SCFS patients, MCFS patients, as well as non-fatigued controls were recruited by emails, newsletters and advertisements in South Eastern Queensland and Northern New South Wales and local CFS supporters groups. All participants completed a health questionnaire and CFS patients were identified based on the CDC symptom criteria for CFS [1]. Subjects were between the age of 25 and 65 years old to avoid any confounding factors that could occur in association with age [2,3]. Eleven SCFS patients (mean age 42.3+ 3.9 years), 11 MCFS (mean age 49.9+ 3.69 years) and 11 non-fatigued control subjects (mean age 50.9+ 2.5 years) were recruited into this study. Participants who had been diagnosed with autoimmune disorders, psychosis, epilepsy, heart disease or who were pregnant or breastfeeding were excluded from the study.

Sample Preparation and Routine Measurements

In order to maintain the wellbeing of participants, a mobile medical team was utilized to visit the “severe/very severe” patient group. This team consisted of a medical doctor (general practitioner) accompanied by the principal researcher. The purpose of this mobile team was to minimise unnecessary physical and mental stress on the SCFS participants. Blood from all CFS participants was taken by the medical doctor while an experienced phlebotomist assisted with blood collections from the MCFS and the non-fatigued group. Vacutainers were used to collect 65mL of blood from each subject during the hours of 9am and 1pm and analysis commenced within 4-6 hours of collection. The blood tubes contained ethylenediaminetetraacetic acid (EDTA) anticoagulant, citrate or lithium heparin (BD Bioscience, San Jose, CA). Prior to experimental assessment routine full blood count was performed with the EDTA blood tubes using an automated cell counter (ACT Differential Analyzer, Beckman Coulter, Miami, FL).

Measurement of Cytotoxic Activity

Density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMC), prior to cytotoxic activity as previously described [4-6]. PBMC’s were labelled with 0.4% PKH-26 fluorescent cell linker dye (Sigma-Aldrich, St Louis, MO) and incubated with K562 tumour cells at an effector to target ratio of 25:1 and 50:1. Annexin V-FITC and 7-AAD were used to stain cells (BD Pharmingen, San Diego, CA) prior to flow cytometric analysis (Becton Dickinson Immunocytometry Systems, San Jose, CA). Lysis was calculated as previously described [4-6].

Quantification of NK Phenotypes

A negative selection method using RosetteSep Human Natural Killer Cell Enrichment Cocktail (StemCell Technologies, Vancouver, BC) was used to isolate the NK cells from whole blood. Phenotypes of the NK cells were assessed using CD56-FTIC and CD16-PE monoclonal antibodies (BD Pharmingen, San Diego, CA) samples were analysed on the flow cytometer. Phenotypic expression of CD56bright and CD56dim was measured as described previously [7].

Assessment of NK Receptors

Following the isolation of PBMCs using Ficoll-Hypaque (GE Healthcare Life Sciences, Milan, Italy) density gradient centrifugation, a negative selection process was performed to preferentially isolate the NK cells from the PBMC samples. This required the use of antibodies that labelled all cells in the PBMC sample except NK cells the unwanted cells were the samples were then passed through a magnetic column (MiltenyiBiotec, BergischGlabach, Germany). NK cells were labelled with CD56-PE or CD56-FITC and monoclonal antibodies for the following receptors CD158a/h (FITC), CD158e (FITC), CD158b (APC), CD158i (APC) (MiltenyiBiotec, BergischGlabach, Germany), NKG2D (FITC) and NKAT2 (PE) at a concentration of 1x107 cells/mL (BD Bioscience, San Jose, CA). Analysis was performed on the flow cytometer.

Investigation of Plasma Cytokines

The quantitative concentration of plasma cytokines was measured using ELISA with antibodies for human IFN-γ, IL-1β/IL-1F2, IL-2, IL-4, IL-17, IL-6 and TNF-α (R&D Systems, Minneapolis, MN) using isolated plasma samples according to manufacturer’s instructions. Each 96 well plate included serial dilutions of the standard, a control and the experimental samples. Samples were measured in duplicates. Absorbance was measured at 450nm and standard curves were used to determine the final concentration (pg/mL) of each sample.

Statistical Analysis

Statistical analyses were performed using SPSS version 18 (SPSS Inc, Chicago, USA) and Graphpad Prism version 6.0 (GraphPadSoftware, Inc., San Diego, USA). The following statistical analysis were performed, a test of normality, multivariate analysis with between and within group test for variances in the data. Additionally, where the distribution of the data was normal or parametric the Bonferroni post-hoc test was used.Multiple comparisons were performed using theTukey’s multiple comparison tests in situations where the data was nonparametric.Results are reported as mean plus/minus standard error of the mean (+SEM) for each group. Criterion for significance in all the tests performed was set at a level of P less than 0.05. Nonparametric correlations were performed using the Spearman correlation tests.


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