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Laurent Pons, PhD, Usha Ponnappan, PhD, Renée A

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1 Soy immunotherapy for peanut-allergic mice: Modulation of the peanut-allergic response 
Laurent Pons, PhD, Usha Ponnappan, PhD, Renée A. Hall, MS, Pippa Simpson, PhD, Gael Cockrell, BS, C. Michael West, BS, Hugh A. Sampson, MD, Ricki M. Helm, PhD, A. Wesley Burks, MD  Journal of Allergy and Clinical Immunology  Volume 114, Issue 4, Pages (October 2004) DOI: /j.jaci Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

2 Fig 1 Symptom scores after peanut challenges of peanut sensitized mice before and after desensitization. PN, Mice killed before undergoing IT; PN/PN, mice treated with crude peanut (0.5 mg of the highest dose); PN/Soy, crude soybean IT (1.0 mg of the highest dose); PN/PBS, placebo IT with PBS; PBS/PBS, naive mice sensitized/desensitized with PBS. Each circle corresponds to 1 animal, and the horizontal line represents the median of the group. Brackets indicate statistical differences between designated groups (∗∗P < .01; NS, not significant). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

3 Fig 2 Core body temperatures after peanut challenges of peanut-sensitized mice before and after desensitization. PN, Mice killed before undergoing IT; PN/PN, mice treated with crude peanut (0.5 mg of the highest dose); PN/Soy, crude soybean IT (1.0 mg of the highest dose); PN/PBS, placebo IT with PBS; PBS/PBS, naive mice sensitized/desensitized with PBS. Values plotted are means ± SDs. Brackets indicate statistical differences between designated groups (∗∗P < .01; NS, not significant). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

4 Fig 3 Peanut-specific IgG1 levels in the serum of peanut-sensitized mice before and after desensitization. PN, Mice killed before undergoing IT; PN/PN, mice treated with crude peanut (0.5 mg of the highest dose); PN/Soy, crude soybean IT (1.0 mg of the highest dose); PN/PBS, placebo IT with PBS; PBS/PBS, naive mice sensitized/desensitized with PBS. Values plotted are means ± SDs. Brackets indicate statistical differences between designated groups (∗P < .05). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

5 Fig 4 Splenocyte proliferative responses to peanut stimulation. Triplicate cultures of pooled spleen cells (n=5) from nonchallenged mice of each group were stimulated with CPE 100 μg/mL, medium alone, or PHA. After 3 days, the cells were pulsed for 6 hours with 1 μCi per well of [3H]thymidine, harvested, and counted for β-radioactivity. Stimulation indexes were calculated as the mean of triplicates. PN, Mice killed before undergoing IT; PN/PN, mice treated with crude peanut (0.5 mg of the highest dose); PN/Soy, crude soybean IT (1.0 mg of the highest dose); PN/PBS, placebo IT with PBS; PBS/PBS, naive mice sensitized/desensitized with PBS. The average stimulation index with PHA for all the groups was 31% ± 5%. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

6 Fig 5 IFN-γ production by splenocytes stimulated with peanut extract. Pooled spleen cells (4 × 105 per well) from nonchallenged mice of each group (n=5) were stimulated with CPE 100 μg/mL. After 24 hours, the supernatants were collected, and IFN-γ levels were determined by flow cytometry with the Cytometric Bead Array method (BD PharMingen). Values plotted are means of triplicate determinations. PN, Mice killed before undergoing IT; PN/PN, mice treated with crude peanut (0.5 mg of the highest dose); PN/Soy, crude soybean IT (1.0 mg of the highest dose); PN/PBS, placebo IT with PBS; PBS/PBS, naive mice sensitized/desensitized with PBS. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

7 Fig 6 TNF-α production by splenocytes stimulated with peanut extract. Pooled spleen cells (4 × 105 per well) from nonchallenged mice of each group (n=5) were stimulated with CPE 100 μg/mL. After 24 hours, the supernatants were collected, and TNF-α levels were determined by flow cytometry with the Cytometric Bead Array method (BD PharMingen). Values plotted are means of triplicate determinations. PN, Mice killed before undergoing IT; PN/PN, mice treated with crude peanut (0.5 mg of the highest dose); PN/Soy, crude soybean IT (1.0 mg of the highest dose); PN/PBS, placebo IT with PBS; PBS/PBS, naive mice sensitized/desensitized with PBS. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

8 Fig 7 Schematic amino acid alignment of peanut allergen Ara h 1 and soybean β-conglycinin drawn from a multiple alignment of the full amino acid sequences by using CLUSTAL W Interruptions in the bars indicate gaps introduced for the purpose of the alignment. Amino acid sequences represented by black (Ara h 1) and white (soybean) rectangles are 15% identical and 31% similar. Amino acid sequences represented by hatched (Ara h 1) and gray (soybean) rectangles are 46% identical and 41% similar. Percentages of identity and similarity for each region were calculated as the average of pairwise comparisons between Ara h 1 and the 3 β-conglycinin subunits. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

9 Fig 8 Amino acid sequence alignment of peanut allergen Ara h 2 and soybean 2S albumin. (A) There are 10 IgE-binding epitopes on Ara h 2. Three of the 10 epitopes are immunodominant (numbered 2, 6, and 7). Immunodominant epitopes 6 and 7 are shown. There are only 3 of 10 identical or similar amino acids between Ara h 2 and the soybean 2S albumin (GenBank AAD09630) in these 2 epitopes. In the nonimmunodominant epitopes (epitope 9, representative example), there is a high degree of identity or similarity (8/10). (B) There are 4 T-cell epitopes on Ara h 2. These epitopes have 67% to 79% amino acid identity or similarity with the aligned soybean 2S albumin sequence. The pairwise alignment was performed on the full amino acid sequences by using CLUSTAL W 1.8 and trimmed to focus on Ara h 2–different epitopes. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2004 American Academy of Allergy, Asthma and Immunology Terms and Conditions

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