Category: Biomarker

Gene expression alterations at baseline and following moderate exercise in patients with Chronic Fatigue Syndrome and Fibromyalgia Syndrome

Abstract:

OBJECTIVES: To determine mRNA expression differences in genes involved in signalling and modulating sensory fatigue, and muscle pain in patients with chronic fatigue syndrome (CFS) and fibromyalgia syndrome (FM) at baseline, and following moderate exercise.

DESIGN: Forty-eight patients with CFS only, or CFS with comorbid FM, 18 patients with FM that did not meet criteria for CFS, and 49 healthy controls underwent moderate exercise (25 min at 70% maximum age-predicted heart rate). Visual-analogue measures of fatigue and pain were taken before, during and after exercise. Blood samples were taken before and 0.5, 8, 24 and 48 h after exercise. Leucocytes were immediately isolated from blood, number coded for blind processing and analyses and flash frozen. Using real-time, quantitative PCR, the amount of mRNA for 13 genes (relative to control genes) involved in sensory, adrenergic and immune functions was compared between groups at baseline and following exercise. Changes in amounts of mRNA were correlated with behavioural measures and functional clinical assessments.

RESULTS: No gene expression changes occurred following exercise in controls. In 71% of patients with CFS, moderate exercise increased most sensory and adrenergic receptor’s and one cytokine gene’s transcription for 48 h. These postexercise increases correlated with behavioural measures of fatigue and pain. In contrast, for the other 29% of patients with CFS, adrenergic α-2A receptor’s transcription was decreased at all time-points after exercise; other genes were not altered. History of orthostatic intolerance was significantly more common in the α-2A decrease subgroup. FM-only patients showed no postexercise alterations in gene expression, but their pre-exercise baseline mRNA for two sensory ion channels and one cytokine were significantly higher than controls.

CONCLUSIONS: At least two subgroups of patients with CFS can be identified by gene expression changes following exercise. The larger subgroup showed increases in mRNA for sensory and adrenergic receptors and a cytokine. The smaller subgroup contained most of the patients with CFS with orthostatic intolerance, showed no postexercise increases in any gene and was defined by decreases in mRNA for α-2A. FM-only patients can be identified by baseline increases in three genes. Postexercise increases for four genes meet published criteria as an objective biomarker for CFS and could be useful in guiding treatment selection for different subgroups.

© 2011 The Association for the Publication of the Journal of Internal Medicine.

 

Source: Light AR, Bateman L, Jo D, Hughen RW, Vanhaitsma TA, White AT, Light KC. Gene expression alterations at baseline and following moderate exercise in patients with Chronic Fatigue Syndrome and Fibromyalgia Syndrome. J Intern Med. 2012 Jan;271(1):64-81. doi: 10.1111/j.1365-2796.2011.02405.x. Epub 2011 Jul 13. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3175315/ (Full article)

 

Assessment of a 44 gene classifier for the evaluation of chronic fatigue syndrome from peripheral blood mononuclear cell gene expression

Abstract:

Chronic fatigue syndrome (CFS) is a clinically defined illness estimated to affect millions of people worldwide causing significant morbidity and an annual cost of billions of dollars. Currently there are no laboratory-based diagnostic methods for CFS. However, differences in gene expression profiles between CFS patients and healthy persons have been reported in the literature.

Using mRNA relative quantities for 44 previously identified reporter genes taken from a large dataset comprising both CFS patients and healthy volunteers, we derived a gene profile scoring metric to accurately classify CFS and healthy samples. This metric out-performed any of the reporter genes used individually as a classifier of CFS.

To determine whether the reporter genes were robust across populations, we applied this metric to classify a separate blind dataset of mRNA relative quantities from a new population of CFS patients and healthy persons with limited success. Although the metric was able to successfully classify roughly two-thirds of both CFS and healthy samples correctly, the level of misclassification was high. We conclude many of the previously identified reporter genes are study-specific and thus cannot be used as a broad CFS diagnostic.

 

Source: Frampton D, Kerr J, Harrison TJ, Kellam P. Assessment of a 44 gene classifier for the evaluation of chronic fatigue syndrome from peripheral blood mononuclear cell gene expression. PLoS One. 2011 Mar 30;6(3):e16872. doi: 10.1371/journal.pone.0016872. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3068152/ (Full article)

 

Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Abstract:

BACKGROUND: There is evidence that myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by activation of immune, inflammatory, oxidative and nitrosative stress (IO&NS) pathways. The present study was carried out in order to examine whether ME/CFS is accompanied by increased levels of plasma peroxides and serum oxidized LDL (oxLDL) antibodies, two biomarkers of oxidative stress.

MATERIAL/METHODS: Blood was collected from 56 patients with ME/CFS and 37 normal volunteers. Severity of ME/CFS was measured using the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale.

RESULTS: Plasma peroxide concentrations were significantly higher in patients with ME/CFS than in normal controls. There was a trend towards significantly higher serum oxLDL antibodies in ME/CFS than in controls. Both biomarkers contributed significantly in discriminating between patients with ME/CFS and normal controls. Plasma peroxide and serum oxLDL antibody levels were both significantly related to one of the FF symptoms.

CONCLUSIONS: The results show that ME/CFS is characterized by increased oxidative stress.

 

Source: Maes M, Kubera M, Uytterhoeven M, Vrydags N, Bosmans E. Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Med Sci Monit. 2011 Apr;17(4):SC11-5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3539515/ (Full article)

Distinct cerebrospinal fluid proteomes differentiate post-treatment lyme disease from chronic fatigue syndrome

Abstract:

BACKGROUND: Neurologic Post Treatment Lyme disease (nPTLS) and Chronic Fatigue (CFS) are syndromes of unknown etiology. They share features of fatigue and cognitive dysfunction, making it difficult to differentiate them. Unresolved is whether nPTLS is a subset of CFS.

METHODS AND PRINCIPAL FINDINGS: Pooled cerebrospinal fluid (CSF) samples from nPTLS patients, CFS patients, and healthy volunteers were comprehensively analyzed using high-resolution mass spectrometry (MS), coupled with immunoaffinity depletion methods to reduce protein-masking by abundant proteins. Individual patient and healthy control CSF samples were analyzed directly employing a MS-based label-free quantitative proteomics approach. We found that both groups, and individuals within the groups, could be distinguished from each other and normals based on their specific CSF proteins (p<0.01). CFS (n = 43) had 2,783 non-redundant proteins, nPTLS (n = 25) contained 2,768 proteins, and healthy normals had 2,630 proteins. Preliminary pathway analysis demonstrated that the data could be useful for hypothesis generation on the pathogenetic mechanisms underlying these two related syndromes.

CONCLUSIONS: nPTLS and CFS have distinguishing CSF protein complements. Each condition has a number of CSF proteins that can be useful in providing candidates for future validation studies and insights on the respective mechanisms of pathogenesis. Distinguishing nPTLS and CFS permits more focused study of each condition, and can lead to novel diagnostics and therapeutic interventions.

 

Source: Schutzer SE, Angel TE, Liu T, Schepmoes AA, Clauss TR, Adkins JN, Camp DG, Holland BK, Bergquist J, Coyle PK, Smith RD, Fallon BA, Natelson BH. Distinct cerebrospinal fluid proteomes differentiate post-treatment lyme disease from chronic fatigue syndrome. PLoS One. 2011 Feb 23;6(2):e17287. doi: 10.1371/journal.pone.0017287. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3044169/ (Full article)

 

Plasma neuropeptide Y: a biomarker for symptom severity in chronic fatigue syndrome

Abstract:

BACKGROUND: Chronic fatigue syndrome (CFS) is a complex, multi-symptom illness with a multisystem pathogenesis involving alterations in the nervous, endocrine and immune systems.Abnormalities in stress responses have been identified as potential triggers or mediators of CFS symptoms. This study focused on the stress mediator neuropeptide Y (NPY). We hypothesized that NPY would be a useful biomarker for CFS.

METHODS: The CFS patients (n = 93) were from the Chronic Fatigue and Related Disorders Clinic at the University of Miami and met the 1994 case definition of Fukuda and colleagues. Healthy sedentary controls (n = 100)) were from NIH or VA funded studies. Another fatiguing, multi-symptom illness, Gulf War Illness (GWI), was also compared to CFS. We measured NPY in plasma using a radioimmunoassay (RIA). Psychometric measures, available for a subset of CFS patients included: Perceived Stress Scale, Profile of Mood States, ATQ Positive & Negative Self-Talk Scores, the COPE, the Beck Depression Inventory, Fatigue Symptom Inventory, Cognitive Capacity Screening Examination, Medical Outcomes Survey Short Form-36, and the Quality of Life Scale.

RESULTS: Plasma NPY was elevated in CFS subjects, compared to controls (p = .000) and to GWI cases (p = .000). Receiver operating characteristics (ROC) curve analyses indicated that the predictive ability of plasma NPY to distinguish CFS patients from healthy controls and from GWI was significantly better than chance alone. In 42 patients with CFS, plasma NPY had significant correlations (<0.05) with perceived stress, depression, anger/hostility, confusion, negative thoughts, positive thoughts, general health, and cognitive status. In each case the correlation (+ or -) was in the anticipated direction.

CONCLUSIONS: This study is the first in the CFS literature to report that plasma NPY is elevated compared to healthy controls and to a fatigued comparison group, GWI patients. The significant correlations of NPY with stress, negative mood, general health, depression and cognitive function strongly suggest that this peptide be considered as a biomarker to distinguish subsets of CFS.

 

Source: Fletcher MA, Rosenthal M, Antoni M, Ironson G, Zeng XR, Barnes Z, Harvey JM, Hurwitz B, Levis S, Broderick G, Klimas NG. Plasma neuropeptide Y: a biomarker for symptom severity in chronic fatigue syndrome. Behav Brain Funct. 2010 Dec 29;6:76. doi: 10.1186/1744-9081-6-76. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3024290/ (Full article)

 

The increase of alpha-melanocyte-stimulating hormone in the plasma of chronic fatigue syndrome patients

Abstract:

BACKGROUND: Despite extensive research, no reliable biological marker for chronic fatigue syndrome (CFS) has yet been identified. However, hyperactivation of melanotrophs in the pituitary gland and increased levels of plasma alpha-melanocyte-stimulating hormone (alpha-MSH) have recently been detected in an animal model of chronic stress. Because CFS is considered to be caused partly by chronic stress events, increased alpha-MSH plasma levels may also occur in CFS patients. We therefore examined alpha-MSH levels in CFS patients.

METHODS: Fifty-five CFS patients, who were previously diagnosed within 10 years of with the disease, were enrolled in this study. Thirty healthy volunteers were studied as controls. Fasting bloods samples were collected in the morning and evaluated for their plasma levels of alpha-MSH, adrenocorticotropic hormone (ACTH), serum cortisol and dehydroepiandrosterone sulfate (DHEA-S). Mean levels of alpha-MSH were compared between the CFS and control groups using Welch’s t test.

RESULTS: The mean plasma alpha-MSH concentration in the CFS group (17.9 +/- 1.0 pg/mL) was significantly higher than that in healthy controls (14.5 +/- 1.0 pg/mL, p = 0.02). However, there was a wide range of values in the CFS group. The factors correlated with the plasma alpha-MSH values were analyzed using Spearman’s rank correlation. A negative correlation was found between the duration of the CFS and the plasma alpha-MSH values (p = 0.04, rs = -0.28), but no correlations with ACTH, cortisol or DHEA-S levels were identified (p = 0.55, 0.26, 0.33, respectively). The CFS patients were divided into two groups: patients diagnosed for <or= 5 years’ duration, and those diagnosed for 5-10 years’ duration. They were compared with the healthy controls using one-way ANOVA and Tukey-Kramer multiple comparison tests. The mean alpha-MSH concentration in the <or= 5 years group was 20.8 +/- 1.2 pg/mL, which was significantly higher than that in the healthy controls (p < 0.01). There was no significant difference between the 5-10 year group (15.6 +/- 1.4 pg/mL) and the healthy controls.

CONCLUSIONS:CFS patients with a disease duration of <or= 5 years had significantly higher levels of alpha-MSH in their peripheral blood. alpha-MSH could be a potent biological marker for the diagnosis of CFS, at least during the first 5 years after onset of the disease.

 

Source: Shishioh-Ikejima N, Ogawa T, Yamaguti K, Watanabe Y, Kuratsune H, Kiyama H. The increase of alpha-melanocyte-stimulating hormone in the plasma of chronic fatigue syndrome patients. BMC Neurol. 2010 Aug 23;10:73. doi: 10.1186/1471-2377-10-73. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933583/ (Full article)

 

A panel of biomarkers accurately identifies CFS/ME patients and contributes to the understanding of the pathophysiology of the disorder

Abstract: 

Background: CFS/ME is a debilitating illness for which no specific biomarkers have been identified, although several immune abnormalities including neuroinflammation have been described. The goal of this study was to assemble a panel of immune and inflammatory markers, with the ability to accurately identify CFS/ME cases.

Objectives: From observations made in clinical practice, four markers were selected (immune and inflammatory). These markers were initially investigated to establish differences between CFS/ME cases and controls. We then evaluated their potential usefulness as a diagnostic biomarker by establishing their specificity and sensitivity.

Methods: Venous blood was collected from 70 male and 70 female CFS/ME patients (mean age 43 and 44 years, respectively – Fukuda case definition was used) as well as 70 male and 70 female healthy controls (mean age 43.5 and 44.5 years, respectively).

Serum Interleukin 8 (IL-8), soluble CD14 (sCD14, a surrogate marker for bacterial LPS), and prostaglandin E2 (PGE2) were measured for all subjects as were absolute CD3- / CD57+ lymphocytes counts (CD57+ lymph), according to accepted clinical laboratory techniques.

We then established median values for all analysed parameters; independent sample t-test, Mann-Whitney test and ROC curve analysis were used to investigate difference linked to gender and age.

Results: ROC Statistics (area under the ROC curve) revealed a significant difference between CFS/ME cases and controls (p <0.001) for the four parameters separately, both in the male and female cohorts. Sensitivity was 74.3 – 80 % (females) and 52.1 – 85.9 % (males). Specificity was 57.1 – 98.1 (females) and 65.7 – 88.6 (males).

Logistic regression analysis for the combination of parameters in our panel (IL-8, sCD14, PGE2 and CD57+ lymph) correctly predicted in 89.36 % of male CFS/ME cases and in 97.14 % of female CFS/ME cases.

Conclusions: This panel differentiates CFS/ME cases from controls with high sensitivity and specificity and therefore represents a potential tool in selecting CFS/ME subjects for clinical studies. Each of these four biological markers relate strongly to the disorder.

PGE2 activates dendritic cells and suppresses their ability to attract T cells. It also suppresses the function of macrophages and neutrophils as well as Th1, CTL-, NK-cell mediated type 1 immunity (e.g. CD3- / CD57+ lymphocytes). PGE2 additionally promotes Th2, Th17 and Tregs and also modulates chemokine production (e.g. IL-8).

When taken together, these data suggest that lipopolysaccharide (LPS), likely from gut bacteria, plays an important role in the pathophysiology of CFS/ME.

This screening panel represents an initial step toward identifying biomarkers to broadly diagnose subjects with CFS/ME.

Subsequent markers will be required to subcategorize CFS/ME subjects in order to tailor therapeutic solutions.

 

Source: Kenny L. De Meirleir1,2, Tatjana Mijatovic3, Eugene Bosmans3, Nossa Van den Vonder2, Vincent Lombardi1. A panel of biomarkers accurately identifies CFS/ME patients and contributes to the understanding of the pathophysiology of the disorder. Abstract from IACFS/ME Conference 2016 Program.

1. Nevada Center for Biomedical Research at University
of Nevada, Reno, USA
2. Himmunitas vzw, Brussels, Belgium
3. RED Laboratories NV, Zellik, Belgium

 

Biomarkers in chronic fatigue syndrome: evaluation of natural killer cell function and dipeptidyl peptidase IV/CD26

Abstract:

BACKGROUND: Chronic Fatigue Syndrome (CFS) studies from our laboratory and others described decreased natural killer cell cytotoxicity (NKCC) and elevated proportion of lymphocytes expressing the activation marker, dipeptidyl peptidase IV (DPPIV) also known as CD26. However, neither these assays nor other laboratory tests are widely accepted for the diagnosis or prognosis of CFS. This study sought to determine if NKCC or DPPIV/CD26 have diagnostic accuracy for CFS.

METHODS/RESULTS: Subjects included female and male CFS cases and healthy controls. NK cell function was measured with a bioassay, using K562 cells and (51)Cr release. Lymphocyte associated DPPIV/CD26 was assayed by qualitative and quantitative flow cytometry. Serum DPPIV/CD26 was measured by ELISA. Analysis by receiver operating characteristic (ROC) curve assessed biomarker potential. Cytotoxic function of NK cells for 176 CFS subjects was significantly lower than in the 230 controls. According to ROC analysis, NKCC was a good predictor of CFS status. There was no significant difference in NK cell counts between cases and controls. Percent CD2+ lymphocytes (T cells and NK cells) positive for DPPIV/C26 was elevated in CFS cases, but there was a decrease in the number of molecules (rMol) of DPPIV/C26 expressed on T cells and NK cells and a decrease in the soluble form of the enzyme in serum. Analyses by ROC curves indicated that all three measurements of DPPIV/CD26 demonstrated potential as biomarkers for CFS. None of the DPPIV/C26 assays were significantly correlated with NKCC.

CONCLUSIONS: By ROC analysis, NKCC and three methods of measuring DPPIV/C26 examined in this study had potential as biomarkers for CFS. Of these, NKCC, %CD2+CD26+ lymphocytes and rMol CD26/CD2+ lymphocyte, required flow cytometry, fresh blood and access to a high complexity laboratory. Soluble DPPIV/C26 in serum is done with a standard ELISA assay, or with other soluble factors in a multiplex type of ELISA. Dipeptidyl peptidase IV on lymphocytes or in serum was not predictive of NKCC suggesting that these should be considered as non-redundant biomarkers. Abnormalities in DPPIV/CD26 and in NK cell function have particular relevance to the possible role of infection in the initiation and/or the persistence of CFS.

 

Source: Fletcher MA, Zeng XR, Maher K, Levis S, Hurwitz B, Antoni M, Broderick G, Klimas NG. Biomarkers in chronic fatigue syndrome: evaluation of natural killer cell function and dipeptidyl peptidase IV/CD26. PLoS One. 2010 May 25;5(5):e10817. doi: 10.1371/journal.pone.0010817. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2876037/ (Full article)

 

Plasma cytokines in women with chronic fatigue syndrome

Abstract:

BACKGROUND: Chronic Fatigue Syndrome (CFS) studies from our laboratory and others have described cytokine abnormalities. Other studies reported no difference between CFS and controls. However, methodologies varied widely and few studies measured more than 4 or 5 cytokines. Multiplex technology permits the determination of cytokines for a large panel of cytokines simultaneously with high sensitivity and with only 30 ul of plasma per sample. No widely accepted laboratory test or marker is available for the diagnosis or prognosis of CFS. This study screened plasma factors to identify circulating biomarkers associated with CFS.

METHODS: Cytokines were measured in plasma from female CFS cases and female healthy controls. Multiplex technology provided profiles of 16 plasma factors including the pro -inflammatory cytokines: tumor necrosis factor alpha (TNFalpha), lymphotoxin alpha (LTalpha), interleukin (IL) – IL-Ialpha, IL-1beta, IL-6; TH1 cytokines: interferon gamma (IFNgamma), IL-12p70, IL-2, IL-15; TH2: IL-4, IL-5; TH17 cytokines, IL-17 and IL-23; anti-inflammatory cytokines IL-10, IL-13; the inflammatory mediator and neutrophil attracting chemokine IL-8 (CXCL8). Analysis by receiver operating characteristic (ROC) curve assessed the biomarker potential of each cytokine.

RESULTS: The following cytokines were elevated in CFS compared to controls: LTalpha, IL-1alpha, IL-1beta, IL-4, IL-5, IL-6 and IL-12. The following cytokines were decreased in CFS: IL-8, IL-13 and IL-15. The following cytokines were not different: TNFalpha, IFNgamma, IL-2, IL-10, IL-23 and IL-17. Applying (ROC) curve analyses, areas under the curves (AUC) for IL-5 (0. 84), LTalpha (0.77), IL-4 (0.77), IL-12 (0.76) indicated good biomarker potential. The AUC of IL-6 (0.73), IL-15 (0.73), IL-8 (0.69), IL-13 (0.68) IL-1alpha (0.62), IL-1beta (0.62) showed fair potential as biomarkers.

CONCLUSION: Cytokine abnormalities are common in CFS. In this study, 10 of 16 cytokines examined showed good to fair promise as biomarkers. However, the cytokine changes observed are likely to more indicative of immune activation and inflammation, rather than specific for CFS. As such, they are targets for herapeutic strategies. Newer techniques allow evaluation of large panels of cytokines in a cost effective fashion.

 

Source: Fletcher MA, Zeng XR, Barnes Z, Levis S, Klimas NG. Plasma cytokines in women with chronic fatigue syndrome. J Transl Med. 2009 Nov 12;7:96. doi: 10.1186/1479-5876-7-96. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2779802/ (Full article)

 

Autoantibodies to lens epithelium-derived growth factor/transcription co-activator P75 (LEDGF/P75) in children with chronic nonspecific complaints and with positive antinuclear antibodies

Abstract:

Autoimmune fatigue syndrome (AIFS) is characterized by chronic nonspecific complaints, consistently positive antinuclear antibodies (ANA), and lack of alternate medical explanations. A newly recognized antibody, named anti-Sa, was detected in approximately 40% of the patients by Western blot (WB) using HeLa extract.

Some patients with AIFS later develop chronic fatigue syndrome (CFS), and most of them are positive for anti-Sa. On the other hand, Muro et al. reported anti-DFS70 in patients with CFS. Anti-Sa and anti-DFS70 were turned out to be same specificities by exchanging studies of blind sera. The target antigen of anti-DFS70 was identified as lens epithelium derived growth factor/transcription co-activator p75 (LEDGF/p75).

The objectives of this study are to confirm whether the target antigen of anti-Sa is also LEDGF/p75, and to develop ELISA system by using recombinant protein. Recombinant protein of LEDGF/p75 was purchased from Protein One (Bethesda, MD, USA). We developed an ELISA system to detect anti-LEDGF/p75 by coating this recombinant protein. 226 sera of AIFS patients (including 36 CFS patients) were applied to this ELISA assay and Western immunoblot, and it was revealed that anti-Sa-positive sera defined by WB and sera positive for anti-LEDGF/p75 on ELISA were identical.

Moreover, reactivities of anti-Sa on WB were inhibited by pre-incubating with recombinant LEDGF/p75, and eluted antibodies from the nitrocellulose membrane could react on the ELISA. These results confirm that the Sa antigen is LEDGF/p75. The ELISA assay using recombinant LEDGF/p75 could be a promising tool for measuring anti-Sa and consequently for diagnosing CFS.

 

Source: Kuwabara N, Itoh Y, Igarshi T, Fukunaga Y. Autoantibodies to lens epithelium-derived growth factor/transcription co-activator P75 (LEDGF/P75) in children with chronic nonspecific complaints and with positive antinuclear antibodies. Autoimmunity. 2009 Sep;42(6):492-6. Doi: 10.1080/08916930902736663. https://www.ncbi.nlm.nih.gov/pubmed/19657776