Синдром хронічної втоми: взаємозв'язок вправ та імунної дисфункції

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Медицина

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was made in PBS-Tween® (0. 1 mL-cm»2 = 15 mL for regular membrane), incubated for 2 h at room temperature while gently shaking or overnight at 4°C, and the tray was covered with a lid or with aluminum foil. The membranes were washed two times with PBS-Tween® (5 min each at room temperature while gently shaking -0. 25 mL-cm-2) and incubated with secondary antibody; a dilution (dependent on the manufacturer's instructions) was made in PBS-Tween® (0. 1 mL-cm-2), incubated for 30 min at room temperature, shaken gently, and the tray was covered with a lid or with aluminum foil. The membranes were washed two times (colorimetric) or four times (chemiluminescence) with PBS-Tween®, 5 min each at room temperature while gently shaking: 0. 25 mL-cm-2. The colorimetric analysis was performed using Opti4CN® (Bio-Rad) by mixing Elix-H20 9/10, Opti4CN®-diluent 1/10, and Opti4CN®-substrate 0. 2/10 (10 mL per blot), incubating while gently shaking until color develops, washing for 10 min with Elix-H20, and drying by blotting the membranes on Whatmann paper. The specific protein bands were quantified by density scanning.

Elastase activity in PBMC was measured using an enzymatic-colorimetric assay: EnzChek® Elastase Assay Kit E-12056 (Molecular Probes). The EnzChek kit contains DQ™ elastin-soluble bovine neck ligament elastin that has been labeled with BODIPY®FL dye such that the conjugate can be digested by elastase or other proteases to yield highly fluorescent fragments. The resulting increase in fluorescence was monitored with a fluorescence microplate reader. First, a PBMC pellet and a PBMC pellet extract (in absence of elastase inhibitor III) were prepared, the protein concentration was measured, and the samples (patient proteins-extracts) were placed on ice and let thawed for 20-30 min. DQ Elastin Substrate (BODIPY®FL) : the new vial was reconstituted by adding 1 mL of dH20 (final concentration 1. 0 mg-mL-') and by mixing thoroughly to dissolve. Five milliliters of the stock buffer (10X) were diluted and 45 mL of dH20 was added to obtain the reaction buffer. To obtain a positive control (porcine pancreatic elastase), the new vial was reconstituted by adding 0. 5 mL of dH20 up to a final concentration of 100 U-mL-1. Then, a standard dilution curve was constructed from the positive control (porcine pancreatic elastase with a starting concentration of 100 U-mL-1) with concentration of 5. 0, 1. 0, 0. 5, 0. 1, 0. 05, and 0. 01 UmL-1. The controls were pipetted in triplicate into the microplate. Samples of 100 fxg of proteins extract (X /xL) were used. The total reaction volume was set at 200 /xL (200 /xL – X /xL = Y /xL buffer IX). Y llL of buffer 1X was added into the samples, and 50 jxL of sample dilution/well was added. The samples were pipetted in triplicate into the microplate. For the substrate, 5 lxL of DQ elastin/sample was added to 145 /xL of reaction buffer/sample. Next, 150 /xL of the substrate/well sample was added. Protected from light, everything was incubated for 2 h at 37°C. Afterwards, the fluorescence intensity was measured in a fluorescence microplate reader (Molecular phospho-imager®FX BioRad and external laser Molecular ImageraFX BioRad). The values were extrapolated from the equation of the curve (fluorescence vs elastase concentration) and multiplied by 200 (U-mg-i). For each sample, the value derived from the no-enzyme control was subtracted to correct for background fluorescence. According to the company supplying the assay, the elastase activity assay had been thoroughly tested before it was brought on the market, but reliability and validity data are proprietary and unpublished (personal communication).

TABLE 1.

The descriptive statistics of the exercise performance variables (/V = 16).

VariableMean ± SO'Range

Exercise duration (min) 14, 8 ± 6, 5[7-26]

HRBESTt (bpm) 78, 6 ± 14, 7[47-110]

HRPEAK (bpm) 158, 6 ±22, 3[125-197]

% target heart rate achieved87, 3 ± 13, 4[62-105]

Workload (W) 148, 3 ± 65, 0[70-260]

Workload per body weight (W-kg~1) 2, 0 ± 0, 7[0, 9-3, 2]

Workload at RER* = 1. 0 (W) 121, 8 ± 52, 6[72-215]

V02PEAK11 (mL-kg-'-min-1) 31, 0 ± 9, 7[16, 2-50, 1]

V02at RER = 1. 0 (mL-kg-'-min»') 26, 0 ± 7, 2[15, 7-40, 2]

RERREST0, 68 ± 0, 10[0, 42-0, 91]

Lactate concentration at rest (mmol-L-1) 0, 9 ± 0, 4[0, 5-1, 8]

Peak lactate concentration (mmol-L-1) 8, 5 ±4, 9[1, 3-21, 2]

« SD, standard deviation; t HRREST, resting heart rate; $ RER, respiratory exchange ratio; H V02PEAK, peak oxygen uptake.

The measurement of nitric oxide level in isolated PBMC was performed using a live cell assay (12, 13). The cells or PBMC were washed once with 500 /xL PBS, and spun for 2. 5 min at 2500 X g. A 15-mM DAF-FM solution (4-amino-5-methylamino 2', 7'-difluorofluorescein diacetate, Molecular Probes D-23844: 3