Date sent: Sat, 10 Nov 2001 Source: Clinical Infectious Diseases Vol 33, #12 Date: December 15, 2001 URL: http://www.journals.uchicago.edu/CID/journal/available.html http://www.journals.uchicago.edu/CID/journal/rapid.html http://www.journals.uchicago.edu/CID/journal/issues/v33n12/010763/010763.html BRIEF REPORT Antiviral Pathway Activation in Patients with Chronic Fatigue Syndrome and Acute Infection -------------------------------------------------------------------------- J. W. Gow,(1) K. Simpson,(1) P. O. Behan,(1) A. Chaudhuri,(1) I. C. McKay,(3) and W. M. H. Behan(2) Departments of (1)Neurology, (2)Pathology, and (3)Immunology, University of Glasgow, Scotland, United Kingdom Received 4 June 2001; electronically published 6 November 2001. Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS. Appropriate informed consent was obtained from all subjects, and the research was carried out within the recommended local and United Kingdom guidelines. Financial support: Myalgic Encephalomyelitis Association, the Linbury Trust, and the Barclay Trust. Reprints or correspondence: Dr. W. M. H. Behan, Dept. of Pathology, Western Infirmary, Glasgow G11 6NT, Scotland, UK (wmb1q@clinmed.gla.ac.uk). ---------------------------------------------------------------------------- Chronic fatigue syndrome (CFS) is a disorder of unknown etiopathogenesis and considerable economic importance, producing disabling fatigue in up to 2% of the general population [1, 2]. Most patients report an acute onset with an influenza-like illness, but no specific virus has been identified (reviewed in [3]). Nonspecific immunologic abnormalities are detectable, however, and the possibility has been raised of defects in general virus-handling mechanisms. Activation of 2 IFN-induced antiviral pathways, the 2,5A synthetase/ ribonuclease latent (RNase L) and RNA-regulated protein kinase (PKR), has been reported in patients with CFS [4-6]. A degradation product of the RNase L pathway, a 37-kDa 2,5A binding polypeptide, has been identified in 88% of patients with CFS, compared with 28% of healthy control subjects [6]. Several laboratories offer detection of this commercially as a diagnostic test, and, if the results are positive, they advise patients that a lengthy and expensive course of therapy to regulate the 2,5A synthetase pathway will be of clinical benefit. Although the 2 pathways are activated during viral and, to a lesser degree, bacterial infections, patients with infections were not included in studies published elsewhere. Our hypothesis was that the reported activation may be related to infection and not specifically to CFS. We studied 22 patients with CFS who were selected according to standard criteria [1]. There were 9 men and 13 women (age range, 19-65 years). The results were compared with those of 10 patients admitted to the Infectious Diseases Unit at Gartnavel General Hospital (Glasgow, Scotland) with severe gastroenteritis (7 men and 3 women; age range, 21-58 years). Twenty-one healthy control subjects were recruited from hospital and university staff (8 men and 13 women; age range, 24-55 years). The mRNA expression of 4 key genes in peripheral blood mononuclear cells (PBMCs) was evaluated. These were the 2,5A-dependent RNase L, the RNase L inhibitor (RLI), the 2,5A synthetase, and the PKR (see figure 1). A housekeeping gene internal control (ableson tyrosine kinase [abl]) was included for comparison. RNA was prepared from 10 mL of PBMCs and amplified by use of a standard reverse-transcriptase PCR technique. PCR products were electrophoresed through 2% agarose and visualized under ultraviolet radiation. Images were stored as TIF/GIF files for quantitative analysis on gel documentation software. The ratio of mRNA of the antiviral pathway genes compared with that of the abl (housekeeping gene) was calculated. Each ratio for gene expression was tested for differences among the 3 groups by applying three 2-sample t tests to each possible pair of groups on the basis of the logarithmic data and the pooled variance of the 3 groups; we compensated for the multiplicity of tests by using Holm's method [7]. The confidence criteria relate to sets of 3 tests considered together. It can be seen from table 1 that there was no evidence of any difference -for any of the genes- between patients with CFS and healthy control subjects (groups 2 and 3). In contrast, the patients with infection had the following evidence of activation of both pathways: gene expression for PKR was increased, with logarithmic values higher by 0.9843, as compared with healthy control subjects, which is equivalent to a factor of 9.6; and gene expression for the inhibitor RLI was higher by a factor of ~17 in the infected patients when compared with the other 2 groups. The latter is likely to be related to the time that the sample was obtained - that is, ~24 h after onset of symptoms, at admission to hospital. No differences in RNase L or 2,5A synthetase gene expression levels were detected between the groups. In studies published elsewhere, the 3 research groups that have examined these pathways in CFS only compared patients with healthy control subjects or with patients who had nonspecific syndromes (fibromyalgia and multiple chemical sensitivity syndrome), but not with patients who had infections. Because these pathways are known to be activated during routine infection, patients with known infection are a very important control group. It was reported that 2,5A levels and RNase L activity were significantly elevated in patients with CFS, and both levels returned to normal after treatment with poly(I)-poly(C12U) [4]. Gene expression for 2,5A synthetase, RNase L, and RL1 were investigated, and a significant decrease in RL1 mRNA was found [5]. This work culminated in the report that a breakdown product, a 37-kDa 2,5A binding polypeptide, was present in a significant proportion of patients with CFS (88% vs. 28% in healthy control subjects), with the proposal that this formed the basis of a diagnostic test [6]. We conclude from our results, however, that patients with CFS showed no significant activation of either pathway. These cascades can remain up-regulated to a degree for months after normal endemic infection, and residual nonspecific increases may account for the results reported elsewhere. Therefore, assay of antiviral pathway activation is unlikely to form a rational basis for a diagnostic test for CFS. Acknowledgment We are grateful to Dr. D. Kennedy (Brownlee Infectious Disease Centre at Gartnavel General Hospital, Glasgow, Scotland) for his help and advice. Figure caption Figure 1. Diagram of key genes in the IFN-induced antiviral pathways. ATP, adenosine triphosphate; elF, eukaryotic initiation factor; P elF, phosphorylated elF; PKR, RNA-regulated protein kinase; RNase L, ribonuclease latent. Table 1. Statistical analysis of ribonuclease latent (RNase L) and RNA-regulated protein kinase (PKR) pathway activation in patients with chronic fatigue syndrome (CFS). ---------------------------------------------------------------- Enzyme, Mean difference (95% CI) P^a group -------------------- Rank-sum test t Test ---------------------------------------------------------------- RNase L 1,2 -0.8890 (-1.79875 to 0.020737) .022 .057 1,3 -0.6897 (-1.54468 to 0.165271) .17 .14 2,3 0.1993 (-0.39075 to 0.789361) .60 .50 PKR 1,3 -0.9843 (-186692 to 0.101759) .08 .024 1,2 -0.9723 (-1.78958 to -0.155010) .08 .024 2,3 -0.0120 (-0.58027 to 0.556180) .94 .97 RLI 1,3 -1.2379 (-2.02793 to -0.447944) .0016 .00092 1,2 -0.8815 (-1.61309 to -0.149996) .0068 .015 2,3 -0.3564 (-0.86501 to 0.152220) .22 .17 2.5A synthetase 1,2 -0.95552 (-2.05246 to 0.14207) .068 .11 2,3 0.4471 (-0.37173 to 1.26590) .64 .43 1,3 -0.5081 (-1.40438 to 0.38817) .64 .43 ---------------------------------------------------------------- NOTE. Enzyme group 1 consisted of infected patients; group 2, of patients with CFS; and group 3, of healthy patients. RLI, RNase L inhibitor. a Adjusted by use of Holm's method [7]. References 1. Fukuda K, Straus SE, Hickie I, et al. The chronic fatigue syndrome: a comprehensive approach to its definition and study. Ann Intern Med 1994; 121:953-9. 2. Frustrating survey of chronic fatigue [editorial]. Lancet 1996; 348(9033):971. 3. Behan WMH. Is chronic fatigue syndrome a muscle disorder? In: Serratrice G, Paget J Azulay J-Ph, eds. Exercise intolerance and muscle contracture. Paris: Springer-Verlag France, 1999:121-32. 4. Suhadolnik RJ, Reichenbach NL, Hitzges P, et al. Changes in the 2-5A synthetase/RNase L antiviral pathway in a controlled clinical trial with poly(I)-Poly(C12U) in chronic fatigue syndrome. In Vivo 1994; 8:599-604. 5. Vojdani A, Choppa PC, Lapp CW. Downregulation of RNase L inhibitor correlates with upregulation of interferon-induced proteins (2-5A synthetase and RNase L in patients with chronic fatigue immune dysfunction syndrome. J Clin Lab Immunol 1998; 50:1-16. 6. De Meirleir K, Bisbal C, Campine I, et al. A 37kDa 2-5A binding protein as a potential biochemical marker for chronic fatigue syndrome. Am J Med 2000; 108:99-173. 7. Holm S. A simple sequentially rejective multiple test procedure. Scand J Stat 1979; 6:65-70. -------- (c) 2001 University of Chicago Press