Methods
Human Study PreDicta
In the European Study PreDicta (Post-infectious immune reprogramming and
its association with persistence and chronicity of respiratory allergic
diseases) we examined healthy and asthmatic pre-school children at the
age of 4-6 years in collaboration with the children hospital in
Erlangen. The study in Erlangen was approved by the ethics committee of
the Friedrich-Alexander University Erlangen-Nürnberg, Germany (Re-No
4435) and it is registered in the German Clinical Trials Register
(www.germanctr.de: DRKS00004914).
Two cohorts of pre-school children (age 4-6 years) with and without
asthma were analyzed. The recruitment of the subjects, inclusion and
exclusion criteria as well as the timescale for clinical visits and data
collection were exactly described recently (Bergauer et al., 2017b; a;
Bielor et al., 2017; Hentschke et al., 2017) along with the clinical
aspects and characteristics and reported in other form in Table
1 and 2.
For gene expression analysis we isolated mRNA from total blood cells of
the children as previously described and performed quantitative
real-time PCR as described below (Bergauer et al., 2017a). The levels of
CRP in the serum samples of the children were measured by turbidimetry
on a Roche Integra 800 Analyzer (CRPL2 reagent, limit of detection 1.0
mg/L, interday CV 1.4% (8.1 mg/L), Roche Diagnostics, Basel). The
detection of rhinovirus in nasopharyngeal swab obtained from the
children was performed at the Department of Virology, University of
Turku (Finland). The description of this procedure is already published
in detail elsewhere (Bergauer et al., 2017a).
FEV1 and PEF
FEV1, FVC and PEF were measured at Baseline visit (B0) by using
spirometry. After a period of normal breathing, the participant should
inhale maximal, directly followed by maximal and fast exhalation. The
volume exhaled in one second is FEV1. The total exhaled volume is FVC.
The ratio FEV1/FVC is stated as FEV1%. The PEF is defined as the
largest expiratory flow, which is achieved with a maximum forced effort
after maximum inspiration.
Human RNA Isolation from Tempus Tubes and quantitative
Real-Time
PCR
At Baseline visit whole blood was collected in Tempus®
Blood RNA Tubes (Life Technologies™, GmbH, Darmstadt, Germany) and RNA
was extracted with the MagMax for Stabilized Blood Tubes RNA Isolation
Kit. For reverse transcription of RNA (1 µg) we used the first strand
cDNA synthesis kit for RT-PCR (MBI Fermentas, Sat. Leon-Rot, Germany).
The resulting template cDNA was then amplified by quantitative real-time
PCR (qPCR) using SoFast EvaGreen Supermix (Bio-Rad Laboratories,
München, Germany). The qPCR itself was performed in a CFX96 Touch
Real-Time PCR Detection System (Bio-Rad Laboratories) with a cycle of 2
min 98°C, 50 cycles at 5 s 95°C, 10 s 60°C, followed by 5 s 65°C and 5 s
95°. The primer sequences used for the for real time PCR are listed in
Table S1. The mRNA of the genes of interest was normalized using the
housekeeping gene HPRT (Hypoxanthine Guanine Phosphoribosyl
Transferase).
Isolation of PBMCs, in vitro cell
culture and analysis of the cell
supernatants
At the time of recruitment (Baseline Visit), PBMCs were isolated from
heparinized blood with Ficoll using density centrifugation. After
isolation, PBMC numbers were adjusted to a concentration of
106 viable cells/mL in complete culture medium. For
cell culture, RPMI 1640 medium supplemented with 25 mmol/L HEPES (GIBCO,
Invitrogen, Darmstadt, Germany) was used. Furthermore, 100 IU/mL
penicillin, 100 µg/mL streptomycin, 50 µmol/L β-mercaptoethanol, 1%
L-glutamine (200 mmol/L), 1% MEM Vitamin, 1% non-essential amino
acids, 1% sodium pyruvate, and 10% FBS were added (complete culture
medium); these reagents were purchased from Sigma-Aldrich (Steinheim,
Germany). The PBMCs were cultured in complete culture medium for 24
hours at 37°C and 5% CO2, whereby parts of them were
challenged in vitro with 10 µg/ml PHA (Sigma-Aldrich, Steinheim,
Germany) or with RV (RV1b). The growth of RV1b and the description of
the RV1b infection itself have been published previously in detail
elsewhere (Bergauer et al., 2017a).
Human IFNβ and IL-10 was detected in the cell-culture supernatants by
using IFNβ ELISA kit from PeproTech (Hamburg, Germany) and IL-10 OptEIA™
sandwich ELISA kit from BD Bioscience (Heidelberg, Germany),
respectively, according to the manufacturer’s protocol.
Statistical analysis
Statistical analysis was performed using Prism version 7 for Windows
(GraphPad, La Jolla, CA, USA). Differences were evaluated for
significance by using the two-tailed Student’s t test or ordinary
One-way ANOVA to generate p-value data (* p ≤ 0.05; ** p ≤ 0.01, *** p ≤
0.001, **** p≤ 0.0001) for all data. Unless otherwise indicated data are
presented as mean values ± SEMs.