ABSTRACT
Genotype-matched vaccines provide ideal protection against infection caused by new Newcastle disease virus (NDV) genotypes or variants even in the vaccinated chickens. In this study, we report a protocol for attenuation and rapid development of a velogenic NDV isolate as an effective vaccine candidate, using a simple and reliable infectious cloning platform. Based on DHN3, a genotype VII velogenic NDV isolate, recombinant rDHN3 was rescued by co-transfection of plasmids expressing the genomic RNA, NDV proteins NP, P and L, and the T7 polymerase without using a helper virus. Subsequently, an attenuated strain rDHN3-mF was produced by substitution of residues from amino acids 112 to 117 in the DHN3 F protein with the corresponding sequence from the LaSota strain. Both rDHN3 and rDHN3-mF are genetically stable during propagation in cell culture and chicken embryos. Further characterization through determination of EID50, MDT and clinical assessments confirmed that rDHN3 is velogenic and rDHN3-mF lentogenic. Vaccination of one-week-old SPF chicks with inactivated rDHN3-mF produced much higher anti-DHN3 antibody response and better protection against live DHN3 challenge than did the commercial LaSota vaccine, providing 100% protection and much earlier viral clearance. This attenuated NDV isolate would merit further development into a vaccine product.
ABSTRACT
PURPOSE: GA-13315 is a gibberellin derivative that reveals antitumor and antineoplastic effects both in vitro and in vivo. In the present study, the chemosensitizing effects of GA-13315 in multidrug-resistant cell lines were examined and the underlying mechanisms were investigated. METHODS: Cytotoxicity and chemosensitizing effects of GA-13315 were determined by MTT assay. Function of ABC transporter was analyzed by measuring intracellular drug accumulation of doxorubicin and rhodamine 123 and by determining the ATPase activity of ABC transporter. Expression levels of apoptosis regulators were analyzed using real-time quantitative PCR and Western blot. RESULTS: GA-13315 selectively killed MCF-7/adr cells that overexpress P-glycoprotein (ABCB1) over the parent MCF-7 cells. In combination with conventional chemotherapeutic agents, GA-13315 at sub-toxic concentrations reversed the multidrug resistance mediated by ABCB1 but exacerbated the resistance conferred by multidrug resistance-associated protein 1 (ABCC1). GA-13315 increased intracellular accumulation of doxorubicin and rhodamine 123 in MCF-7/adr cells and in ABCB1-transfected HEK293 cells but facilitated drug flush-out from cells that overexpress ABCC1. GA-13315 inhibited the ATPase activity of ABCB1 while stimulated that of ABCC1. Moreover, the downregulated expression of Bax in MCF-7/adr cells was restored by GA-13315 markedly. CONCLUSION: These data suggest that GA-13315 sensitizes multidrug-resistant cells at least partially by impeding the efflux function of ABCB1. The upregulation of Bax by GA-13315 may also contribute to the sensitizing action. The opposite effects of GA-13315 on different ATP-binding cassette transporters and their implications in overcoming drug resistance require further investigation.
