Detection and genotyping of human papillomavirus in hypopharyngeal carcinoma samples

Publisher Copyright: © 2021 Sciendo. All rights reserved.The incidence of hypopharyngeal cancer globally is about 0.8 per 100 000. Globally, approximately 38 000 cases of head and neck cancer are considered yearly to be high-risk human papillomavirus (HR-HPV) related. Biopsy material fixation in formalin and embedding in paraffin (FFPE) creates many challenges. The extraction of nucleic acid material requires a more complicated approach, and often the extracted DNA is fragmented. The aim of the study was to compare several HR-HPV detection methods in nucleic acid material extracted from FFPE samples. The extracted DNA was analysed with different molecular biology methods to assess DNA quality and to determine the presence of HPV DNA with various HPV detection systems. The results were compared and statistically analysed. There was good agreement between two real-time PCR methods — Anyplex II HPV28 and Sacace HPV High-Risk Screen Real-TM Quant. We failed to reach a conclusion on agreement between real-time PCR methods and HPV16 type-specific primer PCR. There was moderate positive correlation between Anyplex II HPV28 semiquantitative results and Sacace quantitative results. We suggest that real-time PCR assays detecting smaller DNA amplicons are good and reliable methods for detecting HPV genetic material in FFPE samples.Peer reviewe

Aqueous C60 fullerene solution effects on cell viability

Publisher Copyright: © 2021 Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.. All right reserved.Fullerenes are carbon nanoparticles with the ability to quench reactive oxygen species. The biomedical potential of fullerenes is diminished by their low solubility in water, but many approaches have been developed to bypass this problem, like chemical modification of the carbon cage and the use of the solvent exchange method to transfer fullerenes from one solvent to the other. These two approaches were used in this study. Carboxylated fullerene aqueous solution was acquired using solvent exchange method transferring fullerene nanoparticles (C60) from toluene to water. Effects of varying concentration (0.5, 1, 1.5, 2, 2.5, 3, 5, 10 µM) of aqueous fullerene solution on cell viability and their antioxidative capabilities were evaluated on PC-3 and on monocytes isolated from a blood donor using Resazurin Cell Viability Assay. PC-3 cell viability was drastically affected by the 10 µM fullerene solution but remained relatively stable when treated with other concentrations even after longer periods of incubation with resazurin dye. Elevated cell viability was observed in monocytes treated with various fullerene concentrations, possibly indicative of fullerene protective activity against oxidative stress.publishersversionPeer reviewe

Cellular immunity in human herpes viruses 6 and 7 infected gastrointestinal cancer patients

CD4+ T lymphocytes appear to be the preferential target for replication of HHV-6 (human herpes virus) as well as HHV-7 viruses in vivo. In addition, CD8+ T cells, monocytes/macrophages, natural killer cells, epithelial, endothelial, neural cells and fibroblasts may be infected. By definition, however, even a tumour designated by pathologists to be early stage may be late stage when considered by the immune system. Certainly, even early stage tumours have evaded immune control, suggesting that they have acquired many immunosuppressive characteristics. The aim of the study was to clarify the influence of beta-herpes viruses on cellular immune response. In 95 gastrointestinal cancer patients we determined the immunocompetent cell level CD3, CD4, CD8, CD19, CD38, CD95, CD25 using laser flow cytofluorimeter and B- herpes viruses HHV-6, HHV-7 presence using a nested polymerase chain reaction method. Our data showed no statistically significant difference in immunocompetent cell level between negative, latent and active HHV-6, HHV-7 infection. Patients with immunocompromised immune status (lymphopenia) had a tendency to decreased CD4+, CD19+ absolute count. It may be suggested that virus-mediated immune response inhibition seems to be similar to cancer mediated, but differences in immune response among the same group of individuals had no influence on the average number of the immunocompetent cells in the group. Therefore, to characterise host-virus-tumour interactions, individual interpretation of each case is needed.publishersversionPeer reviewe

Investigation of the Involvement of HHV-6 Encoded Viral Chemokine Receptors in Autoimmune Thyroiditis Development

Funding Information: We have no competing interests to declare. This work was supported by grant No.1.1.1.2/VIAA/1/16/202, Agreement No. 9.-14.5/ 257 “Human herpesvirus-6 involvement in development of autoimmune thyroiditis.” Publisher Copyright: Copyright © 2022 Sultanova et al.The study of human herpesvirus-6 (HHV-6) involvement in autoimmunity development is very challenging, due to the complex nature of this virus. HHV-6 is a ubiquitous, lifelong persistent, and immunomodulating virus, which mainly spreads in solid tissues using cell-to-cell mechanics, and thus can escape from the host’s immune response.Human herpesvirus-6 (HHV-6) contains two genes (U12 and U51) that encode putative homologues of human G-protein-coupled receptors like CCR1, CCR3, and CCR5. It has been shown that these viral proteins can be expressed on the surface of epithelial and some peripheral blood mononuclear cells, suggesting that they could potentially induce autoimmunity. We aimed to investigate the possibility of HHV-6 encoded viral chemokine receptors (U12 and U51) involvement in autoimmune thyroiditis (AIT) development by detecting viral peptide specific antibodies in AIT patient samples. Seventy-nine AIT patients whose thyroid tissues were shown to be positive for HHV-6 and 32 blood donors were enrolled in this study. Twenty-eight synthetic peptides derived from HHV-6 U12 and U51 proteins’ amino acid sequences, as well as recombinant human CCR1, CCR3, and CCR5 proteins were used in suspension multiplex immunological assay to detect specific IgG and IgM antibodies. HHV-6 peptide specific IgG and IgM antibodies were found in patients’ samples. AIT patients' samples were found to be more frequently positive for peptide IgGs in comparison to control group’s samples. Even though peptide antibody cross-reactivity with human CCRs was not demonstrated, our results show a new immunogenic HHV-6 antigen—a possible new player in the HHV-6 induced autoimmunity exacerbation. IMPORTANCE The study of human herpesvirus-6 (HHV-6) involvement in autoimmunity development is very challenging, due to the complex nature of this virus. HHV-6 is a ubiquitous, lifelong persistent, and immunomodulating virus, which mainly spreads in solid tissues using cell-to-cell mechanics, and thus can escape from the host’s immune response. It has been implicated as an environmental factor in several autoimmune diseases. An association between HHV-6 and autoimmune thyroiditis has been demonstrated, yet clear mechanism of involvement remains to be elucidated, since the virus can be detected in nearly all autoimmune thyroiditis patient thyroid glands. Our results show new potentially immunogenic human herpesvirus-6 antigens—possible new players in the HHV-6 induced autoimmunity exacerbation, which could be subjects for further research. Together with previously published results, this study described possible mechanisms which may underlie the induction of autoimmune reactivities against thyroid tissues in AIT.publishersversionPeer reviewe

A novel approach for nucleic acid delivery into cancer cells

Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background. Liposomal magnetofection is based on the use of superparamagnetic particles and cationic lipids and shows better transfection efficiency than other common nonviral gene delivery methods; however, the distribution of aggregate complexes over the cell surface may be ununiform. The use of a dynamic gradient magnetic field could overcome this limitation. A newly developed device for magnetofection under a dynamic magnetic field was used to compare the transfection efficiency of prostate carcinoma cell line PC3 with that obtained by lipofection and magnetofection. Material and Methods. Reporter plasmid pcDNA3.1LacZ DNA was used in combination with Lipofectamine2000 reagent and superparamagnetic nanoparticles CombiMag. The effects of incubation time under a dynamic magnetic field and a rotation frequency of magnets on transfection efficiency for PC3 cell line were determined. Alternatively, lipofection and liposomal magnetofection were carried out. Transfection efficiency of delivery methods was estimated by β-galactosidase staining; cell viability, by acridine orange/ethidium bromide staining. Results. Liposomal magnetofection under a dynamic gradient magnetic field demonstrated the highest transfection efficiency: it was greater by almost 21% and 42% in comparison with liposomal magnetofection and lipofection, respectively. The optimal incubation time under dynamic magnetic field and the optimal magnet rotation frequency were 5 minutes and 5 rpm, respectively. Liposomal magnetofection under a dynamic gradient magnetic field was less cytotoxic (7%) than that under a permanent magnetic field (17%) and lipofection (11%). Conclusions. Our new approach, based on the use of a dynamic gradient magnetic field, enhanced the transfection efficiency and had a less cytotoxic effect on prostate cancer cells in comparison with the standard magnetofection and lipofection.publishersversionPeer reviewe

Possible Involvement of Human Herpesvirus-6 U83 Gene Expression in Autoimmune Thyroiditis Development

Viral infections have been frequently cited as important environmental factors implicated in autoimmune thyroiditis (AIT) development, although no specific virus has yet been conclusively associated with the disease. Some evidence implicates human herpesvirus-6 (HHV-6) in this disease. The aim of this study was to investigate the role of the HHV-6 U83 gene expression in autoimmune thyroiditis development. Fifty-one patients with AIT following thyroidectomy and a control group of 30 autopsied subjects without thyroid pathologies for comparing virology results and 30 healthy blood donors for comparing serology results were enrolled in this study. HHV-6 U83 gene expression was determined using nested PCR with complementary DNA as the template acquired from thyroid gland extracted RNA. Plasma samples of AIT patients and blood donors were tested for IL-2, IL-4, IL-10, sTNF-RII and IL-1beta levels by ELISA. Virology results were compared with pro- and anti-inflammatory cytokine levels to determine possible interaction of HHV-6 with host immune response. HHV-6 U83 gene expression was found only in 24% (12/49) of AIT patient thyroid gland tissue samples and in none of the control group individuals, showing possible involvement of this gene in AIT development. However, no interaction between HHV-6 and changes in cytokine levels was found

Monocytes/Macrophages Act as Mediators for Human Herpesvirus-6 Infection of Thyroid Gland in Patients with Autoimmune Thyroiditis

The aim of this study was to investigate the possibility of using monocytes/macrophages as mediators in human herpesvirus-6 (HHV-6) infection of thyroid gland tissues in autoimmune thyroiditis (AIT). Seventy-three AIT patients were enrolled in this study. The control group consisted of 80 blood donors. Monocyte/macrophage isolation for AIT patient samples was performed by adherence. HHV-6 was detected in peripheral blood mononuclear cell (PBMC) DNA samples using nested polymerase chain reaction (nPCR). Gene expression of HHV-6 active infection marker (U79/80) and chemokine receptors (U12, U51) in patient monocyte/macrophage samples and blood donor PBMC samples was detected using reverse-transcription PCR. HHV-6 viral load was detected by using quantitative-PCR technique. The HHV-6 genomic sequence was found significantly more frequently among AIT patient than control group samples. Markers of active infection were found in 8 AIT patient monocyte/macrophage samples (11%) and in none of control group PBMC samples. HHV-6 U51 mRNA expression was detected only in AIT patient samples (2/24 previously positive for HHV-6). Since HHV-6 genomic sequences were found significantly more frequently in AIT patient samples and active infection markers were found in patient monocytes/macrophages, our results suggest that monocytes/macrophages may be used by HHV-6 as mediators for thyroid gland infection

Effect of HHV-6 and HHV-7 Infection on the Posttransplant Process and the Development of Complications in Patients after Autologous Stem Cell Transplantation

Publisher Copyright: © 2016 by Ilze Trociukas.The relationship between HHV-6 and HHV-7 reactivation and development of post-autologous peripheral stem cell transplantation complications was examined. The presence of viral genomic sequences in whole peripheral blood and cell free plasma was determined by nested PCR, HHV-6 and HHV-7 load by real-time PCR, virus specific antibodies and cytokines in serum by ELISA, and HHV-6 variants by restriction endonuclease analysis. Clinical features, reactivation of viruses and serum TNF-α, and IL-6 concentrations were determined in seventy-six patients with Roseolovirus infection before and after transplantation. Anti-HHV-6 antibodies were found in 62 of 76 (81.6%) patients before transplantation. A significantly higher rate of single HHV-7 infection was found in patients with viral infection in comparison with single HHV-6 infection (p = 0.0003) and concurrent (HHV-6 and HHV-7) infection (p = 0.0017). Complications after transplantation developed in 30.3% of patients and reactivation of viruses was detected in all of these patients. Significant increase of HHV-6 and HHV-7 reactivation with simultaneous increase of pro-inflammatory cytokines serum levels suggests that both viruses may be involved in the development of complications after autologous peripheral blood stem cell transplantation via their immunomodulatory ability. The kinetics of the Roseolovirus reactivation may reflect the potential role of HHV-7 as a co-factor for HHV-6 activation.publishersversionPeer reviewe

High-Risk Human Papillomavirus Infection in Latvian Male Kidney Transplant Recipients

Publisher Copyright: © 2016 by Maksims Cistjakovs.Kidney transplant recipients have higher incidence of human papillomavirus (HPV)-related malignancies, but studies on the natural history of HPV infection are insufficient, especially regarding in male recipients. The aim of this study was to evaluate the course of high-risk HPV (HR-HPV) infection after kidney allograft transplantation in male recipients: to estimate frequency and activity of HR-HPV infection under immune system suppression. Twenty male renal recipients (age 20-68) were enrolled in this investigation and examined in dynamics. Peripheral EDTA-blood samples and urine samples were collected from each patient 2 weeks, 6 months and 12 months after transplantation. Polymerase chain reaction (PCR) with consensus primers was used for initial detection of high range HPV types, a commercial qPCR kit for detection of HR-HPV load in urine samples and ELISA for detection of serum IgG class antibodies to HR-HPV L1-capsid protein. Overall, combining molecular (HR-HPV genomic sequences detected by real-time PCR) and serological studies (IgG class antibodies to HR-HPV L1-capsids' protein), high frequency of HRHPV infection among male kidney transplant recipients (9/20; 45%) was showed. However, the majority of HR-HPV positive recipients (7/9; 78%) showed signs of infection clearance. It means that, despite the applied immune suppressive therapy, the host's immune system is capable of dealing with HR-HPV infection up to the 12th month after transplantation. However, the sample size should be increased to enable through statistical analysis before final conclusions are made.publishersversionPeer reviewe

Semiotic analysis and representation of Batman image in comic books and movies

Bakalaura darba tēma ir „Betmena tēla reprezentācija: komiksu grāmatu un filmu semiotiskā analīze”. Darbs sastāv no teorētiskās daļas, kurā tiks apskatīti nepieciešamie jēdzieni un teorijas par semiotiku, naratīvu, kinematogrāfiju, varoņiem, grafiskajām novelēm un komiksu grāmatām, metodoloģiskas daļas, kurā tiks aprakstītas metodes, semiotiskā un diskursa analīze, un no empīriskās daļas, kurā tiks veikta filmu un komiksu grāmatu un grafisko noveļu analīze. Pētījuma mērķis ir analizēt Betmena tēlu kinofilmās un komiksos, saprast un izdarīt secinājumus par to, kā Betmena tēls un viņa pasaule filmās atšķīras no komiksiem, kā arī saprast, kādas semiotiskas un diskursa nozīmes var atrast filmās un komiksu grāmatās par Betmenu. Darbs beidzas ar secinājumiem. Atslēgvārdi: Betmens, grafiskā novele, komiksi, diskursa uzbūves un semiotiskā analīze.The theme of this bachelor paper is „Semiotic analysis and representation of Batman image in comic books and movies”. This research consist of theoretical part, which includes theory and definitions of semiotics, narrative, cinema, hero, comic book, graphic novel, a methodological part, which describes methods of research that are used in this work: a semiotic and discourse analysis and empirical part, which includes semiotic and discourse analysis of the movies and comic books and graphic novels. The goal of this work is to analyze the image of Batman in comic books and movies, as well as to understand how his image from movies is different from his image in comic books and to understand which semiotic and discourse meanings can be found in every comic book or movie about Batman. The conclusions are provided in the end of the work. Keywords: Batman, graphic novel, comic book, discourse buildings tasks, semiotic analysis