Characterization of PAMAM Dendrimers for the Delivery of Nucleic Acid Nanoparticles.

The protocols described in this book chapter are meant to be used as an outline and guideline to explore the use of a cationic, polymeric, and synthetic carrier-poly (amidoamine) (PAMAM) dendrimers. The amine-terminated, hyperbranched structures have been identified as a vehicle for the delivery of nucleic acids. As such, clear protocols for the optimization of dendrimer usage should be set in place. This chapter details the experiments used to determine the ratio that dendrimers and nucleic acids should be complexed at through the use of binding assays, nuclease protection assays, and competitive binding assays.

Immune defense mechanisms against a systemic bacterial infection in the cat flea (Ctenocephalides felis).

A significant amount of work has been devoted towards understanding the cellular and humoral immune responses in arthropod vectors. Although fleas (Siphonaptera) are vectors of numerous bacterial pathogens, few studies have examined how these insects defend themselves from infection. In this study, we investigated the immune defense mechanisms in the hemocoel of cat fleas (Ctenocephalides felis), currently the most important flea pest of humans and many domestic animals. Using model species of bacteria (Micrococcus luteus, Serratia marcescens, and Escherichia coli), we delivered a systemic infection and measured the following: antimicrobial activity of hemolymph, levels of free radicals resulting from the induction of oxidase-based pathways, number of circulating hemocytes, phagocytosis activity of circulating hemocytes, and in vivo bacteria killing efficiency when phagocytosis activity is limited. Our results show that the antimicrobial activity of flea hemolymph increases in response to certain species of bacteria; yet, a systemic infection with the same bacterial species did not influence levels of hydrogen peroxide (H2O2), a reactive intermediate of oxygen, at the same time. Additionally, the number of circulating hemocytes increases in response to E. coli infection, and these cells display strong phagocytic activity against this bacterium. Moreover, limiting phagocytosis by injecting polystyrene beads subsequently increases flea susceptibility to E. coli infection when compared to injury controls; however, impairing the cellular immune response itself did not increase flea susceptibility to infection when compared to untreated fleas. Overall, this work yields significant insight into how fleas interact with bacterial pathogens in their hemocoel, and suggests that cellular and humoral immune responses cooperate to combat systemic bacterial infections.