The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
This study was approved by the Chinese Clinical Trial Registry with registration number ChiCTR-COC-15006080 and was reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Army Medical University. The methods were carried out in accordance with the approved guidelines. The study was conducted in compliance with the principles of the Declaration of Helsinki. Written informed consent was obtained from the parents of all enrolled subjects. The authors have access to information that could identify individual participants during or after data collection.
Subjects
The subjects were recruited at the Pediatric Outpatient Department of Southwest Hospital. A total of 30 (the number of cases was estimated based on the significance of the expected difference) pediatric patients with allergic asthma (according to retrospective grading of disease severity) older than 5 years were enrolled, along with 30 age- and sex-matched healthy children who underwent a physical examination during the same period (control group). The average age and sex distributions of the patients were considered in the selection of subjects for each group to reduce deviations and ensure a normal distribution as much as possible. The clinical data of the 2 groups are comparable.
The sample size was calculated using the superiority test formula for a two-sample mean comparison based on a two-group parallel controlled randomized block design:
$$ N = \frac{{\left( {Z_{1 - \alpha } + Z_{1 - \beta } } \right)\left( {Z_{1 - \alpha } + Z_{1 - \beta } } \right)^{2} \times \left( {\sigma_{1}^{2} + \sigma_{2}^{2} } \right)^{2} }}{{\left( {\mu_{1} - \mu_{2} - \delta } \right)^{2} }} $$
The observation indicator was IDO activity. According to relevant studies in China and other countries, the characteristic value of IDO activity, which is the ratio of kynurenine to tryptophan content (unit: %), was 12.8 in the experimental group and 7 ± 1 in the control group. Thus, α = 0.025 (unilateral), 1 – β = 0.90, μ1 = 12.8, μ2 = 7, \( \sigma_{1}^{2} = 2^{2} \), \( \sigma_{2}^{2} = 1^{2} \), and δ = 3. The sample size of each group was calculated to be 29. Considering interference factors such as subject dropout during the clinical trial, we determined that each group should have 30 subjects.
Study design
Part I: Collection of complete medical history, physical examination, pulmonary function tests, skin prick test for allergens and eosinophil count (EC).
Pulmonary function tests:
Quality control standard of children’s lung function: Exhale rapidly to PEF, no hesitation, smooth breath descending branch, no cough and glottis closing; Platform to achieve end expiratory flow; If flow at the end > 10%, It can be considered as early termination; deally, at least two acceptable tests should be completed, and the difference between two FVC and FEVT should be less than 0.1 L or 10%.
2. Skin prick test for allergens: allergenicity was defined as a positive skin reaction to more than one of the 12 common inhalant allergens, such as 2 types of dust mites, pollen, cat hair, dog hair, Alternaria and Aspergillus.
Implementation steps: Drop a drop of allergen test solution onto the sterilized skin on the palmar side of the forearm of the subject, and then prick it vertically with a point prick. The negative and positive control groups were set with normal saline and histamine diphosphate solution. According to the papule area ratio caused by pricking solution and positive control, the reaction level was judged: > 25% was positive, < 25% was negative.
Part II: Peripheral blood tests.
After enrollment, blood samples were immediately collected from the subjects in the observation group and the control group, and the samples were processed as follows:
1.
Specimen collection: Four milliliters of venous blood (with heparin as an anticoagulant) was collected from the subjects. Whole blood was centrifuged, and plasma was collected and stored at − 80 °C. Peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation in Ficoll.
1) Flow cytometry was used to detect Th17 (CD4+ IL17+) cells and Tregs (CD4+CD25+Foxp3+).
Antibodies used and isotype controls:
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Human IL–17/IL–17A PE–conjugated Antibody (IC317P); Mouse IgG2B PE-conjugated
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Isotype control (IC0041P).
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Human/Mouse FoxP3 PE– conjugated Antibody (IC8214P-100); Normal Rabbit IgG.
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PE-conjugated control (IC105P)
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Human CD25/IL-2 R alpha APC– conjugated Antibody (FAB1020A-100); Mouse IgG2A APC-conjugated Antibody (IC003A)
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Human CD4 Fluorescein –conjugated Antibody (FAB3791F-100); Mouse IgG2A Fluorescein-conjugated Antibody(IC003F) gating procedures: Th17, the gate was set with CD4, and the proportion of CD4+ IL17+ in the gate was analyzed. Treg set the gate with CD4 and SSc. CD4 cells were selected to analyze the expression of CD25+ and Foxp3+, and the proportion of Foxp3 cells was analyzed.
Cell fixation and or freezing employed:
Machine and software involved:
BD Accuri™ C6 software is Flowjo 7.6.1 Min
2) Enzyme-linked immunosorbent assay (ELISA) was utilized to examine the levels of cytokines, including IL-10, IL-17, IL-6 and TGF-β, in plasma.
Human TGF-beta 1 Quantikine ELISA Kit (DB100B),Human IL-17
Quantikine ELISA Kit (D1700), Human IL-6 Quantikine ELISA Kit (D6050)
Human IL-10 Quantikine ELISA Kit (D1000B) were used. 3) High-performance liquid chromatography (HPLC) was employed to determine tryptophan and kynurenine concentration. IDO activity was calculated as kynurenine concentration (μmol/L)/tryptophan concentration (μmol/L).
It employed a Symmetry Shield RP-C18 column (150 mm × 3.9 mm i.d., 5 μm) and a mobile phase of 15 mmol/L sodium acetate-acetic acid solution containing 2.7% (v/v) acetonitrile (pH 3.6) at a flow rate of 1.0 mL/min. The ultraviolet detector was Operated at 225 nm. Serum samples were first precipitated with a 5.0% perchloric acid solution, then centrifuged to remove protein residue and finally analyzed by HPLC.
Part III: Induced sputum test
1.
Specimen collection: After enrollment, induced sputum samples were immediately collected from the subjects in the observation and control groups. Sputum was aspirated after subjects were given aerosolized 3% hypertonic saline (1.5 mL of 10% NaCl + 2.5 mL of 0.9% NaCl), and the induced sputum supernatant was stored at − 70 °C.
2.
Tested parameters: HPLC was employed to determine tryptophan and kynurenine concentration. IDO activity was calculated as kynurenine concentration (μmol/L)/tryptophan concentration (μmol/L).