Table 1.
Cow’s milk allergenic protein nomenclature and short names as adopted within the present study.
Table 2.
Oral Challenge schedules in patients with reported cow’s milk sensitization.
Table 3.
Ninety two PR water soluble extracts from 46 samples as provided by six producers at different maturation ages.
Figure 1.
Biochemical evaluation of cow’s milk proteins in Parmigiano-Reggiano preparations at different maturation stages.
Panel A: SDS-PAGE of Parmigiano-Reggiano (PR) water soluble extracts at different maturation time (6–41 months). Extracts from different producers, as detailed in Table 3, are shown to give a comprehensive description of maturation effect on (PR); Lane 2: A-6-1-a; Lane 3: D-7-1-a; Lane 4: F-11-1-b; Lane 5: B-12-1-a; Lane 6: C-14-1-a; Lane 7: A-16-1-a; Lane 8: B-18-1-a; Lane 9: B-20-1-a; Lane11: E-24-1-a; Lane 12: B-25-1-a; Lane 13: D-26-1-a; Lane 14: F-33-1-a; Lane 15: A-36-1-a; Lane 16: E-37-1-a; Lane 17: C-38-1-a; Lane 18: E-41-1-b. The molecular weight marker (M, lane 1) and a standard solution (S, lane 10) containing α-lactalbumin, β-lactoglobulin, α-caseins, β-casein, κ-casein are also included. ALA: α-lactalbumin; BLG: β-lactoglobulin; CAS: casein fraction. Numbers above lanes indicate the month of maturation (lanes 2–9 and 11–18); numbers below indicate lanes. Panel B: Amounts of α-lactalbumin and β-lactoglobulin in the PR aqueous extracts having different maturation ages, as measured by UHPLC/ESI-MS. Some of the analyzed samples from different producers are shown. Preparations shown in the graph can be identified in Table 3 as follows, from left to right: 6 months: A-6-1-a; 7 months: B-6-1-a; 12 months: B-12-1-a; 12 months: D-12-1-a; 24 months: A-24-1-a; 24 months: C-24-1-a; 36 months: A-36-1-a; 36 months: B-36-1-a. Values are expressed as mg/100 gr Water Soluble Extract, and reported as average of two independent analyses (standard deviations aprox 10%). Black bars: α-lactalbumin (ALA); Grey bars: β-lactoglobulin (BLG).
Figure 2.
Single Point Highest Inhibition Achievable assay (SPHIAa) results obtained using a pool of sera containing IgE for all milk allergen specificities and Parmigiano-Reggiano preparations at different maturation stages.
Allergen short names are as in Table 1. Error bars indicating variability among samples from 6 dairies are reported above each allergen result.
Figure 3.
In vivo test reactivity distribution and symptoms recorded on DBPCFC with fresh cow’s milk and OFC with 36 months maturated Parmigiano-Reggiano.
Panel A, left graph, includes all 70 patients either tested or not for both milk and PR preparations. Group A includes Cow’s Milk (CM) and Parmigiano-Reggiano (PR) Tolerant subjects: 16 (22.86%); Group B includes CM Reactive PR Tolerant subjects: 29 (41.43%); Group C includes CM and PR Reactive subjects: 21 (30%); Group D includes CM Reactive PR not tested subjects: 4 (5.71%). Panel A, right graph, includes the 50 patients who reacted to CM DBPCFC and differently to PR OFC. Group B includes CM Reactive PR Tolerant subjects: 29 (58%); Group C includes CM and PR Reactive subjects: 21 (42%). Panel B includes the 66 patients tested by both CM DBPCFC and PR OFC; White bars: symptoms recorded after CM DBPCFC in 50 reactive subjects; Hatched bars: symptoms recorded after PR OFC in 21 reactive subjects; The four subjects not tested with PR OFC are not included in the graph. GI Tract: symptoms related to the gastrointestinal tract including vomiting and diarrhea, excluding oral allergy syndrome (OAS). Urticaria, GI tract symptoms and no symptom prevalence differed by the Fisher’s exact test in a statistically significant way for p<0.0017, 0.0297, 0.0001, respectively.
Table 4.
Demographics and in vivo and in vitro diagnostic test results in patients referring to our allergy Centre with a previous diagnosis of cow’s milk allergy.
Table 5.
ROC analyses of performed tests using cow’s milk DBPCFC and PR OFC as gold standards.
Figure 4.
Single Point Highest Inhibition Achievable assay (SPHIAa) IgE experiments with simulated digestions of Parmigiano-Reggiano water soluble extracts at three different maturation stages.
Results are the average of water soluble extracts obtained from four producers. Error bars, indicating variability among four assays, are reported above each allergen result. TCP: Trypsin, Chimotrypsin, Pepsin.
Figure 5.
Additional allergen sensitizations detecting IgE using the ISAC103 microarray system in the three CM sensitised subsets depending on their response to CM and PR oral challenges: A (CM- PR-: white bar); B (CM+ PR−: light grey bar); Group C (CM+ PR+: dark grey bar).
At least one positive IgE result for one of the following allergens has been evaluated for grouping by allergenic molecule groups and allergenic sources: Panel A - LTP: Cor a 8, Pru p 3; Parvalbumin: Cyp c 1, Gad c 1; PR-10: Act d 11, Api g 1, Ara h 8, Bet v 1, Cor a 1.0101, Cora 1.0401, Dau c 1, Mal d 1, Pru p 1, Gly m 4; Profilin: Bet v 2, Hev b 8, Mer a 1, Ole e 2, Phl p 12; Tropomyosin: Der p 10, Ani s 3, Pen a 1, Pen i 1, Pen m 1; Panel B - Anisakis: Ani s 1; Hen’s egg: Gal d 1, Gal d 2, Gal d 3, Gal d 5; Kiwi: Act d 1, Act d 2, Act d 5; Peanuts: Ara h 1, Ara h 2, Ara h 3; Seeds: Ana o 2, Ber e 1, Cor a 9, Ses i 1; Soy: Gly m 5, Gly m 6; Wheat: Tri a 18, Tri a 19, Tri a Gliadin; Panel C - Cat: Fel d 1, Cypress: Cry j 1, Cup a 1; Dog: Can f 1, Can f 3; Grasses: Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 11; Latex: Hev b 1, Hev b 3,, Hev b 5, Hev b 6; Mites: Der p 1, Der p 2, Der f 1, Der f 2, Eur m 2; Moulds: Alt a 1, Alt a 6, Asp f 1, Asp f 2, Asp f 3, Asp f 4, Asp f 6; Olive: Ole e 1; Pellitory: Par j 2; Weeds: Art v 1, Art v 3, Amb a 1, Sal k 1. Detailed information about the nature of additional IgE positive allergens detected by ISAC microarray system can be obtained from www.allergome.org.