Severe, fatal food-induced anaphylaxis is characterized by cardiovascular and respiratory collapse. Mast cell (MC) derived mediators act on target cells to increase vascular permeability leading to fluid extravasation into the tissue, leading to hypovolemic shock and fatal tissue hypoxia. IL-4 has been shown to amplify vascular endothelium (VE) responsiveness to MC-derived mediators, such as histamine, and exacerbate allergic reaction. However, the underlying immune pathways that regulate IL-4 enhancement of fluid extravasation and vascular leakage remain elusive. Herein, we examined the molecular pathway involved in IL-4 + histamine-induced hypovolemic shock in anaphylaxis. In this work, using Abl kinase specific pharmacological inhibition, we demonstrated a requirement of Abl kinases in hypovolemic shock associated with active and passive oral antigen-induced anaphylaxis. Using endothelial C-Abl knockout (KO) mice, we also showed the involvement of VE C-Abl in passive oral Ag-induced anaphylaxis. Notably, we showed that in VE human cells line EA.hy926 IL-4 induced C-Abl kinase activity, which was further enhanced with histamine. Pharmacological inhibition of C-Abl in EA.hy926 cells was protective from paracellualar leakage induced histamine, IL-4 and IL-4 + histamine stimulation. In addition, C-Abl genetic deletion by shRNA was protective from paracellualar leakage induced histamine or IL-4. We employed the specific VE IL-4Ra KO mice to test the requirement of IL-4Ra expression on VE in IL-4 + histamine induced-hypovolemic shock. We showed for the first time that IL-4 enhancement of histamine-induced vascular leak is dependent on signaling through IL-4Ra chain expressed on VE. Moreover, VE IL-4Ra genetic deletion attenuated anaphylactic shock induced by passive anaphylaxis. In vitro, using murine vascular endothelial cell line (mHEVC), we show that IL-4 + histamine paracellular leakage is dependent on H1R and IL-4Ra chain, while it is independent of H2R. To (open full item for complete abstract)

Type I cells are one of two main cell types located within the carotid body. These cells respond to hypoxia, hypercapnia, and acidosis by releasing excitatory and inhibitory neurotransmitters. This causes increased firing of the carotid sinus nerve and restores blood gas levels to their physiological values. While previous studies have shown whether individual neurotransmitters are excitatory or inhibitory, this work demonstrates how the interplay between two neurotransmitters may potentially shape the output of the carotid body. Histamine, which has previously been shown to have no effect on intracellular Ca2+ in type I cells, may function to modulate the actions of an excitatory neurotransmitter such as acetylcholine. Using Ca2+-imaging techniques, this work shows that histamine inhibits the acetylcholine muscarinic receptor-induced rise in intracellular Ca2+. Histamine, acting on the Gi-coupled H3 receptor, may exert its actions by inhibiting the activation of adenylate cyclase and therefore reducing levels of cAMP. The observed inhibition of muscarinic Ca2+ signaling seems independent of protein kinase A (PKA), as two PKA inhibitors did not mimic the inhibition. Data suggest that Gi-coupled receptor activation may inhibit muscarinic Ca2+ signaling in type I cells by the inhibition of Exchange Proteins Activated by cAMP (Epac).

It has been previously shown that Carotid Body Type I cells have the ability to synthesize, package and release histamine in response to hypoxia, thereby contributing to the modulation of respiration within the rat. Here, isolated neonatal rat carotid body type I cells were used to identify the presynaptic effects of histamine and the specific receptor subtypes that modulate them. Although all four histamine receptor subtypes are expressed on the type I cells, and preliminary data showed promising results, further data proved that the activation of these receptors with histamine or selective agonists caused no rise in intracellular calcium ([Ca2+]i) and histamine did not augment calcium entry. Thus activation of histamine receptors on type I cells is unlikely to provide a presynaptic positive feedback mechanism during chemotransduction and any excitatory role attributed to the actions of histamine is likely to come from a postsynaptic effect on the carotid sinus nerve (CSN).

The introduction of N-heterocyclic carbenes (NHCs) to organometallic and inorganic chemistry was achieved by the synthesis and isolation of chromium and mercury NHC complexes by Ofele and Wanzlick in 1968. The synthesis and characterization of numerous carbene metal complexes by Lappert et al were a significant contribution to this area of chemistry. NHCs have become an important area of research after the isolation of the first stable carbene by Arduengo in 1991. In chapter 1, the synthesis and biological activities of NHCs and their silver(I) and gold(I) derivatives are discussed. In chapter 2, the synthesis and characterization of an imidazolium-linked cyclophane is discussed as well as its potential use as a medicinal agent. In chapter 3, the synthesis and characterization of a silver(I) NHC complex is reported. The antimicrobial activity of the silver(I) NHC complex is also reported against clinically important bacteria. In chapter 4, the synthesis of a variety of silver(I) NHC complexes and their carbene transfer reactions to gold(I) reagents are reported. The gold(I) NHC complexes are also investigated for their anticancer properties. In chapter 5, the use of urocanic acid as NHC precursor is reported. The synthesis and characterization of the first silver(I) NHC complex derived from biological molecule urocanic acid is discussed as well as its antimicrobial activity.