Characterizing alterations in IFNγ signaling pathway in human Natural Killer cells following surgical stress

Ahwon Jeong, Immunology with Dr. Dr. Rebecca Auer

 

Surgical resection of solid tumours is an essential component of curative cancer treatment. However, surgical stress can lead to increased metastatic recurrences in patients, and this association has been attributed to the profound immunosuppression following surgery. Natural Killer (NK) cells, in particular, suffer a marked decrease in cytotoxicity and interferon gamma (IFNγ) production. IFNγ released from NK cells exert important anti-tumour effects and recruit components of the adaptive immune response for enhanced tumour clearance. In the post-operative period, lack of IFNγ is associated with increased tumorigenesis. The Auer Lab has demonstrated that NK cells from patient peripheral blood following cancer surgery are completely unresponsive to cytokine stimulation (as measured by IFNγ production). However, the mechanisms of suppression remain elusive. The aim of this study is to identify the alterations that occur in intracellular IFNγ signalling pathways in the dysfunctional NK cells in the post-operative period. To accomplish this, NK cells are isolated from patient blood and incubated with mixtures of cytokines (including IL-2, IL-12, IL-15, and IL-18) to stimulate IFNγ production. We have confirmed that post-surgical NK cells produce negligible amount of IFNγ compared to pre-surgical NK cells. Subsequently, phosphorylation (or activation) of transcription factors and signalling proteins relevant to IFNγ production (including STAT4, STAT5, NF-kB and MAPK) are quantified using flow cytometry. In parallel, we extracted RNA from isolated NK cells before and after surgery for RNAseq. Sequencing will allow comparison of the NK cell transcriptome following surgical stress. Enhancing the immune function after surgery is crucial for improved outcome in surgical patients and for prevention of metastatic disease. By identifying the mechanism of immune suppression, we will gain new insight on developing therapies to mitigate post-operative immune suppression in cancer surgery patients.

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Determining the role of FGL2 in Luteal AngiogenesisDetermining the role of FGL2 in Luteal Angiogenesis

Brendan Kelly, Molecular Biology with Dr. Barbara Vanderhyden

 

The corpus luteum (CL) is a transient endocrine structure found within the ovaries of female mammals after ovulation. Its primary function is to produce progesterone, a hormone that is essential to the maintenance of a pregnancy. The CL is formed from follicular cells (granulosa and theca cells) that remain in the ovary after ovulation, and undergo a process called luteinization. This process is characterized by changes in gene expression at the cellular level, as well as tissue remodeling and reorganization. While a developing follicle is avascular, angiogenesis occurs during luteinization, forming vasculature that is essential to mature CL function. Macrophages of the M2 lineage drive luteal angiogenesis through the VEGF and FGF pathways, while macrophages of the M1 lineage contribute to luteal regression. Fibrinogen-like protein 2 (FGL2) is an immunomodulator that is known to contribute to macrophage polarization to an M2 phenotype and to promote the VEGF pathway. We therefore hypothesize that FGL2 plays an important role in luteal angiogenesis. Preliminary results show that FGL2 expression is specific to theca cells within the ovary, and is highest around the time of ovulation. Components of the VEGF pathway are expressed immediately after ovulation, suggesting the start of vascularization. FGL2 expression preceding angiogenesis markers supports our hypothesis that it may be involved in the regulation of luteal formation. Using superovulation, timed mating and luteal cell collection in FGL2 knockout mice, we aim to further elucidate the role of FGL2 in luteal angiogenesis through techniques such as quantitative RT-PCR and immunohistochemistry.

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Investigation of the impact of a neonatal BPD model on bone marrow MSCs.Investigation of the impact of a neonatal BPD model on bone marrow MSCs.

Xin Xu, Regenerative medicine with Dr. Dr. Bernard Thébaud

 

Extreme prematurity complications are now the leading cause of death in children under 5 years of age. Bronchopulmonary dysplasia (BPD), a chronic lung disease triggered by mechanical ventilation and oxygenation for respiratory failure, is the most common complication of extreme prematurity. Recent insights into stem cell biology offer exciting therapeutic avenues for the prevention and repair of lung injury. Our group demonstrated that bone marrow and umbilical cord-derived mesenchymal stromal cells (MSCs) prevent impaired alveolar growth in a well-established O2-induced BPD model in neonatal rodents. We hypothesize that this therapeutic effect could be due to the fact that BPD causes resident bone marrow MSCs (BM-MSCs) to become perturbed. Our goal is to investigate whether BM-MSC function is impaired in a murine neonatal BPD model. To improve BM-MSC isolation and tracking, a novel transgenic Hic1 mouse line will be used. Hic1 mice contain a tamoxifen-inducible tdTomato (red) tag on the Hic1 gene, which is uniquely expressed in MSCs. BPD will be experimentally induced by exposing Hic1 mice to 85% O2 from postnatal days 0 to 14. BM-MSCs isolated from the BPD model and normal BM-MSCs will be compared in vitro for functional differences. In vivo comparison against healthy BM-MSCs will done by injecting the BPD model BM-MSCs into a non-Hic1 BPD mouse model to investigate whether the therapeutic potential of these cells is reduced. If endogenous BM-MSCs are found to be perturbed during BPD, this could help explain why exogenous BM-MSCs exert therapeutic effects on experimental BPD models.

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Advice and Recommendations given to Pregnant Elite and High Level Recreational Athletes in the Sports Community – A Survey of Coaches

Hiba Haidar Al-Bahrani,  Evaluative Clinical Science with Dr. K Fleming 

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Introduction: A study of pregnancy and reproductive outcomes in elite and high-level recreational athletes (Mahmoud, Fleming) completed in summer/fall 2016 revealed athletes are not receiving adequate information or advice aligned with SOGC guidelines regarding exercise in the antenatal, perinatal and post-partum period. Elite and high-level recreational athletes tended to turn to coaches for recommendations in exercise in pregnancy. Objective: To identify the general knowledge of swimming coaches with regards to exercise in pregnancy, and determine whether advice given to pregnant elite and high-level recreational athletes align with Canadian guidelines. Study design: Retrospective cohort survey study – data will be collected through a link administered by email to Masters Swimming coaches using Qualtrics survey Subjects: Swimming coaches for Masters Swimming (n=300) who have coached pregnant swimmers Observation techniques: Participants were administered a survey of approximately 12 questions. Survey questions will ask about amount of pregnant swimmers, current knowledge of coaches concerning pregnancy, advice and recommendations coaches give their swimmers, and interest in receiving further information on exercise in pregnancy. Results: Preliminary data (n=14) shows coaches in Canada coach on average 5 pregnancy swimmers per year, and 87% of those coaches have been asked for advice on exercise in pregnancy by their swimmers, but feel only moderately comfortable (on a likert scale of 1-10) providing advice. Conclusion: All respondants were interested in receiving further information on exercise in pregnancy, demonstrating a clear absence in dissemation of knowledge of exercise physiology in pregnancy from those providing care, in any form, to pregnant athletes. This study warrants further research with an increased sample size. This study may lead to further attempts at educating trainers and coaches about the exercise and pregnancy guidelines.

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Potential for Akt3 gene upregulation to reduce microcephaly incidence

Olivier Brandts-Longtin and Rishi Gupta, Virology 

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With thousands of cases of microcephaly across Latin America, the Zika virus is well known for causing a wide range of health and birth defects to fetuses infected in utero. Two proteins found in the Brazilian Zika virus (ZIKVBR), known as NS4A and NS4B, are seen to affect the PI3K-Akt3-mTOR pathway found in mammals, which is responsible for inhibiting autophagy, as well as promoting neurogenesis. Interference with this pathway has been shown to result in microcephaly in developing fetuses, who acquire the ZIKVBR infection from their mothers through the placenta. It is predicted that increasing the expression of the Akt3 gene will overcome the effects of, and provide a constant inhibition against, the expression of these proteins, thereby restoring the healthy effects of the pathway, and preventing microcephaly in fetuses. The output of this experiment will be measured based on the expression levels of proteins downstream of the PI3K-Akt3-mTOR pathway. If mTOR (promotes cell growth) levels increase and FOXO (causes autophagy) levels decrease, it indicates that the function of the PI3K-Akt3-mTOR pathway has been restored and thus the potential for microcephaly has been reduced in ZIKVBR affected fetuses. Should this experiment prove successful, future steps could include medication or genetic techniques to reduce the incidence of microcephaly.

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Wood Frogs as a Model Organism

Sue Zhang, Biology with Dr. Vance Trudeau

 

Wood frogs (Lithobates sylvaticus) are widely distributed throughout North America and can be found across Canada. They are early and explosive breeders; adults migrate to temporary ponds as the snow melts and hundreds breed within a few days, resulting in large aggregate egg masses. They reproduce by going into amplexus, which involves a male mounting and clasping a female for several hours to induce oviposition and to fertilize the eggs as they are laid. Like most amphibians, wood frogs are vulnerable to environmental contaminants which are easily taken up through the skin. Compounds found in OSPW (oil sands process-affected water) from bitumen extraction are of particular concern as the wood frogs’ range includes the Athabasca oil sands. In recent years, traditional toxicology assays have begun to include behavioural endpoints; however the sexual behaviours of wood frogs have not been well-described. To characterize these behaviours, 13 males and 9 females were used to mimic the skewed sex ratio and explosive breeding found in the wild. Both sexes were injected with Amphiplex, a combination of GnRH agonist and dopamine antagonist, to induce and synchronize spawning. In addition to amplexus and oviposition, male competitive behaviours were observed (grasp, kick, frog ball, attack) as well as three types of vocalizations (advertisement call, distress call, attack call). The procedure was repeated and video recorded continuously for 8 hours to quantify onset and frequency. In both cases, the behaviours followed a similar progression over time. Certain behaviours often occurred in specific sequences and aggression in males dramatically spiked after several couples had spawned. Detailed description of spawning behaviour helps establish wood frogs as an ecologically relevant lab model.