Anderson, Bambi D.

AbstractLive attenuated Edmonston B strain of measles virus (MV-Edm) is a potent and specific oncolytic agent, but the mechanism underlying its tumor selectivity is unknown. The virus causes cytopathic effects (CPEs) of extensive syncytial formation in tumor cells but minimal damage or cell killing in normal cells. The CPE is dependent on expression of viral proteins and the presence of CD46, the major cellular receptor of MV-Edm. Using a virally encoded soluble marker peptide to provide a quantitative readout of the level of viral gene expression, we determined that tumor cells and normal cells expressed comparable levels of viral proteins. CD46 mediates virus attachment, entry, and virus-induced cell-to-cell fusion. Using engineered cells expressing a range of CD46 densities, we determined that whereas virus entry increased progressively with CD46 density, cell fusion was minimal at low receptor densities but increased dramatically above a threshold density of CD46 receptors. It is well established that tumor cells express abundant CD46 receptors on their surfaces compared with their normal counterparts. Thus, at low CD46 densities typical of normal cells, infection occurs, but intercellular fusion is negligible. At higher densities typical of tumor cells, infection leads to extensive cell fusion. Intercellular fusion also results in enhancement of viral gene expression through recruitment of neighboring uninfected cells into the syncytium, further amplifying the CPE. Discrimination between high and low CD46 receptor density provides a compelling basis for the oncolytic specificity of MV-Edm and establishes MV-Edm as a promising CD46-targeted cancer therapeutic agent.

Abstract Background: Mitochondrial respiratory chain complex (RCC) disorders may occur as commonly as 1 in 8500 individuals. Because of the great variability of phenotypic presentations, measurement of individual RCC enzyme activities is a crucial diagnostic process. Current assay methods are time-consuming and labor-intensive and thus constitute a major impediment to clinical practice. A method with a faster turnaround time would therefore be beneficial. Method: We developed an automated spectrophotometric method for measuring the respiratory chain enzyme activities of complex I, complex II + III, and complex IV with the Hitachi 912, an automated spectrophotometer. Mitochondrial citrate synthase was also determined for normalization of the RCC activities. Results: A blinded method comparison with samples from an external testing center yielded a 91% concordance of interpretations. Mean intraassay imprecision (as CV; n = 20) in a single batch analysis of each RCC was 5.9%. Interassay imprecision, evaluated on 2 samples harvested and analyzed 3 times each, gave mean CVs of 10%–18%. Conclusions: With this automated method, a panel of RCC enzyme activities can be determined in <2 h. In addition, an immunoblot assay using monoclonal antibodies against specific subunits of RCC enzyme complexes can be informative in cases of borderline enzyme activity. Our results suggest that in vitro diagnosis of RCC enzyme deficiencies in skin fibroblasts is an effective alternative to invasive muscle biopsy.