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Browsing by Author "De Luca, Thomas"
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Item Improving the Patency of Jugular Vein Catheters in Sprague-Dawley Rats by Using an Antiseptic Nitrocellulose Coating(Ingenta, 2018-09-01) De Luca, Thomas; Szilágyi, Keely L; Hargreaves, Katherine A.; Collins, Kimberly S.; Benson, Eric A.; Department of Medicine, Indiana University School of MedicinePreclinical studies in animals often require frequent blood sampling over prolonged periods. A preferred method in rats is the implantation of a polyurethane catheter into the jugular vein, with heparinized glycerol as a lock solution. However, analysis of various biologic compounds (for example, microRNA) precludes the use of heparin. We used sodium citrate as an alternative to heparin but observed more frequent loss of catheter patency. We hypothesized that this effect was due to evaporation of lock solution at the exteriorized portion of the catheter, subsequent blood infiltration into the catheter, and ultimately clot formation within the catheter. We therefore tested evaporation and its variables in vitro by using 5 common catheter materials. We used the migration of dye into vertically anchored catheters as a measure of lock displacement due to evaporation. Exposure to dry room-temperature air was sufficient to cause dye migration against gravity, whereas a humid environment and adding glycerol to the lock solution mitigated this effect, thus confirming loss of the lock solution from the catheter by evaporation. We tested 4 catheter treatments for the ability to reduce lock evaporation. Results were validated in vivo by using male Sprague-Dawley rats (n = 12) implanted with polyurethane jugular vein catheters and randomized to receive a nitrocellulose-based coating on the exteriorized portion of the catheter. Coating the catheters significantly improved patency, as indicated by a Kaplan-Meier log-rank hazard ratio greater than 5 in untreated catheters. We here demonstrate that a simple nitrocellulose coating reduces evaporation from and thus prolongs the patency of polyurethane catheters in rats.Item In Vivo siRNA Delivery and Rebound of Renal LRP2 in Mice(Hindawi Publishing Corporation, 2017) Eadon, Michael T.; Cheng, Ying-Hua; Hato, Takashi; Benson, Eric A.; Ipe, Joseph; Collins, Kimberly S.; De Luca, Thomas; El-Achkar, Tarek M.; Bacallao, Robert L.; Skaar, Todd C.; Dagher, Pierre C.; Medicine, School of MedicinesiRNA stabilized for in vivo applications is filtered and reabsorbed in the renal proximal tubule (PT), reducing mRNA expression transiently. Prior siRNA efforts have successfully prevented upregulation of mRNA in response to injury. We proposed reducing constitutive gene and protein expression of LRP2 (megalin) in order to understand its molecular regulation in mice. Using siRNA targeting mouse LRP2 (siLRP2), reduction of LRP2 mRNA expression was compared to scrambled siRNA (siSCR) in mouse PT cells. Mice received siLRP2 administration optimized for dose, administration site, carrier solution, administration frequency, and administration duration. Kidney cortex was collected upon sacrifice. Renal gene and protein expression were compared by qRT-PCR, immunoblot, and immunohistochemistry (IHC). Compared to siSCR, siLRP2 reduced mRNA expression in PT cells to 16.6% ± 0.6%. In mouse kidney cortex, siLRP2 reduced mRNA expression to 74.8 ± 6.3% 3 h and 70.1 ± 6.3% 6 h after administration. mRNA expression rebounded at 12 h (160.6 ± 11.2%). No megalin renal protein expression reduction was observed by immunoblot or IHC, even after serial twice daily dosing for 3.5 days. Megalin is a constitutively expressed protein. Although LRP2 renal mRNA expression reduction was achieved, siRNA remains a costly and inefficient intervention to reduce in vivo megalin protein expression.Item Multiple reaction monitoring profiling as an analytical strategy to investigate lipids in extracellular vesicles(Wiley, 2021) Edwards, Madison E.; De Luca, Thomas; Ferreira, Christina R.; Collins, Kimberly S.; Eadon, Michael T.; Benson, Eric A.; Sobreira, Tiago J.P.; Cooks, R. Graham; Medicine, School of MedicineExtracellular vesicles (EVs) convey information used in cell-to-cell interactions. Lipid analysis of EVs remains challenging because of small sample amounts available. Lipid discovery using traditional mass spectrometry platforms based on liquid chromatography and high mass resolution typically employs milligram sample amounts. We report a simple workflow for lipid profiling of EVs based on multiple reaction monitoring (MRM) profiling that uses microgram amounts of sample. After liquid-liquid extraction, individual EV samples were injected directly into the electrospray ionization (ESI) ion source at low flow rates (10 μl/min) and screened for 197 MRM transitions chosen to be a characteristic of several classes of lipids. This choice was based on a discovery experiment, which applied 1,419 MRMs associated with multiple lipid classes to a representative pooled sample. EVs isolated from 12 samples of human lymphocytes and 16 replicates from six different rat cells lines contained an estimated amount of total lipids of 326 to 805 μg. Samples showed profiles that included phosphatidylcholine (PC), sphingomyelin (SM), cholesteryl ester (CE), and ceramide (Cer) lipids, as well as acylcarnitines. The lipid profiles of human lymphocyte EVs were distinguishable using principal component and cluster analysis in terms of prior antibody and drug exposure. Lipid profiles of rat cell lines EV's were distinguishable by their tissue of origin.Item Novel Quantification of Extracellular Vesicles with Unaltered Surface Membranes Using an Internalized Oligonucleotide Tracer and Applied Pharmacokinetic Multiple Compartment Modeling(Springer, 2021-10) De Luca, Thomas; Stratford, Robert E., Jr.; Edwards, Madison E.; Ferreira, Christina R.; Benson, Eric A.; Medicine, School of MedicinePurpose: We developed an accessible method for labeling small extracellular vesicles (sEVs) without disrupting endogenous ligands. Using labeled sEVs administered to conscious rats, we developed a multiple compartment pharmacokinetic model to identify potential differences in the disposition of sEVs from three different cell types. Methods: Crude sEVs were labeled with a non-homologous oligonucleotide and isolated from cell culture media using a commercial reagent. Jugular vein catheters were used to introduce EVs to conscious rats (n = 30) and to collect blood samples. Digital PCR was leveraged to allow for quantification over a wide dynamic range. Non-linear mixed effects analysis with first order conditional estimation - extended least squares (FOCE ELS) was used to estimate population-level parameters with associated intra-animal variability. Results: 86.5% ± 1.5% (mean ± S.E.) of EV particles were in the 45-195 nm size range and demonstrated protein and lipid markers of endosomal origin. Incorporated oligonucleotide was stable in blood and detectable over five half-lives. Data were best described by a three-compartment model with one elimination from the central compartment. We performed an observation-based simulated posterior predictive evaluation with prediction-corrected visual predictive check. Covariate and bootstrap analyses identified cell type having an influence on peripheral volumes (V2 and V3) and clearance (Cl3). Conclusions: Our method relies upon established laboratory techniques, can be tailored to a variety of biological questions regarding the pharmacokinetic disposition of extracellular vesicles, and will provide a complementary approach for the of study EV ligand-receptor interactions in the context of EV uptake and targeted therapeutics.Item Pharmacogenetics and Practice: Tailoring Prescribing for Safety and Effectiveness(Elsevier, 2018) Fulton, Cathy R.; Swart, Marelize; De Luca, Thomas; Liu, Stephanie N.; Collins, Kimberly S.; Desta, Zeruesenay; Gufford, Brandon T.; Eadon, Michael T.; Medicine, School of MedicineThe promise of pharmacogenomics testing, to find the right medication at the right dose for the right patient at the right time, sits at the heart of precision medicine. Identifying genetic variants that contribute to inter-patient variability in drug disposition and effect allows clinicians to select a more appropriate medication for a patient’s condition by limiting adverse drug events and maximizing beneficial effects. However, as pharmacogenomics is increasingly integrated into prevention-based healthcare, a major obstacle to effective implementation of pharmacogenomics testing is the lack of adequate knowledge of healthcare providers on interpretation of these test results.