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Thesis
The role of obesity-associated inflammation in Clostridium difficile susceptibility
Master of Science (M.S.), Drexel University
Mar 2016
DOI:
https://doi.org/10.17918/00010002
Abstract
Clostridium difficile infection (CDI) causes 15,000 deaths annually, a 300% increase since 1999. The major risk factors for CDI include advanced age, previous antibiotic use, length of hospital stay, and comorbidities like obesity, which affects 35% of US adults. Currently, the mechanistic link between obesity and susceptibility to CDI is unclear, with less diverse intestinal microbiota and underlying chronic inflammation being implicated. Obese individuals suffer from chronic inflammation- a result of adipocytes and surrounding activated immune cells releasing a multitude of inflammatory molecules including TNF-alpha, IL-1[beta], IL-6, and leptin. In this thesis research project, I developed an in vitro model of a polarized monolayer of intestinal epithelium to test the effects of leptin on tight junction permeability and function using 3 methods to measure the integrity of the barrier: transepithelial electrical resistance, lucifer yellow diffusion, and immunofluorescence staining of tight junction markers ZO-1 and F-actin. With the models in hand, I tested the hypothesis that in the presence of leptin, pro-inflammatory cytokines disrupt tight junctions thereby increasing intestinal permeability and subsequent apoptosis caused by CDI leading to more severe disease in obese patients. First, I tested the direct effects of physiological concentrations of leptin on CACO-2 cell tight junction function and showed modest effects comparable to assay controls, suggesting that the effects of leptin on tight junction function is complex. Interestingly, when in the presence of a junction perturbing agent, for example, Clostridium difficile toxin or EGTA, at 24 hours leptin induced a significant synergistic increase in caspase 3/7 activation that correlates with an 2 to 4 fold increase in the frequency of dying CACO-2 cells (Annexin V positive) when compared to Clostridium difficile toxin or EGTA treatment alone. These data suggest the requirement of an inflammatory trigger in order for leptin to mediate its detrimental effects on cellular permeability. To fully address our hypothesis we plan to measure inflammatory cytokines secreted following leptin treatment to determine whether TNF-alpha, IL-1[beta], and IL-6 mediate the leptin-induced changes in intestinal barrier structure and/or apoptosis. Taken together, these experiments will provide an important cellular mechanism by which obese individuals are more susceptible to CDI.
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Details
- Title
- The role of obesity-associated inflammation in Clostridium difficile susceptibility
- Creators
- Ashley R. Curatola
- Contributors
- Michele Kutzler (Advisor) - Drexel University, Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Master of Science (M.S.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- ix, 64 pages
- Resource Type
- Thesis
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991021888731204721