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US physicians in multiple specialties who order or conduct radiological procedures lack formal radiation science education and thus sometimes order procedures of limited benefit or fail to order what is necessary. To this end, a multidisciplinary expert group proposed an introductory broad-based radiation science educational program for US medical schools. Suggested preclinical elements of the curriculum include foundational education on ionizing and nonionizing radiation (eg, definitions, dose metrics, and risk measures) and short- and long-term radiation-related health effects as well as introduction to radiology, radiation therapy, and radiation protection concepts. Recommended clinical elements of the curriculum would impart knowledge and practical experience in radiology, fluoroscopically guided procedures, nuclear medicine, radiation oncology, and identification of patient subgroups requiring special considerations when selecting specific ionizing or nonionizing diagnostic or therapeutic radiation procedures. Critical components of the clinical program would also include educational material and direct experience with patient-centered communication on benefits of, risks of, and shared decision making about ionizing and nonionizing radiation procedures and on health effects and safety requirements for environmental and occupational exposure to ionizing and nonionizing radiation. Overarching is the introduction to evidence-based guidelines for procedures that maximize clinical benefit while limiting unnecessary risk. The content would be further developed, directed, and integrated within the curriculum by local faculties and would address multiple standard elements of the Liaison Committee on Medical Education and Core Entrustable Professional Activities for Entering Residency of the Association of American Medical Colleges. [ABSTRACT FROM AUTHOR]

Conventional therapy for cystic fibrosis has extended the median survival age, but the disease is still fatal. Gene therapy can correct the primary and secondary defects associated with cystic fibrosis, but limited extent and duration of the corrections as well as concerns about the safety of some current delivery systems have prevented gene therapy from being curative. For viral vectors, the main challenges are access to target cells and host immunity, which prevents efficient re-administration. Masking viral particles from the immune system, the use of alternative serotypes, or retargeting have been employed to address these issues. Non-viral vectors have dramatically improved over the past five years but improvements in efficacy are needed. In lung, naked DNA has been inefficient and lipid-based vectors have only achieved efficient gene transfer at doses that elicit limiting inflammatory responses. Molecular conjugates or polymer-based delivery overcomes some limitations, with good ability to transfect non-dividing cells. Improvements of viral and non-viral vectors continue to advance the construction of stable, safe and efficacious vectors that can be re-administered. [Copyright &y& Elsevier]

The activation of mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR), particularly the most frequent mutant allele (ΔF508), is a potential strategy for the treatment of the disease cystic fibrosis (CF). Therefore, it is of great interest that curcumin, a component of the spice turmeric, is reported to restore function to this allele, both in heterologous expression systems and in ΔF508 CF mice. Although other laboratories have not been able to confirm the initial observations, activating ΔF508 CFTR could have such important therapeutic implications that a thorough investigation of the potential of curcumin is warranted. [Copyright &y& Elsevier]

DNA nanoparticles formulated with polyethylene glycol (PEG)- substituted polylysine peptides have minimal toxicity, are non- immunogenic, and can transfect non-dividing cells in vivo at remarkably high efficiencies. We previously reported that compacted DNA nanoparticles labeled with rhodamine are only identified intracellularly in cells that express cell surface nucleolin. Nucleolin is a ubiquitous protein participating in ribosomal biogenesis, and mainly localizes in the nucleolus and cytoplasm. In some cell types, nucleolin is also present on the cell surface, and is the target of multiple extracellular ligands, which include lipoproteins, lipopolysaccharides, viruses, bacterial adhesins, and small peptides. In this report, we test the hypothesis that cell surface nucleolin acts as a receptor for DNA nanoparticles and is essential for their uptake into the cell.Initial experiments tested directly whether purified nucleolin binds to DNA nanoparticles by Surface Plasma Resonance (SPR). We observed tight binding when we immobilized either the naonoparticles or nucleolin on the sensorchip. The calculated KD for a single site is about 1.9 nM. The sensogram also suggested multiple binding sites on DNA nanoparticles for nucleolin. Three nanoparticle preparations manufactured by Copernicus Therapeutics using PEG-substituted polylysine from different vendors were evaluated by SPR for their affinity to nucleolin. Their KD values had the same rank order as the mean luciferase activity detected in mouse lung two days following in vivo administration of the three preparations. No binding was detected between nanoparticles and purified green fluorescence protein.We confirmed the cell surface expression of nucleolin in HeLa cells by both surface biotinylation and immunofluorescence. Cell surface nucleolin was biotinylated and pulled down by avidin-agarose beads, then identified by Western blot. We also labeled cell surface nucleolin with FITC conjugated primary antibody and observed colocalization of nucleolin with a cell membrane marker, concanavalin A. To test the importance of nucleolin in the uptake of DNA nanoparticles, we manipulated expression of cell surface nucleolin and then studied activity of the reporter gene luciferase from DNA nanoparticles. When we treated HeLa cells for 12 hours with serum free media, which is reported to reduce cell surface nucleolin, luciferase expression decreased by 70%. When we used siRNA to reduce surface expression of nucleolin in HeLa cells by 50%, we observed a 50% decrease in reporter gene expression. In addition, we tested whether exogenous nucleolin could prevent uptake and expression of DNA nanoparticles. When we premixed DNA nanoparticles with purified nucleolin protein, then applied these mixtures to HeLa cells, the transfection efficiency decreased by 70%- 90%. So purified nucleolin competes with cell surface nucleolin to bind to DNA nanoparticles, thereby inhibiting their uptake and expression.These data indicate that nucleolin is a cell surface target of compacted DNA nanoparticles and plays an essential role in their cellular uptake.Molecular Therapy (2006) 13, S152–S152; doi: 10.1016/j.ymthe.2006.08.459 [ABSTRACT FROM AUTHOR]

An abstract of the article "C105Y, a Novel Cell Penetrating Peptide Enhances Gene Transfer of Sec-R Targeted Molecular Conjugates," by Melanie Rhee and Pamela B. Davis is presented.

DNA nanoparticles (DNPs) are nonviral gene transfer vectors with excellent in vivo potential. Previously, we reported that cell surface nucleolin directly binds DNPs, and functions as an important receptor for DNPs. However, the fate of the nucleolin-DNP complex following cellular uptake remains elusive. In this study, we examined the role of lipid rafts in the uptake of DNPs, and found that both nucleolin and DNPs are recovered from the low-density raft fractions of the sucrose gradient. Furthermore, nucleolin colocalizes with, and coimmunoprecipitates with a raft protein, flotillin. Disruption of lipid rafts by depleting membrane cholesterol significantly inhibited DNP transfection, while inhibition of other endocytic pathways had little effect. Following the uptake, the nuclear import of the DNPs required microtubules but not F-actin. By coimmunoprecipitation in conjunction with tandem mass spectrometry, we identified glucocorticoid receptor (GCR) as a nucleolin-associated protein, and confirmed this result by western blot. Cortisone or dexamethasone increased nucleolin's association with GCR, and transfection by DNPs. Finally, we detected the expression of nucleolin on the surface of airway epithelia in vivo. Taken together, our findings shed light on important determinants of DNP trafficking in cells and support the notion that nucleolin is a good target for nonviral gene delivery. [ABSTRACT FROM AUTHOR]

Compacted DNA nanoparticles deliver transgenes efficiently to the lung following intrapulmonary dosing. Here we show that nucleolin, a protein known to shuttle between the nucleus, cytoplasm, and cell surface, is a receptor for DNA nanoparticles at the cell surface. By using surface plasmon resonance (SPR), we demonstrate that nucleolin binds to DNA nanoparticles directly. The presence of nucleolin on the surface of HeLa and 16HBEo- cells was confirmed by surface biotinylation assay and immunofluorescence. Rhodamine-labeled DNA nanoparticles colocalize with nucleolin on the cell surface, as well as in the cytoplasm and nucleus, but not with transferrin or markers of early endosome or lysosome following cellular uptake. Reducing nucleolin on the cell surface by serum-free medium or siRNA against nucleolin treatment leads to significant reduction in luciferase reporter gene activity, while overexpressing nucleolin has the opposite effect. Competition for binding to DNA nanoparticles with exogenous purified nucleolin decreases the transfection efficiency by 60–90% in a dose-dependent manner. Therefore, the data strongly suggest that cell surface nucleolin serves as a receptor for DNA nanoparticles, and that nucleolin is essential for internalization and/or transport of the nanoparticles from cell surface to the nucleus.Molecular Therapy (2007); 16 2, 333–342. doi:10.1038/sj.mt.6300365 [ABSTRACT FROM AUTHOR]

Nanoparticles containing DNA compacted with poly-l-lysine modified on an N-terminal cysteine with polyethylene glycol can effectively transfect cells of the airway epithelium when applied by the luminal route. To evaluate the toxicity of these nanoparticles, we administered 10 and 100 μg DNA compacted into nanoparticles suspended in normal saline by the intranasal route to mice and determined the pulmonary and systemic responses to this challenge, compared to administration of saline alone, and in some experiments, compared to administration of naked DNA, Escherichia coli genomic DNA, or lipofectin-complexed naked DNA. There was no systemic response to either dose of nanoparticles in serum chemistries, hematologic parameters, serum complement, IL-6, or MIP-2 levels or in the activity, growth, and grooming of the mice. Nanoparticles containing 10 μg DNA induced responses comparable to saline in all measures, including BAL cell counts and differentials and cytokine levels and histology. However, mice dosed with 100 μg DNA in nanoparticles had modest increases in BAL neutrophils 48 and 72 h after dosing, modest increases in BAL IL-6 and KC beginning 24 and 48 h, respectively, after dosing, and, on histology of the lung, a trace to 1+ mononuclear cell infiltrates about the pulmonary veins at 48 h, which were markedly reduced by 10 days and gone by 28 days after dosing. BAL neutrophil and cytokine responses were no greater than those entrained by naked DNA for up to 24 h. However, compared to administration of only 10 μg E. coli genomic DNA, the response to compacted DNA was much less. A low dose of lipofectin-complexed DNA (5 μg DNA) induced the same response as 20-fold higher doses of DNA nanoparticles. These data indicate that DNA nanoparticles have no measurable toxic effect at a dose of 10 μg and a very modest effect, which is not limiting, at a dose of 100 μg, which gives maximal gene expression. This favorable toxicity profile encourages development of stabilized compacted DNA for airway administration. [Copyright &y& Elsevier]

DNA can be compacted using polyethylene glycol-substituted poly-l-lysine into discrete unimolecular (with respect to DNA) nanoparticles with minor diameter <20 nm that are stable in normal saline for at least 23 months at 4°C. We compared the activity of firefly luciferase in lungs of C57BL/6 mice that received 100 μg compacted plasmid in 25 μl saline (shown to be the optimal dose) via intratracheal or intranasal instillation with levels in animals given 100 μg naked plasmid or in untreated mice. Mice dosed with compacted DNA nanoparticles had peak activity of luciferase in lung at 2 days postinstillation, which declined in log-linear fashion with a half-life of 1.4 days. Luciferase activity in animals dosed with naked DNA was 200-fold less. Addition of polyethylene glycol to the complex was necessary for efficient gene transfer and animals that received DNA compacted with unmodified poly-l-lysine did not exhibit luciferase activity above background. Immunohistochemical staining for bacterial β-galactosidase 2 days after administration of a compacted lacZ expression plasmid (n = 8) revealed expression predominantly in the dependent portions of the right lungs of mice, in alveolar and airway epithelial cells, though macrophages and sometimes endothelial cells also were transfected. No staining for β-galactosidase was observed in uninjected animals (n = 4) or those dosed with naked lacZ plasmid (n = 7). Tissue survey for transgene expression shows expression only in lung and trachea following intranasal administration. Stable compacted DNA nanoparticles transfer exogenous genes to airway epithelium and show promise for lung gene therapy. [Copyright &y& Elsevier]

Molecular conjugates that target the serpin-enzyme complex receptor transfer the cDNA encoding human cystic fibrosis transmembrane conductance regulator (CFTR) to the nasal epithelium of cystic fibrosis mutant mice. These complexes effect partial correction of the chloride transport defect as assessed by in vivo nasal potential difference measurements, produce immunohistochemical staining for CFTR, and restore expression of nitric oxide synthase-2 (NOS-2), which is downregulated in the epithelium of mice and humans with cystic fibrosis. Complexes that lack the receptor ligands were ineffective, so receptor access was essential. Mice treated with receptor-targeted lacZ showed β-galactosidase expression in epithelial cells and submucosal glands, but no electrophysiologic correction or NOS-2 expression, so simply accessing the serpin-enzyme complex receptor was not sufficient to produce the observed electrophysiologic or immunohistochemical changes. Correction of the cAMP-stimulated chloride transport was dose related at days 7 and 12 after complex administration, but, for most animals, nasal potential difference had returned to baseline by day 18. Molecular conjugates targeting the serpin-enzyme complex receptor, used to compact plasmid DNA, hold promise for gene therapy of cystic fibrosis. [Copyright &y& Elsevier]

An abstract of the article "Imaging of sec-R Directed and PEG-Stabilized Gene Transfer Nanoparticles in CF Mice," by Maxwell Kotlarchyk and colleagues is presented.