It is important to know the contribution of specific allergens to a complex allergenic extract and to have a dependable method to assess the effector activity of an extract specifically depleted of that allergen. We have previously shown that removal of the major peanut allergen, Ara h 2, from a crude peanut extract (CPE) minimally altered the effector activity of the extract. Here we describe in detail the methodology used to generate specific rabbit anti-peptide antibodies to remove a related peanut allergen, Ara h 6, from CPE and describe an improvement in the RBL SX-38 cell assay used to assess the effector activity of treated extracts. Our results show that although Ara h 2 and Ara h 6 can be selectively removed from a CPE, removal of each alone from a CPE had no significant effect on the effector activity. However, removal of Ara h 2 and Ara h 6 together significantly reduced the effector activity of CPE.

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Background Peanut allergy has been reported to be transferred to tolerant recipients through organ and bone marrow transplantation. The roles T and B cells play in establishing, and the roles B cell subsets play in maintaining lifelong anti-peanut IgE levels are unknown. Objectives To determine the cellular requirements for the transfer of murine peanut allergy and to determine the role CD20+ cells play in maintaining long-lived anti-peanut IgE levels. Methods We developed a novel adoptive transfer model to investigate the cellular requirements for transferring murine peanut allergy. We also treated peanut-allergic mice with anti-CD20 antibody and measured IgE levels throughout treatment. Results Purified B220+ cells from peanut-allergic splenocytes and purified CD4+ cells from naïve splenocytes are the minimal requirements for the adoptive transfer of peanut allergy. Prolonged treatment of allergic mice with anti-CD20 antibody results in significant depletion of B cell subsets but does not affect anti-peanut IgE levels, symptoms, or numbers of IgE antibody secreting cells in the bone marrow. Adoptive transfer of bone marrow and spleen cells from allergic donors treated with anti-CD20 antibody does not result in the transfer of peanut allergy in naïve recipients, demonstrating that anti-CD20 antibody treatment depletes B cells capable of differentiating into peanut-specific IgE antibody secreting cells. Conclusions and Clinical Relevance Peanut allergy can be established in a naïve hosts with B220+ cells from peanut-allergic donors and CD4+ cells from peanut-naïve donors. However, long-term depletion of B220+ cells with anti-CD20 antibody does not affect anti-peanut IgE levels. These results highlight a novel role for B cells in the development of peanut allergy and provide evidence that long-lived anti-peanut IgE levels may be maintained by long-lived antibody secreting cells.

Background Intermittent fasting (IF) confers pleiotropic cardiovascular benefits including restructuring of the gut microbiome and augmentation of cellular metabolism. Pulmonary arterial hypertension (PAH) is a rare and lethal disease characterized by right ventricular (RV) mitochondrial dysfunction and resultant lipotoxicity and microbiome dysbiosis. However, the effects of IF on RV function in PAH are unexplored. Therefore, we investigated how IF altered gut microbiota composition, RV function, and survival in the monocrotaline model of PAH. Methods and Results Male Sprague Dawley rats were randomly allocated into 3 groups: control, monocrotaline‐ad libitum feeding, and monocrotaline‐IF (every other day feeding). Echocardiography and invasive hemodynamics showed IF improved RV systolic and diastolic function despite no significant change in PAH severity. IF prevented premature mortality (30% mortality rate in monocrotaline‐ad libitum versus 0% in monocrotaline‐IF rats, P =0.04). IF decreased RV cardiomyocyte hypertrophy and reduced RV fibrosis. IF prevented RV lipid accrual on Oil Red O staining and ceramide accumulation as determined by metabolomics. IF mitigated the reduction in jejunum villi length and goblet cell abundance when compared with monocrotaline‐ad libitum. The 16S ribosomal RNA gene sequencing demonstrated IF changed the gut microbiome. In particular, there was increased abundance of Lactobacillus in monocrotaline‐IF rats. Metabolomics profiling revealed IF decreased RV levels of microbiome metabolites including bile acids, aromatic amino acid metabolites, and gamma‐glutamylated amino acids. Conclusions IF directly enhanced RV function and restructured the gut microbiome. These results suggest IF may be a non‐pharmacological approach to combat RV dysfunction, a currently untreatable and lethal consequence of PAH.