Interleukins

We conducted a systematic literature search in the database MEDLINE (1980–2005) to review statistical methods that have been previously applied to cytokine data. Because the objective was to get a crude overview rather than to reveal the exact number of papers published in this area we defined quite sensitive search criteria using the following key words: "cytokine$" or terms to identify specific cytokines (e.g. among others "IL$," "interleukin$," IF$, interferon$, TNF$, etc.) and common univariate and multivariate statistical techniques (e.g. among others "linear regression,""analysis of variance,""cluster analysis,""factor analysis" etc.).
Table 1 shows the results of our search. The most widely used methods found were simple statistical approaches that investigate the relationship between two variables (so called bivariate methods – also called univariate methods when variables are classified as dependent and independent variables). We frequently found standard methods to compare means of immunological parameters between independent groups (e.g. t-test, analysis of variance or their non-parametric equivalents), bivariate correlation analysis (Pearson's or Spearman's correlation coefficients) and univariate linear regression. By contrast, multivariate techniques ( i.e. statistical approaches that consider three or more study variables simultaneously) were less frequently applied to cytokine data. Several studies used factor analysis (to identify groups of correlated immunological parameters) or cluster analysis (to identify groups of individuals with similar immunological profiles) or discrimination techniques such as logistic regression, discriminant analysis (to identify causes or consequences of immunological profiles). We also found a few examples of advanced modelling techniques (path analysis/structural equation modelling) that simultaneously model multiple relationships between the study variables.
In the following section we provide an overview of statistical methods that can be considered for analysing immunological data that should help the applied immunologists without a detailed knowledge of statistics to select the appropriate statistical technique for each particular research question. The definition of which method is the most appropriate is strongly dependent on the research objective, the type of data collected, whether data assumptions are fulfilled and whether the sample size is sufficient. We begin with a short introduction to these topics.

A total of 14 root tendons collected from 2 to 4 weeks were subjected to histological examination, fixed in 10% formalin and embedded in paraffin. The embedded tendons were cut into 4 μm cross-sectional sections and stained with hematoxylin and eosin (HE). An independent examiner completed the histological interpretation at magnifications of ×10 and ×20 and calculated the number and density of Fibroblast (Fb) displacements. In addition, a total of 49 serum samples from 7 blood sampling sites were subjected to Enzyme-linked Immunosorbent Assay (ELISA) to test for the pro-inflammatory factor-rat interleukin IL-1β and the anti-inflammatory factor-growth transforming factor TGF-β1 by double antibody sandwich ELISA, both kits were purchased from Rexin Bio (Size 96T, sensitivity: <0.1 pg/mL, IL-1β test range: 1.25–40 pg/mL, TGF-β1 test range: 2.5–80 ng/mL). Operating procedure All reagents and components are first brought back to room temperature, standards, controls and samples, and it is recommended to do duplicate wells. The operating procedure is as follows:
1) Prepare the working solutions for the various components of the kit as described in the previous instructions. 2) Remove the required slides from the aluminium foil bag and put the remaining slides back into the refrigerator in a sealed self-sealing bag. 3) Set up the standard wells, 0-value wells, blank wells and sample wells. Add 50 μL of standard at different concentrations to each of the standard wells, 50 μL of sample diluent to the 0-value wells, and 50 μL of the sample to be tested to the blank wells. 4) Add 100 μL of Horseradish Peroxidase (HRP)-labelled detection antibody to the standard wells, 0-value wells and sample wells, except for the blank wells. 5) Cover the reaction plate with sealing film. Incubate the plate for 60 min at 37°C in a water bath or thermostat protected from light. 6) Remove the sealing film, discard the liquid, pat dry on blotting paper, fill each well with washing solution, leave for 20 s, shake off the washing solution, pat dry on blotting paper and repeat 5 times. If using an automatic plate washer, follow the operating procedures of the washer and add a 30 s soak procedure to improve the accuracy of the assay. At the end of the wash, before adding the substrate, pat the reaction plate dry on clean, non-flaking paper. 7) Mix substrate A and B in a 1:1 volume and add 100 μL of substrate mixture to all wells. Cover the plate with sealing film and incubate for 15 min at 37°C in a water bath or thermostat protected from light. 8) Add 50 μL of termination solution to all wells and read the Optical Density (OD) of each well on an enzyme marker.

Two 3-mL blood samples were collected from the jugular vein from one pig per pen on the last day of phase 2, as well as on the last day of phase 3. An equal number of barrows and gilts were sampled within treatment, and the same pigs were sampled at each time point. Of the two samples of blood obtained per pig, one was collected in a vacutainer containing ethylenediamine-tetraacetic acid as an anticoagulant, immediately placed on ice, and analyzed for complete blood cell count on a multiparameter automated hematology analyzer (CELL-DYN 3700, Abbott Laboratories, Abbott Park, IL, USA). The second sample of blood was collected in a serum vacutainer containing spray-dried silica as a clot activator. Samples were allowed to clot and then centrifuged at 4,000 × g for 13 min at room temperature. Serum was removed from centrifuged tubes and stored at −20°C until analysis. Total protein, blood urea N, and albumin were analyzed using a Beckman Coulter Clinical Chemistry AU analyzer (Beckman Coulter Inc., Brea, CA, USA). The concentration of immunoglobulin G (IgG) in serum samples was determined by enzyme-linked immunosorbent assay following the manufacturer’s instructions (Bethyl Laboratories, Inc., Montgomery, TX, USA). Concentrations of interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist (IL-1RA), IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-gamma (IFN-γ), and tumor necrosis factor-α in serum samples were measured via a porcine-specific multiplex immunoassay kit (MilliporeSigma, Burlington, MA, USA) and read with a Luminex MagPix instrument (Luminex Corporation, Austin, TX, USA).