Sepsis is defined as a life-threatening organ dysfunction that results in systemic activation of coagulation and inflammation in response to microbial infection. Neutrophil extracellular traps (NETs) have shown to be an important interface between innate immunity and coagulation in sepsis. The major structural components of NETs are nucleosomes (DNA-histone complexes). Although nucleosomes do not modulate coagulation, there are conditions where DNA and histones dissociate from each other in the circulation (e.g. in the presence of heparan sulfate or therapeutic heparin binding histones, or DNase digestion of DNA). In vitro, purified DNA was reported to activate coagulation, but this procoagulant activity has been questioned due to isolation methods that yield DNA that is contaminated with other procoagulant molecules. On the other hand, histones have been shown to not only activate coagulation but are cytotoxic to endothelial cells. However, their contribution to the pathogenesis of sepsis has yet to be determined in an in vivo model. Understanding the contribution of DNA, histones, and nucleosomes to the pathogenesis of sepsis may allow us to develop novel therapies that may prove targeting multiple components of NETs (i.e. DNA and histones) may be beneficial.
Consequently, in this thesis, we (1) identified methods of DNA purification that produce DNA that is free of contamination and confirmed the procoagulant properties of the isolated DNA, (2) determined the harmful effects of DNA, histones, and nucleosomes cytotoxicity, coagulation, and inflammation in vitro and in vivo, (3) and then we explored the possibility of targeting both DNA and histones using a combination approach of DNase I and heparin in a mouse model of sepsis. Since heparin is administered to patients as a thromboprophylaxis and DNase I is a potential therapy in sepsis, it is important to understand any potential drug-drug interactions. / Thesis / Doctor of Philosophy (PhD) / Sepsis is a type of blood poisoning that occurs when the body has an over reactive response to an infection. This can lead to tissue damage, organ failure, and death. Sepsis is recognized as a global health priority. The death rate from sepsis is high between 15% to 30%, suggesting that an improved understanding of how sepsis leads to death may develop into new therapies. Recently, it was discovered that high levels of free-floating DNA and histones in the blood can predict death in sepsis. The DNA and histones are likely released by white blood cells in response to trying to fight off the infection. In test tubes, free-floating DNA can trigger clotting of blood. DNA often exists in blood together with histones. In test tubes, histones can kill blood vessels and make blood thicker. However, no one has confirmed that DNA and/or histones are harmful to mammals and contributes to death in sepsis. Some new studies show that getting rid of DNA with injections of DNase I minimally increases survival in mice. Other studies show that removing histones with a treatment called heparin shows a small increase in survival in mice. Heparin is also a blood thinner and decreases inflammation. No one knows if these drugs used together can improve sepsis survival. Because both drugs on their own show some survival improvement in sepsis, perhaps using them together will cure sepsis.
This thesis has three objectives: (1) to confirm the clotting properties of free-floating DNA, (2) to find out if DNA and/or histones contributes to death in sepsis, and (3) if using a combination of DNase I and heparin can cure sepsis in a mouse model. Finding new therapies for sepsis can save millions of people's lives and decrease the financial burden on society and healthcare systems.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/28454 |
| Date | January 2023 |
| Creators | MEDEIROS, SARAH K |
| Contributors | LIAW, PATRICIA, Medical Sciences (Blood and Cardiovascular) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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