Determining biomarkers for a diagnostic test of Chronic Fatigue Syndrome / Myalgic Encephalomyelitis

Abstract

Chronic fatigue syndrome / Myalgic encephalomyelitis (CFS/ME) is an unexplained chronic multi-system illness, which leads to a lifetime of impairment. Symptoms indicative of the disease include immunological dysregulation, incapacitating fatigue, cognitive impairments, pain in the lymph nodes, and post-exertional sickness. The pathophysiology of CFS/ME is unknown, however several potential causes of development of the disease have been speculated. Consequently, due to a lack of understanding both medically and scientifically, there are no validated laboratory tests for diagnosis or management. Filling this gap of knowledge by finding an appropriate biomarker would aid the medical community in determining appropriate treatment. This pilot study aims to evaluate whether certain molecules in the blood of CFS/ME patients may be biomarkers to aid in diagnosis. This involves analysis of cytokine levels in CFS/ME patients with matched controls, and changes in cytokine levels following a pre-determined exercise regimen, in which patients are known to perform poorly. Additionally, the ratio of translational initiation factor eIF2α, to its stress activated phosphorylated peIF2α derivative, will be examined in white blood cells of patients and controls. We hypothesise that these molecules have the potential to be informative in relation to immune deterioration, a hallmark of CFS/ME. To test this hypothesis we obtained blood samples from 10 CFS/ME patients and 10 matched controls according to the International Consensus Criteria. In addition, following a separate exercise study with 11 CFS/ME patients and 3 MS patients as controls, all samples were fractionated to separate plasma, lymphocyte and neutrophils. The prepared plasma samples from all participants were simultaneously examined for expression of 27 cytokines. Statistical analysis revealed Interleukin-9 (IL9) and vascular endothelial growth factor (VEGF) expressed at significantly higher levels in CFS/ME patients compared to healthy controls (P<0.05), and additionally IL- 13 and interferon gamma-inducible protein (IP-10) expressed at significantly lower levels in CFS/ME patients compared to controls (P<0.05). Cytokine dysfunction was ii confirmed with a longitudinal approach. The exercise-induced cytokine analysis revealed cytokines that were dysregulated directly following exercise stress. IL-7, IL-9 and IL-10 were significantly upregulated directly following a 12-minute exercise task (P<0.05), whilst IL-13 was significantly downregulated (P<0.05). IL-9 and IL-13 were represented in both analyses, which indicates their potential as biomarkers for CFS/ME. Western analysis of proteins isolated from white blood cells detects both phosphorylated and unphosphorylated states of eIF2α with respective purified antibodies. Western analysis showed protein peIF2α to be slightly higher in patient lymphocytes compared to controls, though further experiments will need to be undertaken to determine the value of this result. This work suggests potential biomarkers that can be seen in blood, and Western blot analyses of additional patient and control samples will define whether a change in molecular state of eIF2α could be developed into a diagnostic test. This study gives a means to form a larger cohort for analyses on CFS/ME patients to enhance on current results in terms of identifying a possible biomarker for the disease.