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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.

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.

The current study focuses on finding differential expression between circulating microRNAs in plasma from patients with abdominal aortic aneurysms compared to un-diseased individuals by using a qPCR-based array. In addition, we evaluated the expression of deregulated microRNAs in human tissue samples as well as microarray data from two independent mouse models of aneurysm development. Fifteen miRNAs were found to be significantly differentially expressed, with four of them surviving multiple testing. Interestingly all four of them were substantially different in murine aneurysm development.

According to the cancer stem cell hypothesis a subpopulation of cells within a tumour has the capacity to sustain its growth. These cells are termed cancer stem cells, and are most simply defined as the cells within a primary tumour that can self-renew, differentiate and regenerate a phenocopy of that cancer when transplanted in vivo. Cancer stem cells have now been prospectively identified from numerous human tumours and are actively sought in many cancer types, both clinical and experimental. The cancer stem cell hypothesis remains controversial, with evidence both supporting and challenging its existence in human tumours and in animal models of disease. Here we prospectively identify and study brain cancer stem cells in clinically representative mouse models of the medulloblastoma and glioma. Cancer stem cells from both mouse brain tumour types are prospectively enriched by fluorescent activated cell sorting freshly dissociated cells for the surface antigen CD15, display a neural precursor phenotype, exhibit the hallmark stem cell characteristics of self-renewal and multilineage differentiation, and regenerate a phenocopy of the original tumour after orthotopic transplantation. Additionally, novel mouse medulloblastoma and glioma cancer stem cell lines were established and studied in vitro as adherent cultures in the same serum-free media conditions that support the growth of normal neural stem cells. When mouse and human glioma stem cell lines were compared, many novel molecular mediators of the tumour phenotype were identified, as were chemical compounds that selectively inhibit their growth. Our results have important implications regarding the cancer stem cell hypothesis, the mechanisms that drive brain tumour stem cell growth and the therapeutic strategies that may prove effective for the treatment of glioma and medulloblastoma.

Noonan syndrome (NS) is one of several autosomal dominant “RASopathies” caused by mutations in components of the RAS-RAF-MEK-ERK MAPK pathway. Germ line mutations in RAF1 (encoding the serine-threonine kinase for MEK1/2) account for ~3-5% of NS, and unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with hypertrophic cardiomyopathy (HCM). Notably, some NS-associated RAF1 mutations show normal or decreased kinase activity. To explore the pathogenesis of such mutations, I generated “knock-in” mice that express kinase-activating (L613V) or -impaired (D486N) Raf1 mutants, respectively. Knock-in mice expressing the kinase-activated allele Raf1L613V developed typical NS features (short stature, facial dysmorphia, haematological abnormalities), as well as HCM. As expected, agonist-evoked Mek/Erk activation was enhanced in multiple cell types expressing Raf1L613V. Moreover, postnatal Mek inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice, showing that enhanced Mek/Erk activation by Raf1 mutant is critical for evoking NS phenotypes. D486N/+ female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. In both mouse and human cells, RAF1D486N, as well as other kinase-impaired RAF1 mutants, show increased heterodimerization with BRAF, which is necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also require heterodimerization to enhance MEK/ERK activation. Our results suggest that increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations.

<p>Mycobacterium tuberculosis, the etiologic agent of tuberculosis (TB), is among the leading causes of death from infectious disease world-wide. An intracellular pathogen, M. tuberculosis infects phagocytic cells, and subverts the host immune response, preventing eradication once infection has been established. Even after successful chemotherapy, exogenous re-infection occurs, indicating that sterilizing immune responses are not generated during natural infection. While a TB vaccine exists, it does not alter M. tuberculosis infection rate, rather it prevents the progression from latent TB infection to active TB disease. Vaccines against Haemophilus influenzae and Streptococcus pneumonia protect from bacterial colonization and infection through the induction of antibodies to capsular surface components. This dissertation explores if antibodies to the surface of M. tuberculosis can alter the initial interaction between a bacterium and host cell, leading to a reduction in infection rate. </p><p> When pre-mixed with M. tuberculosis prior to in vitro infection of macrophages, or retropharyngeal instillation of mice, monoclonal surface-binding, but not non-surface-binding antibodies, decrease bacterial burden and the number of infected cells within the first twenty-four hour of infection. If administered retropharyngeally prior to aerosol exposure, surface-binding antibodies decreased pulmonary bacterial burden at twenty-four hours post infection in an Fc&#947;R independent manner. Despite decreasing early bacterial burden, pre-administration of surface-binding antibodies prior to ultra-low dose aerosol infection did not alter infection rate compared to mice instilled with PBS (Chapters 4 and 5). </p><p> Infected humans do not produce high-titer, high-avidity surface-binding antibodies. Plasma from uninfected controls, individuals with latent TB infection, and active TB disease was assayed by ELISA to determine the titer, avidity and IgG/IgM ratio for antibodies to the surface and additional bacterial fraction. In contrast to antibodies to bacterial fractions, individuals with active TB disease had decreased avidity, and no augmentation of the IgG/IgM ratio for antibodies to the live M. tuberculosis surface, as compared to uninfected controls (Chapter 3). </p><p> Overall these findings demonstrate that surface-binding monoclonal antibodies alter early infection events, both in vivo and in vitro, though the magnitude of protection was not sufficient to decrease M. tuberculosis infection rate. Additionally, the failure of humans to generate high-titer, high-avidity surface-binding antibodies after infection indicates and that induction of surface-binding antibodies may be an appropriate target for future vaccines.</p> / Dissertation

It is not known whether pancreatic cancers evolve from a single or multiple cells, or from a particular pancreatic lineage. However, in the Pdx1-Cre; LSL-KrasG12D; LSLTp53R172H mouse model of pancreatic cancer, all pancreatic lineages are susceptible to express mutant KRas and p53. Hence, such mouse model implies a scenario of maximal heterogeneity of cancer cell origins. On this basis, I isolated seven subclones of heterogeneous mouse pancreatic cancer cells from a single tumour; each of them had a distinct morphology and gene expression profile. Notably, they possessed different intrinsic phospho-SMADs downstream of the TGFβ receptor (phospho-SMAD2/3) or the BMP receptor (Phospho-SMAD1/5/8). I discovered that SMAD4, a co-SMAD which is frequently found to be lost in pancreatic caner tissues, upregulated HNF4α via the classical BMP-SMAD1 pathway, when cells were experiencing metabolic stress upon deprivation of serum, or in the presence of excess thymidine. Under serum starvation at a hypoglycemic-like glucose concentration, the HNF4α-expressing sub-clones appeared to be more able to sustain an unstressed morphology than other non-HNF4α-expressing sub-clones. Immunohistochemical staining on pancreatic cancer sections revealed nuclear co-localization of SMAD4 and HNF4α in human (half of the cases) and in mouse samples. As a secondary project conducted during characterization of cells, I also found that three of the subclones more robustly proliferated under anchorage independent conditions, and they relied on the MEK-ERK pathway and the canonical Wnt pathway, to a different degree. Both studies demonstrate for the first time in primary cell culture that pancreatic cancer cells within a tumour could be highly heterogeneous in terms of both morphology and signaling pathways.

Magnetic resonance imaging (MRI) methods thought to assess myelin and axon integrity are improving the understanding of white matter diseases like multiple sclerosis (MS). This thesis improved the understanding of how microstructural tissue changes caused by various pathologies influence MRI metrics by developing and applying MRI methods in a longitudinal study using the cuprizone mouse model of MS. In vivo and ex vivo MRI measurments (T1 and T2 relaxometry, diffusion tensor imaging, and quantitative magnetization transfer imaging) were correlated with tissue measurements taken from electron microscopy images of control and cuprizone fed mice at weeks 2 and 3 of cuprizone feeding. Significant Spearman correlations included mean diffusivity vs. myelinated axon fraction (ρ=0.84), ex vivo T2 vs. myelinated axon fraction (ρ=0.68), and normalized T2-weighted signal vs. myelinated axon fraction (ρ =-0.80). Multiparametric MRI studies show promise in bridging the gap between damage detected in images and clinical status associated with MS. / May 2016

Non-alcoholic fatty liver disease (NAFLD) now affects around one in four adults in the human population and parallels the global increase in obesity. Within the spectrum of NAFLD, simple steatosis is associated with insulin resistance and type 2 diabetes while progression to steatohepatitis (NASH) is associated with an increased risk of liver cirrhosis and all-cause mortality. The molecular pathology of NAFLD is incompletely understood, however observational studies in human cohorts suggest the regulation of DNA methylation may play a role. 5-hydroxymethylcytosine (5hmC) is a cytosine modification generated from 5- methylcytosine (5mC) by the Ten eleven translocase isoenzymes (Tets) as part of a demethylation process. The aim of this project was to examine the role of Tet enzyme activity on the pathogenesis and progression of NAFLD. Detailed characterisation of two established murine dietary interventions allowed the selection of a NAFLD mouse model which broadly recapitulated the metabolic, histological and transcriptional features of human disease. Using DNA immunoprecipitation coupled with whole genome next generation sequencing and RNA micro expression arrays I examined the effect of high fat diet feeding (HFD) on hepatic DNA 5hmC levels within annotated gene regions. Whilst the global 5hmC profile was not altered by HFD, there was profound genic enrichment of 5hmC in upregulated mediators of cholesterol synthesis and transport (Lss, Sc4mol, Fdps, Hsd17b7, Cyp17a1, Mvd, Cyp1a2, Dhcr7 and Apoa4) with no enrichment in genes with other pathological functions (drug detoxification, inflammation, cell cycle regulation). Induced peaks of 5hmC enrichment were subsequently abolished following rescue of the NAFLD phenotype by conversion to control diet. Cross species validation was performed in vitro utilising embryonic stem cell derived hepatocytes challenged with a cocktail of high energy substrates. My in vivo findings were broadly replicated with specific 5hmC enrichment in genes synthesising lipotoxic molecules (PLIN2, CIDEC, APOA4, ACADVL, HMGCS2, APOA5, CYP2J2, IGFBP1, PPAP2C, ACSL1, APOC3, ANGPTL4, NRG1) with no enrichment in upregulated genes of alternative function. To determine whether or not the 5hmC enrichment seen is of functional relevance, I studied Tet1-/- C57BL/6J mice. Tet1-/- mice are grossly normal in appearance, however loss of Tet1 conferred a striking resistance to diet induced obesity with reduced body fat mass, improved insulin-sensitivity and near complete absence of NAFLD compared to wild type littermates. Furthermore, the HFD fed Tet1-/- liver transcriptome showed a ‘protective’ profile, with suppression of genes for lipid synthesis, inflammation and fibrosis. Thus, in multiple cross-species models of NAFLD, over nutrition induces genic hydroxymethylation specifically within activated genes driving the synthesis and transport of lipid molecules. Such changes are reversible with resolution of the NAFLD phenotype strengthening functional association. Tet1 deficiency conveys an obesity and NAFLD resistant phenotype. I therefore introduce Tet1 mediated hydroxymethylation as a novel mechanism for NAFLD pathogenesis.