Michael S. Kolodney

Y target fat. OBJECTIVE To investigate possible mechanisms for the selectivity of DC for fat tissue using in vivo and in vitro models. METHODS AND MATERIALS Histology, drug distribution studies, and cell viability assays were used to examine possible mechanisms contributing to DC selectivity. RESULTS In vitro, DC caused the lysis of all cell types tested within the tested concentration range. DC injected into fat tissue caused adipocyte death, whereas other cell types appeared less affected. Physiological concentrations of albumin or protein-rich tissues decrease the ability of DC to lyse cells. Furthermore, DC relocated to the gastrointestinal tract in animals within hours of injection. This suggests that similar mechanisms may be present in humans. CONCLUSION We report observations that provide a possible explanation for the in vivo preferential fat targeting by DC. Fat tissue, being deficient in cell-associated proteins and interstitial albumin, may be unable to sufficiently neutralize the detergent activity of DC, possibly making fat uniquely sensitive to DC. This study was funded by a grant from Kythera. Drs. Bentow and Knopp are consultants for Kythera, and Nadir Mahmood and Nathaniel David are employees of Kythera....

Detection and sequencing of mutations from clinical specimens is often complicated by the presence of an excess of nonmutated cells. To facilitate the detection and sequencing of minority mutations from clinical specimens, we developed wild-type blocking polymerase chain reaction (WTB-PCR). This technique allows sensitive detection of minority mutations in a tissue sample containing excess wild-type DNA. In WTB-PCR, a nonextendable locked nucleic acid (LNA) oligonucleotide binds tightly to a region of wild-type DNA known to develop point mutations. This LNA sequence blocks amplification of wild-type DNA during PCR while permitting amplification of mutant exon 15. Our results show that the LNA blocking oligonucleotide inhibits amplification of wild-type DNA in a dose-dependent manner. WTB-PCR was able to detect mutant DNA in clinical samples of melanoma tissue containing an excess of nonmelanoma cells. This method was also able to detect small amounts of point mutated or tandem mutated DNA diluted with a much larger concentration of wild-type DNA. This rapid and simple assay overcomes the limitations of current methods to detect minority mutations. The potential applications of WTB-PCR include early diagnosis and prognosis of various cancers.