All research reported here was approved by the University of Utah Institutional Review Board, and written informed consent was obtained from all subjects before participation. Exclusion criteria included active viral or upper respiratory infections, chronic cardiovascular or pulmonary disorders, or other chronic conditions such as anemia, cancer or multiple sclerosis. All subjects were required to refrain from any other continuous exercise greater than 5 minutes of walking, for 2 days before, and 2 days after the scheduled exercise task.
The sample for the present report included 48 CFS patients (33 females) [(15 females) of these are the same patients as in our previous publication [8
]], 49 Control subjects (29 females) [16 (11 females) are the same Controls as in our previous report], and 18 FM only subjects (15 females). This ratio of more females than males is typical in CFS and FM research, and is also consistent with observations from large scale incidence and prevalence studies including the Wichita sample [10
]. The patients reflected the local population with 94% being Caucasian, 6% being minority; thus our findings may not apply to minorities with CFS or FM. All CFS patients met the CDC criteria for CFS [1
], and 46 (96%) met the Canadian Criteria for ME/CFS as well [2
]. Prior screening by an experienced physician (LB) ruled out all other known causes for persistent or relapsing fatigue in these CFS patients. All patients were also screened for FM using the strict ACR research criteria, which includes presence of widespread pain for at least 6 months, and pain reported at 11 or more of 18 sites during tender point examination [3
]. Thirty three of the 48 CFS patients (69%) also met ACR criteria for FM, similar to the high co morbidity of these disorders previously reported [11
]. Eighteen patients met criteria for FM, but did not meet criteria for CFS (principally due to fatigue causing less compromise in normal daily activities), and were classified as FM-only patients (FM).
For primary analyses, all 48 CFS patients were compared to the 49 Controls. All later analyses were done after separating out the CFS subgroup in which the major identifying feature was large decreases in α-2A receptor mRNA at all times following exercise. All 18 FM patients were also compared with this same control group, and with the CFS group.
Both controls and all patient groups included individuals on prescribed antidepressants, and no subjects were withdrawn from these medications because antidepressants require a relatively long washout and patients may be at risk off medications. Also, our prior study [9
] indicated that antidepressants do not substantially alter CFS patients’ expression of the genes in our profile. Thus, 11 of 49 control subjects (23%), 30 of 48 CFS patients (63%) and 9 of the FM patients (50%) were tested while continuing their usual antidepressants. In regard to other prescribed medications, for the first 19 CFS and all 18 FM patients, we requested that these patients be withdrawn from any prescribed pain medications and anticonvulsants for 6 days in order to participate, and only 2 of these CFS patients failed to comply fully. For the remaining 29 CFS patients, in order to increase participation by those with more severe symptoms who were unwilling to interrupt their medications, they were allowed to remain on all their usual physician-prescribed medications. This resulted in 15 (31%) CFS patients being tested on opioid pain medications, and 11 (23%) CFS patients being tested on anticonvulsants. This allowed secondary analyses to be performed comparing CFS patients tested on vs. not on these prescribed medications.
Disorder Onset and Functionality/Severity Ratings
For the CFS patients, CFS symptom onset was reported as sudden by 36 patients (70% of the α-2A increase and 87% of the α-2A decrease subgroups). Thirty five (73%) reported onset to be associated with one or several flu-like, mononucleosis or other viral illnesses or infections. Five patients (10%) associated onset with traumatic injuries and fractures. Three patients (6%) associated onset with surgery, and 5 patients associated onset with life stress or no memorable event. In contrast, the FM only group included only 2 patients (11%) who reported sudden onset of their symptoms, linked to a virus in one and to a herniated disk in the other.
Categorical ratings of disorder severity from 1–4 were assigned for all CFS patients by their physician (LB) based on observations and symptom measurements over multiple visits in the clinical setting, while blind to the gene expression and exercise results. The categories were defined by ability to function in normal daily activities, as follows: 4. Able to work full time 30–40 hours but “nothing left over”, high symptom burden and limitations; 3. Able to do part time school/work/other activities 10–30 hours/week, but easily relapses, and frequent rest needed; 2. Only able to do self-care activities of daily living (ADL), sedentary, <10 hrs/wk of light activities, but could live alone with occasional help; 1. House- or bed-bound, with minimal activity tolerance, marked cognitive dysfunction, and dependent on others for ADL. Thus, lower function scores indicated worse disorder severity.
Exercise testing was always performed at the same time of day (starting between 8 and 9:30 am). Venous blood samples were obtained immediately prior to exercise (baseline) and at 0.5, 8, 24, and 48 hours post-exercise. We assessed the severity of pre-existing and exercise-related fatigue and myalgia symptoms at the time of each blood draw, and at the midpoint and immediately after completing the exercise task; the subject provided numerical ratings of mental fatigue, physical fatigue and overall body pain using a 0–100 scale where 100 was defined as the greatest level of fatigue or pain the subject could ever imagine experiencing.
A combined arm-leg cycle ergometer (Schwinn Air-Dyne) was used for the 25-min, moderate exercise test. In the first 5 min of exercise, subjects were asked to increase pedaling rate until 70% age-predicted maximal heart rate was achieved. Thereafter, work rate was adjusted in order to maintain this target heart rate throughout the sub-maximal exercise protocol. Ratings of Perceived Exertion (RPE) were obtained on a scale of 1–10 every 45 min; heart rate was recorded every minute, and blood pressure was measured at baseline, every 10 min during exercise, and upon completion of the exercise. We elected to use a sustained moderate exercise rather than a maximal exercise test (which typically last only 5–9 min in CFS patients) because of closer similarity to the natural exercise experiences reported to exacerbate CFS symptoms in patients’ daily lives. Our 25 min sub-maximal exercise task did elicit consistent worsening of fatigue and pain symptoms from 8–48 hours post-exercise (see ). In contrast, after a briefer maximal exercise task, reports of worsening CFS symptoms were inconsistent or absent until 5 days after the challenge [14
], a pattern not typically observed in real life. Maximal exercise protocols have demonstrated few differences in cardiorespiratory and perceptual responses; RPE is an exception being consistently higher in CFS patients than controls [15
]. However, it is notable that responses to sub-maximal exercise including VO2
do predict peak exercise performance in CFS patients [16
Figure 1 Behavioral scores for mental and physical fatigue and pain in patients vs. control subjects. A, Average Visual analog scores (maximum 100, minimum 0) for Mental Fatigue for CFS subgroups, FM, and Controls. Data points are at baseline (MF base), half way (more ...)
mRNA extraction and analysis
All blood processing and analyses were performed by personnel blinded to the subject’s group. At each of the 5 blood sampling times, blood was collected in EDTA tubes. Seven minutes after blood collection, the blood was centrifuged at 3200 rpm (1315 × g- Clay Adams Compact II Centrifuge) for 12 minutes, plasma removed, and the white layer carefully collected in RLT+β-ME (Qiagen, Valencia, CA) then quickly frozen using a methanol-dry ice slurry, and stored at −80°. RNA was extracted using RNeasy kits (Qiagen, Valencia, CA), according to manufacture’s directions, and treated with RNase-free DNase-I (Qiagen, Valencia, CA). Immediately following extraction, RNA was converted to a cDNA library using the ABI High Capacity cDNA Archive Kit (Applied Biosystems, Inc., Foster City, CA). The cDNA samples were stored at −2° C until analysis.
RNA integrity was assessed with a Bioanalyzer, and consistently found to have values greater than 9. The cycle counts for the control gene, TF2B) averaged 21.78 ± 1.67 (SD) for control subjects and 22.32 ± 2.09 (SD) for patients. These values and variations indicate consistent, high quality integrity of the RNA, and sufficient amounts of RNA for accurate analysis of the amount of mRNA in these experiments.
The cDNA libraries were analyzed using the ABI quantitative, real-time PCR system on the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Inc., Foster City, CA), using ABI TaqMan Master Mix (Applied Biosystems, Inc., Foster City, CA). Master Mix/primer probe solutions and template solutions were separately loaded onto 96 well pre plates, with robot loading mixing these solutions when placed in the 384 well plates. Plates were centrifuged to remove any air bubbles in the wells. Each sample was run in duplicate with standards being run in quadruplicate. No template control samples were also run. Each 384 well plate contained samples from two subjects/patients, and all genes were analyzed on the same plate. Primer probes (all from TaqMan Gene Expression Assays, Applied Biosystems, Inc., Foster City, CA) were: ASIC3 - Hs00245097_m1; P2X4 - Hs00175706_m1; P2X5 - Hs00175712_m1; TRPV1 - Hs00218912_m1; Adrenergic A2A (α-2A) - Hs00265081_s1; Adrenergic B-1 - Hs02330048_s1; Adrenergic B-2 - Hs00240532_s1; COMT - Hs00241349_m1; IL6 - Hs00174131_m1; IL10 - Hs00174086_m1; TNFβ (Alpha Lymphotoxin or “α-lym”) - Hs00236874_m1; TLR4 - Hs00152937_m1; CD14 - Hs00169122_g1. Control primer probes included TF2B - Hs00155321_m1; β-Actin - Hs99999903_m1; and PSMB6 - Hs00382586_m1. In later experiments, only TF2B was used as the reference gene. All primer probes, except for the adrenergic receptors and CD14 (these genes do not have introns), recognize sequences that cross splice sites, and therefore, make detection of genomic DNA unlikely. In all cases, we quenched the genomic DNA and ran no-template control wells to ensure that genomic DNA did not contaminate the final results. All of these primer probes were designed and tested to be used together, and have similar efficiencies to help eliminate inaccuracy. For the genes that have rarely been described in leukocytes (Adrenergic α-2A, Adrenergic B-1, P2X5, TRPV1, ASIC3) we designed primers that contained 360 to 600 base-pairs, which included the regions ABI indicated the Primer probes listed above spanned. PCR product was generated from our leukocyte samples and sequenced. All of these sequences were 99–100% identical to predicted sequences of these genes. Evaluation of Controls in this and previous experiments indicated that TF2B had less intrinsic variation than other candidates such as β-actin, had a count range that was similar to the genes of interest, and did not increase or decrease due to the exercise protocol. Real-time PCR results were analyzed with SDS 2.1 (Applied Biosystems, Inc., Foster City, CA), and inspected to determine artifacts (loading errors, robot errors, thresholding errors, etc.). Count numbers were exported to an Excel spreadsheet, and analyzed according to the ddCT method described in ABI User Bulletin #2 (Applied Biosystems, Inc., Foster City, CA). Baseline levels for each gene were computed relative to TF2B, and these baselines were used as the comparator for all measures taken after the exercise period (see statistical methods below for further analysis details).
Because the ddCT method used for mRNA analysis necessarily creates a non-normal, rightward skewed distribution, data were log transformed to yield distributions that could be appropriately analyzed with parametric statistics.
Post-exercise values for each gene expression measure were normalized relative to the same subject’s baseline levels (1.00 = baseline). As detailed in our previous report [8
], to reduce false positive findings associated with multiple comparisons, we did not examine each sampling time individually. Instead, the relative mRNA values from the 4 post-exercise time points (0.5, 8, 24, and 48 hour) were summed into a single measure labeled Area Under the Curve (AUC) and then log transformed. Initial MANOVAs were performed for the metabolite detecting, adrenergic and immune markers that differed in our prior study [8
] yielded significant effects of Groups. These were followed by individual ANOVAs, and when significant, by comparisons of baseline and post-exercise AUC of Controls vs. CFS and FM patients determined with independent two-tailed t-tests. Two tailed rather than 1 tailed tested were used despite the predictions of increases as an additional protection against false positive results. This approach to multiple gene comparisons is similar (but on a much smaller scale) to Pathway Analysis as used to explore the many thousands of gene examined in microarray studies. Secondarily, we compared two CFS subgroups (CFS with increased post-exercise α-2A and CFS with decreased post-exercise α-2A) to Controls using one-way ANOVAs. One-way ANOVAs were used for between group comparisons of cardiovascular, work rate and RPE measures obtained during the exercise task.
To examine whether Group differences were related to differences in age, gender, or body mass index (BMI), we performed one way ANOVAs with each of these included in the model as covariates. In no instance did age, gender, or BMI impact the significance of group differences.
Group by Time, Group, and Time differences in ratings of physical fatigue, mental fatigue, and pain were analyzed using 3 × 7 repeated measures (RM) ANOVAs. When significant Group differences were present, within group Time effects were examined with RM ANOVAs with simple contrasts to determine which time points differed significantly from baseline.
In addition to group comparisons, Pearson r correlations were used to examine relationships between exercise variables, pain and fatigue ratings, and gene expression measures. All data are presented as means and standard errors, with significance set at P < 0.05.