Cytotoxicity assessment, inflammatory properties, and cellular uptake of Neutraplex lipid-based nanoparticles in THP-1 monocyte-derived macrophages.

Current antiretroviral drugs used to prevent or treat human immunodeficiency virus type 1 (HIV-1) infection are not able to eliminate the virus within tissues or cells where HIV establishes reservoirs. Hence, there is an urgent need to develop targeted delivery systems to enhance drug concentrations in these viral sanctuary sites. Macrophages are key players in HIV infection and contribute significantly to the cellular reservoirs of HIV because the virus can survive for prolonged periods in these cells. In the present work, we investigated the potential of the lipid-based Neutraplex nanosystem to deliver anti-HIV therapeutics in human macrophages using the human monocyte/macrophage cell line THP-1. Neutraplex nanoparticles as well as cationic and anionic Neutraplex nanolipoplexes (Neutraplex/small interfering RNA) were prepared and characterized by dynamic light scattering. Neutraplex nanoparticles showed low cytotoxicity in CellTiter-Blue reduction and lactate dehydrogenase release assays and were not found to have pro-inflammatory effects. In addition, confocal studies showed that the Neutraplex nanoparticles and nanolipoplexes are rapidly internalized into THP-1 macrophages and that they can escape the late endosome/lysosome compartment allowing the delivery of small interfering RNAs in the cytoplasm. Furthermore, HIV replication was inhibited in the in vitro TZM-bl infectivity assay when small interfering RNAs targeting CXCR4 co-receptor was delivered by Neutraplex nanoparticles compared to a random small interfering RNA sequence. This study demonstrates that the Neutraplex nanosystem has potential for further development as a delivery strategy to efficiently and safely enhance the transport of therapeutic molecules into human monocyte-derived macrophages in the aim of targeting HIV-1 in this cellular reservoir.

Human rElafin Inhibits HIV-1 Replication in Its Natural Target Cells.

Trappin-2/elafin is a novel innate immune factor that belongs to the serine protease inhibitor family and has known antibacterial, antifungal, and antiviral properties. In this study, we further investigated the anti-HIV activity of elafin using different cellular models and both X4- and R5-HIV-1 laboratory strains. We compared the antiviral activity of human recombinant elafin (rElafin) with three well-known antiretroviral drugs, AZT, tenofovir, and enfuvirtide. We have found that when the virus is pre-incubated with rElafin prior to the infection of the cells, HIV-1 replication is significantly inhibited. In target T cells and human peripheral blood mononuclear cells, maximal inhibition was achieved using submicromolar concentrations, and rElafin was found to be as potent as enfuvirtide, showing its potential for therapeutic application. We also show data on the mechanism of the antiviral activity of rElafin. We have demonstrated that rElafin neither binds to CD4, CXCR4, or CCR5 host cell receptors, nor to the viral glycoproteins gp120 and gp41. Furthermore, in our cell-to-cell fusion assays, in contrast to enfuvirtide, rElafin failed to block cell fusion. Altogether our results indicate that rElafin interferes with HIV replication at the early steps of its cycle but with a different mechanism of action than enfuvirtide. This study provides the first experimental evidence that elafin inhibits HIV replication in its natural target cells; therefore, elafin might have potential for its development as a new anti-HIV drug or microbicide.

ABT-869, a multi-targeted tyrosine kinase inhibitor, in combination with rapamycin is effective for subcutaneous hepatocellular carcinoma xenograft.

BACKGROUND/AIMS: Receptor tyrosine kinase inhibitors (RTKIs) and mTOR inhibitors are potential novel anticancer therapies for HCC. We hypothesized that combination targeted on distinctive signal pathways would provide synergistic therapeutics. METHODS: ABT-869, a novel RTKI, and rapamycin were investigated in HCC pre-clinical models. RESULTS: Rapamycin, but not ABT-869, inhibited in vitro growth of Huh7 and SK-HEP-1 HCC cells in a dose dependant manner. However, in subcutaneous Huh7 and SK-HEP-1 xenograft models, either ABT-869 or rapamycin can significantly reduce tumor burden. Combination treatment reduced the tumors to the lowest volume (95+/-20mm(3)), and was significantly better than single agent treatment (p<0.05). Immunohistochemical staining of tumor shows that ABT-869 potently inhibits VEGF in HCC in vivo. In addition, the MAPK signaling pathway has been inhibited by significant inhibition of phosphorylation of p44/42 MAP kinase by ABT-869 in vivo. Rapamycin inhibits phosphorylation of p70 S6 kinase and 4E-BP-1, downstream targets of mTOR, and decreases VEGF. Combination treatment showed synergistic effect on expression levels of p27 in vivo. Dramatic inhibition of neo-angiogenesis by ABT-869 was also demonstrated. CONCLUSIONS: HCC could potentially be treated with the combination treatment of ABT-869 and rapamycin. Clinical trials on combination therapy are warranted.

In vivo activity of ABT-869, a multi-target kinase inhibitor, against acute myeloid leukemia with wild-type FLT3 receptor.

Neoangiogenesis plays an important role in leukemogenesis. We investigated the in vivo anti-leukemic effect of ABT-869 against AML with wild-type FLT3 using RFP transfected HL60 cells with in vivo imaging technology on both the subcutaneous and systemic leukemia xenograft models. ABT-869 showed a five-fold inhibition of tumor growth in comparison with vehicle control. IHC analysis revealed that ABT-869 decreased p-VEGFR1, Ki-67 labeling index, VEGF and remarkably increased apoptotic cells in the xenograft models. ABT-869 also reduced the leukemia burden and prolonged survival. Our study supports the rationale for clinically testing an anti-angiogenesis agent in AML with wild-type FLT3.

Vitamin D2 from irradiated mushrooms significantly increases femur bone mineral density in rats.

A method has been optimized for the conversion of ergosterol in mushrooms to vitamin D2, and the vitamin D-enriched mushrooms have been tested for bioavailability of vitamin D2 using a rat model. Femur bone mineral density (BMD) of the experimental group of animals fed with vitamin D2 (1 microg/d) obtained from irradiated mushrooms was significantly increased. Femur BMD of two groups was significantly higher. Femur BMD of the experimental group was significantly elevated compared to initial femur BMD of the study group. Data indicate that vitamin D2 from ultraviolet (UV)-irradiated mushrooms was well absorbed and metabolized in animals.

Bioavailability of vitamin D2 from irradiated mushrooms: an in vivo study.

Vitamin D2 from irradiated edible mushrooms might present a possible dietary source of this vitamin, subject to its bioavailability. Having previously optimized a method for the conversion of ergosterol in mushrooms to vitamin D2, this paper examines the vitamin D-enriched mushrooms (Lentinula edodes) for their bioavailability of the vitamin, using an animal model. Thirty male Wistar rats were fed for 1 week with a diet deficient in vitamin D. After this 1-week period, six rats were randomly selected and killed for analysis of initial bone mineral density, and serum level of 25-hydroxyvitamin D. A group of twelve rats of the test animals received 1 mug of vitamin D2 from irradiated mushrooms for a period of 4 weeks until being killed. The remaining twelve rats were fed un-irradiated mushrooms at the same level to act as controls. At the end of a 4-week period, the mean serum 25-hydroxyvitamin D level of the experimental group was 129.42 (sd 22.00) nmol/l whereas it was only 6.06 (sd 1.09) nmol/l in the control group. Femur bone mineral density of the experimental group of animals was significantly higher (P<0.01) than the control group. In addition, serum Ca concentrations among groups were shown to be significantly higher (P<0.01). It may be concluded from the results that vitamin D2 from UV-irradiated mushrooms is well absorbed and metabolized in this model animal system. Significant increase in femur bone mineralization (P<0.01) was shown in the presence of vitamin D2 from irradiated mushrooms compared with the controls.