DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8(+) T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

• MeSH
• adjuvancia imunologická aplikace a dávkování   MeSH
• bcr-abl fúzní proteiny genetika   imunologie   metabolismus   MeSH
• biolistika MeSH
• CD8-pozitivní T-lymfocyty účinky léků   metabolismus   patologie   MeSH
• DNA vakcíny MeSH
• experimentální nádory imunologie   patologie   terapie   MeSH
• imunita účinky léků   MeSH
• levamisol aplikace a dávkování   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oligodeoxyribonukleotidy aplikace a dávkování   MeSH
• Papillomavirus E7 - proteiny genetika   imunologie   metabolismus   MeSH
• protinádorové vakcíny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

A series of DNA vaccines based on the bcr-abl fusion gene were developed and tested in mice. Two mouse (BALB/c) bcr-abl-transformed cell lines, B210 and 12B1, which both expressed p210bcr-abl and were oncogenic for syngeneic animals but differed in some other respects, were used as a model system. In the first series of experiments, plasmids carrying either the complete bcr-abl fusion gene or a fragment thereof coding for a 25-amino acid-long junction zone (bcr-abl25aa) linked with genes coding for a variety of immunostimulatory factors were used as the DNA vaccines. A plasmid carrying the complete bcr-abl gene was capable of inducing protection against challenge with either B210 or 12B1 cells. However, the DNA vaccines based on the gene fragment coding for p25aabcr-abl did not induce significant protection. To localize the immunizing epitopes on the p210bcr-abl protein, the whole fusion gene was split into nine overlapping fragments and these, individually or in various combinations, were used for immunization. Although none of the vaccines based on any single fragment provided potent protection, some combinations of these fragment-based vaccines were capable of eliciting protection comparable to that seen after immunization with the whole-gene vaccine. Surprisingly, a mixture of six fragment-vaccines was more immunogenic than the complete set of fragment DNA vaccines. To analyze this phenomenon, the three fragments missing from the hexavaccine were either individually or in various combinations mixed with the hexavaccine. The results obtained suggested that the product of the fragment coding for 197 amino acids forming the N-terminal of the BCR protein was involved in the decreased immunogenicity. However, further experiments are needed to clarify the point. Additional experiments revealed that all the important epitopes were located in the ABL portion of the p210bcr-abl protein. The livers, spleens and bone marrows of the successfully immunized animals were tested for the presence of bcr-abl-positive cells by RT-PCR. The results were negative, this suggesting that these animals were free of any residual disease.

• MeSH
• bcr-abl fúzní proteiny genetika   imunologie   MeSH
• časové faktory MeSH
• chronická myeloidní leukemie genetika   imunologie   patologie   prevence a kontrola   MeSH
• DNA vakcíny genetika   imunologie   MeSH
• HL-60 buňky MeSH
• imunizace MeSH
• lidé MeSH
• mapování epitopu MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• peptidové fragmenty imunologie   MeSH
• protinádorové vakcíny genetika   imunologie   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

B210 cells are murine (BALB/c) cells transformed by bcr-abl fusion gene. After intravenous administration they are capable of inducing leukaemia-like disease in syngeneic mice. From these cells a thymidine-kinase less subline was derived. It was significantly less pathogenic than the parental cells. However, a highly pathogenic clone denoted B210cTK-/cl-2 was isolated from its population. As determined by Western blotting, these cells produced more p210 protein than the parental B210 cells. To successfully transfect these cells a modified electroporation method was introduced. Bicistronic plasmids carrying gene for herpes simplex thymidine kinase (HSV TK) and the gene for either granulocyte-monocyte colony stimulation factor (GM-CSF), interleukin-2 (IL-2) or interleukin 12 (IL-12) were used for the transfection experiments. Gradually, cell lines producing these cytokines were isolated in media supplemented with hypoxantin, aminopterin and thymidine (HAT). All of them were highly sensitive to ganciclovir in vitro confirming that the cells produced HSV TK. The genetic modification of B210cTK-/cl-2 was associated neither with the alteration of p210 bcr-abl production nor with any changes in expression of MHC class I molecules. From populations of each of the three lines several cell clones were isolated and tested for the production of the respective cytokines. The original uncloned population and several clones differing in the cytokine production were administered intravenously into mice. All animals survived without symptoms of the disease suggesting that the gene-modification was associated with the loss of pathogenicity. Keywords: CML, Bcr-Abl, HSV TK, cytokines, gene-modified tumour cells, pathogenicity.

• MeSH
• adjuvancia imunologická genetika   MeSH
• bcr-abl fúzní proteiny analýza   MeSH
• cytokiny biosyntéza   genetika   MeSH
• ganciklovir terapeutické užití   MeSH
• geny abl MeSH
• MHC antigeny I. třídy analýza   MeSH
• MHC antigeny II. třídy analýza   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• thymidinkináza genetika   MeSH
• transfekce MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

For our experiments we selected two oncogenic, bcr-abl-transformed mouse cell lines, viz. B210 and 12B1. Both cell types are capable of inducing leukemia-like disease in syngeneic BALB/c mice after intravenous inoculation. 12B1 cells can moreover form solid tumors after subcutaneous injection. Since immunotherapy would expectedly be most effective in animals in which the tumor mass had been reduced by other therapeutic means, we attempted to develop a combined therapeutic system for suppressing tumor growth. In the present study, mice inoculated with the aggressive 12B1 cells were treated with imatinib mesylate (IM), mouse interferon alpha (IFNalpha) and cyclophosphamide (Cy) in combination with genetically modified tumor cells engineered to produce various cytokines. These cell vaccines had been derived from B210 cells. Therapy with IM or IFNalpha alone or cell immunotherapy alone resulted in partial suppression of tumor growth. Of the different therapeutic regimens tested, a combination of repeated doses of IM, IFNalpha and cell vaccines with one relatively high dose of Cy (200 mg/kg) was the most effective, resulting in tumor-free survival of a large portion of mice. The spleens, livers and bone marrows of the successfully treated animals were tested for the presence of bcr-abl-positive cells by means of RT-PCR technique. Results were negative, this suggesting that the animals had been cleared of residual disease.

• MeSH
• bcr-abl fúzní proteiny imunologie   terapeutické užití   MeSH
• cyklofosfamid aplikace a dávkování   MeSH
• experimentální nádory imunologie   terapie   MeSH
• faktor stimulující granulocyto-makrofágové kolonie biosyntéza   MeSH
• financování organizované MeSH
• imunoterapie metody   MeSH
• interferon gama aplikace a dávkování   MeSH
• interleukin-12 biosyntéza   MeSH
• interleukin-2 biosyntéza   MeSH
• kombinovaná terapie MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• piperaziny aplikace a dávkování   MeSH
• polymerázová řetězová reakce MeSH
• protinádorové látky aplikace a dávkování   MeSH
• protinádorové vakcíny imunologie   MeSH
• pyrimidiny aplikace a dávkování   MeSH
• transformované buněčné linie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

Oncolytic virotherapy is a novel approach to cancer treatment. In the present study we tested the ability of reovirus type 3, strain Dearing, to suppress the growth of tumors induced in mice by HPV16-transformed TC-1 cells. In vitro, these cells are highly susceptible to the virus. In repeated in vivo tests the intratumoral inoculation of the virus resulted in only a minor slow-down of tumor growth, never in a complete cure. The effect of the treatment was not enhanced by the simultaneous administration of non-oncogenic, genetically modified TC-1 cells expressing either IL-2, IL-12 or GM-CSF, and, in fact, the oncolytic effect of the virus was even less expressed in some instances. When cyclophosphamide was used in combination with the viral treatment, a synergistic effect resulting in tumor suppression was observed. In most instances the tumor regression was transitory, however, and was followed by tumor progression. The outcome of these experiments was dependent on the timing of the two treatments.

• MeSH
• cyklofosfamid aplikace a dávkování   MeSH
• experimentální nádory terapie   virologie   MeSH
• faktor stimulující granulocyto-makrofágové kolonie genetika   MeSH
• financování organizované MeSH
• genetická terapie metody   MeSH
• interleukin-12 genetika   MeSH
• interleukin-2 genetika   MeSH
• kombinovaná terapie MeSH
• lidský papilomavirus 16 MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• onkolytická viroterapie metody   MeSH
• protinádorové látky aplikace a dávkování   MeSH
• protinádorové vakcíny aplikace a dávkování   MeSH
• savčí orthoreovirus 3 MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH

• MeSH
• bcr-abl fúzní proteiny MeSH
• DNA vakcíny MeSH
• finanční podpora výzkumu jako téma MeSH
• králíci MeSH
• plazmidy MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH

• MeSH
• down regulace genetika   MeSH
• finanční podpora výzkumu jako téma MeSH
• invazivní růst nádoru MeSH
• leukemie farmakoterapie   MeSH
• myši MeSH
• nádorová transformace buněk MeSH
• piperaziny farmakologie   MeSH
• protinádorové látky farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• stárnutí buněk MeSH
• transformované buněčné linie MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• barvení a značení metody   MeSH
• bcr-abl fúzní proteiny genetika   MeSH
• chronická myeloidní leukemie diagnóza   patologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• histologické techniky metody   MeSH
• imunohistochemie MeSH
• modely nemocí na zvířatech MeSH
• myeloidní leukemie diagnóza   patologie   MeSH
• myši inbrední BALB C MeSH
• transformované buněčné linie transplantace   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH

• MeSH
• alkylační protinádorové látky terapeutické užití   MeSH
• cyklofosfamid terapeutické užití   MeSH
• imunoterapie MeSH
• lidé MeSH
• nádorová transformace buněk MeSH
• nádory terapie   MeSH
• onkogenní proteiny virové metabolismus   MeSH
• represorové proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH