In this study, the Autokit Total Ketone Bodies kit (Wako Pure Chemical, Japan), a total ketone measurement assay using an enzymatic method, was evaluated using a Roche Cobas e702 instrument (Roche Diagnostics, Germany). Precision, linearity, carryover, and reference range verification were evaluated with reference to Clinical Laboratory Standards Institute guidelines. Standard materials provided by the manufacturer and patient samples were used for the evaluation. The precision and carryover of the evaluation result satisfied the acceptance criteria. Linearity was also acceptable at more than 0.99. The quantitative Autokit Total Ketone Bodies kit is precise, and can be widely used in clinical laboratories.
3-Hydroxybutyric Acid ; Evaluation Studies as Topic ; Humans ; Ketone Bodies* ; Methods ; Reference Values

A prerequisite for the development of a cancer cell selective targeting adenovirus is the generation of adenoviral vectors that lack native receptor binding ability and additionally contain domains redirecting the vector to cancer cell specific receptors. Towards this goal, we have generated an E1B 55kDa-deleted oncolytic and coxoackie and adenovirus receptor (CAR)-binding ablated adenovirus, YKL-K420A. This newly engineered adenovirus resulted in a dramatic reduction of transduction efficiency compared to the control adenovirus, YKL-1, in all of the cell lines tested. The malaria circumsporozoite (CS) protein interacts with glycosaminoglycans (GAG) present on the liver cell surface, and plays a prominent role in sporozoite attachment and invasion into hepatocytes. To redirect the CAR binding ablated adenovirus YKL-K420A to hepatocytes, CS protein epitope (EWSPCSVTCGNGIQVRIK) was incorporated onto the C-terminus of the YKL-K420A fiber protein, generating an YKL-K420A-hepa. The In vitro efficacy and specificity of YKL-K420A-hepa was then evaluated by comparing the cytopathic effect in hepatoma and other cancer cells from different origins. In hepatoma cells, YKL-K420A-hepa exerted upto 20-fold higher cytolytic ability compared to the control adenovirus, YKL-1, in hepatoma cell lines. Treatment with YKL-K420A-hepa also significantly suppressed tumor growth in a hepatoma xenograft tumor model when compared to YKL-1. Taken together, these studies demonstrate that the strategy to alter adenovirus tropism may greatly improve adenoviral utilities in gene therapy applications.
Adenoviridae* ; Carcinoma, Hepatocellular ; Cell Line ; Genetic Therapy ; Glycosaminoglycans ; Hepatocytes ; Heterografts ; Liver ; Malaria ; Sensitivity and Specificity ; Sporozoites ; Tropism

PURPOSE: This study has been planned to generate a replication-competent adenovirus which replicates in a cancer cell-specific manner, thus minimizing the side effects and toxicity of cancer gene therapy. MATERIALS AND METHODS: we have generated an E1B 19 kD attenuated recombinant adenoviruses, Ad-TERT-delta19 and Ad-mTERT-delta19, which encode E1A gene driven by the wild type hTERT and modified m-hTERT promoter containing additional c-myc and Sp1 binding sites in the backbone of Ad-deltaE1B19. The in vitro efficacy and specificity of the hTERT and m-hTERT promoter have been evaluated by the comparison of viral replication and cytopathic effect in cancer cells and normal cell lines. To assess anti-tumor effect and safety of hTERT or m-hTERT promoter driven replication competent adenoviruses, tumor regression after subcutaneous injection in subcutaneous C33A xenografts and lacZ expression after systemic injection in organs were examined. RESULTS: The activation of hTERT or m-hTERT promoter was significantly up-regulated only in hTERT-positive cells, but not in hTERT-negative cells. Moreover, the activity of m-hTERT promoter was substantially increased in hTERT-positive cancer cells, but not in hTERT-negative cells. While Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect in cancer cells only. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. CONCLUSIONS: The use of m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of m-hTERT promoter may be a new promising tool for the treatment of human malignancies.
Adenoviridae* ; Animals ; Binding Sites ; Cell Line ; Gene Expression ; Genes, Neoplasm ; Heterografts ; Humans* ; Injections, Subcutaneous ; Mice ; Mice, Nude ; Sensitivity and Specificity ; Telomerase*