This reduction persisted until 4 wks post therapy. anaphylaxis following peanut challenge at 1 day and 4 weeks post therapy. Reduction of peripheral blood basophils began after 1 week of treatment and continued for at least 4 weeks post therapy. The number and FcRI expression of peritoneal mast cells were also significantly decreased 4 weeks post therapy. FAHF-2 treated MC/9 cells showed significantly reduced IgE-induced FcRI expression, FcRI mRNA subunit expression, proliferation, and histamine release upon challenge. Fraction 2 (F2) from FAHF-2 inhibited RBL-2H3 cell and human mast cell degranulation. Three compounds from F2- berberine, palmatine and jatrorrhizine inhibited of RBL-2H3 cell degranulation via suppressing Syk phosphorylation. Conclusion FAHF-2 reduction of basophils and mast cell numbers as well as suppression of IgE-mediated mast cell activation may contribute to FAHF-2 persistent protection against peanut anaphylaxis. Clinical Implications FAHF-2 blocked peanut anaphylaxis. This effect was associated with reduction of mast cells/basophils activation and proliferation. FAHF-2 may be a novel anti-peanut anaphylaxis therapy. effects on murine mast cell (MC/9 cells) proliferation, histamine release, and FcRI subunits expression in response to IgE. Using preparative High Performance Liquid Chromatography (prep-HPLC), we identified and tested the effect of four fractions (F) and 3 major alkaloid compounds on rat mast cells (RBL-2H3) and human skin mast cells. In addition, we determined the mechanism by which the isolated compounds inhibit mast cell degranulation. METHODS Mice and reagents C3H/HeJ mice (female, 5 week-old) purchased from the Jackson Laboratory (Bar Harbor, ME) were maintained on PN-free chow under specific pathogen-free conditions according to standard guidelines for the care and use of animals. Freshly ground, whole roasted PN were prepared as previously described.(7;8) Cholera toxin (CT) was purchased from List Biological Laboratories, Inc (Campbell, CA). MC/9 cells (mouse mast cell line) and RBL-2H3 cells (rat basophilic leukemia cell line) were obtained from American Type Culture Collection (Manassas, Virginia). FAHF-2 is dried powder of aqueous extract of 9 herbs. The formula and process of preparation of the final product are the same as described in previous publications(6;9) and included in repository (Methods E1). To identify active compounds, FAHF-2 was first extracted with butanol, then dried, and then further divided into 4 fractions (F) based on polarity by prep-HPLC. Compounds were identified by liquid chromatography mass spectrometry (LC MS) with standard compounds as described in details in the repository (Methods E2). Antibodies Phycoerythrin (PE) Cconjugated mAbs specific for mouse FcRI mAb (MAR-1), allophycocyanin (APC)-conjugated anti- mouse Gr-1 (RB6-8C5), anti-c-kit (2B8), fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgE (23G3) and FITC-conjugated Amyloid b-Peptide (12-28) (human) control hamster IgG were Amyloid b-Peptide (12-28) (human) obtained from eBioscience (San Diego, CA). FITC-conjugated anti-mouse CCR3 mAb (83101) was purchased from R&D Systems (Minneapolis, MN). PE-conjugated anti-mouse CD3e(145-2C11), PE-conjugated anti- mouse B220(RA3-6B2), APC-conjugated anti-mouse CD49b(DX5), FITC conjugated anti-mouse I-Ak (AbK, 10-3.6) and purified anti-mouse CD16/32 mAb (2.4G2) were purchased from BD Biosciences (San Diego, CA). Mouse anti-dinitrophenol (DNP) IgE was purchased from Sigma-Aldrich (St. Louis, MO). Protocol for PN sensitization/challenge, FAHF-2 treatment and assessment of anaphylaxis Mice were sensitized Amyloid b-Peptide (12-28) (human) intragastrically (i.g.) with ground PN (10 mg/mouse) weekly for 5 wks, and boosted at weeks 6 and 8 (50 mg/mouse) (Figure 1A), as previously described.(4) FAHF-2 was administered 24h after the wk 8 boost, at which time PN hypersensitivity was established.(4) FAHF-2 was administered i.g. at 32 mg in 0.5 ml water, twice daily for 7 wks. The daily dose of FAHF-2 used in this study was calculated by normalizing the daily human dose to mouse body weight as previously described. (4;5)An additional group of PNA mice received equal volume of water (sham). Both groups exhibited equal levels of PN specific IgE prior to treatment. Na?ve mice served as normal controls. All mice were PN GFND2 challenged i.g. (200 mg/mouse) 1 day (wk 14) and 4 weeks post treatment (wk 18). CT as mucosal adjuvant at 20g/mouse was co-administered with PN at all times except at final challenge. Anaphylactic symptoms were evaluated approximately 30 minutes after the challenge dose utilizing a previously described scoring system(4;5) (Repository Methods E3) in a blinded manner. Rectal temperatures were measured approximately 30 minutes following challenge using a thermal probe (Harvard Apparatus, Newark, NJ). Plasma histamine levels were determined 30 minutes after the second challenge and analyzed using an enzyme immunoassay kit (ImmunoTECH Inc., Marseille, France) as described by the manufacturer. Open in a separate window Figure 1 A. Experimental.