MS, University of Cincinnati, 2021, Medicine: Immunology

NETosis is a regulated form of cell death that neutrophils initiate in order to trap and kill bacteria. During this process neutrophil extracellular traps (NETs) are released. The traps consist of extracellularized DNA coated with antimicrobial proteins. Additionally, the release and rupture of granulocytes helps kill foreign bodies. Little is known about NET formation in the context of fetal infection and inflammation. During in utero infection, fetal lungs and other mucosal tissues come in contact with bacteria and bacterial products present in the amniotic fluid. To study the functionality of fetal neutrophils, two Rhesus macaque models of chorioamnionitis were used. Pregnant animals were injected intra-amniotically with lipopolysaccharide (LPS) or Escherichia coli (E. coli), and fetal lung tissues were harvested after 16 or 48 hours respectively. NETs were identified by immunohistochemistry detection of extracellular citrullinated histone in neutrophils, identified as CD68+HLA-DRneg cells. Compared to controls, the IA LPS fetal lungs did have significantly greater mean NET area. To compare the functionality of fetal neutrophils to adult neutrophils, fetal lung samples were matched with their corresponding chorioamnion. In LPS animals, there was a trend towards higher NET formation in the chorioamnion, suggesting that fetal neutrophils may be capable of NET formation but not as much as adult neutrophils. We also compared the LPS treatment to the E. coli treatment since NETs are classically thought to trap and kill live bacteria. We found no difference between the two treatments demonstrating that fetal neutrophils respond to bacterial products.

Committee: Claire Chougnet Ph.D. (Committee Chair); Ian Lewkowich (Committee Member); Jonathan Katz Ph.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, Case Western Reserve University, 2008, Molecular Biology and Microbiology

In the advent of the promise for HIV-1 entry inhibitors to treat patients with drug resistance to available antiretroviral therapy, there has been a surge in studies related to HIV-1 entry. It is clear that the intrinsic susceptibility of primary virus isolates to entry inhibitors varies, which indicates a greater probability for intrinsic resistance to this class of drugs. The studies presented here provide tools and insights into the impact of the envelope (env) gene on HIV-1 replicative capacity, how that capacity influences the emergence of drug resistance, and how easily drug resistance is selected in vivo to a microbicide entry inhibitor. The fitness of a virus is a marker of its replication capability in a given environment. The specific impact of HIV-1 entry on the overall fitness of a virus was tested by cloning a region of the env gene into a common backbone and comparing the resultant recombinant fitness to that of the wildtype virus. The env gene was sufficient to determine the overall fitness of these viruses, which also correlated with sensitivity to entry inhibitors. Specifically, the binding avidity of a virus to the host cell coreceptor contributed the most to fitness. PSC-RANTES is an entry inhibitor that acts on the coreceptor CCR5 to block HIV-1 binding and down-regulate coreceptor expression. When used as a microbicide in a rhesus macaque model, PSC-RANTES failed to block transmission of SHIV¬¬¬SF162 at high doses in some animals. Two mutations were identified in isolates from one of these animals and were cloned into a common HIV-1 backbone to create a chimeric virus. The chimeric virus exhibited resistance to PSC-RANTES and an increase in fitness over that of the wildtype virus used to infect the rhesus macaques. This study showed for the first time the selection of drug resistant viruses to a microbicide in an HIV-1 animal model system. Lastly, a cloning strategy was developed to quickly create replication-competent, fully infectious HIV-1 ch (open full item for complete abstract)

Committee: Eric Arts (Advisor) Subjects: