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Background: Despite the clinical severity and rising prevalence of peanut allergy, there is a marked absence of widespread, practical treatments available for peanut-allergic patients. Peptide immunotherapy, a disease-modifying treatment that uses short peptides recognized by T cells, has been shown to reduce allergic symptoms of allergic rhinoconjunctivitis. This project investigated the ability of peptides from the major peanut allergen Ara h 1 to protect against peanut-induced anaphylaxis and induce immunomodulatory changes in a mouse model. Methods: Mice transgenic for the human leukocyte antigen DRB1*0401 were sensitized to peanut epicutaneously and treated with two intraperitoneal injections of peptides from Ara h 1. Mice were then challenged with intraperitoneal whole peanut and observed for signs of anaphylaxis. Flow cytometry was used to isolate peanut-specific CD4+ T cells labelled with Ara h 1 peptide-loaded tetramers and additional Th1, Th2, and regulatory markers. Results: Peptide-treated mice were protected from severe peanut-induced anaphylaxis. Control mice treated with a sham peptide experienced a mean maximum temperature drop of 3.2°C, while mice treated with Ara h 1 peptides experienced a drop of 1.6°C (p=0.067 vs control). Maximum clinical score was 2.5 in control mice, and 1.4 in treated mice (p=0.0097). Mean hematocrit for control mice was 52.5%, and 47% for treated mice (p=0.013). PD-1+CD4+ T cells were significantly increased in the mesenteric lymph nodes (p = 2.28e-0.05) and spleens (p = 0.014) of peptide-treated mice. MIP1-a+CD4+ T cells were significantly decreased in the peritoneal lavage (p = 0.008). Conclusion: Ara h 1 peptide immunotherapy protected against severe peanut-induced anaphylaxis in a mouse model. Peptide-treated mice experienced significantly reduced drops in core body temperature, clinical signs of allergic reaction, and hemoconcentration. Clinical protection was associated with decreased expression of the pro-inflammatory chemokine macrophage 1-a and increased expression of the surface marker programmed cell death protein 1. / Thesis / Doctor of Philosophy (PhD) / Peanut allergy is a growing public health concern. Its prevalence has doubled in the past 10 years and currently stands at 2%. Reactions to peanut account for the majority of food-induced fatal allergic reactions, termed anaphylaxis. Currently, there are no treatments available for patients with peanut allergy. Healthcare workers can only offer peanut-allergic patients advice on peanut avoidance and rescue medications in case of accidental ingestion. This research project investigated the ability of a new treatment called peptide immunotherapy to prevent severe allergic reactions to peanut in a mouse model of peanut allergy. Peptide treatment uses small portions of the peanut allergen to shift the immune response from pro-inflammatory to anti-inflammatory. After peptide treatment, peanut-allergic mice were protected from severe allergic reactions in response to peanut and their immune cells produced lower levels of pro- inflammatory molecules.

A computer-controlled system, based on the design of S. Brown et al. at the Ontario Cancer Institute, Canada, has been developed for uniform heating of mouse tumors. A steady hyperthermic temperature was maintained in the tumor by combining waterbath heating with RF-heating. The RF field was provided by a transmitter which was connected to two steel capacitive plates via a matching circuit. 50% isotonic saline kept at a temperature 2°C below that in the tumor was circulated in the waterbath. The saline prevented overheating of the skin and provided coupling between the RF field and the tumor. A computer program has been written to measure continually the temperature in the tumor with implanted fine thermocouples. The program also controlled the average RF power delivered to the tumor by switching the transmitter on and off at appropriate intervals. The system has been tested on tumor xenografts growing in the thigh of nude mice. A steady temperature of 42° or 44°c has been maintained in the tumor for up to an hour. The system could be used to study the effect of hyperthermia on the uptake of radiolabelled tumor-associated antibodies and the treatment of tumors by such antibodies. / Thesis / Master of Science (MS)

The upper respiratory tract (URT), including the nasal and oral cavities, is a reservoir for pathogenic and commensal microbial species, collectively known as the microbiota. Microbial colonization of the URT occurs right after birth, and URT microbial composition has been linked to development of respiratory infections, allergy, and asthma, though few direct mechanisms have been uncovered. Thus, I set out to establish animal models for characterizing the URT microbiota, and its role in infections. I found that nasal washes, a predominant method for measuring URT bacterial colonization, were insufficient for completely extracting the URT microbiota. The age and source of mice greatly affected the composition of the microbiota, which could be transferred to germ-free mice via cohousing. I also established that mice colonized with the Altered Schaedler’s Flora in the gut microbiota have no cultivable URT microbiota. To test the function of the URT microbiota, I colonized mice with Streptococcus pneumoniae, the leading cause of bacterial pneumonia worldwide. I show that the presence of a nasal microbiota increases permissiveness to pneumococcal infection in murine models. Addition of a single URT isolate, Actinomyces naeslundii, increased pneumococcal adherence to human respiratory epithelial cells in vitro and increased pneumococcal dissemination in vivo in a sialidase-dependent manner. The microbiota affects expression of several host genes throughout the respiratory tract involved in pneumococcal pathogenesis. Together, this work establishes new models for assessing the URT microbiota, and highlights the contribution of the URT microbiota to pneumococcal pathogenesis and identifies druggable targets to prevent and treat infections. / Dissertation / Doctor of Philosophy (PhD) / Bacteria living in the gut have been shown to benefit our health, but the role of bacteria living in our respiratory tract is relatively unknown. I describe the methods for characterizing the bacteria in the nose of a mouse as a model of the human nose. I found that pockets of the mouse nose are colonized by different bacteria. I also characterized a mouse model that had bacteria in the gut without nasal bacteria. I used this mouse model to understand infections with Streptococcus pneumoniae, the worldwide leading cause of bacterial pneumonia. The mice without nasal bacteria were protected from infections, which was due to a nasal bacteria helping S. pneumoniae escape from the nasal tissue. This work established new models for understanding how bacteria affect respiratory health, and identified new targets for protecting against infections.

The gastrointestinal tract specializes in digestion and nutrient absorption via its mucosal surface. Through this large mucosal surface, interactions between the host and its environment, including food antigens and microbes, occur. Therefore, it is imperative that the gut discriminates between innocuous food components, and potential threats such as infections. On some occasions, this fine-tuned discrimination fails, leading to chronic inflammation. Celiac disease (CeD) is an autoimmune enteropathy triggered by gluten, the name given to a family of storage proteins (prolamins) that are naturally found in wheat, barley, and rye. To develop CeD, an individual must carry the susceptibility genes, the HLA-DQ2 and/or HLA-DQ8 alleles and consume gluten. However, this is not sufficient to cause disease, indicating that environmental co-factors are at play. Individuals homozygous for the HLA-DQ2 allele are at high risk to developing CeD in infancy. Currently, there is no existing transgenic animal model that addresses early life exposure to gluten, co-factors, and their effects on CeD development. Therefore, the overall goal of my thesis is to characterize a mouse model transgenic for the HLA-DQ2 allele with exposure to gluten in early life. I first studied the physiological and immunological responses to gluten at the time of solid food introduction using DR3-DQ2 transgenic mice. I determined that after sensitization to gluten before weaning, mice developed moderate enteropathy and some developed both anti-tissue transglutaminase 2 and anti-gliadin antibodies. I then evaluated the recovery of gluten immunopathology after gluten was removed for an extended period. After 6 months on gluten-free food, enteropathy and intestinal anti-gliadin and anti-TG2 antibody levels improved. These findings show pre-weaning sensitization of DR3-DQ2 transgenic mice reproduces key features of CeD, which can be used in future studies to assess environmental triggers and mechanisms that are of importance during early life. / Thesis / Master of Science in Medical Sciences (MSMS) / Celiac disease is the destruction of the upper gut lining by an immune reaction caused by gluten in people with genetic risk. Celiac patients cannot absorb nutrients well and have many complications. While it can occur at any age, its onset in children is associated with the HLA-DQ2 gene. Because not every child with the HLA-DQ2 gene will develop celiac disease, additional factors are suspected. Understanding these factors could help prevent disease, as the only treatment – a life-long gluten-free diet – is not always effective. Thus, an animal model that mimics early life disease onset would be useful. Therefore, I characterized signs of celiac disease in young mice with the HLA-DQ2 gene. I determined that gluten and a microbial toxin given in early life induces inflammation and positive celiac blood tests. This model constitutes a useful tool to test the role of environmental factors in celiac disease in early life.

No / The study investigated the role of endothelin (ET) and the ET receptor subtypes ETA and ETB in mediating longitudinal contraction in the mouse proximal and distal colon. Cumulative concentration¿response curves to a range of ET agonists (ET-1, ET-2, ET-3, (Ala1,3,11,13) ET and IRL 1620) were established by administering concentrations ranging from 0.01nM to 0.3¿M. Concentration¿response curves to ET-1, which exhibits a high affinity for both ETA and ETB receptor subtypes, were also established in the presence of the ETA antagonist BMS 182874 and the ETB antagonist IRL1038. The addition of the selective ETA receptor antagonist BMS 182874 caused a rightward shift of the concentration¿response curve to ET-1 in both sections of the colon. The ETB receptor antagonist IRL1038 (0.3¿1¿M) did not significantly effect the response to ET-1 in the proximal colon but caused a significant decrease in response towards higher concentrations ranges (3nM) in the distal colon. A comparison of the concentration¿response curves to ET-1, ET-2 and ET-3 showed a rank order of potency ET-1ET-2ET-3 in the proximal colon and ET-1ET-2ET-3 in the distal colon. The selective ETB receptor agonists, (Ala1,3,11,13) ET and IRL 1620 did not produce any response in the proximal sections of the colon but produced a smaller contraction in the distal segments. The data indicate that ET can contract the proximal tissues of the mouse colon predominantly via ETA receptors and in the distal tissues via ETA and ETB receptors.