Drug-induced cardiotoxicity or cytotoxicity followed by cell death in cardiac muscle is one of the major concerns in drug development. Herein, we report a high-content quantitative multicolor single cell imaging tool for automatic screening of drug-induced cardiotoxicity in an intact cell. A tunable multicolor imaging system coupled with a miniaturized sample platform was destined to elucidate drug-induced cardiotoxicity via simultaneous quantitative monitoring of intracellular sodium ion concentration, potassium ion channel permeability and apoptosis/necrosis in H9c2(2-1) cell line. Cells were treated with cisapride (a human ether-à-go-go-related gene (hERG) channel blocker), digoxin (Na(+)/K(+)-pump blocker), camptothecin (anticancer agent) and a newly synthesized anti-cancer drug candidate (SH-03). Decrease in potassium channel permeability in cisapride-treated cells indicated that it can also inhibit the trafficking of the hERG channel. Digoxin treatment resulted in an increase of intracellular [Na(+)]. However, it did not affect potassium channel permeability. Camptothecin and SH-03 did not show any cytotoxic effect at normal use (≤300 nM and 10 μM, respectively). This result clearly indicates the potential of SH-03 as a new anticancer drug candidate. The developed method was also used to correlate the cell death pathway with alterations in intracellular [Na(+)]. The developed protocol can directly depict and quantitate targeted cellular responses, subsequently enabling an automated, easy to operate tool that is applicable to drug-induced cytotoxicity monitoring with special reference to next generation drug discovery screening. This multicolor imaging based system has great potential as a complementary system to the conventional patch clamp technique and flow cytometric measurement for the screening of drug cardiotoxicity.

Sickle cell disease (SCD) is a monogenic red blood cell (RBC) disorder with high morbidity and mortality. Here, we report, for the first time, the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vascular network of increased numbers of highly tortuous arterioles occupying the majority of the BM cavity, as well as fragmented sinusoidal vessels filled with aggregates of erythroid and myeloid cells. By in vivo imaging, sickle and control RBCs have significantly slow intravascular flow speeds in sickle cell BM but not in control BM. In sickle cell BM, we find increased reactive oxygen species production in expanded erythroblast populations and elevated levels of HIF-1α. The SCD BM exudate exhibits increased levels of proangiogenic growth factors and soluble vascular cell adhesion molecule-1. Transplantation of SCD mouse BM cells into wild-type mice recapitulates the SCD vascular phenotype. Our data provide a model of SCD BM, in which slow RBC flow and vaso-occlusions further diminish local oxygen availability in the physiologic hypoxic BM cavity. These events trigger a milieu that is conducive to aberrant vessel growth. The distorted neovascular network is completely reversed by a 6-week blood transfusion regimen targeting hemoglobin S to <30%, highlighting the plasticity of the vascular niche. A better insight into the BM microenvironments in SCD might provide opportunities to optimize approaches toward efficient and long-term hematopoietic engraftment in the context of curative therapies.

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A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia viurs (MLV)-based packaging constructs contained significantly long 5 0 and/or 3 0 untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed. Gene Therapy (2003) 10, 706-711. doi:10.1038/sj.gt.3301892Keywords: retroviral vector; packaging line; homologous recombination; RCR Murine leukemia virus (MLV)-based vectors are the most frequently used gene delivery system, employed in almost 40% of approved protocols and 50% of patients who have been subjected to or are undergoing clinical trials. However, there are many problems that need to be improved in order for MLV-based vectors to become a viable form of gene delivery vehicle in actual clinical settings. In particular, the issue of safety has often been raised because of the possibility of the production of replication-competent retrovirus (RCR). The mechanism of RCR generation still remains poorly understood, but it has been thought to result from homologous recombination between the same nucleotide sequences present in the vector and in the packaging constructs. Indeed, it has been reported that homologous recombination could occur between as very short regions of homology as 7 or 11 bp. 1-3We have recently constructed a series of retroviral vectors that are absent of any viral coding sequences without compromising viral titer or the level of gene expression. 4 These vectors are thought to be safer than other existing vectors harboring viral coding sequences because the probability of homologous recombination between the vector and the packaging genome is much lower with these newly d...