Human subjects
In total of 825 food allergic patients’ serum were collected at the First Affiliated Hospital, Guangzhou Medical University (Guangzhou, China) during January 2015 to November 2016. The diagnosis was conducted by the doctors of this hospital. The clinical features of human subjects are presented in Table 1. This study has been approved by the Human Ethic Committee at Shenzhen University and Guangzhou Medical University. Informed consent was obtained from all subjects and if subjects are under 18, from a legal guardian. All experiments were performed in accordance with the relevant guidelines and regulations.
Table 1
Clinical features of human subjects.
| Food allergy (n = 825) | Healthy subjects (n = 25) |
Sex M/F | 528/297 (M-64%) | 14/11 (M-54.5%) |
Age mean; range | 11.46 ± 0.6051 (1–87) | 17.46 ± 4.761 (1–82) |
Personal history of atopy | 332 (40.24%) | 0 |
Total IgE | 447.7 ± 21.64** | 109.8 ± 17.76 |
Atopic comorbidities | | |
Bronchcial asthma | 223 (27.03%) | 0 |
Allergic rhinitis | 83 (10.06%) | 0 |
Allergic dermatitis | 26 (3.15%) | 0 |
Positive sIgE to HDM | 544 (65.93%) | 20% |
Allergen distribution | | |
milk | 511 (61.93%) | 0 |
egg | 419 (50.78%) | 0 |
shrimp | 223 (27.03%) | 0 |
crab | 114 (13.80) | 0 |
wheat | 57 (6.90%) | 0 |
peanut | 12 (1.45%) | 0 |
soy | 12 (1.45%) | 0 |
fish | 2 (0.24%) | 0 |
Dermatophagoides pteronyssinus Culture and Extracts Preparation
Dermatophagoides pteronyssinus mites were cultured as reported previously,11 dust mites were cultured at 25 °C with 70% relative humidity. Subsequently, mites were isolated from the medium using a modified heat-escape method and the dust mites purity was evaluated by checking mite morphology. Mite bodies were washed with PBS, weigh 2 gram sample adding 1 ml lysate (9M urea, 4% CHAPS, 60 mM DTT, 2% IPG buffer) and homogenized in liquid nitrogen, centrifuged at 15,000 rpm for 20 min under refrigeration. The supernatant was termed HDM extract.
Mice
6 to 8 weeks old female BALB/c mice (weight: 18–20 g), obtained from the Guangdong Experimental Animal Center (Guangzhou, China), were maintained in specific pathogen-free conditions according to standard guidelines for the care and use of animals. The experimental procedures were approved by the Institutional Ethics Committee at Shenzhen University (Shenzhen, China). The study was carried out in compliance with the ARRIVE guidelines.
Induction of experimental food allergy
As shown by Fig. 2A, 18 mice were randomly divided into 3 groups : HDM + cholera toxin (CT) group, HDM group and Control group. Mice were sensitized intraperitoneally with PBS (Control group), HDM extract (1 mg/mouse) and CT (20 µg/mouse) (HDM + CT group), or HDM extract (1 mg/mouse) (HDM group) on day 0 and day 3 respectively. From day 5 on, challenge was performed every other day for 10 days, mice were challenged with PBS (Control group), HDM extract (1 mg/mouse) and CT (20 µg/mouse) (HDM + CT group), or HDM extract (1 mg/mouse) (HDM group) by intra-gastric (i.g) gavage. The body weight of each mouse was recorded every other day. An OVA food allergy followed by HDM exposure were induced in Balb/c mice. Mice were continually exposed to HDM (1 mg, i. g)/PBS for a week. Combined HDM exposure and food allergy was obtained by applying the two protocols successively (Fig. 4A). Control mice were sensitized with PBS alone and challenged with PBS.
Enzyme-linked immunosorbent assay (ELISA)
The levels of specific IgE and IgG1 for HDM were determined by ELISA as described previously.12 Briefly, the ELISA microtiter plates were coated with HDM with at 1 ug/well in 100 µl Carbonate buffered solution (CBS, 15 mM Na2CO3 and 35 mM NaHCO3, pH9.5). After incubation (overnight, 4 °C), plates were washed 3 times with PBST (PBS/0.05% Tween 20) and blocked with 3% bovine serum albumin in PBS (3% BSA-PBS) (1 hour, 37 °C). The serum (1:10 diluted with 3% BSA-PBS) or BSA (using as a negative control) were then added to each well and incubated (2 hours, 37 °C). Subsequently, 100 µL of peroxidase-labeled goat anti-mouse IgE (1:2000) was added to each well. The plates were incubated (2 hours, 37 °C). Following 3 washings, the reactions were developed with TMB (tetramethylbenzidine, 100uL/well) for 20 min and stopped by 50 µl 2 M H2SO4. The plates were read by an ELx808 absorbance microplate reader (BioTek, Shanghai, China) at 450 nm. The splenocytes culture supernatant levels cytikines IL-4 (Boster, Wuhan, China), IL-5 and IFN-γ (Sino Biological Inc, Beijing, China) were determined by ELISA with commercial reagent kits following the manufacturer’s instruction.
Flow cytometry
Spleen cells were prepared according to Gunzer M et al’s report.13 Splenocytes (2x106/well) were labeled with CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester) in dark, incubated with 50 µg/ml HDM or culture medium and 2 µl/ml cell stimulation cocktail (a cocktail of phorbol 12-myristate 13-acetate (PMA) and ionomycin, ebioscience) in 96-well plate (72 h, 37 °C). Following washing with PBS, the cells were stained with CD4-APC (1 h, room temperature). After washing, the cells were analyzed by flow cytometry. The data were analyzed with the software Flowjo.
Assessment of the Intestinal permeability in vivo
This measure is based on the intestinal epithelial barrier permeability to 4,000-Da fluorescent-dextran (Sigma-Aldrich).14 6-h-fasted mice were fed with fluorescein isothiocyanate (FITC)-dextran at 600 mg/kg body weight (125 mg/ml). After 1 h, the mice were sacrificed. The blood was collected from the tip of the tail vein. The blood was centrifuged t 5000 rpm (3 min, 4 °C.) Plasma was diluted in an equal volume of PBS (pH 7.4) and the FITC-dextran concentration in the plasma was determined with a fluorescence spectrophotometer at the excitation wavelength of 485 nm and the emission wavelength of 535 nm. Standard curves for calculating the FITC-dextran concentration in the plasma were obtained by diluting FITC-dextran in nontreated plasma diluted with PBS (1:2 [vol/vol]).
Histology
For assessment of pathologic alterations, jejunum samples were fixed in 4% formalin overnight and embedded in paraffin. The tissues were cut into 4-µm thick sections and stained with hematoxylin and eosin (HE). The eosinophils and mononuclear cells in jejunum were counted under a light microscope. To avoid the observer bias, the observers were not aware of the code .
Statistics
Data are presented as mean ± SD. Difference between 2 groups was determined by Student t test or ANOVA if more than two groups. P < 0.05 was set as a signifcant criterion.