The study was conducted as part of the Kuopio Depression (KUDEP) four-phase general population study involving adults aged 25-64 years in the province of Kuopio in Eastern Finland. A random sample of 3004 participants was selected in 1998 from the National Population Register and was followed up in 1999 and 2001. The baseline sample included 2050 participants, the first follow-up sample (1999) 1722 participants, and the second follow-up sample (2001) 1593 participants. The second follow-up questionnaire was only sent to those subjects who responded in either of the preceding surveys. Questionnaires were re-sent to the non-respondents one month later after each follow-up. Participants responded to questions concerning their age, gender, marital status (married or living with a partner vs. living alone), asthma diagnosis by a physician (yes vs. no), daily smoking (yes vs. no), alcohol use (≥ 2 times per week vs. less), and the use of antidepressant medications (yes vs. no).
In 2005, a sub-sample of 427 participants from the previous study phases reporting either high or low levels of adverse mental symptoms were asked to take part in a clinical evaluation and laboratory testing [27
] as a fourth study phase. For the present study, we selected those participants whose Beck Depression Inventory (BDI) [29
] scores indicated depressive symptoms (BDI scores ≥ 10; depressed) or a euthymic state (BDI scores ≤ 9; controls) consistently at all three earlier data collection points. The mean BDI scores (standard deviation, SD) for the depressed (n = 58) were 18.1 (9.1) in 1998, 21.5 (9.9) in 1999, and 20.9 (11.1) in 2001. The corresponding means (SD) for the controls (n
= 58) were 2.6 (2.2) in 1998, 2.9 (2.6) in 1999, and 2.1 (1.9) in 2001.
Psychiatric interviews were conducted for all participants, including the controls, by a trained, experienced research nurse using the Structured Clinical Interview for DSM-IV (SCID-I and SCID-II) [30
]. Additionally, the 29-item Hamilton Depression Rating Scale (HAM-D-29) [33
] was used for the assessment of depression severity. The HAM-D-29 includes the 21-item Hamilton Depression Rating Scale (HAM-D-21) with a supplementary 8-item subscale (Atypical Depression Supplement, ADS) for measuring atypical features of depression. The ADS includes questions regarding weight gain, increased appetite/eating, carbohydrate craving, hypersomnia, leaden paralysis, and social withdrawal. Subjects with bipolar disorders were excluded and the primary diagnosis for all the rest of the depressed participants was MDD. Data concerning the participants' somatic medications were acquired from the nationwide Social Insurance Institute. Approval for the study was obtained from the Ethics Committee of Kuopio University Hospital and the University of Eastern Finland. The study protocol was in accordance with the Declaration of Helsinki. All participants provided written informed consent before entering the study.
After the clinical interviews, the participants were given a referral to the laboratory, and were then instructed to visit the laboratory at the latest during the following week. The body mass index (kg/m2
) was calculated from height and body weight measured in light clothing without shoes. The venous blood serum samples for the cytokine analyses were stored at -80°C until run. IL-5 (pg/mL), IL-13 (pg/mL) and IFN-γ (pg/mL) levels were analyzed by multiplexing with Bio-Plex Human Cytokine Panel 1 utilizing a Bio-Plex 200 instrument based on Luminex xMAP technology (Bio-Rad Laboratories Inc., CA, US). The Luminex method and the standard enzyme-linked immunosorbent assay (ELISA) are highly correlated [34
]. Before analyses, the samples were centrifuged for 15 min at 3000 rpm, and diluted 1:2 in a sample matrix. The samples were assayed singly, and the assay conditions were standardized and pre-optimized to ensure optimal reproducibility of the assays. The kit instructions and instrument manuals were followed accordingly. The intra-assay and interassay variation for the kit analyses were 4.6-13.8% and 3.7-17.2%, respectively. The results were calculated with BioPlex Manager Software version 4.3 with five-parameter logistic equations [37
]. High sensitivity range standard settings were utilized. The values between zero (blank sample) and the lowest standard (IL-5: 0.52 pg/ml; IL-13: 0.71 pg/ml; IFN-γ: 0.30 pg/ml) were extrapolated from the standard curve by the software. In statistical analyses, the non-detectable samples (ND) were marked as the mean value between zero and the lowest extrapolated value of each cytokine [38
]. There were 17 NDs for IL-5, 30 for IL-13, and 52 for IFN-γ.
The data were analyzed using SPSS version 17.0 statistical software (SPSS Inc., Chicago, IL). Differences between the MDD and control groups were assessed using the chi-squared test for categorical variables, and the Student's t-test and the Mann-Whitney U-test for continuous variables. Parametric tests were used with normally distributed and non-parametric tests with non-normally distributed variables. The differences between the MDD and control groups in the examined cytokines were further examined with logistic regression modeling. The modeling was performed in two steps. First, we formed a basic model consisting of general parameters either showing differences between the groups or known to affect the levels of IL-5, IL-13 or IFN-γ (Model 1: age, gender, marital status, alcohol use [39
], and daily smoking [40
]). Secondly, for more comprehensive evaluation of the effects of potential confounders, we formed models that included the variables in Model 1 together with further adjustments for one of the following covariates: the use of antidepressants (Model 2), the use of NSAIDs (Model 3), and a diagnosis of asthma (Model 4). The cytokines were inserted into the models as continuous variables. The odds ratios (OR) show the likelihood for each 1-point increase in the serum levels of these cytokines, i.e. OR show the odds for a covariate belonging to the MDD group in relation to the control group.