Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins.

We have shown that bacteria injected intravenously into live animals entered and replicated in solid tumors and metastases. The tumor-specific amplification process was visualized in real time using luciferase-catalyzed luminescence and green fluorescent protein fluorescence, which revealed the locations of the tumors and metastases. Escherichia coli and three attenuated pathogens (Vibrio cholerae, Salmonella typhimurium, and Listeria monocytogenes) all entered tumors and replicated. Similarly, the cytosolic vaccinia virus also showed tumor-specific replication, as visualized by real-time imaging. These findings indicate that neither auxotrophic mutations, nor vaccinia virus deficient for the thymidine kinase gene, nor anaerobic growth conditions were required for tumor specificity and intratumoral replication. We observed localization of tumors by light-emitting microorganisms in immunocompetent and in immunocompromised rodents with syngeneic and allogeneic tumors. Based on their 'tumor-finding' nature, bacteria and viruses may be designed to carry multiple genes for detection and treatment of cancer.

Optical imaging: bacteria, viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals.

Early detection of tumors and their metastases is crucial for the prognosis of cancer treatment. Traditionally, tumor detection is achieved by various methods, including magnetic resonance imaging and computerized tomography. With the recent cloning, cellular expression, and real-time imaging of light-emitting proteins, such as Renilla luciferase (Ruc), bacterial luciferase (Lux), firefly luciferase (Luc), green fluorescent protein (GFP), or Ruc-GFP fusion protein, significant efforts have been focused on using these marker proteins for tumor detection. It has also been demonstrated that certain bacteria, viruses, and mammalian cells (BVMC), when administered systemically, are able to gain entry and replicate selectively in tumors. In addition, many tissue/tumor specific promoters have been cloned which allow transgene expression specifically in tumor tissues. Therefore, when light-emitting protein encoded BVMC are injected systemically into rodents, tumor-specific marker gene expression is achieved and is detected in real time based on light emission. Consequently, the locations of primary tumors and previously unknown metastases in animals are revealed in vivo. In the future it will likely be feasible to use engineered light-emitting BVMC as probes for tumor detection and as gene-delivery vehicles in vivo for cancer therapy.

Quantitative analysis of the solubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin.

Necrotic bovine pulp and dentin were used in this study as model tissues to represent the organic and inorganic components of the smear layer present in instrumented root canals. The capabilities of endodontic irrigants to dissolve pulverized forms of these tissues were compared. Lyophilized tissue samples were mixed for 2 h at 37 degrees C with MTAD, three concentrations of sodium hypochlorite (NaOCl), 17% EDTA, or isotonic saline. Undissolved tissues were rinsed with water and lyophilized. The change in tissue weight after exposure to an irrigant was measured to quantify solubilization. The results showed that various concentrations of NaOCl removed organic components of pulp and dentin effectively. As pulp solubilizers 5.25% and 2.60% NaOCl were equal (>90%), and 5.25% NaOCl was capable of dissolving virtually the entire organic component of dentin. EDTA was capable of solubilizing inorganic material in dentin and organic material in pulp and likely also in dentin. It dissolved >70% of the dentin and >51% of the pulp. The solubilizing effects of MTAD on pulp and dentin were somewhat similar to those of EDTA. The major difference between the actions of these solutions was a high binding affinity of doxycycline present in MTAD for the dentin.

Evaluation of PCR-ELISA for determination of telomerase activity in prostate needle biopsy and prostatic fluid specimens.

The conventional TRAP assay will determine telomerase activity in tissue or other specimens. However, methodological disadvantages limit its clinical use. We evaluated a modified TRAP assay, the telomerase PCR-ELISA, as a practical clinical system for measuring its activity in conjunction with prostate cancer (PCa). We examined telomerase activity by both TRAP and PCR-ELISA assays in 48 sextant needle biopsy (SNB) specimens from dye-marked areas of the prostate glands of 7 PCa patients. Each specimen was histologically confirmed as cancerous or cancer-free by examining a paired specimen taken from the same marked area. In addition, prostatic fluid (PF) specimens were analyzed from 18 patients, 9 of whom were diagnosed with PCa while 9 were diagnosed as cancer-free but mostly with BPH. The results on individual SNB specimens matched well for the two methods. The sensitivity (91%) and specificity (69%) for the PCR-ELISA measurements were consistent with those for the conventional TRAP assay, 88% and 81%, respectively. Quantitatively, with the PCR-ELISA assay, the mean telomerase activity (24.5+/-28.4 units) per needle core with PCa cells was significantly higher than that in needle cores without PCa cells (7.2+/-2.2 unit), as it was with the conventional TRAP assay, namely 25.6+/-27.8 units and 7.3+/-1.8 units, respectively. In PF specimens from PCa patients, which had a lower mean telomerase than was found in needle cores containing PCa cells (7.1+/-1.5 units in the PCR-ELISA, 7.2+/-1.8 units in the conventional TRAP assay), statistical analysis showed good matching between the results from the two assays, overall. In conclusion, the PCR-ELISA can be considered a reliable method to determine telomerase activity as an adjunct in the diagnosis and treatment of prostate cancer.