{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001369","ANALYSIS_ID":"AN002285","VERSION":"1","CREATED_ON":"April 29, 2020, 10:46 am"},

"PROJECT":{"PROJECT_TITLE":"Metabolomics analysis of serum samples from patients during 2 years of IAT trial, double blind placebo controled study","PROJECT_TYPE":"Study the changes produced by the use of AIT treatment or placebo","PROJECT_SUMMARY":"47 patients were enrolled in a double-blind, placebo-controlled, multicenter trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 patients who finished the trial. Additionally, serum and PBMCs samples from these samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were grouped in Mono and Poli groups, excluding epithelial allergic patients. Individuals were studied based on their treatment in Active and Placebo and their sensitization level.","INSTITUTE":"Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and Bioanalysis, CEU","LAST_NAME":"Barber Hernández","FIRST_NAME":"Domingo","ADDRESS":"Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España","EMAIL":"domingo.barberhernandez@ceu.es","PHONE":"Tlf: 91 372 47 00 ext. 4662"},

"STUDY":{"STUDY_TITLE":"Grass pollen sublingual immunotherapy treatment induces transcriptomic and metabolic changes due to AIT treatment","STUDY_SUMMARY":"47 patients were enrolled in a double-blind, placebo-controlled, multicenter trial using with GRAZAX® (Phleum pretense) during 2 years of therapy (T2). Immunological assays such as sIgE, sIgG4 and ISAC were carried out to the 31 patients who finished the trial. Additionally, serum and PBMCs samples from these samples were analyzed by metabolomics and transcriptomics, respectively. Based on their sensitization level, 22 patients were grouped in Mono and Poli groups, excluding epithelial allergic patients. Individuals were studied based on their treatment in Active and Placebo and their sensitization level. For metabolomics, samples were analyzed by Liquid and Gas Chromatography coupled to Mass Spectrometry (LC-MS and GC-MS, respectively).","INSTITUTE":"Institute of Applied Molecular Medicine, CEU; The Centre of Metabolomics and Bioanalysis, CEU","LAST_NAME":"Obeso Montero","FIRST_NAME":"David","ADDRESS":"Avda. Monteprincipe s/n 28668 Boadilla del Monte, Madrid, España","EMAIL":"david.obesomontero@beca.ceu.es","PHONE":"Tlf: 91 372 47 00 ext. 4662","NUM_GROUPS":"2 main groups: Active and Placebo, and 2 subgroups: Monosensitized and Polisensitized patients.","TOTAL_SUBJECTS":"22","STUDY_COMMENTS":"https://www.ceu.es;http://www.metabolomica.uspceu.es"},

"SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Subject 1",
"Sample ID":"P1_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P1_t0.d"}
},
{
"Subject ID":"Subject 2",
"Sample ID":"P3_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P3_t0.d"}
},
{
"Subject ID":"Subject 3",
"Sample ID":"P4_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P4_t0.d"}
},
{
"Subject ID":"Subject 4",
"Sample ID":"P8_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P8_t0.d"}
},
{
"Subject ID":"Subject 5",
"Sample ID":"P9_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P9_t0.d"}
},
{
"Subject ID":"Subject 6",
"Sample ID":"P11_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P11_t0.d"}
},
{
"Subject ID":"Subject 7",
"Sample ID":"P12_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P12_t0.d"}
},
{
"Subject ID":"Subject 8",
"Sample ID":"P13_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P13_t0.d"}
},
{
"Subject ID":"Subject 9",
"Sample ID":"P14_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P14_t0.d"}
},
{
"Subject ID":"Subject 10",
"Sample ID":"P15_t0",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P15_t0.d"}
},
{
"Subject ID":"Subject 11",
"Sample ID":"P16_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P16_t0.d"}
},
{
"Subject ID":"Subject 12",
"Sample ID":"P17_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P17_t0.d"}
},
{
"Subject ID":"Subject 13",
"Sample ID":"P20_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P20_t0.d"}
},
{
"Subject ID":"Subject 14",
"Sample ID":"P21_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P21_t0.d"}
},
{
"Subject ID":"Subject 15",
"Sample ID":"P22_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P22_t0.d"}
},
{
"Subject ID":"Subject 16",
"Sample ID":"P23_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P23_t0.d"}
},
{
"Subject ID":"Subject 17",
"Sample ID":"P24_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P24_t0.d"}
},
{
"Subject ID":"Subject 18",
"Sample ID":"P25_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P25_t0.d"}
},
{
"Subject ID":"Subject 19",
"Sample ID":"P26_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P26_t0.d"}
},
{
"Subject ID":"Subject 20",
"Sample ID":"P27_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P27_t0.d"}
},
{
"Subject ID":"Subject 21",
"Sample ID":"P28_t0",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P28_t0.d"}
},
{
"Subject ID":"Subject 22",
"Sample ID":"P30_t0",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P30_t0.d"}
},
{
"Subject ID":"Subject 1",
"Sample ID":"P1_t2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P1_t2.d"}
},
{
"Subject ID":"Subject 2",
"Sample ID":"P3_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P3_t2.d"}
},
{
"Subject ID":"Subject 3",
"Sample ID":"P4_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P4_t2.d"}
},
{
"Subject ID":"Subject 4",
"Sample ID":"P8_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P8_t2.d"}
},
{
"Subject ID":"Subject 5",
"Sample ID":"P9_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P9_t2.d"}
},
{
"Subject ID":"Subject 6",
"Sample ID":"P11_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P11_t2.d"}
},
{
"Subject ID":"Subject 7",
"Sample ID":"P12_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P12_t2.d"}
},
{
"Subject ID":"Subject 8",
"Sample ID":"P13_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P13_t2.d"}
},
{
"Subject ID":"Subject 9",
"Sample ID":"P14_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P14_t2.d"}
},
{
"Subject ID":"Subject 10",
"Sample ID":"P15_t2",
"Factors":{"Treatment":"Active","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P15_t2.d"}
},
{
"Subject ID":"Subject 11",
"Sample ID":"P16_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P16_t2.d"}
},
{
"Subject ID":"Subject 12",
"Sample ID":"P17_t2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P17_t2.d"}
},
{
"Subject ID":"Subject 13",
"Sample ID":"P20_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P20_t2.d"}
},
{
"Subject ID":"Subject 14",
"Sample ID":"P21_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P21_t2.d"}
},
{
"Subject ID":"Subject 15",
"Sample ID":"P22_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P22_t2.d"}
},
{
"Subject ID":"Subject 16",
"Sample ID":"P23_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P23_t2.d"}
},
{
"Subject ID":"Subject 17",
"Sample ID":"P24_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Poli"},
"Additional sample data":{"RAW_FILE_NAME":"P24_t2.d"}
},
{
"Subject ID":"Subject 18",
"Sample ID":"P25_T2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P25_t2.d"}
},
{
"Subject ID":"Subject 19",
"Sample ID":"P26_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P26_t2.d"}
},
{
"Subject ID":"Subject 20",
"Sample ID":"P27_T2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P27_t2.d"}
},
{
"Subject ID":"Subject 21",
"Sample ID":"P28_t2",
"Factors":{"Treatment":"Active","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P28_t2.d"}
},
{
"Subject ID":"Subject 22",
"Sample ID":"P30_t2",
"Factors":{"Treatment":"Placebo","Sensitization":"Mono"},
"Additional sample data":{"RAW_FILE_NAME":"P30_t2.d"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For immunological analyses and metabolomics, serum samples were obtained and stored at -20°C until analysis. In the case of transcriptomics, Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and stored at -20°C in Buffer RLT until analysis.","SAMPLE_TYPE":"Blood (serum)","STORAGE_CONDITIONS":"-20℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Subjects were randomized (1:1) during autumn 2013 to receive either active treatment with GRAZAX® (Phleum pratense, 75,000SQ‐T tablets ALK, Hørsholm, Denmark) or placebo as Investigational Medical Products (IMP) during 2 years. Twenty-five patients were assigned to daily sublingual administration of GRAZAX®, while the rest received placebo tablets that were similar to the active IMP with regard to appearance, smell and taste."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For LC-MS, proteins were removed adding 300 µL of cold (-20 °C) methanol:ethanol (1:1) to 100 µL of sample. Samples were then vortex-mixed and stored on ice for 5 min. Supernatant containing the metabolites was separated from the pellet by centrifugation (16,000× g for 20 min at 4 °C), and put into a LC vial for analysis. For GC-MS, serum samples were first deproteinized using cold acetonitrile (ACN) in a 3:1 proportion (120 μL of ACN were added to 40 μL of serum). Samples were kept on ice for 5 minutes. Afterwards, the metabolites were separated by centrifugation (10 min at 15,400 x g and 4°C). 100 µL of the resulting supernatant were transferred to a GC vial with insert and were evaporated to dryness for 2 hours at 30°C (SpeedVac Concentrator, Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, a methoximation was performed by addition of 10 µL of O-methoxyamine hydrochloride 15mg/mL in pyridine, with the purpose of protecting the reactive oxygen groups in the metabolites. The mixture was vigorously vortex-mixed (1 minute each vial), ultrasonicated (3 times, 20 seconds each) and further vortexed another 2 minutes. Then, the samples were left in darkness at room temperature for 16 h for the completion of the methoximation reaction. Finally, the samples were derivatized by the addition of 10 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS).","PROCESSING_METHOD":"Protein precipitation and metabolite extraction","PROCESSING_STORAGE_CONDITIONS":"On ice"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with triple-Axis detector (5975C, Agilent). Two microliters (2 μL) of the derivatized sample were injected through a GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 μm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent). Carrier gas (He) flow rate was set at 1 mL/min and injector temperature at 250 °C. Split ratio was fixed from 1:5 to 1:10 with 3 to 10 mL/min. He split flow into a Restek 20782 (Bellefonte, PA, USA) deactivated glass-wool split liner. The temperature gradient was programmed as follows: the initial oven temperature was set at 60 ºC (held for 1 min), increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for 10 min. The total run time was 37.5 min. A cool-down period was applied for 10 min before the next injection. Detector transfer line, filament source and the quadrupole temperature were set at 280 ºC, 230 ºC and 150 ºC, respectively. MS detection was performed with electron ionization (EI) mode at -70 eV. The mass spectrometer was operated in scan mode over a mass range of m/z 50-600 at a rate of 2.7 scan/s. Several IS injections, a standard mix of fatty acid methyl esters (FAME C8-C30), extraction blanks and 3 QCs samples were injected at the beginning of analysis, following QCs injections every 5 experimental samples and 1 QC injection at the end of worklist.","CHROMATOGRAPHY_TYPE":"GC","INSTRUMENT_NAME":"Agilent 7890A","COLUMN_NAME":"GC-Column DB5-MS (30 m length, 0.25 mm i.d., 0.25 µm film 95% dimethyl/5% diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent) diphenylpolysiloxane) with an integrated precolumn (10 m J&W, Agilent)","COLUMN_TEMPERATURE":"As Oven","INJECTION_TEMPERATURE":"250","INTERNAL_STANDARD":"Standard mix of fatty acid methyl esters (FAME C8-C30)","SAMPLE_INJECTION":"2 μL","ANALYTICAL_TIME":"37.5 min","OVEN_TEMPERATURE":"The initial oven temperature was set at 60 ºC (held for 1 min), increased to 325 ºC at 10 ºC/min rate (within 26.5 min) and hold 325 ºC for 10 min.","TRANSFERLINE_TEMPERATURE":"280"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","DATA_FORMAT":".D"},

"MS":{"INSTRUMENT_NAME":"Agilent 5975C","INSTRUMENT_TYPE":"Single quadrupole","MS_TYPE":"EI","ION_MODE":"POSITIVE","MS_COMMENTS":"GC-MS analysis was performed by a GC system (Agilent Technologies 7890A) equipped with an autosampler (Agilent 7693) coupled to a mass spectrometer with triple-Axis detector (5975C, Agilent).","HELIUM_FLOW":"1 mL/min","IONIZATION_ENERGY":"-70 eV","MS_RESULTS_FILE":"ST001369_AN002285_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}