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V Vonka

Publications and source records attributed to V Vonka.

At least 37 records · Page 2Linked to original sources
PubMed2002

Freezing and thawing of murine bone marrow-derived dendritic cells does not alter their immunophenotype and antigen presentation characteristics.

The aim of this paper was to assess whether the BMDC after freezing and thawing are capable to retain the immunophenotype and antigen-presenting capacity. BMDC were generated from bone marrow precursor cells as described previously by culturing the cells in medium containing GM-CSF and IL-4. Afterwards, the cells were harvested, counted and used for phenotyping and priming of syngeneic spleen cells. For cryopreservation, the BMDC were frozen in the presence of 10% of dimethylsulphoxide (DMSO) and 90% foetal calf serum. Forty to fifty percent of both samples, frozen/thawed as well as fresh BMDC, exhibited characteristic DC morphology, and the DC obtained from the frozen/thawed samples expressed a similar level of MHC class I-, MHC class II-, CD80-, CD86-, CD11c-, CD11b-, CD54- and CD205-molecule as fresh DC. To examine the in vitro priming effect of cryopreserved BMDC on syngeneic non-adherent murine C57BL/6 (B6) spleen cells, the BMDC were thawed, pulsed with the lysate prepared from HPV 16-associated tumour MK16 and used for 3H-thymidine assay. The findings of the experiments indicate that fresh as well as cryopreserved murine BMDC preparations pulsed with tumour lysate were efficient to prime the mitogenic activity of syngeneic non-adherent splenocytes. Taken together, the results suggest that frozen/thawed BMDC are morphologically, phenotypically and functionally comparable with fresh BMDC and can be used for construction of dendritic cell-based tumour vaccines.

Animals↗
PubMed2001

Modified HPV16 E7 Genes as DNA Vaccine against E7-Containing Oncogenic Cells.

Therapeutic vaccines against tumors associated with human papillomaviruses (HPV) should elicit cellular immune responses against early HPV antigens, primarily the oncoproteins E7 and E6. Because of safety concerns, the direct use of an unmodified oncogene is impossible in human DNA vaccination. Therefore, we introduced three point mutations into the pRb-binding site of HPV16 E7 oncogene to eliminate its transformation potential. The resultant gene was denoted E7GGG. The rates of expression and the cellular localization of E7 and E7GGG proteins were comparable. In immunization-challenge experiments, the efficacy of plasmids containing the E7, E7GGG, or fusion genes of HPV16 E7, viz. L1DeltaCE7(1-60) (M. Muller et al., 1997, Virology 234, 93-111), and Sig/E7/LAMP-1 (T. C. Wu et al., 1995, Proc. Natl. Acad. Sci. USA 92, 11671-11675), was compared. While tumors developed in all animals immunized with the wild-type E7 gene, a significant proportion of mice remained tumor-free after vaccination with the E7GGG gene. The fusion gene L1DeltaCE7(1-60) induced negligible protection, but Sig/E7/LAMP-1 conferred the highest protection. Intradermal immunization by gene gun proved superior to i.m. inoculation. In "therapeutic" experiments, a 1-day delay between inoculation of oncogenic cells and the start of DNA immunization resulted in partial therapeutic effect, but a 3-day delay produced a substantially lower immunization effect. A combination of Sig/E7/LAMP-1 and E7GGG genes did not enhance the immune response. These results demonstrate a significant enhancement of HPV16 E7 immunogenicity after mutagenesis of the pRb-binding site, but the mutated E7 gene did not excel the Sig/E7/LAMP-1 fusion gene.

Administration, Cutaneous↗
PubMed2001

Metastatic MHC class I-negative mouse cells derived by transformation with human papillomavirus type 16.

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by cotransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by the G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 10(5) homologous cells, but not against a higher cell dose (5 x 10(5)). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Animals↗
PubMed2001

Therapy of HPV 16-associated carcinoma with dendritic cell-based vaccines: in vitro priming of the effector cell responses by DC pulsed with tumour lysates and synthetic RAHYNIVTF peptide.

Murine carcinoma induced by MK 16 cells expressing HPV 16 E6/E7 oncogenes was utilized to examine the therapeutic effect of dendritic cell-based tumour vaccines. Mice carrying 5-day MK 16 tumours were injected peritumorally with either dendritic cells (DC) or DC pulsed with MK 16 tumour lysate. Both the unpulsed and MK 16 lysate-pulsed DC vaccines inhibited growth of the MK 16 transplants, the pulsed DC being more efficient than the unpulsed vaccines. In vitro priming of the effector cell-mediated anti-MK 16 responses by DC pulsed with MK 16 tumour lysate and a synthetic HPV 16 E7(49-57) peptide RAHYNIVTF was compared. The priming activity of the lysate was substantially higher than that of the HPV 16 E7(49-57) peptide; the priming activity was similar to that of a standard moderately immunogenic chemically-induced sarcoma. Taken collectively, these results suggest that DC vaccines pulsed with HPV 16-associated tumour lysates represent a prospective modality for treatment of HPV 16-associated carcinomas.

Amino Acid Sequence↗
PubMed2000

Causality in medicine: the case of tumours and viruses.

Clarification of the aetiology of chronic human diseases such as atherosclerosis or cancer is one of the dominant topics in contemporary medical research. It is believed that identification of the causal factors will enable more efficient prevention and diagnosis of these diseases and, in some instances, also permit more effective therapy. The task is difficult because of the multistep and multifactorial origin of these diseases. A special case in contemporary aetiological studies is definition of the role of viruses in the pathogenesis of human cancer. Virus-associated cancer develops only in a small minority of infected subjects, which implies that, if the virus does play a role in the pathogenesis of the malignancy, other factors must also be involved. In this paper the author attempts to review the present methodological approaches to aetiological studies of chronic diseases, discusses the role of criteria for identifying causal relationships and proposes guidelines that might help to determine the role of viruses in human cancer.

Humans↗
PubMed2000

Longitudinal follow-up of antibody response to selected antigens of human papillomaviruses and herpesviruses in patients with invasive cervical carcinoma.

A group of 21 cervical-carcinoma patients was followed longitudinally. All patients had undergone intensive radiotherapy. In the course of a more than 5-year observation period, 2 patients died of cervical cancer, 1 from other causes, 3 were lost from follow-up, and 15 survived without any signs of the disease. Sera taken before, up to 17 months after and more than 5 years after the start of therapy, were tested by ELISA for IgG antibodies reactive with a broad spectrum of HPV-derived antigens, glycoprotein G of HSV 2, whole virion antigen of HCMV, and a synthetic peptide corresponding to the immuno-dominant region of EBNA 1. The therapy was associated with a marked decrease in E2 and E7 antibodies in nearly all patients possessing pre-existing antibodies; the changes in VLP antibody levels in the treated women were more rare and less pronounced. In the course of the observation period, seroconversion to gG HSV2 positivity was seen in 5 patients, while, a marked increase in pre-existing gG HSV2 antibodies was observed in 5 out of 7 originally seropositive patients. At enrollment, only 2 patients were free of HCMV antibody and only 1 was free of EBNA1 antibody; no seroconversion relative to either antigen was seen during the observation period.

Adult↗
PubMed2000

Local cytokine treatment of HPV16-associated tumours results in inhibition of their lung metastases.

Experiments were designed to examine whether local cytokine therapy of subcutaneous (s.c.) tumours results in inhibition of their lung metastases. Moderately immunogenic, major histocompatibility complex (MHC) class I and II negative. B7 negative, metastasizing murine carcinoma MK16 transplantable in syngeneic mice was obtained by co-transfection of human papilloma virus type 16 (HPV 16) E6/E7 and activated H-ras oncogene plasmid DNA into C57BL/6 kidney cells. After s.c. transplantation of the malignantly converted MK16 cells, the majority of the transplanted mice developed lung metastases; the number and size of the lung metastases increased with the increasing size of the s.c. tumour. Therapy of 5-day MK16 tumours by peritumoral administration of recombinant interleukin-2 (IL-2) and recombinant interleukin-12 (IL-12) inhibited growth of the s.c. MK16 tumour transplants and reduced the number of MK16 lung metastases. To investigate the antimetastatic effect of IL-2 and IL- 12 in a clinically more relevant setting, surgical minimal residual tumour disease was utilized. Subcutaneously growing MK16 carcinomas, 8-12 mm in diameter, were removed on day 30 and the operated mice were injected with IL-2 or IL- 12 on days 35-39 and 42-46 at the site of the operation. Treatment with IL-2 significantly reduced the percentage of MK16 tumour recurrences as well as the number of lung metastases, whereas the effect of IL- 12 was substantially weaker and statistically insignificant.

Animals↗
PubMed2000

Peritumoral administration of antigen-unstimulated bone marrow-derived dendritic cells inhibits tumour growth.

Murine BM cells from B6 mice were grown in vitro in medium supplemented with GM-CSF and IL-4 to differentiate DC from DC precursors. After 10 days of culture, approximately 20% of the cell population exhibited the characteristic morphology of BMDC. In cytofluorometric analysis the morphological changes of cells were accompanied by upregulation of the expression of the MHC class II, CD11c, CD80, and CD86 molecules. The BMDC were pulsed with a lysate of syngeneic MK16 carcinoma cells and used for in vitro activation of SC. Co-cultivation of the carcinoma lysate-pulsed BMDC with SC induced a proliferative response of the syngeneic SC. Priming of the proliferative responses was more efficient when the BMDC were grown in the presence of GM-CSF and IL-4 for 10 days than for 7 days. The in vivo effect of mature, tumour lysate-unstimulated BMDC was examined in mice carrying syngeneic MK16 carcinoma transplants. It has been found that local pretreatment with BMDC inhibits growth of a subsequent challenge inoculum of the MK16 cells. Similarly, treatment of mice carrying small MK16 tumours and of those with MK16 surgical minimal residual disease performed with BMDC significantly inhibited tumour growth. It can be concluded from these results that local concentration of mature BMDC at the tumour site can control the development and growth of the transplanted tumour inoculum.

Animals↗
PubMed2000

[Papillomaviruses and human tumors].

The report summarizes the main results obtained in the course of our research project. The results of immunological and epidemiological studies provide further proofs that human papillomaviruses (HPV) are the etiological agents in cervical neoplasia. In addition, they raise hopes that immunological methods may be utilized in diagnostics of cervical cancer and for monitoring the clinical course of this disease in the near future. Since the etiological relationship between HPV and cervical carcinoma seems to be proven beyond reasonable doubt, the development of prophylactic and therapeutic vaccines has become the dominant of the contemporary HPV reseach. For studying immune reactions against HPV-induced tumours we developed a model of HPV16-transformed rodent cells.

Female↗
PubMed1999

Secondary vaccination with vaccinia virus recombinants: role of residual virulence of recombinants and immunogenicity of extrinsic antigens.

ICR mice were immunized intraperitoneally with two doses (10(6) PFU per dose) of vaccinia virus (VV) recombinants of variable virulence expressing either the strongly immunogenic glycoprotein E (gE) of varicella zoster virus (VZV) or weakly immunogenic hepatitis B virus (HBV) preS2-S (S) antigen. Recombinants expressing gE were able to elicit primary and secondary anti-gE antibody irrespective of their residual virulence; after the second dose they did so even in the presence of VV antibody resulting from primary vaccination dose or under other conditions limiting VV replication. As for the S-recombinants, pronounced anti-S antibody development was only observed in mice which had received the more virulent recombinant virus as the first dose. A repeated dose of S-recombinants was unable to elicit a secondary anti-S antibody response. The present findings do not support the assumption that the poor immunogenicity of some extrinsic antigens could be overcome by administering repeated doses of the particular VV recombinant.

Aging↗
PubMed1999

Prospective study on cervical neoplasia IV. Presence of HPV antibodies.

Sera collected in the course of a prospective study carried out in Prague in 1975-1983 were assayed for the presence of human papillomavirus (HPV) antibodies. Women with cervical neoplasia proven by biopsy at enrollment possessed antibodies to peptides derived from E2, E4 and E7 proteins of HPV16 and to virus-like particles (VLPs) of HPV16, -18 and -33 significantly more frequently than matched controls. Women without cervical neoplasia at enrollment who developed the disease in the course of the study differed from matched controls by a higher prevalence of antibodies against VLPs of HPV16 and -18 but not against early antigens of HPV16. In 19 of the latter subjects, paired serum specimens were tested, the first samples having been taken at enrollment and the second at diagnosis. Development of the disease was associated with seroconversion from negativity to positivity to at least one HPV antigen in 11 (57.9%) women.

Adult↗
PubMed1999

Human papillomavirus genotype spectrum in Czech women: correlation of HPV DNA presence with antibodies against HPV-16, 18, and 33 virus-like particles.

Because the biological spectrum of human papillomavirus (HPV) genotypes present in cervical cancer lesions varies according to the geographical region studied, and because little genotype information is available for Central and Eastern European countries, we studied the endemic HPV-genotype spectrum in cervical samples collected from women visiting gynaecological departments of selected hospitals in the Czech Republic. In a series of 389 samples, 171 (44.0%) were positive for HPV DNA using a consensus-primer polymerase chain reaction (PCR). Genotyping of the HPV PCR products was done using dot-blot hybridisation with type-specific oligonucleotide probes and thermocycle DNA sequencing. Twenty-two different HPV types were detected, HPV-16 being the most prevalent type irrespective of severity of the lesions (55.0%). Multiple HPV types were found in 16.4% of our HPV-DNA-positive samples. The prevalence of HPV infection was 23.0% in women with normal findings and 59.4% in patients with cervical neoplasia, and increased significantly with the severity of the disease: 52.9% in low-grade lesions, 58.0% in high-grade lesions, and 73.5% in cervical carcinomas (P for trend < .00001). In the sera of 191 subjects, 89 with normal findings and 102 with different forms of cervical neoplasia, the prevalence of HPV-specific IgG antibodies was tested by an enzyme-linked immunosorbent assay (ELISA) using virus-like particles (VLPs) of HPV-16, -18, and -33. Antibodies were significantly more prevalent in HPV-DNA-positive than in HPV-DNA-negative women and there was no association with age. In agreement with the results of HPV genotyping, antibodies reactive with HPV-16 VLPs were the most frequent and, moreover, their prevalence increased with the cervical lesion severity. About half of the subjects with smears in which either HPV-16 or HPV-33 DNA had been detected possessed antibodies reactive with homotypic VLPs. With HPV-18-DNA-positive subjects, however, fewer than 25% displayed homotypic antibodies. In general, subjects older than 30 years of age had antibodies reactive to HPV-specific VLPs more often than subjects younger than 30 years of age. In women with benign findings, the seropositivity to HPV-16, -18, and -33 VLPs increased with age, whereas in women with cervical neoplasia the seropositivity decreased with age.

Adult↗
PubMed1999

Interleukin 2 gene therapy of residual disease in mice carrying tumours induced by HPV 16.

Experiments were designed to examine the efficacy of IL-2 gene therapy in a surgical minimal residual tumour disease, using moderately immunogenic MK16/1/IIIABC murine cells transformed by activated ras and HPV 16 E6/E7 oncogenes (MK16 cells). Previously we demonstrated that surgical minimal residual tumour disease (SMRTD) could be effectively cured when murine Mc12 sarcoma had been resected and the operated mice were treated with irradiated Mc12 sarcoma cells engineered to secrete IL-2. In this study we performed IL-2 gene therapy of MK16 carcinoma with two types of irradiated MK16-unrelated tumour cell vaccines. One type of vaccine was derived from MHC class I-matched Mc12 sarcoma cells engineered to secrete IL-2 and the other from MHC class I-discordant IL-2 producing plasmacytoma X63-m-IL-2. The vaccines did not share any tumour rejection antigen with the MK16 cells and served exclusively as a local source of IL-2 production. Both vaccines were capable of inhibiting MK16 tumours when administered peritumorally up to 15 days after MK16 tumour challenge. The irradiated MHC class I-matched and IL-2-producing Mc12 sarcoma vaccine was then selected for therapy of MK16 SMRTD. Whereas the recurrence rate in the operated MK16 carcinoma bearers was 80%, so that only 20% of mice were cured by surgery, approximately 65% of the MK16 carcinoma bearers were permanently protected when the surgery was followed by local administration of the IL-2-producing Mc12 sarcoma vaccine.

Animals↗
PubMed1999

The frequency of HLA-DRB1 and -DQB1 alleles in cervical cancer cases in the Czech Republic.

The distribution of HLA class II DRB1 and DQB1 alleles in cervical carcinoma patients was compared with the frequency of these alleles found in healthy population living in the Czech Republic. The RFLP analysis and PCR-SSP were used for DNA typing. Although the differences in the frequency of DRB1*03, DQB1*02 and DQB1*0303 alleles between the cases and the controls were rather large, corrected P values did not reach significance.

Adult↗