Translational Medicine Graduate Theses

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    Evaluating the Protective Capacity of Secreted Products from Bacteroides ovatus against Clostridioides difficile Infection
    (2024-08-01) Greenlaw, Jill; Translational Medicine; Sheth, Prameet
    Clostridioides difficile (CD) is the leading cause of healthcare-associated diarrhea and colitis. CD infection (CDI) is mediated by two exotoxins, toxin A (TcdA) and toxin B (TcdB). The pathogenesis of CDI is closely linked to disruption of the gastrointestinal microbiota; most commonly resulting from antibiotic exposure. Although antibiotics cure 70% of CDI, ~30% of patients do not respond to antibiotics and develop CDI recurrence. Recent treatment strategies have focused on repairing the gastrointestinal microbiota to prevent CDI recurrence using laboratory-cultured defined microbial communities. Our lab recently developed a 4-member microbial community (DMC-4) where we observed that soluble factors from DMC-4 protected mice against CDI. Here, we investigate if soluble factors produced by B. ovatus, a member of DMC-4, protect against CD. Bacterial-free conditioned media (CM) of B. ovatus was created using centrifugation, and filter-sterilization. TcdA/TcdB activity were evaluated using fibroblast cytotoxicity assays and western blots were used to assess the degradation of TcdA and TcdB by CM. CM was tested in vivo using an established CDI mouse model. B. ovatus CM neutralized TcdA and TcdB activity following incubation with CM compared to toxin controls (p<0.05 for both). Size-based separation of CM into products >10 and <10kDa revealed that both fractions were capable of neutralizing toxin activity on cells (p<0.05 for all). Incubation of CM with TcdA and TcdB led to the disappearance of toxins on western blots, suggesting that a protease may be neutralizing CD toxins. Although CM demonstrated promise in vitro, in a pilot study using a CDI mouse model, oral administration of CM did not prevent death, weight-loss, colonic shortening or colonic damage. Furthermore, additional experiments demonstrated the loss of toxin neutralizing activity by CM following incubation at a pH of 3, suggesting that its lack of efficacy in vivo may be attributable to the inability to withstand the low pH of the murine stomach. The in vitro results from this study demonstrate the potential of secreted products from B. ovatus as a therapeutic strategy against CDI and provides insight into the mechanism of action by which DMC-4 protects against CDI.
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    Cannabinoids and novel opioids inhibit visceral nociception in a model of inflammatory bowel disease
    (2024-06-20) Tsang, Quentin Kwin Hong; Translational Medicine; Reed, David; Vanner, Stephen
    Background: Over 70% of inflammatory bowel disease (IBD) patients experience daily pain. Conventional opioids are often needed to manage severe pain but cause serious side effects. Alternative strategies are needed and cannabinoid-use has increased with its legalization. Additionally, a novel pH sensitive opioid, NFEPP, has been developed to be selectively active in acidified microenvironments (e.g. site of inflammation) and may have utility in IBD. However, it is unclear whether these compounds can inhibit visceral pain during colitis. I hypothesize that cannabinoids and NFEPP will inhibit pain during colitis without side effects. Methods: Visceral nociception was evaluated by studying visceromotor responses to colorectal distention, afferent nerve mechanosensitivity and dorsal root ganglia neuronal excitability in patch clamp studies of healthy mice and mice with acute dextran sulfate sodium colitis. Mass spectrometry analysis quantified endocannabinoid (2-AG, AEA) levels. Side effects of cannabinoids and opioids were measured using oxygen saturation, heart rate, locomotion and fecal pelleting assays. Human colonic afferent nerve recordings were also performed. Results: Cannabinoid 1 receptor (CB1R), but not CB2R, agonists inhibited nociception in healthy mice, measured by VMR, afferent nerve mechanosensitivity and DRG neuronal excitability. A combination of sub-analgesic doses of CB1R and MOR agonists synergistically inhibited pain without side effects or tolerance in healthy mice and was mediated, in part, by nitric oxide. During colitis, CB2R agonists inhibited visceral nociception, but CB1R agonists were less potent compared to healthy mice. 2-AG, but not AEA, increased in the colon during colitis. CB1R agonist potency returned after a CB1R antagonist was administered during development of colitis. Both CB1R and CB2R agonists synergistically interact with MOR agonists to inhibit visceral nociception in colitis. NFEPP inhibits nociception during colitis without adverse effects and had no effect in healthy mice, suggesting it is only active in an inflamed acidified microenvironment. CB1R agonists and NFEPP inhibit human colonic afferent nerve mechanosensitivity. Conclusion: Cannabinoids, alone or synergistically with opioids, inhibited visceral nociception in healthy and colitis mice, without adverse side effects or tolerance. NFEPP selectively inhibits nociception in the acidified microenvironment of colitis without side effects. These data demonstrate promising strategies for treatment of IBD-induced pain.
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    A transcriptional analysis of BMPR2 loss and BMP9-induced hyperproliferation in endothelial cells: Implications for pulmonary arterial hypertension
    (2024-06-03) VandenBroek, Michael Martin Anthony; Translational Medicine; Ormiston, Mark
    Background: Pulmonary arterial hypertension (PAH) is a disease of occlusive vascular remodelling, characterized by excessive proliferation of the cells of the lung microvasculature. Genetic, pathological, and experimental evidence all point to endothelial cell dysfunction as the main factor promoting disease pathogenesis. In particular, PAH is associated with mutations in BMPR2, the gene encoding the bone morphogenetic protein (BMP) type II receptor (BMPR-II). Recent work has shown that while BMP9 suppresses cell growth in healthy cells, cells lacking BMPR-II have a hyperproliferative response to BMP9, although the molecular machinery driving this phenotype is not yet understood. Additionally, although BMPR2 loss is known to effect endothelial function via several noncanonical mechanisms, its impact on the endothelial circular RNAs (circRNAs) profile, and their contribution to disease, are not clear. Results: Through RNA sequencing analysis of healthy endothelial cells and those lacking BMPR2, in the presence and absence of BMP9, new mediators of the endothelial response to BMP9 with BMPR2 loss have been identified. These include PALD1, RP11-322e11.5, and N4BP3, the latter of which encodes a disease relevant, novel regulator of BMP9-mediated canonical signalling. In addition, this sequencing dataset was used to identify two novel circRNAs derived from the BMPR2 gene, including circ3218 and circ5078, the latter of which was discovered to regulate endothelial proliferation, stress, and translational responses via an interrelation with the function of linear BMPR2 RNAs. Conclusions: This study has identified several novel mechanisms by which the BMPR2 gene regulates to endothelial function, both at the RNA and protein level. These findings are critical to the understanding of the role of BMPR2 in PAH, especially considering the recent success of PAH therapies that target the genetic causes of disease.
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    A pilot study to assess feasibility of evaluating the safety and efficiency of a simplified diagnostic approach for pulmonary embolism
    (2024-05-23) Morris, Nicole Faith; Translational Medicine; de Wit, Kerstin
    Background: Decision rules designed to reduce the need for pulmonary embolism (PE) imaging are seldom used by emergency physicians because of their complexity and poor credibility. We designed a simple, modified age-adjusted decision rule for PE testing in the emergency department (ED) – the ‘Adjust-Unlikely’ rule. Given increasing pressure on EDs, alternative methods to in-person recruitment are needed to validate the diagnostic performance of the Adjust-Unlikely rule in a full-scale study. Objectives: The primary objective of this research was to assess the feasibility of a protocol consenting patients tested for suspected PE with the Adjust-Unlikely rule by telephone after they left the ED. The secondary objectives were to estimate safety and efficiency of the Adjust-Unlikely rule for diagnosing PE in the ED and to assess whether our methods introduced spectrum bias. Methods: A prospective management pilot study was conducted in one ED and one urgent care centre. Adult patients tested for PE using the Adjust-Unlikely rule were recruited by telephone and followed for 90 days to identify subsequent testing for venous thromboembolism. PE and deep vein thrombosis (DVT) testing during follow-up was independently adjudicated. The feasibility outcomes were recruitment rate, missed eligible rate and overall follow-up rate. To assess for spectrum bias, multivariable logistic regression was used to compare included patients to missed eligible patients and patients who declined participation. Results: Two hundred patients were included. On average, 9 patients were recruited per site, per week and 6 patients were missed per site, per week. The overall follow-up rate was 96.5%. Of the 143 patients with a negative Adjust-Unlikely rule on index visit, 1 patient was diagnosed with PE during follow-up. We found evidence of spectrum bias in our study population. Female patients and older patients were over-represented in our study population compared to patients who were eligible but not included. Conclusions: Telephone recruitment did not meet our predefined thresholds for feasibility. This research has informed the design and planning of a full-scale study that will prospectively validate the safety and efficiency of the Adjust-Unlikely rule for diagnosing PE in the ED across Ontario.
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    Investigating DNA Methyltransferase 3A (DNMT3A) Variants and Clonal Hematopoiesis of Indeterminate Potential (CHIP) in Pulmonary Arterial Hypertension
    (2024-05-16) Emon, Isaac Michael; Translational Medicine; Archer, Stephen
    Background: Mutations are found in 10-20% of idiopathic PAH (IPAH) patients, but none are consistently identified in connective tissue disease-associated PAH (APAH), which accounts for ~32% of PAH cases. TET2 mutations, a cause of clonal hematopoiesis of indeterminant potential (CHIP), predispose to an inflammatory type of PAH. Here, we examine mutations in another CHIP gene, DNMT3A, in PAH. Methods: We analyzed the prevalence of DNMT3A variants in PAH Biobank participants (n=2572) compared to controls (GNOMAD: n=141456, SPARK: n=11198, Additional Pooled Cohort: n=3644). We used exome sequencing (ES) and panel sequencing to examine 2572 and 1659 PAH patients. We also studied DNMT3A mRNA expression in 80 PAH patients and 41 controls. Finally, we evaluated PAH development in a Dnmt3a-knockout mouse model. Results: DNMT3A mutations were more frequent in PAH cases versus control subjects in the ES dataset (OR: 2.60). 33 PAH patients had DNMT3A variants, of whom 21/33 had APAH. While 21/33 had somatic variants (female:male 17:4), 12/33 had germline variants (female:male 11:1). Hemodynamics were comparable with and without DNMT3A variants (mPAP=58±21 vs. 52±18 mmHg). While PAH patients without DNMT3A variants had a 13.4% response rate to acute vasodilator testing, PAH patients with DNMT3A variants were unresponsive. Targeted panel sequencing identified 14.6% of PAH patients with CHIP mutations (242/1659), with DNMT3A accounting for 116/242. There was a significant association between DNMT3A CHIP (OR:25.44, p=4.50e-05) and PAH in analyses adjusted for age and sex, as well as for all CHIP variants combined (OR:23.35, p=2.87e-08). DNMT3A expression was reduced in PBMCs of PAH patients (p<0.0001). DNMT3A variants occur in 7% of all PAH patients and specifically 7.3% of APAH patients. Spontaneous PAH developed in Dnmt3a-/- mice. Dnmt3a-/- mice had increased lung macrophages and elevated plasma IL-13. The IL-1β antibody canakinumab attenuated PAH in Dnmt3a-/- mice. Conclusions: Germline and acquired DNMT3A variants are linked to PAH in humans. DNMT3A mRNA expression is reduced in PAH PBMCs. Hematopoietic depletion of Dnmt3a causes an inflammatory form of PAH in mice. DNMT3A is a putative APAH gene and may be used as a biomarker and therapeutic target.