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Biomedical subjects

M Smahel

Publications and source records attributed to M Smahel.

At least 19 recordsLinked to original sources
PubMed2005

Use of polyclonal rabbit antibodies for detection of the bcr-abl fusion zone in cells transfected with experimental bcr-abl DNA vaccines.

Rabbits were immunized with peptides covering the fusion zone of the chimeric bcr-abl protein in order to prepare antibodies capable of detecting the expression of a selected portion of this fusion zone, by a variety of experimental genetic vaccines. Three peptides of different size covering the b3a2 fusion zone, either unmodified or modified by the omission of alanine at the N-terminal of the a2 section of the fusion zone, and one peptide covering the unmodified b2a2 fusion zone were used. All were capable of eliciting antibodies reactive with the respective immunizing peptides. Their cross-reactivities, especially the results of cross-absorption experiments, strongly suggested that the serum of the rabbit immunized with an octadekapeptide mimicking the b3a2 fusion zone contained antibodies against a novel antigenic determinant created by the chimeric protein, and also against an epitope present in the adjacent a2 section but no antibody reactive with the adjacent b3 region. In Western blotting, these antibodies were capable of detecting the p210bcr-abl or a portion of it (a 25 amino acid-long sequence covering the b3a2 fusion zone) in lysates of 293T cells transfected with plasmids that carried either the full cDNA of the bcr-abl gene or a fragment thereof fused with either the HSP70 gene or certain other genes.

Animals↗
PubMed2003

Local IFN-gamma therapy of HPV16-associated tumours.

We have examined whether peritumoral administration of IFN-gamma can inhibit growth of HPV16-associated, MHC class I- tumour MK16/1/IIIABC (MK16) transplanted in syngeneic mice. It has been found that peritumoral administration of recombinant IFN-gamma performed on days 0-11 after tumour challenge inhibited growth of MK16 s.c. tumour transplants. If the therapy with IFN-gamma was started when the tumours had already reached a palpable size, the IFN-gamma administration was without any effect. To investigate the antitumour effects of IFN-gamma in a clinically more relevant setting, surgical minimal residual tumour disease was utilized. Subcutaneously growing MK16 carcinomas, 8-12 mm in diameter, were removed and the operated mice were injected with IFN-gamma on days 3-14 after the operation at the site of surgery. Treatment with IFN-gamma resulted in a moderate, reproducible, but statistically insignificant inhibition of tumour recurrences. In the next experiments we have addressed the question whether the tumour-inhibitory effect of IFN-gamma was due to the upregulation of MHC class I molecule expression on MK16 tumour cells. IFN-gamma-treated and control mice were sacrificed, their tumours were explanted, and the expression of MHC class I molecules on the MK16 tumour cells was examined. As presumed, the MHC class I expression on the cells of IFN-gamma-treated tumours, as well as on their lung metastases, was upregulated. However, an unexpected moderate upregulation of the MHC class I expression was also observed on MK16 tumours from the control, exogenous IFN-gamma-uninjected mice. Cytofluorometric analysis of the in vivo transplanted MK16 tumours from both groups has excluded that the increased percentage of the MHC class I molecules on the tumour cell populations could be due to the infiltration of the tumours with MHC class I+ leukocytes, since no expression of MHC class II, CD11b, CD80/CD86, and CD11c molecules in the MK16 cell population was observed.

Animals↗
PubMed2003

Immunization with Varicella-zoster virus glycoprotein E expressing vectors: Comparison of antibody response to DNA vaccine and recombinant vaccinia virus.

Immunization with DNA vaccines expressing Varicella-zoster virus (VZV) glycoprotein E (gE) induced formation of specific antibodies in mice. The antibody response correlated with the level of in vitro gE expression if the plasmid was inoculated intradermally (i.d.) with a gene gun but not if intramuscular (i.m.) injection was used. The i.d. vaccination produced a higher antibody level than the i.m. one even though a 100-fold amount of DNA was administered. A plasmid expressing a truncated form of gE was less immunogenic. The magnitude of antibody response induced by immunization with recombinant vaccinia viruses (rVVs) was equivalent to the gene gun vaccination. Administration of DNA by i.m. route or Vaccinia virus (VV) gE by i.d. mute resulted in predominance of IgG2a in the response while the gene gun plasmid inoculation usually elicited similar levels of IgG1 and IgG2a. The antibody response elicited by DNA vaccine was boosted by a secondary immunization with rVV. The boosting effect was highest if the virus was administered intraperitoneal (i.p.).

Animals↗
PubMed2002

[DNA vaccines].

Immunization with plasmid DNA is a new trend in vaccine development that could enhance the safety and efficacy of currently used vaccines. Simultaneously, it will enable preparation of new vaccines that could not be developed by existing procedures. The main methods of plasmid-DNA application are intramuscular injection and intradermal delivery into skin by a gene gun. As a protein antigen is produced inside host cells, both humoral and cell-mediated immunity are significantly activated. The dominant role is played by dendritic cells presenting an antigen. The type and intensity of immune reaction induced can be influenced by various ways. Modification of gene coding for immunization antigen, combination with genes for immunostimulatory factors, and utilization of adjuvant effect of stimulatory CpG motifs are the major methods of improvement of immunization with plasmid DNA. Immune reactions against viral, bacterial, and parasitic infectious agents were successfully stimulated in many experimental systems. Other experiments are under way utilizing DNA vaccines for treatment of malignant tumors, autoimmune diseases, and allergy. The fast progress in DNA-vaccine development resulted in continually increasing number of clinical trials.

Animals↗
PubMed2002

DNA vaccine against Friend erythroleukaemia virus.

Plasmids carrying DNA copies of the gag and env genes of FV, which causes erythroleukaemia in susceptible mouse strains, were prepared. Expression of the cloned genes was confirmed by indirect immunofluorescence in cells transfected in vitro. Immunization experiments were performed in DBA/2 mice. Animals were injected with three doses of plasmid either intramuscularly (100 microg DNA per dose) or intradermally (1 microg DNA per dose); in the latter case, a gene gun was used. The FV type A or P was used as a challenge. The immunization with gag- and env-derived vaccines resulted in protective immunity in a high proportion of mice.

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↗
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

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↗
PubMed1998

Prevalence of antibodies to human papillomaviruses in the general population of the Czech Republic.

Sera from 450 individuals between the age of 1 and 80 years, representing the general population of the Czech Republic, were tested for the presence of antibodies to human-papillomavirus(HPV)-derived antigens. The following antigens were used: (i) HPV1 virions; (ii) HPV16, -18 and -33-virus-like particles (VLP); (iii) peptides derived from L2 open reading frames (ORFs) of HPV16 and HPV6/11; (iv) peptides derived from HPV16 E2, E4 and E7 ORFs of HPV16. The prevalence of antibodies reactive with the capsid-derived antigens was age-dependent, while no clear age dependence was observed in the distribution of antibodies to peptides derived from HPV16 early proteins. In individual sera, high correlations between the presence of antibodies reactive with the 2 L2 peptides, also between the antibodies reactive with different VLPs, were found. While the simultaneous presence of the 2 L2 antibodies was frequently detected in individual sera in all age groups, the simultaneous occurrence of VLP antibodies was detected mostly in subjects older than 20 years. There were no significant differences in HPV-antibody distribution between men and women.

Adolescent↗
PubMed1998

Induction of anti-tumour immunity by suicide-gene-modified HPV-16-transformed hamster cells.

From K3/II, which is a highly oncogenic HPV16-transformed Syrian hamster cell line, thymidine-kinase(TK)-less cells, denoted B 49, were derived. B49 cells were transfected by a plasmid containing the herpes-simplex-virus TK gene (HSV TK) and several sub-lines expressing this gene were isolated from the transfected cultures. The HSV TK+ cells were highly sensitive to ganciclovir (GCV) and other anti-viral substances whose inhibitory effect is based on their phosphorylation by HSV TK. One of the cell lines, denoted KL1/6, exhibited relatively high stability of the HSV TK+ phenotype and was used in subsequent experiments. When KL1/6 cells were co-cultivated in the presence of GCV with various other cell lines of hamster, mouse or monkey origin, the by-stander effect (BE) was observed. GCV treatment of hamsters prevented development of tumours after the administration of KL1/6 cells but not K3/II cells. The treatment of animals with already established KL1/6-induced tumours resulted in tumour regression in all instances, but complete regression was observed only in animals carrying small tumours. The BE of KL1/6 cells on K3/II cells was also seen in vivo. In addition, concomitant immunity was observed in animals simultaneously inoculated with KL1/6 cells and K3/11 cells at 2 separate sites of the body. This effect was evident not only in animals in which KL1/6 tumours developed, but also in those in which tumour outgrowth was prevented by GCV treatment. In other experiments it was demonstrated that one KL1/6 + GCV treatment resulted in partial resistance, 2 such treatments in complete resistance to the challenge with K3/II cells.

Animals↗
PubMed1997

Correlation between the presence of anti HPV33 VLP antibodies and HPV DNA in cervical neoplasia patients.

The L1 capsid proteins derived from human papillomavirus (HPV) type 33 were expressed in a recombinant baculovirus system using Sf9 insect cells. Selected sera originating in women from case-control study carried out in Spain and Colombia found negative and positive for HPV16, 18, 31, 33 and 35 DNA were tested in ELISA for the presence of IgG antibodies to purified virus-like particles (VLP). The reactivity was type-restricted with the possible exception of HPV31.

Antibodies, Viral↗