Recent studies have provided conflicting results in regards to equine intradermal skin testing and its use in defining causative antigens in IgE mediated diseases such as equine recurrent airway obstruction (RAO). This study was divided into two experiments. In the first experiment of this study, the hypothesis tested was normal horses would have minimal variability in the wheals formed by intradermal injection of positive control stimulants. This was evaluated by examining the repeatability of skin test wheals created by 5 concentrations of histamine, compound 48/80, and phaseolus vulgaris (PHA) within a normal horse and between 12 normal horses at 0.5 hours, 4 hours, and 24 hours post injection. Minimal variability was detected within individual horses and between 12 horses for histamine and compound 48/80 at 0.5 hours and for PHA at 4 hours. This information suggests that the intradermal injection of positive control substances is a repeatable test in normal horses. In the second experiment of this study, the hypothesis tested was normal horses react differently to intradermal injection of positive control stimulants (histamine, compound 48/80, PHA) and/or an environmental antigen (Aspergillus) in comparison to horses affected with RAO. This was evaluated by identifying differences in wheal responses between normal horses and RAO affected horses. Concentration response curves were created in normal and RAO affected horses to the aforementioned stimulants at 0.5 hours, 4 hours, and 24 hours post injection. No statistically significant differences were noted in concentration response between normal and ROA affected horses when compound 48/80 and PHA were evaluated. RAO affected horses demonstrated a greater slope at the 0.5 hour time when compared to normal horses suggesting that RAO affected horses are hypersensitive to intradermal injection of histamine. Injection of Aspergillus mix at 4000 protein nitrogen units/ml caused an intradermal wheal reaction at the 24-hour time in 4/5 RAO horses. This reaction was not noted in normal horses. This information suggests that there may be a positive relationship between causative antigens (i.e. Aspergillus) that may induce clinical RAO and positive intradermal skin test results. An additional aspect that was evaluated in both experiments involved histologic examination of skin biopsies taken from wheals created by intadermal injection of histamine, compound 48/80, PHA, and Aspergillus at various times post injection. In the first experiment, intradermal injection of histamine caused severe dermal edema and margination of neutrophils and eosinophils at 0.5 hours. Compound 48/80 demonstrated mild to modest dermal edema at 0.5 hours while PHA demonstrated severe dermal edema, hemorrhage, and lymphactic ectasia at 4 and 24 hours. PHA also demonstrated a neutophilic inflammation at 4 hours that progressed to a mixed lymphohistiocytic and neutrophilic inflammation at 24 hours. In the second experiment, no edema and modest to moderate neutrophilic inflammation was noted in normal horses after intradermal injection of Aspergillus at 24 hours. In contrast, RAO affected horses demonstrated mild to modest edema and a mild to moderate mixed inflammatory response (lympho-histocytic, neutrophilic, eosinophilic) after intradermal injection of Aspergillus at 24 hours suggesting a delayed type response. / Master of Science

<p>Although monophosphoryl lipid A was recently approved by the Food and Drug Administration, more vaccine adjuvants are needed to meet the demand for vaccines against new, emerging, and re-emerging diseases. Additionally, characterizing the mechanisms of action of potent vaccine adjuvants is important for moving toward more rational vaccine design based on the careful selection of antigens and adjuvants to stimulate only the desired immune responses. Two experimental vaccine adjuvants, compound 48/80 (C48/80) and IL-1, were evaluated in these studies. The safety and efficacy of the mast cell activator C48/80 was evaluated when used as an adjuvant delivered intradermally (ID) with recombinant anthrax protective antigen (rPA) in comparison with two well-known adjuvants. Mice were vaccinated in the ear pinnae with rPA or rPA + C48/80, CpG oligodeoxynucleotides (CpG), or cholera toxin (CT). All adjuvants induced similar increases in serum anti-rPA IgG and lethal toxin-neutralizing antibodies. C48/80 induced balanced cytokine production (Th1/Th2/Th17) by antigen-restimulated splenocytes, minimal injection site inflammation, and no antigen-specific IgE. Our data demonstrate that C48/80 is a safe and effective adjuvant, when used by the intradermal route, to induce protective antibody and balanced Th1/Th2/Th17 responses. Histological analysis demonstrated that vaccination with C48/80 reduced the number of resident mast cells and induced an injection-site neutrophil influx within 24 hours. Nonetheless, rPA + C48/80 significantly increased antigen-specific IgG titers in mast cell-deficient mice compared to antigen alone, suggesting that C48/80 has mast cell-dependent and mast cell-independent mechanisms of action.</p><p>IL-1alpha and beta have been shown to have strong mucosal adjuvant activities, but little is known about their mechanism of action. Bone marrow chimeric mice were intranasally vaccinated with Bacillus anthracis lethal factor (LF) with or without 4 µg IL-1alpha or a control adjuvant (cholera toxin) to determine if IL-1R1 expression on stromal cells or hematopoietic cells was sufficient for the maximal adjuvant activity of nasally delivered IL-1alpha. IL-1alpha was not active in IL-1R1-deficient (<italic>Il1r1</italic>-/-) mice given <italic>Il1r1</italic>-/- bone marrow, demonstrating that the adjuvant activity of IL-1 was due to the presence of IL-1R1 and not contaminants. Cytokine and chemokine responses induced by vaccination with IL-1alpha were predominantly derived from the stromal cell compartment and included G-CSF, IL-6, IL-13, MCP-1, and KC. Nasal vaccination of <italic>Il1r1</italic>-/- mice given wild-type bone marrow (WT-->KO) and WT-->WT mice with LF + IL-1alpha induced maximal adaptive immune responses, while vaccination of wild-type mice given <italic>Il1r1</italic>-/- bone marrow (KO-->WT) mice resulted in significantly decreased production of LF-specific serum IgG, IgG subclasses, lethal toxin-neutralizing antibodies, and mucosal IgA compared to WT-->KO and WT-->WT mice (p < 0.05). Our results suggest that IL-1R1 expression in the hematopoietic compartment is sufficient for the maximal induction of antigen-specific adaptive immunity after nasal vaccination adjuvanted with IL-1alpha and that while stromal cells are required for maximal adjuvant-induced cytokine production, the adjuvant-induced stromal cell cytokine responses are not required for effective induction of adaptive immunity.</p> / Dissertation

Otitis media (OM), one of the commonest of childhood diseases, causes much suffering. OM exists in a variety of forms, two of which are acute otitis media (AOM) and otitis media with effusion (OME). The clinical courses of these conditions differ, AOM usually presenting with earache, fever and/or aural discharge, and the OME usually with hearing impairment. The tympanic membrane (TM) mirrors the events in the middle ear cavity, and pars flaccida (PF) is the initial site of inflammatory changes in the TM. PF is rich in mast cells (MCs), which by releasing various mediators, may trigger TM inflammation. The aims of the present studies were to investigate early inflammatory changes in the TM in rat models of OM; after mast cell degranulation, in response to AOM, and OME, after myringotomy in AOM and in normal ears. Furthermore, we developed a new rat AOM model, that excludes surgical trauma and resembles the natural route of infection in man. AOM and OME elicited the first inflammatory response in PF of the TM. The response to OME was discrete, but a slight increase in macrophages was found. During the first 48 hours of AOM, the inflammatory response was intense, following a bimodal pattern. This reaction is similar to that found after MC degranulation. In AOM, macrophages were the predominant cell in PF, while in pars tensa (PT), polymorphonuclear cells (mainly neutrophils) predominated. When myringotomy was performed in AOM ears, the healing time was shorter than that of myringotomy in normal ears. The highly inflamed lamina propria seemed to promote healing. During early AOM, as well as following myringotomy, fibrin extravasates into PF and PT. This fibrin deposition may be involved in regulating the inflammatory response. Repeated nasal challenge with the otitis media pathogen Streptococcus pneumoniae provoked AOM and concomitant TM stimulation reduced the number of AOM cases. This new rat AOM model has the advantage of avoiding trauma in the middle ear cavity, while eliciting an intense inflammatory response in the middle ear cavity (MEC).