p16(INK4a) overexpression predicts translational active human papillomavirus infection in tonsillar cancer.

The causal role of human papillomaviruses (HPV) in squamous cell carcinogenesis of tonsillar cancers (TSCC) depends on the activity of the viral oncoproteins E6 and E7, leading to inactivation of the cellular tumor suppressor p53 and the retinoblastoma gene product pRb. Because of the negative feedback mechanisms, the pRb inactivation causes an increase of the inhibitor of the cyclin-dependent kinases p16(INK4a). In 39 TSCC specimens, genotyping based on the amplification of HPV DNA was carried out using PCR by applying HPV type-specific oligonucleotides. Subsequently, amplicons were hybridised with fluorescence-labeled complementary probes using the Southern blot technology. For HPV E6/E7 mRNA expression, Northern hybridization and RT-PCR were performed, and for p16(INK4a) detection, immunohistochemistry was performed. With 21/39 (53%) HPV-positives, the detection rate is within the range that can be expected in TSCC. The E6/E7 oncogene mRNA was detectable in 11 cases, 10 of which showed positive signals after p16(INK4a) staining. Albeit the small study group was investigated, the correlation of the HPV DNA status with the p16(INK4a) expression was of statistical significance (p = 0.02). Kaplan-Meier estimations revealed better survival outcome for patients with HPV-positive tumors with detectable E6/E7 mRNA and p16(INK4a) overexpression (p = 0.02, median observation time 29 months). As mRNA expression tests are not routinely available in many clinical diagnostic laboratories, and based on the high correlation of p16(INK4a) staining with HPV E6/E7 mRNA expression, in conclusion we suggest for a deeper exploration for the use of p16(INK4a) as a surrogate marker with the potential to impact the standard of care of HPV DNA-positive head and neck carcinomas.

Nasal RANTES and eotaxin production pattern in response to rhinovirus infection.

Tissue eosinophilia is a hallmark of nasal polyposis and its pathogenesis is an area of high interest. RANTES and eotaxin are both known to recruit eosinophils, however, the mechanisms triggering their induction are still uncertain, and viral infections have been suggested to be involved in this process. Therefore, we investigated whether rhinovirus infection is a stimulus for RANTES and eotaxin expression and production. Fibroblasts were cultured from healthy nasal mucosa obtained during endonasal surgery. Cultured cells were infected with human rhinovirus-16 for one to 72 hours. Following total RNA isolation and reverse transcription, RANTES- and eotaxin-mRNA levels were analyzed. In addition, RANTES and eotaxin secretion was measured in culture supernatants by means of an ELISA. Rhinovirus infection induces RANTES-mRNA expression as early as one hour after infection, persisting for up to 72 hours. Eotaxin-mRNA profiles did not alter significantly from control. Protein production was confirmatory for both chemokines, indicating distinct translational latency. Our data suggest that RANTES functions as a host defence mechanism responding to rhinovirus infection, thus supporting a linkage between rhinovirus infections and the pathogenesis of nasal polyposis.

Electrofusion generates diverse DC-tumour cell hybrids for cancer immunotherapy.

BACKGROUND: Hybrid cells generated from dendritic cells (DC) and tumour cells provide tumour-associated antigens (TAA) in a polyvalent mode. The present study was designed to investigate the hybrid cell generation by dendritic cells and different tumour cell lines to establish an electrofusion protocol with an optimal fusion setting. MATERIALS AND METHODS: Hybrid cells from mature DC and tumour cells were generated by electrofusion. Fusion efficiency was determined by flow cytometry, as well as by light and fluorescence microscopy analyses. RESULTS: The gradual electrofusion process constituted different human dendritic cell tumour cell hybrids of high diversity depending on electrical and non-electrical parameters. Factors influencing fusion frequency were determined by specific cell staining with mAbs, FACS analysis and trypan blue dye exclusion. CONCLUSION: Increased fusion efficiency was associated with reduced viability. The protocol presented in this work might be helpful for future fusion studies as a prerequisite for comparable in vitro and human vaccination trials.