A novel murine model of allogeneic vaccination against renal cancer.

OBJECTIVES: To develop a murine model for whole-cell allogeneic vaccination in renal cancer, as such vaccines aim to direct immune responses against patient tumour cells, due to shared antigens between the vaccine and tumour cells. MATERIALS AND METHODS: A novel murine renal cell line, allogeneic to BALB/c, was developed from a C57BL/6 mouse by primary cell culture (RVIK). It was immortalized by HPV16 E6/E7 and transfected with ras in an attempt to improve its immunogenicity. The cell line was characterized and tested as a vaccine in a BALB/c tumour-protection model after subsequent tumour challenge with autologous RenCa tumour cells. RESULTS: RVIK alone, with no ras induced cross-reactive immunity, providing a valid non-tumorigenic allogeneic whole-cell vaccine model for renal cancer. Ras transfection per se did not improve RVIK immunity. CONCLUSIONS: RVIK is a novel immunogenic murine renal epithelial cell line, which confers protection when used as an allogeneic vaccine. It provides proof of principle for the effectiveness of allogeneic whole-cell vaccines and may therefore form the basis of a useful model of allogeneic vaccination to further optimize vaccination schedules, formulation and adjuvants for a clinical setting.

A method for the absolute quantification of cDNA using real-time PCR.

Real-time PCR is an extremely powerful technique, however, often its results are open to interpretation since there is no convention for data presentation. This anomaly has arisen because many applications rely on non-standard calibration genes, which themselves often change in value during experimental manipulation. We present a novel method for absolute quantification of cDNA species using a combination of extremely accurate double-stranded DNA quantification and a plasmid reference curve. PicoGreen and reference standards are used to measure the amount of cDNA present in a sample using fluorescence. Real-time PCR products are cloned into plasmids and then used to calibrate unknown samples. This cloning is achieved using the same primers necessary for real-time PCR and thus does not involve a second design stage. Results are expressed as copy number per microgram of oligo-dT primed cDNA and consequently may be compared between both inter and intra-experimentally. We show results from a sample human system in which absolute levels of interferon-gamma, TNF-alpha, interleukin-2 and interleukin-10 are measured. We further compare the copy numbers of these genes with levels of released protein and find remarkable correlation. Although our interest has been cytokine quantification, we believe that this technique is widely applicable to the majority of real-time PCR applications.