A focus in our lab has been the study of allergic diseases and mast cell proliferative disorders. Even though much is known about mast cell-derived mediators and their potential contributions to these diseases, the identification of mediators (derived or not from mast cells) that critically influence certain aspects of the allergic response and mast cell related disorders remains incompletely understood. In the last two years, we have made some progress in identifying mediators and receptors that are important for anaphylactic responses, an effort related to objective 1 of this project. First, we discovered a contribution for the sex hormone estrogen and the mediator nitric oxide (NO) in increased anaphylaxis susceptibility. Epidemiological studies suggested more frequent anaphylactic reactions in adult women, although a gender bias in anaphylaxis and whether sex hormones can affect anaphylaxis had not been established. We used a controlled mouse model to demonstrate that anaphylactic responses were more severe and longer lasting in female than in male mice and that this was related to the female steroid estradiol. We found that this effect was not due to an enhancement of mast cell related early or late responses. Instead, estradiol increased tissue expression and phosphorylation of endothelial nitric oxide synthase (eNOS), whose activity producing NO is critical for blood vessel responses during anaphylactic shock. Blockage of NOS activity with the inhibitor L-NAME or genetic eNOS deficiency abolished the gender-related differences, linking increased NO production in females with vascular hyper-permeability and intensified anaphylactic responses. Second, in relationship to objective 1 of this project, we interrogated the gene expression profiles that characterize two distinct groups of food-anaphylaxis patients. Food allergens are major triggers for systemic anaphylaxis. Often, induction of anaphylaxis in individuals presenting IgE antibodies to the inciting allergens requires cofactors such as non-steroidal anti-inflammatory drugs (NSAID). However, the reasons for NSAID dependency in the manifestation of anaphylaxis in some individuals but not others are unknown. We collected blood samples of patients with documented NSAID-dependent or independent sensitivity to lipid transfer protein (LTP), variably present in fruits, vegetables and nuts, and used high-throughput sequencing to compare the gene expression patterns in these groups. Gene expression analysis identified constitutive defects in intestinal epithelium renewal and permeability in both groups, and furthermore it evidenced specific gene expression patterns characteristic of each group of patients. Our study pointed towards patterns characteristic of an immunological disease with neutrophil activation in food anaphylaxis patients without the NSAID dependence. In addition these patients had an increased expression of the high affinity receptor for IgG antibodies (FcgammaRI) together with elevated allergen-specific IgG1 and 3 levels. Another canonical pathway exclusively affected in the food anaphylaxis group was Sphingosine-1-phosphate signaling, which will be a focus of further studies. On the other hand, our transcriptome analysis also indicated an increased nucleotide metabolism and increased expression of the nucleoside receptor ADORA3 as a potential mechanism for the sensitivity to the food allergen in the presence of NSAIDs since production of adenosine is promoted by NSAIDs and its receptor ADORA3 is known to potentiate mast cell responses to allergens. This study, in addition to presenting evidence of underlying patterns of gene expression which predispose to the induction of food induced anaphylaxis in the presence of co-factors such as NSAIDS, suggests new and novel mechanisms that may underlie systemic reactions to antigens. Because sphingosine-1-phosphate signaling (S1P) was identified as a pathway dysregulated in food anaphylaxis and S1P is an important lipid mediator for immunity, sensitization to food allergens and recovery from anaphylactic shock, we intend to further explore the role of S1P and its receptors in mast cells and basophils, and models of allergic inflammation. A third goal of this project is the identification and characterization of mediators that are altered in diseases of mast cell proliferation such as mastocytosis. MCs are known to produce reactive oxygen species (ROS) during proliferation and when stimulated with antigen. We find that ROS increases in the serum of patients with mastocytosis. We will further explore how ROS is regulated in mastocytosis, with particular emphasis in a scavenger protein which has been shown to contribute to the elevated ROS levels in atopic dermatitis patients and to modulate mast cell responses. We are also interested in those cellular mediators with a role in the regulation of mast cell proliferation. The exploration of targets other than receptor tyrosine kinase (RTK) has been a focus in the search for treatments in mastocytosis since RTK inhibitors such as imatinib are ineffective in patients carrying the most common activating mutation (D816V) in the RTK for stem cell factor (SCF), KIT. Despite the well-recognized role for the lipid mediator S1P on the growth/survival of many cell types and its association with human cancers, its function on mast cell growth and survival is unknown. We are currently investigating the effect of S1P production on the growth of mast cells.

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Kulinski, Joseph M; Proia, Richard L; Larson, Elisabeth M et al. (2018) S1P? Regulates Passive Systemic Anaphylaxis in Mice but Is Dispensable for Canonical IgE-Mediated Responses in Mast Cells. Int J Mol Sci 19:
Olivera, Ana; Beaven, Michael A; Metcalfe, Dean D (2018) Mast cells signal their importance in health and disease. J Allergy Clin Immunol 142:381-393
Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli et al. (2018) Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT. Front Immunol 9:631
Yin, Yuzhi; Bai, Yun; Olivera, Ana et al. (2017) An optimized protocol for the generation and functional analysis of human mast cells from CD34+ enriched cell populations. J Immunol Methods 448:105-111
Kirshenbaum, Arnold S; Cruse, Glenn; Desai, Avanti et al. (2016) Immunophenotypic and Ultrastructural Analysis of Mast Cells in Hermansky-Pudlak Syndrome Type-1: A Possible Connection to Pulmonary Fibrosis. PLoS One 11:e0159177
Desai, Avanti; Jung, Mi-Yeon; Olivera, Ana et al. (2016) IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3. J Allergy Clin Immunol 137:1863-1871.e6
Boyden, Steven E; Desai, Avanti; Cruse, Glenn et al. (2016) Vibratory Urticaria Associated with a Missense Variant in ADGRE2. N Engl J Med 374:656-63
Hox, Valerie; O'Connell, Michael P; Lyons, Jonathan J et al. (2016) Diminution of signal transducer and activator of transcription 3 signaling inhibits vascular permeability and anaphylaxis. J Allergy Clin Immunol 138:187-99
Kulinski, Joseph M; Muñoz-Cano, Rosa; Olivera, Ana (2016) Sphingosine-1-phosphate and other lipid mediators generated by mast cells as critical players in allergy and mast cell function. Eur J Pharmacol 778:56-67
Muñoz-Cano, Rosa; Pascal, Mariona; Bartra, Joan et al. (2016) Distinct transcriptome profiles differentiate nonsteroidal anti-inflammatory drug-dependent from nonsteroidal anti-inflammatory drug-independent food-induced anaphylaxis. J Allergy Clin Immunol 137:137-146

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