The patient group consisted of 11 patients (6 women and 5 men; mean ± standard deviation [SD] age,: 43.2 ± 13.8 years) fulfilling CFS criteria (2
). The mean (±SD) duration of illness was 7.6 ± 6.6 years. The control group consisted of 14 matched healthy volunteers (10 women and 4 men; mean ± SD age, 39.1 ± 11.6 years). A multidimensional fatigue inventory (MFI) scale (4
) was used to estimate fatigue in both groups. Before the inclusion, biological tests were performed for both groups, which allowed exclusion of organic diseases. Moreover, neither infectious history nor psychiatric illness was noted in the month prior to the start of the study. In the two groups, the mean ages were comparable, and the MFI score (maximal fatigue score = 100) was significantly higher in the patients (53.3 ± 9.4) than in healthy volunteers (6.1 ± 7). The clinical features of the patients are summarized in Table . This study was approved by the local ethics committee of the Hôpital Saint Antoine, Paris, France.
Clinical features of patients in this studya
Venous blood samples were drawn in heparinized tubes. The samples were stored, and PBMCs were isolated within 4 h by density-gradient centrifugation. Blood (diluted 1/1 in phosphate-buffered saline) was layered onto 1 volume of Ficoll (density, 1.080; Gibco BRL, Paisley, Scotland) and centrifuged at 500 × g for 30 min at 20°C. PBMCs at the interface were collected and washed with 5 volumes of phosphate-buffered saline (500 × g, 15 min, 20°C). PBMC pellets were resuspended in 5 ml of erythrocyte-lysing buffer (155 mM NH4Cl, 10 mM NaHCO3 [pH 7.4], 0.1 mM EDTA), centrifuged (500 × g, 10 min, 20°C), and frozen at −80°C until use.
Radiocovalent affinity labeling and analysis of 2-5A binding proteins.
A sensitive assay allowing the specific identification of 2-5A binding proteins in biological samples without a fractionating step was used. PBMC extracts were prepared in the presence of protease inhibitors to avoid artifacts from differential proteolysis.
A 3′-oxidized 2-5A [32P]pCp (3,000 Ci/mmol, 20,000 cpm) probe was incubated with PBMC extracts (200 μg of protein) for 20 min at 4°C and for a further 20 min at room temperature with 20 mM sodium cyanoborohydride, allowing the covalent linkage of the probe to all 2-5A binding protein species. The extract was then fractionated by polyacrylamide gel electrophoresis (PAGE) with sodium dodecyl sulfate (SDS) as a denaturing agent. The size distribution of 2-5A-labeled polypeptides was visualized by autoradiography, and the relative abundance of the various bands was calculated by densitometric analysis with the NIH Image software. NIH Image is a public domain image processing and analysis program developed at the Research Services Branch of the National Institute of Mental Health.
Sensitivity, specificity, and positive and negative prognostic values were calculated with different threshold ratios of RNase L isoforms as a positive diagnosis, particularly ratios of 0.5, 0.4, and 0.3. Confidence intervals (95% CIs) were estimated. The ages, durations of fatigue, numbers of CFS criteria fulfilled, and MFI scores of the groups were compared by Mann-Whitney U test with significance at P < 0.05.