Enhanced plasmid DNA delivery using anionic LPDII by listeriolysin O incorporation

Background A major obstacle to achieving effective DNA-based therapeutics is efficient delivery of the DNA to its site of action in the cell. Upon internalization by endocytosis, the endosomal membrane represents a critical physical barrier preventing access of DNA to the cell cytosol. In order to overcome the membrane barrier and facilitate cytosolic entry, the endosomolytic bacterial protein listeriolysin O (LLO) is a potentially promising agent. Methods LLO was incorporated in an anionic liposome-entrapped polycation-condensed DNA delivery system (LPDII). Plasmid DNA was condensed using protamine sulfate and then complexed to anionic liposomes. LLO was incorporated into the delivery vehicle through encapsulation in anionic, pH-sensitive liposomes. Transfection levels were monitored using a model reporter plasmid encoding luciferase in P388D1 cells, a macrophage-like cell line. Results Transfection using the anionic LPDII delivery platform was enhanced through incorporation of LLO. Additionally, the net charge of the condensate, the lipid composition, and the total amount of LLO-liposomes were all capable of modulating the transfection levels of the vehicle. Importantly, in the presence of serum, transfection levels using the LLO-containing LPDII system were comparable to established cationic lipid delivery systems. Conclusions LLO is capable of facilitating transfection using an anionic LPDII system. This anionic delivery vehicle represents the successful combination of the LPDII system for condensation of the DNA with the unique endosomolytic properties of LLO for improved transfection using plasmid DNA. Copyright © 2005 John Wiley & Sons, Ltd.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48699/1/750_ftp.pd

A Fluorescent hPept1 Transporter Substrate for Uptake Screening

Purpose. To synthesize fluorescent analogues of hPept1 substrates, FITC-Val-OCH 3 , Lys-FITC-OH, and Lys-FITC-OCH 3 , and to characterize their hPept1 transporter-mediated uptake.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41495/1/11095_2004_Article_475835.pd

Targeting and Blocking B7 Costimulatory Molecules on Antigen-Presenting Cells Using CTLA4Ig-Conjugated Liposomes: In Vitro Characterization and in Vivo Factors Affecting Biodistribution

Purpose. CTLA4Ig, a fusion protein of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain, inhibits the essential costimulatory signal for full T cell activation via blocking the interaction between CD28 and B7 molecules and renders T cell nonresponsiveness. CTLA4Ig has been used to control deleterious T cell activation in many experimental systems. We hypothesized that by conjugating CTLA4Ig to liposomes the efficacy of CTLA4Ig could be enhanced through multivalent ligand effect, superior targetability, and modification of the fate of ligated costimulatory molecules.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41500/1/11095_2004_Article_468685.pd

Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation

Human plasmacytoid dendritic cells (PDCs) can produce interferon (IFN)-α and/or mature and participate in the adaptive immune response. Three classes of CpG oligonucleotide ligands for Toll-like receptor (TLR)9 can be distinguished by different sequence motifs and different abilities to stimulate IFN-α production and maturation of PDCs. We show that the nature of the PDC response is determined by the higher order structure and endosomal location of the CpG oligonucleotide. Activation of TLR9 by the multimeric CpG-A occurs in transferrin receptor (TfR)-positive endosomes and leads exclusively to IFN-α production, whereas monomeric CpG-B oligonucleotides localize to lysosome-associated membrane protein (LAMP)-1–positive endosomes and promote maturation of PDCs. However, CpG-B, when complexed into microparticles, localizes in TfR-positive endosomes and induces IFN-α from PDCs, whereas monomeric forms of CpG-A localize to LAMP-1–positive endosomes accompanied by the loss of IFN-α production and a gain in PDC maturation activity. CpG-C sequences, which induce both IFN-α and maturation of PDCs, are distributed in both type of endosomes. Encapsulation of CpG-C in liposomes stable above pH 5.75 completely abrogated the IFN-α response while increasing PDC maturation. This establishes that the primary determinant of TLR9 signaling is not valency but endosomal location and demonstrates a strict compartmentalization of the biological response to TLR9 activation in PDCs

Comparison of Human Duodenum and Caco-2 Gene Expression Profiles for 12,000 Gene Sequences Tags and Correlation with Permeability of 26 Drugs

Purpose . To compare gene expression profiles and drug permeability differences in Caco-2 cell culture and human duodenum.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41490/1/11095_2004_Article_450750.pd

When river water meets seawater: Insights into primary marine aerosol production

The impact of inorganic salts and organic matter (OM) on the production of primary marine aerosols is still under debate. To constrain their impact, we investigated primary aerosols generated by a sea-spray generator chamber using surface water samples from rivers, estuaries, and seas that were collected along salinity gradients in two temperate Korean coastal systems and one Arctic coastal system. Salinity values showed an increasing trend along the river-estuary-coastal water transition, indicating the lowest amount of inorganic salts in the river but the highest amount in the sea. In river samples, the lowest number concentration of primary aerosol particles (1.01 x 10(3) cm(-3)) was observed at the highest OM content, suggesting that low salinity controls aerosol production. Moreover, the number concentration of primary aerosols increased drastically in estuarine (1.13 x 10(4) cm(-3)) and seawater (1.35 x 10(4) cm(-3)) samples as the OM content decreased. Our results indicate that inorganic salts associated with increasing salinity play a much larger role than OM in aerosol production in river-dominated coastal systems. Laboratory studies using NaCl solution supported the conclusion that inorganic salt is a critical factor in modulating the particles produced from river water and seawater. Accordingly, this study highlights that inorganic salts are a critical factor in modulating the production of primary marine aerosols. (C) 2021 The Authors. Published by Elsevier B.V