Forðing av Phosphodiesterase 8A enzym fyri at minka um varandi bruna og krabbakyknusperiðing.
Nicolina Maria Ovadóttir
Verkætlan á Deildini fyri heilsu- og sjúkrarøktarvísindi á Fróðskaparsetrinum
Amanda Vang og Jesper Troelsen
Stuðul úr Granskingargrunninum:
Resistance to chemotherapeutic drugs presents a significant challenge in treating Colorectal Cancer (CRC). Therefore, alternative effective therapies targeting both the inflammation signals driving tissue destruction and the migration of cancer cells are needed. Phosphodieseterase 8A (PDE8A) stands at the crossroads of chronic inflammation and migration with the potential to be a key therapeutic target to treat both the inflammation driving the early stages of CRC development from Inflammatory Bowel Disease (IBD) and the migration of cells during progression of CRC. IBD is an umbrella term mainly used to describe two conditions: Ulcerative Colitis (UC) and Crohn's disease. IBD is a significant risk factor for CRC and is linked to an excessive and abnormal immune response in the gut. Chronically active IBD contributes to the development of low- and high-grade dysplasia, which increases the risk of CRC development. At 74 per 100.000, the incidence of IBD on The Faroe Islands is more than double of what is seen in the rest of Europe. The goal of this project is therefore to determine the inhibitory effect of selective PDE8A inhibition to reduce chronic inflammation and cancer cell migration. Taken together with our collaboration involving the effect of PDE8A inhibition on gene expression in CRC cell lines and our in situ data on PDE8A localization in UC and CRC, this data will contribute to the rationale for clinical trial testing of PDE8A inhibitors in chronic IBD patients at high risk for developing CRC and to provide an alternative therapy for IBD and chemo resistant CRC.
Attenuation of αL and α4 expression on human activated CD4+ T cells with PDE8A inhibition
Background: A normal functioning immune system is crucial for maintaining good health. When the immune system is impaired, diseases may develop. In Autoimmune diseases persistent adhesion and migration of immune cells into healthy tissue can result in inflamed and damaged tissue and endothelium. This state will eventually attract more immune cells to the site of inflammation, resulting in a vicious circle of continuous inflammation controlled by the collaboration between the endothelial cells and immune cells.
Objectives: The aims of this project was to set up a functional cell culture lab and to set up protocols to isolate Peripheral blood mononuclear cells (PMBCs) from whole blood and subsequently to isolate CD4+ T cells from PMBCs. Furthermore, the goal was to activate the isolated CD4+ T cells to mimic an activated adaptive immune system and to treat these cells with the Phosphodiesterase 8A drug, PF-04957325, which has been suggested to interfere with potential cell surface molecules that are crucial for immune cell adhesion to endothelium.
Methods: Whole blood was drawn from volunteers. PMBCs were isolated using a Ficoll-Paque PLUS separation. CD4+ T cell was isolated by CD4+ negative selection. CD4+ activation was achieved with Dynabeads. Activated T cells were treated with PDE8 inhibitor to decrease expression of the cell surface molecules: αL and α4.
Results: The PMBC cell retrieval was 60±20% as expected. The cell viability was >99%, which was above the expected 90%. The CD4+ isolation yielded >95% T cells with viability >98% and a purity of 93.40%, which corresponded with the expected CD4+ yield of 70% with a purity ranging from 90-96%. Dynabeads used specifically for T cell activation proved to be successful but the PDE8A inhibitor did not show noticeable downregulation of αL and α4 expression on activated CD4+ cells.
Conclusion: The cell culture laboratory and cell isolation protocols were successfully established. Changes in the αL and α4 expression were observed but no pattern was seen. Instead, we observed a possible survival mechanism in cells treated with the PDE8A inhibitor, which requires further investigation.
This project resulted in the successful completion of a Bachelor Thesis at the University of The Faroe Islands: Bachelor of Science in Molecular Life Sciences, Fróðskaparsetrið, Award August 1st 2019, Arnfinnur Kallsberg.