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mL of 5-mM stock in 1 mL of PBS) was prepared, and the cells were resuspended in 100 mL of this solution, left untouched for 45 min at room temperature in a dark environment, and spun for 2. 5 min at 2500 X g. A solution of the CD14 staining (Becton Dickinson, BD345785) was prepared (6 /xL CD14 in 60 /xL PBS), and the cells were resuspended in 66 mL of this solution, left untouched for 25 min at room temperature in a dark environment, spun for 2. 5 min at 2500 X g, and resuspended in 500 mL of PBS. Analysis was performed with a flow cy-tometer; the monocyte population was gated and the mean fluorescence (525-nm band pass) of this population was measured. Cells were analyzed quickly, and kept in the dark until processed. For a more detailed prescription of the assay, the reader is referred to references (12) and (13).
Statistical analysis. Data were analyzed using Statis-tica version 5. 1 (Statsoft, Tulsa, OK). Appropriate descriptive statistics were used: frequencies and percentages for the gender distribution; mean, standard deviation (SD), and range for illness duration; age; the exercise performance variables; and the immunological variables. To examine the associations between exercise performance and the immune variables, Pearson correlation analyzes were used. A one sample Kolmogorov-Smirnov (K-S) goodness-of-fit test was used to examine whether the variables entering a Pearson correlation analysis were normally distributed. If a variable was not normally distributed, then the nonparametric Spearman correlation analysis was used. For interpreting correlation coefficients, they were squared to obtain the coefficient of determination. For the correlation analysis, the significance level was set at 0. 01 to help protect against potential type I errors. In case of a «trend» towards a significant association (0. 01 < P <0. 05), a power analysis was performed (22). A power of 80% was considered fair. Finally, the interactions between the exercise performance variables and the immunological variables were further assessed using forward stepwise multiple regression analysis. For the regression analysis, the significance level was set at 0. 05.
RESULTS
The descriptive statistics of the exercise performance variables are displayed in Table 1, the descriptives of the immune variables in Table 2. The mean percentage of monocytes was 18. 9 ± 6. 9 (range [9. 1-32. 2]). All subjects displayed abnormal responses for both the elastase activity and RNase L-ratio, and RNase L activity was abnormal in 15/16 CFS patients. However, the Protein Kinase R activity was within the normal range in the majority of the study sample (11/16), and approximately 50% of the subjects presented with normal NO levels in both monocytes and lymphocytes.
TABLE 2
The descriptive statistics of the immunological variables (N = 16).
VariableMean ± SD'RangeNormal
RangetAbnormal in N
(%) patients
Elastase activity (U-mg-1 extract) 388, 0 ± 469, 5[112-2080]<7, 016 (100, 0)
Protein kinase R activity1, 9 ± 1, 1[0, 6-5, 4]<2, 05 (31, 3)
RNase L activity160, 1 ± 111, 1[46-537]<5, 015 (94, 8)
RNase L ratio1, 8 ± 1, 7[0, 5-7, 7]<5, 016 (100, 0)
[NO] in monocytes38, 0 ± 18, 8[13, 2-72, 9][29, 6-40, 9]8 (50, 0)
[NO] in lymphocytes20, 2 ± 8, 2[10, 4-36, 1][11, 2-18, 5]9 (56, 3)
' SD, standard deviation; t normal range based on laboratory data from 60 healthy volunteers (unpublished data provided by the laboratory).
Apart from the peak RER (K-S z = 1. 49; P = 0. 02), all exercise performance and immune variables were normally distributed. Thus, a Pearson correlation analysis was used for analyzing the majority of associations. For the examination of the associations between RERPEAK and the immune variables, a nonparametric Spearman correlation analysis was used. The outcome of the correlation analysis, examining the associations between the immune and exercise performance variables, is displayed in Table 3. Strong correlations (r ranged between 0. 65 and 0. 73) were observed between four intracellular immune variables (i. e., elastase activity, PKR activity, RNase L activity, and proteolysis) and both the resting RER and the oxygen uptake at RER = 1. 0.
The achieved workload at RER = 1. 0 correlated with the PKR activity, and displayed at trend towards a statistical significant association (0. 01<P < 0. 05) with elastase activity (power = 81%), RNase L activity (power = 81%), and the RNase L-ratio (power = 63%). Likewise, elastase activity (power = 81%) and RNase L activity (power = 63%) displayed a trend towards a correlation with the percentage of target heart rate achieved. Neither the NO concentration in monocytes nor the lymphocytes' NO concentration correlated with any of the exercise performance variables.
TABLE 3. Intracellular immunity vs exercise performance in 16 CFS subjects.
ElastaseRNase