{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002944","ANALYSIS_ID":"AN004829","VERSION":"1","CREATED_ON":"October 27, 2023, 10:00 am"},

"PROJECT":{"PROJECT_TITLE":"Integrated gut metabolome and microbiome fingerprinting reveals that dysbiosis precedes allergic inflammation in IgE-mediated pediatric cow’s milk allergy","PROJECT_TYPE":"(un)targeted MS","PROJECT_SUMMARY":"Background: IgE-mediated cow’s milk allergy (IgE-CMA) is one of the first allergies to arise in early childhood and may result from exposure to various milk allergens, of which β-lactoglobulin (BLG) and casein are the most important. Understanding the underlying mechanisms behind IgE-CMA is imperative for the discovery of novel biomarkers and the design of innovative treatment and prevention strategies. Methods: We report a longitudinal in vivo murine model, in which 2 mice strains (BALB/c and C57Bl/6) were sensitized to BLG using either cholera toxin or an oil emulsion (n=6 per group). After sensitization, mice were challenged orally, their clinical signs monitored, antibody (IgE and IgG1) and cytokine levels (IL-4 and IFN-γ) measured, and fecal samples subjected to metabolomics. The results of the murine models were further supported by fecal microbiome-metabolome data from our population of IgE-CMA (n=24) and healthy (n=23) children (Trial: NCT04249973), on which polar metabolomics, lipidomics and 16S rRNA metasequencing were performed. In vitro gastrointestinal digestions and multi-omics corroborated the microbial origin of proposed metabolic changes. Results: During sensitization, we observed multiple microbially derived metabolic alterations, most importantly bile acid, energy and tryptophan metabolites, that preceded allergic inflammation. The latter was reflected in a disturbed sphingolipid metabolism. We confirmed microbial dysbiosis, and its causal effect on metabolic alterations in our patient cohort, which was accompanied by metabolic signatures of low-grade inflammation. Conclusion: Our results indicate that gut dysbiosis precedes allergic inflammation and nurtures a chronic low-grade inflammation in children on elimination diets, opening important new opportunities for future prevention and treatment strategies.","INSTITUTE":"Ghent University","DEPARTMENT":"Translational Physiology, Infectiology and Public Health","LABORATORY":"Laboratory for Integrative Metabolomics","LAST_NAME":"De Paepe","FIRST_NAME":"Ellen","ADDRESS":"Salisburylaan 133, Merelbeke, Oost-Vlaanderen, 9820, Belgium","EMAIL":"Ellen.DePaepe@UGent.be","PHONE":"0032479081098","FUNDING_SOURCE":"Fonds Wetenschappelijk Onderzoek"},

"STUDY":{"STUDY_TITLE":"Longitudinal polar fecal metabolomics of mice undergoing sensitization to beta-lactoglobulin","STUDY_SUMMARY":"This study is part of a multi-part study, including a. longitudinal polar fecal metabolomics of mice undergoing sensitization to beta-lactoglobulin b. polar fecal metabolomics of a patient cohort c. fecal lipidomics of a patient cohort d. polar urinary metabolomics of a patient cohort e. polar metabolomics of in vitro digestions This specific part is part a. longitudinal polar fecal metabolomics of mice undergoing sensitization to beta-lactoglobulin","INSTITUTE":"Ghent University","DEPARTMENT":"Department of Translational Physiology, Infectiology and Public Health","LABORATORY":"Laboratory for Integrative Metabolomics","LAST_NAME":"De Paepe","FIRST_NAME":"Ellen","ADDRESS":"Salisburylaan 133, 9820 Merelbeke, Belgium","EMAIL":"Ellen.DePaepe@UGent.be","STUDY_TYPE":"(un)targeted MS","PHONE":"0032479081098"},

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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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{
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},
{
"Subject ID":"-",
"Sample ID":"S_21_0066_311",
"Factors":{"Strain":"B6","treatment":"5W","week":"Wk 7"},
"Additional sample data":{"RAW_FILE_NAME":"210611_se398.raw"}
},
{
"Subject ID":"-",
"Sample ID":"iQC57",
"Factors":{"Strain":"-","treatment":"-","week":"-"},
"Additional sample data":{"RAW_FILE_NAME":"210611_se399.raw"}
},
{
"Subject ID":"-",
"Sample ID":"iQC58",
"Factors":{"Strain":"-","treatment":"-","week":"-"},
"Additional sample data":{"RAW_FILE_NAME":"210611_se400.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Fecal samples were collected on different timepoints during sensitization of the mice. Samples were freeze-dried within one week after collection and stored at -80° awaiting analysis","SAMPLE_TYPE":"Feces","COLLECTION_METHOD":"Spontaneous defecation","COLLECTION_FREQUENCY":"1x week","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Two different mice strains and two different protocols were tested: BALB/c (BC) mice and C57Bl/6 (B6) mice. The first sensitization protocol was based on Adel-Patient et al., in which 4-week-old mice received oral gavages containing ß-lactoglobulin (BLG) and cholera toxin (CT) once a week for 6 weeks 20 (treatment W), the control groups received either BLG in PBS (X), CT in PBS (Y) or PBS alone (Z). The second protocol was adapted from Shindo et al. and included intragastrical administration of BLG in PBS or oil emulsion, followed by intraperitoneal injection of sodium salicylate (SA). The latter protocol was performed 5x a week for 3 weeks. Group A received BLG in PBS + SA, group B BLG in oil + SA, group C BLG in PBS + PBS, group D BLG in oil + PBS, group E and F were vehicle controls with group E receiving PBS + SA, and group F oil + SA","TREATMENT_COMPOUND":"Beta-lactoglobulin, cholera toxin","TREATMENT_ROUTE":"oral gavage, intraperitoneal injection","TREATMENT_DOSE":"1 mg ß-lactoglobulin (BLG) per g body weight mixed with 10 µg of cholera toxin (CT) in 0.1 mL PBS, or 1 mg BLG in 0.1 mL individual antigen solution (vehicle), which was either PBS or an oil emulsion (saline, linoleic acid, and lecithin at 5:4:1 by volume). Intraperitoneal administration of 0.1 mL PBS or PBS containing 0.2 mg sodium salicylate (SA)","TREATMENT_DOSEDURATION":"3 or 7 weeks","TREATMENT_VEHICLE":"PBS or oil emulsion","ANIMAL_ANESTHESIA":"CO","ANIMAL_FASTING":"Evening before antigen challenge","ANIMAL_ENDP_EUTHANASIA":"cervical dislocation","ANIMAL_ENDP_CLINICAL_SIGNS":"severe anaphylaxis"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"To extract the polar fecal metabolome, 200 mg of lyophilized homogenized feces was dissolved in 4 mL of ultrapure water, after the addition of 100 mL ISTD mixture (25 ng mL&1 of Dvaline- d8 and L-alanine-d3). Subsequent to 30 s of thorough vortexing, 1 mL of an ice-cold methanol and ultrapure water (80:20, v/ v) mixture was added. The supernatant of the solid-liquid extraction was collected after 1 min of vortexing and 10 min of rotation, followed by a 10-min centrifugation step (13 300 x g, at 4 %C). Next, the extract was passed over a polyamide filter (diameter of 25mm and pore size of 0.45 mm) (Machery-Nagel, Düren, Germany), diluted (1:3) with ultrapure water and transferred to a glass HPLCvial.","PROCESSING_STORAGE_CONDITIONS":"-80℃","SAMPLE_SPIKING":"Internal standard"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile; 0.1% formic acid","FLOW_GRADIENT":"A gradient profile with following proportions (v/v) of solvent A was applied: 0e1.5 min at 98%, 1.5e7.0 min from 98% to 75%, 7.0e8.0 min from 75% to 40%, 8.0e12.0 min from 40% to 5%, 12.0e14.0 min at 5%, 14.0e14.1 min from 5 to 98%, followed by 4.0 min of re-equilibration.","FLOW_RATE":"0.4 mL/min","COLUMN_TEMPERATURE":"45","INTERNAL_STANDARD":"L-alanine-d3, D-valine-d8","SAMPLE_INJECTION":"10 uL"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"MS acquisition: Xcalibur Targeted data processing: Xcalibur Untargeted data acquisition: Compound Discoverer, R studio","MS_RESULTS_FILE":"ST002944_AN004829_Results.txt UNITS:a.u. Has m/z:Neutral masses Has RT:Yes RT units:Minutes"}

}