Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids. We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI). DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells. Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II).

Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids.<p>We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI).<p>DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells.<p>Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II). / Doctorat en sciences biomédicales / info:eu-repo/semantics/nonPublished

Cervical cancer represents an enormous health, psychological and social stress for every woman. The most important risk factor in the development of cervical carcinoma, which the second most common malignant cancer in women, is infection with a high-risk strain of human papillomavirus - a very frequent sexually transmitted disease. More than 100 types of HPV are acknowledged to exist, with HPV 16 and 18 being classified as high-risk types in particular. Worldwide, 500,000 new cases of cervical cancer are diagnosed every year. In the Czech Republic, there are 1,000 new cases of cervical cancer each year, out of which up to 400 women die. It is estimated that there will be up to 1,000,000 new cases of cervical cancer by 2050 unless the prevention is improved. Every woman is at risk of developing cervical cancer. HPV is sexually transmitted, however not only by sexual intercourse but also by skin-to-skin-contact with infected areas. Other risk factors in the development of the disease are: first sexual intercourse at early age, the number of sexual partners, smoking, other sexually transmitted diseases and a long term use of hormonal contraception. Use of condoms, which protects against sexually transmitted diseases, reduces the transmission of HPV by up to 70%. Having regular gynaecological check-ups with Pap smears is crucial for cervical cancer screening, as the screening suggests the presence of cytological abnormalities and pre-cancer. However, it cannot detect all types of premalignant changes and early stages of the carcinoma. Two vaccines have recently been developed, effective against the most frequent oncogenic strains of HPV (16 and 18), which currently cause about 70% of cervical cancer cases. Active immunisation against human papillomavirus is the first vaccination against carcinoma. Together with screening, it represents the best prevention method against cervical carcinoma. Based on the research of technical literature, the first part of the dissertation gives an overall view of the issue of cervical carcinoma. The second part of the dissertation deals with the research, eliciting the knowledge and attitude of women from Southern Bohemian towns in the field of cervical carcinoma prevention in the period of December 2008 - March 2009 and comparing it to technical literature.