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1
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Herpesviruses as possible cofactors in HPV-16-related oncogenesis*

100%
Szostek S., Zawilinska B., Kopec J., Kosz-Vnenchak M.
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issue 2
337-342
EN
Cervical carcinogenesis is a complex problem with papillomavirus widely accepted as a causative agent. Integration of a human papillomavirus (HPV) of the high-risk type into the host cell genome is one of the major contributing factors to cervical malignant transformation. In this study, the correlation of CMV, EBV, HSV-1, HSV-2, HHV-6 and HHV-7 infections with the physical status of the HPV genome in cervical cancer and precancerous cervical lesions was investigated in sixty HPV-16-positive women. Cervical secretion samples were submitted to DNA extraction and analyzed by PCR. HPV-16 DNA was confirmed in genotyping with the reverse hybridization line probe assay. Multiplex PCR with specific primers for the E2/E6 genes was used to assess the viral integration status of HPV-16. Our results show that CMV DNA was more frequently present in samples with mixed forms of HPV-16 than in the episomal form (P < 0.025). Such a correlation was also observed in the case of EBV (P < 0.005). The presence of CMV resulted in a six-fold (OR 6.069; 95% CI 1.91-19.22; P = 0.002), while EBV caused a seven-fold (OR 7.11; 95% CI 1.70-29.67; P = 0.007) increase in the risk of the integrated or mixed HPV-16 genome occurrence. Our data suggest that coinfection with herpesviruses, especially CMV and EBV, may be involved in the integration of the HPV-16 genome and may contribute to the development of cervical cancer.
2
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In situ detection of DNA and mRNA of human cytomegalovirus to distinguish different forms of viral infection in leukocytes

100%
Zawilinska B., Bulek K., Kopec J., Kosz-Vnenchak M.
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2006
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vol. 53
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issue 3
457-461
EN
In situ PCR and in situ reverse transcription PCR (RT-PCR) were applied to discriminate between latent and productive infection of human cytomegalovirus (HCMV) in leukocytes. We investigated 28 samples, in which viral pp65 antigen was detected only in the cytoplasm of leukocytes. Additionally we assayed 12 specimens lacking pp65 antigen. Using nested PCR (nPCR), viral DNA was detected in 27 samples. In six samples the results of nPCR were unreadable due to the presence of polymerase inhibitors. By application of in situ PCR, we were able to confirm the presence of viral DNA in the nucleus and/or cytoplasm. Productive infection was recognized in 20 samples in which transcripts for late viral genes were detected. Among the 20 samples negative by in situ RT-PCR, we recognized phagocytosis of viral particles in eight and the latent form of HCMV infection in five.
3
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HPV16 E6 polymorphism and physical state of viral genome in relation to the risk of cervical cancer in women from the south of Poland

100%
Szostek S., Zawilinska B., Klimek M., Kosz-Vnenchak M.
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2017
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vol. 64
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issue 1
143-149
EN
The aim of this study was to analyse the correlation between HPV16 E6 variants and the physical status of viral genome (integrated, mixed, episomal) among patients with cervical cancer (n=40) and low-grade squamous intraepithelial lesions - LSIL (n=40). The study was performed on 80 HPV16 positive samples. HPV16 E6 variants were identified using PCR and DNA sequencing. Nucleotide sequences of E6 were compared with the prototype sequence (EUR-350T). The physical state of HPV DNA was determined as the ratio of E2/E6 copy number per cell. Twelve different intratypic variants were identified as belonging to European (in 77 samples) and North-American 1 (in 3 samples) sublineages. The most prevalent non-synonymous variant was EUR-350G, which occurred with similar frequency in cervical cancer and LSIL. The frequencies of additional mutations in variants with EUR-350T or EUR-350G sequences differed significantly. For the first time, missense mutations G122A, C153T and G188A were discovered in EUR-350G variant. The integrated viral genome was predominant in women with cervical cancer. The EUR-350T prototype and EUR-350G without additional mutations variants were prevalent in cervical cancer samples with the HPV16 characterized by integrated DNA. In summary, European variants of HPV16 E6 dominated in both cancer and LSIL group. The presence of EUR-350G favoured the occurrence of additional nucleotide changes. We showed that nucleotide changes occur significantly more often in the mixed form of viral DNA and in LSIL group and that the variants without additional mutations may promote integration of HPV16 genome.
4
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Genotype-specific human papillomavirus detection in cervical smears

100%
Szostek S., Klimek M., Zawilinska B., Kosz-Vnenchak M.
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2008
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vol. 55
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issue 4
687-692
EN
Human papillomavirus (HPV) is widely accepted as a causative agent of cervical cancer. The distribution and prevalence of HPV types depend on geographic region and demographic factors. The aim of this study was to investigate the relationship between the presence of various HPV types and the outcome of cytological examination. Cervical smears were obtained from 125 women from southern Poland: low grade squamous intraepithelial lesions (LSIL) - 44, high grade squamous intraepithelial lesions (HSIL) - 12, cervical carcinoma - 27 and 42 women without abnormality in cytology as a control group. DNA was extracted from the smears and broad-spectrum HPV DNA amplification and genotyping was performed with the SPF 10 primer set and reverse hybridisation line probe assay (INNO-LiPA HPV Genotyping, Innogenetics). HPV DNA was detected in approximately 72% cases, more frequently in women with squamous intraepithelial lesions and cervical carcinoma than in the control group (P < 0.0005). The most frequent type found was HPV 16 (37%), followed by HPV 51 (28%) and HPV 52 (17%). A single HPV type was detected in 51% positive cases, more frequently in cervical cancer specimens. Multiple HPV infection was dominant in women with LSIL and normal cytology. Prevalence of HPV 16 increased with the severity of cervical smear abnormality. For women HPV 16 positive, the relative risk (odds ratio) of the occurrence of HSIL and cervical cancer versus LSIL was 14.4 (95% CI, 3.0-69.2; P=0.001) and 49.4 (95% CI, 6.5-372.8; P < 0.001), respectively. Genotyping of HPV will allow better classification of women with cervical abnormalities into different risk groups and could be useful in therapy.
5
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Multiplex real-time PCR to identify a possible reinfection with different strains of human cytomegalovirus in allogeneic hematopoietic stem cell transplant recipients

88%
Zawilinska B., Szostek S., Kopec J., Piatkowska-Jakubas B., Kosz-Vnenchak M.
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2016
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vol. 63
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issue 1
161-166
EN
Human cytomegalovirus (HCMV) infection remains the leading cause of serious contagious complications after allogeneic hematopoietic stem cell transplantation. These infections in HCMV-seropositive recipients can be due to reactivation or reinfection. Different HCMV strains were identified by determining the genotypes isolated from repeatedly tested patients. The UL55 sequences encoding viral glycoprotein B (gB) have been chosen as the target gene. The region, in which the gB precursor protein is cleaved into two fragments by a cellular endoprotease, is characterized by genetic variability, and based on that HCMV is classified into four major genotypes: gB1, gB2, gB3 and gB4. Multiplex real-time PCR assay enabled both, HCMV gB genotyping, as well as simultaneous quantitative assessment of the detected genotypes. This study was carried out in 30 transplant recipients, from whom 105 isolates of HCMV DNA were genotyped. In 40% of recipients, a mixed infection with two or three genotypes was detected. Genotype gB1 dominated in general, and characteristically for mixed infections, the genotype gB3 or gB4 was always present. Although there were no significant differences in the load for each genotype, in case of multiple infections, the number of copies of gB1 genotype was significantly higher when compared to a single gB1 infection. In patients with mixed genotypes, chronic HCMV infections and graft versus host disease were observed more often, as well as antiviral treatment was less effective. It was assumed that these adverse effects can be related to the presence of gB3 and gB4 genotypes.
6
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Physical state of human papillomavirus type 16 in cervical intraepithelial lesions and cancers determined by two different quantitative real-time PCR methods

88%
Szostek S., Biesaga B., Zawilinska B., Klimek M., Kosz-Vnenchak M.
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2015
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vol. 62
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issue 4
923-928
EN
The aim of this study was to analyse the correlation between a new multiplex qPCR assay and a reference qPCR assay for assessment of the human papillomavirus (HPV16) load and the viral genome status. The study was performed on 100 HPV16 positive samples containing premalignant lesions and carcinomas. HPV16 E2 and E6 gene loads were assessed by two PCR methods. The load of E2 and E6 was normalized to the cell number by qPCR targeting the RNase P open reading frame. The physical state of the viral genome was determined as a ratio of E2/E6 copies number per cell. Among 100 samples analysed, there were no statistically significant differences in the E2 and E6 viral load evaluated by multiplex qPCR and qPCR, the correlation coefficients were 0.98 and 0.97, respectively. There were 19% of samples with the integrated, 73% with mixed and 8% with episomal state of viral genome detected by multiplex qPCR and 17%, 79%, 4%, respectively, found by qPCR. Prevalence of integrated and episomal forms estimated by multiplex qPCR was higher than the one obtained by qPCR (Chi2, p < 0.0001), but in samples with premalignant and malignant diagnoses no significant differences were demonstrated regardless of the methods used. Sensitivity and specificity of multiplex qPCR were 93.7% and 100% as compared with qPCR, the positive predictive value was 100%. In summary, the multiplex qPCR assay in respect of HPV16 load and the frequency of viral genome status was shown to be a sensitive and specific reference method. Simultaneous estimation of E2 and E6 genes in one reaction tube reduces the cost of testing.
7
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Detection of human papillomavirus in cervical cell specimens by hybrid capture and PCR with different primers

76%
Szostek S., Klimek M., Zawilinska B., Rys J., Kope J., Daszkiewic E.
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2006
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vol. 53
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issue 3
603-607
EN
The purpose of this study was to compare hybrid capture assay with PCRs using different primers for the L1, E6-E7 regions for the detection of human papillomavirus (HPV) genome. One hundred twenty-five cervical smears with normal (n = 42) and abnormal (n = 83) cytology were investigated. Those at high-risk for HPV were studied by hybridization antibody capture assay and PCR with the pU-1M/pU-2R primers. Target DNA from the HPV L1 region was amplified by SPF10 primer set and home-PCR with MY09/MY11 primers. The presence of HPV DNA in cervical smears was detected by SPF10 (in 72% of cases), MY09/MY11 (58%), hybrid capture (55%) and pU-1M/pU-2R (39%). Results obtained with the SPF10 and MY09/MY11 consensus primer sets as well as hybrid capture and pU-1M/pU-2R specific for high-risk types differed significantly (χ2, P < 0.0005). The correlation between assays with the use of SPF10 and MY09/MY11 was 86% and between hybrid capture and the pU-1M/pU2R technique - 78%. In 49% of samples HPV DNA was detected by the four methods, whereas in 12% only by the SPF10 primers. The most sensitive technique was found to be PCR with the use of SPF10 primers, while the most specific - the MY09/11 PCR method. It seems that home-PCR with MY09/MY11 primers could be applied in screening tests.
8
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Detection of specific lytic and latent transcripts can help to predict the status of Epstein-Barr virus infection in transplant recipients with high virus load

76%
Zawilinska B., Kosinska A., Lenart M., Kopec J., Piatkowska-Jakubas B., Skotnicki A., Kosz-Vnenchak M.
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2008
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vol. 55
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issue 4
693-699
EN
Epstein-Barr virus (EBV), a member of the family Herpesviridae, is widely spread in the human population and has the ability to establish lifelong latent infection. In immunocompetent individuals the virus reactivation is usually harmless and unnoticeable. In immunocompromised patients productive infection or type III latency may lead to EBV-associated post-transplant lymphoproliferative disorder (PTLD). The aim of our research was to investigate the utility of PCR-based methods in the diagnosis and monitoring of EBV infections in bone marrow transplant recipients. Thirty-eight peripheral blood leukocyte samples obtained from 16 patients were analysed, in which EBV DNA was confirmed by PCR. We used semi-quantitative PCR to estimate the viral load and reverse-transcription PCR (RT-PCR) to differentiate between latent and productive EBV infection. In 14 patients we confirmed productive viral infection. We observed a correlation between higher number of EBV genome copies and the presence of transcripts specific for type III latency as well as clinical symptoms.
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