A high-resolution, double-labeling method for the study of in vivo red blood cell aging
ABSTRACT Red blood cell (RBC) senescence is a process that has received considerable study, yet remains poorly understood. This has been primarily due to the difficulty in isolating a RBC cohort of narrowly distributed, well-defined age. Biotin labeling has previously been used to produce an identifiable cell cohort of known mean age; however, the variability of RBC age within the cohort is relatively large for most of its existence. Treatments typically employed on animal subjects to reduce this variability can perturb erythropoiesis and result in abnormal RBC aging.
The objective of this study was to improve on the traditional in vivo biotinylation method by introducing a chemically distinct, second labeling step. In this case, digoxigenin was used to label cells 1 to 2 days before the injection of biotin.
It was shown, in the rat, that two identifiable subpopulations of labeled RBCs can be followed over time: a broad, double-labeled cohort and a narrow, single-labeled cohort, the latter consisting of only those cells created between the first and second labeling steps. The utility of this technique was demonstrated by observing the age-dependent exposure of phosphatidylserine in the single-labeled RBCs.
Its capacity to generate a cohort of narrowly distributed age, without the adverse effects associated with animal treatment, should make this a useful method for the study of RBC senescence.
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ABSTRACT: Different gene delivery systems were developed in this dissertation to promote tissue regeneration by regenerative in vivo gene therapy. A local virus delivery method was developed using a lyophilized adenovirus formulation to restrict viral vector delivery in and around biomaterials. This strategy may reduce the dispersion of virus to avoid unwanted systemic infection and decrease the viral concentration within scaffolds. We also determined that virus bioactivity can be preserved for long-term storage using this method, which allows freeze-dried adenoviruses to be incorporated with biomaterials as a pre-made construct to be use at the time of surgery. This delivery has been applied to successfully repair not only critical-sized craniofacial defects, but also osteonecrosis caused by radiation therapy. To enhance the spatial control of gene delivery, two different strategies were established to effectively bind viral vectors on scaffold surfaces. Avidin-biotin and antibody-antigen interactions were used to mediate virus immobilization. By binding viral vectors to biomaterials, only cells that adhered and proliferated on scaffolds would be transduced to express bioactive signals. Furthermore, a wax masking technique was introduced to control the bioconjugation on defined regions of biomaterials for spatially controlling transgene expression. In order to broadly apply the immobilized gene delivery methods to different biomaterial scaffolds, chemical vapor deposition (CVD) polymerization was utilized to functionalize inert biomaterial, poly-ε-caprolactone (PCL), surfaces for immobilization of cell-signaling viruses. This surface modification was able to be performed on 2-D and 3-D structures. Through these controlled gene delivery systems, bioactive factors may be precisely expressed to engineer distinct tissue interfaces. Ph.D. Biomedical Engineering University of Michigan, Horace H. Rackham School of Graduate Studies http://deepblue.lib.umich.edu/bitstream/2027.42/63755/1/huweiwen_1.pdf
Article: Cell-based drug delivery[Show abstract] [Hide abstract]
ABSTRACT: Drug delivery has been greatly improved over the years by means of chemical and physical agents that increase bioavailability, improve pharmacokinetic and reduce toxicities. At the same time, cell based delivery systems have also been developed. These possesses a number of advantages including prolonged delivery times, targeting of drugs to specialized cell compartments and biocompatibility. Here we'll focus on erythrocyte-based drug delivery. These systems are especially efficient in releasing drugs in circulations for weeks, have a large capacity, can be easily processed and could accommodate traditional and biologic drugs. These carriers have also been used for delivering antigens and/or contrasting agents. Carrier erythrocytes have been evaluated in thousands of drug administration in humans proving safety and efficacy of the treatments. Erythrocyte-based delivery of new and conventional drugs is thus experiencing increasing interests in drug delivery and in managing complex pathologies especially when side effects could become serious issues.Advanced Drug Delivery Reviews 02/2008; 60(2):286-95. DOI:10.1016/j.addr.2007.08.029 · 12.71 Impact Factor
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ABSTRACT: Biotin-labeled peptides are used for numerous biochemical and microbiological applications. Due to the strong affinity of biotin to streptavidin, the detection of biotinylated molecules is very sensitive. A powerful technique for parallel synthesis and high-throughput screening of peptides is the spot synthesis. One example for the use of spot synthesis is the screening of biotinylated peptides synthesized on cellulose membranes, which is particularly favorable for the investigation of protease cleavage sites. Additionally, in combination with biotinylated protein samples, the spot technique can be used for investigations of peptide-protein and protein-protein interactions. Here, we present our results of the use biotin p-nitrophenyl ester (biotin-ONp) in spot synthesis and as a reagent for biotin-labeling of protein samples.Proteomics 03/2008; 8(5):961-7. DOI:10.1002/pmic.200700909 · 3.97 Impact Factor