigue and post-exertional malaise. Its pathogenesis is poorly understood. GDF15 is a circulating protein secreted by cells in response to a variety of stressors. The receptor for GDF15 is expressed in the brain, where its activation results in a range of responses. Among the conditions in which circulating GDF15 levels are highly elevated are mitochondrial disorders, where early skeletal muscle fatigue is a key symptom. We hypothesised that GDF15 may represent a marker of cellular stress in ME/CFS.
METHODS: GDF15 was measured in serum from patients with ME/CFS (n = 150; 100 with mild/moderate and 50 with severe symptoms), “healthy volunteers” (n = 150) and a cohort of patients with multiple sclerosis (n = 50).
RESULTS: Circulating GDF15 remained stable in a subset of ME/CFS patients when sampled on two occasions ~ 7 months (IQR 6.7-8.8) apart, 720 pg/ml (95% CI 625-816) vs 670 pg/ml (95% CI 598-796), P = 0.5. GDF15 levels were 491 pg/ml in controls (95% CI 429-553), 546 pg/ml (95% CI 478-614) in MS patients, 560 pg/ml (95% CI 502-617) in mild/moderate ME/CFS patients and 602 pg/ml (95% CI 531-674) in severely affected ME/CFS patients. Accounting for potential confounders, severely affected ME/CFS patients had GDF15 concentrations that were significantly increased compared to healthy controls (P = 0.01). GDF15 levels were positively correlated (P = 0.026) with fatigue scores in ME/CFS.
CONCLUSIONS: Severe ME/CFS is associated with increased levels of GDF15, a circulating biomarker of cellular stress that appears which stable over several months.
Source: Melvin A, Lacerda E, Dockrell HM, O’Rahilly S, Nacul L. Circulating levels of GDF15 in patients with myalgic encephalomyelitis/chronic fatigue syndrome. J Transl Med. 2019 Dec 4;17(1):409. doi: 10.1186/s12967-019-02153-6. https://www.ncbi.nlm.nih.gov/pubmed/31801546
Immune dysfunction has been posited as a key element in the aetiology of chronic fatigue syndrome (CFS) since the illness was first conceived. However, systematic reviews have yet to quantitatively synthesise inflammatory biomarkers across the literature. We undertook a systematic review and meta-analysis to quantify available data on circulating inflammatory proteins, examining studies recruiting patients with a CFS diagnosis and a non-affected control group. Results were meta-analysed from 42 studies.
Patients with CFS had significantly elevated tumour necrosis factor (ES = 0.274, p < 0.001), interleukin-2 (ES = 0.203, p = 0.006), interleukin-4 (ES = 0.373, p = 0.004), transforming growth factor-β (ES = 0.967, p < 0.001) and c-reactive protein (ES = 0.622, p = 0.019). 12 proteins did not differ between groups.
These data provide some support for an inflammatory component in CFS, although inconsistency of results indicates that inflammation is unlikely to be a primary feature in all those suffering from this disorder. It is hoped that further work will elucidate whether there are subgroups of patients with clinically-relevant inflammatory dysfunction, and whether inflammatory cytokines may provide a prognostic biomarker or moderate treatment effects.
Source: Strawbridge R, Sartor ML, Scott F, Cleare AJ. Inflammatory proteins are altered in chronic fatigue syndrome-A systematic review and meta-analysis. Neurosci Biobehav Rev. 2019 Aug 26;107:69-83. doi: 10.1016/j.neubiorev.2019.08.011. [Epub ahead of print] https://www.ncbi.nlm.nih.gov/pubmed/31465778
Myalgic encephalomyelitis/chronic fatigue syndrome affects at least 2 million people in the United States. Despite its prevalence, there’s no laboratory test for the disease, and its diagnosis is based on symptoms like exhaustion, unrefreshing sleep, and light sensitivity. For patients with this debilitating condition, getting a diagnosis is often a long and expensive process. Now, a long-awaited biomarker-based test for the mysterious disease could be on the horizon.
BACKGROUND: Fibromyalgia syndrome (FMS) is a chronic pain syndrome. A plausible pathogenesis of the disease is uncertain and the pursuit of measurable biomarkers for objective identification of affected individuals is a continuing endeavour in FMS research. Our objective was to perform an explorative metabolomics study (1) to elucidate the global urinary metabolite profile of patients suffering from FMS, and (2) to explore the potential of this metabolite information to augment existing medical practice in diagnosing the disease.
METHODS: We selected patients with a medical history of persistent FMS (n = 18), who described their recent state of the disease through the Fibromyalgia Impact Questionnaire (FIQR) and an in-house clinical questionnaire (IHCQ). Three control groups were used: first-generation family members of the patients (n = 11), age-related individuals without any indications of FMS or related conditions (n = 10), and healthy young (18-22 years) individuals (n = 20). All subjects were female and the biofluid under investigation was urine. Correlation analysis of the FIQR showed the FMS patients represented a well-defined disease group for this metabolomics study. Spectral analyses of urine were conducted using a 500 MHz 1H nuclear magnetic resonance (NMR) spectrometer; data processing and analyses were performed using Matlab, R, SPSS and SAS software.
RESULTS AND DISCUSSION: Unsupervised and supervised multivariate analyses distinguished all three control groups and the FMS patients, and significant increases in metabolites related to the gut microbiome (hippuric, succinic and lactic acids) were observed. We have developed an algorithm for the diagnosis of FMS consisting of three metabolites – succinic acid, taurine and creatine – that have a good level of diagnostic accuracy (Receiver Operating Characteristic (ROC) analysis – area under the curve 90%) and on the pain and fatigue symptoms for the selected FMS patient group.
CONCLUSION: Our data and comparative analyses indicated an altered metabolic profile of patients with FMS, analytically detectable within their urine. Validation studies may substantiate urinary metabolites to supplement information from medical assessment, tender-point measurements and FIQR questionnaires for an improved objective diagnosis of FMS.
Source: Malatji BG, Meyer H, Mason S, Engelke UFH, Wevers RA, van Reenen M, Reinecke CJ. A diagnostic biomarker profile for fibromyalgia syndrome based on an NMR metabolomics study of selected patients and controls. BMC Neurol. 2017 May 11;17(1):88. doi: 10.1186/s12883-017-0863-9. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426044/ (Full article)
Chronic fatigue syndrome (CFS) remains an incompletely characterized illness, in part due to controversy regarding its definition, biological basis and diagnosis. Biomarkers are objective measures that may lead to improvements in our understanding of CFS by providing a more coherent and consistent approach to study, diagnosis and treatment of the illness. Such metrics may allow us to distinguish between CFS subtypes – each defined by characteristic biomarkers – currently conflated under the single, heterogeneous condition of CFS. These delineations, in turn, may guide more granular, focused, and targeted treatment strategies based on more precise characterizations of the illness. Here, we review potential CFS biomarkers related to neurological and immunological components of the illness, and discuss how these biomarkers may be used to move the field of CFS forward, emphasizing clinical utility and potential routes of future research.
Source: Fischer DB, William AH, Strauss AC, Unger ER, Jason L, Marshall GD Jr, Dimitrakoff JD. Chronic Fatigue Syndrome: The Current Status and Future Potentials of Emerging Biomarkers. Fatigue. 2014 Jun 1;2(2):93-109. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052724/ (Full article)
PURPOSE: To study the reliability of a serum angiotensin-converting enzyme (ACE) assay as a marker for the chronic fatigue-immune dysfunction syndrome (CFIDS), and to compare some enzyme characteristics of ACE in CFIDS with that in sarcoidosis.
PATIENTS AND METHODS: Forty-nine patients with CFIDS and 56 endemic control subjects from Lyndonville, New York, and Charlotte, North Carolina; plus 23 untreated patients with active sarcoidosis, 24 with sarcoidosis receiving corticosteroid therapy, and 32 patient controls without sarcoidosis from California. Serum ACE levels were determined with a spectrophotometric method. The effect of freezing and thawing and the effect of storage at 4 degrees C were compared between CFIDS and sarcoidosis samples.
RESULTS: Serum ACE levels were elevated in 80% of patients with CFIDS and 30% of endemic control subjects as compared with 9.4% of nonendemic California control subjects. The ACE activity in CFIDS differed from that in sarcoidosis because of its lability with storage at 4 degrees C in CFIDS and its partial activation with freezing and thawing. Thus, ACE activity was elevated in the majority of CFIDS patients either upon initial assay or upon a subsequent assay after refreezing. ACE activity was elevated in 87% of patients with active sarcoidosis and was not affected by storage or freezing and thawing.
CONCLUSIONS: Serum ACE elevations may be a useful marker for CFIDS, especially if a method can be developed to distinguish ACE in CFIDS from that in sarcoidosis. The sensitivity for CFIDS was 80%, with 68% specificity in an endemic area. The increased prevalence of serum ACE elevations in endemic controls as compared with nonendemic controls suggests that an ACE increase may be an early manifestation of CFIDS and supports the concept that CFIDS is a definite disease state.
Source: Lieberman J, Bell DS. Serum angiotensin-converting enzyme as a marker for the chronic fatigue-immune dysfunction syndrome: a comparison to serum angiotensin-converting enzyme in sarcoidosis. Am J Med. 1993 Oct;95(4):407-12. http://www.ncbi.nlm.nih.gov/pubmed/8213873
Natural killer (NK)3 cells are large granular lymphocytes that appear to play a significant role in the host’s defense against viral infection. We performed an extensive phenotypic and functional characterization of NK cells on 41 patients with the chronic fatigue syndrome (CFS), or “chronic active Epstein-Barr virus infection” syndrome, and on 23 age- and sex-matched asymptomatic control subjects in an attempt to further characterize this illness.
These studies demonstrated that a majority of patients with CFS have low numbers of NKH1+T3- lymphocytes, a population that represents the great majority of NK cells in normal individuals. CFS patients had normal numbers of NKH1+T3+ lymphocytes, a population that represents a relatively small fraction of NK cells in normal individuals.
When tested for cytotoxicity against a variety of different target cells, patients with CFS consistently demonstrated low levels of killing. After activation of cytolytic activity with recombinant interleukin 2, patients were able to display increased killing against K562 but most patients remained unable to lyse Epstein-Barr virus-infected B cell targets. Additional cytotoxicity experiments were carried out utilizing anti-T3 monoclonal antibody to block killing by NKH1+T3+ cells.
These experiments indicated that the NK cell that appears to be responsible for much of the functional activity remaining in patients with CFS belongs to the NKH1+T3+ subset, which under normal circumstances represents only approximately 20% of the NK cell population.
Source: Caligiuri M, Murray C, Buchwald D, Levine H, Cheney P, Peterson D, Komaroff AL, Ritz J. Phenotypic and functional deficiency of natural killer cells in patients with chronic fatigue syndrome. J Immunol. 1987 Nov 15;139(10):3306-13. http://www.ncbi.nlm.nih.gov/pubmed/2824604
All twenty-four cytokines were altered in both long- and short-term patients compared to controls.
Press Release: NEW YORK (Feb. 27, 2015)—Researchers at the Center for Infection and Immunity at Columbia University’s Mailman School of Public Health identified distinct immune changes in patients diagnosed with chronic fatigue syndrome, known medically as myalgic encephalomyelitis (ME/CFS) or systemic exertion intolerance disease. The findings could help improve diagnosis and identify treatment options for the disabling disorder, in which symptoms range from extreme fatigue and difficulty concentrating to headaches and muscle pain.
These immune signatures represent the first robust physical evidence that ME/CFS is a biological illness as opposed to a psychological disorder, and the first evidence that the disease has distinct stages. Results appear online in the new American Association for the Advancement of Science journal, Science Advances.
With funding to support studies of immune and infectious mechanisms of disease from the Chronic Fatigue Initiative of the Hutchins Family Foundation, the researchers used immunoassay testing methods to determine the levels of 51 immune biomarkers in blood plasma samples collected through two multicenter studies that represented a total of 298 ME/CFS patients and 348 healthy controls. They found specific patterns in patients who had the disease three years or less that were not present in controls or in patients who had the disease for more than three years. Short duration patients had increased amounts of many different types of immune molecules called cytokines. The association was unusually strong with a cytokine called interferon gamma that has been linked to the fatigue that follows many viral infections, including Epstein-Barr virus (the cause of infectious mononucleosis). Cytokine levels were not explained by symptom severity.
“We now have evidence confirming what millions of people with this disease already know, that ME/CFS isn’t psychological,” states lead author Mady Hornig, MD, director of translational research at the Center for Infection and Immunity and associate professor of Epidemiology at Columbia’s Mailman School. “Our results should accelerate the process of establishing the diagnosis after individuals first fall ill as well as discovery of new treatment strategies focusing on these early blood markers.”
There are already human monoclonal antibodies on the market that can dampen levels of a cytokine called interleukin-17A that is among those the study shows were elevated in early-stage patients. Before any drugs can be tested in a clinical trial, Dr. Hornig and colleagues hope to replicate the current, cross-sectional results in a longitudinal study that follows patients for a year to see how cytokine levels, including interleukin-17A, differ within individual patients over time, depending on how long they have had the disease.
Stuck in High Gear
The study supports the idea that ME/CFS may reflect an infectious “hit-and-run” event. Patients often report getting sick, sometimes from something as common as infectious mononucleosis (Epstein-Barr virus), and never fully recover. The new research suggests that these infections throw a wrench in the immune system’s ability to quiet itself after the acute infection, to return to a homeostatic balance; the immune response becomes like a car stuck in high gear. “It appears that ME/CFS patients are flush with cytokines until around the three-year mark, at which point the immune system shows evidence of exhaustion and cytokine levels drop,” says Dr. Hornig. “Early diagnosis may provide unique opportunities for treatment that likely differ from those that would be appropriate in later phases of the illness.”
The investigators went to great lengths to carefully screen participants to make sure they had the disease. The researchers also recruited greater numbers of patients whose diagnosis was of relatively recent onset. Patients’ stress levels were standardized; before each blood draw, patients were asked to complete standardized paperwork, in part to engender fatigue. The scientists also controlled for factors known to affect the immune system, including the time of day, season and geographic location where the samples were taken, as well as age, sex and ethnicity/race.
In 2012, W. Ian Lipkin, MD, director of the Center for Infection and Immunity, and colleagues reported the results of a multicenter study that definitively ruled out two viruses thought to be implicated in ME/CFS: XMRV (xenotropic murine leukemia virus [MLV]-related virus) and murine retrovirus-like sequences (designated pMLV: polytropic MLV). In the coming weeks, Drs. Hornig and Lipkin expect to report the results of a second study of cerebrospinal fluid from ME/CFS patients. In separate ongoing studies, they are looking for “molecular footprints” of the specific agents behind the disease—be they viral, bacterial, or fungal—as well as the longitudinal look at how plasma cytokine patterns change within ME/CFS patients and controls across a one-year period, as noted above.
“This study delivers what has eluded us for so long: unequivocal evidence of immunological dysfunction in ME/CFS and diagnostic biomarkers for disease,” says senior author W. Ian Lipkin, MD, also the John Snow Professor of Epidemiology at Columbia’s Mailman School. “The question we are trying to address in a parallel microbiome project is what triggers this dysfunction.”
Co-authors include Andrew F. Schultz, Xiaoyu Che, and Meredith L. Eddy at the Center for Infection and Immunity; Jose G. Montoya at Stanford University; Anthony L. Komaroff at Harvard Medical School; Nancy G. Klimas at Nova Southeastern University; Susan Levine at Levine Clinic; Donna Felsenstein at Massachusetts General Hospital; Lucinda Bateman at Fatigue Consultation Clinic; and Daniel L. Peterson and Gunnar Gottschalk at Sierra Internal Medicine. The authors report no competing interests.
Support for the study was provided by the Chronic Fatigue Initiative of the Hutchins Family Foundation and the National Institutes of Health (AI057158; Northeast Biodefense Center-Lipkin).
About Columbia University’s Mailman School of Public Health
Founded in 1922, Columbia University’s Mailman School of Public Health pursues an agenda of research, education, and service to address the critical and complex public health issues affecting New Yorkers, the nation and the world. The Mailman School is the third largest recipient of NIH grants among schools of public health. Its over 450 multi-disciplinary faculty members work in more than 100 countries around the world, addressing such issues as preventing infectious and chronic diseases, environmental health, maternal and child health, health policy, climate change & health, and public health preparedness. It is a leader in public health education with over 1,300 graduate students from more than 40 nations pursuing a variety of master’s and doctoral degree programs. For more information, please visit www.mailman.columbia.edu.
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Media contact: Tim Paul, Columbia University’s Mailman School of Public Health, 212-305-2676 or tp2111@columbia.edu.
Note: You can read the full text of the Columbia study HERE.
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